Mobile phase as sample

[greg07]

Hello,

I have such problems:

1. I'd like to ask, why I see a peak from methanol, when I injected the sample 20ul (methanol/water 4:1), C18, UV or DAD detector, 240nm, 1ml/min. and mobile phase also methanol/water 4:1? I injected "the same to the same" - from where this peak (h=ca 1mV, tr=4,5min.)

2. I injected a 20ul of standard fluocinolone acetonide (diluted in methanol), mobile phase methanol/water 4:1, C18, 240nm and sometimes I see separated a small peak from methanol and my peak from fluocinolone acetonide and sometimes the peak from methanol is "under" the peak of a fluocinolone acetonide. I have a question are this second situation (no separation) unacceptable and I should not make a quantitative calculation - I thing an area from methanol distorts calculation?

3. a separation methanol as sample solvent and mainly peak with water/methanol mobile phases is a typical problem and only with good condition of column the separation is OK or if this is not so important?

Regards

[tkubowicz]

Hello

It'd be much better if you paste chromatograms. Some people (including me) needs to "see" problem rather than read descriprion of it.

Regards

Tomasz Kubowicz

[mattmullaney]

Hi Greg07,

I'm with Tomasz, but I'll also ask what are the dimensions of the column? That way, we can learn the retention factor of the methanol peak and that of the standard substance you're injecting. Offhand, sometimes methanol may be contaminated out-of-the bottle, or perhaps the injector of the HPLC could use a cleaning, or perhaps this peak represents the "Column Void Volume."

With the flow rate and the column dimensions, we can estimate the column void volume...and the column void time. These will be helpful to solve this mystery, I think.

[greg07]

[img][/http://www.fotosik.pl/zdjecie/d906f8cced85ccf6img]


I've done tests on 3 different columns, resistant tube (20m x 0,2mm) and 3 HPLC sets

[greg07]

http://www.fotosik.pl/zdjecie/d906f8cced85ccf6

http://www.fotosik.pl/zdjecie/8028a3cf9b6e8ea6

For higher peak left max coordinate = 30mV

Column C18, 5um, 250mm, 4,6mm

[tkubowicz]

Hello

First of all check your LC system:
1.Run "no injection" test - simply run method without injection. It will help you to narrow problem dow and exclude carryover from vial or injection system (sample loop, needle/needle seat)
2.If you still see peak check new methanol/solvent batch (perhaps it is contaminated). if you inject pure MeOH you should see solvent peak and nothing else (unless MeOH is not LC grade)

Regards

Tomasz Kubowicz

[greg07]

The sample is take from mobile phase bottle.

Before injection - 20 min. baseline preview.

Even after 10 injection still the same situation and exactly same peak.

I did injections also without column with resistant tube - ca 20m x 0,2mm.

[tkubowicz]

The sample is take from mobile phase bottle.

Before injection - 20 min. baseline preview.

Even after 10 injection still the same situation and exactly same peak.

I did injections also without column with resistant tube - ca 20m x 0,2mm.

1."The sample is take from mobile phase bottle" - what does it mean? sample is sample and mobile phase is mobile phase.
2.If you see peak in 4min with restriction capillary you've got problem either with pump, sampler or solvents. How can you have any retention on restriction capillary if there is no stationary phase?

I'd recommend to inspect LC sampler and pump. Also prepare fresh mobile phase - fresh means from new, unopened solvent bottles.
Check your sampler for carryover - check needle/needle seat (or syringe) and run "no injection" test.

Regards

Tomasz Kubowicz

[uzman]

Hello greg07 ,

As I understand , you are taking some mobile phase from the mobile phase bottle and inject it to your system.

Are you making manual injection ? If so , do not remove your syringe from the injection port , untill you turn the handle to inject position. Otherwise air may enter the loop , and give such a peak.

[greg07]

Yes, you understand me well. I use an autosampler and I maked - as I set - measurements on 3 HPLC sets with diverse column. I make OQ- tests and I see the same situation in carryover test with methanol/water as mobile phase. If I inject naphtalene disolved in mobile phase (methanol/water) under naphtalene peak will be a small peak from methanol. When naphtalene is disolved only in methanol, the peak from methanol is little earlier, before naphtalene. I thing in the situation described in the my post is the same phenomenon. I would like to know whether this is always with methanol/water mobile phases and that phase does as solvent for sample?

[uzman]

Naphtalene must have a good retention on a C-18 column , but not the methanol.

Are you sure is it the methanol peak , under the peak of naphtalene ?

It looks like a carryover , did you try to wash your systems with a stronger solvent ; such as Tetrahydrofurane ; including wash solvents of your autosamplers ?

THF has some negative effects on PEEK materials , but short term exposure is reasonable.

[tkubowicz]

Hello

I would like to know whether this is always with methanol/water mobile phases and that phase does as solvent for sample?

No it is not. Why don't you inject pure methanol and pure water (used for mobile phase) to see where this peak comes from?
If methanol is HPLC grade you should see methanol peak and nothing else.
Again: Check your solvents (use fresh, new batch) and check water - use one for HPLC (Sigma, Merck or other supplier). Perhaps your water system (Millipore) is not perfect and that's where your peak comes from.
Again: check your sampler for carryover (needle/needle seat or rotor seal)

Diagnostic for problem like this is really simple.

Good luck

Regards

Tomasz Kubowicz

[greg07]

Thank you for your attention. I"ll make additional measurments.
Your final request: on a clean system, injection samples taken from the bottle mobile phase can't give any peak, right?

[Peter Apps]

In this scenario the most likely source for the peaks when you inject mobile phase is the sample vial. Try a different make of vial, or a vial with no septum and a vial that you have rinsed repeatedly (at least ten times) with mobile phase.

Is there any difference ?

Peter

[greg07]

I try what you say for ca 14 days, I'll be by customer. I used new vials from Agilent.

Regards