techniques of chromatography part 2. techniques of chromatography a.open-column chromatography b....

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  • Techniques of chromatography

    Part 2

  • Techniques of chromatography

    OPEN-COLUMN CHROMATOGRAPHY PAPER CHROMATOGRAPHY (PC)THIN-LAYER CHROMATOGRAPHY (TLC)Gas chromatographyHIGH-PRESSURE LIQUID CHROMATOGRAPHY SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)ELECTROPHORESIS (Electrochromatography)High Performance Capillary Electrophoresis (HPCE):

  • OPEN-COLUMN CHROMATOGRAPHY

  • stationary phase (silica or alumina) is packed in glass tubes The stationary phase particle size is large (250 jam) to allow the passage of solvent. Disadvantage is the long time needed for the separation of complex mixtures (up to one week or more).

  • B) PAPER CHROMATOGRAPHY (PC)

    The adsorbent is a sheet of paper of suitable texture and thickness

    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.

  • 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.

  • D- Gas chromatographyPrinciple:

    A pressured gas flows through heated tube coated with liquid stationery phase or packed stationery on a solid support.

    The analyte loaded on the head of the column via heated injection port, where it is evaporated.

    The separation of a mixture occurs according the relative time spent in the stationary phase

  • Instrumentation

    Injection of the simples manually or using autosampler usually size of 0.5 2 ul injection volume The sample is evaporated and condensed at the head of the column 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.The oven to heat the column up to 400 oC.The detector usually flame ionization detector FID

  • WWU -- ChemistryThe Gas Chromatograph

  • Gas ChromatographyCapillary columnInjection siteControl panel

  • Gas ChromatographyPlotter

  • WWU -- ChemistryGas Chromatography: Separation of a Mixture

  • Stationery phase for GCTypes of columns

    1. Packed columns:Usually glass columns silanised to remove Si.OH The column mobile phase used is nitrogen at flow rate of 20 ml/min. Limitation, can not be used above 280 oC because of the evaporation of the stationary phase

    2. Capillary column The inner surface is coated with orange silicon polymers which are chemically bonded to silanol groups The mobile phase used usually Helium at low flow of 0.5 to 2 ml/min

  • Columns Packed Capillary

  • Factors governing the retention of compounds in capillary GC;

    Carrier gas type and flow: Nitrogen and helium

    Column temperature: increase of column temperature, decreases resolution between two compounds,

    Column length: increase the column length increases the resolution

    Film thickness phase loading: the greater the volume of the stationary phase the more solutes will be retained

    The column internal diameter: the smaller the diameter the more efficient

  • Gas ChromatogramLowestb.p.Highestb.p.Retentiontime

  • Chromatograms - 551.1

  • Detectors 1. Flame ionization detector FIDCompounds burned at the detector produced ions Detects carbon hydrogen compounds till 10 ng Wide application range up to 10-6

    2. Electron capture detector ECDHighly halogenated compounds can be detected at 50 fg 1 pg Wide application for drugs determination in biological fluids. Have wide application in environmental analysis such as chlorofluorocarbons in the air 3. Nitrogen phosphate collectorsUsed for compounds containing nitrogen and phosphors such as drugs and metabolities in body tissues and fluids High selective

    4. Thermal conductivity detectors TCDResponding to cooling effect of the analyte passing over filament Insensitive, used for determination of water vapour such as in peptides

  • Application of GCDetection of impurities in drug formulation

    used for quantification of drug substances in formulation specially for drugs lack of chromophore

    characterization of some row material used for drug synthesis

    measurements of drugs and their metabolites in biological fluids

  • Limitation of GC only thermostable compounds can be analysed

    the sample may require derivatisation to be volatile

    quantitative sample introduction is more difficult due to the small volume of sample injected

  • Derivatization: GC The technique is extended by the preparation of volatile derivatives of the nonvolatile compounds or of the compounds, which undergo decomposition.used also for improvement of peak shape, relocation of an interfering peak, improvement of sensitivity or improvement of separation of closely related compounds. An example of derivatization is silylation by addition of trimethylsilyl group to carboxylic acids, amines, imines, alcohols, phenols and thiols by treatment with hexamethyldisilazane.

  • ColumnInjectorDetectorHPLCData Processing

  • *Chromatographic Column

  • The system consists of main parts:1-Mobile phase or solvent reservoir.2-A high pressure pump.3-A sample inlet port.4-Column5-Detector6-Recorder

  • 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 "inject position", (6') Switching valve in "load position",

    (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.

  • The pump, capable of maintaining high pressures draws the solvent (mobile liquid phase) from the reservoir and pushes it through the column.

    The sample is injected through a port into the high pressure liquid carrier steam between the pump and the column.

    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.

    The column effluent passes through a non-destructive detector where a property such as :UV absorbance, Rl ormolecular fluorescence

    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.

  • There are two types of HPLC procedures:

    LLC: the column consists of an inert support usually silica gel on which the stationary partitioning phase is adsorbed. 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. 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.

    LSC: The packing may be silica (polar packing) or octadecylsilica, ODS (C18-silica, non-polar packing). Adsorption mechanism is involved here. In the normal phase LSC, the packing is polar (silica) and the mobile phase is less polar (e.g. n-hexane). 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). Again, as under LLC, normal phase LSC is used for polar solutes while reverse phase LSC is used for separation of non-polar compounds.

  • Elution ApproachesIsocratic - constant mobile phase compositionGradient - variable mobile phase compositionstep - change accomplished sharply at a defined point in timecontinuous - change accomplished gradually over time

  • HPLC ColumnsAnalytical columns Made of stainless stele Internal diameter 2.1 4.6 mm column long 30 300 mm Particle size 3 10 micrometer Gourd columnsShorter column 7.5 mm Used to prevent the adsorption of substances on the analytical column

  • Stationary phase in HPLCChemically inert

    Non-soluble in any imaginable mobile phase

    Thermal and chemical stability

    Appropriate physical sorption of analyte

    Shape: Uniform spherical particles

  • Stationary phase in HPLC1. unmodified silica stationary phasesSpherical and regular Particle size 3-10 uM;Polar surface due to the silanol groupsUsesFor the separation and retention of non polar and moderately polar compounds such as poly aromatics fats, oil, isomers

    2. Modified silica stationary phasesSpherical and regular Particle size 3-10 uM: 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 chromatographyUsesFor the separation and retention of wider range of polar and moderately polar substances such as drugs and amino acids

  • Polar phaseNonpolar phase

  • F) SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

    A supercritical fluid: is a substance above its critical temperature and pressure.Critical temperature (Tc): is that above which it is impossible to liquefy a gas, no matter how great a pressure is applied. Critical pressure (Pc): is the minimum pressure necessary to bring about liquefaction at Tc.Critical volume (Vc): 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

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