Retention time shift / Peak height drop

[kristind858]

Hello,
I am running the Vitamin D3 method by USP on an L8 aminopropylsilane column. The peak retention time of the Vitamin D3 should be around ~4.7 minutes.

Mobile phase A 99%: Hexane
Mobile Phase B 1%: IPA.
Flow 1.0 ml/min.

After running the D3 method numerous times and having some consumables changed from time to time, I have seen the retention time shift slightly which is not that unusual to see after continuous usage of the same column.

Recently while I was out from work, our service engineer was called in due to degasser fault. The degasser chambers were flooded and were replaced. The chemists in the lab tried running another sample of Vitamin D3 but the pressure was too high. The service engineer found that the flow cell was the cause of the high pressure so he rebuilt the flow cell. The engineer also replaced the gradient proportioning valve.

The chemist ran a Vitamin D3 standard after the above items were replaced and found that the standard had decreased to half of the normal peak height. Typical peak height for a 3.0 ug/ml standard solution is around ~110,000. We are now getting around ~50,000. Peak retention time for Vitamin D3 has more than doubled what it used to be (Vitamin D3 has been roughly around ~5 min).

The chemist thought that the column was the issue so another brand new column was ordered. The retention time on the new column was around ~11min.

We only run one other test on our HPLC right now. We analyze CoQ10 and the retention time for this has been coming out fine. I initially thought the issue with the Vitamin D3 could be due to the gradient proportioning valve but since the engineer changed that and the CoQ10 mobile phases are mixing well that can be ruled out.

I have been trying to troubleshoot this by doing the following:

1. Checked the delivery volume of the mobile phase reservoirs. They are fine.

2. Checked the system for leaks. Pressure is at the usual ~540 psi (although I know there could still possibly be a leak somewhere even if the pressure is steady)

3. Washing the column with the recommended mfg instructions.

Any other recommendations? I want to get the retention time back to the established time.

[tom jupille]

You are mixing only 1% IPA with hexane; two liquids with very different viscosities. If this were my problem, the first thing I would do would be to pre-mix the IPA and hexane and put both inlet lines in the same reservoir. If the retention returns to "normal", then you have nailed the problem to proportioning system. If the retention time stays long, then you have exonerated the proportioning system.

One scary possibility comes to mind if the system was also used with aqueous mobile phases: some water from the flooded degasser was picked up by your non-aqueous mobile phase. With that problem solved, your solvents are now "dry". If that's the case, the remedy might be to cut back on the IPA (to 0.5% ? -- in which case I would *definitely* pre-mix the mobile phase) or even try water-saturated hexane.

[James_Ball]

If the retention time is now longer, could it possibly be that the old proportioning valve was letting in too much IPA and causing the shorter retention times, or the new one is not letting in enough? Either way with that low of a concentration I would do as said above and just premix if you don't need a gradient. If you need to increase the IPA as a wash step then let it come in at the end of the run on top of the IPA in the premixed mobile phase.

[kristind858]

Thank you for your quick response. I mixed my mobile phases manually (Hex:IPA --> 99:1) and inserted both solvent lines into the same mobile phase reservoir since the Vitamin D3 USP method is isocratic. I was so excited when I saw the retention time of the Vitamin D3 shift back to its orignal RT ~4.7min. The peak height also increased to the usual measured value. I got 3 replicate injections of the Vitamin D3 with the RT ~4.7 min and the peak height around ~130,000. The retention time again started to shift down and take longer. I switched out the column and tried running the D3 standard on each of our columns (we have three L8 columns). RT varies slightly between each column (which I know is not unusual) at about ~9-11 min.

Should I try turning off the degasser(in case it was flooded previously with aqueous mobile phase) as it may possibly be introducing contamination into the GPV? Thanks for your help!!

[tom jupille]

Should I try turning off the degasser(in case it was flooded previously with aqueous mobile phase) as it may possibly be introducing contamination into the GPV? Thanks for your help!!

Presumably the degasser was still in-line when you ran your isocratic test, so that indicates the degasser is OK. That points the finger at the proportioning system (possibly a sticking valve ? ) so you might want to have that tested and fixed. Of course, at 1%, you are on the ragged edge of what that type of system can do. If it's working OK otherwise, I would just use premix mobile phase for this application and let it go at that.

Degassing is particularly important with reversed-phase because the binary mixtures of polar organic + water are very much "non-ideal" in the sense that the properties of the mixture differ from the average properties of the components. Most pertinently, the solubility of air in mixtures of say, methanol + water than the average of it's solubility in the pure solvents. When you mix, the excess air comes out as bubbles. Organic mobile phases are much less of a problem as a result.

[kristind858]

Hi Tom,
Thank you for all your help! Should I have my service engineer come out and change the inlet and outlet check valves? Is it possible we have a pump malfunction? Although the pressure remains steady during our run, it may still be possible to have a small internal leak. This may explain why the diagnostic tests performed by the service engineer checked out. Small internal leaks are harder to detect. Is this correct?

Here is the summary of what I did for troubleshooting:

-Changed in-line filter
-Change mobile phase reservoir filters
-Premixed (Hex:IPA 99:1- Isocratic) and put line A and line B in the same bottle. Results looked promising as the retention time returned to ~4.7 min for three runs of my Vitamin D3 standard. Retention time for Vit D3 peaks slowly began to increase after additional runs. I switched out and tried the other two columns and still observed the retention time increase.
This eliminates the GPV as the problem, correct?

What is really troubling me is that our CoQ10 method is still functioning correctly. We run mobile phase A at 55%(THF:Water 8:1) and mobile phase B at 45%(ACN). The CoQ10 retention time is coming out fine. This would lead me to believe our L8 Aminopropylsilane column used in Vitamin D3 method has been contaminated but we just purchased a new column (although I was not at work to be the first to use it-so it may be possible water has hydrated the column??)

Having the Vitamin D3 return to its normal retention time (even on an older column) for 3 runs gives me some hope that the issue may be somewhere within the machine.


Thanks again in advance