Size-exclusion chromatography is a technique of separation of molecules in the solution according to their size. It is based on the repeated exchange of solute molecules between the solvent of the mobile phase and the same solvent in the stagnant liquid phase (stationary phase) within the pores of the column packing material. The pore-size range of the packing material determines the molecular-size range within which separation can take place.
Molecules small enough to penetrate all the pore spaces elute at the total permeation volume (V,), Molecules apparently larger than the maximum pore size of the packing material migrate along the column only through the spaces between the particles of the packing material without being retained and elute at the exclusion volume (V0 void volume). Separation according to molecular size takes place between the exclusion volume and the total permeation volume with useful separation occurring in the first two thirds of this range.
Related: Principle of Liquid Chromatography
Related: Principle of Liquid Chromatography
Apparatus
A chromatographic column, temperature-controlled, if necessary, packed with a separation material capable of fractionation in the appropriate range of molecular sizes and through which the eluent is passed at a constant rate. The dimensions of the column are stated in the individual monograph as (length x internal diameter). The mobile phase is passed through the column either by gravity or by means of a pump. The outlet from the column is connected to a detector fitted with an automatic reorder that allows the monitoring of the relative concentrations of the components of the sample.
Detectors are usually based on photometric, refractometric or luminescent properties. An automatic fraction-collector may be attached, if required.
Detectors are usually based on photometric, refractometric or luminescent properties. An automatic fraction-collector may be attached, if required.
The packing material may be a soft support such as a swollen gel or a rigid support such as glass, silica or a solvent compatible, cross-linked organic polymer. Rigid supports usually require pressurised systems giving faster separations.
Before carrying out the separation, the packing material is treated, and the column is packed as described in the monograph, or according to the manufacturer's instructions. The temperature of the column, if other than that of the room, the nature of the packing material, the composition and flow rate of the mobile phase and the means of detection are stated in the individual monograph.
Performance
The column efficiency may be derived as described under Gas chromatography but the term in the expression for calculation is called the retention volume (V R) for the component of interest. The retention volume is the distance along the baseline between the point of injection and a perpendicular dropped from the maximum of the peak of interest.
The distribution coefficient (KD), where stated in the monograph, is calculated from the expression
KD = (VR- VO)/(VT-
The values of VR, V0, and VT must be expressed in the same unit of measurement.
Determination of relative component composition
If all of the components of the sample under examination exhibit equivalent responses to the detector, then the relative amount of each component can be determined by dividing each peak area by the sum of the peak areas of the components of interest. If the responses are not equivalent, calculate the relative component composition either from the calibration curves obtained with the calibration standards specified in the monograph or by any other means.
Determination of molecular weight
Carry out the method on the substance under examination and calibration standards using the procedure given in the individual monograph. Plot a graph of the retention volume of the standards as a function of the logarithm of the molecular weight. The curve is almost a straight line within the exclusion and total permeation limits. The molecular weight of the component of interest may be estimated from the calibration curve. The calibration is valid only for the particular system used under the specified experimental conditions.
Materials
Agarose Fe. For the separation of proteins with molecular weights of 6x104 to 2x107 and of polysaccharides with molecular weights of 3x103 to 5x106. They occur as swollen beads 60 to 140 f.U11 in diameter and are available as a 4 percent suspension in water.
Agarose FC, Cross-linked. It is prepared from agarose by reaction with 2,3-dibromo-l-propanol in strongly alkaline conditions. It is used for separation of proteins with molecular weights of 6x104 to 2x106 and of polysaccharides of the same range of molecular weights as with Agarose Fe.
Silica Gel Fe. For the separation of proteins with molecular weights of 1x103 to 3x105. It occurs as a very finely divided powder with an average particle size of about 10 f.U11 with a very hydrophilic surface and an average pore diameter of about 30 nm. It is compatible with aqueous solutions of pH 2 to 8 and with organic solvents.
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