High Performance Liquid Chromatography

1. High Performance Liquid Chromatography Zhang Tao E-mail:zhangt760@nenu.edu.cn

2. How much do you knowabout HPLC?

3. What is HPLC? • High Performance Liquid Chromatography • High Pressure Liquid Chromatography (usually true) • Hewlett Packard Liquid Chromatography (a joke)

4. 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!!

5. LC Origins. Michael Tswett (1906) separation of plant pigments by organsolvent 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 Medium pressure chromatography Ion-exchange chromatography* Size-exclusion chromatography*

8. water methanol caffeine Chromatography II: HPLC compounds that are not dissolved in solution must be removed Hewlett-PackardSeries 1100 HPLC three kinds of chemical compound (very polar) (polar) (fairly nonpolar)

9. HPLC components: Liquid Mobile => Pump => Injection => Separation Phase Valve Column Detector Also an integrator usually records the detectorresponse.

10. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM • Solvent • Solvent Delivery System (Pump) • Injector • Sample • Column • Detectors • Waste Collector • Recorder (Data Collection)

11. 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.

12. 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…

13. 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. HPLC columns

14. Picture of an HPLC column

15. Column Parameters Column Material Deactivation Stationary Phase Coating Material WHAT AFFECTS SYSTEM InstrumentParameters • Temperature • Flow • Signal • Sample Sensitivity • Detector

16. Sample Parameters • Concentration • Matrix • Solvent Effect • Sample Effect

17. Normal phase Reverse phase Size exclusion Ion exchange Several column types(can be classified as )

18. 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 Normal phase

19. 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 . Reverse phase

20. 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. Size exclusion

21. 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. Ion exchange

22. Absorbance (UV with Filters, UV with Monochromators) (widely using ) Fluorescence Refractive-Index Types of Detectors • Evaporative Light Scattering Detector (ELSD) (the most advanced detectors • Electrochemical • Mass-Spectrometric • Photo-Diode Array

23. 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

24. 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….

25. 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) The Mobile Phase in HPLC...

26. Sugar composition analysis Molecular weight analysis

27. 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. Reverse phaseC18

28. Galacturonic acid Rhamnose Galactose Mannose Fucose Xylose Glucuronic acid Arabinose Glucose C18 Column Reverse phase

29. 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). Molecular weight analysis

31. standard dextrans curve

32. Thank You!