Chromatography Forum

I'm working on a method to test various peptides for purity. I have seen various papers that that describe running a gradient to look for impurities, but so far have not found any actual full methods of inj volume flow rate, and sample prep.

My mobile phase if H20 0.1%TFA/ACN 0.1% TFA
I run a gradient starting at 95/5 going to 5/95 over a time period of 35 minutes.

I'm using a wavelength of 214

Currently i'm trying out injection volumes of 2 and 10ul

The issue I am having is figuring out what an appropriate dilution would be. The vials that I need to test contain lyophilized peptide with mannitol as a filler. They range from 1mg-60mg/vial.

What would be the ideal dilution that wont saturate the detector but will still detect impurities at low levels.

Currently my sample prep is to hydrate the vials with 2ml hplc water, and then do a 10x dilution into the autosampler vial.

If I know the mg in the vial before I rehydrate it is there an ideal concentration that I should try to dilute to before injecting?

When I was using LC-MS for impurities detection I would typically inject a solution at 1 mg / mL.

This would typically provide enough material to generate good quality MS spectra.

I suspect that you could also use this concentration for LC-UV work; saturating the detector is OK unless you are also trying to perform quantitative analysis.

Thank you, I will give that a try. My biggest worry was injecting too little of sample and missing a low level impurity.