Chromatography Forum

Hi,

I'm noticing a strange behaviour from my HPLC when I inject the same sample repeatedly.

I have my system calibrated with know standards at:
0.1 ppm
0.2 ppm
0.3 ppm
0.4 ppm
0.5 ppm

The calibration is very accurate and gives good results.

However, when I run any sample, for example I did my 0.3ppm today, repeatedly, my results vary.

Real results from today:

0.3ppm calibration result = 0.3098ppm

But when I run this sample 3 times, I get the following:

1: 0.3020 ppm
2: 0.2910 ppm
3: 0.2830 ppm

If I continue, the sample gets lower and lower. Even if I do sample > blank > sample > blank, the results are the same.

Can anybody offer any input? Thankful for any help at all.

Welcome to the forum.

Can you tell us what compound you are analyzing?

If you reinject a calibration standard after repeated sample injections, does it give a lower area as well?

Welcome to the forum.

Can you tell us what compound you are analyzing?

If you reinject a calibration standard after repeated sample injections, does it give a lower area as well?

Hi Rndirk, thanks for the reply.

I'm analysing formaldehyde. The calibration standard is the re-injection 'sample' that gives a lower result each time.

If you are using NASH reagent for post-column derivatisation , you have to protect your reagent from light , it is light sensitive.

If you are using NASH reagent for post-column derivatisation , you have to protect your reagent from light , it is light sensitive.

Hi uzman,

We do pre-injection derivitisation. The reagent is kept in the dark at all times.

Hi Pear ,

Is your injection solvent weaker than your mobile phase ; if you are using a RP column ?
If not , you may try to dissolve your sample in mobile phase and inject.

Hi Pear ,

Is your injection solvent weaker than your mobile phase ; if you are using a RP column ?
If not , you may try to dissolve your sample in mobile phase and inject.

Hi uzman,

my mobile phase is 0.1% phosphoric acid + ACN: 0.1% phosphoric acid + water (45:55), the sample is added to 9:1 ACN:water then injected.

Hi Pear ,

Your injection solvent is stronger than your mobile phase , you may try mobile phase as injection solvent.

Hi Pear ,

Your injection solvent is stronger than your mobile phase , you may try mobile phase as injection solvent.

Hi uzman,

Thanks again for your help. We tried injections using mobile phase as the sample solvent, yet we are still seeing a decrease in result every injection.

Scratching my head over this one.

Some more suggestions :

First , try to backflush your column with pure acetonitrile , accumulated junk on the column might be the reason.

You may also switch to methanol as your mobile phase , instead of acetonitrile.

Formaldehyde ( as methylene glycol , in aqeous solutions ) is more stable in methanol.

just out of curiosity, if you take two vials, put the same standard in both, put them both in the autosampler, but inject from only one (repeatedly), if after a load of injections you then inject from the other, is it low or normal? I.e., is this a stability problem, or is it connected with making injections from the vial?