1. Prepared by : Mona Abo Hasera
Chromatography
Prepared by :
Mona Abo Hasera

2. Chromatography First application by M. S. Tswett 1903
For the separation of plant pigments. Since the components had different colors the Greek chromatos, for color, was used to describe the process, so it means color writing.
A physical method of separation in which the components to be separated are distributed between two phases:
A stationary phase and a mobile phase that moves in a definite direction.

3. Chromatography is a laboratory technique that
Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass.
Affinity – natural attraction or force between things. (The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column. )*
Mobile Medium – phase which move over or through stationary phase and carries sample along with it, thus resulting in the separation of its component. it is either liquid or gas. (mobile phase)
Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase)

4. Illustration of Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture
Components
Components
Affinity to Stationary Phase
Affinity to Mobile Phase
Blue
Insoluble in Mobile Phase
Black
     
 
Red
    
Yellow 
        
Analyze
Identify
Purify
Quantify

6. Advantages of chromatography
Can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid.
can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed.
Exact quantitative analysis is done even from trace compounds.
Small material consumption.
The quantization has a broad linearity range.
Analyses of several compound can be done during one run.
Chromatography is a fast analysis method.
Well establishes instrumentation with high level automation is commercially available.
*the accuracy of a measurement system is the degree of closeness of measurements of a quantity to that quantity's actual (true) value. The precision of a measurement system, also called reproducibility or repeatability, is the degree to which repeated measurements under unchanged conditions show the same results
* Linearity is the amount of deviation from an instrument's ideal straight-line performance.

7. Types of chromatography Based on stationary phase
Column
Planar
Based on mobile phase
Liquid
Gas

8. Kinds of Chromatography
Liquid Column Chromatography
gel filtration ,ion exchange, affinity, adsorption, reverse phase, metal binding (column)
2. Gas Liquid Chromatography (column)
planar Chromatography (Thin-layer & paper )

9. LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.
With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.

10. ION EXCHANGE
protein interact with stationary phase by charge-charge interaction
Positively charged proteins adhere to negatively charged functional groups (carboxylates, sulfates:cation exchanger)
Sequential elution, change of pH

12. SIZE EXCLUTION/GEL FILTERATION/GEL PERMEATION
porous beads as stationary phase
stroke radius; function of molecular mass and shape
greater the stroke radius faster will be the elution
gel is made of dextran agarose or polyacrylamide

14. AFFINITY sensitivity of most proteins towards ligands
use of resins to attach ligands
elution by competition with soluble ligand or by disruption of interation.

16. Gas liquid chromatography
separation of volatile mixture
stationary phase is non-volatile liquid, coated on an inert solid
mobile phase is a inert gas (ex. Argon or helium ) or an unreactive gas such as nitrogen.

17. Planar chromatography
It is a method for separation and determination of substances, allowing to carry out qualitative and quantitative analysis of chemical components in complex mixtures.
A separation technique in which the stationary phase serves as a plane.
The plane can be either a paper (paper chromatography) or a layer of solid particles sorbent (silica gel, cellulose, aluminum oxide, ion exchange resin) spread on a support such as a glass- or a plastic- plate (thin layer chromatography).

18. For qualitative analysis the different mobilities of substances are used, the distances passed by different substances are different. The distance between the starting line and the center of the spot of substance hx, mm characterizes the substance. Retention factor, RF, provides better way to indentify substances .
For quantitative determination the intensity of the spot is used: the bigger the amount of substance in the mixture, the more intensive is the spot. Also the size of the spot can give quantitative information – the bigger the spot, the bigger the content of this compound in the mixture. Intensity of the spots is evaluated by comparing with the intensities of analyte spots with known amounts visually or using densitometer.
A densitometer is a device that measures the degree of darkness (the optical density) of a photographic or semitransparent material or of a reflecting surface. The densitometer is basically a light source aimed at a photoelectric cell. It determines the density of a sample placed between the light source and the photoelectric cell from differences in the readings.

19. Principles of Paper Chromatography
Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.
Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents.
Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

20. Thin Layer Chromatography (TLC)
Different compound in sample mixture travel different distance according to how strongly they interact with the
stationary phase as compared to mobile phase. ( adsorption)
So it depend on : 1. activity of stationary phase.
2. polarity of mobile phase.
3. structure of substrate .

21. Principle
Different compounds in sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase.
The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.

22. Measuring Rf

23. Instruments, chemicals and glassware:
Procedure:
Instruments, chemicals and glassware:
Eluent. [Mix n-butanol, acetic acid (purity 98 – 100 %) and distilled water in volume ratio 5:1:5. Stir for 10 minutes, then let the layers separate. Use upper layer as eluent].
Developing Solution: Dissolve 0.3 g of ninhydrin in 100 ml n-butanol. Add 3 ml of glacial acetic acid.
0.02 M solutions of different amino acids (e.g. leucine, methionine, alanine and serine) in H2Odd.
Chromatographic paper
Elution chamber, Glass capillaries for spotting the samples.
Drying oven at ~ 60° C.

24. Rubber gloves must be used during this work to avoid contamination of chromatographic paper with amino acids from skin, and for protecting skin from solvents and ninhydrin while working with the sprayer or sprayed paper.
While the paper is being prepared for chromatographic analysis it should be kept on a piece of filter paper.
3. Mark the starting line to the paper mm from the edge of the plate - with graphite pencil (very slight line!). Also mark the locations where the samples will be spotted. The distance between neighboring spots should be about 8 mm and the spots should be at least 5 mm away from the paper’s edge. Usually the spot of unknown substance is applied to the center of the starting line.

25. Before applying samples to the paper and filling the elution chamber fit the length of chromatographic paper with the height of elution chamber.
5. The spots of individual amino acids and sample solutions are applied to the chromatographic paper. Use separate clean and dry glass capillary for each solution. Dip the capillary into solution – some solution is drawn into the capillary. With the filled capillary touch the prepared location on chromatographic paper. The spot on the paper should not be bigger than 2-3 mm. (You can exercise spotting on a sheet of filter paper.)
6. After application of samples let the spots dry. Meanwhile measure with a graduated test-tube 5 ml of eluent into the elution chamber. Cover the chamber with lids and let the chamber atmosphere saturate with eluent vapors for at least 10 min.

27. Elution is stopped when the solvent front has traveled up the plate until 7-10 mm from the lid.
Remove the paper from elution chamber and place it on a sheet of filter paper. After 2-3 minutes mark the eluent front with pencil and dry the paper in oven.
9. When the paper is dry, take it into the fume hood and spray it with solution of ninhydrin until the paper is slightly damp. Chromatographic paper and the paper supporting it should lie at 45° angle while spraying. The chromatographic paper is again put in the drying oven (60 C) for 15 min to speed up the reactions.

28. Remove chromatographic paper from drying in the oven, draw the contours and centers of the chromatographic bands. Calculate RF values by the method described above.
11. Compare retention of standard substances and components in sample and determine which amino acids were present in the sample.

29. Result Determination of amino acids using thin layer chromatography
Adding fluorescence indicators to the sorbent layer during the process of preparation of the plates or spraying the plates with fluorescent solutions and then observing under ultraviolet lamp.
I2 vapor as indicator .
Ninhydrin spray.

30. Ninhydrin spray
Development of Ruhemann’s purple from ninhydrin and amino acid.