AN ASSIGNMENT ON

Chromatography
Chromatography ( Greek word, chroma, color; and graphos, writing) is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase. First ever used by M Tswett for separation of plant pigments There are a number of different kinds of chromatography, which differ in the mobile and the stationary phase used.

Types of Chromatography

Modes of chromatography
Three modes of chromatography: 1) Paper chromatography 2) Column chromatography, -Gel exclusion -Affinity -HPLC -Ion exchange. -gas liquid chromatography chromatography
Paper Chromatography

3) Thin layer chromatography

Thin layer Chromatography

Modes of Chromatography

Column Chromatography

Adsorption chromatography
In this case analyte get adsorbed on the matrix molecules with the help of hydrophobic forces hydrogen bonding and van der Waal forces Matrix used are porous carbon silica (silanol group, Si-OH) alumina Mixture of solvents is used for elution alcohol for OH group acetone ,esters for carbonyl groups hexane, heptane for non polar groups present in analyte

Partition Chromatography
Most popular method Low molecular weight (mw<3000) analytes Polar or non-polar Bonded stationary phase column liquid chemically bonded to support particles 3, 5 or 10 mm hydrolyzed silica particles coated with siloxanes

Partition or distribution coefficient of molecules

Kd=
capacity ratio

conc. In phase A conc. In phase B

K` =

Tr - Tm Tm

 Normal phase HPLC nonpolar solvent- hexane, heptane polar column-alkylamine boded to silica (non polar analyte eluted first polar analyte eluted last) Reversed phase HPLC polar solvent-water, aqueous bufers, methanol nonpolar column- alkylsilane bonded to silica (polar analyte eluted first non polar analyte eluted last)

Gel Permeation Size Exclusion

Separation of molecules occurs on the basis of size and shape of molecules. Components: matrix (polymeric organic compounds, porous glass granules) column solvent Exclusion limit: max. size of molecules that can penetrate the gel pores (in terms of diameter, but generally mol. wt. is taken as diameter is unavailable). Large molecules that are completely excluded from the pores will pass through the interstitial space and will appear in the effluent first. Smaller molecules will be distributed between the mobile phase inside and outside the molecular sieve and will then pass through the column at a slower rate, appearing last in the effluent.

 Used for large mw. Compounds, proteins and polymers Separation mechanism is sieving not partitioning. Stationary phase porous silica or polymer (particlespolystyrene, polyacrylamide) well-defined pore sizes (40-2500 ) Large molecules excluded from pores not retained, first eluted (exclusion limit - terms of mw) Intermediate molecules retained, intermediate elution times Small molecules permeate into pores strongly retained, last eluted (permeation limit - terms of mw)

Ion exchange chromatography

Amino acids proteins etc have the ionizble groups carrying a net positive or negative charge that can be utilized in separation of these materials. Columns are used packed with ion exchanger material. Two types of ion exchanger material are used- Cation exchanger and Anion exchanger

 Ion exchanger matrices used are Cation exchangers: - agarose, cellulose, dextran.
RSO3-..Na+ + NH3+ R`
Exchanger counter ion charged mol.

RSO3- . NH3R+ + Na+

Bound mol exchanged ion

Anion exchangers, cellulose dextran , polystyrine

(R)4N+.Cl

-OOCR`

(R+)4N.-OOR` + Cl-

Affinity chromatography
Components : 1 matrix-agarose dextran polyacrylamide 2 spacer arm- 1,6-diaminohexane, 6-aminohexonic acid 1,4-bis-(2,3-epoxypropoxy)butane 3 ligandsubstrates antigens metal ions e.t.c.

 Matrix is first activated by treatment with agents like cynogen bromide. Then after spacer is attached to matrix, this should be of 6-10 carbon in length. keeps the ligand in a position, facilitating easy binding of macromolecule to ligand. Applications; isolation of mRNA, proteins, antibodies etc.

Paper Chromatography
In paper chromatography, the mobile phase is a solvent, and the stationary phase is water held in the fibres of chromatography paper.

Technique provides an easy way to separate the components of a mixture.

Rf =

Distance the solute moves

Distance the solvent front moves

 A drop of mixture is placed in one corner of a square of absorbent paper. One edge of the paper is immersed in a solvent. (a) The solvent migrates up the sheet by capillary attraction. As it does so, the substances in the drop are carried along at different rates. (b) Each compound migrates at a rate that reflects the size of its molecule and its solubility in the solvent.

After a second run at right angles to the first (often using a different solvent), the various substances will be spread out at distinct spots across the sheet, forming a chromatogram. (c)

 The identity of each spot can be determined by comparing its position with the position occupied by known substances under the same conditions. In many cases, a fragment of the paper can be cut away from the sheet and chemical analysis is run on the tiny amount of substance in it.

Thin Layer Chromatography

The mobile phase is also a solvent, and the stationary phase is a thin layer of finely divided solid, such as silica gel or alumina, supported on glass or aluminum. Thin layer chromatography is similar to paper chromatography in that it involves spotting the mixture on the plate and the solvent (mobile phase) rises up the plate in the chromatography tank.

 Introduction Thin-layer chromatography (TLC) is a very commonly used technique in synthetic chemistry for identifying compounds, determining their purity and following the progress of a reaction. It also permits the optimization of the solvent system for a given separation problem. In comparison with column chromatography, it only requires small quantities of the compound (~ng) and is much faster as well. Stationary Phase As stationary phase, a special finely ground matrix (silica gel, alumina, or similar material) is coated on a glass plate, a metal or a plastic film as a thin layer (~0.25 mm). In addition a binder like gypsum is mixed into the stationary phase to make it stick better to the slide. In many cases, a fluorescent powder is mixed into the stationary phase to simplify the visualization later on (e.g. bright green when you expose it to 254 nm UV light).

Preparing the Plate Do not touch the TLC plate on the side with the white surface. In order to obtain an imaginary start line, make two notches on each side of the TLC plate. can also draw a thin line with pencil. The start line should be 0.5-1 cm from the bottom of the plate.

Spotting the plate

The thin end of the spotter is placed in the dilute solution; the solution will rise up in the capillary (capillary forces). Touch the plate briefly at the start line. Allow the solvent to evaporate and spot at the same place again. This way you will get a concentrated and small spot. Try to avoid spotting too much material, because this will deteriorate the quality of the separation considerably (tailing). The spots should be far enough away from the edges and from each other as well. If possible, you should spot the compound or mixture together with the starting materials and possible intermediates on the plate. They will serve as internal reference since every TLC plate is slightly different.

Developing a Plate
A TLC plate can be developed in a beaker or closed jar. Place a small amount of solvent (= mobile phase) in the container. The solvent level has to be below the starting line of the TLC, otherwise the spots will dissolve away. The lower edge of the plate is then dipped in a solvent. The solvent (eluent) travels up the matrix by capillarity, moving the components of the samples at various rates because of their different degrees of interaction with the matrix (=stationary phase) and solubility in the developing solvent. Non-polar solvents will force non-polar compounds to the top of the plate, because the compounds dissolve well and do not interact with the polar stationary phase. Allow the solvent to travel up the plate until ~1 cm from the top. Take the plate out and mark the solvent front immediately. TLC chamber for development e.g. beacher with a lid or a closed jar after ~5 min after ~10 min after drying

after drying

after ~10 min

after ~5 min

Visualization
There are various techniques to visualize the compounds. 1. spray of ninhydrin is used for visualization of amino acids Sulfuric acid/heat: destructive, leaves charred blots behind2. Ceric stain: destructive, leaves a dark blue blot behind for polar compounds 3. Iodine: semi-destructive, iodine absorbs onto the spots, not permanent 4. UV light: non-destructive, long wavelength (background green, spots dark), short wavelength (plate dark, compounds glow), 5. Circle the spots on the TLC plate to have a permanent record how far the compound traveled on the plate. Also draw a sketch of the developed plate in your lab notebook. Analytes Amino acids (UV) Amino acids Carbohydrates(UV) Reagents Ninhydrin
O-phthalaldehyde Orcinol, H2SO4

2.

Analysis
The components, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials.
Distance the solute moves
Rf =

Distance the solvent front moves

The value should be between 0.0 (spot did not moved from starting line) and 1.0 (spot moved with solvent front) and is unitless.

The Rf (=retardation factor) depends on the following parameters: 1. solvent system

2. absorbent (grain size, water content, thickness)

3. amount of material spotted

4. temperature Due to the fact that all those variables are difficult to keep constant, a reference compound is usually applied to the plate as well.

Advantages
It has an advantage over paper chromatography in that its separations are very efficient because of the much smaller size of the particles in the stationary phase. Thin layer chromatography is particularly useful in forensic work, for example in the separation of dyes from fibres. Gas chromatography and high performance liquid chromatography are more sophisticated chromatographic techniques.

Column Chromatography
Like all chromatographic techniques, column chromatography uses a mobile phase to move a mixture of substances through a stationary phase. The stationary phase and mobile phase are chosen based on the nature of the sample mixture in order to achieve the best possible separation of its components. In most applications in the chemistry laboratory, the stationary phase is either silica (SiO2) or alumina (Al2O3), which is mixed with the solvent being used as the mobile phase to yield a thick white slurry.

 Time taken for each analyte peak to emerge from column is referred as retention time tr. Volume of mobile phase required to elute the analyte is referred to as elution or retention volume VR VR = trFC Fc = flow rate mobile phase

 Chromatography columns vary in size and polarity. A small volume of the sample whose constituents are to be separated is placed on top of the column. Effluent is collected in fractions for different compounds. This elute is analyzed by adding different derivatizing reagents. e.g. ninhydrine for amino acids

 The choice of the eluting solvent should ensure that the sample is soluble. However, if the sample was too soluble the mobile phase (solvent) would move the solutes too quickly, resulting in the non-separation of the different constituents. Matrices used: agarose, cellulose, dextran, polyacrylamide polystyrine, silica.

Advantages of Column Chromatography

Preparative method Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. This is in contrast to paper chromatography, which is solely an analytical method. In fact, it is the most frequently used method of purifying mixtures of products in research laboratories.

Disadvantages of Column Chromatography

Collection vessels must be frequently switched and solvent levels need to be topped up. Unlike paper chromatography, column chromatography is a much more involved and tedious procedure. It is not as simple as sitting back and watching the mixture separate. In short, running a column is time-consuming and tedious, especially for large samples.

High Performance Liquid Chromatography

Basic Components:
1) Solvent Reservoir.

2) The Pump System controls the flow and measures the volume of solvent (the mobile phase). The flow rates of HPLC columns are slow often in the range of 0.5 - 5 cm3 min-1. 3) The Injector System: The sample to be separated is injected into the liquid phase at this point. 4) The Column is made of steel and packed usually with porous silica particles (the stationary phase). Different materials can be used depending on the nature of the liquid. A long column is not needed because separation in HPLC is very efficient. Columns are usually 10 30 cm long, with an internal diameter of 4 mm. Different components of the sample are carried forward at different rates by the moving liquid phase, due to their differing interactions with the stationary and mobile phases.

5) The Detector: When the components reach the end of the column they are analysed by a detector. The amounts passing through the column are small, so solutes are analysed as they leave the column. Therefore HPLC is usually linked to a spectrometer (e.g. ultra violet or mass spectrometry). Once the retention time of a solute has been established for a column using a particular set of operating conditions, the solute can be identified in a mixture. A chromatogram is obtained for the sample. Uses HPLC has many uses such as drug testing, testing for vitamins in food and growth promoters in meat. In each case components of the mixture are separated and detected.

Gas Chromatography
A gas is the mobile phase and the stationary phase can be either a solid or a non- volatile liquid. There are five basic GC components: 1) Pneumatic system gas supply (flow control and measurement). 2) Injection system a heated injector port, where the sample is vaporised if necessary. 3) Column where the separation occurs. 4) Oven The coiled column is wholly contained in a thermostatically controlled oven. 5) Detector integral detector or link to a mass spectrometer.

How does Gas Chromatography Work?

1) A carrier gas, like Helium and Neon flows through the system. A valve controls the flow rate. 2) A sample of the volatile mixture is injected into the carrier gas. The sample is vaporised in the heated injector port. 3) The carrier gas carries the vaporised sample into the column. The columns are stainless steel or glass tubes. They can be up to 25 m in length and are of narrow bore (2-10 mm). Therefore the column is often wound into a coil. The packed columns contain porous support material. The sample mixture undergoes a series of interactions between the stationary and mobile phases as it is carried through the system by the carrier gas. Due to the wide choice of materials available for the stationary and mobile phases, it is possible to separate molecules that differ only slightly in their physical and chemical properties. 4) The coiled column is contained in the thermostatically controlled oven.

5) Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component comes through first. 6) Two types of detector can be used: (1) thermal conductivity detectors (2) flame ionization detector (FID), (3) Mass selective detectors , Retention time is defined as the time taken for a component to go from injection to detection. This varies depending on a) The nature of and the interactions between the solute and the stationary and mobile phases. b) The flow rate of the carrier gas, c) The temperature of the column (shorter retention times are obtained at higher temperatures), d) The length and diameter of the column,

 For unknown compounds the solutes are collected individually and analyzed using another method, e.g. mass spectrometry. For each compound in a mixture one peak is observed on the chromatogram. In the particular set of operating conditions relating to the column, the retention time will increase with the size and polarity of the compound. To find the concentration of a particular compound, the peak height should be measured. Applications : GC is used to analyze blood samples for the presence of alcohol. It is also used to analyze samples taken from athletes to check for the presence of drugs. Water companies test samples of water for pollutants using GC to separate the pollutants, and mass spectrometry to identify them.