Chromatography Forum

I have a Agilent PrepHT 300SB-C18, 21.2x250 mm, 7 um reverse phase column that my PI brought with him. It's severely fouled, but it's significantly larger than any other column available to me. Employment of such would reduce the time of my purification significantly.

I don't know if it was stored properly. I know it was used in the past for synthetic peptide and oligonucleotide purification. The column information says it can be reversed to remove pluggage. I think I've read up on a decent amount with most of what I know coming from a 2003 Column Watch article by an Agilent Employee, Ronald Majors. It has some suggestions on solvents order cleaning but I'd prefer to run it by the collective knowledge here to double check and to save time/solvent.

There are general ordering of organic solvents with IPA at the beginning and end and there are also ones that just call for long flushes of 1% TFA, AcOH, or some propanol mixture there of. It also recommends that you reduce the flow rate for some. The column can handle 21 mL/min? and our system can put out 10 mL/min. So any advice about any of this would be great.

EDIT: This is the ordering the aformention article suggests for protein contamination.

Solvent Composition ( 20 column volumes each )
Acetic acid 1% in water
Trifluoroacetic acid 1% in water
0.1% Trifluoroacetic acid–propanol 40:60 (v/v) (viscous; use reduced flow-rate)
TEA–propanol 40:60 (v/v) (adjust 0.25 N phosphoric acid to
pH 2.5 with triethylamine before mixing)
Aqueous urea or guanidine 5–8 M (adjusted to pH 6–8)
Aqueous sodium chloride, sodium 0.5–1.0 M (sodium phosphate pH 7.0)
phosphate or sodium sulphate
DMSO–water or dimethylformamide–water 50:50 (v/v)

Followed with 40-50 column volumes of HPLC grade water.

Thanks

I would be careful to use TEA or Urea to clean a RP column.
My recommendation is to flush the column with water first, if possible in reversed flow direction without connection to a detector.
After water I would rinse with Isopropanol and than with acetonitrile.
Than try to repeat the QC test of that column. Conditions you can find on the COA delivered with the column, or ask Agilent for a copy.
If the number of theoretical plates is +/- 10% the same as on the original COA you have an excellent column in your hands.
Good luck.

Many thanks. However you totally lost me with COA and plates.

Edit: Do you think it's okay to run the column at 10 mL/min in the reverse flow?

Please start with 2ml/min and than you can increase step by step. Maybe there is a slight dead volume on the column entry. Too high flow rate would kill the packing. Have always an eye on the backpressure.

Column is rated to 5000 psi, forward flow at 10 mL/min gives a back pressure of ~1500, I will however work my way up from 2. Thanks a bunch.

In order to clean a column it's useful to know what has fouled it

I mentioned proteinous waste...

Try injecting several 1 mL slugs of HFIP and flush with 30/30/40 MeCN/IPOH/water. Wear gloves and goggles when handling HFIP. It's definitely not something you want to use as body balm.

If all else fails, flush with 100% MeCN followed by 95/5 DCM/MeCN.

Aside, you need to learn the concept of the "theoretical plate." You will find it on page 1 in any chromatography book. Also, your PI really ought to consider designating one column for oligos, another for peptide/proteins. It's bad luck to use the same column for both