Transfer HPLC method to UPLC method

duna

<p><span>I would like to know if a gradent has to change if it is injected in a HPLC system and in UPLC system. I have done a transfer of method of HPLC to UPLC, I have changed the gradent using the Waters calculator, and in HPLC the bases line up and down and in UPLC, the bases line down and after up. Is it normal? Is it normal to change the bases line when we transfer a method of HPLC to UPLC?.</span></p><p><span>In HPLC the column is a XBridge C18 and in UPLC is ACQUITY UPLC BEH C18. Can be the column?.</span></p><p><span>Thanks,</span></p>

Patty-SystemsMarketingLaboratory

Dear Duna,

The two most important aspects of method transfer after scaling the gradient to the new column dimensions and particle size are (1) system volumes of the LC system and UPLC and the (2) temperature. The system volumes entered into the calculator must be accurate. Also it may be necessary to modify temperature a +/- few degrees C to compensate for slight differences in column heater mechanism between manufactures.

I do not understand the nature of the problem you are seeing; could you possibly send me chromatograms and conditions and point out the differences? Is this a problem of peak spacing (Rs) or baseline drift?

thank you

pat_mcconville@waters.com

duna

Dear Patty,

I send you more information about my transfer of method:

HPLC conditions:

Columna: Waters XBridge C18 15 cm*4,6 mm*3,5 µm

Mobil phase: TFA 0,02% (pH 2,7) / MeOH

Gradent: min %MeOH

0.0 56.0

20.0 56.0

32.0 86.0

47.0 86.0

55. 0 56.0

1 ml/min

T: 30ºC

220 nm

20,0 µl

UPLC conditions (obtained with the calculator):

Columna: ACQUITY UPLC BEH C18 50*2,1 mm; 1,7 µm

Mobil phase: The same as HPLC conditions

Gradent: min %MeOH

0.0 56.0

3.2 56.0

5.2 86.0

7.6 86.0

9.0 56.0

0,45 ml/min

T: 30ºC

220 nm

3,0 µl

I also send two cromatograms, one, UPLC, and the other is a HPLC system but a different version of the method said above.

Please, look at the cromatograms, there are differents, it can be possible in a transfer of a method ? The two cromatograms should be in the same form of the bases line?

Thanks,

AJA

It would be helpful if both chromatograms were in the same Y scale. Currently the HPLC one is at 0.2 AUFS and the UPLC one is at 0.003 AUFS. It is impossible to tell if these are in fact the same, or different when the data is presented like this.

Patty-SystemsMarketingLaboratory

Dear Duna,

Several Chemists have examined the baseline on the UPLC example. It would be helpful if we could see the same scale on the HPLC baseline as well. The UPLC baseline is unusual and we have a number of questions:

1. Is this baseline consistent? Does it look the same from one injection to the next?

2. Is the chromatography consistent in terms of retention reproducibility?

3. How long are you equilibrating the column after each injection?

4. Has the column been used for another application?

5. Have you tried a new column?

6. Have you tried cleaning this column? You will find a cleaning procedure in the care and use manual.

7. Is it possible to balance the TFA concentraion by adding TFA to the methanol to help keep the TFA concentration cosistent across the gradient?

We have a feature in our ACQUITY PDA called "Median Baseline Function" which could be tried to see if it will alleviate the baseline issue but I would rather investigate to eliminate possibility of contamination or equilibration problems before MBF is employed.

Message was edited by: Patty-SystemsMarketingLaboratory

maniarni

Baseline is mostly attributed to the Buffer you used for mobile phase preparation as well the wavelength you selected, you can switch over to different buffer (there is lot of options are available for selecting buffers )

The other thing you can try by switching wavelength as per your sensitivity requirement

you if have still problem let me know the exact analytical method, i would love to help you out

Thanks