You are on page 1of 22

Lecture 2. Chromatographic theory.

Chromatographic Theory
Basis of the separation process
Chromatographic separation process is based on the difference in the surface
interactions of the analyte and eluent molecules.
Component A: interaction with adsorbent is as strong as for eluent molecules.
Component B: strong excessive interaction.
Result:
Component A is moving trough the column with the same speed as eluent
molecules.
Component B is moving slower than the eluent flow.
1
A
B
B
B
A
A
A
A
A
B
B
B
B
B
B
A
A
A
Lecture 2. Chromatographic theory.
General scheme of chromatograph instrument.
Chromatogram.
A graphical or other presentation of detector response, concentration of analyte in
the effluent or other quantity used as a measure of effluent concentration versus
effluent volume or time. n planar chromatography !chromatogram" may refer to the
paper or layer with the separated #ones.
$
%luent &an'
(ump
n)ection valve
Column
*etector
(lotter
+raction collector
,etention time
C
A
B
Lecture 2. Chromatographic theory.
Band Broadening.
&ypical volume of the sample in)ected is -.$/l.
&ypical volume of the sample collected is milliliters.
Broadening is caused by: the noneven flows around and inside the porous
particles, slow adsorption 'inetics, longitudinal diffusion, and other factors. &he
longer the component retained, the more broad its #one.
Separation performance depends on both component retention and band
broadening.
Band broadening is, in general, a 'inetic parameter, dependent on the adsorbent
particle si#e, porosity, pore si#e, column si#e, shape, and pac'ing performance.
Retention does not depend on the above mentioned parameters, but it reflects
molecular surface interactions and depends on the total adsorbent surface.
&wo approaches can be ta'en to explain the separation process.
(late theory 0 proposed in 1121 by 3artin and 4ynge. Based on analogy with
distillation and counter current extraction. ,ate theory 0 accounts for the dynamics
of a separation, introduced by 5.5. 6an *eemter in 11-7.
8
Lecture 2. Chromatographic theory.
Retention Parameters.
Retention time, t
R
. the time between the in)ection point and maximum of the
detector response for correspondent compound. ,etention time is inversely
proportional to the eluent flow rate, F
C
.
Retention olume, V
R
. the volume of the eluent passed through the column while
eluting a particular component.
c
R
R
F
V
t =
!inimum possible retention time, t
M
0 time of unretained mobile phase to travel
trough the column.
Ad"usted retention time, t'
R
0 additional time required for solute to travel the
length of the column, beyound the time required by unretained solvent.
t'
R
= t
R
- t
M
Relatie retention, 0 the ration of the ad)usted retention times of the
components.
where t'
R2
9 t'
R1
, so 91. &he greater is, the greater is separation between two
components. ,elative retention is used to identify the pea's when the flow rate
changes.
Capacity factor, k' 0 also called retention factor, capacity ratio, partition ratio.
Capacity factor is dimensionless and independent on any geometrical parameters of
the column. t could be considered to be a thermodynamic characteristic of the
adsorbent.compound.eluent system.
2
1
$
1
$
R
R
R
R
t
t
V
V

=
M
M R
M
M R
t
t t
V
V V
k

= :
Lecture 2. Chromatographic theory.
#$ample of calculations.
A mixture of ben#ene, toluene and methane was in)ected into a gas chromatograph.
3ethane gave a sharp pea' in 2$ s, whereas ben#ene required $-1 s and toluene
was eluted in 888 s. +ind the ad)usted retention time and retention factor for each
solute and relative retention.
S%&'T(%). &he ad)usted retention times are
Ben#ene: t'
R
; t
R
0 t
M
; $-1 0 2$ ; $/1 s
&oluene: t'
R
; t
R
0 t
M
; 888 0 2$ ; $11 s
&he retention factors are
Ben#ene: k' ; <t
R
0 t
M
= > t
M
; <$-1 0 2$= > 2$ ; -./
&oluene: ': ; <t
R
0 t
M
= > t
M
; <888 0 2$= > 2$ ; 7.1
&he relative retention is
= t'
R
(toluene) /

t'
R
(benzene) <888 0 2$= > <$-1 0 2$= ; 1.81 * + ,,,,,,,,,,,
-
t'
R
t
R
t
M
n)ection time
&ime
*etector responce
3ethane
2$ s
Ben#ene
$-1 s
&oluene
888 s
Lecture 2. Chromatographic theory.
Relation bet-een retention time and partition coefficient.
f the solute is not adsorbed and spend all the time in mobile phase
t
R
= t
M
k' ; /
f the solute spend equal time in mobile and stationary phases
t
R
= 2t
M
k' ; 1
n other words,
k' (time !olute !pen" in !tationary pha!e) / (time !olute !pen" in mobile pha!e)
f the solute spends # times as much time in in the stationary phase as in the mobile
phase, there will be # times as many moles of solute in the stationary phase as in
mobile phase at any time.
or
where C is concentration, and V is volume
7
M
M R
M
M R
t
t t
V
V V
k

= :
&ime solute spends in stationary phase
&ime solute spends in mobile phase
moles of solute in stationary phase
moles of solute in mobile phase
.
k ' =
C
$
V
$
C
M
V
M
Lecture 2. Chromatographic theory.
f column is run in equilibrium conditions
C
$
/ C
M
; %
where K is partition coefficient. (artition coefficients depends on the sorbent,
eluent and analyte nature, as well temperature.
,elation of retention time to partition coefficient
note: V
$
/ V
M
(V
R
& V
M
) / V
M
(t
R
& t
M
) / t
M
Because ad)usted retention time t'
R
, retention factor k' and partition coefficient K
are proportional to each other, relative retention can be expressed as:
The relatie retention of t-o solutes is proportional to the ratio of their
partition coefficients. &he greater the ratio of partition coefficients between mobile
and stationary phases, the greater the separation between two components of a
mixture.
?
k ' =%
V
$
V
M
=
t
R
t
M
t
M
=
t '
R
t
M
o=
t '
R2
t '
R'
=
k '
$
k '
1
=
%
$
%
1
Lecture 2. Chromatographic theory.
#$ample of calculations.
,etention time of methane t
R(methane)
; 2$ s
,etention time of ben#ene t
R(benzene)
; $-1 s
@pen tubular column:
nternal diameter of the column: $-/ m.
&hic'ness of stationary phase layer: 1./ m.
%stimate the partition coefficient of ben#ene, and state what fraction of the time
ben#ene spends in mobile phase.
S%&'T(%).
Cross.sectional area of column ; Ad
$
>2 ; A<$2B=
$
>2 ; 2.B8C1/
2
Dm
$
3iddle diameter of coating d:; $21 Dm
Cross.sectional area of coating ; Ad:Cthic'ness ; A$21C1./ ; ?.BC1/
$
Dm
$
Both phases extend for full length of the column, therefore:
64 > 63 ; <?.B %$ > 2.B8 %2= ; /./171
,etention factor for ben#ene k' ; <$-1 0 2$=>2$ ; -
(artition coefficient for ben#ene K (k' V
M
)>V
$
; ->/./171 ; 81/
+raction of time in mobile phase ; t
M
><t'
R
( t
M
= ; t
M
><k't
M
( t
M
=
1/<k' ( 1= 1<-./ E 1= ; /.1?
#)*+, -n ga! chromatography. retention time! are u!e" more. /herea! in li0ui"
chromatography retention 1olume! are more popular 2or calculation!.
B
column wall
1 m thic' layer
of stationary phase
$2B m
Lecture 2. Chromatographic theory.
Scaling 'p.
3nalytical purposes vs. preparati1e purposes.
%asiest way to achieve this is to 'eep the column length constant and increase the
column cross.sectional area.
&o reproduce the separation conditions, the linear flow rate should be 'ept
constant.
&hat means, the volume flow rate should be proportional to the column cross.
section, and therefore to the mass of analyte.
1
$ample ma!!
Column1olume
=con!t
ma!!
$
ma!!
1
=(
ra"iu!
$
ra"iu!
1
)
$
1olume 2lo/
$
1olume 2lo/
1
=
ma!!
$
ma!!
1
Lecture 2. Chromatographic theory.
#fficiency of separation.
&wo basic factors: distance between pea's <relative retention= and pea' broadness.
&he longer the solute is retained, the broader the pea' is. &he higher the column band
broadening, the smaller the number of components that can be separated in a given time.
&he sharpness of the pea' is an indication of how good, or efficient a column is.
&he pea' width is dependent on a number of parameters <column length, flow rate, particle
si#e=. +low rate is the only parameter which can be changed from run to run on the same
column. &hus, it is better to consider a relative value to express column efficiency.
&he main reason for pea' broadening is the longitudial diffusion. n the ideal case the
chromatographic pea' can be represented by a Faussian curve with the standard deiation /.
&he ratio of standard deviation to the pea' retention time /t
R
is called the relatie standard
deiation, which is independent on the flow rate.
&he values w
1/2
; $.8-/ and - ; 2/ are from Faussian equation.
1/
-.0/
h
t
R
or V
R
h
w
1/2
=2.35
G G
nflection point
<steepest portion
of curve=
*etector responce
t;/
<in)ection= &ime or volume
4( 5)=e

( 55
/
)
$
$c
$
Lecture 2. Chromatographic theory.
1iffusion.
Consider a single band, traveling trough the column.
&he molecules are diffusing across the plane with a concentration gradient d/d!.
+ic':s first law of diffusion describes the number of moles crossing each square
meter per second:
Flu5
(
mol
m
$
!
)
6 =7
"c
"5
where * id diffusion coefficient.
f the solute was in)ected as infinitely sharp layer with concentration of m
moles>m
$
, the Faussian profile of the band is:
where t is time, and ! is distance along the column from the current center of the
band.
&he standard deviation of the band is:

11
c=
m
.
2n7t
e
5
$
2 7t
c=
.
$ 7t
Lecture 2. Chromatographic theory.
Plate 2eight. Column #fficiency.
&wo approaches can be ta'en to explain the separation process.
(late theory 0 proposed in 1121 by 3artin and 4ynge. Based on analogy with
distillation and counter current extraction.
,ate theory 0 accounts for the dynamics of a separation, introduced by 5.5. 6an
*eemter in 11-7.
Plate theory. Performance by one pea3.
&he column is considered to be separated on to a number of "late#, on which the
equilibrium of the solute with the mobile and stationary phases occurs. &he whole
length of the column is divide by this n$%&er of the theoretial "late# to give the
hei'ht of the theoretial "late. As many points of equilibrium are in the column,
and as narrow they are, as efficient the column is.
(late height is introduced by equation <$
!
is a linear flow rate=:
where ( is height of theoretical plate, and
8 9
2
/5
8 i! con!tant proportionality bet/een the 1ariance (
2
)

o2 the ban" an" the
"i!tance ! it ha! tra1ele". For 4C 8 : ;.' to ' mm. 2or 8<LC 8 : '; =m.
1$
c=
.
$ 7t - c
$
=$ 7t =$ 7
5
u
5
=
(
$ 7
u
5
)
5=85
Lecture 2. Chromatographic theory.
Humber of the theoretical plates of the column of ) length is given by:
where t
R
is retention time, and w is pea' broadness in unit! o2 time.
Ising half.height width w
1/2
instead of width at the base:
#$ample:
,etention time is 2/? s.
Base width of the band is 18 s.
Column length is 1$.$ m.
+ind the number of plates and plate height.
18
#=
L
8
=L
5
c
$
=
L
$
c
$
=17
L
$
/
$

17
(
t
R
/
)
$
=
(
t
R
c
)
$
#=-.-2-
(
t
R
/
1/ $
)
$
#=17
(
t
R
/
)
$
=
17 2/?
$
!
$
18
$
!
$
=1.-?1/
2
8=
L
#
=
1$.$m
1.-?1/
2
=/.?Bmm
Lecture 2. Chromatographic theory.
&he real pea's are asymmetric.
Asymmetry factor:
&ailing factor:
Humber of plates:
12
1
>
1
/
1
>
1
/
B A
*etector responce
time
3
2
=
>
(1/1/ h)
3
(1/1/ h)
*
2
=
3+>
$ 3
#=
21.?
>
3
+1.$-
(
t
R
/
)
$
=
21.?
3
2
+1.$-
(
t
R
/
)
$
Lecture 2. Chromatographic theory.
Resolution.
By convention, resolution <,= is expressed as the ratio of the distance between two
pea' maxima to the mean value of the pea' width at the base line:
where ?t
R
or ?V
R
is the separation between pea's in units of time or volume, and
/
a1
is the average width of two pea's in corresponding units.
1-
Re!olution=
At
R
/
a1
=
AV
R
/
a1
=
/.-B1At
R
/
1/ $ a1
$G
8G
2G
7G
,esolution
; /.-/
,esolution
; /.?-
,esolution
; 1.//
,esolution
; 1.-/
Lecture 2. Chromatographic theory.
Relation bet-een plates number and resolution.
,esolution is proportional to square root of plates number or column length.
4electivity vs. efficiency:
17
Re!olution=$
t
R2
t
R'
/
$
+/
1
#=17
(
t
R
/
)
$
-t
R
=
.
#
2
/
Re!olution=
.
#
2
(
o1
o
)(
k '
a1
1+k '
a1
)
#=
.
#
1
#
$
k '
a1
=
k '
1
+k '
$
$
Jow selectivity <K=
Ligh efficiency <H=
Ligh selectivity <K=
Jow efficiency <H=
Mt
,
Mt
,
Lecture 2. Chromatographic theory.
Band broadening outside the column.
&he solute cannot be in)ected as infinitely thin #one. &he band has some finite
width even before entering the column.
f the band is applied as a plug of width *t, the contribution to the final variance is:
&he broadening in the detector holds the same relationship, because some finite
time is required for the sample to pass trough.
#$ample.
%lution rate is 1.8- ml>min.
w
1/2
for the collected band is 17.8 s.
6olume of the sample applied is /.8/ ml.
*etector volume is /.$/ ml.
+ind the variances introduced by in)ection and detection. +ind the width at half.height, which
is caused by column only.
S%&'T(%).
&he observed total variance is:
&he time of in)ection is: *t
in+etion
; </.8/ ml=><1.8- ml>min= ; /.$$$ min ; 18.8 s.
4imilarly, *t
detetor
; </.$/ ml=><1.8- ml>min= ; B.B1 s, and /
4
detector
; 7.-B s
$
.
/
4
obs .
/
4
column
5 /
4
detector
5 /
4
in"ector
/
column
; -.1? s.
&he width due to column broadening alone is: w
1/2
; $.8-Gcolumn 1$.1 s.
1?
c
in@ection
$
=c
"etector
$
=
(At )
$
1$
c
ob!
$
=
(
/
1/ $
$.8-
)
$
=
(
17.8
$.8-
)
$
=2B.11 !
$
c
in@ection
$
=
At
in@ection
$
1$
=
18.8
$
1$
=12.B1 !
$
Lecture 2. Chromatographic theory.
6an 1eemter #7uation.
&he (late &heory is ta'ing the diffusion as only source of the band broadening.
5.5. 6an *eemter proposed in 11-7 the equation, which ta'es into account three
components:
multiple path of an analyte trough the column pac'ingN
molecular diffusionN
effect of mass transfer between phases.
&he plate height is expressed as:
where , is multiple pass term, - is longitudial diffusion term, C is equilibration
time term, and $
!
is linear flow rate.
&he most significant result is that we can find an optimum eluent flow rate where
the column efficiency will be the best.
+or pac'ed columns: 3. >. C O /
+or open.tubular columns: 3 ; /
+or capillary electrophoresis: 3 C ; /
1B
8 3+
>
u
5
+Cu
5
Lecture 2. Chromatographic theory.
, term. !ultiple flo- path.
&he velocity of mobile phase in the column may vary significantly across the
column diameter, depending on the particle shape, porosity, and the whole bed
structure.
Band broadening due to differing flow velocities can be written in form:
, is theoretical plate height <L%&(= pri#ing from the variation in the #one flow
velocityN
d
"
is average particle diameterN
8 is the constant <very close to 1=, describing the particle si#e distributionN the
narrower the distribution, the smaller is 8.
&he term A may be reduced <efficiency increased= by reducing the particle diameter
<which will lead to the increasing of the column bac' pressure= and by narrowing
the si#e distribution.
11
3=8
path
=$\"
p
Lecture 2. Chromatographic theory.
Term -/$
!
. !olecular diffusion.
&he molecules disperse or mix due to the diffusion. &he longitudinal diffusion
<along the column long axis= leads to the band broadening of the chromatographic
#one.
As it was shown before, the variance resulting from diffusion is:
(late height due to diffusion is:
8
"i22u!ion
=
c
$
5
=
$ 7
u
5

>
u
5
#ote, "i22u!ion coe22icient 7 in neat !olution i! "i22erent than that in packe"
columnA
&he higher the eluent velocity, the lower the effect on the band broadening.
3olecular diffusion in the liquid phase is about five orders of magnitude lower than
that in the gas phase, thus this effect is almost negligible at the standard L(JC flow
rates.
$/
c=
.
$ 7t - c
$
=$ 7t =$ 7
5
u
5
=
(
$ 7
u
5
)
5=85
Lecture 2. Chromatographic theory.
Term C$
!
. #ffect of mass transfer bet-een phases.
4ome finite time is required for solute to equilibrate between the mobile and the
stationary phases.
(late height due to finite equilibration time of the mass transfer is:
where C
#
describe the rate of mass transfer trough stationary phase, and C
%
describes mass transfer through mobile phase.
&he equations are different for JC an FC.
GC. %pen tubular column.
k' is the capacity factor,
" is the thic'ness of stationary phase,
7
!
and 7
m
are the diffusion coefficients in stationary and mobile phases,
r is column radius.
*ecreasing the stationary phase thic'ness d reduces plate height and increases
efficiency, because solute diffuse faster across the stationary phase.
*ecreasing the column radius r reduces plate height and increases efficiency by
reducing the distance trough which the solute must diffuse to reach the stationary
phase.
$1
8
ma!! tran!2er
=Cu
5
=(C
!
+C
m
) u
5
C
!
=
$k '
8(k ' +1)
$
"
$
7
!
C
m
=
1+7 k ' +11k '
$
$2(k ' +1)
$
r
$
7
m
Lecture 2. Chromatographic theory.
9inetics of the mass transfer in &C pac3ed column.
3ass transfer for the modern types of pac'ing materials combines two effects:
adsorption 'ineticsN
mass transfer <mainly due to diffusion= inside the particles.
3odern pac'ing materials for L(JC are the spherical, totally porous rigid particles
with average diameter P- Qm and pore diameter P1//R. ,atio of the particle to the
pore diameter is -//>1. &here is no pressure propelled flow inside the particle, and
molecules can move there only by diffusion. t can be shown by analogy: if
consider a tunnel in the mountain which has diameter of $ meters and one 'ilometer
length <same ratio -//>1=, and if there is a storm outside with $// 'm>h wind, there
will be almost no wind in -/ meters from tunnel entrance.
Adsorption 'inetics is almost negligible compare to the diffusion inside the
particles, and band spreading of the pea' may be written in form:

where:
d
"
is the particle diameter,
.
%
is the diffusion coefficient of the analyte in the mobile phase,
/ is the coefficient determined by the pore si#e distribution, shape, and also
particle si#e distribution,
0 is the flow velocity.
&he above equation describes the linear dependence of L%&( on the flow rate.
&he slower the velocity, the more uniformly analyte molecules may penetrate inside
the particle, and the less the effect of different penetration on the efficiency.
@n the other hand, at the faster flow rates the elution distance between molecule
with different penetration depth will be high.
$$
8
m
=o
"
p
$
7
m
1

You might also like