1

Introduction
HPLC Process
Lecture 1
Yuri Kazakevich
Seton Hall University
2
HPLC History
Chromatography
was
discovered by
M.S.Tswett in 1903.

3
Chromatographic Column
4
HPLC Retention
Major parameters,
V
R
is retention volume, depends on the column type,
size, and the instrument parameters
V
o
is dead volume, volume of the liquid phase inside
the column
k is retention factor (capacity factor),



independent of the column size and instrument setup

k
V V
V
R o
o
' =

R R
t F V =
5
Retention Characteristics
Retention factor, k
Selectivity, a
Efficiency, N
General recommendations:
Optimize retention factor between 1 10
HPLC selectivity should be > 1.2
Maximize efficiency
2
'
1
'
2
0
0
16 ; ; '
|
|
.
|

\
|
= =

=
b
R R
w
V
N
k
k
V
V V
k o
R R
t F V =
6
Dead Volume
V
o
, Dead volume is the volume of the liquid phase in the column
Simple rule:
Column dead volume = 65% of the
volume of empty column
4
65 . 0
2
0
L D
V
t
=
e
7
Retention Parameters
8
HPLC Selectivity
9
Efficiency Parameters
10
Efficiency
2
16
|
|
.
|

\
|
=
b
R
w
V
N
N
L
HETP =
Reduced HETP is a measure of how well the column is packed.
p
d
HETP
h =
11
Column Efficiency
Column length is a compromise between the efficiency and
backpressure
Column efficiency is proportional to the column length
Specific efficiency (# of particles per one plate) decreases
with an increase of column length
Length
[cm]
Particle
Dia. [um]
Efficiency,
N
Specific
Efficiency, h
10 3 11111 3
10 5 10526 1.9
15 5 13636 2.2
25 5 15625 3.2
25 10 10000 2.5

12
Efficiency
N
t
R
total
total column inject cell connect
=
|
\

|
.
| = + + +
o
o o o o o
2
. .
13
Resolution
14
Parameters affecting efficiency:
Flow rate
Column length
Particle diameter
Particle size distribution
Parameters affecting retention factor:
Eluent type
Eluent composition
Stationary phase type
Analyte nature
Parameters affecting selectivity:
Stationary phase type
Analyte nature
Eluent additives
Temperature
Eluent composition (ionizable analytes)
Factors Influencing HPLC Separation
15
Reversed Phase
Separation Principle
Nonpolar (nonspecific) interactions of analyte with hydrophobic
adsorbent surface (-C18, C8, Phenyl, C4)
Difference in analyte sorption affinities results in their
separation
More polar analytes retained less
Analytes with larger hydrophobic part are retained longer
Almost no separation of structural isomers
12 11 10 9 8 7 6 5 4 3 2 1 0
Retention Time (min)
50
100
150
200
250
300
350
400
R
e
s
p
o
n
s
e
1
.
6
8
2
.
2
3
5
.
9
4
9
.
0
5
9
.
8
4
16
Retention Process
(Surface Equilibria)
SK V V
R
+ =
0
K
V
S
V
V V
k
R
0
0
0
' =

=
|
|
.
|

\
|
A A
=
|
.
|

\
|
A
=
RT
G G
RT
G
K
eluent analyte
exp exp
|
|
.
|

\
|
+
A A
=
0
ln ) ' ln(
V
S
RT
G G
k
eluent analyte
17
Retention Factor, k
Retention factor is a measure of the analyte
competitive interactions with the stationary phase
|
|
.
|

\
|
A

A
= |
.
|

\
| A
~
RT
G
RT
G
RT
G
k
eluent
analyte
exp exp '
70%

80%

90%

100%
%MeCN
18
Reversed-Phase HPLC Retention:
Neutral Analytes
Dependencies of retention of alkylbenzenes, alkylphenones, and
alkylparabenes plotted against the number of carbon atoms in alkyl chain.
ln(k)= m (#carbon atoms in alkyl chain) + b
19
Selectivity
Eluent composition
Ideally does not have any effect on the selectivity
for neutral compounds
| |
o = = =

|
\

|
.
|
|
\

|
.
|

(
=
|
\

|
.
|
|
\

|
.
|

(
k
k
k k
G
RT
G
RT
G
RT
G
RT
G
RT
G
RT
eluent eluent
'
'
exp ln( ' ) ln( ' )
exp
exp
1
2
1 2
1 2
1 2
A A A A
A A
20
Eluent Composition Effect on
Selectivity
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7
0
5
10
15
20
25
0 1 2 3 4 5 6 7
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5 6 7 8 9
0
2
4
6
8
10
12
0 2 4 6 8 10 12
90% MeCN
80% MeCN
70% MeCN
60% MeCN
21
Eluent Composition Effect on
Selectivity
c% o
Peak numbers
2,1 3,2 4,3
70 1.75 1.6 1.45
80 1.72 1.58 1.4
90 1.7 1.65 1.43
100 1.71 1.62 1.42
1



2


3
4
5
22
Alkylpyridines vs. eluent
composition on Luna-C18
Eluent Composition Effect
MeOH/Water
23
Eluent Composition Effect
MeCN/Water