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Methodology for Separation Procedures

 
     
 
Column chromatography
 
On several occasions, a researcher requires a specific group of macromolecules separated from a biological extract in order to understand the molecule or the process indepth. Chromatography is one of the techniques to separate biological molecules. There are many types of chromatography based on the physicochemical properties of the molecules.
 
These different types of chromatography include gel filtration - ion-exchange - adsorption - affinity chromatography and so on, based on the molecular size and shape, ionic nature, molecular topography and biological specificity of the molecule.
 
 
Essentially, any chromatography consists of two phases: one is stationary phase which may be a solid, liquid or a solid/liquid mixture and is immobilized while the other, mobile phase, is a fluid which flows through the stationary phase.
 
Chromatographic separations in practice may take any one of three modes-paper, thin layer or column chromatography.
 
In column chromatography, the stationary phase is packed in a cylindrical column made of plastic or glass.
 
The concept of gel filtration chromatography is the different molecules are separated based on the molecular size and shape where the stationary gel matrix serves as a sieve.
 
The Principle underlying the ion-exchange chromatography is the attraction between the biological compounds and the stationary phase, each with opposite electrical charges, thus attracting each other. The ionic nature of chemical compounds is hence exploited.
 
Affinity chromatography is based on the attraction of a partially compound specifically to combine with the molecule of our interest. For instance, the inhibitor of an enzyme serves as affinity compound to separate that particular enzyme. However, the inhibitor has to be initially combined with an inert matrix to serve as stationary phase.
 
Majority of the chromatography is routinely carried out Using the column mode. The apparatus and general techniques used for gel exclusion, ion-exchange, adsorption and affinity chromatography have much in common. Gas-liquid chromatography and high performance liquid chromatography each have their own special apparatus, materials and protocols. The column chromatography nowadays been made sophisticated, easier and faster by combining together pumps, detectors, recorders etc. to the columns.
 
As a model, column chromatographic separation of proteins based on their molecular size by gel filtration is described below:
 
 



Principle

The basis of any form of chromatography is the partition or distribution coefficient (Kd) which describes the way in which a compound distributes itself between two immisible phases, such as solid/liquid or gas/liquid.
 
Chromatography columns are considered to consist of a number of adjacent zones in each of which there is sufficient space for the solute to achieve complete equilibrium between the mobile and stationary phases. Each zone is called a theoretical plate and its length in the column is called the plate height. The more efficient the column is the greater the number of theoretical plates involved.
 
 
Materials
Chromatographic column of suitable dimension made up of transparent plastic or glass: Generally, gel filtration is carried out in longer columns (up to 1 m) depending upon the type of gel filtration medium used and the size of the protein to be purified from the bulk
Stationary phase: (ex) Sephadex G 100
There are different types and grades of gel filtration media available. It is to be chosen on the basis of the size of the protein under study
Elution buffer
Fraction collector
Peristaltic pump
Marker proteins (a set of highly pure proteins with known molecular weight (e.g.) cytochrome C, 12,400; carbonic anhydrase, bovine 1,50,000; b-amylase, potato 2,00,000; blue dextran 2,00,000 daltons.
 
 
Procedure
A. Packing the column
1. Suspend the gel (for instance, Sephadex G 100) in a large volume of water or preferably in elution buffer until the gel is fully swollen. The swelling can be done by overnight suspension or by heating in a water bath for 2-4 hr (Follow the manufacturer's instructions for this purpose, carefully).
2. Plug the bottom of column tube with glasswool or sintered filter and stand upright the column.
 
B. Packing the column
3.
Make a good slurry of the gel (stationary phase) in a suitable buffer after proper swelling of the gel.
4.
Pour a small volume of buffer into the column to avoid trapping of any air bubbles in the plug immediately followed by the slurry to the full of column. The top portion may be carefully, gently stirred prior to pouring additional slurry to the growing column, if necessary. Wait until the gel settle down to the desired height by gravitational force.
5.
Place a suitable filter circle on top of the gel bed.
6.
Equilibrate the column thoroughly by passing through the column buffer.
7.
Apply the sample in column buffer onto the top of bed. The sample volume should preferably limited to 1-3% of the total bed volume. The sample can be applied to the top by careful pipetting or conveniently through the buffer pipeline.
8.
Now connect the bufferline to the elution buffer to develop the chromatogram.
9.
Protein molecules pass through the gel space while small molecules distribute between the solvent inside and outside the gel and then pass through the column at a slower rate.
10.
The effluent emerging out of the column can be routed through a suitable spectrophotometer to monitor the absorbance at a
particular wavelength (for proteins either 280, 230 or 210 nm) and the, data recorded. The effluent is then collected using a fraction collector. The effluent is manually collected in the absence of a collector in a fixed time-or volume intervals in tubes and measured subsequently.

11.
The volume of mobile phase required to elute a particular solute is known as the elution volume while the corresponding time for elution of the solute at a given flow rate is known as the retention time.
12.
The elution is continued (usually 2-3 times bed volume of buffer) until the absorbance monitored reached baseline value.
13.
Thereafter the column is extensively reequilibrated with the column buffer for subsequent run.
14.
The reference proteins are loaded onto the bed and the chromatography is carried out as above.
15.
Plot the logarithms of molecular weight of marker proteins against their respective ratios of elution volume to column void volume (Ve/Vo), the column volume being the elution volume of a very large molecule such as blue dextran.
16.
Compute the elution volume of protein of interest and deduce its molecular weight from the above linear graph.
 
 



Notes

1.
The column chromatography is, nowadays, very much modernized/sophisticated and automated. A family of equipment such as pumps, cooling devices, detectors, collector and data processor put together make the technique much interesting and easier.
2.
The column chromatography experiment is an art. Every step in the experiment needs to be carefully done for good results.
3.
The experimenter should choose the right stationary and mobile phases, column etc. for optimal results. It is mostly trial and error when there is no details available on the molecule.
4.
No single column chromatography procedure will result in the complete purification of a molecule except the affinity chromatography procedure. Therefore, chromatography exploiting two or more physicochemical traits of the molecule has to be employed for purification.
5.
Column development using a single solvent is known as isocratic separation. However, in many cases by continuously changing the pH, ionic strength or polarity of the eluent, the resolving power of the eluent could be increased. This kind of elution is called as gradient elution.
 
 



References

1. Gel filtration: Theory & Practice — Pharmacia fine chemicals Hand-Book 97,1974.
2. A Biologists Guide to Principles and Techniques of Practical Biochemistry (eds. Wilson and Goulding) ELBS Publication, 1986.

 
     
 
 
     




     
 
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