CHROMATOGRAPHY. The term "chromatography" is derived from the original use of this method for separating yellow and green plant pigments.

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1 CHROMATOGRAPHY The term "chromatography" is derived from the original use of this method for separating yellow and green plant pigments.

2 THEORY OF CHROMATOGRAPHY: Separation of two sample components in chromatography is based on their different distribution between two non-miscible phases. The one, the stationary phase, a liquid or solid, is fixed in the system. The other, the mobile phase, a fluid, is streaming through the chromatographic system. In gas chromatography the mobile phase is a gas, in liquid chromatography it is a liquid.

3 When present in the stationary phase, they are retained, and are not moving through the system. In contrast, they migrate with the velocity of the mobile phase when being there.

4 TYPES OF CHROMATOGRAPHY: Types of chromatogra phy Planer chromatogra phy column chromatogra phy thin layer chromatograp hy (TLC) paper chromatograp hy liquid chromatograp hy (LC) gas chromatograp hy (GC) Supercritical Fluid Chromatograp hy(sfc):

5 COLUMN CHROMATOGRAPHY

6 FACTORS AFFECTING SOLUTES SEPARATION IN CC Factor Particle size of solid stationary phase (or of support) Column dimensions Uniformity of packing Effect Decrease of size improves separation (but very small particles need high pressure). Efficiency increases as ratio length / width increases. Non uniform packing results in irregular movement of solutes through column & less uniform zone formation, (i.e. band broadning or tailing).

7 Column temperature Eluting solvent Solvent flow rate Continuity of flow Condition of adsorbent Concentration of solutes Increase in column temperature results in speed of elution but does not improve separation (tailing). Solvents should be of low viscosity (to give efficient resolution) & high volatility (to get rapid recovery of the substances). Uniform & low flow rate gives better resolution. Discontinuous flow disturbs resolution Deactivation of adsorbent decreases separation. Substances of high concentration move slowly.

8 Liquid chromatography

9 TYPE OF LIQUID CHROMATOGRAPHY: Liquid chromatography: Adsorption Partition Ion exchange Molecular exclusion chiral bonded

10 ADSORPTION Adsorption was developed first. It has a solid stationary phase and a liquid or gaseous mobile phase. Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent, the least soluble or best adsorbed ones travel more slowly. Liquid chromatography using a column containing silica gel or alumina is an example of adsorption chromatography

12 PARTITION In partition chromatography the stationary phase is a non-volatile liquid which is held as a thin layer (or film) on the surface of an inert solid, and the mobile phase is a gas or a liquid as the mobile phase. The solutes distribute themselves between the moving and the stationary phases, with the more soluble component in the mobile phase reaching the end of the chromatography column first. Paper chromatography is an example of partition chromatography.

14 TYPES PARTITION CHROMATOGRAPHY: According to polarity:

15 ION EXCHANGE: Ion exchange chromatography is similar to partition chromatography in that it has a coated solid as the stationary phase, a resin, and has ions (either cations or anions, depending on the resin) covalently bonded to it and ions of the opposite charge are electrostatically bound to the surface. When the mobile phase (always a liquid) is eluted through the resin the electrostatically bound ions arereleased as other ions are bonded preferentially. E. g : Domestic water softeners work on this principle.

17 MOLECULAR EXCLUSION: also known as: (Size Exclusion Chromatography (SEC), Molecular Exclusion Chromatography (MEC),Molecular Sieve Chromatography (MSC) Gel Filtration Chromatography (GFC),Gel Chromatography)

18 MOLECULAR EXCLUSION: Molecular exclusion differs from other types of chromatography in that no equilibrium state is established between the solute and the stationary phase. but separation is according to particle size. the mixture passes as a gas or a liquid through a porous gel. The pore size is designed to allow the large solute particles to pass. The small particles are slowed down so the smaller the particles, the longer it takes for them to get through the column.

20 CHIRAL CHROMATOGRAPHY: Chiral Chromatography involves the separation of stereoisomers ( enantiomers), these have no chemical or physical differences apart from being three dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes.

21 BONDED PHASE CHROMATOGRAPHY: the molecule acting as the stationary phase is chemically bonded to the solid support.

22 BONDED PHASE CHROMATOGRAPHY: R can be a C18 alkane chain or an amine (NH2) or cyano (CN) group or some other group. The nature of R determines the types of analytes which can be separated.

23 Example for SFC application:

24 PLANAR CHROMATOGRAPHY: In planar chromatography the separation system is a layer. Sample components are separated by a solvent, which is moving through the system, typically by capillary forces.

25 PLANAR CHROMATOGRAPHY: There are two types of planar chromatography: 1. thin layer chromatography (TLC) 2.paper chromatography.

26 THIN LAYER CHROMATOGRAPHY (TLC)

27 THIN LAYER CHROMATOGRAPHY (TLC) TLC is a useful technique because it is relatively quick and requires small quantities of material. Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase. The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase: is a developing liquid which travels up the stationary phase, carrying the samples with it.

28 THIN LAYER CHROMATOGRAPHY (TLC) Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase.

29 PREPARATION OF TLC Preparing a sample to use on TLC bydissolving a small amount of the sample in a small vial

30 PREPARATION OF TLC Adding the volatile solvent to dissolve the sample

31 PREPARATION OF TLC Spotting the sample onto the TLC plate with a microcapillary

32 PREPARATION OF TLC Handling the TLC plate with forceps in order to place it in the developing tank.

33 PREPARATION OF TLC With the lid on, the TLC plate is developed by allowing the solvent to run up the plate. Note that the solvent level is below the pencil line on the plate that marks the origin (i.e. the line where the samples were applied).

34 PREPARATION OF TLC After development, the TLC plate is removed and visualised. Here we see it under a UV lamp.

35 PREPARATION OF TLC If the compounds are colourless, they are made visible by treating the plate with a reagent, such as iodine vapour, that causes colour to develop.

36 SOME COMMON PROBLEMS IN TLC Problem Samples does not show up Samples does not show up Diffuse spots Sample lanes are not straight / parallel Possible causes Not enough sample, different visualisation method required This could be due to the nature of the sample, due to sample overload or a poor choice of solvent mixture. The origin line was below the solvent level or the plate was developed for too long so the solvent front reached the top of the plate. A problem with the way the solvent ran due to the plate being crooked in the solvent, touching the filter paper in the developing

37 . THIN LAYER CHROMATOGRAPHY (TLC) Interpreting the Data: The Rf (retention factor) value for each spot should be calculated. It is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates. Hence, known Rf values can be compared to those of unknown substances to aid in their identifications.

38 THIN LAYER CHROMATOGRAPHY (TLC)

39 Application for TLC:

40 Biochemical analysis: TLC is extremely useful in isolation or separation of biochemical metabolites or constituent from its body (fluids, blood plasma, serum, urine etc). In chemistry: TLC is used in chemistry for the separation and identification of compounds which are closely related to each other. It is also used for identification of cations and anions in inorganic chemistry. In pharmaceutical industry: TLC is used for detection of impurity in a pharmaceutical chemical.

41 Various medicines like hypnotics, sedatives, anticonvulsant tranquillisers, antihistaminics, analgesics, steroid have been tested qualitatively by TLC method. One of the most important application of TLC is in separation of maney pharmaceutical compontents. In food and cosmetic industry, TLC method is used for separation and identification of colours, preservatives, sweetening agent, and various cosmetic products. TLC used in detecting pesticide or insecticide residues in food, also used in forensics to analyze the dye composition of fibers.

42 PAPER CHROMATOGRAPHY is one of the most common types of chromatography. It uses a strip of paper as the stationary phase. Capillary action is used to pull the solvents up through the paper and separate the solutes. A method of partition chromatography using filter paper strips as carrier or inert support. The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases. Partition occurs between the mobile phase and the stationary aqueous phase bound by the cellulose. The isolation depends on partition coefficient of the solute.

43 DETECTION METHODS FOR CHROMATOGRAPHY: 1.UV Ultraviolet light: -most popular. -Lamp Grating/Lens - Wave length nm Flow Celln Differential Light Output 2. RI Refractive Index: Universal analyte detector Solvent must remain the same throughout separation Very temperature sensitive. -Sometimes difficult to stabilize baseline 3.FD Fluorescence: -greater sensitivity, not so popular Excitation wavelength generates fluorescence emission at a higher wavelength Analytes must have fluorophore group. -not very common Very sensitive and selective 4.MS Mass Spectrometry Mass to charge ratio (m/z) Allows specific compound ID

44 CAPILLARY ELECTROPHORESIS (CE) Capillary electrophoresis (CE) is electrophoresis performed in a capillary tube. It is the most efficient separation technique available for the analysis of both large and small molecules. The transformation of conventional electrophoresis to modern CE was occure by the production of inexpensive narrow-bore capillaries for gas chromatography (GC) and the development of highly sensitive on-line detection methods for high performance liquid chromatography (HPLC) Electrophoresis: is defined as the migration of ions under the effect of an electric field.

45 CAPILLARY ZONE ELECTROPHORESIS (CZE) is the simplest form of CE. In this mode sample is applied as a narrow zone (band), which is surrounded by the separation buffer. As an electric field is applied, each component in the sample zone migrates according to its own apparent mobility. Ideally, all sample components will eventually separate from However, neutral molecules cannot be separated because they migrate at the velocity of electroosmotic flow. The separation of charged molecules is most efficiently when differences among the apparent velocities of the components are maximized and random dispersion of the individual zones is minimized.

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