Introduction and principle of glc, hplc

Introduction and principle of glc, hplc
columns of hplc
columns of glc
detectors of glc
detectors of hplc
chromatography
classification of chromatography
gas liquid chromatography
high performance liquid chromatography

1. Introduction and principle of GLC, HPLC Vishnu Vardhan Reddy.P TVM/2015-029 Department of Animal nutrition College of Veterinary Science, Tirupati Sri Venkateswara Veterinary University

2. Chromatography Chromatography is an analytical technique where in a sample mixture under test is separated into different components based on difference in their affinity for a stationary phase and mobile phase.

3. General classification of chromatographic methods Column chromatography Paper chromatography Thin layer chromatography Gas chromatography High pressure liquid chromatography Ion exchange chromatography Gel filtration chromatography Super critical fluid chromatography

4. Gas Chromatography It is a technique where by the components of a mixture (sample) in the gaseous state are separated as the sample passes over a stationary liquid or solid phase and a gaseous mobile phase. Based on stationary phase G.C classified into two types. Gas Solid Chromatography (G.S.C) Gas Liquid Chromatography (G.L.C)

5. GLC Gas Liquid Chromatography

6. In gas-liquid chromatography the mobile phase is an unreactive gas , such as nitrogen (the carrier gas), and the stationary phase comprises of a small amount of non volatile liquid held on a finely-divided inert solid support. Gas Liquid Chromatography (G.L.C)

7. Gas liquid Chromatography Principle of Operation Gas liquid chromatography runs on the  principle of partition. In GLC the components of vaporize samples are fractionated due to partition between a gaseous mobile phase and a liquid stationary phase held in column.

8. Instrumentation Tank of carrier gas Flow regulator and flow meter Injection port Column Temperature controlled device Detector Microprocessor/recorder

9. The Mobile Phase (Carrier Gas) An inert gas such as He or N 2 Function is to transport sample vapors through column No chemical interaction with sample Typical parameters Column inlet pressure: 10-50 psi (above ambient) Flow rate: 25-50 mL/min (packed column) Precise control of carrier gas flow rate is critical to obtaining reproducible retention times.

10. Sample Injection Sample is injected using a syringe into a flowing stream of hot mobile phase High temperature (at least 50 o C above boiling point of sample) causes vaporization of sample Introduces a narrow plug of sample vapor onto the column Various designs For packed columns, inject 1 to 5 L of sample For capillary columns, a split valve is used to introduce a small fraction of sample onto column

11. Columns Column is heart of GC, which decides the separation efficiency. It is made up of glass or copper. Columns are two types based on it’s use: Analytical column: Length 1-2 mts , outer diameter 3-6 mm. Preparative column: Length 3-6 mts , outer diameter 6-9 mm.

12. Columns are two types based on nature: Packed column : Capillary columns:( Golay column) wall coated open tubular (WCOT) column porous layer open tubular (PLOT) column Support coated open tubular ( SCOT) columns

13. Wall Coated Open Tubular (WCOT ) column Internal wall of capillary is coated with a very fine film of liquid stationary phase . Surface Coated Open Tubular (SCOT ) column Capillary tube wall is lined with a thin layer of solid support on to which liquid phase is adsorbed . The separation efficiency of SCOT columns is more than WCOT columns because of increased surface area of the stationary phase coating. Fused Silica Open Tubular (FSOT ) column Walls of capillary fused silica tubes are strengthened by a polyimide coating. These are flexible and can be wound into coils .

14. Columns for Gas Chromatography Selection of appropriate column geometry and dimensions may be critical to a successful separation

15. Column Oven Precise control of column temperature. Column temperature should be slightly below the boiling points of the solutes (but above the dew point; i.e., no condensation ) For complex mixtures with a broad range of boiling points, use programmed temperature Precise control of oven temperature is critical to obtaining reproducible retention times.

16. Detectors Generate an electrical signal proportional to solute concentration or mass flow rate Ideal characteristics High sensitivity Rapid response time Non destructive technique Applicable to wide range of samples Easy to use Stable ,predictable response

17. Detectors for GC

18. May be universal or selective Universal (responds to wide range of solutes) Thermal conductivity detector (TCD) Simple , inexpensive and modest sensitivity. For greater sensitivity or selectivity Flame Ionization detector ( FID) Responds to most organic compounds High sensitivity and wide dynamic range (9 orders of magnitude). Electron capture detector (ECD) Selective and very sensitive for halogenated organics .

19. Thermal conductivity detector Element is electrically heated at constant power. Temperature depends on thermal conductivity of surrounding gas. Measure conductivity with respect to a reference. When analyte comes off, filament temperature goes up, resistance goes down . Thermal Conductivity Detector

20. Mechanism: A detector cell contains a heated filament with an applied current. As carrier gas containing solutes passes through the cell, change in the filament current occurs. The current change is compared against current in reference cell. The difference is measured and a signal is generated. Sensitivity: 5-20 ng . Selectivity: All compounds. Gases: H ydrogen , Helium . Temperature: 150-250 C

21. Flame Ionization Detector Column effluent is passed through a H 2 -air flame produces ions and electrons. Charged particles are accelerated by voltage applied between jet and collector-results in current Less sensitive to non hydrocarbon groups. Insensitive to H 2 o,Co 2 ,So 2 Flame Ionization Detector For most organic compounds

22. Mechanism: C ompounds are burned in a hydrogen-air flame. carbon containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Sensitivity: 0.1-10 ng . Selectivity: compounds with C-H bonds. Gases: combustion –hydrogen and air, makeup-He,N 2. Temperature: 250-300 C .

23. Electron capture detector Carrier gas (and analyte) passes over β -emitter, resulting in ionization and electron production. Produce current between electrodes. Most commonly used for halogenated organics.

24. Mechanism: Electrons are supplied from a 63Ni foil lining the detector cell. A current is generated in the cell. Electronegative compounds capture electrons resulting in a reduction in the current. The amount of current loss is indirectly measured and a signal is generated. Sensitivity: 0.1-10 ng . Selectivity: Halogens, nitrates. Gases: Nitrogen or argon. Temperature: 300-400 C

25. Nitrogen phosphorous detector Mechanism: compounds are burned in a plasma surrounding rubidium bead supplied with hydrogen and air. Nitrogen and phosphorous containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Sensitivity: 1-10 pg. Selectivity: Nitrogen phosphorous containing compounds. Gases: combustion- hydrogen ,make up – helium. Temperature: 250 -300 C .

26. Recorder Recorder is a device that draws the chromatogram that results from a chromatographic process onto chart paper. The device can have a full scale deflection (FSD ) voltage that commonly ranges from 1 mv to 10 v. The time scale of the chart movement normally ranges from about 1 cm per second to 1 cm per hour.

27. Advantages of GLC Both qualitative and quantitative analysis are possible. Instrument is simple ,time of analysis is short . High sensitivity. The method is applicable to about 60% of organic compounds. Very small samples sizes can be used. Analysis can be highly accurate and precise .

28. Factors affecting separation Particle size and surface area. Carrier gas flow rate. Type and amount of stationary phase. Column length. Column diameter. Column temperature .

29. Applications Of GLC In animal feed industry Quantitative and/or qualitative analysis of feed composition that is estimation of: A mino acids, hydroxyl (poly)carboxylic acids, fatty acids, phenolic compounds, sugars, vitamins, and many veterinary drugs, herbicides, and ‘‘natural’’ chemical toxins present in feed. Quantitative and/or qualitative analysis of feed additives. Estimation of flavor and aroma components in feed.

30. Estimation of spoilage components, such as histamine and carbonyls, that cause rancidity. Identification of contaminants , such as pesticides, fumigants, environmental pollutants, natural toxins, veterinary drugs, and packaging materials in animal feeds. Variety of transformation products like polycyclic aromatic hydrocarbons, heterocyclic amines, urethane, nitrosamines, chloropropanols, cholesterol oxides, irradiation products, microbial marker chemicals

31. Only the non-volatile compounds, such as inorganic salts, proteins, polysaccharides, nucleic acids, and other large molecular weight organics, are outside the realm of GLC.

32. HPLC High Performance Liquid Chromatography

33. High Performance Liquid Chromatography HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution . Mobile phase is Liquid Stationary phase is Solid or Liquid

34. Principle The process involves the interaction of the compounds in the analyte or sample across an immobile surface (stationary phase). The compounds bind at specific regions of stationary phase based on certain physical and chemical properties. These bound molecules are then eluted with a suitable buffer and the same are collected with time. The properties are – Polarity Charge Molecular weight Present of functional group

35. Types of HPLC There are many ways to classify liquid column chromatography based on the nature of the stationary phase and the separation process , three modes can be specified . A dsorption chromatography Here stationary phase is an adsorbent (like silica gel) and the separation is based on repeated adsorption-desorption steps .

36. Ion-exchange chromatography Here the stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.

37. Size exclusion chromatography Here the column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. This technique is also called gel filtration or gel permeation chromatography.

38. Concerning the Adsorption chromatography two modes are defined depending on the relative polarity of the two phases: Normal -phase chromatography The stationary bed is strongly polar in nature (e.g., silica gel) The mobile phase is nonpolar (such as n-hexane or tetrahydrofuran ). Polar samples are thus retained on the polar surface of the column packing longer than less polar materials .

39. Reversed-phase chromatography The stationary bed is nonpolar (hydrophobic) in nature. The mobile phase is a polar liquid , such as mixtures of water and methanol or acetonitrile. Here the more nonpolar the material is, the longer it will be retained .

40. Flow chart of HPLC mechanism

41. Instrumentation of HPLC Solvent (mobile phase) Solvent Delivery System (Pump) Injector Sample Column (stationary phase) Detectors (Diode Array) Waste Collector Recorder (Data Collection )

42. Solvent (mobile phase ) In normal phase t ypically non polar solvents such as hexane, heptane, iso -octane are used in combination with slightly more polar solvents such as isopropanol, ethyl-acetate or chloroform. In reverse phase applications water is usually the base solvent. Other polar solvents such as Methanol, Acetonitrile or Tetrahydrofuran are added in fixed or varying proportions . pH is adjusted by buffers to modify separations of ionizable solutes .

43. Pump •The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL /min). • Normal flow rates in HPLC are in the 1-to 2-mL/min range. • Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar ). • During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient ).

44. Injector • The injector serves to introduce the liquid sample into the flow stream of the mobile phase . • Typical sample volumes are 5-to 20-microliters ( μL ). •The injector must also be able to withstand the high pressures of the liquid system . •An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical .

45. Column Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters. The small particles inside the column are what cause the high back pressure at normal flow rates. The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph .

46. Modes of High Performance Liquid Chromatography 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

47. Types of columns in HPLC Guard Column Fast Column Preparative ( i.d. > 4.6 mm; lengths 50 –250 mm) Capillary ( i.d. 0.1 -1.0 mm; various lengths) Nano ( i.d. < 0.1 mm, or sometimes stated as < 100 μm ) Analytical (internal diameter ( i.d. ) 1.0 -4.6-mm; lengths 15 –250 mm)

48. Guard Column These are placed anterior to the separating column. This serves as protective factor. They are dependable columns designed to filter or remove : Particles that clog the separation column Compounds and ions that could ultimately cause “ Baseline drift ”, decreased resolution, decreased sensitivity and create false peaks . These columns must be changed on a regular basis in order to optimize their protective function.

49. Fast Column One of the primary reasons for using these column is to obtain improved sample output ( amount of compound per unit time ). Fast column are designed to decrease the time of chromatographic analysis Here internal diameter is same but length is short and packed with smaller particles , that are 3 μm diameter. Advantages- Increased sensitivity Decreased analysis time Decreased mobile phase usage Increase reproducibility

50. Capillary Column It is also known as micro columns It has a diameter much less than a millimeter and there 3 types: Open tubular Partially packed Tightly packed They allow the user to work with nanoliter sample volume , decreased flow rate and decreased solvent usage volume , led to cost effectiveness

51. Preparatory Column Used when objective is to prepare bulk ( milligrams) of sample for laboratory preparatory application . It has usually a large column diameter , which is designed to facilitate large volume injections into the HPLC system

52. Detector The detector can see (detect) the individual molecules that come out (elute ) from the column. •A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components. •The detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of the detector response).

53. HPLC Detectors

54. Common HPLC Detectors UV-VIS Diode Array Multiple Wavelength Variable Wavelength Mass Spectrometers Refractive Index Fluorescence Light Scattering Electrochemical Radioactivity Conductivity

55. UV-Vis Detectors Characteristics : Specific, Concentration Sensitive, good stability, gradient capability . Special : UV-Vis Spectral capability (Diode Array Technology ). b c Detector Flow Cell I I Log I = A = abc I

56. Fluorescence Detection

57. Electrochemical Detectors Gold for carbohydrates. Platinum for chlorite, sulfate, hydrazine, etc. Carbon for phenols, amines. Silver for chloride, bromide, cyanide .

58. Computer • Frequently called the data system, The computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to: 1. Determine the time of elution (retention time) of the sample components (qualitative analysis ) 2. Determine amount of sample ( quantitative analysis) .

59. 61 How can We Analyze the Sample For example: Carbohydrates 1. fructose 2. Glucose 3. Saccharose 4. Palatinose 5. Trehalulose 6. isomaltose 1 2 3 4 5 mAU time 6

60. 62 Separations Separation in based upon differential migration between the stationary and mobile phases . Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Waste

61. 63 Separations Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time High Performance Liquid Chromatograph

62. 64 Separations Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time

63. 65 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

64. 66 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

65. 67 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

66. 68 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

67. 69 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

68. 70 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

69. 71 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

70. 72 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

71. 73 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

72. 74 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

73. 75 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

74. 76 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

75. 77 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

76. 78 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

77. 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.

78. HPLC uses in Feed industry • Fat soluble vitamins (A,D,E and K) • Water soluble vitamins (B-complex vitamins such as B1, B2, B3, B6, Folic acid, Pantothenic acid, B12, VitaminC ) • Residual pesticides such as 2, 4-D and Monochrotophos . • Antioxidants such as TBHQ, BHA and BHT. • Sugars: Glucose, Fructose, Maltose and other saccharides. • Cholesterol and sterols • Dyes and synthetic colours .

79. Mycotoxins such as Aflatoxins B1,B2,G1,G2,M1,M2and ochratoxin Amino acids Residual antibiotics Steroids and flavonoids Aspartame and other artificial sweeteners. Active ingredients of farm produce such as allin in garlic and catachin in tea extracts .

80. Thank you Vishnu Vardhan Reddy.P TVM/2015-029