SEPARATION SCIENCE SERIES

Editors: Raymond P.W. Scott and Colin Simpson
Quantitative Analysis using
Chromatographic Techniques
Edited by Elena Katz
The Analysis of Drugs of Abuse
Edited by Terry A. Gough
Liquid Chromatography Column Theory
by Raymond P.W. Scott
Liquid Chromatography
Column Theory
by
Raymond P.W. Scott
Chemistry Department, Birkbeck College, University of London, UK
and
Chemistry Department, Georgetown University, Washington DC, USA
JOHN WILEY & SONS
Chichester . New York . Brisbane . Toronto . Singapore
Contents
Preface
Acknowledgements
1 Introduction to the Liquid Chromatographic Column .
The Factors that Control Retention and Selectivity .
Ionic Interactions .
Polar Interactions .
D
· . Itt'
lsperslve n erac Ions .
The Physical Nature of the Column .. , .
The Column Environment .
Chromatography Nomenclature .
LC Column Theories .
References .
2 The Plate Theory .
The Solution of the Differential Equation .
The Retention Volume of a Solute .
The Capacity Ratio of a Solute .
The Separation Ratio .
References .
3 The LC Column Dead Volume .
References _ .
4 Extensions of the Plate Theory .
The Elution Curve of a Finite Charge .
Peak Asymmetry .
IX
Xl
1
4
4
5
5
6
8
9
12
13
15
19
22
25
26
26
27
38
39
39
41
'I
Column Efficiency 45
The Position of the Points of Inflection 48
The Gaussian Form of the Elution Equation 50
References 52
5 Applications of the Plate Theory .. . . . . . .. . .. . . .. . .. . . . . .. . . . . . . . . . . . . .. . . . . . 53
The Maximum Sample Volume 53
Vacancy Chromatography........ 55
The Resolving Power of an LC Column 60
The Effective Plate Number 63
The Peak Capacity of a Chromatographic Column 67
Precision as an Alternative to Resolution 74
A Theoretical Treatment of the Heat of Adsorption Detector 77
References 90
6 Introduction to the Rate Theory . .. . . . . . .. . .. . . .. . .. . . . . . . . . . . . . .. . . . .. . . . . . 93
The Summation of Variances 94
Extra Column Dispersion 95
The Alternative Axis of a Chromatogram 96
The Random Walk Model " . . . .. . .. . . .. . . . . . . . . . . .. . . . .. . . . . . 98
Dispersion Processes that take Place in an LC Column 102
The Multipath Process 102
Longitudinal Diffusion 103
The Resistance to Mass Transfer in the Mobile Phase 105
The Resistance to Mass Transfer in the Stationary Phase . . .. . . . . . 106
References 106
7 The Van Deemter Equation . .. . . . . . . . .. . . .. . . .. . .. . . .. . . . . . . .. .. . . . . .. . .. . . . . 109
References 121
8 Alternative Equations for Peak Dispersion 123
The Giddings Equation 123
The Huber Equation 124
The Knox Equation 126
The Horvath and Lin Equation . . . . . . . . .. .. . . . .. . .. . . . . .. . . . . . .. . . . . .. . . .. . . 128
The Golay Equation 128
References 133
9 Experimental Validation of the Van Deemter Equation 135
The Effect of the Function of k' on Peak Dispersion 149
References 152
10 Extra Column Dispersion .
The Effect of Sample Volume .
The Sample Valve , .
Connecting Tubes .
Low Dispersion Connecting Tubes .
Serpentine Tubes " .
Column Frits .
Dispersion in the Detecting Cell .
Effect of Extra Column Dispersion on Column Radius .
Mass Sensitivity , , .
References .
II LC Column Design - The Design Protocol .
Performance Criteria .
The Reduced Chromatogram , .
Instrument Constraints .
Elective Variables " .
Column Specifications and Operating Conditions .
Analytical Specifications .
12 LC Column Design - The Design Process for Packed Columns .
The Optimum Particle Diameter .
The Optimum Column Length .
The Minimum Analysis Time .
The Optimum Column Radius .
The Optimum Flow Rate .
The Minimum Solvent Consumption .
The Peak Capacity of the Optimized Column .
Maximum Sample Volume .
The Optimum Capacity Ratio .
Packed Column Design Equations .
Computer Program for Packed Column Design .
Gradient Elution .
References .
13 LC Column Design - The Design Process for Open Tubular
Columns .
The Optimum Column Radius .
The'Optimum Length of an Open Tubular Column .
Minimum Analysis Time .
The Optimum Flow-Rate .
VB
153
153
154
154
158
161
164
164
167
172
173
175
176
177
179
181
182
183
185
188
192
194
196
199
201
202
204
204
206
208
212
213
215
218
221
222
224
17111
Maximum Sample Volume and Maximum Extra Column
D
· .
Isperslon .
The Maximum Permissible Detector Dispersion .
Design Equations .
The Open Tubular Column in LC ..
References .
14 Preparative Liquid Chromatography Columns .
Preparative Column Design .
The Efficiency Required from the Preparative Column .
Optimum Particle Diameter .
The Column Length and Analysis Time .
The Column Radius .
The Optimum Flow-Rate .
Solvent Consumption .
Column Wall Thickness .
Preparative Column Design Equations , .
The Use of the Design Equations .
Computer Program for Preparative Column Design
D
· .
ISCUSSlon .
Column Overload as an Alternative to Column Design .
Sample Volume Overload .
Sample Mass Overload .
References .
List of Symbols .
Appendix 1 The Diffusion Coefficients of some Low Molecular
Weight Substances " .
Appendix 2 The Diffusion Coefficients of some Peptides
226
230
231
233
234
237
238
238
239
241
244
247
248
248
250
251
252
253
259
259
261
263
265
268
270
Appendix 3 The Physical Properties of some Solvents
Employed as Mobile Phases in LC 271
Index .. I • .. • • • I I • .. .. • I • • • .. I • .. • • I I • • • • I • • I • .. I ~ • • .. • • • • .. • .. • • .. • • • .. • -II .. • • • • II • • .. .. .. I • • • • ... .. I I .. • • • I • 273
Index
adjusted retention time, definition, 11
retention volume, definition, 11
adsorption, detector, heat, equation for
output, S3
equation for on column detection. 87
theory of, 77
isotherms, 43
alumina, coated stationary phase, 2
limitations as an LC stationary phase, 2
analysis time, approximate for packed
column, 117
minimum, optimized open tube
columns. 222
equation fOL 223
minimum. optimized, packed columns.
194
equation for, 195
minimum, optimized, preparative
columns, 242
equation fOL 242
particle diameter, 121
analytical specifications for optimum
column, 183
asymmetry, peak, 41
available stationary phase, definition, 30
axis of a chromatogram, 96
base line, definition, 10
capacity peak, 67
and capacity factor, 71
equation for, 70
optimized column, 20
equation for, 203
ratio, 25
calculated from, exclusion volume,
151
permeating volume, 150
effect on dispersion, 149
equation for, 25
explicit equation for, 32
cell detector, dispersion effect, 164
design for low dispersion, 166
volume, effective, heat of adsorption
detector. 79
charge, finite, elution curve of. 39
chromatogram, axes of, 96
reduced, 177
chrornatorgaphy. nomenclature, 9
vacancy 55
,
applications. 59
equation of elution curve, 59
column, capillary, 6
characteristics of, 1
dead volume, 10,27
design, open tubular volumns, 215
optimized open tube columns, 231
program for, 232
optimized packed columns, 206
program for. 20R
optimized preparative columns. 250
program for, 252
packed, 185

... _,
optimum particle diameter, 239
diameter versus particle diameter, 171
dispersion. extra, 153
packed. 102
longitudinal diffusion. 103
equation for. 104
L./'f
dispersion (cont.)
mul tipath , 102
equation fOL 10.3
resistance to mass transfer, mobile
phase, 106
stationary phase, 106
environment, 8
efficiency, 45
equation for, 46, 47
frits, contribution to dispersion. 164
flow rate. defini tion , 10
optimum for open tube, 224
optimum for packed, 199
optimum for preparative, 247
heat capacity, 8
length, approximate for packed
column, 117
optimum for open tube column, 221
equation for, 222
optimum for packed column, 192
equation for, 192
optimum for preparative column,
241
equation for, 241
particle diameter for maximum
efficiency, 120
particle limits, 8
material of construction, 6
open tubular, optimum, design, 215
flow rate, 224
equation for, 225
radius, 218
equation, 219
velocity, 217
minimum HETP, 217
limitations. 6, 233
overload, 259
packed, 6
optimum, specifications for. in
column design, 182
physical nature of, 6
properties of Zorbax column, 37
purpose of, 3
quartz. availability, 7
radial equilibrium, 100
radius, dependance on extra column
dispersion, 167
equation for, 169, 170
optimum, open tubular, 218
equation for. 219
packed, 196
equation fOL 198
preparative, 244
equation for, 246
resolving power, 60
equation fOL 62
the ories , 12
thermostat. 8
volume, total, expression for, 28
wall thickness, limitations. 8
preparative, 248
weight, limits, 8
computer program for the design of open
tube columns, 232
report for a packed column design, 233
computer program for the design of
packed columns, 208
report for a packed column design, 210
computer program for the design of
preparative columns, 252
report for a packed column design. 256
connecting tubes, dispersion, cause. 154
equation for, 155
length versus variance, 157
low dispersion, 158
coiled tubes, 159
Hiu curves, 161
serpentine tubes, 161
H!ucurves, 162. 163
pressure drop across, 156
constraints. instrument, in column
design, 179
criteria for column performance. 176
dArcys law. 116
dead, point, definition. 10
time definition, 10
volume, definition, 10
discussion of, 27
kinetic, 31
thermodynamic. 32
design. equations for open tube columns,
231
program for. 232
equations for packed columns, 206
program for. 208
equations for preparative columns, 250
program for, 252
open tubular columns. 215
packed columns, 185
preparative columns. 237
protocol for columns. 175
detector, cell, design for low dispersion,
166
dispersion, effect on, 164
heat of adsorption, theory. 77
equation of output, 83
equation for on column detection,
87
differential equation for the elution
curve, 18
diffusion coefficients, list of, 268
peptides 270
diffusivity relation to, longitudinal
diffusion, 143
minimum HETP, 145
resistance to mass transfer, 147
dipole, induced, 5
interactions. 5
dispersion, detector cell dispersion,
effect on, 164
effect of sample volume, 153
extra column. 6, 95, 153
packed column, 102
radial, 99
equation for, 100
dispersive forces. 5
dispersive interactions, 5
distribution, 4
coefficient. 16
factors controlling, 4
effective. cell volume, heat of adsorption
detector. 79
plates. 63
equation for, 64
relation to equilibrium plates. 65
efficiency, column. 45
equation for. 40. 4.7
particle diameter for a packed column.
119
radius for an open tube column. 132
elective variables in column design. 1K1
elution curve equation, 19.21
derivation. 16
finite charge, 39
Gaussian form, 50
equation for. 52
solution of differential form. 19
gradient. 213
profile. of iow a ~ p e c l ratio LUbe. i65
environment. column. 8
equation. dArcys. fluid flow through a
packed bed. 116
design for optimized packed column,
200
i./.J
efficiency required to effect a given
separation, 62
elution curve. 19.21
derivation. l6
Gaussian form, 50
equation of. 52
finite charge, 39, 40
Giddings. dispersion in a packed
column, 123
Golay, for dispersion on open tubes.
128
heat of adsorption detector, ~ 3
with on column detection. 87
Huber. for dispersion in a packed
column. 124
Horvath and Lin, for dispersion in a
packed column, 128
Knox. for dispersion in a packed
column. 126
mass sensitivity. 172
Poiseuillcs , fluid flow through a tube.
132
Van Deernter, 109
detailed, 110
equilibrium, radial, column. 100
extra column dispersion. 6, 95,153
effect on column radius, 167
factors that control retention and
selectivity. 4
finite charge, elution curve of, 39
flow rate, definition, 10
for optimized open tube column. 224
equation for. 224
for optimized packed column. 199
equation for. 199
for optimized preparative column. 247
equation for. 247
frits. column. contribution to dispersion.
164
Giddings. equation for dispersion in a
packed column. 123
Golay, equation for dispersion in open
tubes. 128
gradient elution, 212
heat of adsorption detector. equation of
output. 84
equation for on-column detection, 87
theory. 77
height. peak. definition. 11
Lib
HETP. curve. for open tubes, 129
for packed columns. III
van Dccmtcr, 138
definition of. 97
equation. for an open tube column. 128
for a packed column. 110
for an open tube. uncoated. 129
minimum, dependance on diffusivitv.
145
packed column. 115
open tube column. U 1.217
Horvath and Lin. equation for dispersion
in a packed column. 128
Huber. equation for dispersion in a
packed column. 124
induced dipoles. 5
inflexion points, of elution curve. 48
equation for. 49
injection point. definition. 9
instrument constraints. in column design.
179
interstitial volume. definition of. and
expression for. 29
effect of ion volume of marker, 34-
static, 29
. )9
movmg. _
ion volume. effect on interstitial volume.
34
ionic interactions, 4
isotherms. adsorptions. 43
effect on peak shape. 44
kinetic dead volume. 31
Knox. equation for dispersion in a
packed column. 126
length. column, approximate for packed,
117
optimum, for open tube. 221
equation for. 221
optimum, for packed. 192
equation for. 1<J2
optimum for preparative. 241
equation for. 241
versus particle diameter, maximum
efficiency. 12l!
versus radius for maximum efficiency.
132
line. base. 10
list of svrnbols. 2h)
longitudinal diffusion. and diffusivity.
I-B
packed column. 103
equation for, 104
low aspect ratio tube. elution profile of.
165
dispersion connecting tubes. 158
coiled tubes. 159
serpentine tubes. Ih I
detector cell design. 16h
mass sensitivity. 172
overload. 261
maximum. efficiency. length and particle
diameter. 12U
ext! a column dispersion. open tube
column.22h
equa tion for. 228
peak. definition. 11
sample volume. 53
equation for. 54
optimized open tube. column. 22h
equation for. 228
optimized packed. column. 204
equation for. 204
minimum. analysis time. optimized
column, 194
equation for. 1<J5
HETP, packed column. 115
relation to diffusivity, 146
open tube column, 217. 131
solvent consumption. for optimized
packed column. 20I
mobile phase. 4
volume of. expression for. 2<J
molecular interactions. 4
size. effect on retention volume. 36
moving phase, definition. 29
multipath dispersion. 102
equation for. 103
and solute diffusivitv, 141. 142
nomenclature, chromatography. t)
open tubular columns. 128
limitations. 233
optimum design. 215
optimum length. 221
equation for. 221
optimum radius. 218
equation for. 219
operating conditions for optimum
column, in column design, 182
optimum. column, capacity ratio.
packed. 204
flow rate, open tube. 224
equation for, 225
packed, 199
equation for. 199
preparative. 247
equation for. 247
length, open tube, 221
equation for, 221
packed. 192
equation for. 192
preparative. 241
equation for. 241
particle diameter. packed. 188
equation for. 189
preparative. 239
equation for. 239
peak capacity. packed. 202
equation for. 202
radius. open tubular. 218
equation for, 219
packed. 196
equation for. 198
preparative, 244
equation for. 246
velocity, open tube column. 130, 217
packed column. 113
approximate. 114
relation to diffusivitv. 146
overload. column, 259
mass. 261
volume. 259
packing. factors, 103. 104. 112
quality of. 127
particle diameter. effect on analysis time,
121
minimum. versus particle diameter.
171
optimum, for packed column, 188
equation for. 189
for preparative column, 239
equation for. 239
versus efficiency for a packed column.
119
peak, asymmetry. 41
capacity. 07
effect of capacity factor. 71
L. I I
equation for. 70
detector sensitivity. effect of. 73
optimized column, 202
equation for. 203
definition. 9
height. definition. 11
maximum. definition. 11
shape, effect of isotherm shape . ..t..t
width. definition. 11
measurement of. 50
equation for . .fO
performance criteria. for column design,
176
peptides. diffusion coefficients. 27D
phase. mobile, .f
stationary . .f
plastic unions. 7
plate. effective plates, 63
equation for, 64
relationship to equilibrium plates, 0)
height. 97
height. reduced, 1.26
theory. 12. IS
theoretical, definition. 17
volume. definition, 18
point. of inflexion. of the elution curve.
48
position. 49
of injection, definition, 9
point. dead, 10
Poiseuilles equation. 132
Poisson equation. form of elution CUrve.
equation for. 21
axes of. 51
polar forces, 5
interactions, 5
pore volume, definition of. and
expression for. 29
power. resolving. of column, 60
eq uation for. 62
precision as an alternative to resolution.
74
preparative columns, 237
design, 238
optimum particle diameter. 239
equation for. 240
optimurn col umn le ngth , 24 i
equation for. 242
program for the design of. open tube
columns. 232
packed columns. 208
program for the design of (cont.)
preparative columns. 252
protocol for column design. 175
Purnell equation, 62. I t l 5 ~
quality of packing and reduced plate
height. 127
quartz columns. availability. 7
random walk model. equation for. 98
radial dispersion. 99
equation for. 100
radial equilibrium. column. 100
radius. column. effect of extra column
dispersion. 167
packed. equation for. 169. 170
optimum. 196
equation for. 19S
optimum. open tubular columns, 218
equation. 219
packed column. 196
equation. 198
preparative column. 244
equation. 246
rate theory. 12. Y3
validation data. 139
equations. 135
ratio. capacity. 25
calculated from, exclusion volume.
151
permeating volume. 150
equation for. 25
explicit equation for. 32
optimum. 204
phase. 25
equation for. 25
separation. 26
equation for. 26
reduced. chromatogram. 177
plate height. 126
velocity. 126
report from computer design. of an open
tube column. 233
of a packed column. 210
of a preparative column. 256
resistance to mass transfer. packed
column. mobile phase. 105
equation. 106
relation to diffusivity. 147
particle size. 148
stationary phase. 106
equation. 106
resolving power, ot column. bU
equation for. 62
Giddings definition. 65
relationship to effective plates. 66
terms of minimum separation ratio.
66
terms of effective plates. 66
resolution. column. see resolving power
retention. factors controlling. 4
time, adjusted, definition. 11
definition. 11
volume. adj usted. definition. II
equation for, 24
volume, definition. 11
derivation of equation for. 22
effect of molecular size of solute. 36
" f '1'1,
equation or. -,-,"
explicit equation for. 31
peak composition. 76
sample volume. effect on dispersion. 153
maximum. 53
equation for. 54
optimized open tube column. 226
equation for. 228
optimized packed. column. 20..+
equation for, 204
overload,25Y
selectivity. factors controlling. 4
sensitivity, mass. 172
separation ratio. 26
equation for. 26
plates required, 6(1
equation for. 62
serpentine tubes. low dispersion, 161
SFC.6
silica gel. importance to liquid
chromatography. :::
specifications. analytical. for optimum
column. 1~ ~ . lX3
solvent. minimum consumption. for
packed columns, 201
eqnation for. 20I
for preparative columns. ~ 4 X
equation for. 2"+8
physical properties of. 271
static phase. definition. 29
stationary phase. 4
summation of variances, 94
symbols, general, chromatographic, 12
list of. 265
thermostat, column, 9
theoretical plate, definition, 17
theory, plate, 12. 15
theory, rate, 12, 93
thermodynamic dead volume. 31
time, analysis, approximate for a packed
column i l l?
minimum for open tube column, 222
equation for. 223
minimum, for packed column. 194
equation for. 195
minimum for preparative column,
241
eq uation for, 242
particle diameter. 121
dead, definition, 10
retention. definition, 11
adjusted rete ntion. definition, 11
Tswett. inventor of chromatography, 1
tubes, connecting, effect on dispersion,
154
length versus variance, IS?
low dispersion, 158
coiled tubes, 159
serpentine tubes, 161
pressure drop across. 156
low aspect ratio, elution profile of, 165
unavailable stationary phase, definition,
30
unions, plastic, 7
vacancy, chromatography, 55
applications, 59
equation for elution curve, 59
validation data for rate equations, 139
valve. sample, elution profile. 152
variance, summation of. Cl4
per unit length, q7
equation for, 97
Van Deemter equation. 109
H versus u curves, 138
\)qlirhtinn nf 1-;"
........... - _.... , , ~ . ... ~ - ~ - ~ -
variables, elective, in column design. 181
velocity. mobile phase optimum, open
tube column, 217,131
optimum, packed column, 113
279
velocity, mobile phase, approximate
value, 114
reduced. 126
volume, finite charge, curve for. 39
column, total, expression fOL 28
dead, definition. 10
discussion of, 27
kinetic, 31
thermodynamic, 31
effective cell. heat of adsorption
detector, 79
interstitial. definition of, and
expression for. 29
effect of ion volume of marker, 34
moving,29
static, 29
mobile phase, expression for. 29
plate, definition, 18
pore, definition of. and expression for,
29
retention, adjusted, definition. 11
equation tor. 2-l
definition. 11
derivation of equation for. 22
effect of molecular size of solute, 36
equation for, 23
explicit equation for, 51
sample, effect on dispersion, 153
maximum, 53
equation for, 54
optimum, open tube, 226
equation for, 228
optimum, packed, 204
equation for, 204
overload, 259
stationary phase , available, definition,
30
expression for, 30
unavailable, definition, 30
wall thickness. preparative column.
248
width, peak. at base, definition, 11
at half height. definition, 11
definition, 11
equation for, 46
measurement of. 50
Zorbax column, physical properties of.
37
Chapter 1
tntrecucnen to the L1qu1d Chromatograph1c Column
The column ts the heart of the llquld chromatograph. It ts a tube, usually a
few centimeters long and less than a centimeter wide, packed with
particles a few m1cron 1n d1ameter. In fact, the column used today is utue
changed from that orlglnally used by Tswett (1), the inventor of
chromatography, nearly a century ago. Tswett would probably recognize the
modern llquld chromatography (LC) column for what tt was, though it 1S
doubtfullf any other parts of the modern chromatograph would make sense
to ntrn However, the contemporary LC column, though smaller and packed
with different mater1als from those used by Tswett, gives a performance
many orders of magnitude greater. The reasons for tnts tmorovernent are
somewhat subtle, but 1t must be ernpnastzeo that 1t has been reauzec as a
result of the development and aopltcatton of ltqutd chromatography column
theory -the suoject of tnts book. The modern LC instrument ts, indeed,
trnpresstve. with its fully automatic multi-solvent programer and sample
injector, together w1th lts vartaole wavelength detector, recorder and
assoc1ated computer. However, all these complex devices are there, merely
to serve the column. Without the column the carefully des1gned ecutornent
would have no purpose and no value. In fact, the clever tnstrument destqn,
that has resulted 1n the h1gh performance that is cnaractertsttc of the
modern llquid chromatograph, has been a direct result of the practical
eppltcatton of LC column theory.
In nts or1g1nal Tswett exam1ned a number of different column
but the vast majority of packlng materials that are in use in LC
today, are based on silica gel. This is true, even for the recently introduced
carbon based packings (2) as they also require silica gel for their
taortcatton Without stuca gel LC would have avery limited performance and
very urntteo areas of appttcatton Furthermore, until some substance is
found that 1s more effect ive than sntca ge1, or some other column type that
requires no packing is developed to a state of general practical use, stltca
gel will remain the one common component of most practical LC column
systems. It is true that alurntna can be employed as a statlonary phase, but
the performance of alumina columns have, until recently, proved to be
grossly inferior to those of silica gel. Moreover, stable bonded phases (the
packing most widely used in LC tooav) cannot apparently, be formed by
direct reaction With alumina. However, a recent product, consisting of
spherical particles composed of fused porous alum1na plates coated wtth
polybutadiene has shown prorntstnq signs of belng the first really effect we
alumina-based LC packing. This is a untcue material that has not been fully
evaluated at this time. Amicrograph of this materia1is shown in figure (1 ).
Figure 1
Micrograph of an LC Packing Consisting of Polybutadiene Coated
By the courtesy of BIOTAGE Inc.
sutca gel has properties that are uniquely suitable for use as a column
packing in LC and consequently, its introduction was one major factor
responsible for the great difference in performance between modern LC
columns and those of Tswett. TIle effect of column dimensions and particle
diameter on column performance is less obvious and, as wlll be seen later,
will require some extensive discussion.
The LC column performs a dichotomy of purpose which must be completely
understood before attempting to start any derivation of LC column theory.
During the development of a chromatographic separation, two processes
occur simultaneously and to large extent independently. Firstly, the
individual solutes in the sample are moved apart in the column as a result of
their different affinities for the stationary phase. Those solutes that
interact strongly wah the stationary phase beIng retained In the column to
a greater extent than those solutes that interact more strongly with the
rnobtle phase. Secondly, as the bands are moved apart, they spread or
disperse and tend to merge together, blurring the separation that has been
obtained. The column, by appropriate design, must minimize this dispersion,
so that, having been moved apart and separated, the individual solutes enter
the detector as individual bands. Thus, to obtain maximum resolution, the
column must move the bands as far apart as possible but, at the same time,
keep each band as narrow as possible.
The factors that control separation and dispersion are qutte different. The
relative separation of two solutes is solely dependent on the nature and
rnaqnttude of the interactions between each solute and the two phases. Thus,
the relative movement of each solute band would appear to be independent
of column dimensions or particle geometry and be determined only by the
choice of the stationary phase and the mobile phase. However, there is a
caveat to this statement. It assumes that any exciuston properties of the
stationary phase are not included in the term 'particle geometry'. The pore
size of the packtnq matertat can control retention directly and exciusiveiv,
as in exclusion chromatography or, indirectly, by controlling the access of
the solute to the statIonary phase in normal and reverse phase
chromatography. As all stattonary phases based on silica gel exhibit some
exclusion properties, the ideal srtuanon where the seiective retention of
two solutes is solely controlled by phase interactions 1S rarely met in
practice. If the molecular size of the solutes differ, then the exclusion
properties of the silica gel will always play some part in solute retention.
The Factors that Control Retention and serecttvtty
The theory of solute retention, as controlled by molecular 1nteractions
between the solutes and the phase system is, in fact, not germane to the
SUbject of trus book. Nevertheless, as otstrtoutton and distribution
coefflc1ents together with retentton volumes and caoacttv ratlos wl1l be
discussed or used in the subsequent theoretical development of column
theory, the basic principles of molecular interactton will be g1ven.
The mechanism of solute retention 1s best cons1dered by rtrst def1n1ng a
chromatographic separatton Def1ned 1n the ciasstcai manner, a
chromatographic separation ts achieved by the distribution of the solute
mixture between two phases, a stationary phase and a mobile phase. Those
solutes d1str1buted preferent1ally tn the mob1le phase w1ll pass through, or
from the system, more rapidly than those substances that are distributed
preferentially in the stationary phase. As a consequence the solutes will be
eluted in the order of the magn1tude of the1r distribution coefficients with
respect to the stationary phase. Th1s dertnttton, although perfectly correct,
tends to obscure the basic process of retention in the term distribution. A
solute is dlstr1buted between two phases as a result of the relative
magn1tude of the molecular forces that exist between the solute molecules
and those of the two phases. Consequently, the stronger the forces between
the solute molecules and those of the stationary phase the more the solute
will be retained. Conversely, the stronger the interactions between the
solute molecules and the mobile phase the more rapidly will the solute pass
through the column. Thus) solute retention is controlled by molecular forces
of which there are three basic types, ionic forces J polar forces and
dispersive forces. There could be considered a third type of intermolecular
interaction, hydrogen bonding" but for the purpose of this discussion, forces
due to hydrogen bond1ng will be classed as strong polar forces,
IQot, J n t e r a c ~
Ionic interactions result from permanent electrical charges that exist on
molecules, for example ionic materials such as salts. Such interactions are
exploited in ion exchange chromatography, in particular, for the separation
5
of organlc acids and bases. It follows that to retain anionic materials in a
chromatographic system, the stationary phase should contatn cations.
Conversely, to retain cationic materials, the stationary phase should
contain anions. The stationary phase can consist of an ion exchanger proper,
such as an ion exchange resin or can take the form of an adsorbed ion
exchanger on the surface of a reverse phase, such as an alkyl sulphonate.
~
Polar interactions between molecules arise from permanent or induced
dipoles existing in the molecules and do not result from permanent charges
as in the case of ionic interactions. Examples of polar substances having
permanent dipoles would be alcohols, ketones, aldehydes etc. Examples of
polarizable substances would be aromatic hydrocarbons such as benzene or
toluene. It is considered that, when a molecule carrying a permanent dipole
comes into close proximity to a polarizable molecule, the field from the
molecule with the permanent dipole induces a dipole in the polarizable
molecule and thus electrical interaction can occur. It follows that to
selectively retain a polar solute, then the stationary phase must also be
polar and contain, perhaps, hydroxyl groups. If the solutes to be separated
are strongly polar, then perhaps a polarizable substance such as an
aromatic hydrocarbon could be employed as the stationary phase. However,
to maintain strong polar interactions with the stationary phase (as opposed
to the mobile phase) the mobue phase must be relatively non-polar or
dispersive in nature.
.DispersiVe l o t e r a c ~
Dispersive interact ions are more difficult to describe. Although electric in
nature, they result from Charge fluctuations rather than permanent electric
Charges on the molecule. Examples of purely dispersive interactions are the
molecular forces that exist between hydrocarbon molecules. n-Heptane is
not a gas due to the collect tve effect of all the dispersive interactions that
hold the molecules together as a liquid. To retain solutes selectively, solely
on the basis of dispersive mteractions, tne stationary phase must not
contain polar or ionic substances but only hydrocarbon-type materials such
as the reverse -bonceo phases now so popular in LC It follows that to allow
dispersive selectivity to dominate in the stationary phase, the mobile phase
6
must be polar and s1gnificantly less dIspersive, Henr.e the use of methanol-
water and acetonitrile-water mixtures as mobile phases in reverse-phase
chromatography systems. It should be pointed out that it is rare, that in any
distribution system, only one type of interaction is present and, if it ts, tt
wm certainly be dispersive in nature. Polar interactions are always
accompanied by dispersive interactions and ionic interact ions will, in all
probablllty, be accompanied by bothpolar and dispersive interactions.
The Physical Nature of the Column
.I
All practtcal, contemporary, LC columns are tuoutar in shape and all
commercially available columns, at the time of writing this book, are
packed. ~ o r k is being carried out to develop a satisfactory open tubular
column for LC but, so far, none has exhibited the necessary loading capacity
or resolving power. They have been used with some success in super critical
fluid chromatography (SFC), however, the subject of SFC is outside the
scope of this book. At present the use of capillary columns in LC is severely
handicapped by, firstly, the lack of a high sensitivity, low dispersion
detector, and secondly, by the lack of associated apparatus that has
sutnctenttv low extra column dispersion. It wi 11 be seen later, that the
width of a solute band, eluted from a capillary column, in terms of volume
of eluted mobile phase, is extremely small. Consequently, any extra column
dtsperston that occurs must be reduced to a fraction of a microliter or less
if the resolution obtained from the column is not to be lost. This problem
will be subsequently discussed in detail when dealing specifically with the
suoject of extra column dispersion. However, at this point, it can be said
that practical capillary LC. columns may not become a viable commercial
product for some time to come and, even when avauaote, may have a very
limited range of appllcatton when compared with the packed column of
today.
The body of the LC column 1S normally made of stainless steel to withstand
the high pressures that will be reoui red to force the mobile phase through
the interstices of the packing. Furthermore, the column is usually fitted at
either end with stainless steel unions which allow connect ion to the
injection system and the detector. However, more recently, columns with
7
inert plast1c untons, have been designed (3) that w111 also cope with high
pressures. These columns employ very fine threads so that the unions can
not only seal satisfactorily at high pressures, but also be nand tightened.
This renders column tnstallatton or replacement a very simple procedure. A
photograph of a column With plastic end fittings is shown in f1gure 2. The
very small diameter columns that are being developed at this time (4,5) are
sometimes made of glass or fused silica. As these columns are often less
than a millimeter in diameter, their walls can be fairly thin and still cope
With the pressure necessary for their satisfactory operation.
Figure 2
An LC Column with Plastic Terminal Unions
By the courtesy of ASTEC Inc.
Some of these columns are pre-packed before drawing and trsrs, as a result
of the drawing procedure, the packing is fairly open. This reduces the flow
impedance and permits relatively low inlet pressures to be employed. Small
diameter glass or Quartz columns are not, as yet, generally avallable and it
may be some time before they can be developed into a commercially useful
product. As the plast 1cs industry develops stronger and more inert plast tcs,
so the totally plastic column may become a realistic possibility. Plastic
.
columns would be much less expensive to manufacture and very easy to pack
and assemble in the chromatograph. Such columns might well become
avallable in the not too distant future, probably initially for use with the
aqueous solvent mixtures used in reverse phase and ion exchange
chromatography.
The length of a packed analytical LC column can vary from a few
centimeters to several meters and the diameter from less than one
millimeter to one or two cent1meters. It w111 be shown later, that fully
coumtzeo, long, analytical columns wlll be relatively narrow in diameter
( 6 ~ 7 ) ~ whereas the shorter columns relatively wide (8). Preparative columns
on the other hand may have diameters of 10 cm or more (9) in order to
accommodate the h1gh sample loads. However, in general, due to practical
constraints, a preparat1ve column wlll usually be not more than two or
three meters in length. The wall-thickness of an LC column must be
appropr1ate for the inlet pressure that 1s to be used and obviously the wider
the d1ameter of the column, the greater will be the wall-thickness. In the
case of large preparative columns, the need for thick walls to handle the
necessary high pressures can make the column extremely massive
(sometimes a ton or more tn weight) whlch can also make packtnq
procedures and handling very difftcult. At the other extreme, small bore
columns, one ml1limeter or less 1n diameter, can have extremely thin walls.
For example, a one millimeter 1.0. column with walls 0.012 inches thick,
wi 11 funct10n sattsractortly at pressures up to 6000 o.s.t.
The Column EnvIronment
It has already been stated that the retention of a solute depends on the
magnitude of the distribution coefficient of the solute between the mobile
and stationary phases. Furthermore, according to Vant Hoff's Law, the
distribution coefficient will vary accordtnq to the exponent of the
reciprocal of the absolute temperature. In addition, the dtspersi on of a
solute band tn a column will be shown to depend on the dtrrustvtty of the
solute in both phases, the viscosity of the mob1le phase and also on the
dtstrtbution coefficient of the solute, all of which vary with temperature. It
follows that, for consistent results, the column must be carefully
thermostated. The column and its contents have a significant aest capacity
and, consecuently, it is of l1ttle use trying to thermostat the column 1n an
air bath; for satisfactory temperature control, the thermostating medium
9
must be a liquid. The thermostat must also bring the temperature of the
mobne phase precisely to the coiumn temperature before enter-jng the
injector and column,
It will be assumed in all subsequent theoretical arguments, that the column
is properly thermostated and, unless otherwise stated, all chromatographic
separations are carr1ed out isothermally.
The purpose of studying the theory of liQui.d chromatography columns is not
merely to understand the function of the column and how a separation can be
achieved, but to provide sufficient knowledge to be able to design a column
that will provide a qtven separat ion, and consequently an analysts, tn the
minimum time.
The 'technical cost' of a separation is paid in units of time and pressure-
both of which are limited in practice. It follows, that there is a llrntt to the
maximum time that can be tolerated before an analysis is completed.
Conversely, there will also be a limit to the complexity of a mixture that
can be separated in an acceptable time. Column theory must allow these
nrntts to be identified. Although, as already stated, only packed columns are
presently in general use, it may be possible that eventually chromatographic
apparatus, particularly the detector and injection system, will be improved
to the point where capillary columns become a viable alternative. Column
theory must, therefore, also ate in capillary column desjgn and be able to
define the specifications of the ancjllary apparatus that will permit the
efficient use of such columns.
Chromatography Nomenclature
Before dealing with the theory of LC columns, however, it would be wise to
define some of the terms used to describe the otrrerent parts of a
chromatogram. Figure (3) depicts a chromatogram showing the resolution of
two solutes.
The envelope of each eluted solute is called a peak. On injecting a sample
onto the column, a mark IS usually made on the chart, sometimes
automatically, and this ts called the injection point, A substance that is not
1Q
retained on the column) if not naturally present in the mixture, is often
added to the sample prior to injectton as it may aid in component
identification. The position on the chromatogram Where this unretained
substance is eluted is called the dead point, The volume of mobile phase
that has passed through the column between the injection point and the dead
point is called the dead volume which, in fact, includes the total volume
between the injection valve and the the detector that is not occupied by the
stationary phase or the support. (It should be emphasized that the dead
volume is llQLmerely the of mobile phase in the column)
However) this does assume that the two Qhases are in equilibrium and that
there ts no mobile phase trapped in the stationarv phase and as a
consequence isolated from the bulk of the mobile phase. The elapsed time
between the injection point and the dead point is called the deadtime and
F1gure J
The Nomenclature of a Chromatogram
Peak
Width
Peak
Mulmum
--- -- ------1
__ __ Peak
I I T Height
I Peak Width I (R)
I at Half Height I W607R
! I I I \ O.5R
I \
I \
Unretained
Solute Peak
If..
I
Peak Width
\
-
--
at Base

-
Dead
Adjusted Retention Volume
f--Volume
(VM)
V'R
Retention Volume
VR
Volume Flow of Mobile Phase
.
Adjusted Retention Time
Time (tM)
t'R
Retention Time

tR
Time ..
Injection Dead
point Point
thus, the dead volume is obtained from the product of the deadtime and the
cotomn /tow-rete . The base line is that portion of the chromatogram
11
recorded when only rnootle phase ts emerging from the column. The point at
the maximun concentration of any eluted peak is the peak maximum . The
distance between the peak maximum and a line joining the base of the peak
by the extrapolation of the base line is called the peak neignt . There are
varlous measurements used for the peak wldtn. The width that has a
fundamental theoretical significance, and which should be used wherever
possible, is the peak width at 0.6065 of the peak height. The importance of
this particular peak width will be discussed later. Throughout the book, if
the peak width is not otherwise defined, then it wtll refer to width at
0.6065 of the peak height. The product of the peak height and peak width
will always give 79.8% of the total peak area for true error function or
Gaussian curves.
For convenience, in Quantitative analysis, the peak area is often taken as the
product of the peak height and the peak width at half peak height, obvtouslv
this has been given the term peak wldtn at naIfneignt . Another measure of
the peak width that is sometimes used, involves constructing tangents to
the points of inflection of the elution curve and measuring the distance
between the1r points of intersection with the base line. This is termed the
peak wldtn at tne base and is, in fact, equivalent to twice the peak width at
0.6065 of the peak height.
Those characteristics of the chromatogram that have theoretical
significance will be discussed later, but some common measurements that
can be taken from the chromatogram are given now. The elapsed time
between the dead point and the peak maximum is called the retention time,
and the time between the dead point and the peak maximum the adjusted
retention time. If the retention time is multiplied by the mobile phase
flow-rate, the product is called the retention volume. Similarly,
multiplying the adjusted retention time by the flow-rate gives the adjusted
retention volume.
In order to obtain a rational relationship between GC and LC the symbols
used for the above parameters of the chromatogram are taken from those
recommended by the Gas Chromatography Discussion Group and IEUPAC.
12
These terms and symbols are now generally accepted for use in both GC and
LC.
dead time, to
dead volume, Vo
retention time, tr
retention volume, Vr
adjusted retention time, tr'
adjusted retention volume, Vr'
One further parameter ought to be mentioned, which to some extent ts
arbitrary, but can be a useful measure of column performance, and that is
resolution. The value taken from the Chromatogram, which is normally
recognized as a measure of the resolut ion of two peaks, is the rat to of the
distance between the peak maxima and the sum of the peak widths at 0.6065
of the peak height.
LC Column Theor1es
There are two basic theories that deal with the processes that occur in a
chromatographic column, the Plate Theory and the Rate Theory, both of
which are essential to understand the function of the column and column
design. The first theory to be developed was the Plate Theory, which was
presented in an exponential form by Martin and Synge (10) in 1945. The
theory was developed further to a more precise and useful form by Said (11).
The publication by Said passed almost unnoticed and this more useful form
of the Plate Theory was not generally known unttl reported by Keulemans
(12). The Rate Theory for packed columns was first developed by Van
Deemter et al (13) In 1956 for (GC) but is directly applicable to LC. Since
that time, a number of other forms of the Rate Theory for packed columns
have been developed and in due course will be discussed. However, it appears
that the form introduced by van Deemter is still the simplest and the most
reliable for use In column design. The Rate Theory for ceptllary columns was
developed by Golay (14) in 1958 and has been used unchanged or modified
since that time, The GOlay equation that arises from his Rate Theory is not
only applicable to capillary columns, but also to injection valves and
connecting tubes and consequent lyI is also used, to evaluate extra column
dispersion.
References
(1) M, S. Tswett, KlJromplJylii Rastitelnom i ZlJivotnoml1ire, Izd
Karbasnikov, Warsaw, 191
(2) J.H.Knox,K.K,Unger and HMueller,JLlqCnromatogr.,6( 1983) 1
(3) II HPLCFlttlngS,T.Upcnurcn,Oak Harbor, Wa., 1988,
(4) V.LMcGuffin and M.Novotny, JClJromatogr.,255( 1983)381
(5) C.Barra,S.M.Han and M.NovotnYJ J ClJromatogr:,385( 1987)75
(6) t.KatZJK.L.Ogan and RP.W.Scott, JClJromatogr:,289( 1984)65
(7) ,. Small Bore LiquidClJromatograplJy ColumnS,(Ed. RP.W,Scott),
John Wi ley and sons.cmcntster, 1984.
(8) E.Katz and RP.W.Scott J ClJromatogr:,253( 1982) 159
(9) E.P,Kroeff,RA.Owens,E.L.Cambe11.,R.D.Jonnson and H.I.Marks,
./ClJroflJatogr:,461 (1989)45
(10) A.J.P.Martin and RL,M,Synge, BioClJem.J
J35,(
1941) 1358
(11) AS.Said, Am.mst. Cnem,EngJ.2(1956)477
( 12) " 6as ClJromatograplJ!(2nd Ed,),A.I.M,Keulemans,Reinnold
Publishing Company,Amsterdam, 1959
(13) J.J.Van Deemter,F.J.Zuiderweg and AKlinkenberg, Cnem.Eng5c/
5( 1956)24
CHAPTER 2
The Plate Theory
Primarlly the Plate Theory provides the equation for the elution curve of a
solute. Such an equation describes the concentration of a solute leaving a
coumn, in terms of the volume of mobile phase that has passed through it.
It is from thiS equation, that the various characteristics of a
chromatograph1c system can be determined using the data that is provided
by the chromatogram. The Plate Theory, for example, will provide an
equation for the retention volume of a solute, show how the column
effic1ency can be calculated, determ1ne the maximum volume of charge that
can be placed on the column and permit the calculation of the number of
theoretical plates required to effect a given separat1on.
There are two methods of developing the Plate Theory, that given by Martin
and Synge (1) and that given by Said (2,3). Both methods were originally
developed for gas chromatography (GC) but are equally appltcable to ltqutd
chromatography (LC), The nrst, developed by Martin and Synge leads to a
binomial expression which, unfortunately, is approximate and has severe
limitations for subsequent use in determining column parameters. However,
it was the first approach to a rational explanation of chromatographic
development.
The Plate Theory, in whatever form, assumes that the solute is, at all
times, in equilibrium with both the mobtle and stationary phase, Due to the
continuous exchange of solute between the mobile and stationary phases as
it progresses down the column, equilibrium between the phases is, in fact,
never actually achieved. As a consequence, to develop the Plate Theory, the
column is considered to be divided into a number of cells or plates. Each cell
is allotted a finite length, and thus, the solute spends a finite time in each
cell. The size of the cell is such that the solute is considered to have
surrtctent time to achieve equilibrium with the two phases. Thus, the
smaller the plate, the more efficient the solute exchange between the two
phases in the column and consequently the more plates there are in a given
column. This is why the number of Theoretical Plates in a column is termed
16
the cokma efficiency. These assumptions are exactly analogous to those
employed in the development of the theory of distillation
Said (2) developed the equation of the elution curve in the following way.
Consider the equtltbrturn existing tn each plate, then ;-
X
s
=KXm (1 )
Where, (X
m)
and (X
s)
are the concentrations of the solute in the mobile and
stat ionary phases respect tvely and (K) is the otstrtoutIon coefficient of the
solute between the two phases.
(It should be noted that (K) is defined with reference to the stationary
phase; t.e. K = Xs/X
m
.tbus the larger the distribution coefficient the more
solute is distributed in the stationary phase)
(K) is a otrnenstonless constant and thus tn liquid/llquid systems, (Xs) and
(Xm) are conveniently measured as mass ofsolute per unit volume or phase.
In llQuid /soltd chromatography an alternative method of measurement could
be, flU/5S of sotote per unit mass of posse, However, in LC the dlfference
between a liquid/liquid system and a liquid/solid system is moot. In fact, in
practice, the reverse phase system in LC is more often considered
liquid/liquid than a liquid/solid system.
Equation (1) merely states that the general distribution law applies to the
system and that the adsorption isotherm is linear. At the concentrations
normally employed in liquid chromatographic separations this will be true.
It will be shown later that the adsorption isotherms must be very close to
linear if the system is to have practical use, since nonlinear isotherms
produce asymmetrical peaks.
Differentiating equation (1),
dX
s
=KdX
m
(2)
Consider three consecutive plates in a column, the (p-l), the (p) and the
(p+ 1) plates and let there be a totaI of (n) plates in the column. The three
plate are depicted in figure (1) Let the volumes of rnobtle phase and
stat ionary phase in each plate be (vm) and (vs) respect tvely and the
concentrations of solute in the mobile and stationary phase In each plate be
Xm(p-J), Xs(p-J), Xm(p), Xs(p), X
m(p+1L
and X
s(p+l)
respectively. Let a
volume of mobile phase, ov, pass from plate (p-1) into plate (p) at the same
time displacing the same volume of mobile phase from plate (p) to plate
(p+ 1>. In doing so, there wlll be a change of mass of solute in plate (p) that
.'" ~ r
w1ll be equal to the difference tn the mass enter1ng plate (p) from plate (p-
1) and the mass of solute leaving plate (p) and entering plate (p+ 1). Thus
bearing in mind that mass is the product of concentration and volume, the
change of mass of solute in plate (p) is:-
dm = (X
m(p-1)
- Xm(p)dV (3)
F1gure 1
Three Consecutlve Theoret1cal Plates in an LC COlumn.
Plate
(p-l )
V
s
Xs(p-l)
Plate
(p)
Xm(P)
Plate
(p+l)
V
m
Xm(p+l)
V
s
Xs(p+ 1)
Now if equilibrium 1S to be maintained in the plate (p), the mass (ern) wtll
dtstrtbute itself between the two phases, which will result in a change of
solute concentration in the the moblle phase of dXm(p) and in the stationary
phase of dX
s(
p).
d
m
=vsdXs(p) + VmdXm(p) , , , (4)
Substituting for dXs(p) from equation (2),.
dm=(vm +Kvs)dXm( p) (5)
1"
. ",
EquatIng equations (3) and (5) and rearranging,
_
--'-'-_ (6)
dV vm+Kv
s
Now, to aid in algebraic manipulation it is necessary to effect a change of
variable. The volume flow of mobile phase will now be measured in units
of, (vm + Kvs), instead of milliliters. Thus the new variable (v) can be
defined where,
V
v;. (v
m
+Kv
s
) (7)
The function (vm + Kvs), is given the name 'plate volume' and thus, for the
present, the flow of mobile phase through the column will be measured in
'plate volumes' instead of milliliters. The 'plate volume' is, and can be
defined as, that volume of mobile phase that can contain all the solute that
is in the plate at the ecuufbrtum concentration of the solute in the mobile
phase.
Differentiating equation (7),
dv- dV (8)
(vm+Kv
s
)
Substituting for dV from (8) in (6)
=X
m
P.l)- Xp) (9)
Equation (9) is the basic differential equation that describes the rate of
change of concentration of solute in the mobile phase in plate (p) with the
volume flow of mobfle phase through it. Thus, the tnteqratton of equation (9)
will provide the equation for the elution curve of a solute from any plate in
the column. A simple algebraic solution to equation (9) is given below and
the resulting equation for the elution curve from plate (p) is as rollows-
19
Where Xm(p) is the concentration of the solute in the mobile phase
leaving tne (pHh plate
and X
o
is the initial concentration of solute placed on the 1st plate
of the column
Thus, the equation for the elution curve from the (nHh plate which is the
last plate in the column (that is, the equation relating the concentration of
solute in the mobile phase entering the detector with volume of mobile
pnase passed through the coumr», 1S given by:-
Xoe-vv
n
X
m
(p 1= n! ( 10)
THE SOLUTION OF THE DIFFERENTIAL EQUATION I
dXmi'»
dv =Xm(P-l) - Xm(P)
First consider the conditions of the above equation when an initial charge of concentration Xo(m)
has been placed on the first plate of the column. but chromatographic development has not
commenced: that is y=O,
Then, Xm(p) = Xo(m) when p=O (t.e. the first plate)
and Xm(p) = 0 when p>O (t.e. for any other plate in the column)
The first condition merely states thet before the cnrometocreptnc development commences, the
concentration in plate p=O is that resulting from the injection of the sample on the column.The
second condition states that the remainder of the column is free of solute.
Thus for plate p=O. and as there is no plate (p-l ),
Integrating, 1000Xo(m) = -Y + constant
20
Nowt when v =0t Xo(m) • Xo(m).
Consequentlyt the constant =l ~ X
o,
or t Xo(m) = Xoe-v "." ,,, ,, , ,, .. ,,, .. ,, (A)
For Plate 1t
dXm(l)
dv =X m(O)- Xm(l) " .. " (B)
Substitut1ngforXm(O) from (A) In(B),
Mu1t1ply1ng throughout bye
V
,
d ~ O ) e' +)(".O,ev =X.
Now tntsequation can be reroJn1zed as the differential of aproduct, Hence.
Integrating.
Now, when v =0, Xm( 1) =0 , thus. k = O. Furthermore,
Inast mller w8'l it can be shown that,
for Plate (2)
21
for PIate ( 3)
Thus, for the nth Plate,
e ~ V v n
Xrntn) =Xo n
l
Equation (10) is the equation of prime importance that arises from tne Plate
Theory, From this equation, it can be shown how the cnaractertst tcs of the
Chromatogram can be used to determine the basic chromatographic
properties of the co lumn and phase system and how various other important
chromatographic requirements can be calculated Equation (10) is a Poisson
function, but it will be shown later that) if (n) is large) the function
approximates very closely to a normal Error function or Gaussian function,
Thus) since in all practical LC systems, (n) is always greater than 100, it
would be expected from the conditions assumed in the derivat ion of the
equation, that all chromatographic peaks will be Gaussian or nearly
Gaussian in snape
Flgure 2
Elution Curves from Columns having 4, 9 and 16 theoret 1ca1
Plates
0.200
10 20
v (Plate vor.:
• n=4
n:09
<> n=-16
30
22
The elution curves, calculated from equation (10), for a solute eluted from
three columns having 4,9, and 15 plates respectively are shown in rtgur-e 3,
With Hie exception of having a different number of theoretical plates all
three columns have identical physical properties,
It is seen that as the number of plates increase (that is the column becomes
longer) the peaks become more symmetrical and, as a result of greater
dilution of the solute) the peaks are also reduced in height It should be
painted out, however, that aIthough the peaks from the co1umn with the
greater number of plates appears the broadest it will be seen Iater that the
resolving power of a column depends on the ratio of the retention distance
to the peak width and thus the column with the larger number of plates will
provide the greater resolving power,
The Retention Volume of a Solute
A chromatogram containing two peaks is shown in figure (4), The different
dimensions along the axis of the chromatogram are described as funct ions
of the volume of moving and stationary phases per plate, the distribution
coefficients of the two solutes and the err ictencv n. These functions will
now be derived from equation (10) and it is appropriate to start with the
simplest derivation) the retention volume V
r,
The chromatogram shown in
figure (4) will be employed again) later in the extension of the Plate
Theory, Other characteristics will be discussed and the appropr iate
functions derived in order to label other dimensions on the chromatogram,
F1gure 3
A Chromatogram Showing the Separation of Two Solutes

Tile retention volume of a solute is that volume of rnobue phase that passes
HIe column between the injection potnt and the peak rnaxtrnun. tt :5
therefore, possible to determine that volume by differentiating equation
(10) and equating to zero and solving for' (v),
Restating ecuat i on ( 10),
or, v =n
Thts means that at the peak maximum, (n) plate volumes of mobile ohase has
passed through HIe co lumn (remembering that the volume flow is measured
in 'plate volumes' and not rnl). ThUS, the volume passed through the column in
rnl will be obtained by multiplying by the 'plate volume', (vm + Kvs)
the retention volume (V
r
) is given bt-
=nV
m
+nKVs
Now tne total volume of mobile phase in the column, (V
m))
Will be the
volume of mobile phase per plate multiplied by the number of plates t.e.
(nvm). In a similar manner the total volume of stationary phase in the
column, (Vs) will be the volume of stationary phase per plate multiolied by
the total number of plates, te (nv)s,
ThuS,
Vr =Vm.. KVs '"'''' "'"'' '"'''''''''''''''''''' " " ( 11)
In practice.tor an unretained peak eluted at the dead volurne.tv-),
Va = Vr(o) + VE
=Vm + VE """"""""""""'''''''''''''''' """ (12)
24
Where, (VE), is the extra column volume contained in the injection system,
connecting tubes and detector cell. In some cases} (VO, may be sufficiently
small to be ignored, but for accurate measurements of retent ion volume the
actual volume measured should always be corrected for the extra column
volume of the system and equatton (12) should be put in the form,
Vr = Vm + KVs VE , (13)
The exact nature of the dead volume is complex} and, at the same time, Is
extremely important parttcularly when it is employed In LC to determine the
thermodynamic properties of a distribution system and to identify the
nature of solute/phase interactions. It is also important in the development
and use of equat ions for the preorctton of the optimum phase systems for
partfcular separations, As a consequence, the subject of the dead volume and
Its measurement in LC systems wi 11 be extenslvely discussed in the next
chapter.
Returning to equation (13) which glves the retention volume of a solute, it
Is now possible to derlve and equatton for the adjusted retention volume,
(V'r),
V'r =V
r
- V
o
Ttv.Js) trom eouat ions ( 12) and ( 13),
and, V'r = KVs ' (14)
To avold any confuslon, it should be reiterated that although the stationary
phase is assumed to have a volume and thus implies a liQuid/llQuid system,
by replacing the rotomec; stationary phase with massof stationary phase,
then the equations can be exactly appl tcable to liquid solid systems,
However, as already stated, the units of concentration must also be
redefined in the measurement of (K).
Ref errrnq back to figure (4) The retention volume of solutes (A) and (8) wll I
be,
and
Vr(A) == Vm+ K(A) Vs + VE .. , , US)
Vr( 8) = Vm+ K( 8) Vs + VE (1 6)
the corrected retention volumes w111 be,
V'r( A) = K(A) Vs (17)
and V' r( 8) = K( 8) Vs " (18)
The Capacity RatIo or a Solute
The capacity ratio of a solute (k') was introduced early in the development
of chromatography theory and was defined as the rat 10 of the distribut ion
coetrtctent of the solute to the phase ratio (a) of the column. In turn the
phase ratio of the column was oertnec as the ratio of the volume of mobile
phase in the column to the volume of stationary phase In the column.
Thus,
and, as a = Vm/Vs,
k':= V'r
a
Note that (Vm) is the volume of rnobtle phase in the column and not Va the
total dead volume of the column.
Consequent ly, in practice
k'::; V'r
Vo-V
E
" (19)
Furthermore, it w111 be shown later that both (V
m)
and (Vs) wll1 not only
vary between different columns, but also between different solutes, due to
the exclusion properties of silica gel. Thus, some caution must be shown in
compartnq (k') values for the same solute from different columns and for
different solutes on the same column. This Wi 11 be discussed in detai1in the
next chapter.
Nevertheless it must be pointed out that, in calculating (k'), the value taken
in practice is often the ratio of the corrected retention distance (the
distance in centimeters on the chart, between the dead point and the peak
maximum) to the dead volume distance (the distance in centimeters on the
chart, between the Inject ion point to the dead point on the chromatogram).
This calculation assumes the extra column dead volume is not significant
and, unfortunately, in almost all cases thts is not true. (k') values calculated
in this way will be In error and should not be used for solute Identification.
Where computer data processing is used and no chart is avai labte the
distances defined above would be replaced by the correspondtnq times.
The separat j on Rat10
The capacity ratio of a solute, (k'), was introduced to develop a
cbrornatoqrapruc measurement, simple to calculate) independent of flow-
rate and one that could be used in solute identifIcation. Although helpful,
the capacity ratio is so dependent on the accurate measurement of extra
column volume and on very umtted solute exclusion by the support and
stationary phase, that it is less than ideal for solute identification. An
alternat ive measurement, the separat1on rat io (a.) was suggested where, for
two solutes (A) and (8),
_ Vr(AJ
a. ---
V'r(B)
K(AJ
V
s
K(B)V s
_K(A)
--
K(BJ
It is seen that the separation ratio IS independent or all colurnn parameters
and depends on lyon the nature of the two phases and the temperature. Thus
providing trlF'> prr1:=,p :=.y=;tem is used on two columns, and the solutes
are cbrornatoqrapheo at Hie same temperature, then the two solutes will
have the same separatIon rat i 0 on both co1umns. The separati on rat i 0 will be
inde{Jendentof the phase rsttas of the two co lumns and the flow-rates. 1t
follows, that tne separation ratio of a solute can be used reliably as a
means of solute identifIcation.
A standard substance is often added to a mixture and the separation ratio of
the substance of interest to the standard 1s used for ident 1rtcat ion. In
practice the separation ratio is taken as the ratio of the distances In
centimeters between the dead point and the maximum of each peak. If
computer data processing is being employed replace distances by
corresponding times.
References
(1) AJ.P. Martin and R.L.Synge
J
8/ocIJem. J. 35( 1941)1358
(2)" Gas CIJromato.qrapl7y <seconaEd/t/on., AI.M.Keulemans (Ed.C.G.Verver)
Reinhold Pubi ishmg Corporatron, New York, (1959) 106
(3)" TheOf;v andflathemat its of Cl7romatography; AS.5aid) Dr. Alfred
HOHlig Veriag GmbdH Heidelberg (1981) 126
Chapter 3
The LC Column Dead Volume
The dead volume of a liquid chromatography column has been the subject of
a considerable number of investigations, dissertations and public
dtscusston-sorne of which have bordered on acrimony. As a consequence, the
subject has been studied extensively from both a theoretical and
experimental point of view. (1-7). The dead votome of a liquid
chromatography column is important in both kinetic and thermodynamic
measurements and is part icularly so, when attempts are made to correlate
solute retention With the composition of the mobt!e phase. In the past, the
pertinent column volumes that have been considered to be important have
been HIe column interstitial volume, (the volume of mobile phase existing
between the particles), the column pore volume, (the volume of mobile phase
within HIe particle) and HIe volume of stationary phase in the column.
However, certain assumptions, tacitly made in early work, may not be
chromatographically acceptable. For example, it has often been assumed
that all the interstitial volume of the column contains moving phase and
none of the interstitial VOlume is static, In addition) it has been assumed
that the pore contents of a column have the same composition as the mobile
phase, Whereas there is considerable evidence that this is not so (8-10).
Another' assumption that has been made, is that all the stationary phase in
the column is chromatographically available and, probably more important)
the exclusion characteristics of the packing material can be completely
ignored. Obviously these assumption are fallacious, and in order to avoid
assumed errors of thiS kind, a stmple rational approach must be made to
identify the nature of all the different volumes that exist in a liquid
chromatography co1umn.
The Internal volume of a column is occupied by three substances, the mobile
phase, the stat ionary phase and the support. The term mobile phase is a
misnomer as it tmoltes that all the mobile phase is moving, Which is not so.
The rnobtle phase within the pores is also stationary and thus constitutes
part of the stationary phase, Nevertheless, it is such a well established
term, it will st111 be used to denote the total mobile phase in the column,
• I ~ . . . .,.. ~ I ...."" I _ _I ~ _ L IlL' L ' ~
rrlovlfly,mu stet tc. IDe rerrn fll()Vlflypflase Will oe useu lUI 1IldL II"dCLIUIl UI
the mobile phase that actually moves, Whereas the term stattc phase wi 11
be used for' that fraction of the mobile phase that is trapped in the pores, or
in the interstices of the support particles, and does not move.
The mobile phase may be a single solvent or a solvent mixture. The support
is usually silica gel, although tn some circumstances) it may be aiumina or)
in ion cbrornatooraphv, perhaps an ion exchange resin, The most common
form of cnromatoqraphy employs a bonded phase) in which case the
stationary phase consists of silica gel with an organic moiety bonded to the
surface. The organic moiety can be aliphatic, aromatic or heterocyclic in
nature and contain a single organic species or a mixture of species.
It follows that.-
Vc =VM + Vs + VSi """.. """"""""'''''''''''''''''''''' (1)
where (Ve) is the total column volume,
(VM) is the total volume of mobile phase in the column,
(Vs) is the total volume of stationary phase in the column,
(VSi) is the total volume of support (in this case silica) in the
co lumn.
It should be noted that (VM) is not the same as (V
m
) from the plate theory as
(V
m
) is moving and some of (VM) will be static, Similarly, (Vs) is not the
same as (V
s
) from the plate theory as (Vs) refers only to the stationary
phase, whereas (Vs)in these circumstances wt 11 also include static mobile
phase.
For simplicity, the two phases will be considered liquid/liquid in nature.
Whether) in fact, a reverse phase does constitute a liquid-like phase or not
is a moot point and even now a SUbject of some controversy, In any event,
the arguments about to be put forward are independent of the exact nature
of the interactions or any solute with the stationary phase, so either form
may be assumed. The volume of stattonarv phase, (Vs) can be replaced by
(Adf), (A is the surface area or the stationary phase in the Column and (df) is
the effective "film thickness" of the stationary phase) if so desired, and the
arguments and the conclusions will remain the same.
Now the vo1ume of the co lumn is given by:-
Vc = nr
2
1 ,,, ,,, .. ,,.,, ,,.,,,,, , , ,,,.,, , (2)
Where, (r) is the column radius,
and (1) is the column length.
Hence,
VM+ V
s
+ VSi =nr
2
1 "" ... "... """ .... ,..... "" .. ".",,,,,,,,,. "," """""" (3)
As some of the mobtle phase exists in the tnterst ices of the packing and
some inside the pores, (VM ) can be initially divided into two parts,
VM =VI + Vp , , " (4)
where, (VI) is the tntersttttai volume between the packing,
and (Vp) ts the pore volume of the packing.
The interstitial volume of the column is also made up of two parts: that
fraction of the interstitial volume that is moving (VI(m», and that fraction
that is close to the points of contact between the individual particles and
away from the flow-stream that is essentially static (VI(s»
Hence,
VI = VI(m) + Vi(s) " (5)
When mixed solvents are employed as the mobile phase, it has been shown
(10) that one solvent can be absorbed preferentially on, or associated with,
the surface of the stat ionary phase, Consequently, some of the solvent
contents of the pore, in close proximity with the stationary phase, will not
necessarily have the same composition as that of the original rnobue phase.
Therefore,
vp '"' Vp( 1) + Vp( 2) (6)
where, (Vp( 1) is that volume of the pore that contains solvent h"aving the
same composition as the original mobtle phase,
and (V
p(2»
is that fraction of the pore volume having a aureren:
composition from that of the mobue phase.
Substituting for (Vi) and (V
p
) from equations (5) and (6) in equation (4),
VM = Vl(m) + VI(s) + V
p(1)+Vp(2)
" ,,, (7)
It is now necessary to consider the volume of stationary phase ill the
column. Owing to the nature of the majority of bonded phases and as a result
of tnetr method of manufacture, some of the smaller pores may become
completely blocked by the stationary phase itself, and thus some stationary
phase may become chromatographically unavailable,
It follows that, Vs =VS(A) + VS( u) " ".............................................. (8)
where, (VS(A» is the volume of chromatographically availablestationary
phase,
and (VS(U» is the volume of chromatographically unavailable stationary
phase.
Substituting for (VM) and (Vs) from equations (7) and (8) in equation (1))
V
c
= VI(m) + VI(s) + V
p(
1) + V
p(2)
+ VS(A) + VS(U) +VSt """'"'''''''''''''' (9)
Now from the plate theory, for solute (A») the retention volume is given by)
Vr(A) =Vo + I<A V
s
" " " (10)
where (Vr(A» is the retention volume of solute (A),
(KA) Is the distribution coefficient of solute (A) between the two
phases,
and (V
o
) and (V
s
) have the mean1ngs previously ascribed to them (V
s
) can be
considered to be (Adf), if adsorption is considered to be taking
place as opposed to part i t i on between two Ii qui ds.
It can be clearly seen from equation (9) that the expression for the
retention volume of a solute, although generally correct, is grossly over
simplified if accurate measurements of retention volumes are required
Some of the stationary phase may not be chromatographically available and
not all the pore contents have the same composition as the mobile phase
and, tneret ore, being static, can act as a second stat tonarv phase. This
sttuat ion is akin to the original reverse phase system of MartIn and Synge
where a dispersive solvent was absorbed into the pores of support to
provide a liquid/liquid system. As a consequence a more accurate form of
the retention equation would be,
V
r
= VI(m) + KVI(s) + K1Vp( 1) + K2Vp(2) + K3VS(A) .. "" .... " .. " .... ,... (11)
wnere, (K) is the distribut ion coefficient of the solute between the moving
phase and the static portion of the mtersttttai volume,
(K1) is the distrlbut 10n coefficient of the solute between the moving
phase and the static pore contents, (V
p
( 1»
(K2) is the distribution coefficient of the solute between the moving
phase and the static pore contents,(V
p(2»
and (K3) is the distribution coefficient of the solute between the moving
phase and the avattable stationary phase (VS(A»
? 1
J&
All static phases will contribute to retention and, as seen from equation
there are a number of distribution coefficients effecting the retention
of the solute. However, the the static interstitial volume (Vi(s») and the
pore volume fraction (V
p
( 1», contain mobile phase having the same
composition as the moving phase and thus,
K=Kl=l
Thus, equation (11) reduces to
V
r
= VI(m) + VI( s) + Vp( 1) + K2Vp( 2) + K3VS(A) ., , (12)
Unfortunately, even this modified equation does not describe the true
pract1cal situation in Le, as it is complicated by the fact that all stltca-
based materials exhibtt exclusion properties. The pore d1ameter of slltca-
based stat ionary phases can range from, perhaps, 2-3 Angstrom to as much
as 1000-2000 Angstrom. Consequently, some, otherwise open pores, are
accessible to the solute while others are not, depending on the size of the
molecule. Therefore, only those pores that have a diameter equal to, or
greater than, that of the solute molecules are accessible and only the
stationary phase within those pores can effect retention. In addition, the
static interstitial volume between the particles can also exhibit exclusion
properties and some of the static interstitial volume may also be
inaccessible to the larger solutes, As a consequence, equation (12) must be
further modified to give,
Vr = VI(m) + \fVI(s) + OVp( 1) + OK2Vp(2) + " ,.. (13)
where. ('l') ts that traction of the static 1nterstitial volume access1ble to
the sotute,
(0) ts that traction of the pore volume accesstbie to the solute,
and (t) is the rractton of the stattonary phase accessible to the solute,
Under some circumstances (0) and can be equal, but the. genera] case w111
be assumed, where they are not equal,
There are two 'dead volumes' that are chromatographically important, viz,
thl\ ..... I;..... ...."'rl th" 11-0"',....... """',,, ........ ,',. rol"..,rol ""1"......... ,, Th" f'" .............. " ... ;,..
1,.11 ... f\ rrrc (. II.. \..II;;U\..I 't V 1\..11111;; G/IIIJ "III;; ,"C:I , II V u)'lIa, " u: \..ICQ\..I V V I \..IIIIC, I r ic I vi IIICt I;:)
used in the study of peak variance. It is used to calculate the linear velocity
of the mobile phase for use in equations that describe the variance per unit
length of a column for any given solute, The latter is used for determining
the thermodynamic properties of a particular solute from retention data.
The kinetic dead votume is represented in equation (13) by (Vi(m»)) and is
solely that volume of mob1le phase tn the column that 1S rnovrnc. The
thermodynamlc dead volume is given, in equation (13)} by}
VI( m) + '¥ VI( s) + OV
p(
1)
The thermodynamic dead volume includes those static fractions of the
mobile phase that have the same composition as the moving phase, and thus
do not contribute to solute retention by d1fferenttal tnteractton in a similar
manner to those with the stattonary phase. It Is seen that, in contrast to the
kinetic dead volume. which by definition can contain no static mobile phase,
and as a consequence is independent of the solute chromatographed, the
thermodynamlc dead volume will vary from solute to solute depending on the
stze of the solute molecule u.e. ts dependent on both ('¥)and (0). Moreover,
the amount of the statiooarv phase accessible to the solute will also vary
with the size of the molecule ( t.e. is dependent on It follows, that for a
qtven stationary phase, it is not possible to compare the retent tve
properttes of one solute with those of another tn thermodynamic terms,
unless ('¥), (0) and are known accurately for each solute. This is
particularly trnportant If the two solutes differ signlf1cantly in molecular
volume. The experimental determination Of ('¥), (0) would be
extremely difficult, If not impossible in practice, as it would be necessary
to carry out a separate series of excluston measurements for each solute
wh1ch, at best, would be lengthy and tedious.
The two functlons tnvolvtnq either K2 or K3 , or both, in equation (13) can, in
theory, contribute to solute retent Ion. This wl1l depend on whether the
solute interacts with the absorbed component of the mobile phase on the
surface or penetrates the layer and interacts with the stattonarv phase
proper. In etther case, the accessibttttv of the solute to the retentive phase
is governed by the magnitude of (0) and Another Important aspect of
equation (13) ts its implication on the accuracy With which the capacity
factor k' can be measured. Now, (k'») is normally calcutated in the follOwing
way,
k' = (VI(m) +,Vl(S)+ nV
p
(1 ) + nK2Vp(2)+
VI(m) +,VI(S) +OV
p
( 1)
or,
k'::: OK2
Vp(2)
+
VI (m) + ,VI(s) +0 Vp(1)
............................... (14)
It can be seen from equation (14) that the value calcuiateo for (k') will
carry the same errors as those associated w1th the measurement of the dead
volume which will change wah the size of the solute molecule.
However, providing that,
OK2Vp(2) + VI(m) + '¥VI(s) + OVp( 1)
the 'corrected retention volume' can be employed for thermodynamic
measurements. or correlated with solvent composition, with acceptably low
errors.
Some or the characteristic column volumes outlined in the orevtous
argument were determined by Alhedai et al (11) in the examination of a
commerctauy available reverse phase column oacktnq, Zorbax C8. These
authors examined the exclusion properties of the interstitial volume of the
column by measuring the retention volume of a number of salts of different
molecular volume.The substances used/ 1n ascending order of ion volume,
were as touows,
Table 1
Data Used for the DetermInatIon of the InterstitIal Volume of the
Column
Substance RetentIon Volume (ml)
Sodium Nitrate 1.85
Sodium Sulfite 1,87
Sodium Thiosul fate 1.83
Sodium Nitropruss1de 1,64
Potassium D1 chromate 1,62
The results obtained are shown as graphs relat ing retention volume against
Ion volume in figure (0, The slope of the curve shown in figure (1) clearly
indicates the exclusion properties of the interstitial volume, It is also seen
that the charge on the ion, be 1t double or Single, has little effect on the
retention. Extrapolation of the linear curve to zero ion volume gives the
nf thp intprc;titi:::.l vnlltmp r_o I ... ••• _ ..... _ ....... __ • .... _ ••• _.
It is seen that the closest measured value to the moving portion of the
interstitial volume is obtatnec from the retention volume of sodium
34
dichromate but this is very little different from the retention volume of
sodium nitro-prusside,
Figure I
Graph or Retention Volume or a Series or Ions against their
tome Volume
Ion Volume (Cubic Angstroms)
The effect of solvent composition on the retention of a series of solutes,
commonly used to measure column dead volumes, was also investigated by
these authors. They employed mixtures of metnanol and water as the rnobue
phase and measured the retention volume of the same salts together with a
silica gel 'dispersion' (containing particles 0.002 micron in diameter), They
also measured the retention volume of the components of the mobile phase
methanol, and water. The silica 'dispersion' was chosen to simulate a solute
of very large molecular size, The results they obtained are shown in figure
(2).
The results indicate that there is little effect of mobile phase composition
on the retention volume of the solutes employed, or the silica 'dispersion',
It should be ootnted out, however, that there are no values included for
methanol between 0% and 10% methanol where adsorption of the methanol
on the reverse phase surface significantly changes the value of its retention
volume,
It is seen that the lowest retention volume is obtained for the silica
'dispersion'. This material, however, is difficult to use as it requires
sontcat ion for at least 5 hours before use, This was necessary, to obtain
complete dispersion of the particles and, thus, ensure symmetrical peaks,
sodium nitro prusstde gives a value, close to that of
dispersed
snica and l'n oracnr- e r- f\lllrl be used tf\ oetermine the total r , IJ \.11.;, I.;VUIU U 1..1 ..... 1..1 I,.V 1..1 1,.1,.,1"IIIII,., .. If .. I
excluded volume or the 'kinetIc dead volume · without incurring serious
error, Values for" the kinetic dead volume measured in this way could be
figure 2
Graph of Retention Volume of a Number of Different Solutes
aqatnst Compos1t1on or the Moblle Phase
a silica smoke
• Sod Nil.Prus.
• Sod Nitrate
o Sod. Sulphite
• Methanol (D)
o water
100 20 40 60 80
v/v Methanol
....--g--
-

[J 0
o
u
0

h
"


II
a
o
I
...
...

-
.... ... .... ....
Q)
E

"0 2
>
c:
o
....
.....
c:
Q)
...
Q)
0::
1
o
-
-
E
-
3
used to determine the linear' mobile phase velocity and capacity factors for
use in dispersion equations such as those of Van Deemter( 12) or Golay (13),
The maximum dead volume recorded was that of water or methano1 at
concentrattons above 10% v/v of methanol. This would appear to represent
the thermodynamic dead volume for small molecules. It should be
emphasized, however, that the thermodynamic dead volurne of the co lumn
for larger molecules will be significantly less due to the exclusion
characteristics of the stltca support.
Alhedat et al also examined the effect of exclusion on dead volume
measurement. A mobile phase consisting of n-octane, the same chain length
as the bonded phase, was employed to ensure no differential interaction
between the solute and the two phases. A range of aliphatic hydrocarbons
Irorn, rr-hexane to [l-t-Iexatr iaconlane were cillornatograpi-led at two
temperature 300C and 500C. The two temperatures were used to ensure that
the retention mechanism was solely exclusion and not partition. If partition
was Hie mecnantsm promoting retentton, then different retention volumes
would be obtained at the different temperatures for each solute. The results
obtained are shown in figure (3)
Figure 3
Graph or Retentton Volume or n-Alkanes against Carbon Number
3 ~ - - - - - - - - - - - ,
-
-
E
-
Q)
E 2
::::J
-o
>
c:
o
-1
,.J
c:
Q)
,.J
Q)
Ct:
o 300C
• 500C
40 10 20 30
Carbon Number
O+-....,.........,...--.......-----------..----f
o
It is seen that an approximately linear relationship exists between the
retention volume of each alkane and its carbon number and that the smaller
molecule exhibits the greatest retention. This is a direct result of the
exc1usi on propert i es of the sil i ca ge1support. The fact that the data, taken
at the two different temperatures, fallon the same straight 1ine confirms
that little or no partition is taking place and that the difference in
retention between the individual solutes is entirely related to their
molecular size.
It follows that retention measurements on silica based stationary phases
for' HIe purpose of obtaining thermodynamic data is fraught with
difficulties. Data from solutes of different molecular size cannot be
comoareo or related to other interact ing vari ables Ideally) thermodynamic
measurements should be made on columns that contain stationary phases
that exhibit no exclusion oropert ies, However, the only column system that
might meet this requirement is the capillary column which, unfortunately
introduces other complicatrons wrncn wlil be discussed later,
The best comororntse for si 1ica based stat ionary phases is to use corrected
retention volume data for solutes eluted at a Ck') of greater than 5 and only
37
compare chromatograph1c data for solutes of approximately the same
molecular size.
A summary of the data for the Zorbax column obtained by Alhedai et al is
shown 1n Table 2.
Table 2
Summary of the Phys1cal and Chem1cal Propert1es of the Zorbax
column
2,79m
a,87ml
2,78ml
2.77ml
a,70ml
0,18ml
1. 91 ml
lAlml
O.SOml
Column Length 25cm
Column Radius 2,3mm
Column Pi£king Zorbax C8 Reverse Phase
carbon Content of Reverse Phase 1O . O ~ w / w
Equivalent Decane Content (dimethyl octene) 11 , 8 ~ w / w
Total Column Volume 4.15ml
Total Volume of Silica in the Column 0,96ml
Total Volume of Stationary Phase in the Column OAOml
Volume of Chromatographically Available Stationary Phase OA9ml
Volume of Chromatographically Unavailable Stationary Phase (by difference)
Total Mobile Phase in the column.
11 By Weighing
21 From the Retention of the alkanes in n-Octens extrapolated
toan alkane carbon number of 3
3/ From the volume fri£tion average of the isotopes retention
volume
Total Interstitial Volume. Value extrapolated from the retention
volumes of ions of different size.
Interstitial Moving Phase Volume. From the Retention of 'SilicaSmoke'
Interstitial Static Phase Volume. By DifferenL1l
Total Pore Volume. By DifferenL1l
Pore Volume Containlng Components of the Moblle Phase of Dlfferent
composition tothat of that of the Moving Phase,( bydifference)
Pore Volume Containing Mobile Phase of the same compostnon as the
Moving Phase, (by differenL1l)

It 1S seen that the otstrtbutron of the vartous chromatograph1cally
important volumes wtthtn an LC column IS netther strr.ole nor' obvtous. it is
also seen that about 70% of the column volume 1S occuotec by rnobtle phase
but only about 50% of that mobile phase 1S actually moving, Furthermore
about 18% of the mobtle phase is tntersttttal but static and about 31 %of the
mobile phase 1S contained Within the pores and 1S also stat 1C. Just over 6%
of HIe mobile phase 1n the pores has a d1fferent composition to that of the
motnle phase proper and UIUS constitutes a second stat i onary phase The
stattonarv chase canstitutes about 12% of the co lumn volume which is
ecuivaient to about 17% of the rnoone phase content of the column.
The values given in table (2) are probably representative of most reverse
phase columns but may differ signHicantly from sihca gel columns.
References
( 1) H. lnge Irlardt, H. Muller and S, Dreyer, aromstoorsonr», 19( 1984)240.
(2) P L, L'lIrorl7atogra,01J13, 20( 1985)425.
(3) J. H, Knox and R. J. kaIi szanJ ./ (lIrorl7atogr , 349( 1985)21 1.
(4) R. J. Smith and C. S. N1eass
J
./ L/q. CIJromatogr: J9( 1986)1387.
(5) H. CoUn, A. <rsturovic, and G. GU1ochon, Anal. CIJem ,54( 1982)2438.
(6) R. A. D]erk.i and R. J. t.auc, ../ Liq Lnromatogr: , 1O( 1987)1749,
(7) E. Grusrlka, ,./ Liq. CIJro07iJtogr: , 5( 1982)1392,
(8) R. P. W Scott and P Kucera, ./ (!Jo.:Jn7atogr 149( 1978)93.
(9) R. M, McConnick and B. L. Karger, Anal. CIJem. ,52( 1980)2249.
(10) R. P. W. Scott and C. F. S1mpson, 5ymp aiem Soc. 15( 1980)69.
(11) A. Alhedat, D. E. Martire and R. P, W. Scott) Analyst) Vol. 14 (1989)869
(12)J
I van Deernter F J 71Iirlorl,l,/on-:lnriA 1I1ir\vol'"\nr",'1"l
• '_. It """"'..... .... •• .c..Yl\••n ....1 \,,0111\.04 ". l'IIIU",-'.I'lIlo,.J\.d:t,
r..neo7, Eng. Sci.,5( 1956)271.
(13) M. .J. E. GOlay, tn "(ids f..nrOfl7iJtogriJ,ofJy /958 ;' (Ed. D,H.Desty),
Butterworths, London, ( 1958)36,
Chapter 4
ExtensIons of the Plate Theory
So far the Plate Theory has been used to determine the equation for the
retentton volume of a solute, calculate the capacity factor of a solute and
identify HIe dead volume of the column and how it should be calculated,
However', the eouatton for the elution curve of a solute that arises dlrectly
from the Plate Theory can do far more than that to explain the
charactertsttcs of a Chromatogram, The equation will now be used ln a
variety of ways to expand our knowledge of the chromatographic process,
The Elutlon Curve of a Finite Charge
In the develooment of the plate theory and the derivation of the equat i on for
the elution curve of a so lute, it was assumed that the mtt lal charqe was
located tn the first plate of the column. In practice, this is dtrr tcutt to
achieve, and any Charge Will, tn fact, occupy a finite column volume and
consequently a spectnc number of the first theoretical plates of the column.
Consider the situation depicted tn flgure 1 where the initial charge is
distributed over (r) theoretical plates.
FIgure ( 1)
The Injection of a Finite Volume of Charge onto an LC Column
123 r _ - ~ n
[ ] ] ] ~ O ~ [
Let the mobile phase flow be arrested, and a volume of sample be placed on
the column such that tt occupies (r) theoretical plates, Now on starting the
_ . , . ........ r, _ , .. 6.:&.... - _ ._ ~ .. '. 4..- .. -& .. _ .. I... ._, _ L .. . ~ " -I - . - , - .- - .- .., .. ~ .: .....1' _ .. _ .1..__
LUIUlllll IIVW, Lilt' LVllLt'11L:J VI t'aUI IJlaLt' WI!'I Ut'Vt'IVIJ all t'IULIVll L.ur v«, LIlt'
ootnts on each curve belng one plate volume away from the cornpltrnentarv
points on trle adjacent CU:-''JE:, The nrst peak 'wlll result from the elution of
40
the contents of the (r)'th plate and the last peak from the elution of the
contents of the first plate. The peak actually monitored by the detector will
be the composite peak formed by the addition Of all the individual peaks. It
should also be noted that on development, the first peak will pass through
all the plates in the column, t.e (n) plates.However, the sample in plate (r)
will only pass through (n-r) plates as it reached the (r)'th plate on injection
Of the sample, It follows that the contents of plate (r) will start
development as though (r) plate volumes of rnobile phase had already passed
through the co lumn.
The elution curve resulting from that portion of the sample on plate one will
be given by the following equation,
and for that portion of the sample on plate 2,
and similarly for that portion on plate 3
Consequently the composite elution curve for the sample on the first three
plates wi 11 be given by,
It follows that the elution curve for a sample initially occupying (r) plates
wi 11 be,
..... ".""" " ".".. (1)
Equation (1) can be employed wtth the aid of a computer to calculate actual
peak prof i les should they be required. It must be remembered, however, that
80 90 100 110 120 130 140
Plate Volume (v)
A1
(v) must be measured In plate volumes and volumes in mf111liters must be
converted to plate volumes in order to use equation (i) saustactoruv.
The curves resulting from a charge covering the first three consecutive
plates of acolumn are shown tn figure (2), The elut ion curve result ing from
the contents of each induvtcua) plate are included together with the
composite curve result ing from the total charge.
Figure 2
Elutton Curves ResUlt1ng from the tnjectton of a Charge that
Occup1ed the F1rst Three Plates of a Column
II'" 4 ....,
lJ
'i
=
..
..
• 3
..
..,
i
..
c
"'""
II 2

:c
• ..
..,

I 1

u
.=
• 0 __.........
i 70
TO strnphty the calculations the column was assumed to have only 100
theoretical plates,
Peak Asymmetry
Tne equauon for the retention voiume of a solute, lhal was der-jved by
differentiating the equation of the elution and given in chapter 2, can be
used to obtain an equation for the retention time of a solute (t
r
) by divldlng
by tne flow-rate (Q),( 1),
42
Thus,
Vr = tr = Vm + KVs
o 0
Now the velocity of a band along the column (n, is obtained by dividing the
column length (]) by the retention time, (tr),
Consequent Iy,
z= _1 = 10
t
r
V
m
+ KV
s
Thus the band velocity (Z) 1S 1nversely oroporttonal to (Vm + KVs) ,and for a
sign1flcantly retained solute,
Vrn << KVs
Consequent Iy, for any given co turnn,
zoc _I, oc _1_ oc 1
V KV
s
K ,.. ", ,., "",.,."" "" ", (2)
r
It is seen from the above equation that the band velocity is inversely
proporttonal to the distribution coefficient With respect to the mobile
phase. It follows, that any changes In the distribution coefficient (K), will
result directly in changes in in the band velocity (Z).
Consider the isotherms depicted in figure (3). Each curve represents a
different isotherm relating the concentration of the solute in the stationarv
phase (X
s
) to that of the rnoblle phase (X
m

PqR represents a linear isotherm and, for this 1tne, the distribut ion
coefflcient is given by,
Since PqO IS a linear isotherm, (Kl) is constant for all values of (Xs)then
from equation (2) all concentrations in the band wi 11 travel at the same
veloc1ty and a symmetrical elution curve will be produced. ThiS symmetrical
curve 1S depicted as the normal peak (a) in figure (4), The symmetrical
nature of the elution curve is to be expected from the plate theory,
FIgure 3
AdsorpUon Isotherms
t
p ..._--------
o
X
s
n 1----1'--1
m
Now consider the situation where a large charge is placed on the column and
the solute molecules, now in a relatively high concentration in the
stationary phase, are no longer surrounded solely by solvent molecules but
by solvent and solute molecules. Under these circumstances the interactive
forces between the solute molecules and the molecules of the stationary
phase are increased because the interactive forces between sotute and
sotute are usually greater than the interactive forces between solute and
solvent Thus (K) wi 11 increase as (X
s)
increases and the result will be an
isotherm of the form depicted by curve PrR The distribution coefficient at
the higher concentrations will now be,
Now as tr >tq and ro =en, then,
K2 >Kl
Thus, from equation (2) It Is seen that the higher concentrations Of solute
\Alilln-::.cc tnrf\llf'ln tho ("f\lllmn -::It -::l clf\\A,or r-::lto·th-::ln tho If\\Alor ("f\n("ontr-::.tif\nc
,. III ,..,u...... ...." ........ \.ot'''''' vVIUtll'. \A\,. ..... "t.J.v.,\..01 1\.011I"',", \,011\,0411 \,.11'-" IV"". ,",VI I"'''f I"" .... \".IVft-.J
of solute, As a result, the peak will be distorted, and the peak maximum will
be moved to a position of higher retention relative to the parts of the peak
....
where the solute 1S at a lower concentration. This results In a peak shaped
of HIe form shown in flgure (4b),
Figure 4
The Effect of Different Absorption Isotherms
on Peak Shape
j
I
+'1'
, I
" 1\
I I "-
I '-
,
,
a.
NamaI Peak
b.
Overload Pea k
c.
Adsorption Peak
Conversely.. in some ltouto-solto systems) hlgh concentrations of solute w11]
cover Hie surrace of the adsorbent with rnuttuavers of solute molecule's As
a result of the screeninq effect of the muttnavers, the forces holding the
outer layers of solute motecutes on the surface will be sign j ncant ly
reduced relative to the forces holding the first or inner layers of solute
molecules. Th1S is because Hie outer layers will be elther, experiencing only
long range torces between them and the adsorbent surface or, be only
mteracunq wlth Hie next layer Of solute molecules. In any event) the effect
wi1l be the oppostte to that of overload and the absorpt ion isotherm will
take HH? form of the curve PsS In figure (3b).
Thus. at high ronrE'ntratiOrlS (if solute.
ts
- = K3
sm
Now since ts <tq and sm =en,
Then
K3 <Kl
Thus, again from equation (2), it follows that those parts Of the solute band
containing high concentrations of solute will move more raPidly through the
column than those parts of the solute band that contain low concentrations
of solute. The resulting elution curve will take the form of that shown in
figure (3c). This peak shape is often called an adsorption peak; this term
arose during the development of GC and is not really an appropriate term for
use in LC.
The major cause of peak asymmetry in LC is sample overload and this occurs
mostly in preparative and semi preparative LC. There are two forms of
sample overload, volume. overload and mass overload Volume overload
results from too large a volume Of sample being placed on the column and
this effect will be discussed later, It will be seen that volume over load
does not, in itself, produce asymmetric peaks unless accompanied by mass
overload. Mass overload which, as discussed above, is accompanied by a
distortion of the normally linear isotherm, can cause very significant peak
asymmetry and, in fact, seriously impair the resotut Ion obtained from the
column.
Column Efficlency
It has already been stated that, in order to achieve the separation of two
substances during their passage through a chromatographic column, the two
solute bands must be moved apart and, at the same time, must be kept
sufficiently narrow so that they are eluted discretely, It follows, that the
extent to which a co1umn can constrain the peaks from spreading wi 11 give a
measure of its efficiency, It is, therefore, desirable to be able to measure
the peak width and obtain from it, some value that can describe the column
performance.
Because the peak will be Gaussian in form, the peak width at the points of
inflexion of the curve (which corresponds to twice the standard deviation of
the curve) will be determined. At the potnts of tnrtexton (2) I
- 0
Thus,
Thus, at the points of inflexion,
Hence, v2 - 2 nv + n(n - J) ;:; 0
and
v = 2n±i(4n
2-4n(n-I»
2
=:;2n±En
2
=n±[n
It is seen that the points of inflexlon occur after n- In and n + In plate
volumes of mobile phase has passed through the column. Thus the volume of
mobile phase that has passed through the column between the lnrlexton
points w111 be,
n + In- n + In = 2Jn " " " (3)
Thus the peak width at the ooints of inflexion of the elution curve will be
2.Jn plate volumes which, inmillillters of mobile phase will be obtained by
multjplying by the plate volume t.e.,
Peak Width = 2 ~ ( v m + Kv
s
) " " (4)
The peak width at the points of inflexion of the elution curve is twice the
standard deviation and thus, from equation (3) it is seen that the var.ance
(the square of the standard deviation) is equal to (n), the total number of
plates 1n the coIumn. Consequent ly, the variance of the band (0
2
) in
milliliters of mobile phase is given by,
NOW,
Thus, V
r
=n(v
rns
+ Kv
s
)
v
2
o2 =---..!:..
n
47
It follows, that the var.ance of tne peak is inversetv oroooruonal to the
number of tneor eucai plates in tile coturnn and larger the r.urnber vi
theoret Ical plates, the more narrow the peak, and the more eff i ci entIy the
column has constrained the band dispersion, As a consequence the number of
theoretical plates tn a column IS given the name Column EffIciency, It 1S,
therefore, important to be able to measure the efficiency of any column and
trus can be carried out in a ver-y sunple manner Let the distance between
the Injection ooint and the peak maximum (the retention distance on the
cnromatooram: be y cm and the peak width at the points of inflexion be x cm
Figure 5
A Chromatogram Showing the Separation of Two Solutes
I
I
I
"
1

I
I
I
I
I
I
INJECTION DEAD
N.:..:.T_ Ym
,..,..-- n(vm·kAvS):VRA
......------n(Vm.kBvS>="R
B
I I>J
I I I
I n 1 I J
""""" I
I =Vo , lL.--nkgS-nkAOWS---",
I I
I : =<k Er 5
I ,
I I
I I
I
I
1
I
I
I
I
Then as the retentron volume IS n(vm + KVs) and twice the peak standard
deviation at the points of lnflexlOn is 2.Jn (vm + Kvs),
Tnen,
RetDistance y n(vm+Kv
s
) In
PeakWi dth =x= 2Jn (v
m
+I<v
s
) =2
.,
n=4(H .. ..dO (5)
Equatlon (5) allows the errtctencv of any solute peak, from any column, to be
calculated from measurements taken direcuy from the chromatogram, The
various characteristics of the chromatogram that have so far been
considered are shown in figure (5) which IS an extension or the
chromatogram shown in figure (3) in Chapter 2.
The Position of the Points of Inflection
SInce the measurement of ernctency Is Important for evaluattng the quality
of a column, it is necessary to know the position of the points of tnrtecuon
jn order to measure the peak wldth, The innecttcn potnts are 111 oertneo on
the chromatogram and it is necessary to know at what fraction of the peak
height they occur. This fraction will be the same as the ratio of the value
of the solute concentration after (n - In) plate volumes Of mobile phase has
passed through the column, to the solute concentration after (n) plate
votumes of mobile phase has passed through the column,
Tbus, if (f) is the rractton of the height (h) at which the points of Inflection
occur, then (3»)
f Xm(n - [n)
- -
h Xm(n)
xoe-(n- .[n)(n- fn)n
n!
xoe-n nn
n!
_ e - ( n - j n ) ( n _ ~ n
-
e-nn
n
= efrt -njr;f
= emf! -Jnf
, • I
Now, 1f X< <1
I
Consequently, since (n) w1l1 always be greater than 100,
(
1) [1 1 1 1 ]
nloge 1- = n - ;; - 2n - - 4n
2
- .........
= -.;;- - 3Jn - 4
1
n-......... .. (7)
Now, SUbstituting for, n 10g
e(1-
from equat Ion (7) in equation (6)
lOge!::; F - --4' - ..
h 3
1
n n
Now,
, 1
- «-
2'
Consequently, the term and above, can be ignored with respect to
Then,
and,
1
f --
h::; e 2 = 0,6065 , " " " " " (8)
It is seen from equation (8) that the points of inflection occur at 0.6065 of
the peak height and the peak width, at that height, is equivalent to two
Many peaks that are measured will be only a few millimeters wide and, as
the calculation of the column efficiency requires the width to be squared,
the distance (x) must be determined very accurately. The width should be
50
measured W1H) a cornuaritor reading to an accuracy of ± 0,1 mrn The
measurements are taken from the inside of one 1tne to the outswe (If t l ~ l e
Figure 6 Measurement of Peak Wldth
x
adjacent line, in order to eliminate errors resulting from the finite wldth of
the Jrlk line drawn on the chart. This procedure 1S shown tn figure (6) The
measurement should be repeated using the alternate edges of the 1me and an
averaqe taken of the two reactnqs to avoid errors arising from any vartation
in nne truckness. At least three replicate runs should be made and the trree
rep1icate values of efficlency should not di ffer by more than 3%, If the data
acoutsttton system has sortware to measure efflclency then tms ran bE='
used orovidmq ltS accuracy 1S carerullv cneckec manually. N01se on tne
detector' can often introduce inaccuracies that are less likely to occur wttn
rnanua1measurement.
The Gaussian Form of the Elution Equation
In order to chance the POisson form of the elution equation into the Gauss1an
form it is necessary to effect a change of origin. Consider the elution curve
shown in I tqure (7), The origin for the Poisson form of the equation is at the
point of inject ion whereas, the origin for the Gaussian ecuat ion wi 11 be at
the peak maximum, (n) plate volumes from the injection point. Thus, a point
A, (v) plate volumes from the injection point) will be (v-n) = w plate
volumes from the peak maximum, The necessary change in origin is
demonstrated tn Figure (7),
Now the POlsson form of the elution equation is given by)
FIgure 7
The Difference in Axes Between The Poisson Function and the
Gaussian runctton
o Axis for Poisson fun etion 0 Axis for
l- _____n _ _ _ _ _ _ _ _ _ _ : Gaussian function
\
I
I
'-------v
'v-n'
, I
,:W,
"'->,
,
)r
)1
SUbstituting Cw+n) for (v)
Now, from Stirlings Theorem, n! ;::;: e-
nnnJ2nn,
Therefore)
It follows, loge Xm(n) = loge ~ - w+ nlOge(l + w)
2nn n
Exp;1nc1ing as the series:
x (w w
2
w
3
)
;::;: 0 _ + +_
loge r;:;::: w n 2 3' - .. "".
,,2nn n 2n 3n
X w
2
W
3
=10ge ~ -w + w- 2n + 3n
2
- "
Now (n) is large and the whole elution curve 1S practically contained
between w = -2jn and w = 2jn (Le. contained wtthtn four standard
deviations of the Gaussian Curve) thus y.f will always be very much
2n
s s
greater than Wz and tnus, Wz and all higher terms can be ignored wHh
3n sn
respect to V; .
2n
Therefore,
w
2
X e
2n
logeXm(n) = loge /2;;n
2nn
........................... " " .. (9)
Equation (9) is the well known Gaussian form of the elution curve equatlon
and can be used as an alternative to the Poisson form in all applications of
the PI ate Theory.
References
1/ .. Contefl7porary LIquIdClJrofl7atograplJY"J (R. P. W. Scott), John Wi ley and
sons, New York, ( 1976)31
2/ " Gas ClJrofl7atograplJy-SecondEdition", (A.I.M.Keul ernans), ReinhoId
Publishing Corporation, Amsterdam, (1959) 122
3/ "Contemporary LiquidChromatography", (R. P. W. Scott), John Wi ley and
Sons, New York, ( 1976)35
Chapter 5
AppHcat10ns of the Plate Theory
The elution curve equation, derived from the Plate Theory, can be used in a
number of ways to describe the chromatographic performance of an LC
column operated under a variety of conditions (1,2). It can also be used to
explain other physical chemical phenomena that occur in a column, such as
the change in temperature of the contents of a theoretical plate during the
passage of solute through it (3) and to examine pressure changes that can
occur in a theoretical plate in GC (4) . In this chapter the elution curve
equation, or its derivatives, will be app11 ed to a number of practicaI
sttuattons in LC to, either expand our knowledge of the physical chemical
processes involved, or to uttuze the process to an analytical advantage.
The Maximum Sample Volume
Any sample placed on to an LC column will have a finite volume, and the
variance of the injected sample will contribute directly to the final peak
variance that results from the dispersion processes that take place in the
column. It follows that the maximum volume of sample that can be placed on
the column must be limited, or the column efficiency will be seriously
reduced. Consider a volume Vi, injected onto a column, which will form a
rectangular distribution on the front of the column. The variance of the peak
eluteo from the column will be the sum of the variances of the injected
sample plus the normal variance of the eluted peak. The principal of the
Summation of Variances will be discussed more extensively in a later
Chapter. at this time it can be stated that,
where, 0
2
is the variance of the elutedpeak,
0'1
2
is the variance of the eluted sample,
and oc
2
is the variance due to column dispersion.
Now the maximum tncrease tn band wtdth that can be tolerated due to any
extraneous dispersion process is obviously a matter of choice but
Klinkenberg (5) suggested a5% increase in standard deviation (or a 10%
increase in peak vartance) was the maximum extra-column dtsperslon that
could be tolerated without sertous loss in resolution, This criteria is now
generally accepted.
Consider a volume (VO, injected onto a column and rorrntnq a rectangular
ctstnoutton at the front of the column. The variance of the final peak will
be the sum of the variances of the sample volume plus the normal variance
of a peak for a small sample. Now the variance of the
distribution of sample volume ate the beginning of the column is and
assuming the peak width is increased by 5% due to the sample volume,
Consequent ly}
Bearing in mind that,
Then,
2( 2 )
= n(v
m
+ Kv
s)
1.05 - 1
It is seen that the that the rnaxtmurn sample volume that can be tolerated
can be calculated from the retention volume of the solute concerned and the
the efficiency of the column. A knowledge of the maximum sample volume
that can be place on a column is important where the column efficiency
available is only just adequate and the compounds of interest are minor
components of the mixture to be analyzed and are only partly resolved.
....
"J)
vacancy Chromatography
Consider HIe situation where an LC column is fed with a mobile phase
containing a solute at a given concentration until equilibrium is acrneveo,
and an injection of pure mobile phase, devoid of the solute, is placed on the
first plate of the column. This will result in a fall in concentration of the
solute in HIe rtrst plate which, mathematically, wul represent the mjection
of a cnarqe of negat ive concentration. Thi s negative concentrat i on prof i 1e
of sample w111 pass tnrouqn the column in exactly the same way as a
postt ive concentration profile and will be eluted at the same retention time
or volume but will be recorded as a negat ive peak by the detector,
The qeneration of a chromatogram of negative peaks by the injection of a
sample of pure mobile phase into a mobile phase containing solutes at
constant and known concentrations has been given the term Vacancy
ChromatograprlY . Vacancy chromatography has interestmq prooerues and
has a number' of useful applications, few of which have exploited to
date. The processes involved in Vacancy Chromatography wi l: be now
considered tneoreticauv employing the equations oertveo from the plate
theory.
If a mobile phase, car-rying a constant concentration of solute (X
o
), is fed
continuously onto a chrornatographic column and equi 1ibrium is allowed to
be established, HIe eluent rrorn the column will also contain the solute at a
concentratton eX
o
) . If a sample of the same solute, dissolved in the mobile
phase at a concentration of (Xi), is now injected onto the column where
either eXo<Xi) ,or eXo>Xi), then this will result in a perturbattor: on the
concentration eX
o
) and) from the plate theory, this perturbation will pass
through the column and be sensed by the detector at the end of the column.
The equation describing this perturbation of solute concentration at the end
of the column will) from the plate theory, be given by:-
where Xen), is the concentration of the solute in the mobile phase
, tho (", 'th ,,1
11l:f '-II\, \IIJ'-II 1-11<..1'-",
V, is the volume passed through the column in plate volumes,
and n, is the number of theoretical plates in the column,
If the sample mjected is solely rnobue phase wlth no solute present then,
XI""O, and,
Thus, the actuaI concentrati on Ieavi ng the (n)th plate and entering the
detector', (XE), wjll be,
Employing the Gaussian form of the elution equation, this can be put in the
form,
.......................... ( 1)
Furthermore, at the peak maximum when v=n , and w=O,
It IS seen from equation (1) that under the conditions considered, where
the charge ts place on the fjrst plate, (X
n
) can never equal zero and pure
mobue phase will never elute from the column. However, the sample rs
rarely injected solely on the f1rst plate. As it occupies a r tntte volume of
mobtle phase when It is Injected onto the column, it wjl1 also occupy a
rintte number of theoretical plates. If (p) plate volumes of pure mobile
phase are injected onto a column that has been ecuiubrtateo wah mobile
phase contairnnq a concentratton (Xo) of a solute, then, on the aooucatton of
a charge of pure rnobue chase.
For- p=l Tne chanqe tn concentration of solute on the r.rst plate will be,
\ J I __ 1 .. '\
1'\0\ e I - I)
for p=2 , The change in concentratlon of charge on the fjrst plate will be,
1:,7
.
and for p=p The change in concentrauon of charge on the nrst plate Wl Jl be,
Xo(e-P -1)
Now, after each plate vo lume of charge has estabI i shed, a new concentration
in plate (1), as contents will be eluted through the column in the normal
manner. If a total of (p) plate volumes of pure mobile phase are mjecteo
onto the colurnn and HIe solute subsequently eluted by a rurtber (v) plate
volumes of equilibrlated mobile phase then; after (r) plate volumes of
sample have been injected, the new concentration of solute in plate (1) wiu
be eluted by a further (p-r) plate volumes of sample and (v) plate volumes of
equi ubrtatec rnobue phase.
Thus the concentration of solute leaving the (n) th plate due to the (r) th
volume of charge will be,
-(v +P-r-n) 2
( )
e 2n
X
n
=X
o
e-f -1 J2Ttn
It follows that the actual concentration of solute tn the (n)th plate, (XE)
result ing from the injection of (p) plate vOIumes of pure mobile phase,
followed by (v) plate volumes of equilibriated mobile phase carrying a
concentration (X
o
) of solute; will be given by:-
Equation (1) can be used to calculate the elution curves that result from
different volumes of pure mobile phase that have been injected onto a an
equilibriated column. Values of (XE) for such curves were calculated for a
column having 500 theoretical plates and for sample volumes of 20, 50,
100, and 200 plate volumes respectively. The curves') relating solute
concentration to plate volumes of mobile phase passed through the column,
are shown in figure (1). It is seen that as the injection volume is increased,
the retention volume of the peak also mcreases. The retention volume of the
small negative peak produced by the smallest charge will be equal to that
for the solute when XpXo and also when chromatographed in the normal way
with the column carrying pure mobile phase only This assumes that the
presence of the low concentration of solute in the mobile phase does not
mr luence the r etent.ve characterlstlcs of the stationarv phase It 1S seen
that HIe concentratton of solute in trle eluteo peak does not fa'll to zero untli
Figure 1
Vacancy Elution Curves for Different Injection Volumes on a
Column of 500 Theoretical Plates
til
30
-
e
:>
>, 25
..
0
L.
-
:E
20
..
0(
I

, 5
0
a
..
c
'0
"
S
u
5
.!
:l

,
0
volyme
meosur£d in plgle
vQlumeS
,
600
AOIN of rnobue phose I n plate vol umes
p. 20
P. eo
P.100
p. ZOO
,
eco
the sarnple volume is in excess of 100 plate volumes, which 1S about itve
time the standard ceviauon of the norrnally loaded peak. Equation (1) can be
developed further' to provide a general equation for a column eouninrtateo
with (q) solutes at concentrations XL X2, X3, ..... X
q
. For any particular solute
(5), if its normal retention volume is RS on a column containing (n) plates,
then the plate volume of the column (Vs) is given by,
R
V
- ..-2
s-
n
If the sample injected is contained in a volume (V) rnl, then the charge
measured in 'plate volumes' is v/vs and if the volume of mobile phase
passed Uwough the column is (y) ml then that will be ecuivalent to y/vs
plate volumes If the sample injected onto the column contains solute (5) at
a concentration X
s
1 then from equation (1) the concentration of this solute
at the (n) th plate in the column (XEs) is qiven by:-
v - (L +Y- -r _n)2
r=- ys ys
vs
XEs .XS+ -It
r=l
59
Thus for a chromatogram of (q) solutes
.................. (2)
2n
V
r·-
s-Q VS
XE =LXs+ L( x ~ - x s ) ( e - r _l)_e--=::----
s=1 r =1
If pure rnobue phase is injected onto the column.then X
s
1
= 0 and equation (2)
becomes,
2n
V
r
e
-
s..,q V5
XE = 2:xs + 2: xs(e-
r
-1 )_e----==--- (})
s= 1 r=l
Equation (3) is similar to that provtdec by Reilly et a/ (5) but the derivation
is simpler, as those authors utilized the approximate binomial form of the
elution curve in their derivation.
vacancy chromatography has a number of apphcatlons areas in practice,
none of which appear to have been extensively exploited. One particularly
interesting aooucatton is that of quality control. If a parttcutar product has
a number of components present, and their relative composition must be
kept constant as in, for example, a pharmaceutical product, Vacancy
Chromatography can provide a parttcular-ly simple analytical procedure for
qualtty control. The mobile phase is made up containing the components of
the product in the specified proportions, but at a low concentration suitable
for LC analysis. A sample of the product Is dissolved in some pure mobile
phase at the same total mass concentration as the standards in the mobile
phase. A sample 1S then injected on the column. If the product contains the
components in the speci f i ed proport i on, no peaks will appear on the
chromatogram as the sample and mobile phase will have the same
composition. If any component is in excess, it will show a positive peak. If
any component is present below specifications, it will show a negatfve
peak. The size of the peak will provide an accurate measure of the
difference between the sample and that of the required standard.
60
The Resolving Power of an lC Column
It has already been stated that for two solutes to be resolved their peaks
must be moved apart in the column and, at the same time, maintained
sufficiently narrow to permit them to be eluted as discrete peaks. The
criterion for two peaks to be resolved is arbitrary, but as stated in Chapter
1, resolution is is usually defined as the ratio of the distance between the
peaks) to the peak width at the points of inflection, The separation of a pair
of solutes on columns of differing resolution is shown in figure (2),
Figure 2
Two Solutes Separated on Columns of Different ResolvIng
Power.
6cr
36N
5cr
25N
4cr
16N
9N
3cr 20"
4N
It is seen that for base-line resolution the peak maxima must be six
standard deviations (60) apart. But for accurate quantitatlve analysis,
employing peak heights measurements, a separation of (40) is usually quite
adequate. Even when peak area measurements are employed, a separation of
(40) will usually provide adequate accuracy, part tcularly If computer data
acquisition and processing is employed with modern software. Therefore}
throughout this book, whenever dealing with resolution, or column design, a
resolution of (40) will be assumed.
It should be pointed out that two adjacent peaks from solutes of different
chemical type or signlficantly different molecular weight will not
necessarily have precisely the same peak widths. However, the difference
will be relatively small and, in the vast majority of cases} will be
bl
negllg1bIe. ConsequentlyJ the peak wtdtns of closely adjacent peaks w111 be
cons1dered the same.
constcer the two peaks depicted in the now well recognized figure (3). The
dlfference between the two peaks, for solutes (A) and (8), measured In
volume flow of mobile phase will be,
n(vm +KBVs) - nCvm +!<Avs) = nCKB + KA)vs -------------- (4)
Now, because it can be assumed that the widths of the two peaks are the
same, then the peak width in volume flow of mobile phase will be,
20 = 2Jn(vm +KAVS) ------------------- (5)
FIgure 3
A Chromatogram Showlng the Separation of Two Solutes
,. ....--ri"m·lcAvS):VRA
......-------n(vm.lcSvs>=\Rs I tl
I I I
..... ..., -n :
: =Vo !'---nkgls-nlc
A
'5--'"
I =<lc Er 5

I
I
I
I
I
I
,
• ,
INJECTION DEAD
----:Ftl-=.:.:..' N;.,;..T_ Ym
I
I
I
lit
I

I
I
I
I
I
I
Rearrangj ng,
in== 4(vm+KAv~ )
(KB -KA)vS
Dividing through by (vm),
,
k
Now as (0) is defined as, 0 = ~
k
B
Then,
........................................ (6)
**********************************
Equation (6) is extremely important and was first developed by Purnell (6)
in 1959. It allows the necessary efficiency to achieve a given separation to
be calculated from a knowledge of the capacity factor of the first eluted
peak of the pair and their separation ratio. It is used extensively throughout
this book and is particularly important in the theory of column design,
It is of interest to determine from equation (6) how the required efficiency
to achieve a separation varies with the separation ratio (0) and the capacity
factor of the first eluted peak of the pair (k'A), In figure (4), curves relating
(n) and (k'A) are shown for a column separating sotute pairs having
separation rattos of 1.02, 1.05 and 1.10. It is seen that the necessary
efficiency (n) Increases as the separat ion becomes more difficult. That is,
when the peaks are closer together and (0) is small. This to be expected, but
what is not so obvious is the dramatic increase in (n) as the value of the
capacity factor becomes small. It follows, that to reduce the number of
b3
theoretical plates needed, and thus reduce the necessary column length and
analysis ttme , the phase system should be chosen such that the pair of
solutes that are closest together in the chromatogram, are not eluted at
very low (k') values.
F1gure 4
Graph of Log. Efflclency against Capacity factor for Solute Pairs
havIng DIfferent separatton Ratios

=
u
c
I)

4
....
LI.I
C
E
::I
-o 3
t.J

CD
Q
...
o 1.02
• 1.05
• 1.10
30 20 10
2
o
k"
The trrecnve Plate Number
The concept of the effective plate number was introduced and employed in
the late nineteen fifties by Purnell (7), Desty (8) and others. Its
introduction arose directly as a result of the development of the caplllary
coumn, which, even in 1960, could be made to produce efficiencies of up to
a million theoretical plates (9). It was noted, however, that these high
eff1c1enc1es were were only reauzed for solutes eluted close to the column
dead volume, that is, at very low k' values. Furthermore, they in no way
reflected the increase in resolving power that would be expected from such
L'_L -&&,-,---: 6-1-._ P..... "'".... F .......""' ....I"\,... ........ f rV':lrVan r"lllNlnc Thi
IIlyll VII lilt:' ua;:ll;:l VI u ic "'CI IVII11QlI\.,c; VI tJU\",I, ....... \",VI ....I"...J. ,,,,5
poor performance, relative to the high efficiencies produced, can be shown
theoretically ( and indeed will be, later in this book) to result from the high
phase ratio of capillary columns made at that time. That is the ratio of the
mobile phase to the stationary phase in the column. The high phase ratio was
due to the fact that there was very little stationary phase in the capillary
column (the film was very thin). It has already been shown that the
corrected retention volume of a solute is directly proportional to the
amount of stationary phase there is in the column and) consequently) solutes
wer'e eluted trorn a capillary column at relatively low k' values. The thin
t ilrns gave rise to very high efficiencies but) as was shown in the previous
section, at low k' values ver'y hlgh err ictenctes are needed to acmeve
relatively simple separations.
To compensate for, what appeared to be very mtsleaotnq efficiencies values,
the 'effective plate number' was introduced. The 'effective plate number'
uses the corrected retention distance, as opposed to the total retention
distance to calculate the efficiency. Otherwise the calcutatton is the same
as that used in the normal calculation of theoretical plates, In this way the
'effective plate number' becomes siqruucant ly smaller than the true number'
of theorettcal plates for solutes eluted at low k' values At high k' values,
the the two measures of efficiency tends to converge. In trus way the
'effective plate number' appears to more nearly correspond to the column
resolving power, In fact, it is an indirect way of trying to define resoluuon
in terms of the number of 'effect ive plates' in the co lumn.
The efficiency of column (n) in number of theoretical ht:l <hown to
be q.ven by tr.e followmg eouation,
'I
n=4 y'"
x
2
Y1S the reter.tion distance,
x is the peak width.
Now the number of 'effective plates', (N), by definition, is given by,
2
(Y-Yo)
N= 4 2 """ " (7)
x
where (Yo) is HIe retention distance of an unretamed solute (the position of
the dead poi nO
Now from the plate theory,
y_ +Kvs)
x- +Kv
s)
and .thus,
Y-Yo _ n(v
m
+Kvs)-nvm
x - 2Jn (v
m
+Kvs)
By divid1ng through by (vm )J and noting that, Kv
s
= k'
v
m
Jnk'
-
2( 1+ k1
Consequently,
4((Y-:olf = n ( ( I : ~ . J =N (8l
Equation (8) describes the relationship between the efficiency of a column
in theoretical plates and the efficiency given in 'effective plates. It is also
seen that the calculation of the number of 'effective plates in a column does
not provide an arbitrary measure of the column performance, but is directly
related to the number of theoretical plates 1n the column as defined by the
plate theory. It should be noted that as (k') becomes large, (n) and (N)
converge to the same value.
The 'effective plate number has an interesting relationship to the function
for the resolution of a column that was suggested by Giddtngs (10), Giddings
put forward the function : ~ , as a means of defining the resolving power (R)
of a column. He employed this function in an analogous manner to the
function used in spectroscopy to define resolution, that is, ;'A The value
taken by Gidd1ngs for Ak' was the band width at the base of the eluted peak
which is eoutvalent to twice the peak width.
Trurs from t.he olats theorv.
. . _. - , -.- t #'
66
Aqatn, divlding through by (vm ), and noting that, Kv
s
=k'
v
m
_ jnk'
R - 4(1+k')
IN
- 4 ., (9)
It is seen from equat ion (9) that the resolving power of the co Iurnn, as
defined by Giddings) will be directly proportional to the square root of the
number of 'effective plates', As a consequence (R) can be used by the
chromatographer to directly compare the resolving power of columns of any
size, or type. However, the value of (R) will vary with the value of (k') for
the solute, and so comparisons between columns must be made using solutes
that have the same (k') va lue.
It is also of interest to the chromatographer to know the minimum (a) value
of a pair of solutes that can be separated on a particular column. In fact,
this has been suggested, (11 ), as a basis for compar1ng the resolving power
of different columns. The d1sadvantage of this type of crtteria is that the
value of (a) becomes smaller the h1gher the resolving caoacttv of the
column. Nevertheless, the minimum value of (a) 1S important in practice and
it is of interest to see lf it can be related to the 'effective plate' number of
the column.
Now the minimum value of (a) of apair of solutes that can be separated on a
given column will be given by the ratio of the retention distance of the first
peak, plus its width at the base, to its normal retention distance, assuming
satisfactory resolution is obtained when the peak maxima are separated by
(40),
Thus,
4 (1+k')
=1+ r:
~ n k '
Again, bearing tn mind that,
l(\J _
". ~ - k'
--
vm
O(
4 1
Therefore, a = 1+ = 1+- (10)
R
It is seen that the chromatographer can arrive at the minimum (a) value for
a pair of solutes that the column resolve, directly from either the
resolution, as defined by Giddings, or from a simple function of the number
of 'effective plates'. However, again it must be emphasized, that this will
not be a omaoe value for any colurnn as it will aIso depend on the (k') of the
eluted solute.
The Peal( Capacity of a Chromatographic Column
The peak capacity of an LC column ts the number of peaks that can be fi tted
into a chromatogram between the dead volume and the 'last peak', each peak
being separated from its neighbor by (40). The 'last peak' can be arbttrarl ly
defined, or determined, from the properties of the column and/or the
chromatograph with which it is used. Most chromatographers have
experienced difficulties, when attempting to separate mult i-comoonent
samples, even when employing gradient elution.
Under isocrattc development, if the early peaks of the mixture are
adequately separated, then the Iate peaks are often broad, and consequent ly,
at concentrations so low that they are hardly detectable. Conversely, if the
late peaks are eluted at a sufficiently low k' values to improve detection
limits, the early peaks become bunched together and are not resolved. This
problem is obviated considerably by employing gradient elution, but if there
are a large number of individual solutes present in the sample.tnen the same
problems will arise. These difficulties are caused by the column having a
limited peak capacity and it is, therefore, important to determine how to
calculate peak capacity and how to control it. From the Plate Theory, the
peak width at the base is given by:-
and the retention volume (V
r
), given by:-
Tkllc c i rrirv l o f"rm "f tho. re:.' ('''lllrl hp t1ivpr'l hv'-
•••U'4.JJ ..... .....J "11"'" '''''' t VI II. VI ..... t"'" t-''''Y., \'-'J --_.- - ... :::J'. _ .. -I·
66
(5), is the number of peaks, having the same width as the last peak, that can
be f1tted into the chromatogram up to and tncludtnq the last peak. Th1s
function does not, of course, give a true value for the peak capacity, as all
the peaks that are eluted before the last) will have s1gn1f1cantly smaller
widths. Consequently, a considerably greater number of peaks can be fitted
into the chromatogram than the value of (5), calculated in this way,
suggests. In order to evaluate a true number for the peak capacity of a
column a d1fferent approach must be used.
Consider the chromatogram 1n figure (5), which d1agrammatlcally
represents (r) resolved peaks, each peak be1ng separated from its neighbor
by (40).
The base width of the (r)th peak will be)
FIgure 5
D1agram of a Chromatogram havIng (r) Resolved Peal(s
·1···· .. ······
ra-················ r(\fu. ~ •.......•....•.......•.•..•••••••.••.•"1
•s·. r(\fn +I«r-tt's> it: :
• •
• •
• •
• •






•,
, ,
: 4 ~ (Vm +!«r-l}Js) : 4I'\(\tn + ~ )
Consider apoint where the last two peaks merge. At this point the retention
volume of the last peak, minus half the peak width at the base, will equal
the retention volume of the last but one peak, plus half its peak width at the
base.
consecuent Iy,
n{vm+K(r-nVs)+2Jn(Vm+K(r-l)V
s)
=n(vm+K
rVs)+4Jn(Vm
+Krv
s)
ThUS,
Therefore, for peak w1dth (W( r-l» of peak (r-l ),
In a stmtlar way tt can be shown that the w1dth (W( r-2» of peak (r-2) will
be:-
Thus, lf the number of peaks that can be nttec tnto the chromatogram
between the 'dead time' and the time for the complete elution of the last
peak is t r ), then,
1U
Not ing that Kvs ;: k' I and rearranging
v
m
(
(r-1 ) ( r: tr-2 ) (r:)2 ( r: )
k' _ 4 n-2.jn n-2; n n-2,n n-2,n
l+k' - jn n+2jn) + n+2[n) +".""."." + n+2jn + n+2[n +05
Replacing the geometric series by the expression for its sum)
(
n- 2
fn
)r
k' _ 4 1- n+2jn
l+k' - jn n-2.jn) - 0.5
1- --"=
n+2jn
Rearranging to provide an expression for ir )
.............................................. (12)
.......................... (13)
EQuat ion (12) is very similar to that produced by Giddings (12). However,
Giddings makes certain assumptions in his derivation, not made in the above
argument, that render the peak capacities quoted by him for l1quid
chromatographic systems somewhat less than those given by equation (13),
However, the difference will not be practtcauy significant. Equation (13)
Shows that the peak capacity depends on the column efficiency and the
capacity ratio of the last eluted peak. Empioymg ecuanon (13)) the peak
capacity of a series of columns having different efficiencies were
catculated for a range of peak capacity factors and the results are shown as
curves relating peak capacity to capacity ratto in flgure (6)
71
F1gure 6
Graph of Peak capacttv aqatnst Capactty Factor
80 -r--------------------,
60
:=
..,
...
u
a
t 40
U
~
a
CD
e, 20
o n-2000
• n=6000
• n-12000
12 10 8 6 4 2
O__--..,..--_r_-......--r---r--....,..----/
o
Capaclty Factor (kO)
It ts seen that, as one would exoect, the peak capacity increases wtth the
column errtctency, but the overriding factor is the capacity ratio of the last
eluted peak. It follows that any ltrnttatton to the value of (k') for the last
peak will also ltrntt the peak capacity. Davis and Giddings (13) have shown
that the theoretical peak capacity will be an exaggerated value of the true
peak capacity due to the statistically irregular dlstrtbutton of the
indlvldual (k') values of each solute in a realistic rnuttt-cornoonent mixture.
In fact, they pointed out that solutes do not array themselves conveniently
along the chromatogram four standard deviations apart to provide the
maximum peak capacity. Nevertheless, the actual nature of the distribution
of (k') values for any given solute mixture is unpredictable and will vary
from mixture to mixture depending on the source of the sample.
Consequently, the values for the theoretical peak capacity of a column given
by equation (13) will be a good basis for the comparison of the peak
capactttes obtainable from differenlcolumns
J
albeit the theoretical values
............... l •., , · , ~ ••• lll ....,.. l", ,..",...,..,...,... "f' th" "",,-:lIV f"-:lIn"::lor-itioc I"O":l117ori 11"1 nr'ArtirA
VULc:lIlICU W III VI;; III CI\\,C.;).;) VI \.1 Ie "'Cc.l" ",,"ul-'uv I; ,........ I ....... ' ,............ '" .... ' ,.n.. ". "''''.
Now any characteristic of the column system that places a limit on the
max1mum value of (k'), will also place a limit on the maximum peak
capacity. One factor that controls the maximum (k') value of the last eluted
.,?
.-
peak is the detector senstnvtty. As the (k') of a solute increases, the peak
becomes more dtsoersed, and consequently, the peak helght 1s reduced, and
eventually at a given (k') the peak will disappear into the detector 'noise'. If
tt is merely necessary to unamb1guously 1dent1fy the presence of a peak,
then the peak maximum would need to be about five time the noise level. The
detector senstttvtty or the rntntrnum detectable concentration (Xo) ts
usually certneo as equal to twtce the noise level and consequently the peak
height at the maximum (k') value must be 2.5.Xo. Now.the concentration of
solute at the peak maximum can be taken as twice the average
concentration. Thus 1f a a mass (m) ts placed on the column and the peak
width is 4Jn(v
m
+Kv,) I
Then,
2m =2.
5Xo
4Jn(V
rn
+KVs)
m;;5XDJ"n(V
rn
+KV
s
) (14)
Now the max1mum sample volume (Vj) that can be placed on the column
Without tolerating a band width increase of more than 5% has been shown to
be,
Furthermore, the effect of sample volume on peak width wtll be greatest for
the early peaks (the most narrow peaks) and wm progressively decrease for
all SUbsequent peaks. As the resolution of both late and early peaks must be
qiven equal priority, the sample volume must be chosen such that the
increase in the wtdth of the rtrst unretainedpeak ts restrtctec to 5%.
ThUS, for an urretalned peak)
Consequently, 1f the solute concentration in the sample is (Xj),
....... ~ , . ( 15)
Equat lng eouat Ions ( 14) and ( 15),
fj
D1vld1ng both sides by (vm), remembering ( k'= K ~ ) and simpl1fying,
vm
1.
1X
l 1 '
-= +k
5Xo
and thus,
k' =0.22 Xl _1 ".......................... (16)
Xo
It is seen that the maximum value of the capacity factor is inversely
proportional to the detector sensitivity or, the minimum detectable
concentration. It follows, that the detector sensitivity also sets an
ultimate urntt on the peak capacity that can be realized from a column. This
lima however ts fairly high as can be seen from the data qtven in Table (1)
The capacity ratios and peak capacities were calculated for a column having
an efficiency of 10,000 theoretical plates, a dead volume of 6.7 ml and a
sample concentration of 1%v/».
Table 1
Capactty Rattos and Peak Capacttles for Detectors of Dtfferent
Senstttvtttes.
Detector Maximum k' Retention Peak
senstuvtty Time Capacity
(min)
10-6 g/ml 220 73.6 134
10-7 g/ml 2,200 736 177
10-8 g/ml 22,000 7,360 194
It is seen from table (1) that by increasing the minimum detectable Quantlty
by an order, from 10-7 q/rn: to 10-
8
q/rnl, the rnaxtrnurn (k') value is also
increased by an order but the peak capacity is only increased by about 10%.
Furthermore, this lncrease in peak capacity is achieved at an increase in
retention time from twelve hours to about five days. It follows that,
attainment of increased peak capacity by increased detector sensitivity, is
extremely costly in time and higher peak capacities are best achieved by
the use of columns of tntrtnsic high efficiency.
74
Prectston as an Alternative to ResolutIon
Providing retention tirnes can be measured precisely they can be used to
deterrntne the composition of a rntxture of two substances, that, altnough
having finite dtrrerences tn retention times, are eluted as a slngle peak on
the column employed. This can be acrneved, providtnc the Standard Devtation
of the measured retention time is small compared with the dlfference ln
retention times of the two solutes.
Consider two solutes eluted close together such that a single composite
peak is produced. From the Plate Theory the concentration profi te of such a
peak can be descrtbeo by the fol1owlng equation-
................................ (17)
Where, (XAB) Is the concentration of solutes (A) and (5) in the cornoostte
peak,
(XA) ts the initial concentratton of solute (A),
(XB) ts the tnttral concentratton of solute (B),
(nA) is the column efficiency for solute (A),
(nB) is the column errrctencv for solute (B),
(VA) ts the volume of rnobue phase passed through the column tn
'plate volumes' of solute (A),
(VB) ts the volume of rnobue phase passed through the column in
'plate volumes' of solute (B).
If (tA) and (tB) are the retent 10n times of solutes (A) and (B) respecttvely by
simple proportion equation (17) can be put in the rorrrr-
where the vartable (v) ts replaced by the vartable (0 the elapsed time.
It ts seen from equation (18) that when only (A) is present, the function will
exhibit a maximum at t =tA, and if only solute <B) is present, It will exhintt
a maximum at t = te . 1t follows that the compos: te curve will give a range
75
of maxtrna between (tA) and (tB) for different oroooruons of solute (A) and
(B), Thus from the value for the retention time of the composite peak the
composttton of the ortqtnal rntxture can be determined.
For closely eluted peaks nA = nB and thus, (J2nnA) and (J2nnB) are, in
effect, average dtlution factors resulting from the peak disperston and can
be replaced by a constant. The efficiencies (nA) and (nB) in the exponent
runctton, however, can only be considered equal lf the peak is symmetrical.
This is because in that part of the composite peak that determines as
maximum, the rear part of the first peak merges wtth the front part of the
second peak. In Le, the concentration ororues of eluted peaks are rarely
completely symmetrical, and, thus, (nA) must represent the efficiency of
the rear half of the nrst peak (solute A) and (nB) the efficiency of the front
half of the second peak (solute B). Efficlencies are catculated in the normal
way except, when deterrntrnnq the errictencv of the front half of the peak,
twice the front half peak width is used tnstead of the total peak wtdtn
SimilarlYJ to deterrntne the errictencv of the rear half of the peak, twice the
rear half width is employed instead of the total peak width. In addttton the
response of the detector to the specific solutes (14 ) must be taken lnto
account.
Thus lf (D) is the voltage output from the detector, equation (18) can be put
in the rorrn-
............... (19)
Where, (e) Is a constant,
(a) is the response of the detector to solute (A),
and (p) is the response of the detector to solute (B).
Equation (19) was examined by Scott and Reese (15) employing mixtures of
nttrooenzene and fully deuterated nitrobenzene as the test sample. Their
retention times were 8.927 min and 9.061 min respecttvetv givlng a
difference of 8.04 seconds. The separation ratto of the two so lutes was
1 f"I0? .... "I'l "h" "f'f'i ..... i "" ; " ..... "f' t ..... " f'""""t .... "'I'l """ .... "" ",,,,,,,t; ""''"' "f' th" .... "..,,[,.,..., "'"''''''
I .VL..... GllJu \..111; I; I I 11... 11;11 1'1;.;) VI \..111; II VII\.. GlIIU .I;;;GlI ""VI \..IVI I.;) VI \..11<.:; Yl;Uf\.,,) ¥'I' <':;1 c
5908 and 3670 theoretical plates respecttvely. The detector was, not
surprtsinqly, found to have the same response to both SOlutes, i.e. a =p. Thus
inserting these values in equation (19),:-
76
A range of concentrations of the two substances were inserted in equation
(20) and a curve constructed relating retention time of the composite peak
(calculated by means of a computer) to mixture composition. The results are
shown as the curve in figure (7)
FIgure 7
Graph of Retention Time or Composite Peak against Composttton
or M1xture
9-061 -- -- - - - - - - - - - -- - - - - - - - - - - - - • - - - - - - - - - --
7-5
&927mn 0
NITROBENZENE 10 20 30 40 50 60 70 80 90 100
%w/w DEUTERo-NITR08ENZENE
The retention time of a series of mixtures of nitrobenzene and deuterated
rutrobenzene were determined by Scott and Reese and are shown plotted as
points in figure (7). It is seen that close agreement is obtained between the
experimental points and the theoretical curve, The procedure described is an
interesting alternative for the analysis of mixtures of closely eluted
SOlutes, to the difficult and tedious construction of columns of extremely
high efficiencies. With great care and the of modern sophtsttcatec
computer programs the required accuracy can be easily obtained, and it is
suprising that this approach is not used more often,
77
A Theoret1cal Treatment or the Heat or Absorption Detector
The Heat of Adsorption Detector, devtsed by Claxton (16) 1n 1958 has been
trwesttqatec by a number of workers (17,18, t 9) but although once
commercially available, has not been extensively employed as an LC
detector. One reason for tnts is the curious and apparently uaoredtctaoie
shape of the temperature-time curve that results from the detectton of the
usual sausstsn or P01sson concentration peak profile. The shape of the
curve changes with detector geometry, the operat1ng conditions of the
chromatograph, the retention volume of the solute and for closely eluted
peaks, it produces a complex curve that is extremely diff1cult to interpret
The detector consists of a small plug of adsorbent, usually silica gel,
through which the eluent passes after leav1ng the column. Embedded tn the
silica gel is either a thermocouple or a tnermtstor which continually
monitors the temperature of the adsorbent and ns trnrnedtate surround1ngs.
When a solute eluted from the column enters the cell, some IS adsorbed onto
the surf ace of the s11 i ca ge1, the heat of absorption is evo1ved and the
temperature rises. When the peak maximum has passed through the cell, the
solute desorbs from the silica, aosorbmq heat with a consequent fall tn cell
temperature. The output of the temperature measurtnq device w11l tnus,
f1rst record a rise in temperature, and subsequently a decrease in
temperature relative to tts surround1ngs. The form of the temperature
profile Will depend on the heat loss during the adsorption/desorption
process and the re1ative plate capaci ttes of the detector ce11 and the
associated column. The elution curve equation, as derived from the plate
theory, can be used to obtain an expresston for the change of temperature of
the detector cell with the volume of mobile phase flowlng through it.
Consider a sma11 cell, containing the adsorbent, srtuated at the end of the
column through Which the column eluent passes. It IS assumed that the
eluent is brought to a constant temperature by a suttable heat exchanger
situated between the cell and the column and that the exchanger does not
contribute signif1cantly to band dispersion. Let the cell have internal and
external radii of (rl) and (r2) respectively and length (I), The following
postulated wlll be rnaoe.-
t / The flow of rnootle phase through the cell 1S constant.
.
2/ The temperature of the ce11 surroundings is constant
3/ The cell is of surrtcient size that solute equilibrium between the
two phases can be assumed,
78
4/ The cell itself does not contribute significantly to band dispersion.
Consider the heat balance of the cell,
[(Heat Capacity of Cell) X(Change in Ternperatureil =(Heat Evolved in Cell)
- (Heat Convected from Cell by Mobile Phase) - (Heat Conducted from Cell)
The volume flow of mobile phase wIII be measured in 'olate volurnes' (v) of
the attached column and the column plate volume will be designated as
(ca) for solute (a). Let a volume dv of rnobue phase pass through the cell
carrying solute that is absorbed onto the silica wlth the evolution of heat,
and let the resulting temperature change be (de).
Then assuming the heat capacity of the solute is negligible,
Hda =(Heat EvaIved In Ce1])- (Heat Convected from Ce1I)
- (Heat Conducted from Ce 11) .
Where, His the heat capac: ty of the ce11,
t.e H= VmdmS
m
+ V
5dsSs
+ VgdgS
g
and V
m
, V
s
, and Vg are the volume of rnobue phase in the cell, the
volume of aosorbant in the cell and the wall volume
respect ively,
d
m
• d
s
, and d
g
are the densities of the rnobue phase, absorbent
and cell walls respectively,
Sm155, and Sg are the specific heats of the mobtle phase,
absorbent and ce11 walls respectively.
Note the Italtc form, ( V), is used to distinguish it from volume, (V), and
plate volume, (v).
The heat evolved in the cell during the passage of a volume (dv) of mobile
phase will now be derived. Let a volume (dv) of mobile phase, equivalent to
(cadv) ml, enter the cell, and let the concentration of solute (a) in the
incremental volume be (X
n
). Let an equivalent volume (cadv) of rncbuc prlQS€
be displaced from the cell, and let the concentration of solute In the mobile
phase contained by the cell prior to the introduction of the volume (dv) be
(X
m
).
79
Now the net change of mass of soIute (dm) 1n the ce11 w111 beI
dm =(Xn-Xm)cadv " " " (21)
As equi ltbrturn ts assumed to occur tn the detector ce 11 the tntrocucnon of
the mass of solute (ern) w111 result tn a change tn concentration of solute tn
the rnobue phase and adsorbent of (dXm), and (dXs) respectively, where (X
s
)
ts the concentratton of solute in the absorbant
and as,
ern = VsdX
s
+ VmdXm
dX
s
=KdX
m
dm > VsKdX
m
+ VmdX
m
=( VsK + Vm)dXm (22)
Equating eouat ions (21) and (22) and rearranging)
(
~ s K + ~ m )dXm X - X
-+ m- n
c
a
dv
Now, VsK + V
m
IS the 'effective cell volume' tn much the same way that (ca)
ts the column 'plate volume'.
(K Vs+Vm) C "errective plate volume" f t a)
--- = :; or solu e (
ca a "column plate volume"
dX
m
C
a
- + X
m
=X
n
" " ". (23)
dv
v
MUltlplylng throughout by (e
Ca
),
v v v
-C dX
m
-C C
C
a
e a - + Xmea = X
n
e a
dv
ou
dv
v
~ - - - - - - ~ =xne
ca
or,
Integrating}
v v
CaeCaX
m""
JXneGadv + R
Now, when v =0 the solute has not moved from the point of injection on the
column.
Thus, when v =0 ,X
m
= Xn =0 and consequent lv, R=0 and,
v
--
e Ca
X
m
=
C
a
v v
JX
n
eGa dv """ "."".".. ".. "". (24)
o
Equation (24) provides an expression for (Xm). Continuing; if the change in
mass of solute on the absorbant due to a volume flow of rnobt!e phase cadY,
is (dms), then the consequent heat evolved dG tn the cell will be qtven by:-
dG == g(d:
s
)dV
where (g) 1S the heat of adsorption of the solute tn cal. per gram of solute.
Hence,
dG == 9V
s
( ~ ~ )dV and, as dXs =KdXm.
From ecuat ion (23),
(
dXmJ
dG =KgV
s
dV dv
dX
m
= (X
n
-X
m
)
dv C
a
VI
SUbstituting for ( dX
m
),
dv
Thus, suostttvttnq for (X
m
) from eouatton (24) and rearranqtnq,
Now,
Therefore,
Let,
Hence,
dG =KgV
s
X
o
e-vvn_(X
o
) Jee: (e-vv
n
) dv dv
C
a
nl C
a
nl
dG = KgV
s
f(v)dv (25)
C
a
Now, the heat conducted from the cell will be considered to be controlled by
the radial conductivity of the total cell contents and not by the cell
walls alone. Furtnerrnore, the axial conductivity of the cell will be ignored
as tts contribution to heat loss will be several orders of magnitude less
than that lost by axial convection. Consequently, as the cell is
the heat conducted radially from the cell has been shown to be (20)
2nlEedt
L o g ~
rt
where, (E) is the thermal conductivity of the cell contents,
(8) is the excess temperature of the cell above its surroundings,
(rt) is the radius of the senstnq element
and (dO is the time taken for a volume cadv of rnootle phase to pass
through the ce 11,
Now (dt) refers to the time interval durlng the Introductton of the volume
(cadv) of mobile phase and tnus, if the flow rate is (0)1
dv = ~ or dt = ca
dv
dt c
a
Q
2n1EC
a
Sdv
Thus, heat conducted from the cell ts,
OLog e!l
rt
and the heat convected from the ce11 lSI dmS
m
SC
a
dv
Thus) inserting the above expressions for the heat conducted from the cell
and the heat convected from the ceII, together w1th the heat evolved from
the cell, from equation (25) in equation (20A))
HdS =KgV
s
f(v)dv- 2nlEc
a
Sdv dmS
m
SC
a
dv
C
a
QLoge.!l
rt
where,
A =KgV
s
and
C
a
...... ,.."" ....... ,,"... ," ..""" .... """, ....... "",... (26)
A - Lni tCa dv d S
..,- - mm
OLoge.!l
rt
R C 'i.
e
t" aH t
MUltiplying equation (26) throughout by and in egrating..
Now) on tnjectton of the sample, when v=O, 8 = °and f(v) = 0 and
consequent ly, R= 0,
Therefore, """'''''''''''''''' (27)
Letting
Then,
A d pC
a
=~
H=cp an H 't'
V
B
v
=.e-
t v
JetVf(VldV (28)
o
It is seen from equation (28) that the constant (cp) merely effects the
magnitude of (8) but the constant (q,) and rev) condition the shape of the
temperature profi Ie and produces the curious shaped peaks recorded by the
detector. The constant (q,) can be considered as the heat loss factor of the
cell. It should be noted that the magnitude of f(v) will depend on the value of
(C
a
) the ratio of the 'effective volume of the cell' to the 'plate volume' of the
column,
Inserting the full expression for f(v) in equation (28),
J
v e-vvn (X) -v JV .s: (. e-vv
n
J' lJ
B
v
= .e-
t V
e
t v
X
o
n! - c: eGa e C. n! dv dv
o 0
....... (29)
Equation (29) is the expltctt equation for the temperature of a detector and
can be used to synthesis the different shaped curves that the detector can
produce. Employing a computer in the manner of Smuts et al (21) Scott (22)
calculated the relative values of (8) for ( v= 74 to 160) for a column of 100
theoretical plates) and for ([a) ranging from 0.25 to 4 and (ctl) ranging from
0.01 to 1.25. The curves obtained are shown in figure (8).
FIgure 8
Theoretical Temperature Curves and the Integral Temperature
Curves Obtained from the Heat of Absorption Detector




AIntegral Curves
The twenty curves shown are graphs of (9) versus (v) and the integral of
(9) versus (v) for different values of «(a) and (ctl) and are all normalized to
the same peak height. The curves cover the practical range of heat loss
factors that might be expected from an heat of adsorption detector cell.
They demonstrate the effect on the shape of the (9) versus (v) curves of
changes in 'detector cell cacacttvvcolurnn plate capactty' ratios that would
result from different cell designs but when detecting a peak of constant
width. The curves for different values of ([a) would also represent the
effect on peak shape of solute oJ different retention, and consequently,
having different peak widths passing through a cell of fixed dimensions. It
is seen that the major effect on peak shape is the 'detector cell
capacttvv'coturnn plate capacity' ratio, (C
a
). 'When tne capacity of the
detector cell is less than the plate capacity of the column, ([a< 1), the
negative part of the signal dominates and when the detector cell capacity
exceed HIe column plate capacity, ([a) 1), the positive part of the signal
dominates. For this reason when (C
a
>1), the integral curve nses to a
maximum but does not return to the baseline. Conversely.. when «(a< 1), the
integra1curve first rises and and then falls below the base1ine and does not
return. Only when (C
a
= 1) does the detector slgnal simulate the differential
form of the elution curve and consequently the integral curves describes a
true Gaussian peak,
The effect of the heat loss factor (.)1 on peak shape is small but the
magnitude of the signal varies tnversely as (4l) although, this is not apparent
in figure (8), as all curves are normalized, It should also be noted that for
low values of ( ~ ) , where the maximum senstttvtty is realtzed the peak
maximum is displaced. However, for large values of ( ~ ) J the maximum of the
integral curves for (C
a
= 1), is almost coincident with the maximum of the
elution curve,
It follows, that if the detector was to be effective and produce the true
Gaussian form of the eluted peak then, (C
a
), must at all times be unttv and
consequently the detector must have the same plate capacity as that of the
column. This means that the detector must employ the same absorbent, have
the same geometry and be packed to give the same plate height as the
column. It is obvious to accomplish this, the column must also be the
detecting cell and the temperature sensing element must be placed in the
column packing itself,
Restating the expression for f(v),
Now, if the sensor is in the column packing, C
a
= 1
Consequent ly,
86
T h u s ~
Recalling the basic differential equation for the elution curve (9) given on
page 18 in chapter (2)
f(v) = dX(n+1) (30)
dv
Now, as there is no (n+ 1) plate that constitutes the detector, the sensing
element can be considered to be placed in the (n)th plate.
Thus,
_ dX
n
f(v) - - ,... (3 1)
dv
If the (n)th plate of the column acts as the detecting cell, there can be no
heat exchanger between the (n-1)th plate and the (n)th plate of the column.
Thus) there will be a further convective term in the differential equation
that will take into account the heat brought into the (n)th plate from the
(n-l)th plate by the flow of mobile phase (dv).
Thus the heat convected from the en-1)th plate to plate (n) by dv will be
d
mSm
9(n-l )cadv " " (32)
substttuunq for f(v) from equat ion (31) ion equat ion (26) and insert ing the
extra convection term from (32)
dan =(A
p)
dX
n
_(Ppc
a
) a +(dmSmc
a)
a
dv H dv H n H (n-I)
p p p .
where the subscript (p) accounts for the change from the already defined
physical charactertstrcs of the detecting cell to those of the last plate of
the colurnn.
ThUS,
den dX
n
- = a--Be
n
+y9Cn-l'
dv dv I
A7
(33)
where)
A
a =--.£.
H
p
A solution to the differential equation (33) is given by:-
r
ran
a
= a ",\;,[CY-D] dXCn-r)
n CB-D.L.. (8-D dv (34)
r-O
The validity of this solution can be confirmed by differentiation as
follows:-
Now if,
Thus)
e-vv
n
X
n
= X
o
- -
n!
d
2X
n
_ dX{n-l) dX
n
--- --
dv
2
dv dv
d
2XCn-r)
SUbstituting for 2 in equation (35)
dv
r. n r
dan ~ ~ r C Y - D l (dXCn-l-r)
dv - CB-D~ L CB-DJ l dv
r.O
dXCn-r) I
dv )
or,
r- n r
den = a dX(n_1-r)
dv (B-O (B-O dv
r-O
r- n r
.s., dX(n_r)
(B-O.L.. (B-O dv
r=O
NowJ for the series,
dX(_l) =0
dv
Thus,
Consequent IyJ
or = (l-Y) eCn-l) +(B-1 )9
n
(36)
From equation (34) and using its expanded form,
(B-1)9
n
= a
dXn
+a(Y-1)dXCn-O +a[(y-,)]2 dX(n_2) + ..
dv (B-1) dv (B-1) dv
and,
Thus,
(l-y)eCn-l) = _ a(y-1) dXCn-l) _ a[(Y-1)]2 dX(n-2) ...
(B-1) dv (B-1) dv
_ dX
n
(1-Y)9Cn-O + (B-1)9
n
- a dv (37)
Substituting ad:: from equation (37) for (1-y)8(n_l) + (B-(B
n
in eouation
(36) and rearranging)
d9
n
dX
n
- = a--B9
n
+y9(n-O " " " " ..(38)
dv dv .
It Is seen that equattons (37) and (36) are tcentrcai wruch suostanttates the
validity of equation (35) for can), It is also seen from equation (35) that (o),
only effects the magnitude of the curve while (V) and (8) effect its shape as
well as its magnitude. Scott (22), assumed practical values for' the various
physical parameters of the system and calculated the temperature and
integral temperature curves for a sertes of different practical values of (8)
and (y), The results are shown in figure (9). In figure (9) the values of (V) are
represented as(G),
figure 9
Theoret1cal Temperature and Integral temperature Curves
Generated by a Temperature Sensor Situated in the Column
Packing
G=1.40 8=1.40 G=1.40 8=1.50 G=1.40 8=1.52 G=1.40 8=1.5 G=lAO 8=1.58
G-2.66 8=2.57 G",2.66 8",2.58 G=2.668=2.70 G=2.66 8=2.74 G-2.668=2.86
A' INTEGRAL CURVES
It is seen from the curves in figure (9) that the heat convected into the
detector cell or plate also distorts the curves. It is apparent that, unless
the heat lost radially is extremely high, so that little heat is convected to
the sensor, symmetrical integral peaks will not be obtained, This heat loss
appears Impossible to acrueve tn practtce and thus, the heat of absorption
detector does no seem Viable for LC.
It is now clear that the plate theory has a wide field of aooucatron Its use,
however is not restricted to LC For example, the plate theory can be used to
investigate temperature changes that take place in a GC column.G),
I ., I • I " ' __ '" I' A\ tl ~ ~ . ~ , L
pressure cnanges tnat take place In a \JL COIUrTH\) ,Lf)) ute ell ell 01 SOIUle
decomposition on band profile and other strnt tar effects that can take place
in a chromatographic system, The plate theory has many areas of appltcat lon
that still remain to be explored,
90
References
( 1) R P. W. Scott,c. G, Scott and P. Kucera, Anal Chem.}44,No 1(1972) 100.
(2) R P, W. Scott, Anal. 0' 36,No8( 1964)1455.
(3) R. P. W. Scott} J Chromatogr. ScI. , July (1973)349.
(4) A. Klinkenberg, in" Gas Chromatography /960"(Ed. RP.W.ScotU,
Butterworths Scientiftc Publtcations<London,England,( 1960)194.
(5) C. N. Reilley,C. P. Hildebrand and J, W, Ashley, AnaIChem,,34( 1962)1198,
(6) J H, Purnell} Nature)(London), 184,Suppl. 26( 1959)2009,
(7) J. H. Purne11 and J,Bohemen} J Chem. Soc} (1961 )2030.
(8) D.HDesty and AGoldup," Gas Chromatography /960"(Ed. RP,W.Scott),
Butterworths Scient! ftc Publ teat tonset.onoon.Enqlano.t 1960)162.
(9) R.P,W.Scott, Nature (London), 183( 1959)1753,
(1 O)J.C.Glddlngs," The. Oynan7ics of Chromatography"Marcel Dekker,
New York,( 1965)265.
(11) J.C.Giddings, ../Chron7atogr. so., 12( 1974)1753,
(12) J.C.Glddi ngs, Anal Chen7. 39 No. 8( 1967)1027,
(13) J.MDavls and J.C.Glddings, Anal. Chem, 55( 1983)418.
(14) RP.W,Scott," LiquIdChromatography Oetectors",Elsevler,Amsterdam
New York, ( 1986)18.
(15) RP.W.scott and C.E.Reese, ../Chron7atogr.138( 1977)283.
(16) G.C.Claxton, ,,!Chromatogr.2( 1959)136.
(17) A
AI t ///1nn/1nJ 18,)(1nE::O'11t:'1")
r-» lola (J, t,..- ,1'-'-""'-1'-"11/, I L\' -;!..JU/I
(18)K.P.Hupe and E.J,Bayer, JGas Chromatography. Apr11 (1957) 234.
91
(19) J.L.Cashaw,RSegura} and A. Zlatkfs} JCnromatogr SelB( 1970)363.
(20) RH.PerryJC.H.Chi Iton and S,D.Kirkpatrick}" Cnem. Eng Handbook".
McGraw Hfll ( 1975)1O.
(21) T.W.Smuts)P.W.Rickter and V.J.Pretorious) J Cnromatog Sci 9( 1971 )457.
(22) RP.W.Scott) JCnromatog. Se/,11 (1973)349
Chapter 6
Introduction to the Rate Theory
Solute equi 1lbrtum between the rnobue and stationary phases is never
achieved in the chromatographic column except possibly (as Giddings points
out) at the maximum of a peak (1), As stated before, to circumvent this non
equilibriurn condition and allow a simple mathematical treatment of the
chromatographic process, Martin and Synge (2) borrowed the plate concept
from dtsuuatton theory and considered the column consisted of a series of
theoretita1pI ates in which equtl ibrium could be assumed to occur. In fact
each plate represented a 'dwell time' for the solute to achieve eoun rerum
at that point in the column and the process of distribution could be
constcerec as incremental. It has been shown that employing this concept an
equation f or the elution curve can be easily obtained and, from that basic
equat ion, other'S can be developed that oescrl be the vanous properti es of a
chromatogram, Such equations wl1l permit the calculation of efficiency, the
calculation of the number of theoretical plates required to achieve a
specific separation and among many applications, elucidate the function of
the heat of absorpt ion detector,
The Plate Theory, however, does little to explain how the efficiency of a
colurnn may be chanqeo or, What causes peak dispersion in a column in the
first place, It does not tell us how dispersion is related to column geometry,
properties of the packing, mobile phase flow-rate, or' the physical
properties of the distribution system, Nevertheless, it was not so much the
limitations of the Plate Theory that provoked Van Deemter et a/ (3) (who
wer'e cherntcal engineers and rnathernat tctans) to develop, what is now
termed Hie Rate Theory for chromatographic dispersion, but more to explore
an alternative mathemat ical approach to explain the chromatographi c
process. Virtually all basic chromatography theory evotved over the twenty
five year's between 1940 and 1965 and it was in the middle of this period
that Van Deernter' aneJ ~ l i s colleagues presented their Rate Theory concept in
(1956), Since that time, other Rate Theories have been presented, together
With accompanying dispersion equations and in due course each will be
discussed, but most were very similar in form to that of Van Deemter et et .
It is tnterestmq to note, rlowever, that, even after trnrty five years of
chromatography devetoprnent, the equation that best describes band
dispersion in practtce is still the Van Deemter equation, This is particularly
true for columns operated around the mobile phase optimum velocity where
the maximum column efficiency is obtained,
The purpose of the Rate Theory is to aid in the understanding of the
processes that cause dispersion in a chromatographic column and to identify
those factor's H\3t control it. Such an unoerstandinq will allow the best
column to be designed to effect a given separation in the most efficient
way. However', before discussing the Rate Theory some basic concepts must
be introduced and nlustrated.
The Summation or Variances
The widU\ of the band of an eluted solute relative to its oroxtrnttv to its
nearest neighbor determines whether two solutes are resolved or not. The
ultimate band width as sensed by the detector is the results of a number of
indiVidual dtsperslon processes taking place in the chromatographic system,
some of which take place in the column itself and some in the sample valve,
connecting tubes and detector', In order to determine the Ultimate otsperston
of the solute band it is necessary to be able to calculate the final peak
variance, This is achteved by taking into account all the individual
oisoerston processes that take place in a chromatographic system. It is not
possible to sum the band wtoths resulting from each individual dispersion
process to obtain the final band Width, but it is possible to sum all the
respect ive variances. However, the surnmatl on of all the variances result ing
from each process is only possible if each process is non-interacting and
random in nature. That is to say, the extent to which one dtsperston process
proqresses is independent of the development and progress of any other
otspersion process.
Thus, assuming there are (n) non-interacting, random dispersive processes
occurring in the chromatographic system, then any process (p) acting alone
will produce a Gaussian curve having a variance o ~ )
2 2 2 2_ 2
Hence, 0 1+0 2+0 3 0 n- 0
where, 0
2
is the variance of the solute band as sensed by the detector.
The above equation is the alqebraic enunciation of the ortnctote of the
summation of variances and is fundamental1y important. If the tndlvicuaI
dtsoerston processes that ar'e taking place in a column can be identified, and
the variance that results from each dispersion determined, then the
variance of the final band can be calculated from the sum of all the
tndtvldual vartances. Thts is hOW the Rate Theory provides an equation for
the flnal variance of the peak leaving the column, ,A.s an example the
principle of the summation Of variances will be applied to extra column
dispersion
Extra Column Dispersion
Extra column dispersion is that contribution to the total band dispersion
that arises from spreading processes taking place outs/de the column.
There are five major sources of extra column dtspersion and these are as
rouows-
1/ Dispersion resulting from the sample having a finite volume which can
be considered to contribute a variance,(o;)
2/ Dispersion occurring tn the channels of the sample valve Itself which
can be considered to contribute a
3/ Dispersion occurring in the sample valve/column and column/detector
cornecttons.Tnese can be considered to contribute avariance, (0 f)
4/ Dispersion occurring in the detector cell, or in the sensing volume of
the detector, which can be considered to contribute avari ance,
5/ Finally, there will be dtsoersion resulting from the response time of the
electronic system and sensor of the detector contributing avariance,
ThUS, summing all the variances,
Where is the the variance of the eluted peak,
It follows that the allocation of all the permitted extra column ctsperston
to semote votome dispersion, as defi ned by Kl inkenberg (4) and suggested
on page (54), is not permissible. Other sources of otsoersion must be taken
into account and take a share of the permitted 10% increase in column
variance.
Unfortunately) the magnitude of the vanance contrioutton from each source
will be different and the ultimate minimum size of each is often dictated by
the limitations in the physical construction of of the different parts of the
apparatus and consequently not controllable. It follows that equijJartitlon
of the perrmtted extra column dispersion is not possible. It will be seen
later that the the maximum sample volume provides the maximum
chrornatocrephtc mass and concentrat ion senstttvtty. Consequently) all other
sources of dispersion must be kept to the absolute minimum to allow as
large a sample volume as possible to be placed on the column wtthout
exceeding ttie permitted limit. At the same time It must be stressed, that
all the permitted extra column dispersion can not be allotted solely to the
sample volume.
The Alternative Axis of a Chromatogram
An elution curve of a chromatogram can be expressed using parameters
other than the volume flow of mobile phase as the independent variable,
Instead of using milliliters of mobile phase) SOlute concentration in the
mobile phase can be plotted against) time I or distance travelled by the
solute band along the column and proporUonally the same chromatogram
will be obtained. This is illustrated in figure (1)
Figure 1
An Elution Curve Plotted on Different Axes
Concentration
of Solution In
Mobile Phase
--. Volume Flow Through Column
- Olatance AJongColumn at.
A Fixed Time
---/ '-- ....._-
- Time at a Fixed Olatance AJong
The Column
97
As the curves are describing the same chromatogram, by proportion, the
rat 10 of the vanance to the square of the retent i on, in the respect tve uni ts
in which the independent variables are defined, will all be equal.
Consequent ly,
where crv. ax and at are the standard deviations of the elution curves
when related to the volume flow of rnobue phase,the
distance travelled by the solute along the column and
time, respectively,
and Vr', 1and tr refer to the retention voturne.colurnn length and
retention time, respectively.
Now, from the Plate Theory it has been shown that,
Thus,
and
Therefore,
0
2
1 _ x
---
n I
The ratto, (1). the column length divided by the number of theoretical
n
plates in the column, has for obvious reasons become termed the Height
Equivalent to the Theoretical Plate (HETP) and has been given the symbol
a 2
(H). However, it is seen that (H) Is numerically equal to, +J which is, in
')
fact, the variance per umt length of the column. Thus, the function, a
xl-,
is
the variance that the Rate Theory will provide an explicit equation to define
and can be experimentally calculated for any column from its length and
QA
column efficiency. It follows that the equations that give a value for, (H),
the variance per unit length of the column, have been termed HETP equations.
To develop an HETP equation it is necessary to first identify the dispersion
processes that occur in a column and then determine the variance that will
result from each process per unit Iength of co lurnn The sum of a11 these
variances Will be (H), HIe Height of the Theoretical Plate or the total
variance per unit column length. There are a number of methods used to
arrtve at an expression for the variance resulting from each dispersion
process and these can be obtained from the various references provtoed
However, as an example, the Random-Walk Model introduced by Giddings (5)
will be ernployed nere to illustrate the procedure.The theory of the Random-
Walk processes itself can be found in any appropriate textbook on
probability (6) and will not be given here but the consequential equation
wi 11 be used.
The Random Wa1k. Mode1
The random-walk model consists of a series of steplike movements for each
molecule which may be positive or negative the direction being completely
random. After' (p) steps, each step haVing a lenqth (s) the average of the
molecules will have moved some distance from the starting position and
wi 11 form aGaussian type dtstrtout ion curve with avariance of 0
2
.
Now according to the random-walk model,
o =s.JP " " , "............ (1)
Equation (1) can be used in a general way to determine the variance
resulting from the different dispersion processes that occur in an LC
column. T ~ l e application of equation (1) is simple, the problem that often
arises is the identification of the average step and sometimes the total
number of steps associated with the particular process being considered. As
an illustration of its use, equation (1) will be nrst applied to the problem
of radial dispersion that occurs when a sample is placed on an LC COlumn in
HIe manner Of Horne et 131 (7).
When a stream of mobile phase carrytnq a solute impinges against a
particle, the stream divides and flows around the particle. Part of the
divided stream then joins other split streams from neighboring particles,
impinges on another and divides again. When a sample is placed on the
column at the center of the packing, initially it is in a condition of non-
99
radial equilibrium, but as a result of this process the sample spreads across
the column durtng passage through the column and eventually acriteves
radial equtubrturn In the very early work in liquid chromatography,
relatively low inlet pressures were employed and thus, samples could be
tnjectec on the column by turntng off the pump and injecttng the sample
with a syringe through an approprtate septum device. Thts method of
injection often resulted tn radial equtubrturn never being achieved by the
solutes before they were eluted. The tntrocuctton of the sample valve)
however, aids in establishing radial equilibrium early In the separation but
unless some special spreading device is employed at the front of the
column, tt wtl1 not necessartly occur at the potrit of tnjection The stream
sputttnq process ts deoicteo in Figure (2A).
Figure 2
The Mechanism of Radial Dispersion
y
A B
AI The Stream Splttting Process B/lllustration of the Typtcal Radial
Step
If a particular rnotecule is considered passing round a partrete, it wIll
suffer a lateral movement that, from figure (28), can be seen to be given by,
d
Lateral Movement/Particle = ;eoS9
It follows that the average lateral step will be,
1t
+-
dn rrosA dn
2J -- ~ -d9 = ~
1t
2
100
Employing the random walk function, the radial variance is given by:-
2_ 2
o - (Number of Steps) X(Step Length) .... "..... "........ ". (2)
Now assuming one lateral step is taken by a molecule for every distance
(jdp) that it moves axially, then) (n) the number of steps is given by:-
1
n=-
jd
p
where (I) is the distance travelled axtally by the solute band.
Thus, substituting for' (n) in equation (2),
In practice the value of (j) will lie between 0.5 and 1.0, but, for simplicity
the value of (J) will be taken as uruty.Thts implies that one lateral step will
be taken by a given molecule for every step travelled axially equivalent to
one particle diameter.
Thus)
or,
(1 d
p
)0.5
on = " ", (3)
n
Consider a sample injected precisely at the center of a 4 mm diameter LC
column. Equation (3) allows the calculation of the distance traveled axially
by the solute band before the radial standard deviation of the sample of
solute is numerically equal to the column radius. That is, the band has now
spread evenly across the column and the solute is in radial equilibrium.
For the conditions qiven above OR = 0.2.
Thus, SUbstituting this value in equation (3),
( )
0.5
ld
p
0,2 =--
n
101
(O.2n)2
1 = . .. ... ".... ",,,,,,," .. ,,,,,,,, .... ,, ....... , (4)
d
p
Using equation (4) the distance that a solute band must pass along a column
before a sample, injected at the center of the packing) is evenly spread
across its diameter, was calculated for columns packed with different sized
particles.
The results are shown as a graph relating length against particle diameter
in figure (3).
Figure J
Graph of Column Length Traveled by Solute against Particle Diameter
800
'"
E
u
600
.....,
c
E
~
-C
400
U
~
C
~
..,
200
=
C
CD
~
0
0 20 40 60 80 100 120
Particle D1ameter (micron)
It is seen that with the particle diameter range norrnalty employed in liquid
chromatography (t.e. 2-25 micron) it is likely that radial equtl tbrturn would
never be achieved for those column lengths commonly in use in LC today) lf
the sample was placed symmetrically at the center of the packing. However)
if the column packing 15 complptply homogeneous throughout the column
length) then the column efflclency should not be trnpaired Unfortunately)
ideal packlng condltlons are not always achtevec and channellng often
occurs} under which circumstances lack of radial equilibrium could result rn
the column efficiency being reduced with consequent loss in resolution.
102
Thus, to ensure radial equil1brium, either it must be achieved on
tnjection by the use of a suitable sample distribution device, or by
employing narrow bore columns where radial equilibrium is more quickly
achieved. The latter alternative, however, will depend on the resolution
required and will be discussed later under LC column design.
Dispersion Processes that take Place tn an lC Column
There are four basic dispersion processes that can occur in a packed
column that will account for the final band variance. They are namely,
The Multipath Effect, l.onqttudtnal Diffusion, the Resistance to Mass
Transfer in the Moblle Phase and the Resistance to Mass Transfer in the
Stat ionary Phase. All these processes are random and essent ially non-
interacting and, therefore, provide individual contributions of variance
that can be summed to produce the final band variance. Each process will
now be discussed individually.
Ib.tl1uJt IDath Process
In a packed column the solute molecules will describe a tortuous path
through the interstices between the particles and obviously some will
trave1 shorter paths than the average and some longer paths.
Consequently, some rnolecutes will move ahead of the average and some
will lag behind thus causing band dispersion. The multipath effect is
illustrated in figure (4)
Figure 4
Dispersion by the Multipath Effect
The MUltipath effect can also be used to demonstrate the l J ~ P . of the
Random Wa Ik Mode 1.
10'
The average path length rs equtvalent to the d1ameter of the particle (dp)
and thus the number of steps will be equivalent to the column length
1
d1vided by the average step t.e n =d'
p
( )
0.5 (ldp)Q5
Thus, applying equation (1), o = d
p
dip =
Dividing by the column length, (1), the variance per unit length or, the
multipath contribution (HM) to the overall height of the theoretical plate
(H), is obtained,
In fact, Van Deemter introduced a constant 2A to account for the
inhomogeneity of packing so his expression for the Mult1path contrtoutton
became,
Longitudinal Diffusion
If a local concentration of solute is placed at the midpoint of a tuoe
filled with either a liquid or a gas the solute will slowly diffuse to
either end of the tube. it will f1rst produce a Gaussian dtstrtbutton with
a maximurn concentration at the center and finally, when the solute
reaches the end of the tUbe, 'end' effects occur and the solute wi II
continue to diffuse untu there ts a constant concentration throughout the
length of the tube. The process is illustrated in Figure 5.
Ordinary diffusion is the result of random molecular movement in f1rst
one dtrectton and then another and thus, resembles the Random Walk
Model. Uhlenbeck and Ornstein (8), derived the following expression for
the overall standard deviation (0) arlsing from diffusion process,
05
o = (2D
m
t)
where (t) is the time period over whlch the process occurs,
and Dm is the DHfusivity of the solute ln the mobile phase.
F1gure 5
01spersion by long1tudinal D1ffus1on
H
conc·lL
Position
, bmd
Fi
---.. -
~
In fact (t) IS the time the solute spends tn the moving phase and thus, ts
qtven byI t=l ) where rn and (u) ts the length of the column and the ltnear
u
moblle phase velocity respectively.
Thus,
(
1)°5
0= 2D
mlj
Therefore, d1vld1ng by I to obtain the var1ance per una length,
0
2
=H
L=
2D
m
(5)
1 u
Van Deemter also tntrocucec a constant (y)into the t.onqttudtnal Dispersion
contr1but ion to variance to account for some pack ing tnhornoqenel ty and so
the exoression for the Diffus10n contnbutton to the var1ance per onrt length
of the column became,
H
- 2yD
m
L -
U
...................................................... (6)
To be wholly accurate there should be a second longitudinal otrruston
contrtbutton to the overall variance that would arise from the stationary
105
phase, The same method of derivat ion can be used but the ti me the solute
1
spends in the stat 1onary phase is now, ).
u
Thus, the contribution to HIe variance per unit length from diffusion in the
stationary phase (HL(S»lS given by,
where, Os is the Diffusivity of the solute in the stationary phase
The total contrioutton to (H) from longitudInal diffusion w111 thus be:-
HL = 2Yl
0
m + 2Y2
k
'DS
u u
HL = 2Yl k'Om (1 + k') """, ".",,,,,,,.,,, .. (7)
u
It is seen rrorn equation (7) that the longitudinal diffusion term ts a
function of (k') the capacity factor of the solute. Whlle trus may be a
siqntr tcant effect in LC capillary column systems, where the rurn of
stationary phase is conttnuous along the length of the column, it wl11 not be
so tn a packed column, The stationary phase in a packed column is not only
broken into segments between each particle but also between each pore ln
each particle so free diffusion would be impossible, It follows that the
lonqttudtnal diffusion term for packed columns will be independent of the k'
of the solute or, very nearly so, and the experimental support for this will
be discussed in the next chapter,
The Resistance to Mass 1n The MObl1e Phase
Ouring the movement of a solute band along a column, the solute molecules
are continually transferring from the mobile phase into the stat 1onary phase
-. ............ -.,.1.1 ......... .,... _ .... __ .... __ .... _ ..... __ ".. ... ,." """""'A .... .......... .....",...,,, Th;,... +"""""" ...... _1"",.....
QIIU l)Q\..1\ II VIII lilt: ::llQlIVIIQI Y IJIIQ::lC IlllV lilC IIIVUIiC IJ"Gl;:lC, 1111,:) 1,.1 GlII;:lICI
process is not instantaneous, because a rtntte time is required for the
molecules to traverse (by diffuslOn) through the mobile phase in order to
reach, and enter the stationary phase, Thus, those molecules close to the
stationary phase will enter it almost immediately, whereas those rnotecules
106
some distance away from the stationary phase will find theIr way to it a
significant interval of ttrne later. However, as the mobile phase is moving,
during this time interval while they are diffusing towards the stationary
phase boundary, they will be swept along the column and thus dispersed
away from those molecules that were close and entered it rapidly, Van
Deemter deduced an expression for the contribution to variance due to this
effect as,
.""."..,,,,,.,,, .. ,,,,,, .... ',....... ,,,,,,,,.,,,,.,, (8)
The Resistance to in The Stationary Phase
The dispersion resulting from the resistance to mass transfer in the
stat tonary phase is exact ly analogous to that in the mobile phase. Those
solute molecules close to the surface of the stationary phase, will leave the
surface and enter the mobile phase a significant time before those that have
diffused farther into the stationary phase and have a longer distance to
diffuse back to the surface. ThUS, as those molecules that were close to the
surface w111 be swept along by the moving phase, they will be dispersed
from those molecules sttu diffusing to the surface. Van Deemter deduced an
expression for the contribution to variance due to this effect as/
2 _
°RS - Ds u "... """."""....... ",."""."."." .. " ."". (9)
where (dt) is the effective film thickness of the stationary phase.
By summing all the respective contributions from the different sources of
dispersion the final Van Deemter equat ion is obtaineo,
2yD
H;;; 2Ad
p
+ u m(1+ + D
m
P U + Ds u ".".""". (10)
Equation (10) is the basic form of the Van Deemter equation and will be
expanded and discussed with other HETP equations in the next chapter,
References
( 1)J.C.Giddings," Dynamics orCIJromafograp/)y Part I" ,MarceI Dekker Inc.New
York.t 1965)22.
107
(2) A.JP.Mart In and R.L.M.Synge, BioclJem. J.,3S( 1941 ) 1358.
(3) J. J. Van DeemterJ. J. Zuiderweg and A. Klinkenberg, Chern, Eng. Sci.
5( 1956)271
(4) A. Klinkenberg, "Gas Chromatography 1960",(Ed. RP,W.ScotU,
Butterworths, London,( 1960)194.
(5) J.C.Giddings, " Dynafl71(s or t.:lIron7atograplJy Part /" ,( 1965)29
(6) W. Feller," Probabili(r TlJe(Jryand Wiley,New York,
(1950) Chapt.l4.
(7) D.S.Horne,J.H.Knox and Ertct.aren, "Separation Tecnniques 1/7 ClJefl71stry
andBlot:1'Jen71str;v" , (Ed.RAKeller), Marcel Dekker, New York,
(8) G. E. Uhlenbeck and L. S. Ornstein, PlJysRev 36( 1930)823
Chapter 7
The Van Deemter EQuation
The Van Deernter squat ion (1) was the first rate equation to be developed
and this took place as long ago as 1956, However, it is only relatively
recently that HIe equat ion has been va1idated by careful expertrnenta1
measurement (2), As a result, the Van Deemter equat ion has been shown to
be the most appropriate equation for the accurate oreotcuor of dispersion
tn nqutd cbrornatoqraorw columns, The Van Oeemter equation is particularly
pertinent at mobile phase velocities around the ooumom vetoctt« (a
concept that will shortly be explained) Furthermore, as al1 LC columns
should be operated at, or close to, the optrmum velocity for maximum
eructencv, the Van Deernter equation is oarttcularty important in column
design, Other rate equations that have been developed for liquid
chromatography wi 11 be drscussed In the next chapter and compared W 1trl the
Van Deernter equation
Restating the Van Oeemter ecuation,
In fact, in Hle original form, eouauon (1), was introduced by van Deemter
for packed GC columns and consequently, the longitudinal ourusron term for'
the liquid ohase was not included and 2yD
m
( l + ~ k ' ) , was replaced by, 2yD
m
,
u u
FurUlerrnor'e, as the equation was developed for Ge, where the otrtustvtty of
the solute in the gas was four to five orders of rnaqmtude greater than ln a
liqUid, Van Deernter considered the resistance to mass transfer' in the
fl(k')d
2
mobi1e phase to be negligible. As a result the function, P u was also
Om
not included,
110
The equation actually developed by van Deemter took the form,
2yD
m
f 2 ( k ' ) d ~
H=2Adp+ u + Os U
......................... " (2)
The form taken by f2(k') was considered by Van Deemter to be,
and thus, the complete HETP equat ton became,
2yD
m
8 k' df
H =2Ad
p
+ u + 2" 2 0 u ...... """"", .. '" (3)
n (hk') S
Equation (3), however, was developed for a gas chromatographic column and
in the case of a liquid chromatographic column, the resistance to mass
transfer in the mobile phase should be taken into account. Van Deemter et al
did not derive an expression for f I Ck') for the mobile phase and it was left
to Purnell (3) to suggest that the function of (k'), employed by Golay (4) for
the resistance t.o mass transfer in the mobile phase in his rate equat ion for
capillary columns, would also be appropriate for a packed column In LC. The
form of flCk') derived by Golay was as follows,
1+ 6k' + 11k,2
24(1+k,)2
(It should be noted that the Golay equation for capillary columns wlll be
discussed in detail in the next chapter.)
Thus, Van Deemter's equat ion for LC becomes,
111
Equation (4 ) can put in a strnplttted form as follows,
B
H=A+-+Cu , , (5)
u
where, A =2Ad
p
and
B=2yOm
[
I +6k' +11k ~
2
d~ 8 k' df)
c= 24(1+kl Dm+ n
2
(1+kl
Ds
Equattcn (5) ts a hyperbollc function which indtcates that there will be a
mtrnmum value of (H) for a particular value of (u). That ts a maximum
efficiency will obtained at a particular linear mobile phase velocity. An
example of an HETP curve obtained in practice is shown in figure 1.
Figure 1
An Experimental HElP Curve for Hexamethylbenzene
0.003
0.002

~

H(FiU
E


H
Co)
• ....,

A
=
<:>
B/u
0.001
x
Cu
-() .000 ~ ~ ~ - = = : ~ : ; : : : ~ ~ ~ = ; = = : : ; : : : : : : ! = = = r = = = = r = ~
-o.ooo 0.100 0.200 0.300 0.400 0.500 0.600
u(em/sec)
112
The upper' curve, which is the result of a curve fitting procedure to the
points snown, is the HETP curve. The column was 25 em long, 9 rnrn in
diameter and packed wHh 8.5 micron (nominal 10 micron) Partlsil silica gel.
The mobile chase was a solution of 4.8%w/v ethyl acetate in n-oecane The
minimum of the curve is clearly indicated and it 1S seen that the fit of the
potnts to the curve is fairly good. As a result of the curve fitting procedure
the values of the Van Deemter constants could be determined and tile
separate contriout ions to the curve from the muIt ipath d1 spers ion,
longitudinal dispersion and the resistance to mass transfer calculated.
The three contribut ions to dtspersicn are also shown as separate curves in
figure 1. It is seen that the major contribution to dispersion at the optimum
vetocity, where the value of CH) is a minimum, 1S the multipath effect. Only
at rnuch lower velocities does the longitudinal diffusion effect become
SIgnificant. Conversely, the mobile phase velocity must be increased to
about 0.2 ern/sec before the resi stance to mass transfer begms to become
relatively siqrurtcant compared to that of the multipath effect.
The values for the Van Deemter constants were found to be,
and
A=O.OO 117
8=0.0000175 cm
2/sec
C=0.00250 sec.
It is seen that if the mean particle size was 0.00085 c m ~ thus as,
0.00117 = 2A 0.00085 or, A= 0.69
Giddings (5) determined theoretically that , for a well packed column (}..)
snoulo take a value of about 0.5 and thus the column used was reasonably
well packed.
In a similar manner, 8 = 2yD
m
ThUS, 0.0000175 = 2yDm
Now Katz et a/ (2) determined the otrrustvttv of hexamethyl benzene in
4.83%w/v ethyl acetate in n decane and found it to be 1.17 10-5 cm2/sec,
Consequent ly, y=0.747
1 1 ~
Giddings (5) also determined theoretically that for a well packed column (y)
should be about 0.6 so again the lonqttodtrial diffusion effect confirms that
the column was reasonably well packed.
The HETP curve clearly shows, that for a packed column, the part tcle size
has a profound effect on the minimum value of the HETP of a column and
thus the maximum efficiency attainable. It would also appear that the
highest efficiency column would be obtained from columns packed with the
smallest particles. This wjll in due course be shown to be a fallacy, but
what is true, ts that the smaller the part rcle diameter the smaller wj 11 be
the minimum HETP and thus, the larger the number of plates per unit length
ootair.aote from the column. At this time it will suffice to point out that the
total number of theoretical plates that can be obtained will depend on the
lengtll of tile conmn which, in turn, must take into account the available
inlet pressure.
The optimum mobile phase velocity can be obtained by different tating
equation 5 with respect to (u) and equating to zero.thus,
B
H==A+-+Cu
u
and
Equatlng to zero
Thus,
dH B
- == -- + C
du u
2
dH B
du = 0 and consequent ly) - u
2
+ C= 0
Uopl = ~ (6)
Subst1t.uting for (6) and (C»)
r 2yD
m
0.5
114
and lett ing D
s
=
"" ,,(7)
0.5
48Yn
2
(1+k' )2
n
2
(1+ 6k' +11k,2 )d +192 k'd )
Uopt =D
m
..,..-------------:-
It is seen from equation (7) that the optimum velocity is , directly
proportional to the diffusivity of the solute in the mobile phase. To a lesser
extent it also appears to be inversely dependant on the particle diameter of
the packing (the particle size is an optional choice) and the film thickness
of the stat ionary phase. The fi Im thickness of the stat ionary phase is
determined by the physical form of the packing, that is, in the case of
si1 tea gel, the nature of the surface and in the case of a reverse phase, on
the bonding chemistry.
Now, if it is assumed that df « d
p
(which in practice will always be true)
then,
0.5
48y

Uopt = D
m
....,.....--------
Furthermore, when k:» 1, that is, for well retained peaks,
[ ]
0.5
4.36y
Uopl = D
m
-
Under these circumstances, it is seen that the optimum velocity is directly
proportional to the solute diffusivity in the mobile phase and inversely
proportional to the particle diameter of the packing. The quality of packing,
obviously also plays a part, but to a very less significant extent.
It is now possible to determine the factors that control the rnaqnttuoe of
Hmin. SUbstituting the function for Uopt from equation (6) in equation (5) an
express: on for Hmin 1s obtained.
8 r- 18
Hmin = A + + '1'c
or, , ""'''''' (9)
SUbstituting for A,B, and C,
[
1+ 61<' + 11k,2 d 8 k' d7]
Hrnin = 2Adp+2 2y
O
m 2 0 + 2" 2 D ..... ". (10)
24 ( 1+k' ) m n (1+k' ) S
Noting that Os =;D
m,
and simplifying,
Hmin = 2Ad
p
+2 ........ ( 11)
Again) assuming that, df « d
p
, and k» 1, that is for well retained peaks,

2Yd
p
Hrnin = 2Adp + 2 24
Bearing in mind the values of (A) and (y) for a well packed column are 0,5 and
0.6 respect ively,
Hmin. = 2.48dp " ( 12)
Thus, the approximate value of Hmin. for a well retained solute eluted from a
well packed column and operated at the optimum linear mobile phase
velocity, can be expected to be about 2.48d
p
. Furthermore} to the first
approximation, this value will be independent of the nature of the solute,
mobile phase or stationary phase, For the accurate design of the optimum
columns tor a oarucutar separation however, this aoproximat ion can (lot be
made, nevertheless, the value of 2.48 for Hmin is a useful gUide for
assessing the quality of a column.
110
From d'Arcy's Law for nuto f low through a packed bed, at the optimum
rnobile phase velocity the length of the column ts given by,
where P is the inlet pressure to the column,
TI is the viscosity of the mobile phase}
ftI is the o'Arcy's Constant.
Now, 1= nHml
n
thus, substituting for l, and rearranging,
2
cpPd
p
nHmin Uopt = ---'-- .... ",,,,, "" .... """" ..... "..... ".... ,, ..
TI
SUbstituting for Uopt and Hmin from equations (8) and (12) respectively,
( 13)
cpPd 2
n2.48d
p
1 6 d ~ m - n p (14)
fPPd 2
4.02nD
m
:: p " " " "" " "."." " (15)
TI
or, " ",," ", .. , ", (16)
It is seen from equation (16) that, if an LC column is operated at its
optimum linear velocity, the maximum efficiency obtainable for we11
retained peaks wlll be directly proportional to the inlet pressure available
(P) and the square of the part tete di ameter of the pack ing. Thus, the larger
the oarucle diameter, the greater etf iciencv attainable at a qiven pressure.
This is because, as the particle diameter is increased the permeability is
also increased allowing a longer column to be used, The permeability
increases as the square of the pa
r
Ucle diameter but as sho\4.'n by equation
(12) the variance per unit length only increases proportionally to the
particle diameter. Thus, doubling the particle diameter will allow a column
four times HIe length to be used but the number of plates jJer unit lengtl?
will be halved. Consequently, the column efficiency will be increased by a
1 1 (
factor of two. It ts also seen that the higher erricienctes will be obtameo
with mobile phases of low viscosity and for solutes of low diffusivity
Solvent Viscosity and solute diffusiv1ty tend to be inversely proportional to
each other and so the sensttivttv of the the maximum obtainable efficiency
to either solvent viscosity or solute ctrrustvttv will generally not be large.
The approximate length of a column that will provide the maximum column
efficiency when operated at optimum velocity is gwen by, 1=nHmin.
Thus, from equat ions (12) and (16))
[

2
1
1 = (2.48d
p
) 4.0;m'l

p
. .. (1 7)
It is seen that HIe column length varies inversely as the product of the
solute dtrrustvtty in the mobile phase and the mobile phase viscosity in
much the same way as the column efficiency does when operating at the
optimum velocity. As would be expected the column length is directly
proportional to the inlet pressure but, less obviously is also proportional to
the cube of the particle diameter.
The analysis time for a solute mixture in which the last peak is eluted at a
capacity ratio of k'F is given by,
t = (1 +k'F)to = (1 + k' F)_1_
Uopt
SUbst.ituting for (uopt) and (D from equations (8) and (17),
or,
110
It is seen from equation (18) that the analysis time 1s proportional to the
fourth power of the particle diameter and inversely proportional to the
square of the diffusivity in HIe mobile phase. In a similar manner to column
length, the analysis time is also directly proportional to the applied inlet
pressure an(J inversely proportional to HIe mobile phase viscosity.
Summarizing, if a column is operated at its optimum velocity and the solute
concerned is eluted at a relatively high k' value, and assuming the nirn
thickness of HIe stationary phase is small compared with the particle
diameter (a condi t ion that is met in almost all LC separat ions) then,
_ 1.62D
m
Uopt = d (8)
p
Hmin. ~ 2.48dp ( 12)
q»Pd 2
n :: 4. 0a:
m
Tl , ( 16)
O.617q»Pd
3
I ::: p
- DmTl
( 17)
As already stated, these equations are insufficiently precise to be used for
accurate column design but, they can give a general indication of the more
important performance specifications that could be expected from an
approximately optimtzeo chromatographic system.
It would be of interest to use the equations to calculate the rnaximurn
efficiency, column length and analysis time that would be obtained from
columns packed with particles of different diameter under conditions that
commonly occur in LC analyses.
The diHuSivity of solutes having molecular weights ranging from about 60
to 600 vary from about 1X10-
5
to 4X 10-
5
cm
2/sec.(6)
ThUS, an average
value of 2X10-
5
cm
2/sec
will be taken in the following calculations. In a
similar way a typical vtscostty value for the rnocue phase Wli"1 oe assumed
as that for acetonitrile (7), t.e. 0.375X 10-
2
poises. The inlet pressure taken
will be 3,OOOlb/p s.i. or about 200 atmospheres. Many LC pumps available
today will operate at 6,000 o.s.i or even 10,000 o.s.i., however, it is not the
pump that determines the average operating pressure of the chromatograph,
but the sample valve. Sample valves can, for a limited time, operate very
satisfactorily at very high pressures but their 1trettrne at maximum
pressure is significantly reduced. Most valves, designed for high pressure
operation will have extensive lifetimes when operated at 3,000 psi. and so
this is the pressure that will be employed in the using the above equations.
The value assumed for the d'Arcy constant.te) was 35 when (P) is measured
in p.s.t. which had been determined experimentally (8). The capacity factor
of the last eluted peak was taken as 5 for calculat tnq the elution time.
Figure 2
Graph of Maximum Efficiency against Particle Diameter
7
:::D 6
u
c
CD
-
Co)
-
.... 5
....
~
.
=c
-'
4
30 20 10
3 + - - ~ - - ~ - ~ - - - r - - - - - - r - - - - - - .
o
Particle Diameter (micron)
Employing the above values, equatton (16) was used to calculate maximum
efficiency attainable for columns packed with particles of different
diameters. The results obtained are shown in figure 2.
1? (\
-- ~
It is seen from figure 2 that changing the particle diameter from 1 to 20
micron results tn an efficiency change from about 3500 theoretical plates
to nearly 1.5 million theoretical plates and furthermore, this very high
efficiency is achieveo at an inlet pressure of only 3000 o.s.t. It is also seen
that HIe maximum available efficiency increases as the particle diameter
increases, Trlis is because, as already discussed, if the pressure is limited,
in order to increase the column length to accommodate more theoret tcai
plates the perrneaotltty of the column must be increased to allow the
optimum mooile chase velocity to be realized. It is possible to increase the
inlet pressure to some extent, but ultimately the pressure will be limited
and the effect of particle diameter will be the same but at higher efficiency
levels.
The column lengths necessary to achieve these efficiencies can be
calculated employing equation (17). the results obtained shown as curves
relating maximum column length to olarttcte diameter 1S shown In figure 3.
FIgure 3
Graph of Column Length for Maximum Efficiency IParticle Diameter
4
""'"
E
Co) 3
.....,
.c
..-I
=c 2
m
...I
c
E
:"
-~
u
= 0
~
...I
30 10 20
Particle Diameter (micron)
-1 +-----r---,------,-----r---.------,
o
It is seen from figure 3 that the colurnn length ranges from just under a
centimeter, which orovides an efficIency of about 3500 theoretical plates
to nearly 70 meters which provides an efficiency nearly 1.5 muuon
1? 1
theoretical plates. Seventy meters is a long column) but not an impractical
length, Th ouesuon remains ts how long will tne separation take?
The t irne taken to achieve these efficiencies when elut i ng the last peak at a
k' value of 10 can be calculated employing eouat ion (18). The results
obtained are shown in figure 4 where the elution time obtained from
columns of maximum efficiency are plotted against part tcle diameter.
F1gure 4
Graph of Log. Elution Time against Particle D1ameter
5
,...,
~
m
4 ..,
~
c
-
E
3
.....-
m
E
2
- ...
c::
e
-
..,
~
-~ 0
.
'=
C
...J
-1
0 10 20 30
Particle Diameter (micron)
On examination of the curve in figure 4 the problem associated with high
efficiencies becomes apparent. The elution time for the short column having
about 3500 theoretical plates is only just about one half minute. The elution
time from the 1.5 million plate column) however) is about 54 days) a rather
long time to wait for a cnrcmatooraontc separation. It is also seen that the
higher the efficiencies that are required (the more difficult the separation
problem) the longer the separation time and this is inevitable as a result of
practical limits to the column inlet pressure.
References
1/J.J. Van DeernterJ.J.Zuiderwg and AKI inkenberg) LiJern.Eng ..,"ci,5( 1956)271
122
2/E.Katz,K.L,Ogan and R. P, w.Scot t, J CIJromatogr
J
270( 1983)51
3/J.H.Purnell and C.P.Qui nn, in "Gas Chromatography 1960" (eo. R.P. W.Scot t),
Butterworths London ( 1960)184
41 M. J. E. Golay, "Gas CIJromatograjJlJy /958) (ed.D H.Desty) Butterworths,
London) ( 1958)36
5/J.C.Giddlngs,"Dynarnics of Chromatography",Marcel Dekker,New York,
(1965) 56
6/ED,K.atz and RP.W.5cott, ,.lLnrOf17atogr )70( 1983)29
71 J.ARiddick and W.B.Bunger,"Oraganic Solvents"Wi ley-l nterscience,New
York,( 1970)399
8/E.Katz,K.L.Ogan and RP.W.Scott,,,/ClJrolnatogr,289( 1984)65
Chapter 8
Alternatlve Equations for Peak. ntsperston
The Van Deernter equation remained the estaohshed equation for describing
the peak dispersion that took place in a packed column unti 1 about 1961.
However, when experimental data that was measured at high linear mobile
phase velocities was fitted to the Van Deemter equation it was found that
there was often very poor agreement. In retrospect, this poor agreement
between theory and experiment was probably due more to the presence of
experimental artifacts, such as those caused by extra column dispersion,
large detector sensor and detector e1ectroni c time constants etc. Ulan the
inadequacies of th Van Deemter equation. Nevertheless, it was this poor
agreement between theory and experiment, that provoked a number of
worker's in the field to develop alternative HETP equations in the hope that a
more exact relationship between HETP and linear mobile phase velocttv
could be obtained that would be compatible wlth experimental data.
The Giddlngs Equation
In 1961, Giddings (1) developed an HETP equation of which the Van Deemter
equation appeared to be a special case. Giddings was dissatisfied with the
Van Deernter equation insomuch that it predicted a finite contribution to
dispersion independent of the solute otrrustvtty in the limit of zero mobile
phase velocity. Trus concept, not surprisingly, appeared to him unreasonable
and unacceptable. Giddings developed the following equation to avoid this
irregularity.
A B
H= - + - + Cu " """., """ .. """"" """ (1)
1 + ~ u
u
It IS seen that when u » E, eouat jon (1) recuces to the Van Deemter
equat ion,
B
H = A+ - + Cu
u
1 2 ~
It is also seen that at very low velocities, where u « E, the first term tends
to zero, UIUS meeting the requirements that there ts no rnuluoatn dispersion
at zero mobile phase velocity. Gjddings also suggested that there was a
coupling term that accounted for an increase in the 'effect ive diffusion' of
the solute between the particles. The increased 'diffusion' resulted from the
tortuous path followed by the molecules as they twisted and turned through
the interstices of Hie packing. Trlis process was considered to produced a
form of microscopic turbulence that induced extremely rapid transfer of
solute in the tnterparttculate spaces. However, again at velocities Where u
» E, this mixing effect could be considered complete and the resistance to
mass transfer in the mobile phase between the particles becomes very
small and HIe eouat ion again reduces to the Van Deemter equat ion. However,
on constcerattor, there is a difference; the C term 10 the Van Deemter
equation would now only describe the resistance to mass transfer' in the
mobile pnase contained in tile pores of HIe part tcles, and tnus, would
const itute an addit ionai resistance to mess transfer in tne stationary
(static mootte!IJnase. This concept has some indirect experimental support
in the developrnent of the rorrn of f 1(k') from experi menta1data given in the
next chapter. The form of f lCk') is shown to be closer to the original form
given by Van Deernter for f2(k') tnat is appropriate for the resistance to
mass transfer in HIe stationary phase. It is not known for certain, but it is
possible and likely, that this was the reason why Van Deemter et al did not
include a resistance to mass transfer term for the mobile phase in their
original form of the equation.
The HUber Equatton
The next HETP equation to be developed was that of Huber and Hulsman in
1967 (2). Tbese autnors introduced a modified multipath term somewhat
similar in form to that of Giddings and a separate term describing the
resistance to mass transfer in the mobile phase contained between the
part icles. rne form of their equat ion was as Iollows.-
A
E
1+ u
1j 2
It is seen that Hie first term differs from that in the Giddings equation, in
that it now contains the rnobiie pnase velocuy to the power of one half.
Nevertheless, again when u
l 12
» E, the first term reduces to a constant
similar to HIe Van Deemter equation. The additional term for HIe resistance
to mass transfer in the mobile phase is an attempt to take into account the
'turbulent mixing' that takes place between the particles. Huber's equation
Implies but, tn face was not exp! tcit ly stated by the authors, that the
mixing effect between the particles (that reduces the magnitude of the
resistance to mass transfer in the mobile phase) does not commence untII
the mobile phase velocity approaches the optimum velocity (as defined by
the Van Deernter equation). Furtnerrnore, it is not complete until the mobile
phase velocity is well above the optimum velocity. Thus, the shape of the
HETP/u curve will be a little different from that predicted by the Van
Deernter equat ion.
The form of the HETP curve that is produced by the Huber equat ion is shown
in figure ( 1).
Flgure 1
H versus u Curves Resultlng from the Huber Equation
0.
O12
l
G
Coupling Term

Long.Diff

Mas.Trans Mobil
0.010
1)
Mass Trans stat

Composite curve
0.008
"""
E
Co)
0.006 ot",;
:c
0.004
0.002
0.8 0.2 0.4 0.6
linear VeloeHy (em/sec)
-0,000 ~ ~ ~ : : = : ~ = ' - - - - - t - - - - t ~ - - - . . - - . . . . . . - - - - ,
0.0
It is seen that the composite curve obtained from the Huber equation is
indeed similar to that obtained from that of Van Deemter but the individual
contribut ions to the overall variance are different. Although the
contrtbuttons from the resistance to mass transfer in the mobile phase and
longitudinal diffusion are common to both equations} the (A) term from the
Huber equation increases with mobile phase flow-rate and only becomes a
constant value} similar to the rnulttpatn term in the Van Deemter equation,
when the mobile velocity is sufficiently large. In practice, however, it
120
would seem that the magnitude of the rnootte velocity, where the (A) term
gives a constant value, ts qutte low, relative to the normal range of
operating vetoctttes employed in practical LC. The portion of the composite
curve shown at the h1gher veioctt tes is not quite 1tnear due to the non-l inear
form of the term for the resistance to mass transfer tn the rnobi le phase
and this becomes more apparent at higher mobile phase velocities. At, and
around the optimum velocity. however, the form of the two curves differ
only sligrltly.
The Knox Equat ion
During 1972 and 1973 Knox and his co-workers (3), (4), and (5) carried out a
considerable amount of work on different packing materials with particular
reference to the effect of particle size on the reduced plate hejght of a
column. The concept of reducedplate lie/gilt (II) and reduced velocity (V)
was introduced by Giddings (6) and (7) in 1965 tn an attempt to form a basis
for the comparison of different columns packed with part teres of different
diameter. The reduced plate height is oertnec as,
H
II = d
p
." ".." " ,,, ,, .. (3)
In fact the reduced plate height merely measures the normal plate height in
units of particle diameters. It is also seen that the reduced plate height is
dimensionless.
The reduced velocity is defined as,
ud
p
v = - " ".".. " " ,. (4)
D
m
The reduced velocity compares the mobile phase veloc1ty with the veloctty
of the solute crrrusron through the pores of the particle. In fact, the mobile
phase velocity is measured In units of the 1ntraparticle diffusion velocity.
As the reduced velocity is a ratio of velocities, like the reduced plate
height, it also is dimensionless.
Employing the reduced parameters the equation of <nox takes the followlng
form,
II = B + Av
1
/
3
+ Cv "" " (5)
v
127
It should be noted that the constants of the equation were arrived at by a
curve f1tting procedure and not derived theoretically from a baste
dtsperston model; as a consequence the Knox equation has umiteo use in
column design. It is, however, extremely valuable in accessing the Qual ity of
the packing. This can be seen from the diagram shown in figure 2.
F1gure 2
Graph or log. Reduced Plate heIght against log. Reduced VelocHy
for Poor and Well Packed Columns
1.4
..,
..:
..
1.2
EI Well Packed
-
I)

Poorly Packed
:
m 1.0
.-
II
-a.
0.8
"
m
u
~
0.6
"
m
CK
• 0.4
..
c
-'
02
-1.5 -<>,5 0.5 1.5 2.5
log.Reduced Velocity
The curves represent aplot of Log.( 1l)I(Reduced Plate heighUagainst Log.(v ),
(Reduced vetocttv) The lower the Log.( Il) curve versus the Log.(v) curve the
better the column is packed. At low veloctttes the (B) term dominates and at
high veloctttes the (C) term dominates as In the Van Deemter equation, The
best column efficiency is achieved when the minimum is' about 2 particle
diameters and tnus, Log (Il) is about 0.35. The minimum value for (H) as
predicted by the Van Deemter ecuatton has also been shown to be about two
particle diameters. The optimum reduced velocity is in the range of 3 to 5
that IS Log.(v ) takes values between 0.3 and 0.5. The Knox equation is a
simple and effective method of examining the quality of a given column but,
as stated berore, is not nearly so useful In column design due to the
empirical nature of the constants.
The Horvath and lin Equation
In (1976) Horvath and Lin (8) and (9) introduced yet another equation to
describe the value of (H) as a function of the linear mobile phase veioctty
(u). Their equation is given as follows,
H = A
E
1+ 173
u
B 2/3
+ - + Cu + Du """ ...... "" .. """ ... ".".. (6)
u
The equation of Horvath and Lin was very strn i lar to that of HUber and
Hulsman and, in fact, only differed in the magnitude of the power function of
(u) in their (A) and (D) terms, These workers were also trying to address the
problem of a zero (A) term at zero velocity and the fact that some form of
"turbulence' between particles aided in the solute transfer across the voids
between the particles. All the above equations will be tested against
experimental data in the next chapter.
The Golay Eouat ion
The basic equation describing the dispersion that takes place in an open
tubular column was developed by Golay (8) for gas chromatography but IS
equally, and directly, applicable to liquid chromatography. The GOlay
ecuation differs in one important aspect from the equations for packed
columns in that, as there is no pack tnq, there can be no mu1tipath term or
coupling factor and thus, contains only two functions. One function
describes the longitudinal diffusion effect and the other the combined
resistance to mass transfer terms for the rnobi le and stat ronary phases. The
Golay equation takes the following rcrrn'-
Where (H) is the variance per unit length of the column for the given solute,
(k)' is the capacity factor of the eluted solute,
(K)the distribution coefficient of the solute between the two phases.
(OM) 1S the ottrustvttv of the solute in the mobile phase)
(DS) is the diffusivity of the solute in the stationary phase,
(r) is the radtus of the column,
and (u) is the linear velocity of the mobile phase.
129
If the solute is unretained ( i.e. k'=O ) then the the Golay equation reduces to}
20 r
2
H = -l!!. + 2 0 u (8)
u 4 m
Taking a value of 2.5 Xl 0-
5
for D
m
(the diffusivity of benzyl acetate in n-
heptane) equation (8) can be employed to calculate the curve relating (H) and
(u) for an uncoated capillary tube. The results are shown in figure (3).
F1gure J
Graph of H aqatnst U for a Capillary Column ( Unretalned Peak)
0.06
0.05
0.04
""'"
E
Co) 0.03
'-'
::c
0.02
0.01
o Long.Diffusion
• Mass Transfer
• Composite Curve
0.04 0,03 0.02 0.01
o.00
0.00
Linear Velocity (em/Sec)
It is seen that the Golay equation produces a curve identical to the Van
Deernter equation but with no contribution from a multipath term. It is also
seen that. the valUe of (H) is solely dependent on the diffusivity of the solute
in the mobile phase and the 1inear mobile phase velocity !t is clear that the
capillary column can} therefore} provide a simple means of determining the
diffusivity of a solute in any given ltqutd
The Golay (PCllli1tlon 7) can be out in a simplified form in a similar
manner to the equat.ions for packed coumns.-
B
H= - + Cu " (9)
u
1'2.'"
-.., .
Where,
and
B= 20
m
u
(1 +6k' + 11k'2)r
2
C=-----
. 24 (1+ k' f D
m
k'3 r
2
+ ----.----
6(1 + k,)2
K
2
Ds
The form of the HETP curve for a capillary column ts the same as that for a
packed column and exntbtts a minimum value for (H) at an optimum velocity.
Differentiating equation (2) with respect to (u):-
dH B
- = -- + C
du u
2
Thus} when
H= Hmin,
6
then -- + C =0
, 2
u
and Uopl. =~ (10J
sobstuut lng for (6) and (C) In ecuat Ion ( 10)
0.5
20
m
uopt. =
(1 +61<' +1lk·
2
)r
2
24 (1 +k' )2 Om
k'3
r2
+-----
6( 1+ k,)2 K
2D
s
( 11)
131
It 1S seen that, in a stmtlar manner to the packed column, the optimum
mobile phase velocity is directly propcrnoral to the diffusivity of the
solute 1n thernobile phase, However, in the capillary column the radius (r)
replaces the particle diameter (d
p
) of the packed column and consequently,
(uopt) is inversely orooorttonal to the column radius,
Now,
or,
B
Hmin. = - + CUopt
Uopt
Hmin. = 2JBC , "..... . (12)
Again substituting for (B) and (C),
Hm1n. = 2
H
m1n
= 2r
Equation (13) shows that the rntrnrnurn value of (H) is solely dependant on
the column radius (r) and the thermodynamic properties of the solute/phase
system. As opposed to the optimum velocity, the minimum value of (H) is not
dependent on the solute diffuSivity.
In addition it is seen, from equation (12), that the expression for Uopt is very
similar to that for a packed column but the expression for Hmin. 1S much
simpler' as it is devoid of the (A) term from the multipath effect.
Due to the relative simple, and precisely defined, geometry of the capillary
column it contains no arbitrary constants involved with the packing quality
of the co lurnn such as (y) and OJ. As a consequence, the propert res Of the
1~ ?
-
capillary column can be explored further in a relatively straightforward
manner.
From Potseutlle's Equation, describing the flow of liquid through an open
tube.
Pr
2
1=--
811
U
opt
Where, (I) is the length of the column,
(P) is HIe pressure drop across the column,
and (11) is Ule viscosity of the flowing liquid.
NowI employing equations ( 10) and ( 12),
I =nHmin
Thus,
From the Golay equation}
2
2nB= ~
811
B =2D
m
Pr
2
n ;:: 32T)D
m
".., (14)
It is interesting to note from equation (14) that when a column is run at its
optimum velocity, the maximum efficiency attainable from a capillary
column is direct.ly proportional to the inlet pressure and the square of the
radius and inversely proportional to the solvent viscosit.y and the
diffusivity of the solute in the mobile phase. This means that the maximum
efficiency attainable from a capi llary column increases with the column
radius. Consequently, very high efficiencies will be obtained from relat/ve(y
large diameter columns.
Employing equation (14) it is possible to calculate the range of efficiencies
attainable under operating conoitions commonly used for packed columns.
The conditions assumed are as follows,
133
Inlet. Pressure IOOOlb/sQ.1n.
vtscositv of Mobile Phase 0.00397 Poises
DiffuSivity of the Solute in the Mobile Phase 2.5 X 10-
5
cm
2/sec
The results obtained are shown as a graprl relating column efficiency
against column radius in figure 4
7
8
Figure 4
Graph of Log.Efficlency against Log.Radius for a Capt nary Column
10 ...,..--------------"'7"'"""0
9
= u
c
CD
...
U
...
..
..
1.1.1
.
f 6
.J
5
2,5 1.5 0.5
4 + - - - - - ~ - - - - . . , . . . . . . . - - - _ _ _ _ _ 1
-0,5
Log.Radius (Radius in micron)
It is seen that HIe 1 micron column can provided an efficiency of over two
hundred thousand plates whereas the column 100 micron in radius can
provide an efficiency of over two billion theoretical plates (assuming an
inlet pressure of 1000 o.s.n It wi 11 be seen later) however, that the
practteal Iimitations of present day chromatography equipment render the
reauzat ton of even a modest performance from LC cap111ary columns
extremely cit t tcult.
References
(1) J C. Giddings, .j CI'IrOf!7ato
v
qr. 5( 1961 )46.
(2) J. F. K. Huber and J. A. R. J. Hulsman, Anal C!}/!n. Acta.)38( 1967)305.
(3) G. ,..1. Kennedy and J. H. Knox, ..J Chromatogr: Sci, 1O( 1972)606.
(4) J. N. Done and J. H, Knox) JCIJromatogr: Sc); 1O( 1972)606,
(5) J. N, Done) G. J. Kennedy and J. H. Knox) in" Gas Chromatography /972")
(Ed. S. G. Perry) Applied Science PUBLISHERS, Barking, (1973)145,
(6) J. C Giddings, Anal Lnenl ,J5( 1963)1338.
(7) J. C. Giddings)" Oynanllts or MDekker (New York)
(1965) 125.
(8) M, J E.Golay, in "6as cnn.1fl7dtograpny 1958", (edD.HDesty), Butterworths,
London, (1958)36
Chapter 9
ExperImental VaHdatlon or the Van Deemter EQuat10n
The equations discussed in HIe previous chapter, that described the variance
per unit lengtrl of a solute band after passing through an LC column, were all
significantlY different. It is) therefore, necessary to Identify the specific
equation that most accurately describes the dispersion that takes place, so
that it can be employed with confidence in the design of optimized columns,
The different eouattons wer'e tested against an extensive set of accurately
measured experimental data by Katz et al (1) and) in order to ioentify the
most pertinent equation, their data and some of their conclusions will be
cons toerec in thi 5 chapter.
Reiterating the equations that were examined) they are as follows,
B
H;; A+ - + Cu
u
A B
H = - + - + Cu
1+ ~ u
u
The Van Deemter equation,(2)
The Giddings equation, (3)
H::;;;: A
E
1+-
u
1
/
2
+ ~ + Cu + [)j 1/2
u
The Huber equation, (4)
h =~ + Av
1
/3 + Cv
v
The Knox equat ion, (5)
H = A
E
1+ -
1/3
U
B 2/3
+ - + Cu + Du
u
The Horvath equat ion, (6)
At first sight, it might appear adequate to apply the above equations to a
number of experimental data sets of (H) and (u) and to roentuy tnat equanon
that provides the best fit Unfortunately, this is of little use as, due to their
nature, all five equations would provide an excellent fit to any given
exoenmentauv derived data set, provided the data was obtained with
sufficient precision, However, all the individual terms in each equation
purport to describe a soectr«: di!:.>/Jersiv& errect . That being so, if the
dispersion effect cescrtbed is to be physically significant over the mobile
phase velocity range examined, all the constants for the above ecuations
derived from a curve fitting procedure mos: be poslt/ve and real. Any
equation, that did not consistently provide positive and real values for all
the constants, would obviously not be an appropriate and explicit equation
to describe the dispersion effects occurring over the range of velocities
examined. However, any equation that aoes orovtoe agood fit to a series of
experimentally determtnec data sets and meet the requirement that all
constants were posit ive and real would still not the
correct equation f or co lumn design.
When a satisfactory fit of the experimental data to a particular eouation, is
obtained the constants, (A), (B), (C) etc. must then be replaced by the
explicit tunct ions derived from HIe respective theory and which incorporate
the respective prlysical properties of solute, solvent and stationary phase.
Those physical properties of solute, solvent and stationary phase must then
be varied in a systemat tc manner to change the rnaqrntude of the constants
(A), (B),(C) etc. Trle cnanoes predicted by the equation under examination
must then be compared wlth those obtained exoertrnentauv. The equation
that sat tsr ies ootn requirements can then be considered the true equation
that describes band dispersion in a packed column.
The identification of the pertinent HETP equation must, therefore, be
arrived at from the results of a sequent ial series of experiments. Firstly,
all the equations must be fitted to a series of (H) and (u) data sets and those
equations that give positive and real values for the constants of the
equations identified. The explicit form of those equations that satisfy the
preliminary data, must then be tested against a series of data sets that
nave been obtained from dinerent Chromatographic systems. Such systems
migrlt involve colurnns packed with different size particles or employ
mobile onases or solutes having different but known phYSical properties.
Katz, et a! (1) measured the efficiency of two different solutes (benzyl
acetate and nexametrwi benzene) on a si Itea co lumn 2S cm long and 9mm I.D.
packed with Partisil 10 (actual mean particle diameter employing six
1 ~ 7
different solvent mixtures, Measurements were made in triplicate and
further replicate measurements made rr the mean of the three differed by
more than 3% from the extreme, They employed a specially designed
chromatographic system with low extra column dead volume to ensure that
the contribution of extra column dispersion to the values of (H) was less
than 2%. The propert tes of the system are shown in table l
Table 1
Propert1es of the Moblle Phase and Solutes
MOb i Ie Phase Benzyl Acetate Hexamethyl Benzene
k'
k'e
D
m
( 10-
S
cm
2
/ sec) Om ( 10-
S
cm
2
/ sec)
1/4.6%(w/w) EtAc. 2.05 4.07 3.61 3.51
in n-oentane
2/4.9%(w/w) ELAc. 1.97 3.94 3.06 2.73
in n-nexaoe
3/4.32%(w/w) ELAc. 2.04 4.06 2.45 2.23
in n-heptane
4/4.5%(w/w) Et.Ac. 2.01 4.01 2.01 1.71
in n-octane
5/4.4%(w/w) ELAc. 2.12 4.20 1.65 1.35
In n-nonane
6/4.8%(w/w) ELAc. 2.01 4.01 1.46 1.17
in n-decane
Values for k' were obtained using the retention time of nexametrwl benzene
as to. Values for k'e were obtained employmg tne retention time of the
completely excluded solute polystyrene (t(e)O) (Mol. Wt.83,000). t(e)O was
also employed for the measurement of the linear mobile phase velocity.
An exarnpIe of the results they obtained for the solute benzyl acetate
crvcmatoqrapneo with the solvent mixture 5.4%(w/w) ethyl acetate in n-
hexane is shown In figure (1). The curve through the points is the fitted
curve to the Van Deemter equation and the contributions from the mutttpath
term, the lonqrtuomal diHuslOn term and the total resistance to mass
transfer, term ceriveo from the curve fit, are inciuded.
Figure 1
Graph of (H) versus (u) (Partisil 10) n-nexane +5.4XCw/w)Et. Acetate
Cornposi te (H)
• (H)
• MUltipath Term
Q Long.Diff.Term
:z: 0.000 --f--........-r----r----r---......-r----r---r------i
-0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700
Velocity
""'"
% 0.004
ow
e
..,
C 0.003
e
-
..,
::I
~ 0.002
~
..,
c
e
U 0.001
~
e
ow
e
u
0.005 _r__-----------------.,
It is seen that an excellent fit is obtained with the Van Deemter equation
with an Index of determination, for that particular fit, of 0.999885
However, the results from testing all the data to each of the dispersion
equations need to be known in order to identify the equation that, overall,
shows HIe best fi t.
The value of (H) for each solute was determined in each solvent mixture
over 10 differ'ent linear velocities that covered the normal practical range
of velocities used in LC. Measurements at each velocity were taken in
tripl reate wrlich resulted in a minimum of 180 values of (H) being taken for
each solute. Each data set, from each solvent mixture) was fitted to each
dispersion equation and the values for the respective constants (A), (8), (C),
etc. calculated, together' Witil the index of deterrninal icn for each fitting. it
ta seen that the data was sufficient in both quantity, and quality to be able
to dentify HIe most appropriate dispersion equation with some confidence.
The results obtained are shown in table 2.
I j ~
Table 2
ExperImental Values for the ntsperaron Equatton Coeff1c1ents
Obtained by a Curve Fitt1ng Procedure
n-pentane
4.7%w/w
Et.Acet.
n-hexane
4.9%w/w
Et.Acet.
n-heptane
43%w/w
Et.Acet.
n-octane
45w/w
Et.Acet.
n-nonane
44%w/w
Et.Acet.
n-decane
485w/w
Et. Acet.
The Van Deemter ecuat ion H= A+~ + Cu
u
A 0.001189 0.001144 0.001144 0,001210
B 0.000108 0.000091 0.000081 0,000065
C0.002525 0.003008 0.003362 0.003661
0,001208
0000049
0004298
0,001237
0,000045
0.004786
0,001257
0.000053
0,004754
0,008100
0.001144
0.000091
0,003008
o
The Giddings equation H= ~ + ~ + Cu
. E u
1+ -
U
0.00 I 123 0.001210 0001407
0.000086 0.000065 0.000067
o003348 0.003661 0.004001
0.005243 0 0,0337
A 0.001189
B0,000108
C0.002525
DO
0.1612
0,000056
0,002310
-0,06804
2,13100
0.002626
0,000061
0.002304
0.005583
0.000051
0.009169
-0,008577
97.30
0.000702
0,000057
0,002769
0.001775
o
0,000825
0.000056
0,001948
0.002454
o
0,001197
0.000065
0,003585
0.000080
o
0,001507
0,000092
0,002495
0,000541
o
The Huber equation H= A + ~ + Cu + Ou
1
/
2
1+_E_ U
u
1
/
2
0.000986 0,00 1196
0.000084 0 000065
0.002979 0,003622
0.000447 0.000046
o 0
The Knox equatron h =~ + Av
1/3
+Cv
v
0,002390 0.002545 0.002608
0.000096 0.000080 0,000064
0.001754 0.002003 0,002518
A '"'
The Horvath equat ion H= + ~ + Cu + Du
2/ 3
1+_E_ u
1/3
u
0.001013
0.000083
0.002744
0.000647
o
A 0,002509 0.002422
B 0,000123 0.000105
C0.008720 0.001407
A 0,001366
B 0.000104
C0,003572
D-O.OO110
EO
A 0001455 0.001408
B 0.000104 0.000092
C0.003302 0.002728
o-0,00092 0,000331
EO 0
l ~ U
Examination of the results obtamec by Katz et a/as given in table 2 show
rattonal fits between the experimental data and the equations of Van
Deemter, Giddings and Knox. The fit of the data to both the HUber and
Horvarth equations gave alternating positive and negative values for the D
constant which is the coefficient of the term involving a fractional power
of (u). Furthermore, for the HUber equation) the value of coefficient (E) is
consistently zero and for the Horvath equation, is zero for four solvents
rmxtures out of six) with an extreme value of 97.3 for one solvent.
On HIe basis of the irrational fits of the data to the Huber and Horvath
equations, these equations will not be consl derec to sat tsractori ly describe
HIe relationship between (H) and (u). According to Katz et al the same
irrational behavior of the Huber and Horvath equation was observed if the
data for' hexamethyl benzene was also fitted to them,
It is seen that, although a good fit is obtained to the Giddings equation, the
value of (E) is numerical1y equal to zero, Thus, the Van Deemter equation can
be considered to be a speCial case of the Gjddings equation, where at the
linear velocities employed (t.e those normally employed in practical LC) the
constant (E) was zero. It might we11 be, however, that at mobile phase
velocities outside the range studied, the Glddings equation might be more
appropr'iate, To date, sufficiently precise data has not become available to
test thts oosstm1ity. In any event, the Van Deemter ecuat ion and the Knox
equation are the two that must be further considered as they do describe the
experimental data accurately.
To proceed further and determine which of the two equations are the most
appropriate the explicit eouat ions must be used.
The Van Deemter equation in explicit form lSI
and that of Knox,
,... \
................... \4J
where) (g) and (g') are constants
and the other symbols the meanings previously ascribed to them.

Cornparinc equations (1) and (2) it is seen that there is a significant
difference between them, In that. only the Van Deemter eouattons should
provide an (A) term that is independent of both the linear mobile phase
velocity and the solute diffusivity, The flt of the Van Deemter equation to
the expertrnental data confirms the former condition and the plot of the (A)
term against Hie solute diffusivlty, data taken from tables (1) and (2) and
shown in f igur"e 2 conf inns Hie latter,
Figure 2
Graph of (A) Term against Solute 01ffuslvlty for Benzyl Acetate
2.500e-3
".., 2,OOOe-3
C
....."
u
o
o D

E
l; 1.500e-3

..::
-
., 1.000e-3
a.
-
-
-::s
I: 5.000e-4
4e-5
l
3e-5
I
2e-5
,
le-5
o,aaoHo -t--.-_r--------r---.--"""'T"""-----1
Oe+O
Dlffuslvlty
It is seen from figure 2 that the magnitude of the (A) term appears, Within
experimental error, independent of the diffusivity of the solute in the
mobile phase. On close examination, however, there could be a slight
resicual dependance of the (A) term on oirrustvttv indicating, perhaps, that
the velocity ranee over which the data was taken was not quite sutucient ly
high enougrl to ensure that the first term of the Giddings equation was
reduced to a constant value resulting in the simple Van Deemter equation.
This can be examined further by considering the detailed expression for the
Itrst term of Hie Giddings equation.
The expanded expresston for the first term in the Giddings equation (7) is as
roltows:
It is seen that when the value of (u) is sufficiently large the right hand
function in Hle denominator becomes negl igible and the term simplifies to
2Ad
p
, the same as the first term in the Van Deemter equation. However) if
(u) takes values, where HIe right hand side of the denominator was not cutte
zero, then the value of HIe (A) term would show a Slight decrease with
increasing values of Om. This effect may well be substantiated by the slight
slope of the straight line in figure 2
Figure 3} which is the same plot of the value of the (A) term against solute
diffuslvlty for hexamethylbenzene gives even stronger confirmation of this
possjonttv.
Figure 3
Graph of CA) Term against Solute Di ffusivity for Hexamethylbenzene
0.0015
y : 0.0012 - 9,607x R: 0,95
0.0010
E
~
CD
~
""'"
c
""" 0.0005
4e-5 3e-5 2e-5 le-5
o . o o o J ~ - - - . . - - - - r - - - ~ - - - - r - - - _
Oe+O
Diffusiv1ty
Thus, altJlougrl the Data of Katz et al shows some s1i ght dependance of the
(A) term on Om, by definition, and as a result of the curve fitting procedure
to the equation H= A+ +CU it is shown not to be dependent on (u) and
u
thus, supports the Van Deemter eouanon as opposed to the Knox equat i on. It
does, however, also support the idea that the Van Deemter equation is a
special case of the Giddings equation.
Further examination of equations (1) and (2) indicates that both the Knox
equation and the Van Deernter equation predict a linear relationship between
the value of the (B) term (the longitudinal diffusion term) and solute
dlffuslvlty. Aplot of the (B) term against otrrusivuv for benzyl acetate and
hexameUlyl benzene is shown in figure 4.
Figure 4
Graph of (6) Term against DiffusivHy
0.0001
o Benzyl Acetate
• Hexamelhylbenze
E0.0001
CD

0.0000
4e-5 3e-5 2e-5 le-5
-0.0000
Oe+O
DlffuS1v1ty
It is seen that HIe predicted linear relationship is indeed realized. However,
it can be shown that the values for the (8) term from the Knox ecuat ion
curve fit also give a linear relationship with solute diffusivity so the linear
curves shown in figure 4 do not exclusively support the van Deemter
ecuat ion.
The stationary phase) for a silica gel column would consist almost entirely
of that portion of the 'mobile phase' that is 'static' and trapped in the pores
of the silica gel. As a consequence, in equation (1) it would appear
reasonable to assume that
)
Dm =Os
However, this would not be quite true as) if a binary mixture of solvents is
used for the mobile phase, then a layer of the more polar solvent would be
adsorbed on the surface of the silica gel (8) and the mean composition of the
solvent in the pores of the silica gel would not be exactly the same as that
of HIe rnobne phase. Nevertheless) it would be reasonable to assume that,
Om = £Ds
where (c) is a constant) probably close to unity,
It must be emphasized, however that there are two ways in which the
diffusivity of the solute in the mobile phase can be changed. The change can
result from changing the solute which is being cnromatoc-apneo, in which
case the above assumptions are valid as (Os) is likely to change linearly
Witrl (Dm), However) the solute oirrustvtty in the rnobue phase can also be
changed by the use of an stteraeuve moorte onsse in which case (Om) will
be crlanged but (Ds) will remain the same, Under these circumstances the
above assumptions are not likely to be precisely correct. Nevertheless) if
the resistance to mass transfer in the stationary phase makes only a small
contribution to the overall value of (H) due to the magnitude of (dO being
much smaller than (d
p
) (see equation (I» then the assumption Dm = dJs wi]]
be approximately true.
Assuming that the diffusivity is varied by changing the nature of the solute
and not HIe mobile phase, then Os can be replaced by (Os and equation (1) can
be simplified to)
2yD
m
bu
H;: 2J..d
p
+ u + Om ,.. "" ..... "... """""","........ ,,, .. (3)
where,
2
(
') 2 d
f
b = fl k d + f2-
e p (
1 4 ~
Differentiating equation (3) with respect to (u), equating to zero and solving
for uopt., it can be seen that,
(
2)0.5
Uopl. = D
m
bY
..................................................... (4)
Further tJy Substituting for (uopt.) 1n equation (3) it can be seen that the
minimum plate rleight (Hrnin.) is given by,
Hmin. ;; 2Ad
p
+ 2J2b (5)
Now, it is seen rrorn equations (4) and (5) that the optimum linear velocity
should be linearly relateo to the diffusivity of the solute in the mobile
phase, whereas the minimum value of the HETP should be constant and
tndeoendent of HIe solute dlffuSivity. Trl1s , of course, will only be true for
solutes eluted at the same (k').
Figure 5
Graph of H (minimum) against Solute Diffuslvlty
0.0025 ~ - - - - - - - - - - - - - - - - - - ,
0.0020
Q 0
8
o
8
~
E
:s
0,0015
E
-c
-I:
0.0010
~
:z:
0 BenzyI acetate

Hexarnel.benzene
0.0005
0.0000 j
Oe+O le-5 2e-5 3e-5 4e-5
Dlffuslvny
In table (1) it is seen that tr1e values of (k') and (k'e) are approximately the
same for all HIe mobile phase mixtures employed and jf it is assumed that
146
the contribut ton of the resistance to mass transfer in the stat ion phase to
(H) is small then equations (4) and (5) can be tested.
In figure (5) the values of (Hmin) are plotted against solute diffusivity and
it is seen that the independence of (H
m
l n) on diffusivlty is largely
connrmed Close exarntnatton, however,shows that neither of the 1tries for
the two solutes are completely horizontal with the baseline, but the
dependance of (H
rn
ln) on diffus1vny ts extremely small for the solute benzyl
acetate. The sllght slope of the line for the solute hexamethylbenzene might
well result from the fact that either the (A) term Is not completely
independent of the diffusivity (D
m
) as shown by the results in figure 3, or
the resistance to mass transfer in the stationary phase does make a small
but significant contribution to the the value of (H).
The curves r'elat1ng the optimum veloc1ty with solute d1ffuslv1ty are shown
in figure 6 and it is seen that the straight lines predicted by the Van
Deemter equation are realized for both solutes, It is also seen tnat the
curves extrapolate to values close to zero also substantiating the validity
of equat ion (4),
Figure 6
Graph of Optimum Velocity aga1nst Solute Dttrustvtty
0.300
......
~
CD
co

"E
~
~
0.200
=
-
-~
0
-CD
>
0.100
E
::s o Benzyl Acetate
E

hexameLbenzene
-
-
~
Q
0.000
Oe+O
t_ r:'
2e-5 3e-5 4e-5 I r:;:-",
Dtrrustvtty
It should be noted that similar treatment of the Knox equation does not
predict that values of H(min) should be independent of the solute diffusivity
1"1 r
neither does it predict that (uopt) should vary linearly with solute
otrrostvttv. Thls is strong evidence, supporttno the vauottv of the Van
Deernter equation, as opposed to the Knox equation.
Returning to equation (1), it is seen that the Van Deemter equation predicts
that the total resistance to mass transfer term must also be linearly
related to the reciprocal of the solute diffusivity. Furthermore, it is seen
from eouattoru 1), that, if there is a significant contrtbut ion from the
resistance to mass transfer in the stattonarv phase, the curves will show a
positive intercept.
In figure 7 the Resistance to Mass Transfer term (the (C) term from the Van
Deemter curve fit) is plotted against the reciprocal of the diffuSivity for
both solutes.
o Benzyl AceLaLe
• HexameL.benzene
0.002
E 0.003

Q)

u
0.001
0,004
Figure 7
Graph of the C Term against the Reciprocal of the Dtrrustvtty
0.005..,.--------------'7""---.,
8e+4 6e+4 4e+4 2e+4
o,000
OHO
l/D1ffus1vUy
It is seen that the expected linear curves are obtained and that there is a
small, but significant, intercept for both solutes. This indicates that there
i,.... .... ,.... .......... 11 I'\"'\#"' ...th,...l"' ............ f""'l'\trihll+;"'I'\ f r- "'1'Y"\ tho r-oC';c+"':)I'\ r- O +'"
'.:1'1.:1111'111 u ...... , I II;. Y 1;.1 "'Il;;II:,,J..J, ,-VI 1"-1 luU,,'V'1 II VIII "-"'" ''''""""",,"u,,,-,,, "-v
mass transfer in the stationary phase for these two particular
solvent/stationary phase/solute systems. Overall, however, all the results
in figures (5),(6) and (7) support the Van Deemter equation extremely well.
.... v
Katz et al (1) also examined the effect of particle diameter on the value of
the overall resistance to mass transfer constant (C) They employed columns
packed with 3.211, 4.4IJ., 7811, and 17.511, and obtained HETP curves for the
solute benzyl acetate in 4.3%wIw of ethyl acetate in n-heptane on each
column, The data was curve fitted to the Van Deemter equation and the
values for tne A, Band Cterms for a1l four columns calculated. According to
the Van Deemter equation trie (C) term should be linearly related to the
square of the part tete diameter.
A graprl relating the value of the (C) term with the souare of the particle
diameter is shown in figure (8).
Figure 8
Graph of (C) Term against Particle Diameter Squared
0.015 . . . , . . . - - - - - - - - - - - - - ~ - - - .
0.010
E
~
CD
~
......
u
......
0,005
0.000 + - - . . . . - - - - r - - - . - - ~ - - . . - - - _ r _ - . . . - - ~
o 10 20 30 40
Particle Diameter Squared ex 0.0000001)
Ftqure 8 shows the predicted linear relationship between the resistance to
mass transrer term and HIe square of the particle diameter. The unear
correlation is extremely good and it is seen that therp. is, indeed an
intercept on the (C) term axis, at zero particle diameter. which confirms
the existence of a small, but significant, contribution from the resistance
to mass transfer in the stationary phase.
149
The Effect of the Function of (k") on Peak Dispersion
Rei terat ing the Van Deemt.er eouat ion,
where the symbols used have the meaning previously ascribed to them
If it is assumed that the column is operated at relatively high velocities,
such that the contribution from ronqituctnal dlffusion is no longer
srqntftcant, then
........ .. .... ........ (7)
Assuming that the otrrustvlty of the solute in the stationary phase (Os) 15
s1mply related to tne diffuSivity in the mobile phase (Om), i e. DS = ~ D r n
Subst Hut ing for (OS) in equation (7)
............................. ...... (8)
Inserting the expression for' fl(k') recommended by Purnell (9) and the
expression for' f 1(k') as oeriveo by Van Deemter (2) and rearranq'nq,
1 [1 +6k' +11k,2 2 8 k' d~ ]
H-2Ad
p
'" Om 24(I+k,)2 d
p
+ n2(I+k. 2)T Uh", (9)
1+ 6k' + 11k,2
Dividing throughout by ') j and rearrangmg,
24(1+k't
f'I ( ?L1(1.dt',2 1 _ (") 1_' d?
(
". ) vrn l- '\' ."/ J LV" I
H-2J..dp - 2 = d +2( ) ......... (10)
u 1+6k' +11k' p 11 1+ 6k' + 11k,2 ~
k'
against -------,-
(1 +6k' + 11k'2)
l)V
I ;rD:U(I + t w;s a:;i,l d
u 1+ 6k' +11k' 1+ 6k' +11k'
a straight line, Katz and Scott (10) in their work involving the development
of a rnethod for the measurement of solute molecular weight from
crrornatojraptuc data, generated sufficient data to test the relationship
given in equation (10), Furthermore.tne equation could be tested against the
two alternattve values for' tne capacity factor (k') calculated by employing
the fully permeating dead volume, or' (k'e) derived by employing the excluded
Dm( 24 (
1
+k' )
2
1
dead volume. Trle graprl relating, (H -2Ad
p
)- , to,
u I +6k' +11 k,2
( k' _) should provide a straight line.
1+ 6k' +llk'L
D (24(I+k')2 'J
Figure 9 Graph of (H - 2Ad
p
)-!I!. 2
u 1+6k' +1Ik'
1.5
(k' values calculated from the retention time
-
1.2 of the fully permeating unretained solute)

- 0
u

• ....
0
0
..
E
0.9 0 Co>

I
C
.-
0
JiC
-
0
:.
0.6
eo
-

-
00
°
...
Q
olbi
0
-
0
C
• rpff°'b f
0
I
%
0.3
-
°aA'orJI8 I 0
0
0 0.02 0.04 0.06 0.08 .1
f.(k) k'
fm(k; =1 + 6 k' + 11 kIf
11:, 1
-
It is seen that a linear curve is not obtained and the use of (k.') values
derived from the fully permeaunq dead volume can not be used in the kinetic
studies of LC columns. In contrast, the linear curve shown in figure (10), is
the same basic graph but, in this case, the ordinate values were calculated
using (k'e) values based on the excluded dead volume.
k
e
1+6k' +11 k' 2)
e e
against -r------
24(1+k'e)2
, .2
1+6k
e
+ lIke
0.50
Graph of (H - 2Ad
p
) D
m
u
Figure 10
0.40
-
u

..
....
..
E
0.30
I
C)
....
(ke-values calculated from the retention lime
of the fully excluded solute)
.1 0.08 0.06 0.04 0.02
0+---+----+-----+--+---+--..........---;--+----+--""""4
o
fs(ke)
1 + 6 + 11
The straight I tne confirms that the excluded dead volume must not only be
used for measuring mobile phase velocities but in kinetic studies of LC
columns and LC column design it must also be employed for the
measurement of capacity rectors.
In summary, it can be said that all the dispersion equations give a good fit
tl"\ rl:::lt:::l hilt {)rd\l thp \/:::In f)ppmtpr Pflll:::ltion thp Girlrlinl1c;
..... ..., ",., ... ..... _ .... ,.. ••• , ..... 1..... - •• ---"'''--' --,-'"_ .... _. J _.- ... :::;_
equation and the Knox equation give positive and real values for the
constants in the respective equations. The basically correct equation
appears to be that of Giddings but, over the range of mobile phase velocities
normally employed in LC
J
the Van Deemter equation is the simplest and most
152
approprtate to use. The Van Deemter equation appears to be a special case of
the Giddings equation) which simplifies to the Van Deemter equation when
the mobile phase velocity is close to, or around} the optimum mobile phase
velocity. The rorrn of the Van Deemter equation and} in particular) the
individual tunct ions contained in 1t. are well substanti ated by experiment.
The Knox equation is obtained from an empirical fit to experimental data
and the individual functions contained in the equation are not all
suostant i ated by experl ment.
It would appear' from the data available at this time, that the Van Deemter
equation would be the most appropriate to use in column destqn.
References
(1) E.Katz) KLOgan and RP.W.scott, ../CnrOfi?atogr. 270( 1983)51.
(2)J.J.Van DeernterFzutderweq and AKlinkenberg) Cl7em.Eng5ci,5( 1956)271.
(3)J.C.Giddlngs) .j r..71rofi7atogr.,5( 1961 )46.
(4)J.F.K.Huber· and J.ARJ. Hulsman, Anal Cl7irl7. Acta.,38( 1967)305.
(5) G.J.Kennedy and J.H.Knox
j
.j Cl7rOfilatogr sci, 1O( 1972)549.
(6) Cs. HorvaUI and H.J.Lin, J Cnrofi7atogr. 149( 1976)40 1
(7) J Calvin Giddings," TI7e. 0rnafilics of Cnrofi7atograpl7y" Dekker, New York,
(1965)55
(8) R P. W. Scott and P, Kucera, ,./ Cl7rOfi7atogr., 149( 1978)93.
(9) J. H. Purnell. Nature.,(L ol7dol7)SuPPl. 26( 1959) 184} .
(10) E. D. Katz and R P. W. Scott, J Cl7romatogr. 270( 1983)29
Chapter 10
Extra Column D1spers1on
The resolutton of two solutes on a chromatographic column depends on the
extent to which the dispersion of the individual solute bands are
constrained and this can be described by the Van Deemter equation. However,
as it has already been mentioned, that there are other dispersion processes
that can occur outside the column that can contribute to the overall band
variance as measured by the detector. The sum of the individual variances
that occur outside the column is called the extra column dispersion and it
has been shown in aprevious chapter that this should not exceed 10% of the
band variance contributed by the column. Dispersion in LC equipment has
been discussed by Scott and Kucera, (1) Martin et al (2) and Knox and Gi lbert
(3) and the measurement of extra column dispersion in some commercially
avauaote chromatographs has been carried out by Scott and Reese (4).
Excluding the response time of the detector sensor and detector electronics
(which today. as aresult of modern electronic technology has been rendered
virtually insignificant> there are six main sources of extra column
dispersion; VIZ. the sample volume; the sample valve; the connecting tube
between inject ion valve and column; the column fri ts: the connecting tube
between column and detector cell (ttns will include connect ing tubes
exterior and mter/o: to the detector ); and f] na lly the the detector cell
itself, These sources of dispersion wi 11 now be discussed in detai 1.
The Effect of Sample Volume
The dispersion effect of the sample volume was discussed in Chapter 4 and
little more needs to be said about it It will be seen later that the sample
volume controls both the concentrat ion and the mass senstttvtty of the
chromatographic system and thus} should be made as large as possible. This
means that all otner sources of band dispersion must be kept to an absolute
minimum to permit the maximum possible sample volume to be used. A
better understanding of the causes of band dispersion has resulted in
154
improved design of the chromatographic apparatus and) as a consequence)
high mass senstttvtttes can be realized from modern LC systems.
The Sample Valve.
The elution proflle of a solute band leaving a sample valve is controlled
largely by the geometry of the sample volume itself (t.e whether it takes
the form of an exterior tube or a groove in the spigot of the valve). However)
a does also depend to some extent on the geometry of the exit ports to the
valve-column connecting tube. Today) low dead volume unions have virtually
eliminated dispersion in unions and thus) these will not be considered as a
significant source of extra column dispersion. If the sample volume consists
of an external tube. then any dtsperston that takes place in it will be the
same as that, which would occur in a connecting tube of equivalent length)
and will be considered later. Dispersion resulting from an internal sample
volume consisting of grooves cut in the valve spigot together with
dispersion arising in the sample valve exit ports are extremely dif ficult to
treat theoretically and need to be determined experimentally. Such
information should be provided by the manufacturer but, unfortunately) even
if known, it is rarely, if ever, made available to the customer. This leaves
the onus on the user to determine the extra column dispersion and a method
for doing so will be given later. Theoretically) it should be possible to
design exit ports to orovide secondary flow and thus, increase the effective
diffusivity of the solute, and consequently, reduce band spreading.
Unfortunately, there appears no evidence to show that this approach has
been adopted by the manufacturers in the design of modern sample valves
Connecting Tubes
The dispersion that takes place in the sample-valve/column connecting tube
and the column/detector cell connecting tube will both result from the
parabolic velocity profile that occurs in open tubes and thus, will be
considered together.
The dispersion that takes place in an open tube is described by the Golay
equation (S),(ref. chapter 8) under condttrons where there is no stationary
phase and thus) k'<O. As a resu It, the vari ance due to an open tube (Otu
2
)w i 11
be
-- ')
LLJ
m
r"'u
=--+--
u 240
m
..." (1)
where (u), (r) and (Om) have the meanings previously ascribed to tnem,
155
If u » Dm/r then eouatton (1) reduces to,
, (2)
Now) from the Plate Theory) the peak Variance in volume units (a;(V) is)
2
(Tube Volume)2
at(v)
-
n
(llI" 21)2
=
n
., (3)
Where (n) is the number of theoretical plates in the tube. Now, l = H,
n
Thus) replacing (l) by (H») and SUbstituting for (H) from equation (2),
n
2
°t(v) -
Bearing in mind that the flow rate Q = nr
2u
2
at(v) -
....... ",., .. ,.. ,..... ".................... (5)
Eauation (5) clearly indicates the orocedore that must be followed to reduce
. ,
the dispersion that arises from connecting tubes. However, for maximum
efficiency, the column should be operated at its optimum mobile phase
velocity and consequently, the flow rate, (Q), is already defined, and cannot
be used to control tube dispersion. In a similar manner the diffuSivity of the
15()
solute, (D
m
), 1s determ1ned by the nature of the sample and the rnooue phase
that is chosen and so th1s is also not a variable available for dispersion
controi. The major factor effecting disoerston, is, in fact, the tube radius. It
is seen that the dispersion increases as the fourth power of the tube radius
and thus} a reduction in the tube radius by a factor of 2 will reduce the
dispersion by a factor of sixteen.
Unfortunately, there is a limit to the process of reducing (r) as, from
Poiseuille's equation the pressure drop across the tube ts given by,
Thus as Q = nr
2u
J J
~ p = 8n
lQ
(6)
nr
4
It is seen from ecuat ion (6), that the pressure drop across the connect ing
tube increases inversely as the fourth power of the tube radtus, Thus} as it
is tnaovtsable to dissipate a significant amount of the available pump
pressure across a connecting tUbing, there will be a lower limit to which (r)
can be reduced in order to minimize dispersion,
It is also interesting to note that changing the length of the connecting tube
has the same effect on both dispersion and pressure drop. Thus, reducing (I)
will linearly reduce dispersion and at the same time proportionally reduce
the pressure drop across the connecting tube. It follows that the length of
the connect ing tube iS
I
by far, the best method of control Iing dispersion and
by making (I) as small as possible both the dispersion and the pressure drop
can be minimized.
In practice the diameter of the connecting tube should not be made less than
0.0\2 ern, (O.OOSin.L DJ, not merely because of the pressure drop that will
occur across it, but for amore mundane, but very important reason. If tubes
of less d1ameter are employed, they will easily become blocked. Employing
equation (S) the volume variance and the volume standard deviation
contribution from connecting tubes of different lengths were calculated and
the results are shown in figure 1A and \ B. The tube radius is assumed to
be 0.0 12cm, the flow rate 1ml per minute, and the diffusivity of the solute
C' ..,
in the motnle phase 2.5 X 1o:: crn-zsec.
It is seen in figure 1A that) as would be expected, the variance increases
linearly wah the tube length. These values for variance must be added to
1) (
that of the column and other extra column dispersion variances to arrive at
the final variance of the peak. It might be possible to reduce the tube
diameter to about 0.008cm (0.003 in) I.D., and so decrease the tube variance
by about a factor of eight, but this would increase the chance of tube
blockage very significantly. The curve in figure 18 is more informative
Flgure 1A
Graph of Tube Variance against Tube Length
30 20 10
Oe+O ~ - - - - ~ - - - - _ r _ - - - - - - i
o
3e-5
G)
Co)
c
a
1: 2e-5
a
>
G)
"Q
:II
1e-5
...
4e-5 ,..------------;r--------,
from a practical point of view as, although standard deviations are not
additive as already discussed, they do give an idea of the actual band Width
that a tube alone can cause. It is seen that a tube 10 cm long and 0.012 cm
I.D. can result in a peak with a standard deviation of 4 Ill. This would be
equivalent to a peak with a base width of 16~ l and, as it wlll be seen later,
many short columns packed with particles, 3 11m (or less) in diameter will
produce peaks of commensurate size. This means, that if high ernctencv
columns are to be used, With small plate heights, then connecting tubes
should either be eltmtnateo altogether, or reduced to the absolute minimum
in length. In practice tt is sometimes extremely dIfficult to achieve
short lengths or a connecting tube. parttcutartv for column detector
connections. This is because manufacturers often design detectors such that
the sensor cells require sign1f1cant lengths of tUbing to connect them to the
exterior union.
FIgure 1B
Graph of TUbe Standard Devtatton (ml) aqatnst Tube Length (ern)
7
"""
~
Q)
6
'-
.-
-
-
5
C
'-
U
-
4
E
"""

:. 3
CD
Q

.-
2
en
CD
.a
:I
~
0
0 10 20 30
Tube Length (em)
low Dispersion Connecting Tubes
It is ObV10US that the icea: situation where the sample valve and the
detector sensor cell is coupled directly to the column is virtually
irnoossiote in practice and thus) a connecting system that provided little or
no dtsperston would be hlghly desirable. The dispersion in simple open tubes
results from the parabolic velocity profile that exists in such tubes causing
the solute contained in the mobile phase close to the wall to move very
slowly and t.hat at the center to move at the maximum veloctty. This effect
causes the band dispersion that is described by the Golay equation. In order
to reduce this dispersion) the parabolic velocity profile of the fluid flowing
through the tube must be disrupted to allow rapid radial mixing. The
parabolic velocity profile can be disturbed, and secondary flow introduced)
into the tube, by cetorrntnq its regular geometry.
The dispersion in geometrically deformed tubes (squeezed) twisted and
coiled) has been extensively studied by Halasz (6; 7 and 8») and the effect
of radial convection (secondary flow) on the dispersion introduced in tightly
tc;Q
coiled tubes has been examined both theoretically and experimentally by
rtjssen (9). The effect of secondary flow produced by ernploytnq serpent tne
shaped tubes has also been examined by Katz and Scott (10). It was found
that the dispersion characteristics of serpentine tUbing were far superior
to those or coiled tubes. Furthermore, the dispersion that takes place in
serpent me tubtnq is practically independent of the mobile phase 1tnear
velocity and consequently, such tubes can be used over a wide range of flow
rates. The authors flrst examined the effect of secondary flow on band
disperslon that took place in tubes of different radius coiled to different
diameters. The theory of Ttjssen (9), was successfully employed to
qualltatively describe the relationship between the variance per unit length,
(H), and tne mobile phase velocity.
For the sake of simplicity, the equations that Ttjssen derived for radial
dispersion in coiled tube are given in terms of conventional chromatographic
terrmnotoqy. At relatively low linear velocities (but not low relattve to the
optimum velocity for the tube) Tijssen derived the equation,
jr
2
u
H = Om , ,.................... (7)
where (j) is a constant over a given velocity range,
and the other symbols have the meaning orevrouslv ascribed to them,
It Is seen that the band variance is directly proportional to the square of HIe
tuoe radius and the relationship is very similar to that derived by Golay (5)
for a straight tube.
At high linear velocities, Tijssen deduced that,
H =
bOO.
14
m
qJu
..... " """ ""."" "" (8)
where (b), is a constant for a given mobile phase
and (,), is the ratio of the tube radtus to the coil racios, and was given
the term the coil aspect ratio.
Consequently}
lp = rtube
reoi]
160
It can be seen from equation (8) that at the higher ltnear mobile phase
velocities, the value of (H) depends on CD
m
) taken to the power of 0,14 and
Inversely dependent on the cou aspect rat 10 and the 1inear veloctty.
Accord1ng to equations (7) and (8) at low velocities the band dispersion
increases with (u), whereas at high veroctttes the band dispersion
decreases with (u), It follows that a plot of (H) against (u) should exhibit a
maximum at a certain value of (H), By combining equations (7) and (8), an
equation can be obtained (5), that predicts the value of (u) at which (H) is a
maximum, and is qtven by,
c
u=-
rf'
where (c) is a constant for a given solute and given mobile phase
The above equations were employed to investigate the effect of tube radius
and coil aspect ratio on the onset of radial rmxinq in coned tubes, The
dimensions of the coiled tubes examtned are given 1n Table 1 and the curves
relating (H) and (u) in figure 2,
Table 1
Tube
1
2
3
4
PhYSlcal Dlmenslons of Coiled Tubes Examlned
(r xrn. (L)cm, rtco: Dcrn
(,) L(coi1)cm,
0,019 365.8 0.5 0,038 18,5
0.020 365.0 0.085 0.235 65,8
0,0127 998.0 0.0765 0,166 128,0
0,0127 337,5 0,0498 00255 73.7
It can be seen that at low linear velocities where radial mixing is still poor)
the values of (H) increases as (u) increases Furthermore, the dispersion in
coil ed tubes ( 1) and (2) of 1arger radi t, 1S greater than that in tubes (3) and
(4) wrucn had smaller radii. At high linear velocities) were radial mixing
commences, the values of (H) decrease as (u) increases, As the the range of
linear vetocittes is approacneo where radial mixing dominates the solute
dispersion becomes independent of the linear velocity (u). It is also seen the
maximum value of (H) for any particular coil occurs at different values of
(u) depending on the combineo values of rand (111"). In g ~ n ~ r f : l l , it WOlJ1rl spem
that a high coil aspect ratio reduces both the maximum value of (H) and the
value of (u) at which it occurs,
H>l
It is interesting to note that, although the straight tube theory of GOlay is
not applied to coiled tubes, his equation can be employed to qualitatively
explain the shape of the curves given in figure 2. At low values of the
mobile phase velocity the effect of longitudinal diffusion dominates) but as
the velocity tends to approach the optimum, the resistance to mass transfer
term begins to increase and the value of (H) also rapidly increases.
Figure 2
Curves Relat tnq (H) agaInst (u) for DIfferent Col1ed Tubes
MOBILEPHASE,' 5%ETHYL ACETATEin n - HEXANE
SOLUTE,' BENZYL ACETATE
2.5
E
u 2.0
a..
I-
w
I 1.5
1.0
u
COIL£O TUB£ .,I
COILED TUBE # 2
COILED TUBE #3
COIL£O TUBE #4
However, at higrl velocities the effective value of the ctrrustvtty of the
solute dramatically increases as a result of induced radial flow} eventually
. reducing HIe resistance to mass transfer factor to Virtually zero, This
results in a corresponding dramatic reduction in the value of (H), Finally) at
very high velocit ies, the greatly reduced longi tuci naI diffus ion effect agai n
dominates. At this point, the value of (H) is very small and, in fact,
decreases even further as the mobile phase velocity is turther Increased,
Serpent ine Tubes
The 10\,,-/ dtsperston serpentine tube developed by katz ft 8/. (10) was an
alternative approach to the coiled tube and was designed to increase
secondary flow by actually reversIng the direction of flow at each
serpentine bend, Adiagram of a serpentine tube is shown in figure 3. In fact}
the serpentine tUbing shown in figure 3 was designed to be an interface
162
between a liquid cbromatoqrapb and an Atorntc Adsorption Spectrometer.
The serpentine tube is encased in an outer sheath to protect the tube and
provtde some r·igidity.
Figure 3
The Low Dtsperston Serpentine Tube
Dimensions: (r), (internal), 0.0127 cm (0.010 in. 1. OJ
(n, (externa1), 0.025 cm (0.020 in O. DJ
(U, length (linear) 42.5 cm.
(]), length (cotl) 38.5cm.
(5), (serpenti ne ampl i tude) 0.05cm.
Figure 4
Graphs of Peak Variance aga1nst Flow Rate for Coiled and
Serpentj ne Tubes
o Coiled Tube Cr=O.O13 em,I=337.5 em.d(coll)=O.996 mm)
• Zlg·Z.g Tub. (r=O.O13 em.I=42.46 em )
-
E
u
........
N

-
I:
-
D
: 0·20
.,.,
..

Do


u
c:

':: 0-05

>

o

1·0 2-0 3-0-
Fla. R.te Iml/mln\
4'0
16'
A graph relatlng the variance per untt length of the tube (H) against flow
rate is shown in figure 4} for a serpentine tube having the dimensions given
in figure 3.
The flow rate is employed as the mdepencent variable) an alternative to the
more usual linear velocity, as the flow rate is defined by the column with
which the low dispersion tUblng 1S to be used It will be shown in due course
that the column flow rate is independently defined by the nature of the
separation that is to be achieved by the column. It is seen that a similar
curve is obtained for the serpentine tube, as that for the coiled tuoe. but the
the maximum value of (H) is reached at a much lower flow rate than that
with the coiled tube. Furthermore) the variance remains more or less
constant over a wide range of flow rates that encompass those usually
employed in normal LC separat ions.
Figure 5
Graphs of Peak Variance against Flow Rate for a straight and
Serpentine Tube
',5
-
E
u
<,
N
-
:a-
-
~
-D I.
e:

~
-
e:
~
,.

Do 0.5

u
e:

';

>
Straight Tube 0.007 In. I.d.
Zig-Zag Tub. 0.010' In 1.41.
,,0 2·0 3'0 4<>
Flow R.t. (ml / min)
It is now interesting to compare HIe dispersion charactertsttcs of a straight
tube with that of a serpentine tube. The variances of a straight tube and
serpentine tube are plotted against flow rate in figure 5. The values of the
variance for the straight tube were calculated from the Golay equation It is
clearly seen that the dispersion resulting from the serpentire tube ts
drastically reduced in comparison with the straight tube. According to the
graph, the numerical value of the peak variance per unit length for the
serpentine tube (,010 in I. D.) is 0 . 0 5 ~ L 2/ cm and consequently a tube 10 em
1 " ~ • w •
long would contribute a variance of 0.511- 1
2.
In contrast, the dispersion of
a straiont tube of the same same mternal diameter and only one centimeter
in linear length would be 5.5,. 1
2
, which is an order of magnitude larger.
Low dispersion connecting tubes are sti 11 not in common use in LC
equipment today although, at least one manufacturer provides serpentine
tUbing as a standard column/detector connection in a combined sample valve
/column/detector system. Low dispersion tUbing has a another feature that,
in fact, could be anticipated from its principle of operation. The secondary
flow, that results from its serpentine form, also greatly improves its
thermal conduct lng properties and thus, serpent Ine tubes can be used as
highly efficient heat exchangers. As a consequence, another instrument
manufacturer utilizes serpentine tUbing as a heat exchanger between a
thermostatlng medium and the inlet tube carrying rnobue phase to the
column. It was found that only a few centimeters of serpentine tUbing were
necessary to achi eve complete thermal equi Itortum between the
tnerrnostannq medium and the mobile phase.
Column FrHs
Dispersion in column frits was originally thought to be large and thus, made
a significant contribution to the overall extra column variance. It was not
until the introduction of low-dispersion unions that it was found that most
of the dispersion that was thought to occur in the trtts, actually occurred in
the unions that contained the fri ts. Scott and Simpson (11) measured the
dispersion that occurred in some commercially available column rr tts and
demonstrated that their contribution to dispersion to be insignificant
compared with other sources of extra column dispersion.
Dtsperston 1n the Detect1ng Cell
Dispersion that takes place in detector cells can make a large contribution
to the overall extra column dispersion of a chromatographic system. This is
because the detecting cell must have a significant volume (which in some
cases may be quite large) in order that the detector may have adequate
sensitivity. The dispersion that takes place in a detector cell also results
from the parabolic velocity profile that occurs 1n all tubular flUid conduits.
However, due to the fact that the aspect ratio of the cell (length/radius) is
relatively small the simple GOlay equation does not accurately describe the
dispersion that takes place. Atwood and Golay, (12), examined the dispersion
that takes place in tubes of small aspect ratios. If (n) is the number of
It>5
theoretical plates in the cell and n >1they employed the elution equation as
derived from the Plate Theory or one of a similar type t.e )
where the symbols have the meanings previously defined.
For values of ) (Xm(n))) where n< 1 ) however) they developed a separate
mathematical argument
Some examples of the type of curves predicted by the theory of Atwood and
Golay are given in figure 6.
Figure 6
Elution Prot tles as a Functlon of Tube length for Low Aspect
Ratio Tubes
z
Q
ti
a:
!z
lLJ
U
Z
o
U
lLJ
.....J
Q..
:E
«
VI
o 1.0
/ ~
/
I
/ / /
~
/ / /
2.0 3.0
/ /
/
/
/
/
/
NORMAUZEO ELUTED VOLUME V/VT
It is seen that from a short tube) of relatively large diameter) a broad
asymrnetrlca1peak is obtai ned and as the diameter of the tube is reduced
(resulling in an increase in the effiei ency of the tube in theoretteaI plates)
(n)) the peaks become sharper in the front but with an extended tai 1. It
should also be noted that the tube diameter must be decreased to a point
where the efficiency, in theoretical plates) approaches the value of 30
166
before the elution profile becomes reasonably symmetrical. In practice such
a recucnon may not be possible as 1t would reduce the sensitivity of the
detector to an unacceptably low level. Fortunately, the peak shapes,
illustrated in figure 6, are not usually realized in practice as the tubes
leading the mobile phase into the cell and away from it are designed to
produce the maximum secondary flow in the cell and thus, reduce dispersion,
An example of a typical detector cell design that introduces secondary flow
in the cell and reduces dispersion to an acceptable level is shown in figure 7
Figure 7
lC Detector Cell DesIgned to Introduce Secondary Flow and
Reduce Dispersion
Optical x
WinOOw /' .
1nlet from Column

.< .••.
...... ....... /
- .'"

..............
..,' "
Exi1
. ./ Optical
¥ Window
It is seen that the entrant and exit tubes are set at an angle which, as the
flow direction must be reversed for the mobile phase to flow through the
tube, results in a radial flow across the face of the tube window. This not
only increases the errecttve diffusion, and, consequently, reduces dispersion
but also has a cleaning action on the face of the tube. The exit tube is also
set at an angle to the cell which causes the reverse effect. The mobile
phase, passing axially along the cell, must reverse direction in order to
leave the cell and, thus, radial flow is again introduced into the cell.
Dispersion of solute bands in cells of different dimensions have also been
expertrnentallv measured by Scott and Simpson, (11) but with concentric
inlet and outret tubes their nOl"'tinol"\t t" ,..."""',, +..... " ...
___ . , ..... " ............... , ....... "v"''::''' .... "', ,-'''1,,1'''' ..v .;;lVII'1; J..Yj.JC.;;l VI
detectors, (for example conductivity detectors) are not applicable to optical
detectors where inlet and outlet tubes can not be axially oriented.
11\7
In general, it can be sa1d that) often of necessity) the detector cell may be
relative large wah a low aspect ratio and thus} would theoretically produce
serious band dispersion. In practice the predicted dispersion is reduced by
deign of the inlet and outlet tubes) as discussed above) to ensure maximum
secondary flow in the cell and thus) minimize dispersion. The success of the
procedure to reduce detector cell dispersion depends on the type of detector
and the principle of detection. For example,it is far easier to design a low
dispersion electrical conductivity cell than a low dispersion UV absorption
cell
Effect of Extra Column Dispersion on Column Radius
The total extra column dispersion that takes place in a liquid
chromatographic system places a 1trntt on the minimum column radius that
can be employed for a given separation. The effect of extra column
dispersion on the minimum column radius was examined by Reese and Scott
(13) who derived an equat ion that allows the minimum column radius to be
calculated for any particular separation.
The maximum extra column dispersion, (OE), that can be tolerated) has
already been shown to be given by,
OE =O.32oc ............ , " ,.. , (9)
where 0E 1s the total extra column dispersion)
and Oc is the column dispersion.
The limitation of (OE) to (O.32oc») restricts the increase in variance of the
peak eluted from the column to a maximum of 1O ~ and the increase in peak
width to 5%.
Now) from the Plate Theory) Oc =In(vo +Kvs) " ( 10)
and, V
r
= n(vo -Kvs)
Thus) wo +KvS) = Vr/n " " (11)
SUDstltutmg for) ((vo +KVS)), In (10) from (i i)
Oc = Vr / In (12)
161\
V
r
= v» + KVS = Va( 1-k')
Where, V
r
, v». K, Vs, and k', have the meanings previously ascribed to them.
Now,
ac = Vo( 1 +k')/,m (13)
v» = rnr
2
1 ." " "." "" "" .. "". (14)
where, (r) 1s the column radius,
(I) is the column length,
(c) IS the fraction of the column occupied by the rnobue phase,
and 1=nH ", " "" " (15)
Furthermore, if the column is run under optimum conditions and,
consequently at the optimum mobile phase velocity, (H) will be at its
mlntrnurn, t.e.,
H =2dp " " " (16)
Substituting for (H) in (15) from ( 16) and for (I) in (14) from ( 15)
a( = 2rnr2dp (1 +kI ),m "... ... .. .. .. (1 7)
Now, if the column is to effect a particular separation, where the pair of
solutes that are eluted closest together have a separation ratio of (a) and
the first of the pair, ( solute A), is eluted at a capacity ratio value of (k'A),
then the value of (n) is given by the Purnell Equation, which was discussed
in the chapter on The Applications of the Plate Theory, viz
............. " " " (18)
Substituting for (,m), in equation (17) from equation (18), and Simplifying,
Rr1tr
2
ri ... (1+k' A )2
--_. -1-'\' ""/
ac = k'A(a-1) " " " "" (19)
169
The optimum value for (k'A), that will provide the maximum resolution and
consequently, the mtmrnurn elution time, (for an inopttmtzeo column) has
been deduced by Grushka and Cook, (14) and Katz et a/ (15) to be about 2.5.
As a r'esult, the composition of the mobile phase is usually adjusted to
achieve this value for (k') if it is at all possible.
tnos, taking the accepted value of (c) to be c.ao.z and rearranging,
Then,
d
0e =86.16r 2_D
a-I
(20)
SubstHuting for (oe) in equation (9) from ecuation (20),
d
0E =27,6r
2_D
a-I
Rearranging to obtain an expression for (r),
Equation (19) shows that the minimum radius will increase as the square
root of the extra column dispersion and as the square root of (a-I) but,
increase inversely with the square root of the particle diameter.
(However,it will be shown later that, that if the column is packed with
partieles of optimum diameter for the particular separat ion then the
column radius will become linearly related to the function (a-I)).
Nevertheless) for unoottmtzeo columns) and for simple separations) the
minimum column radius will be reiattvelv large and for difficult
separations the minimum column radius will be relatively smal1. It will be
seen later) that tt ts highly desirable to operate with a column of minimum
diameter as this will provide the maximum mass sensitivity from the

.... 4.'''......... :::''. _ ..... ,. t r:> ... < ••
To calculate the minimum diameter for a column of length (1) packed With
particles of diameter (dp) which has not been optimized for a particular
170
separation lt IS necessary to return to equation (17), From equations (15)
and (17) it is seen that,
.[n =J (22)
SUbstituting for (In) from equation (22) in equation (17) and rearranging,
lJc = , (23)
Again, taking the accepted value of (E) to be c.a. 0.7, substituting for (od in
equation (9) from equation (23) and rearranging, it is seen that,
( )
0.5
r - °E
- .J%(1+ k') , .
(24)
Equation (24) allows the minimum column radius to be calculated from its
length} the particle diameter of its packing and the extra column dispersion
of the chromatographic system. Unfortunately, the extra column dispersion
IS rarely known and very few rnanuracturers even provide data on the overall
dispersion of the detector. When values are given for the detector
dispersion, it is often for the sensing cell alone and does not include
internal connecting tubes and, as a consequence, can be very misleading.
The total extra column dispersion can be easily measured by removing the
column and connecting the tube from the sample valve directly to the tube
leading to the detector. Avery small sample ( 0.2p.1 or less) is then injected
into the system and the dispersion calculated as follows,
0E = o«j
where, (Q) is the flow rate in rnt/rntn,
(x) is the standard deviation of the eluted peak in ern,
and (j) is the chart speed in em/min.
Employing equation (24), the minimum column diameter was calculated for a
column 10em long packed with particles of different diameters. The
standard cevtatton of the extra column dispersion was assumed to be 5, 10
and 15rmcrolttres respectively, values that embrace those that would be
1 ( 1
expected in practice from a well oestqneo Chromatographic system. The
results obtained are shown in figure (8)
Figure 8
Graph of Minimum Column Diameter against Particle Diameter
12
"
E
E
10
~
~
I)
8
~
I)
E
b
.,
...
Q
c: 4
E
~
-
2
Q
c.J
0
0 10 20
Particle D1ameter (micron)
El S.D.O.005ml
• S.D.O.OlOml
• S.D.O.015ml
It is seen that if the length of the column is constant, the minimum column
diameter does not change much with particle diameter once it exceeds about
7 microns. However) the rntntmum column oiameter increases rapidly and
becomes very large when the particle diameter is reduced to 3 micron or
less. It is also seen that the extra column ctsperslon has a profound effect
on the minimum column diameter for particles of all sizes,
As the extra column a/soersta: becomes l a r g ~ the column diameter must be
mcreesea to ensure that its variance contrtouttoa remains no more than
10%of toe t!Jat of the elutedpeak
As a finite mass of solute is placed on the column, any increase in peak
volume necessary to compensate for high extra column dispersion will
dilute the solute concentration as sensed by the detector. Consequently, as
the senstttvtty, or minimum detectable concentrat ion of the detector, has a
limit, increasing the column diameter will result in a reduced mass
sensitivlty.
172
Mass Sensitivity
The mass sensitivity of a chromatograplJlc system is that mass of sotate
(m) that will provIde a peak with a height equivalent to twice the noise
level
The sensitivity of the detector (Xo) (or minimum detectable
concentration) is defined as that concentration of solute that will
provide a signal equivalent to twice the noise level. Now the
concentration of solute at the peak maximum ts approximately twice the
average concentration of the solute in the peak volume. ThUS, the
minimum detectable mass will be that mass (rn) that, when dissolved in
a volume of mobile phase equivalent to the peak volume, will produce a
concentrat ion of Xo/2 .
Now the peak vOIume can be taken as 4oc,
Thus, m/40c =Xo/2
or, m= 2ocXo (25)
Now, from equat ion (9) 0t =0.320c
or, Oc = oE/0.32
SUbstituting for (oc) in equation (25),
m= 6. 250E Xo (26)
The importance of the extra column dispersion now becomes apparent, as
equation (26) shows that the minimum detectable mass increases
11nearly with the extra column dispersion, It is also becomes obvious
that it is of little use designing a detector for increased sensitivity (Xo)
if this is achieved (as is often the case) at the expense of increased
extra column dispersion (OE). Conversely, if the chromatographic system
is designed to have very low extra column dispersion, a proportional
reduction in the minimum detectable mass will be achieved even if the
actual detector concentratton sensitivity remains the same. It follows,
that In the desion of an optimized column for a particular analysis, the
extra column dispersion will determine both the radius of the column and
the mass sensttivtty that will be available.
References
(1) R. P. W. Scott and P. Kucera, ,j Chromatogr Sci,9( 1971)641
(2) M. Martin, C. Eon and G. sutccron, J. CIJromatogr., 108( 1975)229
(3) J. H. Knox and M. 1. Gi lbert, J. CIJromatogr, 186( 1979)405
(4) C. E. Reese and R. P. W. Scott, J.CIJromatogr. Sci, l8( 1980)479
(5) M. J. E. Golay, in" Gas Chromatography /958'',(Ed. D. M. Desty)
Butterworths, London} ( 1958)36
(6) I. Halasz, H, O. Gerlach, K. F. Gutlich and P. Walking, U 5 peten;
3,820,660, (1974)
(7) K. Hofmann and I. Halasz, .j Cliromatogr 17J( 1979)211
(8) K. Hofmann and I. Halasz, J CIJromatogr. 199( 1980)3
(9) R. Ttjssen, Separ. Sci recmot; 1J( 1978)681
(10) E. D, Katz and R. P. W, Scott, J CIJromatogr 268( 1983)169
(11) R. P. W. Scott and C. F. Simpson, J CIJromatogr. Sci 20 ( 1982)62
( 12) J. G. Atwood and M. J. E. Golay, J. CIJromatogr
j
218( 1981 )97
( 13) C. E. Reese and R. P. W. Scott J. Chromatogr. Sci, 18( 1980)479,
( 14) E. Grushka and W. D. Cook} J. CIJromatogr. Sci,9( 1971 )310.
( 15) E. Katz, K. L. Ogan and R. P. W. Scott J. 1984)65
173
Chapter 11
LC COLUMN DESIGN-THE DESIGN PROTOCOL
All chromatographic analyses involve the use of a separation procedure that
is associated with a large number of interacting vartetnes, some of which
are under the controI of the chromatographer and some of which are not. The
nature of the sample presented for analysis, the sample throughput of the
analytical service and the cost effect tveness of the laboratory, all make
their own individual demands on the crrcrnatoqrapntc system, and, In
particular, the design of the chromatographic column. These exiqenctes,
(which, unfortunately, are sometimes in conflict with each other), result
from the various and different requirements expected from the system and,
for clerttv, need to be incorporated into a design protocol From such a
protocol a procedure can be developed that can identify the basic
characteristics of the fUlly optimized column.
The Design Protocol contains three different sources of data which will be
termed the column design Data Bases, The needs of the analyst will
constitute the first data base which will be given the title Performance
Criteria. The performance criteria must state explicitly, in numerical
terms, the quality of the separation that is required in order to achieve an
accurate anaivsts.
The apparatus employed for a given analysis will have operating
specifications that are unique to the particular instrument that is available
or that is selected. These specifications will be determined by the design
and method of manufacture of the instrument and wi 11 probably differ
significantly from one instrument to another. They will controt, and limit,
the ultimate performance achieved by any column with which the instrument
is used. However, it is likely that as a result of careful design by the
rnaruracturer, the important instrument charactertsttcs effecting column
design w111 remain sensibly constant over the 1trettrne of the instrument.
This wIll allow any column that is designed for optimum use with the
instrument to also have a reasonable life span. The instrument
176
soectrtcauons provide the second data base necessary for the design of the
opt imum column and this data base is given the term Instrument constraints
.It is important to realize, and it will become increasingly apparent during
the development of the design procedure, that it is the instrument
constraints that ultimately control and limit the optimum performance of
the column.
Finally, the analyst is left with some choice in the strategy that can be used
in the analysis by way of tne chromatographic media selected, and in the
level of some operat ing variables that may be considered appropriate or
necessary. The range of variables left to the choice of the analyst
const itutes the the third data base necessary for opt imum column cesi gn
and this wlll be termed the elective variables, However, as most of the
conditions that need to be specified will be defined under performance
criteria and determined under instrument constraints) the analyst is not
left with a very wide choice of variables from which to choose. This might
be considered advantageous, however, as the fewer the decisions that are
left in the hands of the operator, the less skill and experience will be
required and fewer mistakes wi 11 be made.
The information provtded by the three data bases allow the column to be
designed and the colufl7n soectrtcsttons to be calculated. Furthermore, once
the column has been designed, and its properties defined, a complementary
set of ana!yticalspeclfic:-:alions can also be determined.
The column design orotocoi. therefore, consists Of three data bases,
perf'orfllance criteria, elective variables and instruments constraints.
These data bases win provide, firstly, the column soectrtcstroos and
final1y, the analytical soectrtceooas . A diagram representing the overall
design protocol is shown in figure (1). The four different components of the
column design protocol win now be discussed in detail.
Performance Crtter ta
Chromatography is a separation technique and, consequently, a satisfactory
chromatographic analysis demands, a prtort , that an adequate separation of
the constituents of the sample is obtained. The separation must be such that
an accurate quantitative measurement of each component is possible. In
order to achieve this separation, an approor iate phase system must be
chosen so that the individual components can be moved apart from one
another in, the column. The column must, therefore, be designed to have
sufficient efficiency to separate all the components of the mixture. To do
this, the concept of the ReducedChromatogram must be introduced,
J f f
Figure 1
The Column Oes1gn Protocol
Performance Elective Instrument
Criteria Variables Constrarnts

... ~ t
.. ....
't
Column
Spec j fl cations
't
Analytical
Specifications
The Reduced Chromatogram
Any chromatogram that represents the separation of a complex mixture of
solutes can be reduced to a relatively simple separation that will concisely
and accurately represent the urntts and extent of the separation problem.
The simple separation can be depicted in the form of a reduced
C/JrO/773togra.lJ7) an example of which is given in figure (2).
The reduced chromatogram consists of four peaks) the first the dead volume
peak) (which has been shown previously must be the fUlly excluded peak
determined from the retention volume of a salt or solute of large molecular
178
welghU, the pair of peaks In the mixture that are eluted closest together
and thus, the most difficult to separate ( the crrttce! pair), ana the last
peak that is eluted from the mixture which will define when the analysis Is
complete and determine the total analysis time. The column must be
constructed with sufficient efficiency to separate the crittcal pair and, if
this Is achieved, all other peaks will also be resolved
Figure 2
The Reduced Chromatogram
I
k' =0
k
1
=2.0
ex =1.0t.
k =6
However, it should be pointed out that any given column, operated at a
specific flow rate, can exhibit a range of efficiencies depending on the
nature of the solute that 1s chosen for efficiency measurement.
Consequently, under exceptional circumstances, the predicted conditions
for the separation of the crttical pair may not be suitaote for another pair
and the complete resotutton of all solutes may not be obtained. This could
occur 1f the separation ratto of another solute pair. although larger, was
very close to that of the critical pair but contains solutes, for example, of
wideIy dl fferent molecular weight. However, the oosstt» Iity of this
situation artstnq, In practice, is extremely remote and will not be
considered in this discussion. It follows, that the efficiency required to
separate the critical pair, numerically defined, Is the first performance
criterion
The efficient laboratory manager will also require the maximum throughput
of samples from the equipment and tnus, the second criterion is that the
analysis must be achieved in the minimum time. It should be pointed out
179
that separation is not just required to be separated rapidly but, that it
should be ach1eved In the absolute minimum time that an optimized
column can achieve. In fact, the column must be be designed such that with
the apparatus avatlable and the phase system chosen no otner cotomo can
effect the separalionmorerapIdly.
Another aspect of cost reduction would be the need to employ the minimum
amount of solvent per analysis and this would be the third performance
criteria. F1nally} to conserve sample and to have the capability of
determining trace contaminants, the fourth criterion would be that the the
combination of column and detector should provide the maximum possible
mass sensitivity. The performance criteria can therefore be summarized as
follows,
Performance Criteria
1/ Adefined resolut ion must be obtained.
2/ The analysis must be completed in the minimum time,
3/ The analysis must be completed with the minimum solvent consumption.
4/ The maximum mass sensit tvtty must be real i zed,
Instrument ConstraInts
Certain operating limits are inherent in any liquid chromatograph and these
limits will vary with the purpose for which the instrument was designed.
For example, the preparative chromatograph will have very different
operat ing characterist tcs from those of the analytical chromatograph. The
rtrst, and obvious operating limit, will be the maximum column inlet
pressure that the solvent pump wi 11 provide. It wi 11 be seen that the
maximum inlet pressure that is available (or can be tolerated) will
determine the optimum column length, the optimum particle diameter of the
packing material and, as a consequence, the the minimum analysis time, It
should also be noted that it is not the avatlable pressure that the pump can
provide, that usually limits the available column inlet pressure} but more
often, the maximum pressure that the sample valve can tolerate under
cont1nlJolJs opp.r;ttion Most pumps can provide pressures of at 1P.3St
6,OOOp.s.i. but the sample valve operating pressure wl1l often limit the
column tnlet pressure to as little as 3,OOOp,s,i. Although, it is claimed by
many menutactures, that their sample valves w111 operate at 6,OOOp.s,i, or
even 10,000 p.s.t. the life time of the valves, when operated at these
11\0
pressures, is often relat ively short. As a consequence, for successful ana
cont1nuous operation over an extended period of time, the operating pressure
of the chromatographi c system may we11 be 1trn tted by the long term
pressure tolerance of the sample valve and not by the available pressure
from the solvent pump.
The maxtmum and minimum flow rate available from the solvent pump may
also, under certain circumstances, determine the minimum or rnaxirnurn
column diameter that can be employed and, as a consequence, place limits on
the mass sensltlvity of the chromatographic system as well as the solvent
consumpt ion. However) in practice, almost all commercially available LC
solvent pumps have a range of f low rates that wi 11 embrace the optimum
flow rates that may be required for most LC analyses.
Another extremely important instrument specification is the total
dispersion that takes place in the sample valves, cornecttnq tubes and
detector cell of the chromatograph. The SUbject of extra column dispersion
has already been discussed in the previous chapter. It has been shown that
the extra column dtsperston determines the minimum column radius and
thus, both the solvent consumption per analysts, and the mass senstttvttv of
the overall chromatographic system. The overall extra column variance,
therefore, must be known and quantitatively specified.
Finally the speed of response of the detector sensor and the associated
electronics once played an important part in oottrnurn column desron The
speed of response) or the overall time constant of the detector and
associated electronics, would be particularly important in the analvsts of
simple mixtures where the analysis time can be extremely short and the
elution of each peak extremely rapid. Fortunately) modern LC detector
sensors have a very fast response and the associated electronic circuits
very small time constants and tnus, the overal1 time constant of the
detector system does not signiflcantly influence column destqn The
Instrument constraints can therefore be summarized as follows,
Instrument Constraints
1/ The maximum operat lng pressure
2/ The extra coturnn dtspersl on
3/ The rnlntrnurn flow Rate
4/ The maximum Flow Rate
101
5/ The response tlrne of the detecting system,
Elective Variables
The choice of variables rernairunc with the operator, as stated before, is
somewhat restrtcteo and is usually confined to the selection of the phase
system, Pre1lmtnarv experiments must be carr: ed out to identify the best
phase system to be used for the particular analysis under consideration. The
best phase system wi 11 be that, which provides the greatest separat i on
ratio for the critical pair of solutes, and at the same time ensures a
minimum value for the capacity factor of the last eluted solute.
unfortunately, at this time, theories that predict the optimum solvent
system that will effect a particular separation are largely empirical and
those that are available can be very approximate to say the least.
Nevertheless, there are commercially available experimental routines for
selection of the best phase system, the results from which can be analyzed
by supporting computer software, The program may then suggest further
routines based on the initial results and, by an tterat lve procedure,
eventually orovtdes an optimum phase system as defined by the computer
software,
Whether the optimum phase system is arrived at by a computer system, or
by trial and error experiments (which are often carried out, even after
computer optimization), the basic chromatographic data needed in column
design wtl 1be identified. The phase system wi 11 define the separation rat to
of the cr tttcal pair) the capacity ratio of the first eluted peak of the
crttical pair and the capacity ratio of the last eluted peak. It will also
define the viscosity of the mobile phase and the diffusivity of the solute in
the mobile phase.
There remains little more for the operator to decide. Sometimes,
alternative but similar solvent mixtures that have a lower viscosity or
higher solute otrrustvtty could be selected. For example, a n-
hexane/methanol mixture might be chosen as an alternative to the more
viscous n-heptene/tsopropvj alcohol mixture as it has' similar elution
properties, However, it will be shown later, that if a fully oottrmzeo column
is employed HIe viscosity of the mobile phase does not seem to effect the
column performance as it is taken into account in the optimization
procedure. The operator would, under some circumstances, be free to choose
less toxic or' less costly solvents; for example) in reverse phase
Chromatography the operator could select methanol! water solvent mixtures
as opposed to acetonitrile/water mixture on the basis of lower cost or 'less
182
toxicity'. However, it must be remembered that the elutton characteristics
of methanol and acetonttrue, although strntlar, are certainly not identical.
Furthermore, it should be emphasized that the solvent optimization
procedure must take place after the indlVi dual solvents have been chosen
and, if subsequently changed again, then the optimization procedure must be
repeated. The elective variables can be summarized as follows,
Elective Variables
1/ The separation ratio of the critical pair.
2/ The capacity ratio of the nrst solute of the critical pair.
3/ The capacity ratio of the last eluted solute.
4/ The viscosity of the solvent
5/ The durustvtty of the first solute of the cr tttcal pair in the mobtle phase
Column Specifications and Operating CondHions
Employing the condiUons defined in the three data bases and the appropriate
equations derived from the Plate and Rate Theories the physica: properties
of the column and column packing can be determined and the correct
operating Conditions identified. The precise column length and particle
diameter Hlat will achieve the necessary reso turton and provide the analysis
in the mmnmsn lirn&can be calculated. It should again be emphasized that)
the specifications will be such, that for the soectnc separation carried out)
on the phase system selected and the equipment avai laale, the minimum
analysis time will be absolute No other column is possible that wlll allow
the analysis to be carried out in less time.
The optimum mootte phase vetocttr will also be determined in the above
calculations and, as the minimum radius will also be calculated in order to
achieve minimum solvent consumption and maximum mass sensttrvttv, the
oottmu» llowrate can also be identified. The column specifications and
operating conditions can be summarized as follows.
Column Soecificat ions and Operat ina ronda ionc;

1/ The optimum column length.
2/ The optimum column radius.
3/The optimum particle diameter.
41 The optimum mobile phase linear ve loc ity.
5/Mobile phase flow rate.
Analytical spectr tcattons
The analytical specifications must prescribe the ultimate performance of
the total chromatographic system, in appropriate numerical values.. to
demonstrate the performance that has been achieved. The separation of the
critical pair would require a minimum column efficiency and the number of
theoretical plated produced by the column should be reported. The second
most important requisite is that the analysis should be achieved in the
minimum time and thus the analysis time should also be given. The analyst
will also want to know the maximum volume of charge that can be placed on
the column, the solvent consumption per analysts, the mass sensitivity and
finally the total peak capacity of the chromatogram. The analytical
specifications can be summarized as follows.
Analyticql Specifications.
1/ Column efficiency in theoretical plates.
21 Analysis time.
31 Maximum volume of charge.
41 Solvent consumption per analysis.
51 Overall mass sensitivity.
6/ Total peak capacity.
It is obvious that such aprotocol would not be employed to design a column
for a single analysis or even for a few dozen analyses. The optimization
procedure entails a considerable amount of work and therefore.. would only
be justified for a routine analysis that was repetitive and would be carried
"'"+ ... ",.,,,1 ........ 1............"' .. +i ......rcr» .... ........... I I,., ..... " .... """l"'h tho "CO ,.,f tho
VUI. Il;;'yUI<.l1 11 IllQl11 1.1I11\;;;:) c.I uc.l1' VIIU\;;' .,)U\..-II \..-11 \..-VIII-J ... UII ........ "", ... , ..... u"".... VI \,11 ....
protocol to construct the fUlly optimized column could be economically
advantageous, increasing the sample throughput dramatically and
signif1cantly reducing operating costs.
184
Unfortunately, sorne of the data that is required to calculate the
spectt icattons and operating conditions of the ootimum column, tnvoives
instrument spsc.rtcations which are often not available from the
instrument manufacturer, In particular, the total dispersion of the detector
and its internal connecting tubes is rare1y given, In fact, under some
ctrcunstances the dispersion or- volume of the sensing cell alone is
reporteo, implYlng that the over-all detector dispersion results solely from
the detector cell. T ~ l i s is particularly misleading as, in many detectors, the
major dispersion takes place in the tnternal connecting tube to the cell not
in HIe cell itself. In a sirnnar manner, a value for the dispersion that takes
place in a sample valve is rarely provided by the manufactures. The valve, as
discussed in a previous chapter, can make a significant contribution to the
extra column cisperston of the chromatographic system which) as has also
been shown, will deterrrune the magnitude of the column radius,
Unfortunately, it is left to the analyst to measure this data and a procedure
for- measuring the extra column dispersion has already been given in the
previous chapter.
Chapter 12
LC COLUMN OE.SIGN- THE. DESI GN PROCESS
Packed Columns
The design process tnvolves the use of a number of spectrrc equattons (most
of whtch hav1ng been prevtously derived and/or discussed) to tdent 1fy the
column carsmeters, the operating conditions and the resulting analyt tcal
specifications necessary to achieve a particular separatron The
charactertsttcs of the separation w11l be defined by the reduced
cllrofl7atografn of the part lcul ar sample of 1nterest.
The first equation to be employed will be that of Purnell (I), Wh1Ch is used
to calculate the efficiency required to separate the sample into its
constituents. The data used is the separation ratio of the critical patr and
the capac i ty rat! a of the first eluted peak of U1e erit 1ca1 oatr. The Purne11
equation is retterateo as follows,
............... '"........ .... .. ........ (1)
where (a) is the separation ratio of the crrttcal pair,
and (k') is the capacity ratio of the first eluted peak of the crttrcal patr.
The next equation of importance 1S the retatronsntp between the column
length and HIe fleight of Hie theoretical plate of the column (H),
I =nH , , ,.,.,., ,., , """', "."" ., , ,., .. ,,(2)
Now) as has been previously shown. from the Van Deemter equation (2), the
value of (H) 1S qiven by}
B
H ;;; A+ - +Cu " (])
u
where, A ;;; 2Ad
p
(4)
IA6
B =2yDm (5)
and
Now, in Le, (Om) and (Os) are commensurate and, if the stationary phase is
silica gel or a bonded phase, then dr « d
p
, and thus, the second function in
the equation can be ignored,
Consequent ly,
Differentiating equation (3) and equating to zero) to obtain an expression for
the optimum velocity it has been shown that)
Uopl = C8J
SUbstituting for (B) and (C), from equations (5) and (7) in equation (8) and
strno: Hying,
0,5
, .",,,.,, ..... ,,,,.,,.,,, (9)
By substituting for (Uopt) from equation (8) in equation (3) and simplifying
an expresston for (Hmin) can be obtained,
(10)
Again substttuttnq for (A)l (B), and (C) from equations (4), (5) and (7)
I ( '"' .-l2 \

l
1+6k' +l 1kIL UPJ
.Hrnin = 2Ad
p
+2 2yD
m
2 D ",( 11)
24 (1 + k') m
187
Thus,
..........................." ... (12)
Now) Giddings (3)} has calculated that for a we11 packed co lumn (A) and (y)
would take values of 0.5 and 0.6 respectively.
Thus) when k' = 0, Hmin = 1,45d
p
and) when k' =inflnity Hmin =2,48d
p
It can be seen that the numerical value of (k'), the capacity ratio of the first
eluted peak of the critical oatr, can make a stqntr icant difference to the
value of Hmin . To stmpury expressions in chrornatocraprr c theory it is often
assumed that, to the first approxtrnatton, (Hmin) can be taken as 2d
p
. It is
clearly seen that, in column design, such assumptions could lead to
significant errors particularly at extreme values of (k').
Substituting for (Hmin) and (n) from ecuat ions (12) and (1) respectively) in
equation (2), an expression for the column length (1) can be obt atned
1=r
4
(1+k' ))2[2A + [)1+6k'+lW2l10'51d (13)
Lr'fno 1\ .;«, .. \? P
\,q-... "I l l )\ I +K r ) )
( 14)
188
It snouto be pointed out that equation (13) does not give an expression for
the Of)tl/77UIl? column length as the cournorn particle otarneter rias yet to be
i dent if i ed
The Optlmum Partlcle Dlameter
The column lengHI is also defined by the D'Arcy equation that cescrjbes the
flow of a fluid trrouch a packed bed in terms of the oart tcle diameter, the
pressure aopuec across the bed, the Viscosity of the fluid and the linear
velocity of the fluid. The D'Arcy equation is qiven as follows,
2
lpPd
p
1=--
TJu.
where (P) is the inlet pressure to the column,
(TJ) is the viscosity of the motnle phase,
(qJ) 1S the D'Arcy's constant which for a well packed LC column takes
a value of about 35 when the pressure is measured in p.S.1 ..
Equat j ng eouat ion (2) to ecuat i on ( 14),
2
qJPd
p
_
-- - nHmin
TJ Uopt
Substituting for (uopt) and (Hmin) from equattons (9) and (10) respectively,
2
qJPd
p
T J ~
~ C
and rearranging,
Substituting for (A), (8) and (C) from equations (4), (5) and (7) respectively,
in equation ( 15)
tn
P
d
p
2
0 3y (1+ k,)2
_T--.;.._ =n 2Ad 4 m _
11. p dp (1 + 6k' + llk,2)
0.5
+ 4yD
m
., .. (16)
1 ~ 9
It is seen that there will be a unique value for (dp), the optimum particle
dterneter, that wlll allow the minimum HETP to be realized when operating
at an inlet pressure (P). Rearranging and solving for dp(opt)
]
0.5
4nllOm 3y (1 +k'f
dp:opt) ;::: P 2A ( 2) + Y
cp 1+ 6k' + 11k'
0.5
''''',", .. ,,,,,,, ( 17)
Substituting for (n) in equation (17) from equat ion (1)
05 0.5
8(1 + k') TlD
m
3y (1 +k,)2 ).
dp(opt) = ( ) - 2A ( 2) + y .........,( 18)
k' a - 1 cpP 1+ 6k' + 11k'
Equation (18) allows the optimum particle diameter to be calculated that
will allow the separation to be achieved in the minimum time by utilizing
the maximum available inlet pressure and operating at the optimum mobile
phase velocity, It is one of the most important equations in column design.
The characteristics of many of the equations discussed in this Chapter will
be tested against realistic Chromatographic conditions and the typical
conditions chosen are given in table 1,
Table 1
Typical Chromatograph1c Operat lng Condtt Ions
Separat10n Ratio (a) (Critical Pair)
Capacity Ratio (first eluted peak of the Crtttcal Pair)
Viscosity of the mobile phase (11)
otrrustvtty of Solute COm)
O'Ar'cy's Constant (cp)
Inlet Pressure (P)
Packing Factor ()J
Packina Factor (v)
OJ •
CapacHy Ratio of the last eluted peak (k'2)
1.05
25
0.023 Poises
3.5X 10-
5
cm
2/sec
35
3000 p.s.t
0.5
0.6
5.0
Graphs of optimum particle diameter aga1nst separation ratio were
calculated using equation (18), for a range of (a) values between 1.01 to
l":'V
1.10 and inlet pressures of 2000, .:1000, and 6000 p.s.t. The resul ts obtained
are shown in figure C1), The calculations were carried out ernptovtnq the
basic cnromatocraphtc data given in table CI)
Figure 1
Graph of Opt1mum Particle D1ameter aga1nst separatton Ratto
50
f) 2000 D.S.1.
• 4000 D.S.i.
b 6000 o.s.i:
1,14 1.02 1.04 1.06 1.08 1,10 1.12

1.00
10
'- 30
CD
.-
CD
E
c 20
-Q
G)
-
Co)
-
t
c
Q.
"" c:
c
'- 40
Co)
-
E
....
Separat ion Ratto
It is seen trorn f igur'e (1 ) tr.at the optimum oarttc: e di ameter ranqes trorn
about 2 rrucron for very sirnple separations Ca= 1.12) carrteo out at an inlet
oressure of 6000 psi. to about 40 micron for dtrr rcu.t separations (a=10 1 )
carried out at an inlet pr'essure of only 2000 p S,l Furtherrnore, the curves
shown in figur'e (1) appear to be in conflict with popular opinion, in that, the
more difficult separations are best achieved w.th particles of relatively
larqe diameter, whereas. stmple separations require particles or small
diameter' f or optirnum pertorrnance Trn s apparent paradox will be discussed
more fUlly later In Hie cnaoter. EquatlOn (18) also discloses some
inter'esting properties of the optimized column.
Firstly it is Si?en that that the optimum particle diameter 1S
proportional to (a-1 ),
.. _ • . .... _. __ 1.
IIIVt'1
1. e.
1
dp(opt) ex (a-1)
1Q 1
Consequently as shown in figure (1) the optimum particle diameter will
rapidly increase as (ex) becomes smaller; i e as the separation becomes more
difficult.
Referring back to equation (18), it is seen that the optimum particle
diameter is inversely oroport i anaI to the square root of tile 1nlet pressure.
Thus, the larger the available inlet pressure the smaller the optrmum
particle diameter can be. Nevertheless, as a result of the square root
function, the sensittvtty of HIe optimum particle diameter to the magnitude
of the inlet pressure is much less than it is to the separation ratio of tbe
critical pair. This is confirmed by the curves shown in figure (1) where it is
seen that, providing the inlet pressure is above 2000 p.s.t., the effect of
pressure on the optimum particle diameter is not nearly as significant as
might be expected.
However, it nas been mentioned berore, and must be bourne 1n mine, that It
is not the pressure cacaon tty of the solvent pump that normally determines
the maximum pressure that can be employed, but the maximum pressure the
whole chromatographic system can tolerate. Although valves have been
designed to operate at 10,000 p.s. i., or even higher, their useful 1ifet irne at
that pressure is orten retat tvely short, usually as a result of sample
contamination scoring the valve seats. For toop-term contl17UOU50peration
the maximum trilet pressure a valve can tolerate is often only about 3000
o.s.t.
It is seen from figure (1 ) that, over the range of separation rat i as chosen,
the magnitude of HIe optimum particle diameter extends from about I urn to
about 30 Jl-m and simple separations are best achieved uSing particles of
small diarneter. Whereas, dtrrtcuit separatrons require the use of particles
of large diameter. As stated above, these conclusions appear to be in
conflict With traditional ideas on the effect of particle size on column
performance. Popular assumptions are that for fast separations) velocities
above tne optimum should be employee and for higr1 resolution emu high
efficiencies) parttcle diameters should be made as small as possible. These
misconceptions have arisen, partly as a result of disregarding the fact that
there is a limit to the pressure available from the pump or that can be
employed with a particular apparatus) and partly from attempting to achieve
192
fast separattons from a column of fixed length, As a consequence of ltrntteo
inlet pressure the particle diameter can not be reduced beyond the 1imit
that provides sufficient permeability to allow the optimum velocity to be
realized, If higher efficiencies are required, the column must be made
longer, an<J to achieve this, the column oerrneabu ity must be increased by
making the particle diameter larger. Mobile phase velocities rligher or lower
than HIe optimum would increase the HETP and thus the required efficiency
would not be obtained.
However, if for some reason the length of the column can not be changed,
and HIe column has excess efficiency to that required, the analysis time can
be reduced by increasing the mobile phase velocity above the optimum, and
consequent ly discarding the excess efficiency. Nevertheless, it must be
emohas: zed that, under these circumstances, although the analysis time
will be reduced, it will not be tne mm/mo». The analysis time would be
reduced further, if the particle diameter was reduced to the optimum, the
coumun column length reduced and the column operated at the optimum
velocity. Unfortunately, for very strnole separations the optimum particle
diameter may be smaller than that commercially available, or below that
which can be packed successfully wlth known pack ing tecnrr ques. Under
such circumstances, non-opt i mi zed columns with excess effi ciency,
operated at higrl velocit tes, may be the only way to reduce HIe analysis time
to an accepted level. Such a set of limiting conditions, however, is rarely
met in practice,
The Optimum Column Length
Substituting HIe optimum value for the part rete diameter from equation (18)
in equation (13) the optimum column length can be obtained,
(
2)10.5 )0,5
40+k') 2 1+6k'+11k' 8(I+k') ll
D
m 3y (1+k,)2
I opt = 2A+ Y - 2A +Y
( ) kca-ll) 3(I+kl; k'(a-I) qJP (1+6k'+1Ik,2)
or,
0.5
(
05\f r 05 11°,5
(
'40+k'fJ3l l( (1+6k'+11k
I2
)J\ 'J'l
l1 Dm
l l( 3Y(1
t
k,)2 \J' jj
I opt = 2 2A+ Y - 2A +Y
l I k'(Il-J) 3(1+ki qJP (1+6k'+11k'2)
... C1g)

Eouatron (19) StlOWS that the optimum column length is inversely
proportional to the truro power of the function (1)-1) and tr'IUS, wiJ1 increase
very r'apldly with the difficulty of the separation It is also seen that the
lengUI is inversely proportional to the square root of the avatlable inlet
pressure and, consequently, has a similar sensitivity to pressure as the
optimum column radius.
A graprl relating optimum column length to the separation ratio of the
crit tea: pair f or inlet pressures of 2000, 4000, and 6000 o.s. 1. is shown in
figure (2).
F1gure 2
Graph of Log. Optimum Column length against Separat ion Rat to
"
E
CJ
3
.....
.c
-
=C
2

...J 0
2000 p.s.i
C

4000 p.s.: .
E
0
6000 psi,

-e
U
• 0
=C
...J
-1
1.00 1.02 1.04 1.06 1.08 1 10 1.12 1,14
seoarat i on Rat 10
4-r--------------------,
It is seen from figure (2) that the length of the optimum column ranges from
nearly 30 meters for HIe separation of solute pairs having a seoar ation ratio
of 1.01 at an inlet pressure of 2000 o.s.t., to less than 1cm for a solute pair
rlaving a separation ratio of 1 12 at an inlet pressure of 6000 o.s.t It is also
Sf:r.1I that dr.soUr. thr. lonar tthrntr c;f.i1lp for r.olurnn lr.noth chanotnn thp
,. .J - ..J v.;;)
inlet pressure from 2000 o.s.t. to 6000 psi. does not have a profound effect
on the optimurn colurnn length. This of course is because the optimum length
is inversely proportional to the square root of the inlet pressure,
194
The Minimum Analysis Time
The minimum analysis time is that achieved by employing the column of
optimum length, packed wttn particles of optimum diameter and operated at
HIe opttrnurn velocity. Thus, HIe minimum analysis time, (t(min)), is given
by,
(
') lopt
t
rn 1n
;:; 1+ k
2
-
Uopt
where k'2 is the capacity ratio of the last eluted peak
suostttuttnq for (uopt) from equation (9) and the column length (j) from
equation (13) t.hen analysis time is seen to be given by,
( 2)]0.5 [ ]0.5 2
_ ,4(1+k') 2 1+6k' +11k' 1+6k' +11k' 2 dp
t - (l+k
2)(k'(
I)] 21.. + Y 2 2 -D- .".""". (20)
a - 3(1+ k') 3y(I +k' ) 4 m
SUbstituting for dp(opt) from equation (18),
_( ')(4(I+k')J
2,.
(1+6k'+11k.
2))0.5
[1+6k'+1Ik'2]0.5
trnin - 1+k
2
I LA + Y----- X
k(Q-j) 3(1+k,)2 3y(l+k,)2
Simplifying and rearranging,
r
_( ')(4<1+k')J
4
'1 [Y(I+6k'+11k.
2))
t.min - 1+k
2
- 21.. +
k' ( a - 1) cp P 3(1+ k' )
2
(21 )
It is interesting to note from equation (21) that the analysis urns does not
depend on HIe magnitude of the diffusivi ty of the solute in the mobile phase
but only on the viscosity of the mobile phase, It does, however, increase
o 2000 psi,
• 4000 p.s i
lJ. 6000 PSI,
2
4
3
,...
5

-.".,t
m
E
-

-
=
-
a
Ie
C

=
e

Figure 3
Graph of log. Minimum Analysis Time against Separation Ratio

l+--.....,.---.----.,.--.-....--opo--r----"'T"""---.----,---.---I
1.00 1.02 1.04 1,06 1.08 1,10 , , '2 1' , 4
Separation Rat 10
dramatically as the inverse of the fourth power of the expression (a-I) The
analysis time is now inversely related to the inlet pressure and not the
square root of the inlet pressure and so pressure rias a more Sl gnl ncant
effect on the analysis time than on the column length. Employing equation
(21) curves were constructed relating analysis time to the separatton ratio
Af th.::> rv-a i r- fAr- i rvl o f nr.::>ccl,r-.::>c r..f 0/1/1{'\ r"\ c i /1{'\1l1l r"\ C i ':lnt1 h/11l1l
Vt I",ll\...- ' ........... Ul tJ..... ll IVI Iltl .......... tJl ...... ...J...J\... u \.;.,J VI L-VVV 1 ................... !'J ........ I. l",,41 ...... v ...... vv
p.s. L and are shown in figure (3)
It is seen trorn figure (3) that there is a very wide range of analys1s trrnes
extenrJing frorn just over ten days) wrncn was required to analyze a rmxture

where Hie critical pair had a separation ratro of 1.01 and with an inlet
pressure of 2000 p.s. i. to about 15 seconds where the seoar at! on r at io was
1.12 and the inlet pressure 6000 p.s.t. It is also seen that, again bearing in
mind the logarithmic scale for the analysis time in figure (3), the effect of
pressure on analysis time is not as great as might be expected, Increasing
the inlet pressure from 4000 ps i. to 6000 p.s.i only reduces the analysis
time by about one third.
The Optimum Column Radius
Tile optimum column radius has been discussed 1n chapter (1) but the
expression obtained was for an.v column and not specifically an
column. Consequently a Sllghtly different derivatton wiu be employed here,
Starting with equation (9) rrorn chapter (10) The maximum value the extra
colurnn di spersi on, (OE), trlat can take pIace is given by,
OE =0320c
where (OE) is the total extra cotom» dtsperston,
and (oe) is Hie cotomo dl spersion
The limitation of CaE) to (0.32oc), allows the variance of the peak eluted
from the column to be increased by a maximum of 10% as a result of the
extra column dsoersion and the width of the peak by a maximum of 5%,
Now, from Hie Plate Theory, Oc = (vo +Kvs) and (vo +Kvs)= Vr/n
Where, (n) is the efficiency of the column,
(VO) IS the volume of mobile phase per plate)
(vs) is Hie volume of stationary phase per plate,
and (K)is the distnbution coeff icent of the solute between the two
phases,
Thus,
Now,
0E =0.32Vr/;n
Vr = Vo + KVS = Vo( 1 +k')
Where, (Vo) is the total volume of mobile phase in the column,
(Vs) is the total volume of stationary phase in the column
Thus,
Now,
... ,. f
0E = o32VO( 1 +k')/.Jn " " "" " (23)
v» = rnr2loot .. '''' , "."" '''' (24)
where, (r), is the optimum column radius,
(lopt) ts the optimum co lumn length,
and (c) ts the tract-on of the column occupied by the mobile phase,
and for an optimized column,
1
op
t ::o nHmin :.".. ""'''''''''''''''' (25)
Thus, substituting for (lopt) from equation (25) in (24)
v» = enr
2
nH
m
i n " " " (26)
Substituting for (Va) from equation (26) in equation (23),
0E =O.32rnr
2
J; Hmi n( 1 +k')
Substituting for (In) from equation (1) and for (Hmin) from equation (12) in
equation (26),
,.. 2 4 ( 1+ k' )2 I(I + 6k' + I lk,2 )]0.5
(JE :0 032cnr k'(a -1) n + y 3(1+ k} dp ...... ... . ,. (27)
Substitutmg for (d
p
) in equation (27) from equation (18),
........... " (28 )
lQA
Rearr'anging and so lving for rope
025
J
2 ]0.5
3y(1+k')
+y
(1+61<'+ 11 k'2)

rapt = 05
0.5 .
3 (1+6k'+ l1k'2)] D
p(l+k}2 2A+ y 2 1') m 2
3(1 +k') «pP
L
(29)
Figure 4
Graph of opttrnurn Column RadIus aga1nst Separation Ratio
o 2000 p.s.l
• 4000 p.s.i.
• 6000 p s.i.
0.3 -r-------------------,
-
...
Q,
Q 0.0 .......--r"-__,.--_r__....-_r_--_r_.......--/
1.00 1.02
"....
E
CJ
....,

-
i 0.2

c
E

-o
U 0.1
E

E
1.04 1.06 1.08 1.10 1 12 114
Separat i on Rat 10
It is seen from equation (29) that (rapt) is directly proportional to (a-1) and
mverselv orcooruonai to the fourth root of the pressure, Thus, durtcult
separations (where a=1.0 1) wou1d be carried out on long, narrow diameter
colurnns packed with relatively large particles. In contrast, strnote
seoarations (Iuhr,,,,,,, 1'Y-1 1 ")\ ClJAII)rl 10-.", "' ..... h;,..,,,,,'" ....... .... ... ....
l.1 11.:1 \ ...... 111;11; ,.... -1. ILl VVVUIU UI; QI.,IIII;VI;U VII t.., VVIUt: UIQIIIt:t..t:1
colurnns packed with very smaII part teres. Employing equation (29)} the
values of (rapt) were calculatec for different values of (a) and the results
plotted as curves relat ing (rapt) to (0) in figure (4), It is seen that the
linear relationship between (r(opt» and the function (0-1) and the small
dependance on the in let pressure is confirmed,
The Optimum Flow Rate
The optimum flow rate is obviously the product of the fraction of the cross-
sectional area occuotec by the mobile phase and the opttrnum mobile phase
velocity, t.e.
Substituting for (ropt) and (uopt) from equations (29) and (9) respectlvely,
and simplifying,
0.5
.... 0.5
, 2 3y (I + k't
4Dm(O.312k(a-1)) °E ( )
1+6k
'
+11k'
2
3 (1+6k
1+1Ik
I2
))05 'lOrn 3y(l+kf )0,5
dp(opt' (l+k') 2).+ y 2 - 2A ( ) +y
J 3(I+k') cpP 1+6k'+1Ik'2
L J
Qopt := --------------'------"'------
Substituting for dpopt from equation (18) and simplifying,
QoPt = -----------:-
", , (30)
It 15 seen from equation (30) that the optimum flow-rate is also
proportional to the extra column dispersion and, as a consequence, the total
volume of mobile phase employed in an analysts will also depend on the
extra column dispersion. I t follows that the economy of the analysis I ies in
the hands of t.he designer of the chromatograph, a responsibi Iity for Which,
many instrument makers are not aware. Steps taken in the design of the
chromatographic system that would reduce the extra column dispersion by a
factor of two would also halve the volume and cost of solvent used in the
?OO
analysis. Most instrument makers, at this time, do not publish a value for
the extr a coturm ctsoersion of tne ir mstrurnents and inoeec, many may not
even know their true value, Ttle progressive concern over waste disposal of
solvents and Hleir potential toxic hazard may well change this attitude in
HIe future. It may wen be that, in future, the analyst may also be more
concerneo in employing optimized chromatographic systems for the very
same reason,
The optimum flow rate is also proportional to the applied pressure, the
tourth power of (Q-l) and the inverse of the viscosttv It follows, somewhat
surpr'isingly that by selecting a solvent system of low viscosity would also
permit a higtler flow rate. Whether this will also increase the solvent
consumption will be seen In due course. Employing equation (30) curves
were constructed relat ing optimum coIumn flow-rate to the separation
ratio of the critical pair and these are shown in figure (5).
Figure 5
Graph of Log. Opt1mum Flow-Rate aga1nst Separation Ratio
2
3
4.------------------,
n 2000 p.s.i.
• 4000 p.s.i.
t:. 6000 p.s.i

.-
Q,
- 1+-_....,..._--r--.....---r"__
CD
C) 1.00 1.02 1.04 1.06 1.08 1, 10 1.12 1.14
.J
,..,

e
.-
-
-e

(.)
-
E
""'"
m
.-
D
a:
I

e
L:: 0
Separation Ratio
It ts c:ppn that thp f\ntilYtllrYl ;nrro';lCOC r''::Inlr1111 -xc- thn r- An"'}r"')ti""
.... - •................ 14 L.oll'-rt ... 11 ..... Vt-" .... I IV" • Ulv--.... II I\.;I "" ....... --""""-' I U.J t..tl\, .J\"tJUI Ul.IVII
becomes less ctrrtcutt and, in fact, there is a flow-rate Change that extends
over- three orders of magnitude. Again the trend in column design becomes
more apparent, simple separations are carried out on snort, wide columns,
packed WiUI relatively large oartrc!e and operated at at high flow-rates.
201
Converse Iy, difficult separat ions should be carried out on long, narrow
columns, packed wHh relat1vely tarqe cart rcles and operated at very Jaw
flow rates.
The Mlnimum Solvent Consumptlon
The minimum solvent consumption will be obtained from the product of the
optirnurn f low-rate and the analysts t i me,
Vsol = Qopt tm\n
Substituting for (tmin) from equation (21) and for COopt) from equation (30),
OS 2
00486<pPk,3 (0_1)30E (' '1 V(I+6k'+ 11k'2)]
V
sol=-----------I+k
t - 21..+ ----
(
0.5 2 2 kCa-1) IpP 3(I+k,)2
4 (1+6k'+ I 1k'2 ))
'l(1+k') 21..+l v
3(l+k,)2
The above equation reduces to a surprisingly simple function.
It is seen that for' a fully optimized column, the solvent consumption
recutreo to complete an analysis is !ndejJenden! of the inlet pressure,
viscosity of the mobile phase and even the diffuS1Vity of the solute in the
mobile phase. Equation (31) aqatn emphasizes the importance of the extra
column dispersion in the control of the solvent consumption and
consequentially) the economy of the analysts. The need to adjust the phase
system to ensure that) not only is the separation achieved) but that the last
peak is not eluted at too high a value of (k'z) is also demonstrated by
ecuat ion (31) .Employing eouatron (31») a graph was constructed showing the
relationship between solvent consumption and the function (a-l) and is
shown in figure (6). It is seen form figure (6) that the solvent consumption
.... ' .. '" I r 3"'·... 1\10'"',1"' r,"'''r4 r,,,r h", 1""r- no if -:l flllh, flntimi7t:lrt rflllimn <:::\.I<:::tt:lm 1'<:::
lUI QII LI,.. IIC.lIy.:lI..J 11<;;";'U I IV I,. 1..1 .... " "" IU", """"""'"'' ._
employed. In fact for most analyses the consumptwn snoulc lie between 1
and 5 011 per analysis, Even for very difficult analyses) due to the cpt-rnurr
columns having small radii, the solvent consurnpuon is stiu restricted to
about 7.5 rn:
202
F1gure 6
Graph of Solvent Consumption aga1nst SeparatIon Rat10
8.....-------------------,

,...,
~
E
...,
c 6
c
-
-
Co
E
~ 4

c:
c::l
c.J
-
c 2
m
~
~
=
en
0-+-----..--------------,.----1
1.00 1.02 1.04 1.06 1.08 1,10 1.12 1.14
separat Ion Rat 10
Figure (6) allows the solvent consumption of any analysis to be compared
with which would be obtained from a fUlly optimized column, The data used
is obtained from the reduced chromatogram and the extra column dispersion
of the respective apparatus. It should be bourne in mind that the extra
column dispersion assumed in the above calculations was equivalent to a
standard deviation of 2.5 rntcrotttres This value for (OE) could be expected
from a well designed chromatographic system.
The Peak Capacity of the Optimized Column
The peak capacity of a given column system is given by equation (15) in
chapter (5) on page (69) and is reiterated here)
r -
203
substHuting for (n) from equation (1) and simplifying
l
[ (
k ~ ( I + k ' ) ](2k'(a-l)))
log 1- , + 0.5 (,
(1 + k
2)k'
(a - 1) 2 +k + kex
I (2+3k'-k'CI)
og 2 + k' +k'o
...................... (32)
Equation (32) shows that the peak capacity of the optimized column is only
dependent on the separation ratio of the crttrcat pair (Q) and the magnitudes
of (k') for the first eluted peak of the pair and for that of (k'2)J the last
eluted peak, A graph of peak capacttv against separation ratio, calculated
from equation (32) is shown in flgure (7)
Figure 7
Graph of Peak CapacIty against Separation Ratio
300 -.---------------...,
200
100
a+--.,.---r----..,---...,...........-...,..........,.--r----I
1.00 1.02 1.04 1.06 1.08 1.10 1.12 1.14
Separation RatIo
it is seen from figure (7) that the peak capacity of the chromatographic
system increases very rapidly as the separation becomes more difficult, For
simple separattons, where (a) ranges from 1,06 to 1.12, the capactty lies
between about 21 and 42. However, between (ex) values of 1.05 to 1.01 the
204
peak capacity changes from 50 to about 125. It should be remembered,
nowever > HIe peak capacity as calcul atec, ts a oct: rn 1st i c figure, as it
assumes that all the components are equally spaced. Giddings has pointed
out (5), HIe actual effectlve peak capacity for real samples may be less
Ulan half the values calculated from the above theory.
Maximum Sample Volume
The maximum permissible charge that can be placed on an LC colurnn was
discussed in chaoter 5 on page (54) and is given by the f 0 11 owing equat ion,
V
r
Vsemo =1.1 In .H (33)
Now (Vr-) is equal to the total solvent used to elute the fi rst peak of the
critical pair' and is given by equation (31), by substituting (1 +k') for (I +k'2),
substttuttnq for (V
r
) from equation (31) and for (n) frorn equation (1)
in equation (33),
Simpufying,
Vsamp
12 44
0
E(1 +k' )
k' (a-O
=1 1-----:-----:---
. 4 (1 + k,)
k'( a -I)
V
samp
= 3.420E ", ,., "................ (34)
It is seen from equation (34) that for a fUlly optimized column the maxtrnorn
sample volume depends solely on the extra column dlspersion (00. Trus
again emphasizes the importance of not only using equipment with low extra
column dispersion but, also, knowing the value of (OE) for the particular
cbrornatoqraph being used.
The optimum Capaclty naue
Througtlout the optimization procedure, the role played by the capacity ratio
has
n()t hpPrl (It') hoar. treatec '":>'" r.,.."'.... +......... + ....
........ _...,....,.1 ...... i ... ........... , .......,•• \,JI 'I\. I U"""I' ..... "'" "'\"u ("I...) U \..vll;:J .... al'l. II' Lilt:'
equations. As a consequence, the effect of the magnitude of (k') on
cbrornatoqraohtc performance has not been considered. This is because, in
choosing the phase system to provide the maximum separatton ratio for the
critical pair, the value for (k') is automatically defined by the phase system

selected and thus} (k') is normally not considered a variable Nevertheless, it
is of tnterest to determtne jf the value of the capacity rato or the first
Figure 8
Graph of Log. Analysis Time against Capacity Ratio
9
,...,
8
Co)
CD
7

.....,
CD
6
E
-
5
0 Sep Rauo1,01
Cj)


SepRatio 1.06
- •
Sep Ratio 1,12
Cj)
4
=

• •

-It
C 3
II(
• •

= 2
e

o 2 3 4 5 6
Capactty Rat 10
peak of the eluted pair does have a profound effect on the performance
of the optimized column. AS the critical parameter in the efficient
operation tne cnrornatoqraotIc system ts the analysis time which should be
made a mtntmum, the effect of (k') on the analysis time will be determined,
Employing equation (21)1 that allows the minimum analysis time to be
calculateo, HIe values of trnin were calculated for (k') values ranging from
0.1 to 51 for separation ratio values of 1.01) 1.05 and 1.10 and for an
operating pressure of 4000 p.s. i.. Ine results obtained are shown as graphs
relating rntrnrrum enatysis time to capacity ratio values in figure (8),
It is seen from figure (8) there is no true optimum value for (k) that will
produce a minimum analysis time} but the shortest times will occur when
.LL. _ ...... AlA ........ +A "I-.", ,,,,,,+ ",1,,+,..'-'/ <"'1'\1"tac I\f tr.o lVlivtllro
Lilt' 1...1 IllLal lJall al \:; !,V !,Ile; -.,JVIUL .... ..J v, "'"" '''''''"'-'' v, "l,,,",
situation will be rarely oosstbte in practice. However, it is also seen from
the curves in figure (8), that if the phase system is adjusted such that the
value for (k') is 2.5 or greater, then the (k') value will not greatly effect the
analysis time. trns is because HIe curves are very flat subsequent to a (k')
~ v v
value of 2.5. In general when selecting the phase system, the operator
should make it a ttrst priority to obtain a minimum value for (0). Furtner
adjustment of HIe phase system to make the (k') of the first eluted peak of
the critical pair 2.5 or greater srlould be carried out only rr the rrnrnrnurn
value of (0) is not compromised.
It now oosstble to summarize the design equations for a packed column
wrlicrl are given as follows.
PACKED COLUMN DESIGN EQUATIONS
The Optimum Particle Diameter
d
p
(opt)
0.5
The Optimum Co lumn length
_ (4(1+k.»)3 [ (1+61<'+ 11k'2 ))0.5
lopt - 2 k'( -1) 2A + y 2
a 3(1+k')
The Minimum Analysis Time
0.5
- ( ')(8(1+k·))4 TJ r f
V
(1+6k' +11k' 2)1o.
5
1
2
tmin - l i k - 2A+ -----
2 k'( a -1) ~ p 3 {1 + k' ~ 2
L \ ' , J J
207
The Optimum Column Radtus

The Optimum Flow-Rate
Qopt ::::
Minlmum Solvent Consumption
12.440E( ')
Vsol = ,( ) 1+k
2
k a-I
025
Peak Capac I ty
reap =
[ (
](2k'(a-l))]
log 1- + 05
o+k )k' (a - 1) 2+k' +k' a
10 (
2
+
3
k'-k' exl
9 2+k'+k'a
Maximum Sample Volume
Vsamp =3.42 0E
The design equations can be used in a simple computer program to report the
basic data and print the column and analytical specifications for any
particular analysis carried out on a specified liquid chromatograph. The
program is written In the M1crosoft ouick BasK language that can be used on
20(}
any Macintosh computer or with some slight modification on other types of
computers. The program is written in a 'very simple form so tnat those
unfamiliar with computer pr'ogramming can still enter the oroqrarn and use
it and turthermore.It is interroqative, in that the requtreo basic data is
asked f or, and then entered by the operator.
Certain values can be assumed. For example, for a well packed column the
packing factors (A) arrs (y) can be taken as those recommended by Giddings!
(3) namely, 0.5 and 0.6 respectively. The value assumed internally in the
program for the D'Arcy constant is 35 as previously discussed Values for
the viscosttv of the mobile onase can be obtained from "orqanic Solvents ..
by Riddick and Bunger' (4) and values for solute dHfusivity can either be
estimated from the data given in the appendix or calculated from the Arnold
equation (5). Ttle program for packed column design is given as follows,
Computer Program for the Design of Packed Columns
LPRINT
LPR1NT' PACKED COLUMN DESIGN FOR LC"
LPRINT
LPRINT
LPRINT"PERFORMANCE CRITERI A"
LPRINT
LPRINT" 1I A defined resolution must be obtained"
LPRINT"2/ The analysts must be completed in the minrmurn time"
LPRINT"31 The analysis must be completed with HIe mtmrnum solvent"
LPRINT
PRINT"Enter Separation Ratio of the Crical Pair":INPUT A
PRINT"Enter Capacity Ratio of the First Peak of the Pair".INPUT K1
PRINT"Enter Capacity Ratlo of the Last Eluted Peak".INPUT K2
PRINT"Enter' utrrusivnv of Solute in Mobile Phase":INPUT Dl
PRINT"Enter Viscosity of Mobile Phase (Poises)":INPUT M
PRINT" Enter Column Inlet Pr'essure (p.s.i.),,:INPUT P
PRINT" Multipattl Packing Factor":INPUT L1
PRINT" Longitudinal DiffuslOn Packing Factor":INPUT G
PRINT" Column Moblle Phase Fraction":INPUT E
PRINT" Extra Column Dispersion": INPUT 5
LET Xl =2*L 1*«3*G*( 1+K 1t2)/( 1+6*K1+11 *K1
LET D2=8*( 1+K 1)*«M*D1*x 1!(3S*r)r.S)/(K1*CA-1))
LET B=2*G*D I:A1=2*L1*D2:C=« I +6*K1+11 *K 1A2)*D2
A2)/(24*D
1*( 1+K 1f2)
LET H=A 1+2*(B*Cr.5:U=(B/CrS:N=(4*( 1+K 1)/(K 1*(A-l ))t2:L=N*H
LET T=( I +K2)*L/U:R:::(S/( 1.00S3*E*H*( I +K 1
LET Q= 188495
1*E*U*R
A2
209
"
"N
I
"
" P" s 1'"
l p",
" S "ul"
I
")L I
" G
)
"E
I
" A
}
" KI
I
" K2
I
"10 I "so.ern per sec"
" M"Poises ,
LET Y=Q*T160000!:V=3.12*5
LET J I =(2*KI *(A-I ))/(2+K i +K i *A):J2=(2+3*K i -K I *A)/(2+K i +K 1*A)
LET J3=1-((K2*( I +K 1»«I +K2)*KI *(A-1 ))+.5)*(J I)
LET NI =LOGC..J3)/LOG(J2)
LPRINT "INSTRUMENT CONSTRAINTS"
LPRINT
LPRINT"Maximum Column Inlet Pressure
LPRI NT"Extr'a Co lurnn Dispersion
LPRINT"Multipath Packing Factor
LPRINT"Longitudinal Diffusion Packing Factor
LPRINT"Column Mobile Phase Fraction
LPRINT
LPRINT "ELECT I VE VARI ABLES"
LPRINT
LPRINT"Separation Ratio of the Critical Pair
LPRINT "Capacity Ratio of the First Peak of the Palr
LPRINT ''Capacity Ratio of the Last Eluted Peak
LPRINT"Olffus;vlty of Solute in Mobile Phase
LPRINT "Viscosity of Mobile Phase (Poises)
LPRINT
LPRINT "COLUMN SPECIFICATIONS"
LPRINT
LPRINT "Opt irnumColumn Radius
LPRINT USING ":Jt,###"; R}
LPRINT"cm"
LPRINT "Opt imumColumn Length
LPRINT USING "#####,#";L,
LPRINT"cm"
LPRINT "Opt imurn Particle Diarneter
LPRINT USING "###.##"; 20000 *D2
1
LPRINT"um"
LPRINT
LPRINT "ANALYTICAL SPECIFICATIONS"
LPRINT
LPRINT "Minimum Analysis Time
LPRINT USING "######.#11; T)
LPRINT"sec"
LPRINT "Column Efficiency
~ r\ r\. ~ IT . , ~ , ...... ..: __ I • -....... r 1 ~ I • I rl_"'",..,
LI""t'\II\11 VlJl,.llllUllI r IVVV-r\al.1:"
LPRINT USING "#.###"; 0
1
LPRINT"urn/min"
LPRINT "Maximum Sample Volume
LPRINT USING "#,###"; V*IOOO,
210
LPRINT"ul J
LPRINT U:->ING "#.###"; V,
LPRINT"mI"
LPRINT "Peak capacity
LPRINT USING n#:tt##". NI
I
II
If the proqrarn is run on aMacintosh computer, then it will start processing
immediately, However, it will obviously not complete the program until the
last entry is made (the value of the extra colunn otsoerstoni The data is
entered sequent ially on request from statements given on HIe computer
screen. On completion, HIe results are sent to the printer and an examp te of
a computer print out is given be low.
The Computer Report
PERFORMANCE CRITERIA
II A defined resolution must be obtained
2/ The analysts must be comp1eted in the minimum time
3/ The analysis must be completed wah the mlnimum solvent
"INSTRUMENT CONSTRAINT
Maximum Colurnn Inlet Pressure
Extra Column Dispersion
MUltipath Packing Factor
Longitudinal Diffusion Packing Factor
Column Mobile Phase Fraction
"ELECTIVE VARIABLES"
"Separat ion Rat to of the entical Pair
"Capacity Ratio of the First Peak of the Pair
"Capacity Ratio of the Last Eluted Peak
"Dlffusivity of Solute in Mobile Phase
"Vi scosity of nobue Phase
COLUMN SPECIFICATIONS
Opt imurn COl umn Radius
oottmum Column Length
Optimum Particle Diameter
6000 o.s. i.
0.0025 ul
.5
.6
.65
101
2.5
5
0,000035 so.ern per sec
0.023 Poises
0.0197 cm
1577 cm
46.2urn
? 11
ANALYTICAL SPECIFI CATIONS
Minimum Analysis Time 306089 sec
Column Efficiency
313600
Optimum Flow-Rate 1.47 ul/rntn
Maximum Sample Volume 8.59 ul
Solvent Consumption per Analysis 7.50 ml
Peak Capacity 251
Table 2 Design Data on a Series of Optimized Columns
Separation Parti cle Column Analysis Peak
Ratio Diameter Length Time Caoactty
(micron) «rn)
1,006 385 7300 27.3 days 419
1.008 28.8 3079 8.6 days 314
1.010 23.1 1576 3.5 days 251
1.012 19.3 913 41 hrs 210
1.014 16.5 574 22 hrs 180
1.016 14.4 385 13 hrs 157
1.018 12.8 270 8.1 hrs 140
1.020 11.6 197 5.3 hrs 126
1.02 10.5 148 3.6 hrs 115
1.026 8.9 112 111.6 min 97
1.030 7.7 58A 58,4 min 84
1.035 6.6 36.8 34.0 min 72
1.040 5.8 24.6 19.9 min 63
1.050 4.6 12.6 8.2 min 51
1.060 3.9 7,3 3.9 min 42
1.070 3.3 4.6 128 sec 36
1.080 2.9 3.1 74.7 sec 32
1.100 2.3 1.6 30.6 sec 26
1.120 19 0,9 14.8 sec 21
Note
k'=2.5, k'2=5, DlffuSivlty of solute =3.5X 10-
5
, viscoSity=0.023 POlses
).,=0.5, y=0.6) £=0.65 and extra column dtspersion 1S taken as 0.0025 ~ l
i L j ~ ~ e e r l lhal by the appropriale use 01 1illUid cluomatoqrapnv column
theory it is possible to design the optimum packed column for any specific
separation. In HIe next chapter a similar procedure will be adopted to
develop a regime for designing the optimum open tubular column. The
212
conditions used to obtain the data given in table 2 ts fairly typical for most
LC analyses of relauvetv small molecular weight substances, It is seen)
that there is a ttmtted range over which the operation of LC columns would
be practical. A nine meter column, packed with 20 rmcron particles) 'would
separate a critical pair with a separation ratio of only 1.012, but it would
take 41 hours to complete an analysis. It is possible that there might be a
sample of sufficient importance to spend this amount of time on the
analysis, but It 1S more likely that the analyst would seek an atternative
method At the other extreme particles havlng diameters less than 3 micron,
although available, are curtcutt to pack, Assuming that the practical peak
capacity is about half the theoretical peak capacity) a column 3.1 ern long
packed with particles 2,9 micron in diameter would separate a mixture
containing about 16 components In just over 30 seconds. This particular
column is commercially avatlaole in ctose-to-oottrntzeo form (known
colloquially as the 'three by trvee': and would probably be the most suitable
for n)e rapid analysis of simple mixtures, The two other common,
cornmerctauy available) particle sizes) 5 and 10 micron, should be packed
into columns 12.6 and 148 centimeters 10ng respectively for optimum
oertormance Such columns would provide a 'column family' for general use
which would encompass the vast majority of LC appl tcattons that face the
analyst tooav It shoulo be emphasized, however, that for a repetitive
analysis that is repeated many times every day) as in a quality control
laboratory, HIe construction of the fully oourmzeo column that is spectr tc
for HIe particular analysis would always be econormcauv wortnwrule.
Gradient Elution
The design procedure described above will, in theory, be applicable only to
samples that are separated by .socrauc development. Under gradient elution
conditions HIe (k') values of each solute are continually cr,cmging, together
with the viscosity of the mobile phase and the ourusivitv of each solute in
the mobile phase, As a consequence the equations derived from the p1ate and
rate theorles will only be approximate at best and in most cases give very
misleading values particularly for column efficiency. As the effiCiency
required to separate the critical pair is crucial to column design, the
optimum column for use 'in gradient eluttor deveiopment 1S almost
impossible to calculate,
However, qradient elution is often employed, as an alternatjve to isocrauc
development, to svota Hie design and construction of the optimum column
which is seen as a procedure Whl en can be ted lous and time consuming.
Samples that contain solutes that cover a wide ootar tty range, when
separated with a solvent mixture that elutes the last component in a
213
reasonable time, often fails to provide adequate resotution for those solutes
eluted early in the chrornatogram. For the occasional sample, gradient
elution provides HIe best and immediate solution to this problem. However,
for oualttv controt, where there is a h1gh dally throughput of samples,
tsocratic development, employed in conjunction with an oourntzec column.
1S likely to be more economic In many applications, the optrmizcd column,
operated tsocrattcauv, will provide a shorter analysis time than that
obtained by qradrent elution used With an ad ooc column Furthermore,
tsocratic developrnent eliminates the time required after gradient elution to
bring the column back to equillbrium with the inlt1al solvent mixture before
commencing the next analysis. Excluding samples of biological orq!n,
isocratic development is the preferred method for routine LC analysis as,
winl an opurntzeo column. it is usually faster, utuizes less solvent and
reouires 1ess expensive appar atus.
Samples of biological origin fall in a class of their own. Many blologica1
samples can be separateo by gradlent elution particularly the
rnacrornotecules, polvpeptides, proteins etc However, due to the nature of
the samples. the gradient is often very small indeed as a suqnt charqe in
mobile phase composition can make a dramatic change in elution rate of
many rnacrorno1ecui es. Consequently, the solvent viscosity and solute
diffusivity does not ctianqe significantly during the program and, for' the
purposes of column design, the crromatocrarn can be treated a though the
separatton was deve1ope(j isocratical1y and separation ratios capac-tv
ratios and efficiencles calculated in the normal way
References
(1) J. H. Purnell, Nature.No.4704,Dec, 9,( 1959)2009,
(2)J.JVan DeernterJ.J.Zuider'weg and AKlinkengerg, CIJemEng,5ci,S( 1956)24
(3)J C. G1ddJngs, olf.77romatograjJlJ,J1, Marcel Dekker,
New York.t 1965) 56,
(4) J A. Riddick and W. 8. Bunger, O(qan/c .Jorm VIney and Scns,
New York,Sydney and Toronto (
Chapter 13
LC COLUMN DESIGN-THE DESIGN PROCESS
Open Tubular Columns
In a sirrnlar manner to the design process for lx/eked COIUf17n.5., the p
rlYSlcal
cnaractensucs and the oertorrnence soecincattons can bE' calculated
tneoret i cally for the ope() tubular COIUfl7/)S Again, tne proceour e lnvolves
the use of a number of ecuattons that have been previoustv oer iveo and/or
dtscussed (1). However! it wi 11 be seen that as a resul t of the qeometr i c
stmphcttv of the open tubular column, there are no packing factors and no
rnultipatf term and so HIe equations that result are rar less complex and
easier to manipulate amJ to understand,
The baSIC starting equation 1S again that of Purnell (2) wrncn auows the
number of theoreti cal plates requi red to separate the cr tt i ca1 pair of
solutes to be caicutated
...... ."" ' ,... (1)
where (0) is the separation rat i 0 of the crit tea1pair,
and (k'p) is the capacity ratio of the first eluted peak of the critical pair.
The next equation of importance is the relationship between the column
lengt.h (J), and tlle height of the theoretical plate (H),
1 =nH , , , " (2)
where (n) 15 the column etr ictency as defined in eouauon (1)
216
J '-J 11 I t:\ ,r\ i r l-J"I -; (' ; \ 1.'", ,'" r-.,
" ,", .• 'r- .', • I I I I -, \ j I L' ''J
_ ••'" J ,I, ..... .j I 1
B
H=- + Cu
u
where: (u) is trw ltnear vetoc.tv of the mooue phase
and (8) and (C) are constants
(3)
Now, B = 2D
m
( 4)
where (Drn), is Hie DlffuSivlty of the solute m the mobile phase
and
c=
(1+6k+11k'2)r
2
24(1+k,)2
0m
1<,3 r
2
+
6(1 + k,)2 k
2
o,
(f') 15 U"IE' dlstribut ton coerrcient of solute between tlle two
or -xc ''1C'
t
CD:) ts the dtr f us ivlty of the so1ute In the st at 1onarv phase
anrj (() is HIe r ac.us of tne tube,
Restating the second exoression in equatror (5), which ts for the res.stance
to transfer in Hie stationary phase,
Now, from the Plat E' Tneorv,
k' - I<V
s
_ k2nrdf1 _ 2Kdf
" - V
rn
- n r21 r
wriere is the volume of stat ronary phase in the open tuoe cO"lumrl,
( \!rn) j S trl e VOl urne 0f rn0bi 1e phase In the 0pen t U0e col urnn,
(, df) 1S tne T11m HI I CkN:<3S of the stat i onar y phase,
and ("I) 1:, HIe Ier.qtr. (if trle open tube co1urnn
217
Substituting for k'2 in the expression for the resistance to mass tr ar.srer 1n
the stat ionary phase,
=
2k'dr
2
3(1 +k,)2 o,
Replacing the modified express.on for the resistance to mass transfer In the
stat i onary phase in equat: on (5),
(1 +6k' + l1k'2)r
2
C= ------
24(1+k,)2
0m
Now, df« r and In LC, Os 1S commensurate wlth Om, Consequently, the riqht
hand expression in equation (5) can be consrdereo negl1gible
Thus)
C=
(1 + 6k' + 11k' 2)r
2
24 (1 + k,)2
0m
. (6)
If equation (}) is d1fferent1ated and equated to zero, the expression for the
optimum velocity (uopt) can be shown to be}
Uopt. =n .
(7 )
Furthermore. if HIe value for (uopt) from equation (7) 15 substituted for (u)
; - ,... -"'"'t 1 J.-.'" "~ , : t " ' " . {7 \ __",",.-J ,-,.;. ................ 1 ; ... ; ".".-J .-......... ~ ...... I • __ .. ~ ,.., '" ... _..... II J , ~ r . . "- h ... ......" "'I """' '" ......I
II I C 1..1 U Q L I VI j I. .•J J (JI I U ::J II I , fJ I I I leu, Q I I C 1..1 u Q L I U I I I V I 'I 1mm) I J U U L Gl I I It;" u,
H
rnm
::;: 2JBC . . . . " (8)
The Optimum Column Radius
Tne column length, (U, is also defined by the Poiseujlle equation that
descrioes the flow or a fluid through an open tube in terms of the tube
radius, HIe pressure applied across the tube (column), the vrscosttv of the
fluid ano HIe linear vetocrtv of the fluid, The Poiseuille equation is qiven as
follows,
Pnr
2
1=---
411U
op
t
"." , " "........ (9)
where (P) Is the inlet pressure to the COlumn;
and (11) is the Viscosity of the mobile phase)
( It snouldbe emonsstrea tnat the dirnensions of the applied pressure J ( P ~
most be apjJroprt'ate tol' tile dimensions in wrucr: the vtscosrtr. (I//' IS
meesireasnaalso the rsatus. (r) ~
It snouro also be pointed out that the pressure (P), 15 the pressure drop
across the column, and not merely the /ntet pressure. However, as inmost
LC equipment the only device ahead of the column is the detector, and the
pressure drop across the detector is usuaily sma11 compared with that
across the co lurnn, the term in1et pressure w111 be used synonymously wjth
pressure drop across the column in this chapter.
ThUS, substttuttnq for (1) in equation (9) from eouauon (2) and usmg the
optimum velocity and the minimum HETP,
( 10)
Substituting for (uopt) from equation (7) and for (Hmin) from equation (8),
S1mpltfy1ng,
Pnr
2
n2B = opt.
4Tl
Substituting for (B), from equation (4), and rearranging,
2 _ 16nD
m
Tl
r
opt
- Pn
suostttuttnq, for en) from equation e1) and SOlving for (r),
ropt = 16t +k'] (Dpm11 ]05,..................... (11)
k' a-I 1t
Equation (11) is the first important equation for open tubular column design,
It is seen that the optimum radius) with which the column will operate at
the optimum veloctty for the given inlet pressure) increases rapidly as an
inverse function of the separation ratio (a-I) and inversely as the square
root of the inlet pressure. Again it must be remembered that, when
calculating (ropt), the dimensions of the appl ied pressure, (P), must be
appropriate for the dimensions in which the viscosity, (Tl), is measured.
As in the chapter on packed column design, the cheractertsttcs of many of
the equations discussed in this chapter w111 be examined employing
realistic chromatographic conditions and the typical conditions chosen for
an open tubular column are given in table 1.
Table 1
Typical Chromatographic Operating Cond1tlons
Separation Ratio (a, range 1.01-112) (Critical Pair)
CapacHy Ratio ( Itrst eluted peak of the Critical Pair)
Viscosity of the mobile phase ell)
Diffusivlty of solute, (D
m
),
I n lot Droccllro (D r-::lnno 1 - 1(\(\(\1"\ C' i ,
1.11"'"' ... lt""...,...,YI ...... IVVV,.., .......... 1
Caoactty Ratio of the last eluted peak (k'2)
1.05
2.5
0.023 Poises
3.5X 10-
5
cm
2/sec
1 (\(\(\ r. C' i
lJ.tJ.l.
5.0
The conversion factor used to change pounds per square inch to dynes per
square centimeter was taken as 68948 (3).
220
The ootrmum column radius for the separ at ron of solute mixtures rangmg In
dlfi Ilult'y' from (Q = 1.0·i) to (r.t::o 1.1':2) ana for rruet pressure:! Of 1,1 U, 1oo.
and IGOr) psi. respect tvel'y, were caicul at.ed emp1oymQ equation (11) The
results obtameo are shown as curves relatlng tr.e optimurn column radius to
separat "N: r at.l a j n figur e I,
Flgure I
Graph of Optimum Column Radius against Separation Ratio
2
"...,
c
0
~
C,,)
-
E
......
en
:::J
-
~
c
~
c
E
0
:::J
-0
u
.
en
0
..J
-1
1.00
o 1 p.s.i.
• 10[.i.5.1,
• 100Ps.:
o 1000p 5 I
1.02 104 1.06 1.08 1.10 1.12 114
Separation Ratio of the Critical pair
It lS seen that tile opumum column radius for an open tubular column varies
widely W1trl inlet pressure and the difficulty of the separation. Considering
a separation of some difficulty> for example (a = l.02)} it is seen tr.at at an
.nlet pressure of 1000p.S.i., the optimum Column diarneter would be about 4
micron whereas, at. an rntet pressure of only 1 psi! it would be about 43
mi cron Trle f orrner would be quite difficult to coat with stat i onary or.ase
ar.o would demand detectors and rr-jectron systems of almost impossibly
lOW dlspersion A column of 43 micron rn diameter, on the other nano, would
be pI det. ir.al from the comt of vIew or both ease of coating and an
accept.abte system extr a column dispersion. However, H1e lenqths of such
colurrms and the resulting analysis urnes remains to be determined and may
orec1ude nlei r use.
221
The Optimum Length of an Open Tubular Column
The column lengt.h is given by ecuatton (2), viz,
lont = nHmln
Substituting for (n) and (Hmin) from equations (1) and (8) respectively,
(
4(1+k,)]2
lop! = k(a-I) 2jBC
1+ 6k' + 11k,2 Omll
3 Pn (12)
Equation (12) shows that the optimum column length is inversely proportion
to the third power of (ex-I), This is an analogous relationship to the length
of a packed column. In a similar manner the column length is inversely
proportional to the square root of the pressure drop across the column.
USing equation (12) the optimum column length was calculated that was
necessary to separate a series of mixtures having (ex) values ranging from
1.01 to 112 for mlet pressures of 1,10,100,1000 p.s.i. respectively. The
results are shown as graphs relating column length (1) to separation ratio
(ex) in figure 2.
It is seen from flgure 2 that, operating at an inlet preSsure of OI-lly 1 o.s.: ,
HIe optimum column length varies from about 15 meters for HIe separation
of Solute pairs rlaving a separation ratio of 1.01 to only 2cm for solutes
i f\ (If 1 1? At tllp IIthpr p'I(trpmp IIf nrpc;c;! rrp 1nnn
I,,,,,,, .. """ ........... .,..."-"1. Y .... l ....... ' 1 '0001II .... ,_ ..... 4 •• 1_' i '''' .......... _ ... ' ..... ' _"_' _ ••• __ • ('"'" ) _
p.s. t., the column length varies from about 65 cm for a separat ion ratio of
1.01 to only 0.3 of a millimeter for a separation ratio of 1.12 it 1S
ObVl0US that HIe optimum conditions for open tubular columns are not
pract i ca1 for the seoarat i on of simple mixtures at high pressures as both
the column radius would be too small for the extra column ctsperston of
the ecuipment and the column would be far too short to construct. At low
pressures, and for dlfficult separations, open tuoutar columns might be
practical from the point of view of column diameter and column length.
Figure 2
Graph of Log. Column Length against Separation Ratio
~
E
CJ.
"""
I:.
+J
-=
C
CD
..J
C
E
:=I
-o
u
-=
o
..J
4-r-----------------,
3
2
o
-1
-2 ~ . . . . . . . - _ r _ _ - o o o o r - - - - - - - r " - - - . - _ r - _ _ r - ~ - _ _ _ _ j
1.00 1.02 1.04 1.06 1.08 1.I 0 1.12 1.14
Separation Ratio
EJ 1p.s.i,
• 10 p.s.l.
• 100p.s.i.
o 1000 p.s.t.
Minimum Analysis Time
The analysis t1me is given by the followlng equation,
, I opt
t
m
1n = (1 + k2)-
Uopt
Substitutlng for (lopt) from equation (2), for (uopt) from equatton (7) and
for (Hmin) from equation (8),
. . 2Jf3C
t
m1n
= (1 + k2)n ~
Simplifying
J
tmin = ( 1+k'2) n2C
SUbstituting for (C) from equation (6))
2
, 1+6k'+11k,2 rapt
trnin =(l+k 2)n 2 0
12(I+k') m
and for (n) and (ropt) from eouat: ons ( 1) and ( I I ) , respect tve Iy,
Simpl irying)
Equation (13) gives the minimum analysis time that can be obta.ned from an
open tubular column) when separaunq a mixture of defined dlfflcultYI under'
given ctromatocraoruc condtttons It is seen that, in a strnuar: manner to trie
packed column, the analysis time IS inversely oroportronal to the fourth
power of HIe runction (0-1) and inversely proportional to HIe inlet pressure
The contribution of HIe function of (k'), to the analysis time is not clear and
can be best seen by calcutauon It 1S also seen (perhaps a utue
surpr:slngly) that the analyS1S time is comp1etely mcepenoent of the
diffuSivltyof the solute in the rnobne phase but is direcuv proportional to
the viscosity of the mobile phase.
USing equation (13) HIe minimum analysis time was calculated for the
separation of a series of mixtures havmg (ex) values ranging from 101 to
1.12 and for in1et pressures of 1J 10, 100
1
1000 Ps. i. respectively. The resul ts
are shown as graphs relating analysis time (D to separation ratio (0) lr1
figure 3.
Figure 3 shows that, there might mdeed, be a limited practical range of
coturnn ounensions and ooerat mq conoiuons that WOUld make tne open
tubular column a possible alternative to the packed column in LC. To
separate a solute mixture with the separation ratio for the critlcal pair of
1.01 and an inlet pressure of 1 p.s.i would require an analysis time of 6.5
days whicrl even for this dIfficult separation IS tediously long, However,
the analysis times wouic be commensurate to tbose obtained (rum an
optimized packed coiumn, separating a rmxtor e of the same difficulty and
would nave a similar lengtrL Furtherrnore, it would be far easier to rabrrcatc
and coat a 15 meter capularv column than It would to etr tcient lv prepare a
packed co turnn of cornoerable 1ength.
Figure 3
Graph of Log. Analysis Time against separt ton Ratio
6--,------------------..,
4
CD
E
-
....
ct) 2
-
ct)
=-
-
c
c:
< 0
.
'=
o
..J
-2
1.00 1.02 1.04 1.06 1.08 1,10 L12 1.14
secarat ion Rat to
DIp 5 1
• 10 p 5 i
• 100 psi
c 1000 o.s ,I.
At 10 p.S.1 the diameter of the column is down to 26 rmcror which 15 a
little small for easy practtcal use, but the ar.alvsrs time would be reduced
to about 15 hours. However, it is now necessary to calculate the mobile
phase flow rate and, in particular, the maximum extra column dispersion
that can be tolerated to determine the ultimate oracttcauty of open tubular
columns in LC.
The Optimum Flow-Rate
The optimum flow-rate (Qopt) will be qiven by:
225
suost Hut i ng few (8) and (C),
r ,'; .,0S
l
30
2
(1+
Qopt = 4nr
op
t m 2
1+ 6k' + 11 k' J
FinaIIy, sUos tit uti n9 for (r op[ ) I fromequat ion ( I 1)
(1 +k,)2 3nD
3
'1
o t =64--- m
op k'(a - 1) P( 1+ 6k' + I 1k' 2)
05
( 14)
It is seen that (Oopt), vartes mversely as (a-I) and the square root of the
inlet pressure but directly as tre diffuSiVlty of the solute to tne power of
3/2. Curves re13ting the optimum flow-rate to the separation ratio of triP
cr'itical pair- are shown in figure 4 for' inlet pressures of 1) 10, 1oo.eno i (JO(j
p.s.:
Figure 4
Graph of Flow-Rate aqainst Separat i 00 Rat 10
0....------------------.,
o 1p.s.i.
• 10 p.s:
• 100 o.s.i.
o 1000p,S.I.
1 14 1.02 1.04 1.06 1.08 1 10 1.12 1,00
-1
-2
i

·
=e
..J
"..,
·
c
-
E
......
·
ctl
L.
..
-
-
·
CD
..
f'J
a.::
I
f;
e
-
Separation Ratio
It 15 seen from figur"e 4 Hlat the coumurr flow rate is extremely small
irresoecttve of the applied oressore. The maXlrnurn floV'i-rate for a co.umn
optrrrnzeo to separate a criticat pair having a separ auon ratio of 101, witrl
an inlet pressure of 1 o.s.: , is only about 05 rrucrolttres per minute. For a
column opt.rntzeo to separate a relatIvely simole mixture (ex =1,07) with an
inlet pr'e::;sure of 1000p,s,i" the optimum flow-rate would be only 0 00224
mtcrohtr es per rmnute and for simpler' rmxtores even smaller The small
flow rates ere a otrect result of the very small coumon cotisrm rad/! tnat
an? nece-::)sary f or open tuou tar colurnns 1f used In LC. Peferrmq to equat ior:
(11) It is seen that tne opurrum rac.us 15 drrert lv oropor t ronat to tile
square root of the diffusivlty of the solute in the mobue phase It is because
HIE' (jlffu,:,ivity of a solute in a liquid is so srnall. In fact, four- or nve oroers
of macnttuoe less than 1n a gas, that causes in the opt1mum co lumn radi i,
and consequently the optimum flow-rates, to also be so small For" the same
reasons, HIe optimum column radn and the opt.mum flow rates employed
witr open t.u[Jular columns in GC are several orders of rnaqrutuoe greater
than LC .
Maximum Sample Volume and Maximum Extra Column Dispersion
In a paclred cotumn the HETP depends on the oartrc!e oiameter and 15 not
relateo to HIe column radius As a result, an expression for the optimum
part icle diameter is independently derived, and then the column radius-
determined from the extra column otsoer sion Tms is not true for trie open
tuoutar column) as HIe HElP IS determined by the column radius It rollows
that a converse orocecure must be employed. FIrst ly the opt: mum co lurnn
radius 15 deterrruned and then the maximum extra column disperston that HIe
column can tolerate calculated ThUS, Winl open tubular columns, the
cbromatoqraphtc system, in oart rcular the detector dispersion ar.d the
rnaxirnurn sample volume, is dictated by the column design wruch. in turn, is
qoverned by the nature of the separ'ation.
As already stated, tne maxunun extra column oispersion tr.at can be
tolerated, such that tne resotunon is not slgmflcantly reduced, wll1 be
equivalent to an increase of 10% of the column variance
and (oc
2
) is the variance of tne peaf< eluted from the colurnn
227
If the total extra column orsperston 15 shared equally (letwpen HIe
volume and HIe detector,
OD
2
= 0 OSoc
2
where (oS2) IS HIe extra column dtspersion resulting from the sample
volume
and (,00
2)
is the extra column vanance resu.t mq trom ctsoersron 11"1 the
detector
or
and
os = O.220C
0D = o220C
( i c:; i
, J,
( 16',
,I I
Oeve loptnq, the expression for the maximum nerrntssble sample volume as
in chapter (5)1
Consioer a volume (Vi) .njecteo onto a column, rorrmnq a rectangular'
di strtbutton at the front of the co turnn. rre var. anee of the f ma1 peak will
rJ(;> HIe SUrf! of HIe vanances of HIe sarnple volume, plus Hie nor-mal
of a peak for 3 srna11 sarnple Now the var 1ance of tr.e ret tan(Ju 1a(
distrtbutton of sample volume at the begmning of the column \5 V\2/ 1=: J and
the colurnn var iance trrom the Plate Theory) is where (V
r
) is the
In)
retention volume of the solute. Assurnmo the peak variance is Increased by
5% due to the sample volume, (Vi) wi]] then be the maximum sample volume,
and
')
v
2
(V \'-
;;:: O. OSl f
L... ,.In
,. ,
( 17)
SUbstltutmg the tuncnon (or the retennon volume for an open tUbe,
( 18)
Substitut.ing in eQuatlOfl (18) for' ( t oot ) and ( loot ) rrorn equat.l0r6 (11) ano
(12) respecuvety and slrnpl1fYlng,
Sutlstltutmg in equatlOn ( 17) for- ( l ) from equaucn (l9)j
, In
(
') ( ( '"' 3\ cS
v, == 12616 1+k2 1+ k ) 1+ 6k' + 11k' L l' D
m
11 J J
k'4(a_I)4 31l P
(20)
Furtrlerrnore, sUbstitutmg for' ( 'YL )from ecuanor (29) In equat.ion (16)
In
(
,.., 1 )
,"'" s: I
Samp Ie Va lume
The expression for the maximum permissible sample volume, given by
equat ion (20)} shows a strong dependence on the product of the solute
(liffusivity and the viscosity of Hie mobile phase It 15 also seen to vary as
t '? : nver-:: '? r) f he f (\ IJ r r\ pt, wpr of (ex - 1) sothat for ver y dIff 1cult
separattoos, wr\ere (ClJ is small, the sample volume will be a rnaxtrnorn
Tne curves in figure (5) show the retat tonstuo between rnaximurn sample
volume and Hie separation r at io of the crucai oair for a funy oourmzeo
column and were obtame(j USlr1Q equation (20) The curves give the nrst
m(l1 cat IOn of the Im?!t.)! tons 0/ open toaots: columns 117 1. C and the reason
wrlY tnev have not acrueved tJle popularity and success of the packed uJlumn
22G
Figure 5
Graph of Log. Sample Volume aqainst Separation Rat10
2.,...........----------------,
o 1 p.s l.
• 10 PSI
• 100 p.'S.I.
o 1000 J;.S 1
1 14 I 02 104 106 1.08 1 10 1 12
o
Separation Ratio
"-6 -
-2
-4 -
-8 -
-10 + - - - , . . . . . - - - - , . - . . . . . - , . . . - - - - . . . , . . . - - ~ - - _ _ _ , . - . . . . . . . . . . ,
1 (1)
.
=e
..J
CD
E
=-
-e
>
CD
-
Q,
E
c
tn
"...
CoO
CD
..
...,
-
-Cl
..
CJ
-
E
"""
Bearing in rnind that t.he the sample volume is measured in I77J(lc
l//tFi?5,
it
is seen tnat the largest sample volume calculated was for the separation of
the rnost difficult mixture where the separation ratio of the cr tticai pair 1 ~ )
101 at an mlet oressore of 1 o.s I Even then, the sample volume is only
about 1.0 rntcroutre. For a seoaratron rat to of 104 (a moder at.elv 'Jlfficuit
separation) HIe maxirnurn sample volume is only about 80 nanoutrcs and
SUCrl a small sample volume would be difficult to inject, even Wltrl a stream
splitting system. Furtrlennor'e, a charge so srnalt, would certainly reduce the
dynamic ranqe that could be covered by the detector, particularly as the
sensing volume of the detector would also have to be very small, A recuct ion
In HIe sensing volume wilt. as a consequence, reduce the saso/ut» detector
seostttv/r« It follows that by ernplovmq a charge of oniy a few nanoutres.
HIe total concentrat ion range that could be sensed by the detector would be
restricteo to about two orders of maomtude or even Jess. It should be noted
that HIe dynamic range of a detector lies between HIe maximum
... ~ . . . . . ... ........ ... _ ... ~ ... ". ,-. ,. - r- ,. rl '" L-.... _ _ ... Lo-.,., __ .-... . . . . . , . ~ e-, 1,.... " e-, 11 .....'¥'"'\. ...~ r-: " r-- r- ..; .-.. ~ " ..-.I .-.. r-. +'\ r r. ~ \ i r-.. f n:l j r {'\
l.VIH..-t'lll1 ell-IVII :JCII:Jt"U Wlltll Lilt :JQIIIf.)11:; VVIUIIII:; I.-VII:;JI..:Jt-\:,:\J c i i c u \.-Iy VI t-J\..ll '-
solute, and the detector noise level (5)
From the curves shown tn figure (5) it would appear that the very small
oerrmssiote sample volume would limn the use of tne open tubular column

to HIe separati on of f al rly dlf f 1cult rfl1 xtures (rn1xtures where a< 104 ) and
for mlet pressures jess than "iCJ p.s 1. wnere tne analysls t imes tan be
extremely long,
(0 D) the Maximum Permissible Detector Dispersion
The expression for' the maximum permissible detector ctsoer sion. glven by
equation (21)} also shows it's strong dependance on the product of the solute
ourusivttv and HIe viscosity of the mobile phase together with the mverse
of the tourtn power of (a-1) A graph relating (OD) to the separation rano of
HIe crrttcal pair is shewn in flgure (6)
Figure 6
Log, Maximum Detector Dispersion against Separat ion Rat to
o--r-------------------
DIp 5 I
• 10 P,S I
• 10(1 pSI
o 1000D5 i
1 14 1.02 1.04 1.06 108 1.10 1 12
-2
Separation Ratio
-4
-6
-8
-10
1.00
.
I:')
Cl
...J
".,....
.
c
e
-
...
CD
Q,
ct)
-Q
...
Cl
..
(,,)
CD
..
CD
Q
..
-
-
Cl
...
CJ
-
E
.......
The resul ts in figure (6) ernonasi zes the stringent demands that open
tubular' columns place on system design. Even when separat mq solute pairs
an (a) value of 1.01, ano operating at an inlet pressure of only 1
p.s.i., HIe rnaxtmurn value for (OD) will only be about 0.16 rmcroutres If it
is assomeo that in practice (OD) can not be reduced to mucn less tnan 5
nanolitres (and thls would be extremely difficult) the use of open tubular
231
columns would be restricted to intet pressures below 10 p.s.t. Fur tnerrnore.
they 'would tr.en be only suita[Jle for the separation of retauvetv difficult
mixtures where HIe seoaration of the critical pair is less than 104.
Design [Quations
basic design ecuat ions for the design of open tubutar columns In LC can
be summarized as follows
Column Radl us fopl - rd................. (II)
Column t.enqtr
1+ 6k' + l1k,2 Drn'l
3 Pn ." (12)
Analysis Tlrne
0.5
.. " ...... '"'''' ( 14)
Maximum Sample Volume
Detector DIspersion
Employing the design equations, a strnole program can be written that will
allow the optimum parameters to be calculated for any given set of
chromatographic conditions. Such a program is given in table (2) and is
written in nicrosort QUick Basic for the Macintosh Computer The program is
interactive and the user is asked sequentially, by a series of input
statements, the value of the pertinent variables required by the equauons
232
Table 2
OPTIMISATION PROGRAM FOR OPEN TUBULAR COLUMNS
PRI NT'OPEN TUBULAR COLUt1N DE51 (iN FOR Le"
PRINT
PRINT'lnter Separation RatlO of the Crittcal Pair" INPUT A
PRINT'Enter Capacity Ratlo of the First Peak of the Pair-" INPUT f< 1
PRINT''Enter Capacity RatlO of the Last Eluted Peak" INPUT K2
PRINT'Enter DiffuSivitv of 501ute In 1"1obile Phase":INPUT D1
,
PRINT"Enter Viscosity of (Poises)".INPUT M
PRINT" Enter Column Inlet Pressure (o.s i )"'INPUT P
PRINT"BASIC CHROMAT06RAPHIC DAT A'
PRINT"Sepa,"'atl0n Ratio of the Crrtical Pair"JA
PRINT'Capacity Ratio of the Flrst Peak of the Pair";K1
PRINT"Capacity Ratio of the Last Eluted Peak"K2
PRINT"DjffuS1Vlty of Solute in rtobue Phase"Dl
PRINT"Visco::;ity of rtooue Phase
PRI NT"Co1urnn InJet Pr'essure (p s.i.)"P
LET N=( 16*( 1+K 1r2)/«(A-1 Y2)*(K 1
LET R=( 16*( 1+K 1)*«(D1*M)/(3 I 42*P*68948&)rS)/(K 1*(A-l))
LET B=2*D I,C=« 1+6*K1+11 *1<. 1"2)*R
h2)/(24*D
1*( 1+K 1f2HJ=(B/CrS
LET L=N*2*(B*Cr,5T=( 1+K2)*LI U,Q=3.
LET V=({3 1416*0 7746*L 1 5
LET S=C22*L*3 142*( 1+i<2)*R
h2)/(Nr.5
PRINT "DESIGN SPECIFICATIONS"
PRINT "Optimum Column Radius" R"ern
PRINT "Optimurn Column t.enqtn" L "ern'
PRINT "Minirnum Analysis Time" T "sec"
PRINT "Column Efficiency" N
PRINT "Optimum Flow-Rate" 0
PR INT "rtaxirnum Sarnple vo1urne' V 'rn1"
PRINT "Maxlrnum Extra Column Dlspersion" 5 "rnl'
In a sumter manner' to the program for opttmizatror of a packed cOIUrnn, the
program written in table (2) is .n a very simple form that can be translated
easily to the bas«, language USed by other computer's. lIlt output will be
sent directly to HIe monitor' screen In Hie output window. However', by
replactnq HIe PRINT statements by LPRINT statements tr.e output will be
sent to a printer. If the cnpboard 1S defined a file; and thp. nr1nt
statements are rectacec by wrlte statements, then the results can be sent
to trIP. CItoboaro and pasted 1nto Woro processmg text. An examp1e of an
output trom the program is shown in Table (3)
233
Table 3
OPEN TUBULAR COLUMN DESI GN FOR LC
BA::iIC CHROMATOGRAPHIC DATA
Separation Rat10 of the Crrt real PaIr
Capacity Rat io of the Last Eluted Peak
DlffuSivity of Solute in
VISCOSlty of !'1obi le Pilase
Colurnn Inlet Pressure (p Si)
DESIGN SPECIFICATIONS
ooumom Column Radius
Opt irnum Colurnn Lenqth
Mir,lrnurn Analysis Tlme
Column Efficiency
optirnurn Flow-Rate
rtaximurn :)ample Volume
f
v
1aXl rnurn Extra Column Dispersion
1.01
5
o000035cm
2/sec
0023 POIses
1 p 51
4 318E -03) crn
2056 cm
577893, sec
313601
1.251 E-06 rni/sec
1,OOE-03, rnl
2.84E-04, ml
It is seen from Table (3) that an open tubular column with an 10 of about 86
micron and '20 meters long, operating at an inlet pressure of only 1 pS.l. can
complete the very dIfficult separanon in about a week. A very long analysis
time, perhaps, but not much longer than would be required by an oourmzeo
packed column and in thts case the separation 1S carred out Wln1 a sunpie
tube to coat and witn no hlgh pressure pump recurred
The Open Tubular Column in LC
The properties of open tubular columns shown in figures (1) to (6) indicate
that the areas where such columns would have oract leal use is very
restricted. At pressures in excess of io p 5.1.) and whatever the nature of
the separation, whether simple or difficult, the optimum column diameters
are so small that t.hey would be exceedingly difficult to fabricate or coat
with statronarv phase, The maximum samo!e volumes and extra column
oispersron that COUll; be tolerated would also be well below that phys1cal1y
possibIe at this tl me. At retat lyeIy i ow that is at pressures less
than 10 o.s. i. the diameter of the opt unum column is Iarqe enough to
rabrtcate ami coat wltl) stationarv phase providmg trie separations requireo
....if'f'i ...."H-. i,.., t-hCl C'Clr\-:lr':lt-i,"ln r'AtH' r'lf thp rritir::::ll n/l\r mlJst be less
UIIIII."..UIl-
J
1.\..· ....... 1' ..........'\.·t-'u1v ..... v •. 1\,,04 ..... _ ""l ... ',...., _. ,_.- ...... 1--
than 1.03. However) even under these condttions the sample volume wu
be extremely small) the ext.ra column cisoersron restr.cteo to an almost
impossibly low limit and the analysis t trne would be very long
Nevertheless) open tubular columns used for very difficult separations
mIght be preteraole for blochermcal sepa-auons, 1f made of suitably
inactive rnateriats. Capillary coumns could be easilY made tno-companote.
Furthermore, the fact that open tubular columns can be operated at very low
inlet oressures and thus, do not require a pump make them economically
attractive. Nevertheless, unless the separation is extremely difficult, and
there are special conditions required that arise from the nature of the
sample, open tubular' columns. operated by pressure mduced flow, should be
avoided If posstbte.
Examining equations (l1)j (20) and (21) that algebraically describe the
rnaqnttudes of the ootrrnum column radius, maximum sample volume and
maximum detector dispersion, it is seen, that it is the magnitude of
diffuSlvity of HIe solute tn the mobile phase, that 1S responsible for the
tmpracttcaltty of open tubular columns. The success of open tubular columns
in GC is due solely to the relatively high solute dif tusrvttv in a gases. In LC
the problem of low solute diffusivity is not so severe in packed columns due
to the aided dutuston between the part tete resutttnq from secondary flow,
In fact the resistance to mass transfer in the mobile phase in a packed
column 15 largely located within the porous particles not between them,
Where the flow of mobile phase is driven by electro-osmosis there is no
parabolic velocity profile to cause band dtspersion and thus.captuarv
etectroonloresrs can be carried out very successtultv.
To render the open tuoular column suitable for normal LC analysts it 1S
necessary to increase the dlffusivlty of the solute by supplying external
energy in a suitable form. The posstbthty of supersonic agitation of the
liquid core of the tubular co lumn might we11 be worth mvest igat ion, The
column could be situated as flat spiral between two plates one fixed, and
the other attached to a otezoetectric driver, In this way It migrlt be possible
to qenerate sonic waves across HIe diameter of the tube and thus,
artifiCially increase the dtrrustvuy in a rather a dramatic manner. If such a
system could me made to transfer sonic energy to the 1i quid core of the
column, it could make open tubular columns a very attractive alternative to
packed columns. Another approach would be to use low dispersion tUbing for
LC columns such as serpentine tuotnq (6). Such tuotnq, as has already been
discussed, lntroduces strong secondary flow and thus acrueves rapid radial
mixing. To date, the autnor is unaware of serpentine tubinq being used for LC
columns but they have been used as low dispersion connect ing tubes and for
heat tran,:.f er tubes very successful !y.
References
(1) R W. P. Scott, .../ t..17rofnatogr:, 517( 1990)297,
(2) J. H. Purneu. Nature (London), 184 (Supp1. 26)( I 959)2009,
(3) M....J, E. Golay) in ''6as Chromatography 1958", (Ed. D.ADesty))
Butterworths, t ondon.t 1958)36)
(4)" Handbook of Chemistry andPhyslcs"Jhe Chemical Rubber Cornoarw.on:o,
( I 97 I )F- 241)
(5) R. P, W, Scott, "LiquId oetector«, Elsevier, Amsterdam,
( 1986)7
(6) R. P, W, Scott and E, Katz US Patent (t 991)
Chapter 14
Preparative liquid Chromatography Columns
The use of the adjecttve, 'preparative', to describe a part icular form of
chromatography does not convey a specific rneantnq to most modern
chromatographers, nor does it suggest a particular sea te of separation. The
term 'preparative' chromatography has been applied to sample loads rangjng
from a few milligrams, to grams or even hundreds of grams of material and
to columns having diameters ranging from a few mill imeters to hundreds of
centimeters. preparative chromatography can be employed to isolate a few
rn ill iqrams of a substance for structuraleiuctcatton by appropriate
spectroscopic techniques or, perhaps, to obtain 50 gram of a pure
intermediate for subsequent synthesis, At the extreme, preparative
chromatography can be carried out in columns over a meter in diameter to
purify a biosynthetic product on the kilo scale.
In previous chapters, liquid chromatography column theory has been
developed to explain solute retention, band dispersion} column properties
and optimum column design for columns that are to be used for purely
analytical purposes. The theories considered so far) have assumed that
solute concentrations approach (for all practical purposes) infinite dilution,
and, as a consequence, all isotherms are linear. In the specific design of the
optimum preparative column for a particular preparative separation,
Initially, the same assumptions wi 11 be made,
The theory of preparat ive columns must show how the optimum preparative
column should be constructed, and how 1t should be operated to provide the
throughput required, However, preparative columns can be expensive to
construct and often the operator has an already existing column with which
to work. The theory must, therefore, be extendable to columns that are not
specifically designed for a given preparative separation but for general
preparative use, As a consequence, the theory must also help to determine
the maximum possible volume and mass of charge that can be placed on an
exisunq corumn ana suu permit the rsolauon of apure sample of tile SOlute
of interest. If the preparattve column is not spectrtcauy designeej for the
separation then, when working efficiently from the point of view of
maximum column loading, it may operate under conditions where the
Isotherm Is not ltnear and the column is overloadea. In ract, the theory must
indicate the 1imits to whiCh a column can be 'abused' to achieve the
maximum sample load and satisfactory resolution for a particular
separat ion.
In the first instance, the design of the optimum column for a given
preparat tve separat ion will be consi dered .
PreparatIve Column Design
Many of the equations that have been previously employed in the design of
analytical columns (1,2) are pertinent to preparative columns with certain
provisos. Normally, there is only one component in the mixture that needs
to be isolated. ThUS, In preparative columns, the critical pair is no longer
the pair of solutes most difficult to separate, but consists of the solute of
interest and its closest neighbor; the resolutton of other components of the
mixture becomes irrelevant to the problem, In almost all cases, this means
that the separation will be fairly Simple as the critical pair will probably
not be the most difficult in the mixture to resolve. ThUS, the phase system
can be chosen With greater nexintutv, the operator focussing on the solute
of interest and its nelgIJlJor and ignoring the proximity of all other so lutes
in the mixture. Furthermore, the column radius Will no longer be oetermtneo
by the extra column dispersion of the chromatographic system but will be
the direct and major factor controlling the maximum sample load.
The EfficienC'i Reguired from the Preparative CQlumn
The first equation to be employed will again be that of Purnell (1), which is
used to calculate the efficiency recut red to separate the crttical pair. The
data used is the separation ratio of the crtttcal pair and the capacity ratio
of the first eluted peak of the critical pair, The critical pair now being the
peak for the solute of interest and as closest neighbor. The Purnell equation
Is re1terated as follows,
"" " " " "." (t)
where (a) is the separation ratio of the critical pair,
and (k') is the capacity ratio of the first eluted peak of the crtttcal oatr.
In the operation of preparative columns, it is necessary to obtain the
maximum mass throughput per unit time and, at the same time, ach1eve the
required resolution, Consequent lv, the column wtu be operated at the
optimum velocity as in the case of analyt1cal columns, Furthermore, the
D'Arcy equation will still hold and the equation for the optimum particle
diameter can be established in exactly the same way as the optimum
'partlcle diameter of the analytical column, The equation is fundamentally
the same as that given for the optimum particle diameter for a packed
analytical column) t.e. (18) in chapter 12, except that (a) and (k') have
different meanIngs.
8(1 +k') 'lD
rn
dr(opt) = k'(a -1 ) fPP
05
,,,.,,,,,,,, (2)
where (n) Is the vtscostty of the mob1le phase)
(Om) is the ctrrusivttv of the solute in the mobile phase,
(fP) ts D' Arcy's constant,
(P) Is the column tnlet pressure
and (Aandy) are packing constants as previously defined.
As in the previous chapters on column design, the characteristics of many of
the equations discussed in this chapter Will be examined employing
realistic chromatographic conditions and the typical conditions chosen for a
preparative column are given in table 1.
The conversion factor used to change pounds per square inch to dynes per
square centImeter was aga1n taken as 68948 (3), 1t is seen that there are
some changes from the data used for analytical packed columns. Due to the
difficulty in packing preparative columns the packing factors will be larger
and values of 0,65 and 0.80 are taken for GU and (y) respecttvelv as typ1cal
for preparative columns. The separation ratio values are also larger as in
preparative Chromatography there is more freedom to adjust the phase
system to achieve a larger (a) value for the critical pair. Furthermore,
240
columns of large d1ameter must have very thick walls to withstand high
pressures and often the limited mechanical strenqtn of tile column wali will
restrict the maximum inlet pressure that can be app: teo to the column. For
this reason the applied pressure is constdereo to be about 500 o.s.t .
Table 1
Typical Chromatographic Operatlng Conditions for a Preparative
Column
Separation Ratio (a, range 1.10-1.50) (Crtttcal Pair) 1.20
Capacttv Ratio ( t trst eluted peak of the Critical Pair) 2.5
CapacHy Rat10 of the last eluted peak (k'2) 5.0
Viscosity of the mobile phase (h) 0.023 Poises
DiffusivHy of Solute (Om) 3.5X 10-5 cm2/sec
Inlet Pressure (P), (range 1-6000p.s.1.) 500 p.S.1.
Multipattl Packlng Factor ('-J 0.65
LongHidlnal Diffusion Packing Factor (y) 0.8
Fraction of Column Cross-Section Containing Mobile Phase (£) 0.65
Concentration of Solute in Sample Solution, (l) 5.0 %w/v
SampIeLoad 25g
Employing Equation (2) and the data given 1n table 1, the optimum particle
diameter was calculated for- the preparative separat i on of a solute where
HIe separation ratio of the critical pair ranged from 1.0 I to 1.50 at inlet
pressures of 1,10,100, I ,000 and 10,000 p.s t. The results obtained are
shown in figure I
It is seen from figure 1 that the optimum part1cle diameter covers a large
range including many, that in practice, would be extremely difficult to pack
efficiently. In general, it is difficult to pack preparative columns with
particles having diameters less than 5 micron and closely graded particles
greater than 100 micron are not, at present, readily avai table. As a
consequence, there is a relatively narrow window of particle size, between
5 and 100 micron, that can be used in preparative LC. It is also seen that if
the separat ion of the critical pair is greater than about L 15 (which may
often occur jn practice) ve(y lOW rnlet pressures may nave to be used tn
conjunction with particles rlaving relatively large diameters.
Such ltrnltattons that are imposed on the preparative column design is
inevitable. There will be practical limits to the column lengH!, thE' analysis
time and the column diameter. There will also be more subtle limitations,
a 1 p.s.t.
• 10p.si.
• lOOp,s,;,
o 1000P.s.i.
• 10,000 p.s.i.
MINIMUM
MAXIMUM
1,6 1.5 1.4 1.3 1.2 1.1
2 ~ - - - - . . ~ - - - I : ; I - - __---------l
o
""'"
c
e
~ 3
-
E
""""
L.
CD
...
CD
E
c
-
Q
CD
...
~
-...
L.
C
a..
Figure 1
Graph of log. Opt Imum Part tcle Diameter against Separat10n Ratto
4-r------------------,
Separation Ratio
such as the column aspect ratio i.e. the ratio of the column length to the
column radius, It is obvious that a very large diameter column, a few
centimeter's in length, would also be difficult to pack.
The Column Length and Analysis Ilm.e.
By using exact ly the same procedures as those used in the design of packed
columns the same equations can be derived for the COlumn lengtr. and the
anaiysis ttrne Namely,
05 05 0.5
loot - 2 , 2A+ y - 2A + Y (3)
k(Q-l) 3(1+k,)2 rpP (1+6k'+11k,2)
2
(
2))0.5
_ ( . )(8(1 +k·))4 11 y 1+ 6k' + 11k'
t min - I +k
2
k'( 0 -P 2A+ 2
a - lp 3( 1+k' )
..................... , , (4)
Where (k'2) is the last eluted peak of the mixture and the other various
symbols have the meanings previously ascribed to them.
Employing Equation (3) and the data given in table II the optimum column
lenqth was calculated for the preparative separatton of a solute where the
separation ratio of the critical pair ranged from 1.0 1to 1.50
j
again at inlet
pressures of 1) 10
1
I 00) 1,000 and 10jOOO p. s.t. The resul ts obtained are
shown in figure 2
Figure 2
Graph of Optimum Column length aga1nst Separation RatIo
a 1p.S,1.
• 10 p.5.1.
• 100 p.si.
Q 1000 p.s.l.
• 10,000 o.s.t:
MINIMUM
MAXIMUM
1.6 1.5 1.4 1.3 1.2 1.1
6 ~ - - - - - - - - - - - - - - - "
-
..,
a.
C! -2 +---.---,..._-.,..-_--r-_-_-__- . . . . ~
c:a
C) 1.0
-'
......
E
u
..."
.I:
.., 4
c:a
C
CD
-'
c
E 2
~
-
C)
c.J
E 0
~
E
Separat10n Rat10
It is seen from figure 2 that the optimum column length ranges from over
500 meters to a fraction of a millimeter, It is also obvious t h ~ t there must
be further lim1tations placed on the design of the column to ensure its
pract1cal use. Apreparative column less than 5 cm in length would be very
difficult to pack as would a preparative column that had a length greater
than 150 cm particularly H it had a diameter in excess of 20 em. It should
be pointed out that the limits that have been placed on the physlcal
properties of the preparative column are somewhat arbttrarv and can be
modified to sutt the user.
In figure 2 the 1trnlts of length are included in the diagram and it is seen
that, for the particular sample consioereo, an optimum column can not be
used for the separation of mixtures where the separation ratio of the
crtticai patr exceeds 1.3. Furthermore, at a separation ratio of 1.3,
extremely low inlet pressures must be employed to ensure the use of a
column 5cm or more in length. ThUS, ror simple mtxtores, columns with
excess efficiencies may have to be used which) as wi 11 be seen later, can be
overloaded and the excess resolution traded in for increased sample load,
1n a sirn i lar manner, by employing equat ion (4), the separat ion time can be
calculated for a series of mixtures where the separation rat to of the
critical pair ranged from 1.0t to 1.50, and, again, at inlet pressures of
1, 10,1 00,1,000 and 10,000 p.s.t. The results obtained are shown in figure 3
Figure 3
Graph or log. Analysis Time against Separat ion Rat 10
B-r-----------------,
,.....
u 6
I)
• .....
I) 4
E
-...
• 2 ......
-

=
'i 0
I:
C
. -2
Q
CI
...I
n tp.s.t
• 10 p.s.i.
• 100 o.s.l
e 1000 p.s.i.
• 10,000 p.s.i.
MINIMUM
MAXIMUM
1.6 1.5 1.4 1.3 1.2 1.1
-4+-........ ........-----.-...,.-........
1.0
Separat i on Rat i 0
It is seen from figure 3 that under optimum conctttons.tne range of analysis
times is exceedingly Wide, extending from many days to a few mi 11 tseconds.
244
In a strntlar way to particle diameter and column length, there are also some
practical limits that must be unposed on the analysis time. It must be again
emphasized that these limits are arbitrary in nature, and may be changed by
the user if so desired, A minimum analysis time of one minute 1s
recommended to allow time for sample rnampulatton and fraction collection
and at the other extreme a maximum anatvsts time of one hour is considered
acceptable. It is seen that a practical window exists between a separation
ratio of 1,01 and 1,3. Furthermore, at a separation ratio of 1.3, extremely
low lnlet pressures must be employed to ensure the analysts time is not
less than 60 seconds. ThUS, the urnttatton of analysts time to a minimum of
60 seconds, means that for very simple mixtures, columns with excess
efficiencies must be used. As has already been suggested, such columns can
be overloaded and the excess analysis time also traded in for increased
samp1e load.
The Column RadiUS
The radiUS of an column is determined, among other factors, by
the extra column dispersion of the chromatographic system, For preparative
columns, however, the radius is determined by the sample load that is
required to be placed on the column to obtain the necessary throughput.
Now the maximum charge that can be placed on the column (Vi) has been
shown to be, (Chapter 5 page 54),
Now from the Plate Theory,
Vr = V
a
( 1+k') ,,., ,, , , ,, ,, ,,". (6)
Where (V
o)
is the dead volume of the column.
Furthermore, Va = cn r
2
lopt " " " " (7)
where (c) is the fractional cross-section of the column occupied by the
mobile phase and usually equivalent to 0,65,
(r) is the column radtus,
and (lopt) is the optimum column length.
SUbstituting for (n) from equation (1), for (V
r
) from equation (6) and for (V
o)
in equat ion (5),
1.1 (1 +k') cnr 2
lopt
V1 :; 4( I +k' ) ,.' ,.,., ,,, ,.. .. (8)
k'(a -1 )
Substilut1ng for (loptl from equation (3) and stmpurytnq,
[
(
2))05
40+k') 2 1+6k'+ I Ik'
Vi :;;2.2(I+k')cnr
2(k'(
») 2A+ y 2
o-l 3( 1+k')
[
2 ]0,5
nDm 3y(1 +k')
- 2A +y
fPP (1+6k'+11k'2)
tt"I •• t ••• t .. t " ' J J f ~ " " " ' 1 (9)
0.5
It is seen from equation (9) that the maximum sample volume depends on the
square of the radius and 1nversely on the square root of the column inlet
pressure. NowI although (r) and (P) are not mathemat 1cally interdependent,
there is a practical dependance of (r) on (P), The column must) physicauv, be
able to withstand the the pressure (P) and thus, the column walls must be
sufficiently thick to accommodate the pressure for any given radius (r). The
aspect of column strength, and weight will be discussed further in due
course, Now, if the mass of the selected solute that is required per
separatton is (M) and is placed on the column in the maximum perrrnssibte
sample volume (Vi),
M= ooVi/ 100 ------------------ (10)
where (00) is concentration of solute tn the sample solvent %w/v
In oractice the value of «(a) wtll vary between about 2 and 5 ( t.e sample
concentrations will lie between 2%w/v and 5%w/v) before mass overload
becomes a significant factor 1n band dtsperslon .A rurnertcat value for (00) of
5 will be taken in subsequent calculations. The correct value of (00), for the
part1cular solute concerned) can be experimentally determ1ned on an
analytical column carrying the same phase system 1f so required.
Rearranging equatton ( 10),
Vi =100M/w -------------------- (1 I)
Substltuting for (V
1
) from equation (11) in equation (9) and solvinq for (r)}
05
k'{a-l) 45.5M
r = - ~ ----------------------
4(1+k') 0.5 05 0.5
(1+6k'+ 1lk'2)] n
D
3y(1 +k,)2 ]
Ca)(1+k') E1t 2A+ Y ----.!!l. 2A +Y
3(I +k,)2 tpP (1+61<' +11k,2 )
L J
...... "" " , ".. (12)
EmploYIng equation (12) the oottrnurn column radius can be calculated that
would be required to separate 25 9 of solute for a range of separation ratios
for the critical pair of 1.01 to 1.50. Curves were obtained relating opttmurn
column radius to separation ratio for trilet pressures of 1,1 O} 100, 1000, and
10,000 p.s. i. respect ively. The resuIling curves are shown in figure 4
D 1p.s.t .
• 10p.s.t.
• 100p.s.l.
e 1000o.s.t
• 1000p.si,
D 10000p.s.i.
MAXIMUM
45
""'"
40
E
u
35
....,
..
30
:::I
-~
25
CI
~
20
C
E
15
:::I
-
0 10
U
5
0
.,.,
j, i
1.2 1,3 I,V
Separat I on Rat I 0
Figure 4
Graph of Column Radlus/Separallon Rallo for a Load of 25 gram
50 ~ - - - - = - - - - - - r - - - - - - - - - .
It is seen from figure 4 .that the optimum column radius ranges from a few
m111imeters to over 50 centimeters, It is also seen that, for a sample load
of 25g, where the maximum the diameter of the column is restricted to
about 30cm (c.a.: root), the optimum radius can only be employed where the
separation ratio of the critical pair 1s 1.3 or less. Above 1.3, a longer
column Ulan opttrnum would have to be used in conjunction with the
maximum column raotus, and the excess resolution could then be discarded
for an even larger sampIe load, There will of course aIso be an increase in
separat ion time. It should also be pointed out that there will be another
oracttcal restriction to the column length and radios. The column radius
should not be greater than the column length if the column is to be
eHiciently packed with the standard packing procedures avallanle tocay.
Employing normal packing procedures, short wide columns tend to leave a
gap across between the top of the column packing and the flange cap. This
results in poor sample distribution across the column, channelling in the top
of the packing and a consequent loss of column efficiency and resolution
The Optimum [Jow-Rate
Having determined the optimum column radtus, the optimum velocity being
known then the optimum flow-rate volume is given by,
Qopt = En r20pt Uopt " "" .. ,., ", ,., .. , ,.. ,.. ,., .. , ,. ,., (13)
Substituting for (uopt) from equation (9) in Chapter 12 ,
2 4D
m
3y( 1+ k,)2
Qopt ::; EJtr
opt
-d
p
( )
1+ 6k'+ l1k,2
05
............. (14)
Substituting for (dp(opt) from equation (18) in Chapter 12 page 183 and for
(ropt) from eouati on ( 12),
Q _ [k'(a_l)]3 91MlpP
opt - 4(1+k') 05 2
. J (y(1+6k' +11k'2)) . l
"'CAl (1 + k' Jl2 A. +l
3(1 +k,)2
... (15)
It is seen that equation (15) is very stmtlar to that for the optimum flow-
rate for an analytical column except that (OE) is replaced by the expression
(110M/CAl) as the extra column dispersion no longer controls the column
radius,
Solvent Coosumpt1Qn
The solvent consumption (Vsol)is equal to the product of the analysis time
and the optimum flow rate,
vsol = Qopl Tmin .. , ,.. , '" " " ( I 6)
Substituting for (Tmin) from equation (4) and for COopt) from equation (15) ,
k'(a-1) 3 (' )(80+k·»)4 11 05 2
4(I+k') 91MqtP 1+k2 k'(a-1) qtP V(1+6k'+ l1k'2)]
VSol = .100--_-...1.... 2A+ -----
05 2 3(1+k,)2 ...... ( I 7)
y(1+6k'+1Ik'2)]
tlCAl(I+k') 2A+ 2
3(I +k')
Simplifying,
5824M(I+ k ~ )
VSol = wk'(a -1) , , , , (18)
It is seen from equation (18) that the solvent consumption is directly
proportional to the charge placed on the column and the capacity ratios of
the first peak of the critical pair and the last eluted peak respectively. It is
also seen that, as with the optimized analytical column, the diffuSivity of
the solute and the viscosity of the mobile phase play no part in controlling
the solvent economy. It should be oointec out, however, that this 1S only true
for a completely optimized column
Column Wall Thickness
It has become apparent that preparative columns will have signi ncant Iy
greater diameters than analytical columns in order to accommodate the
greater sample loads, As a consequence) the wall thi ckness of the columns
249
wlll also be large in order to wttnstano the high pressures employed Tne
relat ionsrlip between wall thtckness (ttl), Column radius and tntet pressure
for safe operatton, is given by the following equatron, whicrl is an arbitrary
relat tonsntp derived trorn experimental data (4»
th = 2 ( ~ D + o py) + C (131
Where (P) is t.he inlet pressure in lb/sq. In,
(Do) is the exterior diameter of the tube in inches,
(C) is the sum of the allowances for corrosion and thread cutting
and it is assumed that) for preparative columns, there 15 no
corrosion and the connections are made to the pipe by flanges)
as a consequence (C) is taken to be zero,
(S) is the allowable stress and for Stainless Steel, TP 316 is taken
to be 18750 ibs/su in. over the temperature range of 0-1 000C)3)
(E) is the longitudinal weld ootnt factor and 1S taken for double
welded butt joints as 0.85(3)
(V) 15 t.he "metal coefficient" (4) which can be taken as 0 4
It should be noted that equation (13) is for a specific stainless steel and a
particUlar type of tube. Before employing the equation to fabricate a column the
original reference should be reviewed to ascertain the correct constants or
function to employ for the type of steel and tube that 1s Intended for use.
SUbstituting the pertinent values for) (Do), (S)} (E) and (V) in equation (13)
and simplifying) • •
PR
th = 31875-0.2P (14)
One further property of the preparative column is often requirec and that is
its unpacked weiqht (W(Col). This is simply given by,
where (d) is HIe denstty of the column material and the other symbols have
the meaning previousty ascribed to them.
Taking the densttv of stainless steel to be 7.8g/ml and simplifying,
WCol = 7,8 (2ropt th + th
2
) lopt , 15)
2 ~ O
The equation for the optimization of a preparative LC column can be
summarized as follows.
PREPARATIVE COLUMN DESIGN EQUATIONS
Optimum Particle Diameter
2 ]0,5
8(1+k') 'lD
rn
3y(1+k')
dot) = - 2A +y
P( P k'{a -1) Ipp (1+ 6k' +l1k,2)
Optimum Column length
0.5
(
4(1 +k,))3 (1 +6k I +1 I k,2 )]0.5
lopt =2 , 2A+ Y ' - - ~ ~
k (a -1) 3(1+ k' )2
The Minimum Analysis Time
nDm 3
Y
(1+k
l
)2 ]0.5
-2A---- +y
Ipp (1+6k'+ 11k'2)
0.5
(
.)(80 +k'))4 n y( 1+6k' +l1k'2 )]0,5 2
tmin = l+k I - 2A+ -----
2 k(Cl- 1) «pP 3(1+k' )2
The Optimum Column Radius
0.5
k'(a-l) 45.5M
r=--I--------------------
4 (l+k') ( 05'If r 05 11°·5
l
l
(l (1+6k'+11 k'2)'J 1
1
nD li( 3y (1+ k,)2 \J j J
Cal(l+k')cJt 2A+ V ---!!l 2 +V
3(1+ k,)2 ,P (1+6k'+11k'2) J
The Maximum Sample Volume
Vi = 100M/fa)
The Optimum Flow-Rate
3
k' (Q - 1) 91M~ P
Q -
opt - 4 (1 +k') 05 2
TJOO( I + k') 2l + 2
3(1+k')
The Solvent Consumption
5824M( 1+k ~ )
VSol:: ook'( Cl-1)
The Column Wall Tntckness
t = PDo + C
h 2(SE+PV)
The Weight or the Column
WCoI = 7.8 (2roptth + th
2
) lopt
THE USE OF THE DESI GN EOUATIONS
The design equations can be incorporated into a simple program to calculate
the dimensions and properties of a preparative LC column) together with the
oottrnum operating conditions that would allow the isolation of a specific
mass of a solute from a given mixture in the minimum time. In a similar
lTli:mne( to t I U : ~ previous pr oqr arns, this program lias a150 been Wit tten Iii
Microsoft Quick Basic that 1S appropriate for a Macintosh computer. The
program can be easily modified to be suitable for other types of computers
that} perhaps) use a different basic language. The program is not
sophisticated and not designed to be 'user friendly' and thus, It is
recommended that it be only used by someone with some knowledge of
computer programmlng, The program can be entered as typed be low, the
interrogative procedure wlll be carried using the computer screen and when
the run is complete the results wlll be printed as an output from the
associated printer.
COMPUTER PROGRAM FOR PREPARATI VE COLUMN DESIGN
2 LPRINT
3 LPRINT
4 LPRINT
5 LPRINT" PREPARATIVE COLUMN DESIGN FOR LC"
6 LPRINT
7 LPRINT
8 LPRINT"PERFORMANCE CRITERIA"
9 LPRINT
10 LPRINT"I! A oettneo resolution must be obtained"
11 LPRINT"2/ The analysis must be completed in the minimum time"
12LPRINT"3/ The analysis must be completed with the minimum solvent"
13 LPRINT
14PRINT"Enter Separation Ratio of Crltical Pair( 1.05 or greater)":INPUT A
15 PRINT"Enter Capacity Ratio of the First Peak of the Pair":INPUT Kl
16 PRI NT"Enter Capac: ty Rat to of the Last Eluted Peak": INPUT K2
17 PRINT''Enter Diffusivity of Solute in nobne Phase":1 NPUT D1
18PRINT"Enter Viscosity of Mobile Phase (Poises)":INPUT M
22 PRINT" Enter Minimum Column Inlet Pressure (p.s.i.),,:INPUT P1
23 PRINT" Enter Maximum Column Inlet Pressure (p.s,L)":INPUT P2
24 PRINT" Enter Column Pressure Increment (p.s.i.),,:INPUT P3
25 PRINT" Multipath Packing Factor":lNPUT L1
30 PRINT" Longitudinal Diffusion Packing Factor":INPUT G
31 PRINT" Column Mobile Phase Fraction":INPUT E
32 PRINT" Column Load (gram)": INPUT MI
33 PRINT" Sample Concentration %w/v":INPUT F
37 T9"" 1000000000#:09=019=0:R9=0
38 FOR P=P 1TO P2 STEP P3
39 PRINT A,P,T9
50 LET Xl =2*L1*«3*G*( 1+K1r2)!( 1+6*K1+I 1*K 1~ 2 ) r , 5 + G
60 LET D2=8*( 1+K 1)*«M*D1*x 1/(35*p)r.5)/(K1*(A-1»
70LETB=2*G*Dl
75 LET AI =2*L1*D2:C=« 1+6*K1+11*K1"2)*02"2)/(24*01 *( 1+K 1)"2)
80 LET H=A1+2*(B*Cr.5:U=(B/Cr.5:N=(4*(1+K 1)/(K 1*(A-I ))),,2:L=N*H
",P9"p s.t."
",M 1"g"
,. L1
,
",G
.. E
,
90 LET T=( 1+K2)*L/U R=(28.93*M 1*(N".5))/(L*E*F*( 1+K 1»r 5'V=100*1"11/F
100 IF 02<.0005 THEN 180
110 IF 02> .01 THEN 180
120 IF L<5 THEN 180
130 IF L> 150 THEN 180
140 IF L/R< 1 THEN 180
150 IF T<60 THEN 180
160 IF T>3600 THEN 180
165IFT>T9THEN 180
170 LET 09=02:L9=L:T9=T:R9=RP9=P. V9=V
171 LET TH=P9*R9/(31875-.2*P9): Q=
172 LET W=49*TH*(R9+TH/2)*L9:Y=Q*T9/60
180 NEXT P
260 LPRINT "INSTRUMENT CONSTRAINTS"
270 LPRINT
280 LPRINT
290 LPRINT" Inlet Pressure
300 LPRINT"Maxirnum Column Load
310 LPRINT"MultipaHl Packing Factor-
320 LPRINT"Longltudinal Diffusion Packing Factor
330 LPRINT"Column Mobile Phase Fraction
340 LPRINT
350 LPRINT "ELECTIVE VARIABLES"
360 LPRINT
370 LPRINT"5eparation Ratio of the Critical Palr ",A
380 LPRINT "Capacity Ratio of the First Peak of the Pair ",K1
390 LPRINT "Capacitv Ratio of the Last Eluted Peak ,. ,K2
395 LPRINT"Sarnple Concentration ?ow/v "J
400 LPRI NT"Diffusivity of Solute in nobue Ph::ise ,o,D 1"sq. em per
sec"
410 LPRINT "Viscosity of Mobile Phase
420 LPRINT
421 LPRINT "LIMITING CONDITIONS"
422 LPRINT
4
'")3 LPRINT "Partir"e Diarneter Rance S-1 (1() mirrnn L. ...... \,,\..., l \All':' ..... ) f .......... ....... • _ ••
424 LPRINT "Column lengHI 13 less tJlan 150cm "
425 LPRINT "Column Aspect Ratio L/R must be greater than umtv"
426 LPRINT "Ihe Analysis Time must lie between 1 minute and 1 hour' ,.
427 LPRINT
430 LPRINT "COLUMN SPECIFICAT10NS"
440 LPRINT
450 "Opt irnurn Co lurnn Radius
451 LPRINT USING "##,#"; R9,
?c:,4
-., -
452 LPRINT "ern'
460 LPRINT "Optimum Column Length
461 LPRINT USING "###. #"; L9
l
462 LPRINT "ern"
470 LPRINT "Optimum Particle Diameter
471 LPRINT USING "###.#"; 10000 *D9,
472 LPRI NT "urn"
480 LPRINT
490 LPRINT "ANALYTICAL SPECIFICATIONS"
500 LPRINT
510 LPRINT "Minimum Analysis Time
511 LPRINT USING "##,.tt"; T9/60,
512 LPRINT "min
520 LPRINT "Co1umn Eff i ct encv
521 LPRINT USING "######"; N
530 LPRINT "Optimum Flow-Rate
531 LPRINT USING "###,#"; Q,
532 LPRINT "rnt/rntrr
540 LPRINT "Maximum Sample Volume
541 LPRINT USING "#:Jt.##"; V9,
542 LPRINT "ml"
550 LPRINT "Solvent Consumption per Analysis
551 LPRINT USING "######"; Y,
552 LPRINT "rnl"
560 LPRINT "Column Wall Thickness
561 LPRINT USING "##,##,#"; TH
l
562 LPRINT "ern"
552 LPRINT "Column Weight
553 LPRINT USING "####,#"; W,
554 LPRINT "q"
570 CLOSE # 1
580 END
Discussion
"
"
On running the program,the Performance Criteria are first printed out and
then the separation ratio of the critical oatr is asked for on the monitor
screen. After typirtg tn the aooropriate value, the capacity ratio of the first
peak
f\f tho ..... ,..1+i r- -:l1 carr f"l1(\\.,on hu tho r- -:In-:l r- i t l l l ' ' ~ t i , , l')ct 011ltorlno";lv nO-:lv
VI \,1 I\, \",1 1\,II.oYI ~ II IVIIV1"'II.o\.l vr .. II\, ""YtJU""'l-r lUI-IV IU..." .. "'''''I.""" tJ"'-'" tJ" .... "
are then requested, The request procedure conttnues until all the necessary
tnrormat ion has been entered after which the program runs, Some care must
be taken when entering the minimum pressure, maximum pressure and
pressure increment. If the range is made too large and the increment too
small the program make take a very long time to run. Assuming that the
pump has a maximum aval table output pressure of 6000 o.s.t. then the
minimum pressure might be set at 1000 o.s. t., the maximum pressure at
6000 p.s.t. and the increment 100 p.s.t. The program would then identify the
column and performance data that are necessary to attain the minimum
separation time. When an approximate value for the operating pressure is
obtained, the program can then be run again with a more narrow pressure
pressure range and an appropriately smaller pressure increment. The packing
factors (1) and (y) can, for preparative columns, be taken as 0.65 and 0.8
respectively as, in general, preparative columns can not be packed as
efficiently as analytical columns. The diffusivity of the solute in the mobile
phase may be calculated from the Arnold Equation (5)1 or estimated from the
diffusivity data provided in the Appendix. The viscosity of the mobile phase
can be best obtained from asuitable reference book on solvents (6).
The program assumes anumber of limit ing corditions,
1/ The practical range of particle diameters is between 5 and 100micron,
program lines 100and 110.
2/ The practical range of column length is between 5 and 150ems, program
lines 120 and 130.
3/ The column length must not be shorter than It's radius (the aspect
ratio) must not be less than unity, program line 140.
4/ The separation time must be greater than one minute and less than one
hour program 1tries 150 and 160.
These conott ions can be Changed, if so required, by modifying the
appropriate program lines, but care should be taken not to place impractical
limits on the separation. During the operation of the programI the current
separation ratio, current pressure and current minimum analysis time will
continuaiiy appear on the monitor screen. When the minimum analysis time
has been found it wn1 not change on the screen but the program will
continue to the maximum pressure has been reached. The optimum conditions
will then be printed out. An example of the computer print out is given as
follows:-
COMPUTER REPORT
PREPARATIVE COLUMN DESIGN FOR LC
PERFORMANCE CRITERIA
1/ Adef1ned resolution must be ootatnec
2/ The analysis must be completed in the minimum time
3/ The analysis must be completed with the minimum solvent
INSTRUMENT CONSTRAINTS
InIet Pressure
Maximum Column Load
Multipath Packing Factor
Longitudinal Diffusion Packing Factor
Column Mobile Phase Fraction
ELECTIVE VARIABLES
Separation Ratio of the Critical Pair
Capacity Ratl0 of the Flrst Peak of the PaIr
Capacity Ratio of the Last Eluted Peak
Dtrrustvttv of Solute in Mobile Phase
Viscosity of Mobile Phase
LIMITING CONDITIONS
690 p.s. i
5g
0,65
0,8
0.65
1.09
2.5
5
0,000035 sq.crn per sec
0.023 Poises
Particle Diameter Range 5-100 micron
Column length Is less t.han 150cm
Column Aspect Ratio L/R must be greater than unity
The Analysis Time must lie between 1 minute and 1 hour
COLUMN SPECIFICATIONS
Optimum Column Radius
Optimum Column Length
Optimum Particle Diameter
9.20 cm
("\ 7(\ ~ ' " ' "
~ .JV ....111
9,00 um

-,.,.
ANALYTICAL SPECIFICATIONS
Minimum Analysis Time
Column Efficiency
Opt Irnurn Flow-Rate
Maximum SampIe Volume
Solvent Consurnptton per Analysis
Column Wall Thickness
Column WeIght
1010min
3871
961 ml/rntn
100 ml
9695 ml
020 cm
852g
(It shouldbe noted that the column weight doesnot include flanges)
The program can be modified slightly, to calculate the minimum analysis
time required to isolate 5 gram of material, at a qiven tnlet pressure, from
a series of mixtures of differing separation difficulty. The results obtatnec
are shown as curves relating separation time to separation ratio in figure 5.
FIgure 5
Graph of Analysts TIme agatnst separatton Ratio for a Load of 5g
60

I)
50
..,
:::I
C
-
J: 40
c:
-

30
E
-to-

20
...

-
10
D
c:
C
0
1.0 1.1 1.2 1.3
Separat10n Rat10
Figure 5 Shows that here ts a rnintrnum in the analysis ttrne at a separanon
__ .... ... #" _""- .... i AI: l ......I., 1- __ "..,.. ................ "' .......... IIlI"'oL. "I" ...... lI"' ...... l ..... ...... """" ,.."1,."",,,, ..
1 G1I..1V VI G1UVIJI. t,VU, I'tVVV. II VIII I.IIC 1.1ICVI Y VI GlIIGlIYI.I\;QI \"VIUIIIII Il,.
wouro be expected that, lf the column was fully opttrmzed, the analysis
time would decrease conttnuouslv as the separation ratio of the critical
pair increased, The reason that a minimum exists in figure 5 arises from the
ltrnttattons place on the column by the rntmrnum aspect ratio of unity and
258
the minimum column length of 5 em. Thus, as the load of 5 gram requires a
column of specific ciameter to avoid undesirable peak dispersion, the
column can not be reduced to the optimum length without rendering the
column aspect ratio less than unity. As a result the column length must be
greater than the optimum causing an extension of the analysis time. ThiS
situation is exacerbated as the separation ratio increases.
The program can also be modified to show how the optimum pressure
required to effect the preparative separation of 5g of material in the
minimum time, changes with the separation ratro.or the critical pair. The
results are shown in figure 6 as curves relating the logarithm, of the inlet
pressure to the seoarat i on ratio.
Figure 6
o Column 5
1.3 1.2 1,1
-
.
=e
...I 0
1.0
.
4
Graph of Log. Inlet Pressure against Separation Ratio for a Load of 59
5.,....-------------------.
separat Ion Rat 10
Figure 6 Shows the effect of the three ltrruts Imposed on the column oesicn,
the need to maintain an aspect rat to greater than unity) restricts the column
length to a minimum of 5 cm and confines the analysts time to between one
minute and one hour. without the restriction, the column with the mtrnrncm
analysis time would be operated at the maximum pressure available.
However, as a result of the limitation on column length) the optimum
pressure must be reduced to allow the optimum particle diameter to be
trcreased This, in turn, allows the column length to be increased to
21)9
maintain a column aspect of unity while achieving the rntnimum practical
analysis time and the required column loading. !t is seen that the
interactions between the column properties, and their effect on analysis
time, are inherently complex and, unfortunately, become more so, as turther
external limitations are applied to the column deSign. It should also be
noted that tne more simple the separation U. e. the smaller (ex)) the lower
the optimum operat ing pressure).
Column Overload as an Alternative to Column Design
Prior to the development of a rational procedure for the design of
preparative columns, large scale separations were carried out by employing
the technique of column overload. Column overload, although not providing
optimum separations, was partly successful, but could only function well
where overload was possible, 1.e. where the separation ratio of the critical
pair was large e.g. greater than I 5 and preferably 2) 3 or even 4
Unfortunately, with the majority of LC separation problems met in oractice,
this is not possible, due to the cornplexitv of the mixture. Even with very
careful mobile phase selection, the necessary large values for (ex) are not
often attainable. Nevertheless) the technique of column load can be useful
under some circumstances and thus, the basic principles of column over load
will be briefly discussed. The theory of column overload has been
considered by a number of workers in the field (7-13) and more recently in a
paper by Knox and Piper ( 14) and in a number of papers by GUiochon and his
co-workers (15-18).
For any given chromatographic system, there is a limiting charge that can be
placed on a column before the resolution is impaired, Loss of resolution
from column overload can arise from two causes, either excessive sample
feed volume or excessive sample mass, The theory of moderate sample
volume overload has already been considered in the applications of the Plate
Theory. The theory of excessive sample volume overload will now be
discussed.
Sample Volume OverlQad
Consider the situation deptcteo in figure 7, To determine the band dispersion
tnat results from a significant, but moderate) sample volume overload the
princtple of the summation of variances CG1l\ be appl ieu However, Wi-len HIe
sample volume becomes excessive, the band dispersion that results from the
overload becomes equtvatent, to a first approximation, to the sample volume
itself. In figure 6, two solutes are depicted that are eluted from a column
under' conditions of no overloac If the excessive sample volume (whiCh now
also acts as part of the eluting mobile phase) is of such a value that it Just
allows the peaks to touch at the base, the peak separation 1n ml of rnobue
phase passed Ulr'ough the column will be ecutvalent to the sample volume
(Vj) plus nalr the base width of both peaks shown rn fjgure 7.
ThuS,
where (n) is the co turnn errciency and 1s assumed to be the same for
bOUI peaks,
(Ka and Kb) are the distribution coefficients of the two solutes (a) and( b),
and (cd is the separation ratio of solutes (a) and (b).
Figure 7
Two Solutes Eluted Under Condtt tons or No Volume Overload
i"'---,
I
1J,; (vm+KaAs) •
2b1
Noting that Vo =rwrn and
Then,
.
v ,
=k
Va a
I
Vb' , ,
- =k and k =ak
V
o
b b a
Noting also that (n) is large, and thus, to the first aoproxtmatton,
V
1
=Va (a - 1 .. " ' .. , , , , '"'''' (15)
It is seen from equation (15) that the maximum overload volume is linearly
related to the (k') value of the first eluted solute of the critical pair, the
function (0-1) and the column dead volume, Consequently, tne larger the
coturnn, either in length and/ or radius) the larger the sample volume can be,
This assumes that the column is of such a size) that it can be efficiently
packed with practical techniques and that the partrcte size of the packing JS
chosen such that the pump pressure avai table can provide the necessary
mooue phase fl ow-rat.e.
In the authors opinion volume overload employing a solution of the material
in the mobile phase at a level of about 5% w/v is the recommended method
of sampling for preparative columns if the system is not oottrntzec.
However, as will be seen later' a combination of volume overload and mass
over load has also been suggested as an aiternat tve procedure. An example of
progressive volume overload ts qtven tn figure 8.
Sample Mass Overload
Band dispersion from sample mass overload is a direct result of the
chromatographic process proceeding under cone: ttons, where the acsorpt ion
isotherm of the solute on the stationary phase, is no longer linear, The
rlf>vf>lonrnf>nt of ;:m that rjpc:;trihpc:; thp pxtpnt of h?lnrl ;=:\c:;
- - - -.- .. - - - -, - - - - >... - -- - - - - - - - - - - - - _. - - r- - - _. .;,;) - - -
function of mass of sample placed on the column, Is complex. This problem
has been elegantly approached by Gulochon and his co-workers (15-18) from
the basis of the adsorption isotherm of the solute on the stationary phase.
202
The form of the isotherm need not be t.ancrmnr in nature, but in any event)
must be experimentally determined in order- to identify Ute true profile of
HIe overloaded peak. In practice, the determination of the adsorption
isotherm of each compound to be separated by a preparat ive
cbrornatoqraprnc procedure can be arduous and time consuming, A better
alternattve micnt be to design the fUlly optimized column from basic
prmctples in tne manner previouslv described
Figure 8
Progressive Volume Over Load of an LC Column
FLOW RATE Iml/(11j(I
SAMPLE VOLUME 10 1.11
500ul
A B C
2ml
c A
3ml
e c
SCUlTES A ANISOLE
e BENZYL ACETATE
C AI:£. TOPHENONE

Knox and Pyper (14) assumed that the majority of the adsorption isotherms
were, indeed, Langmuir in form and then postulated that an the peaks that
were overloaded would be approximately triangular in shape As a
consecuence, Knox and Pyper assumed that mass overload could be treated in
a similar manner to volume overload, Whether all sotute/stat ionarv phase
isotherms are LangmuIr in type, is a moot point, and the assumption should
be taken with some caut ion. Knox and pyper then suggested that the best
cornororntse was to utilize about half the maximum sample volume as
certneo by equation (15) which would then reduce the distance between the
peaks by hal r They then recommended that the concentrat j on of the solute
was increased unt: 1 dispersion due to mass overload just caused the two
peaks to touch. Knox summarized his recommendations in the following way
( 19),
1/ Develop an analytical separation which qtves the best possible
resolution between the crtttcal solutes.
2/ Determine the difference between the retention volumes of the two
solutes (l1V) (equation (15)).
3/ Employ sample volumes of about O.4tJ..V containing increasing
concentrations of solute unttl the gap between the crtttcal pair 1S just
fill ed
unfortunately, this procedure assumes that the critical pair can be well
resolved and column overload is a practical solution to the problem, More
often, due to the complex nature of the rntxture values for (a) of 1, I, 1 or
even less, are the best that can achieved, Under such circumstances, the
optimum column must be designed which, in the desmn procedure, wll1
automat tcauv be given a radius that wi 11 accommodate the load that is
required.
References
( 1) Exatz, K,L.Ogan and R.P,W.Scott, J 289( 1984)65,
(2) RP,W. Scote J cnromatogr.) 468( 1989)99.
(3) J.H.Purnell, Nature" No 4704 , Dec.9,C 1959)2009.
(4) RH.Perry and C.H.Chi lton, Chemical EngIneers HandBook, McGraw-Hill
Book Company, New York (1973)6-12,
264
(5) J. H. Arnold, J Amer: cnen Soc., 52( 1930)3937.
(6) ..J.A.Riddick and W. B. Bunger, U Organic sotveats , John Wiley
and Sons, New York, Sydney and Toronto ( 1970).
(7) I Halasz, H. Schmi dt and P. vogteI, J Chromatogr 126( 1967) 19.
(8) J. H. Knox and MSaleen. J Cnrmatogr. Sci 7( 1969)614,
(9) R. P. W. Scot t and P, Kucera, j 11 9 ( 1976)467.
( 10) L. R. Snyder, ,./ Lnrofi7atogr. Sci, 15( 1977)441.
( 11) B. COQ, G. Creti er and J. L. Rocca, J 186( 1979)457.
(12) R. Rosset, M, Claude, J. Desbares and E. SChmidt, B( 1980)213.
(13) K. P. Hupe and H. H. Lauer, J Chromatogr, 203( 1981)41
(14) J. H. Knox and H. M. Pi per, J. Chromatogr., J63( 1986) 1
(15) G. GUlochon and A. Katt I, Chromatograph/a, 24( 1987) 165
( 16) S.Gotsnan-srnrazr, AJaulmes and G.Gulochon, Anal Chem)
60( 1988) 1856.
( 17) S.Golsnan-Srnrazt, AJaul mes and G,Guiochon, Anal Chenl,61C 19891276.
(18) S.Golshan-Shlrazl, AJaulmes and G.Guiochon, Anal Chem,
6H1988) 1368.
(19) J. Knox, The Chromatographic Society Bullet!n, 29( 1988) 18.
LIST OF SYMBOLS
a separation ratio
c fraction of column occupied by the mobile phase
~ proportionality constant
y longitudinal diffusion packing factor
11 viscosity
cp D'Arcy's constant
AmuIt Ipatn packIng f act.or
v reduced rnoone phase velocity
etemperature
ostandard deviation
ec standard deviation due solely to column dispersion
Os standard deviation due solely to sample volume
Ov standard deviation due solely to dispersion from sample valve
0t(u) standard deviation due solely to connecunq tubes
oD standard deviation due solely to detector
oR standard deviation due sOlely to detector
of standard deviation due to total extra conmn dispersion
e concentratton of solute in sample
~ proportional ity constant
,proport1onallty constant
nproporti anaIi ty constant
A multipath term in van Deemter equation
As surface area
B longitudinal diffusion term in the Van Deemter equation
C resistance to mass transfer term in the Van Deemter equation
Ca detector cell capacity factor/column capacity factor for a given solute
o dispersion equat 10n constant
Dm d1ffusiv1ty of solute in the mobl1e phase
Ds dtrrusrvttv of solute in the stationary phase
E dispersion eouatton constant
H height of theoretical plate or variance/unit length of column
HM contribution to Hfrom mutttpatr, dispersion
HL contribution to Hfrom longitudinal diffusion ion rnobue phase
HL<s) contribution to Hfrom longitudinal diffusion ion stationary phase
K distribution coefficient
K(A) distribution coefficient of solute (A)
M mass of solute to be separated
N number of 'effective' theoretical plates
P pressure
Q flow rate
R resolution
Speak cepacl ty
Sm specific heat of mobile phase
5s specific heat of adsorbent
Sg soecinc heat of detector cell walls
V volume in convent ionaI units
Vo system dead volume
Vr retention volume
V'r corrected retention volume
Vm volume of mobile phase in the column
Vs volume of stationary phase in the column
VE extra co Iumn voIume
Vm volume of mooue phase in the heat of adsorption detector ceu
Vs volume of adsorbent in the heat of adsorption detector cell
V6 volume of the wall of the heat of adsorption detector cell
Vc total column volume volume
VSi volume of stuca in the column
Vp pore volume of the packing
Vi sample volume
VI interstitial volume or totally excluded volume
VI(m) portion of the Interstitial volume that is moving
VI(s) portion of the tntersttttai volume that is static
Vsoi volume of solvent used in the separation
Xm concentration of the solute in the mobile phase
Xs concentration of the solute in the stationary phase
Xo concentration of the solute in the first plate of the column on injection
Xo minimum detectable concentration
Z peak velocity
dm density of mobile phase
ds density of stat tonarv phase
dg density of detector cell wall
dp particle otarneter
df rum thickness of stationarv phase
f height of points of inflexion of the elution curve
g heat of aosorpt ion of solute
h peak height
!J reduced plate height
k' capacity factor of solute with respect to (Vm)
k'E capacity factor of solute wtth respect (VI) the interstitial volume
1 column length
m mass of solute
n number of theoretlcal plates
r column or tube radius
r peak capacity
to dead time
tr retention time
t'r corrected retention t1me
u linear veloci ty
v volume measured in plate volumes of the respective column
vrn volume of mobile phase per theoretical plate
vs volume of stationary phase per theoretical plate
267
Append1x 1
The D1ffusion Coeff1cients of some Low Molecular Weight
Substances
Substance Denslty Mol Weight Diffusion Coefficient
g.cm-
3 cm2
sec-l
Xl 0-
5
benzene 0.874 78 4.20
benzonttrtle 1.001 103 3,77
p-xylene 0.861 106 3.76
benza1cerwce 1.046 106 3.84
anisole 0.989 108 3.77
anthranll 1.183 119 3.52
acetophenone 1.028 120 355
nl trobenzene 1.207 123 3.73
oenzn chloride 1.103 126 3.42
naphtha1ene 1.145 128 3.69
phenyl-2-propenone 1,028 134 3.14
z-rnettwlacetoonenone 1,005 134 3.42
2-benzothiazole 1,248 135 3.29
O'ni troto1une 1.168 137 356
p-dimethoxybenzene 1.053 138 3A9
c.o,c. trifluorotoluene 1.199 146 3.63
4-phenyl-3-butene-2-one 1.020 146 3,05
anethole 0.991 148 3.28
benzyl acetone 0.989 148 3.02
2-rnethyl benzothiazote 1.203 149 332
benzyl acetate 1.057 150 3.13
ethyl benzoate 1.051 150 3.06
p-tolyl acetate 1.049 150 3.06
biphenyl 1.041 154 3.12
i-chloro- 3-ni troberizene 1.534 157 3,35
2-metoxynaphthaIene 1.013 158 3.10
1-3-5-triethylbenzene 0.863 162 2.59
n-propyl benzoate 1,021 164 2.86
ettlyl phenylacetate 1,031 i64 L.e4
p-diethoxybenzene 1.008 164 2.98
2,3-dirnethoxy-
-benzatoerwoe 1.019 166 2.94
carbazole 1, 100 167 2.48
L O ~
Substance Density Mol Weight Diffusion Coefficient
g.cm-
3
cm
2sec- 1
X10-
5
m-di nitrobenzene 1.575 168 3.14
acetonaphthone 1.147 170 2.97
l-ni tronaontnaiene 1.331 173 2.92
ethyl cinnarnate 1.049 176 2.83
-t-btphenvtcartomtri le 1.041 179 2,81
2',5'-dimethoxy
acetophenone 1,126 180 2.81
benzophenone 1,080 182 2.75
2,4-dinltrototuene 1,521 182 2,81
btbenzv 0.978 182 2.76
azobenzene 1,203 182 2,69
1,2-dirnethoxy-4-nitro- 1.384 183 2,82
benzene
(rnl tro-o.o.o-tr: fl uoro- 1,281 191 2.94
toluene
dtbenzvl ether 1,036 198 2.62
pheny1benzoate 1.235 198 2.65
p-bromoacet oohenone 1,647 199 3.14
9-cyanoanthracene 1.097 203 2,51
benzu 1.23 210 2.50
benztt benzoate 1. I 12 212 2.59
1,2-diphenoxyethane 1,098 214 2.48
2,5-diphenyloxazole 1.152 221 2.51
trtoherwtene 1.302 228 2.50
p-terphenyl 1.221 230 2.42
diethyl oherwtmatonate 1.095 236 2,25
2-naphthyl benzoate 1.160 248 2.36
dipropyl phthalate 1.059 250 2.22
oerviene 1.35 252 2.35
tri decy1benzene 0.881 260 2.06
dobutylphthalate 1,043 278 2.02
hexachlorobenzene 2.044 285 2.60
1,2,4,5-dlbenzopyrene 1,288 302 2.18
m- quaterpheny1 1,206 306 1.89
otoctv: oritnalate 0.981 390 1.64
dldecyl phthalate O . 9 6 ~ 446 1.46
E. Katz (Ph.O.Inests) Birkbeck College, University of London, United Kingdom,
AppendIx 2
The Dtffuslon Coefficients of some Peptldes
SUbstance
tetraglyclne
hexaqlycme
tnsuttn B(22-25) peptide
cholecystok1nl n (CCK)
tetrapeptide (30-33)
lsulln (21-26) peptide
anqtotenstn III
cholescystokintn (CCK)
neptapeptlde (27- 33)
anqlotenstn II
anqtotensts I
gastrin releasing
peptide (1-16)
neurotenstn
Insulin chaln A, oxiotseo
glucagon
tnsuun chain B, oxtdised
parathyroid hormone
(39-68)
corttcotrootn releasing
factor
insulin
Mol WeIght
246
360
525
598
758
931
948
1046
1297
1547
1673
2530
3460
3496
4109
4758
6000
Dtrrusion coerrtctent
cm
2
sec-
1
XI0-
5
0.479
0.434
0.366
0.381
0.295
0.283
0.291
0.264
0.240
0.227
0.233
0.183
0.170
0.174
0.163
0.152
0.144
E. Katz (Ph.D.Thesls) Blrkbeck COllege) Un1verslty of London, United K1ngdom.
Appendix J
The Phystcal ProperUes of some Solvents Employed as rtonue
Phases in LC
Solvent
hexane
heptane
n-chlorobutane
dichloromethane
chloroform
methyl acetate
ethyl acetate
acetone
tetrahydrofuran
acetonttrtle
propanol
ethanol
methanol
water
Mol. weight
86
100
92.5
84.9
119.4
74.1
88.1
58.1
72.1
41.1
60.1
46.1
32
18
Density (200C)
(g.cm-
3)
0.659
0.684
1.402
1.325
1.488
0.934
0.901
0.790
0.889
0.782
0.804
0.789
0.791
0.999
vtscosttv (200C)
(dyn.sec.cm-
2)
0.00312
0.00418
0.00444
0.00423
0.00574
0.00385
0.00522
0.00321
0.00550
0.00355
0.0225
0.00991
0.00551
0.0100
J.A.Riddick and W.B.Bunger, "Organic Solvents", Techniques of Chemistry,
Volume II, wuey-tnterscterce, Division of John Wlley and Sons, Inc. New
York) London, Sydney and Toronto.
Note. As association between water and miscible soiventts) often occurs
the viscosity of aqueous mixtures can not be calculated by simple
proportion from the viscostty of the components.

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