Applications of Size Exclusion Chromatography in Pharmaceutical Analysis

APPLICATIONS OF SIZE EXCLUSION CHROMATOGRAPHY IN PHARMA-CEUTICAL ANALYSIS

The size-exclusion-chromatography may be used for two specific purposes in the analysis of pharma-ceutical substances, such as :

(i) Determination of relative component composition, and

(ii) Determination of molecular weight.

1. DETERMINATION OF RELATIVE COMPONENT COMPOSITION

The assay method along with specific experimental parameters are duly stated in the official mono-graph. Here, two situations arise, namely :

(a) Equivalent Responses : In case, all of the components of the sample exhibit equivalent responses to the detector, then the relative quantity of each component may be determined conveniently by dividing each peak area by the sum of the peak areas of the components of interest, and

(b) Non-equivalent Responses : In case, the responses achieved are not equivalent, calculate the relative component composition either from the calibration curve obtained with the calibration standards specified in the official monograph or by any other method stated in the official monograph.

2. DETERMINATION OF MOLECULAR WEIGHT

The following steps may be followed in a sequential manner to determine the molecular weight of a pharmaceutical substance :

(i) Follow the method on the sample by employing the specified procedure laid down in the official monograph,

(ii) Plot a graph of the retention volume of the calibration standards as a function of the logarithm of the molecular weight,

(iii) The curve, thus obtained, normally approximates to a straight line within the exclusion and total permeation limits,

(iv) The molecular weight of the component of interest may be determined from the calibration curve, and

(vi) The calibration is valid only for the particular system employed under the specified experimental parameters.

The techniques of size-exclusion chromatography has been used effectively in checking the purity of the following pharmaceutical substances for their respective impurities, such as :

(i) Corticotrophin                   : For impurities of higher molecular weight,

(ii) Insulin                              : For proteins of higher molecular weight,

(iii) Human Insulin                 : For proteins of higher molecular weight, and

(iv) Plasma Protein Solution  : For polymers and aggregates.

3. CORTICOTROPHIN : FOR IMPURITIES OF HIGHER MOLECULAR WEIGHTS

Materials Required : Corticotrophin : 1 mg ; acetic acid (1 M) [prepared by dissolving 57 ml of

glacial acetic acid in 1000 ml of DW] : 100 ml ; sodium dodecyl sulphate (1% w/v) : 10 ml ;

Procedure : Dissolve accurately weighed 1 mg of corticotophin in 1 ml of 1 M acetic acid containing 1% w/v of sodium dodecyl sulphate. Heat the solution at 100 °C for 10 minutes and allow to cool.

The chromatographic procedure may be performed using (a) a column (about 85 cm × 10 mm) packed with polyacrylamide or cross-linked dextran for chromatography having a fractionation range for peptides with relative molecular weights of approximately 1000 to 10,000 ; (b) 1 M acetic acid as the mobile phase with a flow rate of 7 ml per hour, and (c) a detection wavelength of 276 nm. Now, connect the detector, fitted with a flow-cell suitable for liquid chromatography having a volume of not more than 1 ml, to a strip-chart recorder. Set the detector and chart recorder at a full-scale sensitivity of 0.5 absorbance unit.

Equilibrate the column with 1 M acetic acid. Apply the cold solution to the top of the column using 0.4 ml per cm² of column cross-sectional area. The sum of the areas of any peaks eluted before the principal peak is not greater than 5.0% of the sum of the areas of all the peaks in the chromatogram.

4. INSULIN : FOR PROTEINS OF HIGHER MOLECULAR WEIGHT

Materials Required : Solution (1) : Dissolve 10 mg of insulin in 1 ml of the mobile phase ; Solution (2) Dilute 100 μ l of solution (1) to 10 ml with the mobile phase ; and Solution : (3) Dissolve 10 mg of procine insulin EPCRS* of bovine insulin EPCRS, as appropriate, in 1 ml of the mobile phase.

Procedure : The chromatographic procedure may be carried out using (a) a column (60 cm × not less than 7.5 mm) packed with silica gel for chromatography (10 μ m ; pore size about 13 nm) Water 1-125 ; Toyo Soda TSK 2000 SW ; and Zorbax GF 250 are suitable ; (b) as the mobile phase with a flow rate of 0.5 ml per minute a filtered and degassed solution prepared by mixing 20 volumes of glacial acetic acid and 50 volumes of water, adjusting the pH to 3.0 by the addition of a 25% v/v solution of ammonia, adding 30 volumes of acetonitrile and mixing 1 and (c) a detection wavelength of 276 nm.

Inject 50 μ l of each solution. Adjust the sensitivity of the detector so that the height of the principal peak in the chromatogram obtained with solution (2) is 50-70% of full-scale deflection. In the chromatogram ob-tained with solution (1) the sum of the area of any peak eluting before the principal peak is not greater than the area of the principal peak in the chromatogram obtained with solution (2) (1.0%).

5. HUMAN INSULIN : FOR PROTEINS OF HIGHER MOLECULAR WEIGHT

Materials Required : Solution (1) : Dissolve 10 mg of human insulin in 1 ml of the mobile phase, Solution (2) : Dilute 100 μ L of Solution (1) to 10 ml with the mobile-phase, and Solution (3) : Dissolve 10 mg of human insulin EPCRS in 1 ml of the mobile-phase.

Procedure : The chromatographic procedure may be performed using (a) a column (60 cm × not less than 7.5 mm) packed with silica gel for chromatography (10 μ m ; pore size about 13 nm) Water 1-125 ; Toyo Soda TSK 2000 SW and Zorbax GF 250 are suitable, (b) as the mobile phase with a flow rate of 0.5 ml per minutes of a filtered and degassed solution prepared by mixing 20 volumes of glacial acetic acid and 50 volumes of water, adjusting the pH to 3.0 by the addition of a 25% v/v solution of ammonia, adding 30 volumes of acetonitrile and mixing, and (c) a detection wavelength of 276 nm.

Inject 50 μ L of each solution. Adjust the sensitivity of the detector so that the height of the principal peak in the chromatogram obtained with solution (2) is 50 to 70% of full-scale deflection. In the chromatogram obtained with solution (1) the sum of the areas of any peaks eluting before the principal peak is not greater than the area of the principal peak in the chromatogram obtained with solution (2) (1.0%).

6. PLASMA PROTEIN SOLUTION : FOR POLYMERS AND AGGREGATES

Materials Required : Plasma protein solution : 2.0 ml ; mixed phosphate buffer pH 7.0 with azide [To 1000 ml of a solution containing 1.8% w/v of disodium hydrogen orthophosphate and 2.3% w/v of sodium chloride and sufficient of a solution containing 0.78% w/v of sodium dihydrogen orthophosphate and 2.3% w/v of sodium chloride (about 280 ml) to produce a pH of 7.0 Dissolve sufficient sodium azide in the resulting solution to give a 0.02% w/v solution] : 1000 ml ;

Procedure : The chromatographic procedure may be carried out at room temperature using (a) a column (1 M × 25 mm) packed with a cross-linked dextran suitable for fractionation of globular proteins in the range of molecular weights from 5,000 to 350,000 (Sephadex G-150 is suitable), (b) mixed phosphate buffer pH 7.0 with azide as the mobile-phase with a flow rate of about 20 ml (4 ml per square centimetre) of column cross-sectional area) per hour, and (c) a detection wavelength of 280 nm.

Collect the eluate in fractions of about 4 ml and combine the fractions corresponding to each peak. For each combined fraction carry out the determination of nitrogen as per BP (1993). Not more than 10% of the total nitrogen is present in the combined fraction associated with non-retained proteins.