Chapter 4 High Performance
Liquid Chromatography
（HPLC）
高效液相色谱
1952，A.J.Martin and R.L.M.Synge
received the Nobel Prize for the
discovery of partition chromatography.

In 1941, they wrote:
The mobile phase need not be a liquid but may be
a vapour. We show below that the efficiency of
contact between the phases is far greater in the
chromatogram than in ordinary distillation or
extraction columns. Very refined separation of
volatile substances should therefore be possible in
a column in which a permanent gas is made to flow
over a gel impregnated with a nonvolatile solvent in
which the substances to be separated
approximately obey Raoults’ law.
4.1 Introduction of HPLC



Development of HPLC
From LC to GC
From LC to HPLC
Application of HPLC
Special characters of HPLC
High pressure
High Flow
High performance
Others
Types of HPLC
Chemical-bond (distribution), Adsorption, Ion
exchange*, Exclusion
4.2 Discussion of LC from the Rate
Theory

H=A+B/u+Cu

A=2λdp

B→0

C=?
Mass transfer resistance causing by
the flowing mobile phase
Mass transfer resistance causing by
the stagnating（滞留）
Equation of Rate-theory of HPLC
Cm d p2
Cd Dm
H  2d p 
(
u
Dm
涡流扩散项
质
纵向扩散
流动相传质

C s d p2
Dm

滞留区传质
C s d 2f
Ds
)u
固定相内传
Conclusion

Fine stationary phase

big Dm

Low flow

Extra-volume of the column

Temperature
Glossary






HPLC
Chemical-bond phase chromatography
Ion exchange chromatography
Exclusion chromatography
Stagnate
Isocratic and gradient elution
4.3 Instrument of HPLC





Typical HPLC chromatographs
Pumping and Solvent System
Sample Injection System
Columns
Detectors
4.3.1 Diagram of HPLC instrument
Picture of HPLC instrument
4.3.2 Pumping and Solvent System
Principle and Picture of reciprocating pump (往复泵）
Gradient elusion （剃度洗脱）

Reason for
gradient elusion

Compared with
GC

Two types of gradient elusion
溶剂A
溶剂B
溶剂A
溶剂B
电磁阀A
电磁阀B
高压泵A
高压泵B
混合室
混合室
泵
Low-pressure mixing
High-pressure mixing
4.3.3 Injection system
Injection valve
4.3.4 Column
 Size
 Packing
materials
 Packing technology
4.3.5 Detectors

UV-detector (紫外检测器）

Differential
refractive
index
detector
RI
（示差折光检测器）

Fluorescence Detector (荧光检测器)

Electrical conductivity detector (电导检测器)

UV detector
Liquid flow cell for UV detection in HPLC
 Single
wavelength detector
（固定波长的紫外检测器）
 Multiwavelength
detector
（可变波长的紫外检测器）
 Photo-diode
array detector
（光电二极管阵列检测器）
Single wavelength detector
Multiwavelength detector
DAD (二极管阵列) detector
3-D figure of photodiode-array detector
(二极管阵列检测器之三维图)
Example of Selecting different
wavelength
Characters of UV-detector

Sample should be UV-absorption

High sensitivity

Large linear range

Suitable for gradient elution

Most widely used
 Refractive index detector
(折光检测器)
Properties of RI detector

Non-specific

Low-sensitivity

Easily affected by temperature

Unsuitable for gradient elution
 Fluorescence detector
Properties of Fluorescence detector

High selectivity ( for protein, drug)

High sensitivity
 Electrochemical detector
(电化学检测器)
Properties of Electrochemical detector


Selective to electro-active subject
Easily affected by some surfaceactive substances
 Comparison of the detectors
RI
UV
Flu.
EC
selectivity
General
Special
High
Special
Special
Gradient
elution
no
yes
yes
no
Linear range
104
105
103
104
Detection limit
μg
ng
pg
ng
high
low
low
middle
Unlimited
limited
limited
limited
Sensitive to
temperature
Property of
mobile phase
Glossary








Pump
Valve
Loop
Gradient elution
Refractive index
Fluorescence
Cell
DAD (photodiode array detector)
4.4 Types of HPLC (HPLC modules)
 Chemically
bonded phase HPLC
 Adsorption HPLC
 Ion pair chromatography
 Ion exchange and ion
chromatography
 Size exclusion chromatography
4.4.1 Chemically bonded
phase chromatography
(化学键合相色谱法)

Liquid-liquid
Partition
(液-液分配色谱）
chromatography

Normal phase liquid chromatography ： polar
stationary phase and less polar or nonpolar
mobile phase

Reverse phase liquid chromatography: Polar
mobile phase and less polar or nonpolar
stationary phase

Disadvantage of the partition chromatography

Solid phase of chemical-bonded phase Chro.
(1) Reversed phases: silica gel surface is
modified with alkyl group(non-polar, such
as n-octadecyl，C18), becoming hydrophobic.
Si
固
定
相
表
面
OH
O
Si
OH
O
Si
O
OH
C18H37SiCl3
固
定
相
表
面
Si
O
Si
O
Si
O
O
O
O
Si
C18H37
(2) More about the solid phase of RP-HPLC
• Principle of the retaining of the analyte
• Alkyl group and other groups
• Stability of the solid phase
(3) Normal phase: polar functional groups
chemically modified on the surface of
silica gel
The group could be diol, cyano, amino, etc.
 Mobile phase of HPLC
①
General rule
Mobile phase play an important role on k
Suitable k is between 2 and 5
②
Eluotropic series
Polarity and polarity index of solvents
Polarity of solvent mixtures
③
Elution strength
Relationship between polarity of solvent
used as mobile phase and solid phase
Elution behavior of reversed phase
chro. and normal phase chro.
④
Other properties of solvent used as mobile
phase:
chemical stable
compatible to detector
low viscosity (粘度)
others (boiling point, toxicity and
expense)
⑤
Strategy of choosing mobile phase
Optimizing k and α
through experience or systematic
procedure according to some rules
(typical solvents trial).
⑥
In reversed-phase Chro.: mixture of
the solvents methanol, acetonitrile
and tetrahydrofuran with water.
In normal-phase chro.：mixture of
diethyl ether, methylene chloride
and chloroform with n-hexane
⑦
Isocratic elution and Gradient elution
Troubles of gradient elusion (column,
detector and pump)
4.4.2 Adsorption chromatography of
HPLC (liquid-solid chromatography)
Mechanism of the separation
By using solid absorbent as stationary phase and solvent as
mobile phase, the solute of being analyzed could be absorbed
by the active centers on the surface of the stationary phase and
then desorbed by the solvent.
Difference of this procession
could cause the separation of different analyte.
Solid phase
Polar phase：silica gel，alumina etc.
non-polar phase：active carbon, polymer
micro-bead
Mobile phase
 ε°
of the solvent
 Mixture
of the n-alkyl( 正 构 烷
烃）and methylene chloride
 Effect
solvent
of
water
and
other
Effecting of water to the adsorption HPLC
（4）Application of adsorption HPLC

Suitable to nonpolar substances which are
difficult to dissolve in water, such as
fattiness and oil

Positional isomers or stereoisomers
Glossary









Chemically bonded phase HPLC
Reverse Phase HPLC (RP-HPLC)
Normal phase HPLC
Liquid-liquid Partition HPLC
C18,C8 ,-NH2, -CN
Alkyl group
Elution strength
Methanol, acetonitrile
absorbent
4.3.3 Ion pair chromatography
(离子对色谱法)
（1）Mechanism

If the analyte is ion（A+）, which can not be
retained by the solid phase, an counter ion (B-)
is added into the mobile phase to combine with
the ion（A+）,forming an neutral molecule
（ion-pair complex) and then retained by the
solid phase.

W

W


A  B  ( A  B )O
E AB
[ A  B  ]O
 
,

[ AW ][ BW ]


[ A  B ]O

K

E

[
B
AB
W ]，分配系数

[ AW ]
k

W
E AB  [ B ]

（2）Solid phase, moble phase and
ion-pair reagent

Solid phase：C18,C8

Mobile phase：water solution

Ion-pair reagent：(C4H9)4N+(四丁基铵
正离子)、十六烷基三甲基铵正离子，
ClO4-，十二烷基磺酸根等
4.4.4 Ion chromatography (离子色谱法)
and ion exchange chromatography
(1) Mechanism

Exchange equilibrium of the ion-exchangers：
cation exchange：resin—SO3-H++M+ = resin—SO3-M ++H
+
anion exchange ： resin—NR3+Cl-+X- = resin—NR3+X -+Cl

Retention
differences
depend
on
the
tendency
existing on the resin matrix, while the stronger
the tendency the bigger tR of the ion.

The detection of ion chromatography
Problems of detection of IC
Suppressor column：Adding a suppressor to decrease
the background conductivity of the elution and
increase the signal-noise ratio。
canion:
R—OH-+H+ CL-= R—Cl-+H2O
R—OH-+M+ CL-= R—Cl-+MOH
anion:
R—H+ + Na+OH-= R—Na++H2O
R—H+ + Na+Br-= R—Na++HBr
Single column：No need of the suppressor column, but
with special ion-exchangers and eluents.
（2）Solid Phase
ion-exchanger: Coating on supporter
or kind of ion-bonded phase ， the ion
exchangers
are
divided
exchangers
(strong/weak)
exchangers (strong/weak).
as
and
cation
anion
（3）Mobile phase

Suppressor column
Cation: HCl，HNO3
Anion: NaOH,NaHCO3/NaCO3。

Single column
Cation： HCl，HNO3(low concentration)；
Anion: Salt of benzoic acid(苯甲酸),
citric acid(柠檬酸) etc.
（4）Application of IC

Inorganic anion

Inorganic cation and organic acid, organic
base, sugar etc.

Name of the ion chromatography and
ion exchange chromatography
（5）Definition of
ion chromatography and
ion exchange
chromatography
Example of application
4.4.5 Size-exclusion
chromatography
（体积排阻色谱法）（SEC）
(1) Mechanism

Special retention reason: the analyte is
trapped into the pore of stationary phase.
Separation of molecules is due to their
different size --- excluded or retained
according to the diameter of gel-pores.
Separation of Size-exclusion Chromatography

Elution volume: VR
VR=V0+KD·Vp
K=0, completely excluded
K=1, free permeation
0<KD<1.0

Sample with different size (more than
10%).

Water soluble: Gel filtration
chromatography(凝胶过滤色谱法）（GFC），
analyzing protein.(多肽、蛋白质、核酸、多
糖).

Water insoluble: Gel permeation
chromatography (GPC) (凝胶渗透色谱法)，
analyzing Mole Weight of high polymer (高
聚物)（如聚乙烯、聚氯乙烯等）.
（2）Solid phase

Porous glass, silica particles,
polymers, polysaccharides(多聚糖）

Pore size of packing materials
and exclusion limit

Being hydrophobic or hydrophilic

Pressure limit
（3）Mobile phase

Sample-soluble

Solid phase and detector compatible

Buffers of water for GFC
Tetrahydrofuran for GPC
（4）Application of SEC


Separation and Analysis of big molecules
Distribution of Molecular Weight of polymer
example
Polyethylene glycol （聚乙二醇）
1.40000 2. 10000
3. 3000
4. 1000 5.500
4.4.6 Guide to liquid chromatography
mode selection

According to the analytical task

Concentration

interference (composition)

Information of the analyte

structure

molecular weight

acidity

solubility
 Guide to liquid chromatography mode
selection
water soluble---SEC, aqueous mobile phase
MW
＞2000 water insoluble---SEC，nonaqueous mobile phase
water insoluble ---bonded phase (RP), adsorption
Sample
MW
＜2000
water soluble, nonionic---bonded phase (RP)
acid---anion exchange/ion pair
water soluble，ionic
basic---cation exchange/ion pair

HPLC chromatographic conditions

Column

Mobile phase

Detectors and conditions

Preparation of sample
Glossary











Ion pair chromatography
Counter-ion
Ion pair reagent
Ion chromatography
ion-exchange chromatography
Resin
Cation
Anion
Suppressor column
Size-exclusion chromatography
gel-filtration and gel permeation















Displacement pump 活塞泵
diaphragm 隔膜
Hydraulic 水力的 液压的
Cartridge 柱 桶
Alkyl 烷基
Elutropic 洗脱的
Chiral 手性
isophthalic acid 异酞酸
Polysaccharid [生化]多醣,聚糖,多聚糖
Divinylbenzene [化]二乙烯基苯
Copolymerisate 共聚物
Homolog 同系物
Oligomer 低聚物
dextrane [化][药] 右旋糖苷
Phosphorescence 磷光



Aliphatic〈化〉脂肪族的
Cuvette 小玻璃管, 透明小容器, 试管
Phenanthrene [化]菲