Introduction to High Performance Liquid Chromatography-HPLC

1. Presented by: Mehdi Soleymani Goloujeh Supervisor: Dr. S. Davaran

2. Outlook:  History  Introduction  The components of the high performance liquid chromatograph  What affects system  The separation process  The chromatogram  The most common modes of HPLC  HPLC Applications  The differences between HPLC and GC  Advantages of High Performance Liquid Chromatography Mehdi Soleymani 2 Medical Nanotechnology Department

3. 3 Ether Chlorophyll CaCO3 Chromatography Colors Mehdi Soleymani Medical Nanotechnology Department

4. 4 Water flow Base Light leaf Heavy stone Mehdi Soleymani Medical Nanotechnology Department

5. 5 Chromatography in which the mobile phase is a liquid.  The liquid used as the mobile phase is called the “eluent”. The stationary phase is usually a solid or a liquid. In general, it is possible to analyze any substance that can be stably dissolved in the mobile phase. Mehdi Soleymani Medical Nanotechnology Department

6. 6 Chromatography: Analytical technique Chromatograph: Instrument Chromatogram: Obtained “picture” Chromatographer: Person Mehdi Soleymani Medical Nanotechnology Department

7. 7  Higher degree of separation!  Refinement of packing material (3 to 10 μm) Reduction of analysis time!  Delivery of eluent by pump  Demand for special equipment that can withstand high pressures The arrival of high performance liquid chromatography! Mehdi Soleymani Medical Nanotechnology Department

8.  HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir 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. Mehdi Soleymani 8 Medical Nanotechnology Department

9. Mehdi Soleymani 9 Medical Nanotechnology Department

10. 10 Isocratic system Constant eluent composition Gradient system Varying eluent composition  HPGE (High Pressure Gradient)  LPGE (Low Pressure Gradient) Mehdi Soleymani Medical Nanotechnology Department

11. 11 Mixer High-pressure gradient Low-pressure gradient unit Mixer Low-pressure gradient Mehdi Soleymani Medical Nanotechnology Department

12. 12 Principle Pattern An Example Solvent Cabinet Vacuum Degasser Binary Pump Autosampler Thermostatted Column Compartment Detector Solvent Reservoirs Controller Mehdi Soleymani Medical Nanotechnology Department

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.  Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, is inert to most compounds and has a high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible. Mehdi Soleymani Medical Nanotechnology Department 13

15. 15 Problems caused by dissolved air in the eluent  Unstable delivery by pump  More noise and large baseline drift in detector cell In order to avoid these problems, the eluent must be degassed. Mehdi Soleymani Medical Nanotechnology Department

16.  The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column. When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered to the vial. Mehdi Soleymani Medical Nanotechnology Department 16

17. 17 Front View Inject Rear View Load - Inject Sample Loop Mehdi Soleymani Medical Nanotechnology Department

18.  Absorbance (UV with Filters, UV with Monochromators)  IR Absorbance  Fluorescence  Refractive-Index  Evaporative Light Scattering Detector (ELSD)  Electrochemical  Mass-Spectrometric  Photo-Diode Array Mehdi Soleymani 18 Medical Nanotechnology Department

19. 19 Carbohydrates 1. fructose 2. Glucose 3. Saccharose 4. Palatinose 5. Trehalulose 6. isomaltose Zorbax NH2 (4.6 x 250 mm) 70/30 Acetonitrile/Water 1 mL/min Detect=Refractive Index 1 2 3 4 5 mAU time 6 Mehdi Soleymani Medical Nanotechnology Department

20. Waste Mehdi Soleymani 20 Medical Nanotechnology Department Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Separation in based upon differential migration between the stationary and mobile phases. Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column.

21. 21 Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time High Performance Liquid Chromatograph Mehdi Soleymani Medical Nanotechnology Department

24. 24 Injector Detector Column Pumps Solvents Mixer Chromatogram Start Injection mAU time High Performance Liquid Chromatograph Mehdi Soleymani Medical Nanotechnology Department

38. 38 to Injection tR mAU tR time to - elution time of unretained peak tR- retention time - determines sample identity Area or height is proportional to the quantity of analyte. Mehdi Soleymani Medical Nanotechnology Department

39. 39 Mobile Phases Flow Rate Composition Injection Volume Column Oven Temperature Wavelength Time Constant Mehdi Soleymani Medical Nanotechnology Department

40. Column Parameters  Column Material  Deactivation  Stationary Phase  Coating Material Instrument Parameters  Temperature  Flow  Signal  Sample Sensitivity  Detector Mehdi Soleymani 42 Medical Nanotechnology Department

41. Sample Parameters  Concentration  Matrix  Solvent Effect  Sample Effect Mehdi Soleymani 43 Medical Nanotechnology Department

42. Normal phase Reverse phase  Size exclusion  Ion exchange Mehdi Soleymani 44 Medical Nanotechnology Department

43.  Normal Phase. - Polar stationary phase and non-polar solvent. • Reverse Phase. - Non-polar stationary phase and a polar solvent. Mehdi Soleymani Medical Nanotechnology Department 45

44. 44 Types of Compounds Mode Stationary Phase Mobile Phase Neutrals Weak Acids Weak Bases Reversed Phase C18, C8, C4 cyano, amino Water/Organic Modifiers Ionics, Bases, Acids Ion Pair C-18, C-8 Water/Organic Ion-Pair Reagent Compounds not soluble in water Normal Phase Silica, Amino, Cyano, Diol Organics Ionics Inorganic Ions Ion Exchange Anion or Cation Exchange Resin Aqueous/Buffer Counter Ion High Molecular Weight Compounds Polymers Size Exclusion Polystyrene Silica Gel Filtration- Aqueous Gel Permeation- Organic Mehdi Soleymani Medical Nanotechnology Department

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

46. I need a quantitative separation of carbohydrates in some of our products as soon as possible. I’ll need a separation technique. Mehdi Soleymani 46 Medical Nanotechnology Department I’ll get on it!

47. I have two separation techniques in my lab, High Performance Liquid Chromatography and Gas Chromatography. Which one should I use? Mehdi Soleymani 47 Medical Nanotechnology Department

48. 48  High separation capacity, enabling the batch analysis of multiple components  Superior quantitative capability and reproducibility  Moderate analytical conditions  Unlike GC, the sample does not need to be vaporized.  Generally high sensitivity  Low sample consumption  Easy preparative separation and purification of samples Mehdi Soleymani Medical Nanotechnology Department

50. 50  http://192.215.107.101/ebn/942/tech/techfocus/1071main.html  http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.html  Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed. Orlando: Harcourt Brace & Co., 1998.  http://weather.nmsu.edu  http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm  http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html  http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLCHP1090/H PLCINJ.HTM  http://test-equipment. globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytical_Instru ments/Chromatographs/HPLC_Columns  http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm

Introduction to High Performance Liquid Chromatography-HPLC

Royan Institute

Introduction to High Performance Liquid Chromatography-HPLC