Chromatography Forum

I am hoping someone can provide some insight regarding a problem I am having with my retention times changing.

I am trying to develop an assay for quantitating morphine, and I am using a slightly modified version of a method I found on the Restek website (they used MS detection, I am using UV). Unfortunately, I cannot get reproducible RTs for my morphine standard from day to day.

Day 0 - Mobile Phase prepared
Day 1 - No analyses
Day 2 - RT = 5.8 min
Days 3-6 - No analyses
Day 7 - RT = 6.4 min (same batch of mobile phase as Day 2)
Day 8 - No analyses
Day 9 - RT = 6.6 min (same batch of mobile phase as Day 2)
Day 10 - New mobile phase prepared
Day 10 - RT = 5.4 min
Days 11-12 - No analyses
Day 13 - RT = 6.0 min (same batch of mobile phase as Day 10)

Experimental Parameters are as follows:
Allure PFP Propyl column - 150 mm x 4.6 mm x 5 µm
Mobile Phase - 50:50 A:B
A: 0.1% Formic Acid in H2O
B: 0.1% Formic Acid in 80:20 MeOH:ACN

I'm thinking this is some sort of mobile phase issue, but since I'm relatively new to LC, troubleshooting things like this isn't my strong suit yet... Any suggestions would be greatly appreciated. If you need more info, please let me know.

Thanks

When retention time changes occur, the first suspect should always be flow rate. Is t0 (the "solvent front") changing by about the same percentage as the retention time of your analyte? If it is, you have a flow rate problem.

That said, your symptom (a gradual increase in retention as the mobile phase ages) is consistent with evaporation of organic solvent. You didn't specify, but from the context I'm guessing the you are blending the A and B solvents "on-line". Problems with the proportioning valve *can* cause retention variations, as can temperature changes, but the fact that retention decreases again with fresh mobile phase argues against those causes.

If this were my problem, I would make up fresh mobile phase every day and see if that helps.

Hi EJG,

You do not mention the column temperature. Are you using a column heater/chiller?

Kind regards,

Ade Kujore

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Cecil Instruments Limited
Milton Technical Centre, Cambridge CB24 6AZ
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email:- ade.kujore@cecilinstruments.com
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I also facing same problem. The mobile phase that used is methanol 70% and water 30% to detect concentration nitrate and nitrite by using column ODS hypersil. Is this show that I used wrong mobile phase?

interesting!

how many injections you made each day? how many of them were std ? and how many Samples?

did you have RT shift within each day injections?

did you really mean "B: 0.1% Formic Acid in 80:20 MeOH:ACN" ? or "H2O:ACN"?

I using standard solution sodium nitrate and sodium nitrite. The retention time is different for various concentration standard solution. Injection volume that I used is 5 microliter and flowrate is 1 ml/min.

I had tried 85% ACN and 15% water (no gradient) but the RT still different for each. Methanol and water also give the same results. so, what should I modified?

weitze: Are you using a buffer or just water? When analyzing ions it is critical to control at least the ionic strength of the mobile phase, and preferably also the pH.

Thanks to all for the replies.

It appears as though it is a mobile phase evaporation issue. I've monitored the retention time all week and it changes on a daily basis - quite linearly in fact.

For those who asked:

Yes, I am using a column oven - set to 30 deg C, just above room temp.
RTs are constant during any given day, changes are only noticed from day-to-day.
Yes, "B" really is 80:20 MeOH:ACN

Looks like I'll be making up new mobile phase every day...

Or get your hands on a quaternary pump and mix everything online...

there is a lot of talk of those new safety caps these days

we got some for the safety issue and also because for UHPLCs we use a lot more TFA and formic for the buffers
they prevent evaporation no doubt about it and they prevent those issues also

we use the vaplocks from western fluids
you can find them on the web easily

also if you can use smaller bottles, that means a lot less volume goes to the vapor phase as the liquid volume decreases. this also helps alot