High Performance Liquid
Chromatography
Zhang Tao
E-mail:zhangt760@nenu.edu.cn
How much do you know
about HPLC?
What is HPLC?
 High Performance Liquid Chromatography
 High Pressure Liquid Chromatography (usually true)
 Hewlett Packard Liquid Chromatography (a joke)
What is HPLC?
 High Priced Liquid Chromatography (no joke)
 HPLC is really the automation of traditional liquid
chromatography under conditions which provide for
enhanced separations during shorter periods of time!
 Probably the most widely practiced form of
quantitative, analytical chromatography practiced
today due to the wide range of molecule types and
sizes which can be separated using HPLC or variants
of HPLC!!
LC Origins.
Michael Tswett (1906) separation of plant
pigments by organ solvent mobile phase & chalk
stationary phase.
Martin and Synge (1941) liquid-liquid partition
chromatography, 1952 Nobel Prize in chemistry.
Other variants – Paper chromatography
Thin-layer chromatography
(TLC)
Preparative column
chromatography
CHM 101/102
Chromatography II: HPLC
Hewlett-Packard
Series 1100 HPLC
compounds that are not
dissolved in solution must be
removed
three kinds of chemical compound
water
methanol
caffeine
(very polar)
(polar)
(fairly nonpolar)
Laboratory Manual
HPLC components:
Liquid
Mobile => Pump => Injection => Separation
Phase
Valve
Column
Detector
Also an integrator usually records the
detector response.
COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM








Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors
Waste Collector
Recorder (Data Collection)
Introduction
 HPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution.
HPLC instruments consist of mobile phase, a pump,
an injector, a separation column, and a detector.
 Compounds are separated by injecting a sample
mixture onto the column. The different component in
the mixture pass through the column at
differentiates due to differences in their partition
behavior between the mobile phase and the
stationary phase. The mobile phase must be
degassed to eliminate the formation of air bubbles.
Uses of HPLC
This technique is used for chemistry and
biochemistry research analyzing complex
mixtures, purifying chemical compounds,
developing processes for synthesizing
chemical compounds, isolating natural
products, or predicting physical properties.
It is also used in quality control to ensure
the purity of raw materials, to control and
improve process yields, to quantify assays
of final products, or to evaluate product
stability and monitor degradation.
And more using…
HPLC columns
The column is one of the most important
components of the HPLC chromatograph
because the separation of the sample
components is achieved when those
components pass through the column.
The High performance liquid
chromatography apparatus is made out
of stainless steel tubes with a diameter
of 3 to 5mm and a length ranging from
10 to 30cm.
Picture of an HPLC column
WHAT AFFECTS SYSTEM
Column Parameters
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Column Material
Deactivation
Stationary Phase
Coating Material
Instrument Parameters





Temperature
Flow
Signal
Sample Sensitivity
Detector
Sample Parameters




Concentration
Matrix
Solvent Effect
Sample Effect
Several column types
(can be classified as )
Normal phase
Reverse phase
Size exclusion
Ion exchange
Normal phase
In this column type, the retention is
governed by the interaction of the
polar parts of the stationary phase
and solute. For retention to occur in
normal phase, the packing must be
more polar than the mobile phase
with respect to the sample
Reverse phase
In this column the packing material is
relatively nonpolar and the solvent is
polar with respect to the sample.
Retention is the result of the
interaction of the nonpolar
components of the solutes and the
nonpolar stationary phase. Typical
stationary phases are nonpolar
hydrocarbons, waxy liquids, or
bonded hydrocarbons (such as C18,
C8, etc.) and the solvents are polar
water-organic mixtures such as
methanol-water .
Size exclusion
In size exclusion the HPLC column is
consisted of substances which have
controlled pore sizes and is able to be
filtered in an ordinarily phase
according to its molecular size. Small
molecules penetrate into the pores
within the packing while larger
molecules only partially penetrate the
pores. The large molecules elute
before the smaller molecules.
Ion exchange
In this column type the sample
components are separated based
upon attractive ionic forces between
molecules carrying charged groups of
opposite charge to those charges on
the stationary phase. Separations are
made between a polar mobile liquid,
usually water containing salts or
small amounts of alcohols, and a
stationary phase containing either
acidic or basic fixed sites.
Types of Detectors
 Absorbance (UV
with Filters, UV
with
Monochromators)
(widely using )

Fluorescence
 Refractive-Index
 Evaporative Light
Scattering
Detector (ELSD)
(the most
advanced
detectors


Electrochemical
MassSpectrometric
 Photo-Diode Array
What does the analyst do?
Select the correct type of separation for the
analyte(s) of interest, based on the sample
type (among other factors).
Select an appropriate column (stationary
phase) and mobile phase
Select an appropriate detector based on
whether universal or compound-specific
detection is required or available
Optimize the separation using standard
mixtures
Analyze the standards and sample
Optimization of Separations in HPLC
 Correct choice of column so the above equilibrium
has some meaningful (non-infinity, non-zero)
equilibrium constants.
 Correct choice of mobile phase
 Decision on the type of mobile phase composition
 constant composition = isocratic
 varying composition = gradient elution
 Determination if flow rate should be constant
 usually it is
 Decision on heating the column
 heating HPLC columns can influence the above
equilibrium….
The Mobile Phase in HPLC...
 Must do the following:
 solvate the analyte molecules and the solvent they are in
 be suitable for the analyte to transfer “back and forth”
between during the separation process
 Must be:
 compatible with the instrument (pumps, seals, fittings,
detector, etc)
 compatible with the stationary phase
 readily available (often use liters/day)
 of adequate purity
• spectroscopic and trace-composition usually!
 Not too compressible (causes pump/flow problems)
• Free of gases (which cause compressability problems)
Sugar composition analysis
Molecular weight analysis
Reverse phase C18
Sugar composition analysis
1、Polysaccharide samples (2 mg) were hydrolyzed
first with anhydrous methanol containing 1 M HCl
at 80 ℃for 16 h
2、2 M TFA at 120 ℃ for 1 h.
3、The resulting hydrolysates were derivatized with
1-phenyl-3-methyl-5-pyrazolone (PMP) according
to the method in the literature
4、Analyzed on a DIKMA Inertsil ODS-3 column
(4.6×150 mm) connected to a Shimadzu HPLC
system(LC-10ATvp pump and SPD-10AVD UV–VIS
detector).
5、The PMP derivative(20µL) was injected, eluted
with 82.0% PBS (0.1 M, pH 7.0) and 18.0%
acetonitrile (v/v) at a flow rate of 1.0 mL/min
and monitored by UV absorbance at 245 nm.
40
20
9
10
11
12
13
14
120
100
23.690
15
16
16
17
17
18
18
19
19
20
时间(min)
340
300
280
260
240
220
200
180
160
140
60
0
时间(min)
20
21
22
21
22
23
24
25
25
26
26
27
27
28
28
29
29
30
36.223
24
34.223
23
31.157
30
25.865
28.898
23.765
40
28.890
28.157
Glucose
24.632
9.815
Mannose
25.757
10
20.565
Glucuronic
acid
22.657
320
20.457
15
18.923
14
17.790
13
16.673
12
15.698
15
15.173
20
14.398
电压(mv)
16.698
12.632
Rhamnos
e
15.690
80
11
13.598
25
14.357
10
13.607
9
11.798
8.982
8.182
7.148
7.315
7.698
6.432
3.690
70
12.657
2.890
75
11.773
电压(mv)
5.490
5.982
5
9.807
4.057
4.390
3.007
3.207
35
11.007
3.698
3.015
45
8.957
7.807
7.373
6.415
5.490
5.857
4.190
4.390
4.757
4.965
2.890
C18 Column Reverse phase
2.640
80
Galacturonic
acid
65
60
55
50
Galactose
Fucose
Xylose
Arabinose
0
30
Molecular weight analysis
High performance gel permeation chromatography was
carried out at 40 ℃ using a TSK-gel G-3000PWxl
column (7.8×300 mm,TOSOH, Japan) connected to a
Shimadzu HPLC system. The column was pre-calibrated
with standard dextrans. Twenty microliters of sample (5
mg/mL) was injected, eluted with 0.2 M NaCl at a flow
rate of 0.6 mL/min and monitored using a refractive
index RID-10A detector (Shimadzu, Tokyo, Japan).
standard dextrans curve
y = -0.2903x + 1.8843
R2 = 0.9965
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Kav
线性
(Kav)
0
2
4
6
8
Thank You!