2. Chromatography is one of widely used separation techniques of chemical mixtures, exploiting differences in their physical/ chemical properties. All chromatography relies on the differential distribution of compounds between two phases, that are called stationary phase and mobile phase. Chromatography is used for qualitative and quantitative analysis and is also used for isolation.

5.  Two Phases are involved in a adsorption LC process: Stationary Phase -Solid porous, surface-active material in small- particle form. Mobile Phase -Liquid  Separation is achieved as a result of differences in the interaction between the different target compounds with the stationary phase

7.  Normal Phase Mode  Reverse Phase Mode  Reverse Phase Ion Pairing Mode  Ion Exchange Mode  Chiral Separation Mode

8. Normal Phase Mode zColumn (Stationary Phase): Polar zSolvent (Mobile Phase): Non polar

9. Normal Phase HPLC Column zSilica gel Type: General use zCyano Type: General use zAmino Type: For Sugar Analysis zDiol Type: For Protein Analysis

10. Stationery Phase for Normal Phase (Silanols) HPLC Column

11. Mobile Phase for Normal Phae yPrimary solvents(non-polar) -Hydrocarbons (Pentane, Hexane, Heptane, Octane) -Aromatic Hydrocarbons (Benzene, Toluene, Xylene) -Methylene chloride -Chloroform -Carbon tetrachloride ySecondary solvents -Methyl-t-butyl ether (MTBE), Diethyl ether, THF, Dioxane, Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-propaol, ethanol, methanol 4A primary solvent is used as mobile phase. Addition of secondary solvents is to adjust retention time.

12. What is the Interaction

14. Hydrogen Bonding  If the sample has -COOH, -NH 2 and -OH Hydrogen bonding become strong  If the sample has balk group -due to stereo Hindrance, Hydrophobic group, non-polar part, long chain of hydrocarbon. -Hydrogen bonding become weak.

15. Retention Time and Hydrogen Bonding

16. Reverse Phase Mode zColumn (Stationary Phase): Non Polar zSolvent (Mobile Phase): Polar

17. Reverse Phase HPLC Column.C18 (ODS) Type.C8 (Octyl) Type.C4 (Butyl) Type.Phenyl Type.TMS Type.Cyano Type.Amino Type

18. Stationary Phase for Reverse Phase (C18)HPLC Column CH 3 | -0-Si-(CH 2 ) 17 -CH 3 | CH 3

19. Mobile Phase for Reverse Phase zWater (buffer) + Organic Solvents -When buffer is used, the concentration and pH are important factors -Methanol, Acetonitrile or THF are common organic solvents for RP HPLC 4Optimization of water (buffer) and organic solvents ratio is very important

20. What is the Interaction

21. Hydrophobicity zIf the sample has balk group -due to stereo Hindrance, Hydrophobic group, non-polar part, long chain of hydrocarbon. Hydrophobicity become strong. zIf the sample has -COOH, -NH 2 and -OH Hydrophobicity become weak.

22. Retention Time and Hydrophobicity

23. Ion Pairing Reverse Phase Mode

26. Effect of pH on analysis

30. Choice of LC mode

31. System Suitability Repeatability,%RSD (CV) Capacity factor, K’ Selectivity/Relative Retention Time Theoretical Plates, N (Efficiency) Resolution Asymmetry/Tailing Factor

32. Repeatability, CV (%RSD) X=(X1+X2……..Xn-1+Xn)/n N=: Number of Analysis X1..Xn: Retention time (or area or heights) X: Average C.V: Coefficient of Variation

33. Capacity Factor, K’

34. Selectivity/Relative Retention Time, 

35. Theoretical Plate

36. Resolution

37. Tailing Factor,T T=W 0.05 /2f

38. HPLC Configuration Generally two types of HPLC configuration are possible *Isocratic System *Gradient system

39. Isocratic system

40. High Pressure Gradient system

41. Low Pressure Gradient system

42. Facilities of Gradient system zSome analysis required gradient mode zSave the solvents zSave labour zMethod development zAccuracy of Analysis

43. Instrumentation of HPLC Main ModulesAccessories zPump- Column Oven zInjector- Degasser zColumn zDetector zData Processor

44. Desirable HPLC  High Sensitive -Low pressure fluctuation -High sensitive detector  Reliable: Reproducibility - Precision of flow rate, injection volume, oven temperature, wavelength or applied potential and data transfer  Flexible: Modular Type

45. Solvent delivery pump

46. Desirable Pump performance oHigh Pressure Resistance Shimadzu LC-10ATvp- 400Kgf oPrecise Flow Rate Shimadzu LC-10ATvp- Within  0.3 (%RSD<0.1) oLow Pressure Fluctuation Shimadzu LC-10ATvp- 0.5 Mpa oHigh-accuracy gradient performance oPulse-free solvent delivery oValidation and Productivity

47. Desirable Pump performance

50. Automatic Sample Injector

51. Desirable Auto Sampler

53. z Performs various pretreatment functions, including addition of sample or reagent, mixing and diluting z Performs derivatization, addition of internal standard or sample dilution z transfer reagents and sample to an empty vial for pretreatment mixing

54. Desirable Auto Sampler

55. HPLC Detectors  Ultraviolet/Visible Detector (UV/Vis)  Photodiode Array Detector (PDA)  Fluorescence Detector (RF)  Conductivity Detector (CDD)  Refractive Index Detector(RID)  Electrochemical Detector (ECD)  Mass Spectrophotometer Detector (MS)

56. Desirable HPLC Detectors zHighly sensitive zLow Noise Level zWavelength Reproducibility zSimultaneous dual- wavelength measurement zWavelength programming zWavelength Scan mode

57. Desirable HPLC Detectors

60. Desirable Column Oven

61. Desirable On line Degasser

62. Desirable Chromatographic Workstation Software  Windows based  User friendly  Excellent Graphical Interface  GLP Compliance  Automated Sequential Run  System Suitability, QC Check  Auto baseline Check  Auto Validation Run  Auto Shutdown  Customize Report  Automation of Analytical Works  Lot of way to calculations  Easy to Upgrade  Easy to reinstall by the customer

63. Desirable Chromatographic Workstation Software