Different RF with differing MeOH in sample.

[Brando]

Problem: Our analyte response increases with increasing methanol content in the sample. The difference in response factor is around 40% from zero MeOH to 80% MeOH. This difference is too large to be explained by evaporation. Could this be an autosampler problem?
Any suggestions.
Thanks,
Brando

[Consumer Products Guy]

Need more information, including column size, flow rate, mobile phase composition, column type, etc. For example, if your MP is higher in aqueous, maybe your injection size is OK when there's less methanol in the sample, but causes issues when there's more methanol in the samples as the methanol injected can affect the chromatography by acting as a "strong" solvent.

[4ugirish]

Problem: Our analyte response increases with increasing methanol content in the sample. The difference in response factor is around 40% from zero MeOH to 80% MeOH. This difference is too large to be explained by evaporation. Could this be an autosampler problem?
Any suggestions.
Thanks,
Brando

Response factor can be found out by linearity experiments, Are u getting the linearity? (i.e. Reg. coeff. 0.999) If yes, then there is no issue of evaporation, because if evaporation is there you will never get the linearity?

[gbalock]

Are you sure that there is not a reaction happening in your sample prep? Normally unless your chromatography stays the same, your RRF stays the same no matter what your sample prep is.

[Brando]

Thanks everyone. Some additional info:

The MP is 40% acetonitrile in phosphate buffer (10 mM pH 5), column Phenomenex C18, 140 x 4.6 mm, flow rate 1 ml/min, detection 295 nm.
My calibration curves are perfectly linear whatever MeOH conc. I use but they differ in response (smaller response in lower MeOH). I even tried this with another analyte and got a similar effect.
UV scans of the peaks demonstrate identical lambda profiles irrespective of MeOH conc. which I would think ruled out any chemical reaction in the sample. Am I wrong?
Further suggestions?
Brando

[juddc]

Absent further information as to the nature of the analyte, it sounds a bit like an extraction issue to me. Lower methanol concentrations will extract less analyte; higher methanol concentrations will yield higher analyte concentrations...

Am I missing something?

[Peter Apps]

Hi Brando

This might be a solubility issue. If the analyte is more soluble in methanol than in water, and if you filter your samples into the vials, you get more sample dissolved at higher methanol concentrations.

Is this effect apparent with only one analyte ? - if so it cannot be anything to do with the autosampler.

Peter

[Brando]

Hi Peter and JuddC
This is not a solubility issue as we observed this effect with another analyte. It should not be an extraction problem, we are just dissolving our compound in different MeOH conc. and according to the literature it is souble in all proportions MeOH/H2O.
Brando[/quote]

[Peter Apps]

Hi Brando

Are the analytes with the changing RFs chemically similar ?

Are there any analytes that you do not see the RF effect on the same HPLC ?

Do you see the same effect with e.g. acetonitrile or IPA in the sample instead of methanol ?

What hardware are you using, and what is the injection volume ?

Do you have any carryover ?

Peter

[Bruce Hamilton]

What happens when you inject twice the amount of a sample that has the low methanol concentration, and half the amount of a sample with a high methanol content?.

I'd also try changing the sample and column temperatures as part of an investigation.

Good luck,

Bruce Hamilton

[yangsh]

Hi, Brando

Are there any changes in its peak shape when you used different diluent to prepare your samples? Peak shape always broadens if higher methanol content be used in the sample solution, then peak response maybe change.

[Brando]

Hi everyone.
The problem has been solved. It turned out a combination of a slightly better solubility in methanol AND an autosampler issue which we solved by using a fixed loop for the injections. It seems like that the needle in our HPLC unit is drawing more sample volume when the methanol conc. is higher.
Thanks,
Brando

[Peter Apps]

Hi Brando

Now this is really interesting - with higher methanol concentration, your sample viscosityis lower, so it is easier for the autosampler to suck out of the vial. What model of autosampler are talking about here ?, is there any chance that you have an obstruction in the vial needle - say a bit of vial septum stuck inside it ?

Peter

[HW Mueller]

I see a problem with the solubility effect and calibration linearity.
Brando, how did you prepare your standards used in the calibration?

[ym3142]

Brando,
like most of people here, I am not satifactory with your explanation. How could higher methanol content cause suck more than the lower? If it is due to viscocity did you see least response at 50% MeOH since where it has the highest viscocity.
I believe there are other reasons.
If you do not want reveal your detail sample property/preparation could you answer this question: is it true that somehow your sample dissolves better/faster at higher MeOH contained Diluent?

thanks for sharing.