Chromatography Forum

Hi LC forum experts

We are performing testing of Doxycycline Hyclate related substances API and tablets as per the EP.

The column specified is 25 cm x 4.6 mm, 8um styrene-divinylbenzene copolymer, we are using an Agilent PLRP-S 100A 8um, part no. PL1512-5800.

The mobile phase is made up of 60 g 2-methyl-2-propanol, 400mL of pH buffer 8 (0.2M potassium dihydrogen phosphate, adjusted with NaOH), 50 mL 1% tetrabutylammonium hydrogen sulphate (adjusted to pH 8 with NaOH), 10 mL of 4% disodium edetate (adjusted to pH 8 with NaOH), made to 1 L with purified H2O. Then vaccum filtered through 0.45 um HVLP membrane filter.

HPLC conditions
1 mL/min
254 nm
60 C column temp
20 uL injection

We are using a Dionex RSLC to run this testing.

The chromatography is good with a new column but, even after only one run the plate count and resolution deteriorate significanly, sometimes the coulmn will last only 4 - 5 runs before system suit can no longer be acheived. The blue chromatogram is from the second run using this column, the plate count of the Doxy peak has gone from 1117 to 539, and the resolution of Impurity A has gone from 2.8 to 1.3.



After running the column is washed using ACN/H2O from a low % ACN upto 90 % ACN for storage.

Does anyone else have experiance with these coulmns and can suggest a way to regenerate or wash them to extend the life that we are getting.
From what I have read these columns should be robust when it comes to these high pH high temperature applications, but the pores of the particles may be getting blocked. All mobile phase and samples are filtered through 0.45 um, is a finer filter required?
I have tried washing the column in reverse but it doesn't seem to improve the chromatography?

Any suggestion much appreciated as we are going through columns at a fast rate

What shows the back pressure? Any changes?
On Agilent website you will find the column care information.
It is strongly recommended that a minimum of 1% organic
modifier is maintained in the mobile phase. Column performance
may be reduced when reintroducing organic modifier after prolonged
use in 100% aqueous eluents. PLRP-S adsorbents have
minimal swell and so may be used with all common organic
modifiers in both isocratic and gradient elution modes.
Please try to get in contact with Agilents services.
Do you use a guard column?

Thanks for your response Gerhard

The pressure doesn't change as the chromatography changes, for the two chromatograms that I posted the first one (good chromatography) has pressure 79 bar , the second one (later elution) has pressure 82 bar.

We have tried many diffenent washing/regeneration procedures and its only makes the chromatography worse, the peaks eluting later with less separation and more tailing.

We have tried washing the column as per the Agilent column care info, including using 1M NaOH + 1 % ACN, this has not made any improvement to the chromatography.

We are using a Phenomenex security guard cartridge with PolymerX RP-1 insert.

Have you tried to reproduce the QA?

You may have a void. Are you able to look at the bed on the inlet end of the column (of you open the outlet, the resin will ooze out.. keep your guard up for this)

Hi DJ
Yes I have tried to reproduce the QA as per the column performance report that came with the coulmn.
This is run with 7:1 ACN:H20 and acetone as the test substance, 1.0 mL/min 254 nm.
The peak comes out at the correct RT but plates are significantly lower at 7000, compared to 45000 of the column performaance report.

I have now tried to wash the column with NaOH at elevated temp (85 C) this appears to be improving the chromatography, I will continue to wash the coulmn even at higher temps to see if systemn suit can be met.

I have taken the end nut off the coulmn but there is a peek inser that a can't get out without damaging so I can't see the packing.