high performance liquid chromatography

High Performance Liquid

Chromatography Zhang TaoE-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. LC Origins. Michael Tswett (1906) separation of plant pigments by Michael Tswett (1906) separation of plant pigments by

organorgan solvent mobile phase & chalk stationary phase. solvent mobile phase & chalk stationary phase. Martin and Synge (1941) liquid-liquid partition Martin and Synge (1941) liquid-liquid partition chromatography, 1952 Nobel Prize in chemistry. chromatography, 1952 Nobel Prize in chemistry. Other variants – Paper chromatography Other variants – Paper chromatography Thin-layer chromatography (TLC) Thin-layer chromatography (TLC) Preparative column chromatography Preparative column chromatography Medium pressure chromatography Medium pressure chromatography Ion-exchange chromatography* Ion-exchange chromatography* Size-exclusion chromatography* Size-exclusion chromatography*

CHM 101/102CHM 101/102

Laboratory ManualLaboratory ManualLaboratory Manual

Chromatography II: HPLCChromatography II: HPLC

Hewlett-PackardHewlett-PackardSeries 1100 HPLCSeries 1100 HPLC

caffeinecaffeinewaterwater methanolmethanol(very polar) (polar) (fairly nonpolar)

three kinds of chemical compound

compounds that are not dissolved in solution must be re

moved

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 columnPicture of an HPLC column

WHAT AFFECTS SYSTEM

Column Parameters

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

Mass-Spectrometric

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 C18Sugar composition analysis1 、 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.

(mi n)时间3029282726252423222120191817161514131211109

(mv)

电压

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

5

0

2.8

90 3

.007

3.2

07

3.6

90

4.0

57 4

.390

5.4

90

5.9

82

6.4

32

7.1

48 7

.315

7.6

98

8.1

82

8.9

82

9.8

15

11.7

98

12.6

32

13.5

98

14.3

98

15.6

98

16.6

98

20.5

65

23.7

65

24.6

32

25.8

65

28.8

98

Rhamnose

Glucose

Galacturonic acid

Glucuronicacid

Galactose

Xylose

Arabinose

FucoseMannose

(mi n)时间3029282726252423222120191817161514131211109

(mv)

电压

340

320

300

280

260

240

220

200

180

160

140

120

100

80

60

40

20

0

2.6

40 2

.890

3.0

15

3.6

98

4.1

90 4

.390

4.7

57 4

.965

5.4

90

5.8

57

6.4

15

7.3

73

7.8

07

8.9

57

9.8

07

11.0

07

11.7

73

12.6

57

13.6

07

14.3

57

15.1

73

15.6

90

16.6

73

17.7

90

18.9

23

20.4

57

22.6

57

23.6

90

25.7

57

28.1

57

28.8

90

31.1

57

34.2

23

36.2

23

41.0

90

C18 C

olumn R

everse phase

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

00. 1

0. 20. 30. 4

0. 50. 60. 7

0. 80. 9

0 2 4 6 8

Kav

线性(Kav)

Thank You!