Chromatography Forum

Sadly, I need some more information before I can make any sensible suggestions.

1. Is this a USP method ( sounds like it with GF and IF ), if so which one?
Does the method specify or permit dilution with mobile phase. I would, if I could - refer 5 below.

2. What sample volume are you injecting?. Are you ensuring that it is cooled before dilution and filtration.

3. What happens when the system is just running with no injection, is the pressure is increasing - you suggest that it is, and it isn't. I would confirm that the pressure is stable with injections of mobile phase. If it isn't, then you mobile phase requires better filtration.

4. What happens to the pressure when you inject appropriately diluted, and cooled - if necessary, samples of each of the components of the sample solution ( Gastric Fluid, Intestinal Fluid, Buffer ), and then the unused blend used for sample solutions. My suspicion is that they should be OK in 70% aqueous, but you should check.

5. It seems that your samples have something that is precipitating in the mobile phase, so the next step may be to dilute your samples with mobile phase, allow to stand for a few minutes ( 5 - 10 ) before filtering. Then ascertain if the pressure still increases on injection.

6. If you have not got aguard column, I would be useful to add one, just to protect the column for dissolution media junk.

Please keep having fun,

Bruce Hamilton.

Koko, I don´t understand this, you say that your analysis has nothing to do with proteins, yet you use body fluids?? Proteins are not taken out by a 0.45µm filter.
Also, did you say /mean that pressure increases without ever injecting your samples, or did you refer to pressure staying high after flushing when you got a pump stop?



Shahis77, why could gelatine not be a problem if present?

What is the strength of your phosphate buffer, in mM. Did you filter your phosphate buffer prior to adding Methanol.

I suspect either precipitation (excess buffer conc) or particulates from the buffer are clogging up your column.

The buffer composition is NaH2PO4:Na2HPO4 pH 5.8, 50mM, and filtered through 0.2 um membrane

Koko,

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I have found the problem, seems like some of gelatin in the sample got stuck in the column,then I flushed the column, using water 100% 2ml/min at 55 centigrades for 2 hour, that resolved the problem,I dont saw any precipitate in the buffer with sample but the pressure gone down. T hank you for your help.