High- Performance Liquid Chromatography (HPLC)(1).pptx

High performance liquid chromatography

1. High- Performance Liquid Chromatography (HPLC) Early liquid chromatography was performed in glass columns having inside diameters of 10-50mm, 50-500 cm lengths and using packing particle size of 150-200mm. RECALL : relationship between Resolution and height equivalent to a theoretical plate ( HETP). Increased column length improves band separation (resolution)

2. These types of columns were characterized by slow flow rates . In late 1960s, technology was developed for producing and using packings with particle diameters as small as 3 – 10mm . This technology required instruments capable of much higher pumping pressures than the simple devices that preceded them . HPLC is the most versatile and widely used type of elution chromatography . The technique is used for separating and determining species in a variety of organic, inorganic, and biological materials .

3. HPLC uses high pressure to force solvent through closed columns containing fine particles that give high-resolution separations . The name high-performance liquid chromatography (HPLC) is often used to distinguish this technology from the simple column chromatographic procedures that preceded them

4. Types of HPLC The types of HPLC are often classified by separation mechanisms or by the type of stationary phase. Partition or liquid-liquid chromatography Adsorption or liquid-solid chromatography Ion exchange or ion chromatography Size-exclusion chromatography Affinity chromatography Chiral chromatography

5. Forms of HPLC There are two forms of HPLC – normal phase HPLC and reversed phase HPLC. In normal phase HPLC , the stationary phase is silica and the mobile phase is a non-polar solvent like hexane . In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar.

6. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an n - octyl (C8) or n - octyldecyl (C18) hydrocarbon chain . Most reverse phase separations are carried out using a buffered aqueous solution as a polar mobile phase . Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5

7. HPLC Instrumentation Pumping pressures of several hundred atmospheres are required to achieve reasonable flow rates with packings in the 3- to 10-mm size range. Because of these high pressures, the equipment for high-performance liquid chromatography tends to be considerably more elaborate and expensive .

8. HPLC components include: Solvent reservoirs and treatment systems Pumps Injector Columns Detectors

9. Solvent Reservoirs & Treatment Systems A modern HPLC instrument is equipped with one or more glass reservoirs , each of which contains 500 mL or more of a solvent. Dissolved gases can lead to irreproducible flow rates and band spreading . In addition, both bubbles and dust interfere with the performance of most detectors. HPLC equipment therefore usually provide for removal of dissolved gases and dust from the liquids .

10. Degassers can consist of vacuum pumping system, distillation system, a device for heating and stirring, or a system for sparging – in which dissolved gases are swept out of solution by fine bubbles of an inert gas that is not soluble in the mobile phase.

11. An elution with a single solvent or solvent mixture of constant composition/concentration is termed an isocratic elution . In gradient elution , two or more solvent systems that differ significantly in polarity are used and varied in composition during the separation. The ratio of the two solvents is varied in a preprogrammed way, sometimes continuously and sometimes in a series of steps.

12. Pumping Systems The requirements for liquid chromatographic pumps include: The generation of pressures of up to 6000 psi ( lb /in 2 ) Pulse-free output Flow rates ranging from 0.1 to 10 ml/min Flow reproducibilities of 0.5% relative or better Resistance to corrosion by a variety of solvents NOTE : Usually high pressures generated by liquid chromatographic pumps are not an explosion hazard because liquids are not very compressible.

13. Pumping Systems Many commercial instruments are equipped with computer-controlled devices for measuring the flow rate by determining the pressure drop across a restrictor located at the pump outlet. Any difference in signal from a preset value is then used to increase or decrease the speed of the pump motor.

14. Sample Injection Systems Many HPLC instruments incorporate an autosampler with an automatic injector. These injectors can introduce continuously variable volumes from containers on the autosampler.

15. HPLC Columns Liquid chromatographic columns are usually constructed from stainless steel tubing , although glass and polymer tubing, such as polyetheretherketone (PEEK), are sometimes used. Stainless steel columns lined with glass or PEEK are also available.

16. Analytical Columns Most columns range in length from 5 to 25 cm and have inside diameters of 3 to 5 mm. Straight columns are used . The most common particle size of packings is 3 or 5 mm. Columns of this type provide 40,000 to 70,000 plates/ metre .

17. Precolumns Two types of precolumns are used. A precolumn between the mobile phase reservoir and the injector is used for mobile-phase conditioning and is termed a scavenger column . The solvent partially dissolves the silica packing and ensures that the mobile phase is saturated with silicic acid prior to entering the analytical column. This saturation minimizes losses of the stationary phase from the analytical column.

18. A second type of precolumn is a guard column , positioned between the injector and the analytical column. A guard column is a short column packed with a similar stationary phase as the analytical column. The purpose of the guard column is to prevent impurities, such as highly retained compounds and particulate matter, from reaching and contaminating the analytical column. The guard column is replaced regularly and serves to increase the lifetime of the analytical column.

19. HPLC Detectors The ideal detector for HPLC should have all the characteristics of the ideal GC detector except that it need not have as great a temperature range. The detector should also be small and compatible with liquid flow. Unfortunately, no highly sensitive, universal detector system is available for high-performance liquid chromatography. Thus, the detector used will depend on the nature of the sample .

20. The most widely used detectors for liquid chromatography are based on absorption of ultraviolet or visible radiation . Both photometers and spectrophotometers, specifically designed for use with chromatographic columns, are available from commercial sources. However, spectrophotometric detectors are considerably more versatile than photometers. The combination of HPLC with a mass spectrometry detector (LC/MS) produces a very powerful analytical tool.

21. HPLC Detectors Detector Applications Detection Limits UV-Visible Absorbance For compounds that absorb in the UV or visible range pg quantities Fluorescence For compounds capable of fluorescence (especially polyaromatic hydrocarbons) fg quantities Refractive Index (RI) For alcohol, sugar, saccharide, fatty acid, and polymer analysis with refractive indices different from the solvent ng quantities Electrochemical For analyzing a wide range of compounds high pg quantities Conductivity for IC Mainly for inorganic ions ~ng quantities

22. Detector Applications Detection Limits Evaporative Light Scattering (ELS) For a wide variety of compounds that lack UV/Vis chromophores including triglycerides, sugars and natural products μ g quantities Fourier Transfer Infrared For compounds with vibrational functional groups μ g quantities Mass Spectrometry A universal detector μg to pg quantities depending on the type of mass spectrometer

23. Separations Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time High Performance Liquid Chromatograph

24. Separations Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time

25. 26 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

26. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

27. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

28. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

29. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

30. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

31. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

32. 33 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

33. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

34. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

35. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

36. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

37. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

38. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

39. 40 The Chromatogram Injection t o t R mAU time t R t o - elution time of unretained peak t R - retention time - determines sample identity Area or height is proportional to the quantity of analyte.

40. The Chromatogram Injection t o t R mAU time t R t o - elution time of unretained peak t R - retention time - determines sample identity Area or height is proportional to the quantity of analyte.

41. Comparison of HPLC and GC Sample Volatility Sample Polarity HPLC No volatility requirement Sample must be soluble in mobile phase GC Sample must be volatile HPLC GC Separates both polar and non polar compounds PAH - inorganic ions Samples are nonpolar and polar

42. Comparison of HPLC and GC Sample Thermal Lability Sample Molecular Weight HPLC Analysis can take place at or below room temperature GC Sample must be able to survive high temperature injection port and column HPLC GC No theoretical upper limit In practicality, solubility is limit. Typically < 500 amu

43. Comparison of HPLC and GC Sample Preparation Sample Size HPLC Sample must be filtered Sample should be in same solvent as mobile phase GC Solvent must be volatile and generally lower boiling than analytes HPLC GC Sample size based upon column i.d. Typically 1 - 5 L

44. Comparison of HPLC and GC Separation Mechanism Detectors HPLC Both stationary phase and mobile phase take part GC Mobile phase is a sample carrier only HPLC GC Most common UV-Vis Wide range of non- destructive detectors 3-dimensional detectors Sensitivity to fg (detector dependent) Most common FID, universal to organic compounds

45. HPLC Applications Chemical Environmental Pharmaceuticals Consumer Products Clinical polystyrenes dyes phthalates tetracyclines corticosteroids antidepressants barbiturates amino acids vitamins homocysteine Bioscience proteins peptides nucleotides lipids antioxidants sugars polyaromatic hydrocarbons Inorganic ions herbicides