After obtaining pellets by centrifugation of ethanol precipitated mixture, I dissolved it in de-ionized water and done characterization studies by using colorimetric assay from Blyscan the data confirmed the presence of glycosaminoglycan both O-sulfated type and N-sulfated type and then I placed the sample in -80 degree for two days and then I placed the tubes for drying in freeze dryer. I got very less amount of brown color sticky material in my tubes. I want to know, that the brown color obtained after freeze drying signifies what?
Hello
have you used a dialysis step at any point in your sample preparation? It sounds a bit like the samples may have hydrolysed. If the sulphate groups do not have a counter ion such as sodium or calcium but are in the acidic form your polysaccharide may have auto hydrolysed..... It happened to us once with a sulphated polysaccharide. We just had some small black spots in our powder. To resolve the issue we re-dissolved and neutralised. From the sounds of it, your sample did not remain frozen during the freeze drying step either. To avoid that you should shell freeze your sample rather than sitting it in a freezer for a couple of days. To shell freeze put your sample in a cold bath and keep rotating your vessel to form a thin frozen shell round the sides of the vessel. This gives you a bigger surface area for sublimation and should help to avoid that your sample liquifies.
Thank You
No I not use dialysis step yet but I use DNA digestion at pH 5 by using sodium acetate and magnesium chloride and after 24 hour of digestion I just filtered my sample then kept it in -80 and then freeze dried but after freeze drying I found that it become brown color and its not even properly dried still have some moisture in it.
That means my sample still can work if i just neutralize it, so if the pH is acidic will I use phosphate buffer or NaOH to neutralize it.
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