GPC/SEC–MALLS Analysis of Hyaluronic Acid

Introduction

Hyaluronic acid is a polymer of disaccharides and can have up to 25 000 disaccharide repeat units. The molar mass range is from 5000 Da to 20 000 000 Da. Hyaluronic acid is mainly used in medical and cosmetic applications.

GPC/SEC is the method of choice for measuring the molar mass distribution of hyaluronic acid. True molar masses can be obtained when online multi angle light scattering is used in combination with a refractive index detector. The refractive index increment (dn/dc) for light scattering data evaluation can be determined online, measured offline using dedicated instrumentation, or taken from literature.

Experimental Conditions

Eluent:                       0.005 M phosphate buffered saline, 0.069 M NaCl

Columns:                   PSS SUPREMA Lux 10 µm 30 Å, 10 000 Å and 10 000 Å (8 × 300 mm) + precolumn

Data acquisition:         PSS WinGPC Unity with Compliance Pack

Detectors:                  SECcurity GPC1200 RI SECcurity MALLS SLD7000

Flow-rate:                  0.5 mL/min

Concentration:           0.5 g/L

Injection volume:         100 µL

Results and Conclusions

High molar mass samples require lower flow-rates and concentrations in GPC/SEC. The GPC/SEC conditions and columns have been optimized for high molar mass hyaluronic acid with respect to loading, flow-rate, column particle size and porosity. For sample preparation the water content of approx. 12% has been taken into account.

Due to the lack of high molar mass calibration standards the use of light scattering detection is recommended. This allows also to measure true molar masses.

Figure 1: Slice concentration and molar mass obtained with a SLD7000 and an RI detector.

A refractive index increment (dn/dc) for hyaluronic acid of 0.165 mL/g has been used to evaluate the light scattering data.¹ Figure 1 shows the slice concentration measured by the RI detector as well as the online measured molar mass. Sample recovery (compare Conc. Calculation vs Given) was nearly 100% showing that all material eluted from the column.

Figure 2: Molar mass distribution, molar mass averages and cumulative distribution for a hyaluronic acid obtained using GPC/SEC-MALLS.

Table 1 shows the molar mass averages of the hyaluronic acid sample obtained using a Pullulan calibration (RI only, apparent molar masses) and using light scattering. The light scattering M_w molar mass is three times lower than that obtained with a pullulan calibration curve.

Table 1: Comparison molar mass averages: Only light scattering gives absolute molar masses.

References

1. Lavrenko, Linow and Gornitz in Analytical Ultracentrifugation in Biochemistry and Polymer Science, Royal Society of Chemistry, Cambridge, 517–531 (1992).

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