The Analytical Scientist / Issues / 2013 / Apr / GPC/SEC-MALLS analysis of Hyaluronic Acid
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GPC/SEC-MALLS analysis of Hyaluronic Acid

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.

04/16/2013 1 min read

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

Eluent:
0.005M phosphate
buffered saline, 0.069 M NaCl Columns:
3 x PSS SUPREMA
Lux 10 µm
Ultrahigh (8 x 300 mm) + precolumn  Data acquisition:
PSS WinGPC
UniChrom with Compliance Pack Detectors:
SECcurity
GPC1260 RI
SECcurity MALLS
SLD7000 Flow-rate:
0.5 ml/min Concentration:
0.5 g/l   Injection volume:
100 µl

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 and column particle size and porosity. For sample preparation the water content of approx. 12 % has been taken into account.

* Highest molar mass standard: Mp = 2 500 000 Da, P/N PSS-dpul2.5m

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

Due to the lack of high molar mass calibration standards the use of light scattering detection is recommended. This also allows the measurement of true molar masses.   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 on-line measured molar mass. Sample recovery (comparing Concentration Calculation vs. Given) was nearly 100% showing that all material eluted from the column. 

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

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