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Size-Exclusion Chromatography
Size-exclusion chromatography is a high-pressure liquid chromatographic technique that separates molecules in solution according to their size. Methods for size-exclusion chromatography are divided into gel permeation chromatographic methods, which utilize nonpolar organic mobile phases and hydrophilic packings, and gel filtration chromatographic methods, which utilize aqueous mobile phases and hydrophobic packings. The sample is introduced into a column, which is filled with a gel or a porous particle packing material and is carried by the mobile phase through the column. The size separation takes place by repeated exchange of the solute molecules between the solvent of the mobile phase and the same solvent in the stationary liquid phase within the pores of the packing material. The pore-size range of the packing material determines the molecular-size range within which separation can occur.
Molecules small enough to penetrate all the pore spaces elute at the total permeation volume, VT. On the other hand, 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, VO (void volume). Separation according to molecular size occurs between the exclusion volume and the total permeation volume, useful separation usually occurring in the first two-thirds of this range.
Apparatus— The components of the chromatograph are described under High-Pressure Liquid Chromatography.
Column—If necessary, the column is temperature-controlled. It is packed with a separation material that is capable of fractionation in the appropriate range of molecular sizes and through which the eluant is passed at a constant rate. One end of the column is usually fitted with a suitable device for applying the sample, such as a flow adaptor, a syringe through a septum or an injection valve, and it may also be connected to a suitable pump for controlling the flow of the eluant. Alternatively, the sample may be applied directly to the drained bed surface, or, where the sample is denser than the eluant, it may be layered beneath the eluant. The packing material may be a soft support such as a swollen gel or a rigid support composed of a material such as glass, silica, or a solvent-compatible, cross-linked organic polymer. Rigid supports usually require pressurized systems giving faster separations. The mobile phase is chosen according to sample type, separation medium, and method of detection.
Detector—The outlet of the column is usually connected to a suitable detector fitted with an automatic recorder that enables the monitoring of the relative concentrations of separated components of the sample. Detectors are usually based on photometric, refractometric, or luminescent properties (see Detectors under High-Pressure Liquid Chromatography). An automatic fraction collector may be attached, if necessary.
Procedure— Before carrying out the separation, the packing material is treated and the column is packed, as described in the individual monograph or according to the manufacturer's instructions. Where necessary, procedures for verifying the suitability of the system are described in the individual monograph. The column efficiency may be evaluated from the number of theoretical plates, N (see the section Interpretation of Chromatograms). The elution characteristics of a compound in a particular column may be described by the distribution coefficient, KD, which is calculated by the formula:
(VIVO) / (VT VO)
in which VO, V T, and VI are the retention volumes for the nonretained component, the component that has full access to all the pores in the support, and the compound under test, respectively. Each retention volume is measured from the time of application to the time of the peak maximum.
Determination of Relative Component Composition of Mixture—Carry out the separation as directed in the individual monograph. Monitor the elution of the components continuously, and measure the corresponding peak areas. If all the components under test exhibit equivalent responses to the physicochemical property being monitored (for example, if they exhibit corresponding absorptivities), calculate the relative amount of each component by dividing the respective peak area by the sum of the peak areas of all the components under test. If the responses to the property used for detection of the components under test are not equivalent, calculate the content using calibration curves obtained from the calibration procedure specified in the individual monograph.
Determination of Molecular Weights—Size-exclusion chromatography is used to determine molecular weights of components under test by comparison to calibration standards specified in the individual monograph. Plot the retention volumes of the calibration standards versus the logarithm of their molecular weights. Draw the line that best fits the plotted points within the exclusion and total permeation limits for the particular separation medium. From the calibration curve, molecular weights of components under test are estimated. This calibration is valid only for the particular macromolecular solute-solvent system used under the specified experimental conditions.
Determination of Molecular Weight Distribution of Polymers—The material used for calibration and the methods for determination of the distribution of molecular weights of polymers are specified in the individual monograph. However, sample comparison is valid only for results obtained under identical experimental conditions.