RT changing in three replicates from the same sample

[Rui]

Greetings

I am writing to know if it is normal to have differences of 30 seconds in the retention time of a compound in the three replicates of the same sample.

Our lab work with plants, and in the analysis of the phenolic profile of these plants, namely phenolic acids and flavonoids.
We have a HPLC-DAD method for the screening of these compounds.
We use an Gilson HPLC equiped with an Agilent DAD 1100 series, an Waters shperisorb ODS2 250X4.6 mm cartridge (RP C18).
Our gradient is:
Flux: 0,9 ml/min
0 min: 5% MeOH (A), 95% Formic acid 5% (B)
3 min: 15% (A), 85% (B)
13 min: 25% MeOH, 75% (B)
25 min: 30% (A), 70% (B)
35 min: 35% (A), 65 (B)
39 min: 45% (A), 55% (B)
42 min: 45% (A), 55% (B)
44 min 50% (A), 50% (B)
47 min: 55 % (A), 45% (B)
50 min: 70% (A), 30% (B)
56 min: 75% (A), 25% (B)
60 min: 100% (A), 0% (B)

Normally the extractions are made with MEOH, Water or a mxture of MeOH:H2O.

I tried everything, changed cartridge, filter, pre-column, checked the pumps, the mixer and didn't solve the problem.

Hope you could help

Thank you in advance for your reply

Best regards
Rui

[Rui]

I tried to upload a figure to try to demontrate the problem

[Noser222]

Are you overlaying back to back injections of the same sample there?
Whats your injection volume? If you are injecting directly from methanol that could cause problems.

[Consumer Products Guy]

That's not normal, even for running a gradient. What is your equilibration/conditioning time/volume before your next injection? I see that error (insufficient re-equilibration volume) frequently, guidelines are about 5 to 10 column volumes for that.

[mbicking]

First, remember that a 0.5 minute change in retention time over 19 minutes is only about 2-3 %. In some systems this may only slightly larger than normal.

You have a long column and a slower flow rate. Your void time is almost three minutes. You should allow about 15 - 30 minutes for equilibration between injections. If you not waiting until the column is equilibrated, then you could see some problems. Is the trend consistent; are the times always increasing?

Finally, you have a very complex gradient and it may be difficult for the system to reproduce this exactly for each injection.

[Rui]

Thank you all for the quick reply

The chromatograms are 2 injections from the same sample.
My injection volume is 20 micrL., usually the samples are dissolved in MeOh or H2O.

I normally wait about 15 minutes to inject once again.

The RT's don´t encrease more then ~30 seconds. When i inject the same sample four times i can get two with same RT's.
Our gradient was made to get the most complete phenolic profile, since there are more polar and less polar phenols we tried to made a gradient that could detect the diffrerent phenolic compounds.

Thank you for your advice, i will wait a little longer to re-equilibrate the column and hope the problem disappears.

Best regards
Rui

[tom jupille]

If the retention times are varying randomly (as opposed to continually increasing or decreasing), the most likely problem is a poorly functioning gradient proportioning system. Run the manufacturer's recommended qualification test on the pumping system and look for inaccuracies.

The second most likely issue is temperature variations, but that tends to cause systematic drift in retention times (e.g., decreasing during the day as the lab warms up and then increasing again in the evening).

[jiang295]

I think 15 min is enough for equilibrium.
change inlet check valve to see if there is any change.

Thank you all for the quick reply

The chromatograms are 2 injections from the same sample.
My injection volume is 20 micrL., usually the samples are dissolved in MeOh or H2O.

I normally wait about 15 minutes to inject once again.

The RT's don´t encrease more then ~30 seconds. When i inject the same sample four times i can get two with same RT's.
Our gradient was made to get the most complete phenolic profile, since there are more polar and less polar phenols we tried to made a gradient that could detect the diffrerent phenolic compounds.

Thank you for your advice, i will wait a little longer to re-equilibrate the column and hope the problem disappears.

Best regards
Rui

[jiang295]

ps, this gradient method is really strange.....
you probably spend a lot time in developing this method.

Greetings

I am writing to know if it is normal to have differences of 30 seconds in the retention time of a compound in the three replicates of the same sample.

Our lab work with plants, and in the analysis of the phenolic profile of these plants, namely phenolic acids and flavonoids.
We have a HPLC-DAD method for the screening of these compounds.
We use an Gilson HPLC equiped with an Agilent DAD 1100 series, an Waters shperisorb ODS2 250X4.6 mm cartridge (RP C18).
Our gradient is:
Flux: 0,9 ml/min
0 min: 5% MeOH (A), 95% Formic acid 5% (B)
3 min: 15% (A), 85% (B)
13 min: 25% MeOH, 75% (B)
25 min: 30% (A), 70% (B)
35 min: 35% (A), 65 (B)
39 min: 45% (A), 55% (B)
42 min: 45% (A), 55% (B)
44 min 50% (A), 50% (B)
47 min: 55 % (A), 45% (B)
50 min: 70% (A), 30% (B)
56 min: 75% (A), 25% (B)
60 min: 100% (A), 0% (B)

Normally the extractions are made with MEOH, Water or a mxture of MeOH:H2O.

I tried everything, changed cartridge, filter, pre-column, checked the pumps, the mixer and didn't solve the problem.

Hope you could help

Thank you in advance for your reply

Best regards
Rui

[Rui]

Thank you all for your advice

Hope one day I can help you too

Rui