Chromatography Forum

Hello,
I am trying to switch a method from a UV assay to an HPLC assay for the quantitation of Polyvinyl alcohol (PVA). In the previous method the sample is complexed with iodine so as to give a uv absorbance, however i don't believe this to be very accurate as it is dependent on free hydroxyl sites.I have tried the following using an RI detector.
1) TSKGel G400PW 7.5mm ID * 30cm with a potassium phosphate buffer
2) C8, C18, CN with variuos Acetonitrile/ water concentrations
3) CN column with ACN/ H2O (pH2.20)/ 2-Propanol (58/33/9) flow 0.7 mL/min

In 3) i have a problem in that my retention time is approx. 2-3 mins and i am worried that when stressing my samples and placebo (containg Povidone) for specificty studies that i will have excipients coming out at this short retention time. Changes in mobile phase (conc. and pH) does not seem to affect the retention time significantly. Is it being retained at all on the column?

4) I have also tried a Hillic column with various conc. of acetonitrile/ water

Am i on the right path using an RI detector- is this sensitive enough for detecting possible impurities?

I am new to this "game" of method development and i apologise if i am missing the obvious but any suggestions would be very much appreciated. Thanks.

The best method for the quantitation of a polymer is a SEC method like your first method. I do not know why you walked away from this.

Isocratic retention chromatography of a polymer is something that does not work. You will get eays shifts in retention. Only gradient elution will work for polymers.

Since you got a peak on your CN column, it is probably unretained, but nobody can tell you since you did not tell us the column dimensions. You can calculate this actually yourself: if the peak elutes at about 66% of the column volume or earlier it is not retained.

Dear D reape:
Did you find a solution for this separation problem?

According to Waters' catalog, PVA is a 'nonionic' material, and can be run on an Ultrahydrogel column, with 0.1M NaNO3 as MP. Adding buffer may not improve resolution, since SEC is based on 'effective size.' Usually, SEC is used to 'characterize' a polymer (e.g. MW distribution).
A SEC column from Waters may cost ~ $1200.

Best wish,
Alfred.

I don't think SEC method can be used for quantitation purpose for PVA. It is only for MW distribution. We're using UV method too, however, the high throughput 96-well UV can help for quantitation. I'm wondering if there is HPLC method for PVA quantitation

Why should one not use SEC for quantitation? You get a peak, you get an area... Insulin is commonly quantitated using SEC...