Chromatography Forum

hye,

I have been investigating detection of insulin in plasma using HPLC.

what is the suitable internal standard for bovine insulin?

now im using lectin as my internal standard, the peak did came out earlier than my standard which is pure bovine insulin. the problem now is that my bovine and lectin came out 2.3 min and 2.1 min respectively as they combined together and it is really hard for me to separate them.

i already added up some methanol so that the bovine peak would come out late, change the mobile phase, but still they didnt get separated. I have used 15ppm of bovine insulin and final concentration is 200ppm. as for lectin is 75ppm.

now im using monolithic column, RP18 from merck (chromolith performance RP 18-e 100-4.6mm), HPLc 2010 from shimadzu, 0.05M Na2SO4 anhydorus(76%) and ACn (26%)
wavelength 214,200nm. injection volume -20uL.

since the compound is endogenous, im using charcoal to suppress the insulin available in plasma but it didnt worked at all.
so when i extracted the blank plasma the peak insulin came out at retention time 2.3 mins.

Could you tell me how can I overcome such problems? huhuu really need help now......

Do you have human insulin or porcine insulin to try as an internal standard? Look here:
http://www.thermo.com/eThermo/CMA/PDFs/ ... e_9708.pdf

Without knowing all the pertinent details, it's hard to make reasonable suggestions.

What is the dead time (t0)? How wide was the insulin standard peak?

With a 10 percent difference in retention, you should get a reasonable separation if you have reasonable efficiency.