Chromatography Forum

i am using 700 volumes of buffer (KH2PO4 and Cetrimide) and 300 volumes 1-propanol for frusemide analysis. what washing solvent can i use to effectively clean the column after the analysis. column: c8 250mm x 4.6mm x 5um. i see a increasing trend in back pressure after each use.. kindly advice. thanks in advance.

Joe

Hello

Just replace buffer with water. So you can wash your column with 70:30 water/1-propanol.

Regards

Tomasz Kubowicz

I made good experience by washing the column with 100 % isopropanol. Please avoid precipitation of the buffer and make an additional washing step as described by Tomasz. Hopefully it will work for your problem to. Can you please provide us more detailed data about the increasing back-pressure?

The problem is maybe caused by particles or impurities of your ion pair agent or buffer salt. Do you use the best available quality for your salts and solvents? Nevertheless the best quality of ion pair agents is usually not very good. I am surely not a big friend of filtration of mobile phase, but in your case it might be necessary.

Thanks for the replies. All chemicals and solvents used are of Merck. The mobile phase was filtered with 0.22um filter. the initial back pressure of the column was 3100psi on waters alliance hplc. after around 60 injections the back pressure is 4000psi. i think the washing i have done is not sufficient, usually hours at 1.0mL/min.

The higher pressure from injection to injection could be also caused by adsorption of placebo or matrix substances. Maybe you should think about instaling the precolumn for this analysis if the solution of furosemide is with high concentration of some kind of matrix substances, or if you dont filter you solutions maybe you can try it also.

I would also recommend changing from the 100% isopropanol to 100% acetonitrile - higher elution strgh, better cleaning.