Molar Mass Determination of Gelatins Using GPC/SEC

Gelatin is a polypeptide produced from hydrolysis of collagens in skin and bones. It is obtained from acidic or from basic hydrolysis and contains poly amino acids. Gelatin is used in the food and beverage industry and in the pharmaceutical industry as a stabilizer for tablets and for many other applications.

Sample Preparation

The isoelectric point of the basic produced gelatin is between 4.7 and 5.2; the acidic produced gelatin has an isoelectrical point of 7.5 to 9.3. Basic produced gelatin can be easily measured using standard conditions (see PSS Application notes 10271 and 10272). Gelatin with an isoelectric point between 7 and 9 must be measured using low pH values or at pH-values > 9 (not recommended) in order to achieve sufficient solubility.

The following experimental conditions can be applied to gelatins independent of their isoelectric point and the hydrolysis process.

However, exact sample preparation following a strict protocol is a crucial step for the GPC/SEC analysis of gelatins.

Figure 1: Overlay of 3 different gelatins from Carl Roth Chemicals, Karlsruhe, Germany.

Experimental

GPC/SEC analysis was performed on a PSS SECcurity GPC System comprising:

• an isocratic pump

• an autosampler with variable injection volume

• a DAD detector operated at a fixed wavelength 214 nm +/- 4 nm

The analysis conditions were:

Columns: PSS PROTEEMA, 5µm, 1000 Å, 300 Å, 100 Å (8 × 300 mm each) + precolumn

Solvent: Phosphate buffer pH 5.5 (acidic gelatin) to 6.6 (basic gelatin) + 0.2 M NaCl + 0.5% SDS

Flow-rate: 0.5 mL/min – 1 mL/min

Temperature: Room temperature

Calibration: PSS Pullulan kit, 10 standards: 342–710000 Da (alternative: PSS Poly(styrene sulfonate) sodium salt kit, 10 standards: 100–1000000 Da)

Concentration: 2 g/L

Inject volume: 100µL

Data acquisition, calibration and evaluation: PSS WinGPC UniChrom 8.0

Results and Conclusion

Figure 1 shows the measured molar mass distribution for three different gelatins. Reproducible and easy-to-use GPC/SEC analysis for all types of gelatins can be done using a SECcurity GPC system, PROTEEMA columns and a solvent with a pH of 5.5 for acidic and 6.6 for basic produced gelatin. Data analysis was done using WinGPC UniChrom.

Software solutions for advanced detection (e.g., triple detection), simultaneous molar mass analysis of up to 8 regions and 21CFR11 compliant analysis are also available.

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