CHROMATOGRAPHY Chromatography - PowerPoint Presentation

1. CHROMATOGRAPHY

2. ChromatographyA technique for the separation of a mixture by passing it in solution or suspension through a medium in which the components move at different rates.

3. PrincipleIt is based on the principle of the partition of the solute between two phases/solventsIt consists of a mobile phase and a stationary phaseThe mobile phase is usually refers to the mixture of the substances to be separated dissolved in a liquid or a gasThe stationary phase is a porous solid matrix through which the sample contained in the mobile phase perculates

4. The interaction between the mobile and the stationary phases result in the separation of the compounds from the mixtureThese interactions include the physico chemical principles such as adsorption, ion-exchange, molecular sieving and affinity

5. Chromatography- Classification1. Based on interactions between sample component and stationary phasePartitionAdsorptionIon-exchangeGel-filtrationAffinityHigh performance liquid chromatography

6. 2. Based on nature of stationary phase or mobile phase , it is of two typesPlanar- It may be paper or thin layerColumn- It may be gas or liquidPlanar chromatography is a separation technique in which the stationary phase is present as or on a planeColumn chromatography is a separation technique in which the stationary bed is within a tube

7. Chromatography classification

8. Partition ChromatographyThe principle of partition chromatography is that the separation of components of given sample occurs due to partition of components between two liquid phasesIn this process, the stationary phase is coated with a liquid surface. The solid surface must be immiscible in the mobile phase.

9. Distribution coefficientsThe basis of all forms of chromatography is the distribution or partition coefficient (Kd), which describes the way in which a compound (the analyte) distributes between two immiscible phases.For two such phases A and B, the value for this coefficient is a constant at a given temperature and is given by the expression 

10. It is used for the separation of mixture of amino acids and peptidesThe molecules of a mixture get partitioned between the stationary and the mobile phase depending on the relative affinity of each one of the phasesIt is undertaken in two waysPaper chromatographyThin layer chromatography

11. Adsorption ChromatographyIn this technique the separation is based on difference in adsorption at the surface of the solid stationary mediumThe adsorbents such as silica gels, charcoal powder and calcium hydroxyapatite are packed in to a column in a glass tube. This serves as the stationary phaseThe sample mixture in a solvent is loaded on this columnThe individual components get differentially adsorbed on to the adsorbent

12. PrincipleIt involves the competition of components of sample mixture for active site on adsorbent. These active sites are formed in molecule due toCracksEdgesThe electrostatic forces present in the molecule, which hold together the crystal lattice, are directed outwardThese forces and electrostatic forces of solute molecule cause separation

13. Separation occurs because of the fact that an equilibrium is established between molecules adsorbed on stationary phase and those which are flowing freely in mobile phaseThe more the affinity of the molecule of particular component, less will be its movement

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15. Paper ChromatographyAn analytical technique for the separation and identifying mixtures that are either coloured or can be made colouredIt is a liquid partition chromatographyUsed for the separation of amino acids, sugars, sugar derivatives and peptides

16. The stationary phase is water held on a solid support of filter paper (cellulose)The mobile phase is a mixture of immiscible solvents which are mixtures of water, a non polar solvent and an acid or base

17. Mobile PhasePure solvent, buffer solutions or mixture of solventsHydrophilic mobile phaseIsopropanol: ammonia: water 9:1:2Methanol: water 4:1N-butanol: glacial acetic acid: water 4:1:5Hydrophobic mobile phaseDimethyl ether: cyclohexaneKerosene: 70 % isopropanol

18. ProcedureA small spot of sample is applied to a strip of chromatography paper about two centimetres away from the base of the plate The paper is then dipped in to a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed containerThe solvent moves up the paper by capillary action and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample

19. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to difference in their attraction to the fibres in the paperPaper chromatography takes from several minutes to several hours

20. Ascending and Descending Paper ChromatographyAscending Chromatography- In this method, the solvent is present at the bottom of the vessel in which the paper is supportedIt rises up the paper by capillary action against the force of gravity

21. Descending Chromatography- In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow the paperThe liquid moves down by capillary action as well as by the gravitational force

22. Ascending and Descending Paper Chromatography

23. Paper chromatography- AnalysisThe paper remaining after the experiment is known as the chromatogramThe components which have been separated differ in their retention factorRatio of distance travelled from the spot or origin by the solute component to that of the distance travelled from the spot or origin by the solvent

24. Rf ValueRf is Distance travelled by sample/ distance travelled by solventIf Rf value of a solution is zero, the solute remains in the stationary phase and thus it is immobileIf Rf value is 1, then the solute has no affinity for the stationary phase and travels with the solvent frontRetention factor can never be greater than one

25. Detecting/Visualising AgentsIf the substance are coloured they are visually detected easilyFor colourless substances, physical and chemical methods are used to detect the spotPhysical methods (Non-specific methods)Iodine chamber methodUV chamber for fluorescent compounds at 254 or at 365 nm

26. Chemical method (Spraying or specific method)Ferric chloride - Phenolic comp. & tanninsNinhydrin in acet. - Amino acidsDragendroff’s Reag.- Alkaloids3,5 dinitro benzoic acid - Cardiac glycosides

27. Two Dimensional ChromatographyIt is used to separate a complex mixture of substances which was not resolved by a single runs with one solvent systemIn two-dimensional chromatography, a second run is carried out by a different solvent system, in a direction perpendicular to the first run which enhances the separation of a mixture in the individual components

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29. Significance of Paper ChromatographyPaper chromatography is a very easy, simple, rapid and highly efficient method of separationCan be applied even in microgram quantities of the sampleCan also be used for the separation of a wide variety of materials such as amino acids, oligopeptides, sugars, oligosaccharides, glycosides, purines and pyrimidines, steroids, vitamins and some alkaloids such as penicillin, tetracyclin and streptomycin

30. Not preferred for separating proteins because they are not soluble in many of the solvent systems and are denatured by themPaper chromatography is inferior to thin layer chromatography in resolving power

31. Thin Layer ChromatographyIt is the one of the simplest, fastest, easiest and least expensive of several chromatographic techniques used in qualitative and quantitative analysis to separate organic compounds and to test the purity of compounds

32. Principle of TLCIt is based on the principle of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent, its treatment and nature of solvents employedThe components with more affinity towards stationary phase travels slowerComponents with less affinity towards stationary phase travels fasterQualitative and quantitative analysis of the eluted substances

33. TLC requiresA mobile phase (developing solvent)A stationary phase (a plate or strip coated with a form of silica gel)Analysis is performed on a flat surface under atmospheric pressure and room temperature

34. In TLC, a solid phase, the adsorbent, is coated onto a solid support (thin sheet of glass, plastic or aluminium) as a thin layer (about 0.25 mm thick)Mostly, a small amount of a binder such as plaster of Paris is mixed with the absorbent to facilitate the coating

35. Selection of Stationary PhaseThe choice of the stationary phase for a given separation problem is the most difficult decision in TLCTo obtain satisfactory separation efficiency, the mean particle size, the particle size distribution and the morphology of the particle must be considered

36. Stationary phases used for thin-layer chromatographySilica gel, modified silica gels, alumina, cellulose powder – adsorption chromatographyCellulose powder, Kieselguhr – partition chromatography

37. Selection of adsorbentsSolubility of compound (hydrophilic or lipophilic)Nature of substance to be separated (acidic, basic or amphoteric)Adsorbent particle sizeAdsorbent should not adhere to glass plateReactive of compound with the solvent or adsorbentChemical reactivity of compounds with binders

38. Plate preparationTLC plates are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and waterThis mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foilThe resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °CThe thickness of the absorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC

39. Mobile phaseNature of the substance to be separated (polar or non-polar)Nature of stationary phase usedMode of chromatographyNature of separation (analytical or preparative)Solvents used should be of high puritySolvents should be miscible

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41. Development of a TLC plate

42. TLC plate visualization

43. Factors affecting Rf valueIt depends upon the following factorsNature of adsorbentMobile phaseActivityThickness of layerTemperatureEquilibriumLoadingDipping zoneChromatographic Techniques

44. Advantages of TLCShort analysis timeAll spots can be visualizedAdoptable to most pharmaceuticalsLow costUses small quantities of solventRequires minimal trainingReliable and quickMinimum amount of equipment is needed

45. Column Chromatography

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48. TermDefinitionMobile phase or carriersolvent moving through the columnStationary phase or adsorbentsubstance that stays fixed inside the columnEluentfluid entering the columnEluatefluid exiting the column (that is collected in flasks)Elutionthe process of washing out a compound through a column using a suitable solventAnalytemixture whose individual components have to be separated and analyzed

49. Ion-exchange chromatographyIon chromatography (or ion-exchange chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. The stationary phase is an ion exchange resin that carries charged functional groups which interacts with oppositely charged groups of the compound to retainedIt works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids.

50. In this type of chromatography, a resin (the stationary solid phase) is used to covalently attach anions or cations onto itSolute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forcesIon exchange mechanism separates analytes based on their respective charges

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53. MechanismTo optimize binding of all charged molecules, the mobile phase is generally a low to medium conductivity (i.e., low to medium salt concentration) solution. The adsorption of the molecules to the solid support is driven by the ionic interaction between the oppositely charged ionic groups in the sample molecule and in the functional ligand on the support.

54. The strength of the interaction is determined by the number and location of the charges on the molecule and on the functional group. By increasing the salt concentration (generally by using a linear salt gradient) the molecules with the weakest ionic interactions start to elute from the column first. Molecules that have a stronger ionic interaction require a higher salt concentration and elute later in the gradient. The binding capacities of ion exchange resins are generally quite high. This is of major importance in process scale chromatography, but is not critical for analytical scale separations.

55. Buffer pHAs a rule, the pH of the mobile phase buffer must be between the pI (isoelectric point) or pKa (acid dissociation constant) of the charged molecule and the pKa of the charged group on the solid supportFor example, in cation exchange chromatography, using a functional group on the solid support with a pKa of 1.2, a sample molecule with a pI of 8.2 may be run in a mobile phase buffer of pH 6.0. In anion exchange chromatography a molecule with a pI of 6.8 may be run in a mobile phase buffer at pH 8.0 when the pKa of the solid support is 10.3.

56. Salt GradientsAs in most other modes of chromatography (SEC being the exception) a protein sample is injected onto the column under conditions where it will be strongly retained. A gradient of linearly increasing salt concentration is then applied to elute the sample components from the column. An alternative to using a linear gradient is to use a step gradient. This requires less complicated equipment and can be very effective to elute different fractions if the appropriate concentrations of salt are known, usually from linear gradient experiments.

57. Varying pHMany chromatographers also use changes in pH to affect a separation. In cation exchange chromatography, raising the pH of the mobile phase buffer will cause the molecule to become less protonated and hence less positively charged. The result is that the protein no longer can form a ionic interaction with the negatively charged solid support, which ultimately results in the molecule to elute from the column.

58. In anion exchange chromatography, lowering the pH of the mobile phase buffer will cause the molecule to become more protonated and hence more positively (and less negatively) charged. The result is that the protein no longer can form a ionic interaction with the positively charged solid support which causes the molecule to elute from the column.

59. ProcedureA mixture of arginine and aspartic acid is passed through a cation exchange column.Arginine has extra positive charge and so adheres to the column, but the negatively charged aspartic acid molecules will not adhere and come out first from the columnWhen a weak NaOH is passed, the positive charge of arginine is neutralized.Na+ will replace arginine in the column, thus arginine is eluted finally

60. Types of Ion exchange resinsCation exchange resinPolysterene sulfonate resins, CM-Sephadex gel, CM-celluloseThese bear acidic groups and immobilize cations from adjacent solutionsAnion exchange resinDEAE cellulose, Trimethyl amino polysterene, DEAE-SephadexThese bear basic groups ionizing into fixed positions and immobilze anions from neighbouring solutions

61. Size-exclusion chromatographySize-exclusion chromatography (SEC), also known as molecular sieve chromatography, in which molecules in solution are separated by their size, and in some cases molecular weightThis technique is also referred to as molecular exclusion chromatographyThe apparatus consists of a column packed with sponge like gel beads (usually cross-linked polysaccharides) containing poresThe gels serve as molecular sieves for the separation of smaller and bigger molecules

62. PrincipleThe solution mixture containing molecules of different sizes (say protein) is applied to the column and eluted with a bufferThe larger molecules can not pass through the pores of a gel and therefore move fasterThe smaller molecules enter the gel beads and are left behind which come out slowly.By selecting the gel beads of different porosity, the molecules can be separatedThe gel filtration chromatography can be used for an approximate determination of molecular weights

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64. Procedure A mixture of molecules dissolved in liquid (the mobile phase) is applied to a chromatography column which contains a solid support in the form of microscopic spheres, or “beads” (the stationary phase). The mass of beads within the column is often referred to as the column bed. The beads act as “traps” or “sieves” and function to filter small molecules which become temporarily trapped within the pores. Larger molecules are “excluded” from the beads . Large sample molecules cannot or can only partially penetrate the pores, whereas smaller molecules can access most or all pores. Thus, large molecules elute first, smaller molecules elute later, while molecules that can access all the pores elute last from the column. Particles of different sizes will elute (filter) through a stationary phase at different rates.

65. Affinity ChromatographyThe principle of affinity chromatography is based on the property of specific and non-covalent binding of proteins to other molecules, referred to as substrates or cofactorsThe technique involves the use of ligands covalently attached to an inert and porous matrix in a columnThe immobilized ligands act as molecular hooks to selectively pick up the desired protein while the remaining proteins pass through the columnThe desired protein captured by the ligands, can be eluted by using free ligands moleculesAlternatively, some reagents that can break protein ligand interactions can also be employed for the separation

66. SignificanceIt is useful for the purification of enzymes, vitamins, nucleic acids, drugs, hormone receptors, antibodies, etc.,It is widely used for the estimation of Glycated Hb.Normal Hb does not bind and comes out first, while glycated Hb binds with the Boronic acid is used as a ligand.Sorbitol is then added to elute the Glycated Hb which can be quantitated then

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68. HPLC (High Performance Liquid Chromatography)It is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column

69. HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller sorbent particles (2–50 μm in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.

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75. HPLC Instrumental setupThe schematic of an HPLC instrument typically includes a degasser, sampler, pumps and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components.

76. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry Most HPLC instruments also have a column oven that allows for adjusting the temperature at which the separation is performed.

77. Types of HPLC ChromatographyBased on modes of chromatographyNormal phase modeReverse phase modeBased on the principle of separationAdsorption chromatographyIon exchange chromatographyPartition chromatographySize exclusion

78. Based on elution techniqueIsocratic separationGradient separationBased on the scale of operationAnalytical HPLCPreparative HPLCBased on the type of analysisQualitative analysisQuantitative analysis

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81. Advantages of HPLCHigh sensitivityHigh performanceRapid process and hence time savingCan be used for qualitative as well as quantitative estimationCane be used for both analytical and preparative purpose

82. Normal-phase liquid chromatographyIn this form of partition chromatography, the stationary phase is polar and the mobile phase relatively non-polar. The most popular stationary phase is an alkylamine bonded to silica. The mobile phase is generally an organic solvent such as hexane, heptane, dichloromethane or ethyl acetate. These solvents form an elutropic series based on their polarity. Such a series in order of increasing polarity is as follows:

83. The order of elution of analytes is such that the least polar is eluted first and the most polar last. Indeed, polar analytes generally require gradient elution with a mobile phase of increasing polarity, generally achieved by the use of methanol or dioxane.The main applications of normal-phase liquid chromatography are its use to separate analytes that have low water solubility and those that are not amenable to reversed phase liquid chromatography.

84. Reversed-phase liquid chromatographyIn this form of liquid chromatography, which has many similarities with hydrophobic interaction chromatography, the stationary phase is non-polar and the mobile phase relatively polar, hence the name reversed-phase. By far the most commonly used type is the bonded-phase form, in which alkylsilane groups are chemically attached to silica. Butyl (C4), octyl (C8) and octadecyl (C18) silane groups are most commonly used

85. The mobile phase is commonly water or aqueous buffers, methanol, acetonitrile or tetrahydrofuran, or mixtures of them.Reversed-phase liquid chromatography differs from most other forms of chromatography in that the stationary phase is essentially inert and only non-polar (hydrophobic) interactions are possible with analytes.

86. Reversed-phase HPLC is probably the most widely used form of chromatography mainly because of its flexibility and high resolution. It is widely used to analyse drugs and their metabolites, insecticide and pesticide residues, and amino acids, peptides and proteins.

87. Gas ChromatographyThe principles of gas chromatography (GC) are similar to those of HPLC but the apparatus is significantly different. It exploits differences in the partition coefficients between a stationary liquid phase and a mobile gas phase of the volatilised analytes as they are carried through the column by the mobile gas phase. Its use is therefore confined to analytes that are volatile but thermally stable.

88. The partition coefficients are inversely proportional to the volatility of the analytes so that the most volatile elute first. The temperature of the column is raised to 50-300 °C to facilitate analyte volatilisation.

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92. The stationary phase consists of a high-boiling-point liquid material such as silicone grease or wax that is either coated onto the internal wall of the column or supported on an inert granular solid and packed into the column. There is an optimum flow rate of the mobile gas phase for maximum column efficiency. Very high resolutions are obtained hence the technique is very useful for the analysis of complex mixtures.

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94. Gas chromatography is widely used for the qualitative and quantitative analysis of a large number of low-polarity compounds because it has high sensitivity, reproducibility and speed of resolution. Analytically, it is a very powerful technique when coupled to mass spectrometry.

95. Apparatus setupThe major components of a GC system are:a column housed in an oven that can be temperature programmed;a sample inlet point;a carrier gas supply and control; anda detector, amplifier and data recorder system

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97. High-Performance Thin-Layer Chromatography (HPTLC)HPTLC is the most advanced form of TLC and comprises the use of chromatographic layers of utmost separation efficiency and the employment of state-of-the-art instrumentation for all steps in the procedure: precise sample application standardized reproducible chromatogram development and software controlled evaluationqualitative and quantitative analysis.

98. It involves the same theoretical principle of thin layer chromatography (adsorption chromatography) The mobile phase solvent flows through because of capillary action.The components move according to their affinities towards the stationary phase travels slower.

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100. HPTLC Instrumental Setup

101. Mobile phaseSolvents used areMethanolChloroform: Methanol Ethyl acetate: MethanolChloroform: Methanol: AmmoniaMethylene chloride: Methanol1% Ammonia or 1% Acetic acid

102. Stationary phasePre-coated platesThe plates with different support materials and sorbent layers with different format and thickness are usedPlates with sorbent thickness of 100-250 µm are used for qualitative and quantitative analysis

103. Sorbents used in HPTLCSilica gel 60F (Unmodified)Aluminium oxideCellulose (Microcrystalline)Silica gel chemically modified

104. 80 % of analysis takes place by silica gelAmino acids, dipeptides, sugars and alkaloids- celluloseNon-polar substances, fatty acids, carotenoids, cholesterol – RP2, RP8 and RP18Preservatives, barbiturates, analgesic and phenothiazines – Hybrid plates (RPWF254s)

105. Pre-washing of pre-coated platesThe main purpose of the pre-washing is to remove impurities which include water vapours and other volatile substances from the atmosphere when they get exposed in the lab environmentSilica gel 60F is most widely used sorbent. The major disadvantage of this sorbent is that it contain iron as impurity. This iron is removed by using methanol : water in the ratio of 9:1. This is the major advantage of the step of pre-washing

106. Pre – washing methodsAscending methodDipping methodContinuous methodSolvents used for pre – washing MethanolChloroform: methanol (1:1)Chloroform: methanol: Ammonia (90: 10: 1)

107. Activation of platesPlates exposed to high humidity or kept in hand for long time require activationPlates are placed in oven at 110 - 120° C for 30 min prior to the sample applicationAluminium sheets should be kept in between two glass plates and placing in oven at 110 - 120° C for 15 mins

108. Chamber SaturationUn – Saturated chamber caused high Rf valuesSample applicationUsual concentration range is 0.1 – 1 µg / µlAbove this causes poor separation Linomat IV (automatic applicator) – nitrogen gas sprays sample and standard from syringe on TLC plates as bandsBand wise application – better separation

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111. Chromatographic development and dryingAfter development, remove the plate and mobile phase is removed from the plate – to avoid contamination of lab atmosphereDry in vacuum desiccator

112. Post Chromatography StepsDetectionPhoto documentationDensitometry measurements

113. Detection and VisualizationDetection under UV light is first choice – non destructiveSpots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length)Spots of non fluorescent compounds can be seen in fluorescent stationary phase is used – silica gel GFNon UV absorbing compounds like ethambutol, dicylomine etc – dipping the plates in 0.1 % iodine solution

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115. QuantificationSample and standard should be chromatographed on same plate after development chromatogram is scannedConcentration of analyte in the sample is calculated by considering the sample initially taken and dilution factors

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117. Advantages of densitometer scannerThe purpose of scanner is to convert the spot/band on the layer into densitogram consisting of peaks similar in appearance to HPLCThe position of the scanned peaks on the recorder chart is related to Rf valuesQuantitation is faster, reliable accurate & reproducible

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119. Application of HPTLCPharmaceutical industry: Quality control, content uniformity, uniformity test, identity/purity checkFood Analysis: Quality control, additives, pesticides, stability testing, analysis of sub-micron levels of alfotoxins etc.Clinical applications: Metabolism studies, drug screening, stability testing etc.Industrial applications: Process development and optimization, in-process Q.C. check, validation etc.Forensic: Poisoning investigations