Chromatography Forum

Dear all;

I have response variation problem. When injections made from same vial responses of the analyte and IS changes but not with same proportionation.

extraction procedure:liquid-liquid sample preparation
column:x-terra c18
mobile phase:Acetonitrile:10mM ammonium acetate:formic acid (50:50:0.530 ,vv) pH:4.6
reconstitue solution:water+25mM ammonia+ACN (50:50) ,pH:9.9
ınjection volume:15 ul
flow rate:0.25ml/min

Also after every six injections in a batch mobile phase is injected four times
and results get better with this procedure.But,I can not provide reproducibility
between batches.Results of one batch could be good with MP cleaning,but the other ones results could be bad.

I have written my conditions above,without any changes in my conditions,what can you suggest me solve response difference problem?

Hale Aydonat

A bit more inoformation is needed:

Is this MS detection? If so, is ISTD isotopically labeled? If isotopically, labled where is it labeled (C, N, O?) and with what isotope?

What are your column dimensions? 2.0mm ID x ?

Do you sonicate after adding reconsitution solvent?

Is it necessary to reconstitute with pH 9.9?

It is msms detection and internal standard is not isotopically labelled.

Column dimension is 3.5um,3.0x100 mm

I do not sonicate but vortex for 1 min after adding reconstitution solution.

Unfortunately I can not change my conditions pH has to be 9.9.

By the way thank you for your answer

After reconstituting the sample, try centrifuging it at high xG for 5minutes?

Without information regarding the ionizability of your compounds you can not expect anything better than these "blind" suggestions. Also how do you maintain a sample pH of 9.9 with aqu. ammonia?