Retention Time Issues

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hello everyone,
I am having some retention time issues where the peak may shift from 4.937min to 5.478min for one example (this is enough of a shift that chemstation cannot identify it as the same compound)

I am 100% confident this is the same analyte because the runs are just repeats of the same vial.

I am using full reports so I can see that the initial and final column temp is the exact same, flow is the exact same, pressure changes by at most 0.3bar.

Some background info:
- Using an isocratic eluent 75% Acetonitrile 25% water both are obviously HPLC Grade
- Sample is 95% solvent matched (950uL 25/75 H2O/ACN + 50uL Methanol)
- Column is basically brand new minus calibration runs and a handful of samples
- All the peek tubing in the HPLC is new
- The HPLC is an Agilent 1100: Units: G1322A, G1311A, G1315A, G1316A, G1329A, G1330A
- Flow rate: 0.700mL/min
- Average pressure: 173bar
- Column temp: 35C
- Injector: 10uL
- DAD: 225,4 Ref 370,80, range 190-700, step 0.5, slit 4, response 0.1s.
- Agilent Chemstation Rev. B.04.03-SP1 [87]
- Analyzing cannabinoids for potency 0-500ng/uL = 0-100%
- Problem seems to persist independent of analyte concentration (ie. a 5% control will be just as inconsistent with respect to retention time as a 100% control)

What could be causing this kind of shift in retention time?
Thank you for providing almost all details. You just forgot the column and its dimensions.

Could it be, that this peak is a cannabinoid acid?
Then it may be an ionisation issue as your mobile phase is not buffered or at least acidified with e.g formic acid.
Maybe try to add 0.1% of formic acid (or phosphoric acid may be better for UV-detection) to your mobile phase. Hopefully the separation stays ok, otherwise you know it's an issue and you need to find the right pH with a real buffer
Sorry forgot to add that, it is a restek raptor c-18, 150mm x 3mm 2.7um

And the solvents I listed both have 0.1% formic acid and the water also has 0.05% ammonium formate

I am testing both acidic cannabinoids and neutral cannabinoids, problem is consistent with both
Also, I don't know if I made this clear or not previously:
This issue is happening with all peaks, not just the one example I made that's around 5min. If I run a 9pt Std. the RT of all 9 peaks will vary from run to run where sometimes 5 of the 9 will be able to be quantified, and then the next run a different 7/9 will, etc. So I end up doing multiple runs to get every peak, and average the values from the peaks that were able to be quantified.
Your possible variables are flow rate, column temp., mobile phase composition, sample diluent, injection volume and condition of column/frits (although the three latter can probably be eliminated at this stage).

If I had a nasty, suspicious mind (which I think that you need in this situation) I would not necessarily take the printed reports (constant temp/pressure/flow rate etc) as gospel.

1) I would manually, check flow rate by collection in a measuring cylinder

2) I would visually, check pressure over some consecutive runs

3) Is the column in an oven or can temp. be affected by lab. air conditioning?

4) Is your mobile phase (75:25) in a single bottle or in two separate reservoirs?
If in a single bottle, it does away with possible mixing issues. However, in a single reservoir there may be an issue of sparging which may preferentially blow out the MeCN.......

If issues 1-4 above all check out, perhaps raise temp to 40 C and drop flow rate to 650-675 uL/min concurrently; does this resolve the problem?

If it does, change them independently to see if one change alone is sufficient.

Good Luck with this nasty problem!

EDIT: I see from your second post that your mobile phase is in two separate reservoirs; also that formic acid and ammonium formate are present. Beware of sparging which may blow out volatile components.........!!! and thus change the composition of the mobile phase just enough.
As HOLLOW has suggested, perhaps use phosphoric acid (or a phosphate buffer).
Hi GabeS,
if your pressure does not fluctuate, your flow should be ok. If it is not the mobile phase, then it is probably the stationary phase. Since you are using isocratic conditions, it could possibly be that you are accumulating some kind of material on the column, that then causes RT shift. Some injections later this might come off the column and your RT's are back to normal.
You could try to add a rinsing step with 100% ACN to your method. Maybe you even need something stronger (like ACN/IPA).
Thanks for the advice everyone!

I manually checked flow by putting the outlet into a grad. cylinder while its running and sure enough at 0.7mL per minute for 5 minutes yielded 3.5mL exactly, doing it for 10 minutes gave 7mL, and for 15 minutes gave 10.5mL. So I don't think the pump is the issue.

As for mixing, I did the same test (bypassing the column so that I'm not running 100% water through it) but pumping either 100% ACN or 100% Water and both had accurate volume outputs. I think this means that it isn't a mixing issue since the pump is pushing the right amounts of both.

I ran the agilent lab advisor software and did a pressure test and everything seems to be fine there.

The column is in the G1316A compartment, so it is semi-closed off to the lab environment, plus the temperature comes from running the sample through the column oven, not just against it so I believe it to be accurate.

I haven't tried raising the temp and lowering flow rate yet but will do that soon!
Just for my own reference, what would be an okay shift in RT? 2% or so?

Eventually I'd like to recalibrate for a lower flow rate anyways, since CBC and THCA retention times are only different by 13.2 seconds in the cal. table. Hoping to separate them a bit more.
As for mixing, I did the same test (bypassing the column so that I'm not running 100% water through it) but pumping either 100% ACN or 100% Water and both had accurate volume outputs. I think this means that it isn't a mixing issue since the pump is pushing the right amounts of both.

I'm glad that you checked flow from each pump head independently, but I don't think that it necessarily means that the 75:25 ratio is guaranteed. I think that there is a piece of hardware (a proportioning valve or similar name) that actually generates the ratio of A:B, followed by a mixer volume. This needs to function correctly (at all A:B ratios for a gradient method); I don't know if there is any maintenance required for this valve.

For RT variation, I would say certainly less than 5% at RT 10.0 min, probably around 1-2% for your isocratic method.

What happens if you premix 75.25 and let it run 100% on each channel?
Hollow wrote:
What happens if you premix 75.25 and let it run 100% on each channel?

This would be what we did at my workplace, if we wanted to isolate whether it was a pumping or mixing issue.
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