Chapter 4 chromatography_b_2010


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  • Packed - As suggested by the term, it is filled with a coated inert solid support such as fire brick, alumina, and graphite with a specific mesh size. The coatings are called phases and for best results are chemically bonded to the support. Chemical bonding provides for longer column life and less bleeding (major source of background noise) contributing to lower sensitivity. Column dimensions 1/8” - 1/4” ID x up to about 6’ using glass or stainless steel. Advantages - higher capacity (higher conc). Disadvantages: low resolution and low S/N. Capillary - Here the phase (film) is coated on the inside diameter of the capillary wall with film thickness range of 0.1 to 5μ where the ticker film provides for better resolution but also allows for more bleed. Typical dimensions .25mm - .53mm ID x up to 60m made of fused silica coated with polyamide. Advantages: high resolution and better S/N. Disadvantages: low capacity and cost.
  • Note peaks 15, 16 17 & 18 on the DB-5 column and note the same peaks on the DB-1701 column. This shows the need for confirmatory columns (columns with different phases) so that separation of the compounds can be verified.
  • Chapter 4 chromatography_b_2010

    1. 1. Techniques of chromatography Part 2
    2. 2. Techniques of chromatography <ul><li>OPEN-COLUMN CHROMATOGRAPHY </li></ul><ul><li>PAPER CHROMATOGRAPHY (PC) </li></ul><ul><li>THIN-LAYER CHROMATOGRAPHY (TLC) </li></ul><ul><li>Gas chromatography </li></ul><ul><li>HIGH-PRESSURE LIQUID CHROMATOGRAPHY </li></ul><ul><li>SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) </li></ul><ul><li>ELECTROPHORESIS (Electrochromatography) </li></ul><ul><li>High Performance Capillary Electrophoresis (HPCE): </li></ul>
    3. 3. <ul><li>OPEN-COLUMN CHROMATOGRAPHY </li></ul>
    4. 5. <ul><li>stationary phase (silica or alumina) is packed in glass tubes </li></ul><ul><li>The stationary phase particle size is large (250 jam) to allow the passage of solvent. </li></ul><ul><li>Disadvantage is the long time needed for the separation of complex mixtures (up to one week or more). </li></ul>
    5. 6. B) PAPER CHROMATOGRAPHY (PC) <ul><li>The adsorbent is a sheet of paper of suitable texture and thickness </li></ul><ul><li>Development may be ascending in which case the solvent is carried up the paper by capillary forces, or descending , in which case the solvent flow is also assisted by gravitational force. </li></ul>
    6. 7. C- Thin-layer chromatography Separation is based on migration of the sample spotted on a coated (stationary phase) plate with one edge dipped in a mixture of solvents (mobile phase). However, it is not usually as accurate or sensitive as liquid chromatography.
    7. 8. D- Gas chromatography <ul><li>Principle: </li></ul><ul><li>A pressured gas flows through heated tube coated with liquid stationery phase or packed stationery on a solid support. </li></ul><ul><li>The analyte loaded on the head of the column via heated injection port , where it is evaporated. </li></ul><ul><li>The separation of a mixture occurs according the relative time spent in the stationary phase </li></ul>
    8. 9. Instrumentation <ul><li>Injection of the simples manually or using autosampler usually size of 0.5 – 2 ul injection volume </li></ul><ul><li>The sample is evaporated and condensed at the head of the column </li></ul><ul><li>The column either capillary or packed column , the mobile phase is a gas to carry the sample through the column which is Helium or nitrogen gases . </li></ul><ul><li>The oven to heat the column up to 400 oC . </li></ul><ul><li>The detector usually flame ionization detector FID </li></ul>
    9. 10. The Gas Chromatograph WWU -- Chemistry
    10. 11. Gas Chromatography Capillary column Injection site Control panel
    11. 12. Gas Chromatography Plotter
    12. 13. Gas Chromatography: Separation of a Mixture WWU -- Chemistry
    13. 14. Stationery phase for GC <ul><li>Types of columns </li></ul><ul><li>1. Packed columns: </li></ul><ul><li>Usually glass columns silanised to remove Si….OH </li></ul><ul><li>The column mobile phase used is nitrogen at flow rate of 20 ml/min. </li></ul><ul><li>Limitation, </li></ul><ul><li>can not be used above 280 o C because of the evaporation of the stationary phase </li></ul><ul><li>2. Capillary column </li></ul><ul><li>The inner surface is coated with orange silicon polymers which are chemically bonded to silanol groups </li></ul><ul><li>The mobile phase used usually Helium at low flow of 0.5 to 2 ml/min </li></ul>
    14. 15. Columns <ul><li>Packed </li></ul><ul><li>Capillary </li></ul>
    15. 17. Factors governing the retention of compounds in capillary GC; <ul><li>Carrier gas type and flow : Nitrogen and helium </li></ul><ul><li>Column temperature : increase of column temperature, decreases resolution between two compounds, </li></ul><ul><li>Column length : increase the column length increases the resolution </li></ul><ul><li>Film thickness phase loading: the greater the volume of the stationary phase the more solutes will be retained </li></ul><ul><li>The column internal diameter: the smaller the diameter the more efficient </li></ul>
    16. 18. Gas Chromatogram Lowest b.p. Highest b.p. Retention time
    17. 19. Chromatograms - 551.1
    18. 20. Detectors <ul><li>1. Flame ionization detector FID </li></ul><ul><li>Compounds burned at the detector produced ions </li></ul><ul><li>Detects carbon – hydrogen compounds till 10 ng </li></ul><ul><li>Wide application range up to 10-6 </li></ul><ul><li>2. Electron capture detector ECD </li></ul><ul><li>Highly halogenated compounds can be detected at 50 fg – 1 pg </li></ul><ul><li>Wide application for drugs determination in biological fluids . Have wide application in environmental analysis such as chlorofluorocarbons in the air </li></ul><ul><li>3 . Nitrogen phosphate collectors </li></ul><ul><li>Used for compounds containing nitrogen and phosphors such as drugs and metabolities in body tissues and fluids </li></ul><ul><li>High selective </li></ul><ul><li>4. Thermal conductivity detectors TCD </li></ul><ul><li>Responding to cooling effect of the analyte passing over filament </li></ul><ul><li>Insensitive, used for determination of water vapour such as in peptides </li></ul>
    19. 21. Application of GC <ul><li>Detection of impurities in drug formulation </li></ul><ul><li>used for quantification of drug substances in formulation specially for drugs lack of chromophore </li></ul><ul><li>characterization of some row material used for drug synthesis </li></ul><ul><li>measurements of drugs and their metabolites in biological fluids </li></ul>
    20. 22. Limitation of GC <ul><li>only thermostable compounds can be analysed </li></ul><ul><li>the sample may require derivatisation to be volatile </li></ul><ul><li>quantitative sample introduction is more difficult due to the small volume of sample injected </li></ul>
    21. 23. Derivatization: GC <ul><li>The technique is extended by the preparation of volatile derivatives of the non­volatile compounds or of the compounds, which undergo decomposition. </li></ul><ul><li>used also for improvement of peak shape, relocation of an interfering peak, improvement of sensitivity or improvement of separation of closely related compounds. </li></ul><ul><li>An example of derivatization is silylation by addition of trimethylsilyl group to carboxylic acids, amines, imines, alcohols, phenols and thiols by treatment with hexamethyldisilazane. </li></ul>
    22. 24. Column Injector Detector HPLC Data Processing
    23. 25. Chromatographic Column
    24. 26. The system consists of main parts: 1- Mobile phase or solvent reservoir. 2- A high pressure pump. 3- A sample inlet port. 4- Column 5- Detector 6- Recorder
    25. 27. Schematic diagram of an HPLC unit (1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in &quot;inject position&quot;, (6') Switching valve in &quot;load position&quot;, (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.
    26. 28. <ul><li>The pump, capable of maintaining high pressures draws the solvent (mobile liquid phase) from the reservoir and pushes it through the column. </li></ul><ul><li>The sample is injected through a port into the high pressure liquid carrier steam between the pump and the column. </li></ul><ul><li>The separation takes place on the columns , which vary, from 25-100 cm length and 2-5 mm in internal diameter. Typical flow rates are 1-2 ml/min with pressures up to several thousand psi. </li></ul><ul><li>The column effluent passes through a non-destructive detector where a property such as : </li></ul><ul><ul><li>UV absorbance, </li></ul></ul><ul><ul><li>Rl or </li></ul></ul><ul><ul><li>molecular fluorescence </li></ul></ul><ul><li>To increase the efficiency of separation, the mobile phase may be altered by changing its polarity, pH or ionic strength . HPLC offers the advantages of speed, resolution and sensitivity. </li></ul>
    27. 29. <ul><li>There are two types of HPLC procedures: </li></ul><ul><li>LLC: the column consists of an inert support usually silica gel on which the stationary partitioning phase is adsorbed . </li></ul><ul><ul><li>In the normal phase mode , the stationary phase is polar (e.g. methanol, acetonitrile or water) while the mobile phase is less polar (e.g. iso-octane, chloroform or n-hexane). This mode is usually used for the separation of polar components. </li></ul></ul><ul><ul><li>In the reverse phase LLC , the stationary phase is less polar and the mobile phase is polar. It is usually used for the separation of non-polar components. </li></ul></ul><ul><li>LSC : The packing may be silica (polar packing) or octadecylsilica, ODS (C 18 -silica, non-polar packing). Adsorption mechanism is involved here. </li></ul><ul><ul><li>In the normal phase LSC, the packing is polar (silica) and the mobile phase is less polar (e.g. n-hexane). </li></ul></ul><ul><ul><li>In the reverse phase LSC , the packing is non-polar (eg. ODS) and the mobile phase is polar (e.g. acetonitrile-water or methanol-water). </li></ul></ul><ul><ul><li>Again, as under LLC, normal phase LSC is used for polar solutes while reverse phase LSC is used for separation of non-polar compounds. </li></ul></ul>
    28. 30. Elution Approaches <ul><li>Isocratic - constant mobile phase composition </li></ul><ul><li>Gradient - variable mobile phase composition </li></ul><ul><ul><li>step - change accomplished sharply at a defined point in time </li></ul></ul><ul><ul><li>continuous - change accomplished gradually over time </li></ul></ul>
    29. 32. HPLC Columns <ul><li>Analytical columns </li></ul><ul><li>Made of stainless stele </li></ul><ul><li>Internal diameter 2.1 – 4.6 mm </li></ul><ul><li>column long 30 – 300 mm </li></ul><ul><li>Particle size 3 – 10 micrometer </li></ul>Gourd columns Shorter column 7.5 mm Used to prevent the adsorption of substances on the analytical column
    30. 34. Stationary phase in HPLC Chemically inert Non-soluble in any imaginable mobile phase Thermal and chemical stability Appropriate physical sorption of analyte Shape: Uniform spherical particles
    31. 35. Stationary phase in HPLC <ul><li>1. unmodified silica stationary phases </li></ul><ul><li>Spherical and regular Particle size 3-10 uM; </li></ul><ul><li>Polar surface due to the silanol groups </li></ul><ul><li>Uses </li></ul><ul><li>For the separation and retention of non polar and moderately polar compounds such as poly aromatics fats, oil, isomers </li></ul><ul><li>2. Modified silica stationary phases </li></ul><ul><li>Spherical and regular Particle size 3-10 uM: </li></ul><ul><li>Non-polar due to the modification of silanol groups by chloroaloxy silane produces stable stationary phases, Ex ODS Octadecylsilane the most used stationary phase in reversed phase chromatography </li></ul><ul><li>Uses </li></ul><ul><li>For the separation and retention of wider range of polar and moderately polar substances such as drugs and amino acids </li></ul>
    32. 37. Polar phase Nonpolar phase
    33. 38. F) SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) A supercritical fluid: is a substance above its critical temperature and pressure. Critical temperature (T c ): is that above which it is impossible to liquefy a gas, no matter how great a pressure is applied. Critical pressure (P c ): is the minimum pressure necessary to bring about liquefaction at T c . Critical volume (V c ): is the volume occupied by one mole of gas or liquid at the critical temperature and pressure. SFC , is a column chromatographic technique in which a supercritical fluid is used as a mobile phase .The used mobile phase is frequently cooled to be maintained in a liquid state for easier pumping to the column. Carbon dioxide is the most frequently used mobile phase .Other mobile phases include ammonia, nitrous oxide , and xenon .SFC ,is an intermediate between GC and HPLC and offers the advantages of both .
    34. 39. Compound T c , °C P c , atm. Carbon dioxide 31.05 72.9 Nitrous oxide 36.4 71.5 Ammonia 132.4 111.3 2-Propanol 235.1 47.6 Methanol 239.4 79.9 Acetonitrile 274.8 47.0 Water 374.1 217.6
    35. 40. <ul><li>Advantage of supercritical fluids as mobile phases in chromatography compared with liquid chromatography is that </li></ul><ul><li>solutes generally have much higher diffusion coefficient in them than in liquids. This leads to enhanced speed of separation and possibly greater resolution with complex mixtures, especially for large molecules. </li></ul><ul><li>SFC possesses also advantages over GC in that solutes do not have to be volatile or thermally stable . </li></ul>
    36. 41. C) ELECTROPHORESIS (Electrochromatography) Introduction : Electrophoresis is a technique in which solutes are separated by their different rates of travel through an electric field. - commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids The rate of migration (electrophoretic mobility) of each species is a function of its charge, shape and size .
    37. 43. Gel electropherograms
    38. 44. – another type of zone electrophoresis – It involves high voltage electrophoresis in narrow bore fused-silica capillary tubes and on-line detectors similar to those used in HPLC. - On passing through the detector, they produce response profiles that are sharper than chromatographic peaks. High Performance Capillary Electrophoresis (HPCE):