Chromatography Forum

Im working with a column that has 'in house' synthesized packing, the base material is porasil silica from waters and these particles were functionalized with mixture of short oligo ethylene glycol silane and octyltrichlorosilane. I ran the column with 50 mM phosphate buffer and various concentration of Na2SO4 or (NH4)2SO4. The test sample is lysozyme, dissolve in pure water. The first set of run is fine, i get good tR and peak shape. I noticed a decrease in recovery especially at high salt concentration (max 1 M). After several hr of runs, I left it alone, the next day I failed to run the column. The recovery was extremely low, peak was very flat, tR shifted a lot. I think I have some protein left over in the column. I tried to clean the column using various solutions such as 6 M guanidine HCl, 0.1% TFA/isopropanol, 30-70% ethanol, 0.1% TFA/acetonitrile. All failed, can anyone suggest me a cleaning method? Thanks.

http://www.lcgceurope.com/lcgceurope/da ... rticle.pdf - Table 3, page 4


1. Acetic acid 1% in water
2. Trifluoroacetic acid 1% in water
3. 0.1% Trifluoroacetic acid–propanol 40:60 (v/v) (viscous; use reduced flow-rate)
4. TEA–propanol 40:60 (v/v) (adjust 0.25 N phosphoric acid to
pH 2.5 with triethylamine before mixing)
5. Aqueous urea or guanidine 5–8 M (adjusted to pH 6–8)
6. Aqueous sodium chloride, sodium 0.5–1.0 M (sodium phosphate pH 7.0) phosphate or sodium sulphate
7. DMSO–water or dimethylformamide–water 50:50 (v/v)

Adapted from R.L. Cunico, K.M. Gooding and T. Wehr, “Basic HPLC and CE of Biomolecules,â€

Yep, that's the reference that I have, that apparently doesnt really help my case. Thanks anyway

Have you considered that your stationary phase may be damaged? Do you have a separate, small molecule organic (e.g., toluene) that you can inject to check for peak shape?

Yes, I thought of that, right now Im in the process of verifying whether the coating on the particles is damaged, thank you.