when I use C18 Reverse phase column , TFA and acetonitrile as mobile phase , gradient elution at a flow rate of 0.5 ml/min , the retention time is around 3.58 mins and I am not sure about the peaks that are eluted , when i compare it with the whey I get the same pattern of peaks but at aroun 5.45 mins. Can someone help me how to analyse with the same. Thank you
Retention time depends on multiple instrumental factors and is thus not reproducible enough to answer the question, even if you would give more details like column size, temperature etc. If you observe a time offset between otherwise identical and characteristic peak patterns of two subsequent runs on the same system I would guess that the equilibration time was insufficient for one of the runs. To be sure: have the column equilibrated with at least ten dead volumes of initial eluent before injection and have an empty gradient run before injecting the first sample. Be also sure that all samples have the same solvent and the same injection volume. I would also recommend to check the pressure profiles if available. They should be identical for two subsequent runs, lack periodic fluctuations and allow for some diagnostics if not.
Could you write please. Who is the producer of your column? What is the size of column? What the origin of sample and which you need to separate?
I agree with Jandirk. Do you have authentic standard of lactoferrin? I think for assay using HPLC we need to have authentic standard. If you have standard than you can compare Rt and UV spectrum of your target peak in sample with UV spectrum and Rt of the standard. If you have MS as detector than it will be nicer, you can compare to MS spectrum of standard. If you have MS/MS than you can use MRM to confirm your target peak by identification point system (n = 4) of Commission Decision 2002/657/EC.
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