We can now offer a series of analyses specific to charged and uncharged dextran and Polysucrose derivatives. This includes molecular weight determination using GPC (gel permeation chromatography), quantitative determinations various substituents (sulphates carboxyls, amines etc.), optical rotation, spectrophotomeric and spectrofluorimetric studies, viscosity etc. Feel free to inquire if you have specific demands.
We can also run GPC according to GMP requirements.
Gel Permeation Chromatography (GPC)
For customers who require guidance regarding molecular weight characteristics of their products, we can now offer GPC services which resolve polysaccharides and their derivatives from 1000 to 4 million. The chromatography is performed using specially selected gels which are run in series according to the molecular weight range in question. The parameters for the system including flow rates, eluents, and sensitivity are carefully controlled to obtain optimal resolution. The following data can be obtained:
- Weight average molecular weight (Mw)
- Number average molecular weight (Mn)
- Peak molecular weight (Mp)
- Polydispersity (PD)
- Ranges (e.g. percentage of product within defined molecular weight ranges)
The columns are calibrated with dextran standards and the optimal polynomial fits are calculated.
For those that are working with polysaccharides or their derivatives, an understanding of mean molecular weight and polydispersity is essential. The most common parameters for defining polymer products are:
Weight average molecular weight (Mw) - a calculation with emphasis on the molecular weight of each molecule in the fraction.
Number average molecular weight (Mn) - a calculation with emphasis on the number of molecules of a given molecular weight in the fraction
Polydispersity (PD) - as expressed by the ratio of Mw/ Mn
Peak molecular weight (Mp) – the molecular weight at the peak of the distribution curve.
The parameter PD is valuable as it reflects the broadness in the distribution (polydispersity) of the fraction. Whereas a normal fraction may have a value of 1.5, a very broad fraction may have values over 5. If the sample only consisted of one molecular species, the PD would be 1. For purposes aimed at establishing the permeability of a membrane or cell, it is desirable to have a narrow fraction.
How do we determine molecular weights?
For molecular weight determination, several methods are available; e.g. light-scattering, viscosity, sedimentation or gel permeation chromatography (GPC) – also known as size exclusion chromatography (SEC). For routine manufacturing control, we prefer GPC as it provides a clear image of the distribution of molecules in the product. This image may then be converted into nominal values. Charged or lipophilic derivatives may give different elution volumes (Ve) than neutral dextrans of the same size (see next paragraph). Providing the calibration curve covers the range required and the chromatography gel system resolves the fraction under study, GPC can be used for products from 1000 to 10 million Dalton. The disadvantages of light-scattering are that (a) the solutions studies must be absolutely free from particles (b) must not be strongly coloured and (c) molecular weights less than 10000 may not give reliable results.
What to think about when ordering dextran derivatives
The Degree of Substitution (DS) of dextran derivatives is of considerable importance for many applications. DS is interpreted as the number of a particular substituent per glucose molecule in a dextran chain. Thus DS 0.1 means 1 substituent for every 10 glucose molecules. The substitution of dextran with anionic, cationic or lipophilic substituents may lead to changes in the hydrodynamic volume in solution, most often expansion. This may give an increase in the nominal molecular weight determined by GPC by 50% or more. It is therefore important that the molecular weight specified is determined on the final product and does not refer to the starting dextran. In low-substituted products, e.g. FITC- and TRITC-dextran, the differences are minimal. For dextran sulfates with high DS, the increase is substantial so that, at TdB Consultancy, a lower MW of dextran starting material is selected, whereby the final product will then have the specified MW.
Products with high fluorescence and high levels of substitution may well affect the permeability of the derivative owing to interactions with charged membranes. TdB Consultancy limits DS levels of its products to 0.002-0.008 in order to provide products with properties as close to those of the starting polysaccharide as possible.
Are you satisfied with the polydispersity (PD) of the product?
The Certificate of Analysis (CoA) for our products, with a few exceptions, gives the Mw and the Mn of the product and one can easily calculate the PD. Generally the PD of products with molecular weights from 5 to 200 kDa will lie in the range 1.5-2.5, which is reasonable for most purposes. Narrow fractions of the highest molecular weight products (>500 000) are difficult to prepare.
Using our premium products with controlled narrow fractions will give you reliable and reproducible results. If you have special requirements, please contact us.