Chromatography Forum

I am about to run a calibration for podophylotoxin, a drug used in the treatment of certain types of cancer.
The method I am following uses 35% acetonitrile/65% Water as the mobile phase with a C18 column. The sample solvent used in extraction is methanol. Should I be mixing my standards in ACN/WATER or methanol?
We have run a real time sample (methanol) several times and all peaks are well resolved.
Thank you in advance for your response(s).

Ricardo

In general, standards and blanks should always be the same solvent as the sample.

Ideally, the sample solvent should also be as close to the mobile phase as possible, as this prevents deposition of analyte or junk in the injector or at the front of the column.

However, if the method works well for samples, then just make up the sample, standards, and blanks with methanol.

Given that your mobile phase is nice and simple, it would also be a very good idea to spike some samples with standard to confirm recovery is acceptable.

Please keep having fun,

Bruce Hamilton

whenever your sample solvent is different then your mobile phase you run the possibility of having peak shape distortion. either fronting or tailing. it is due to a difference in the solvent strenghts.

check that all your peaks have a good shape and that assymetry by EP/USP is well with in the range of 0.8 to 1.5.

if one of your coumpounds is not in this range then i would go for an additional dilution step.

I agree with the above responses. That said, we more often than not use solvents for the sample extractions other than those in the mobile phases, such as methanol or DMF to extract because it works with our products, and water-ACN for the HPLC. We usually inject 5ul so that also helps us out.