Chromatography Forum

Hi All,

I have this dilemma here...I am studying total protein charateristic using C-18 column. After some injections (50 or so)...the column has lost its retention time and the back pressure increased significantly. I cleaned itwith Water/ACN (95/5); 100%ACN, 100% THF, 100%Methanol, then 95%Water/ACN, then reverse flushed the column and back using mobile phase. . Although it brought the back pressure to slightly more than normal but the retention time still off.

I am thinking this is because some lipids or hydrophobic proteins/ materials got adsorped onto the column packing. I haven't tried to clean it with 6M urea...but any suggestions how to fix the problem? or prevent the problem in the future? the dilemma is...I need to see the total protein

My next question is I have another study to detect some cryoprotectant chemicals in animal tissues. After tissue homogenization What is the best way to remove all proteins other than acid precipitation? Because I don't want to clog my column like the one above. In this study, I want to see the cryoprotectant chemicals and I don't care about the proteins and lipids.
any SPE methods?

Your input is greatly appreaciate it!


Ernest Basarah
Research Scientist

In order to understand the retention loss on your protein column, we need to have more information. What column did you use, what was the mobile phase etc.?

6M urea won't do any good on a C18 column.

You can free samples readily from proteins. You can look up suitable procedures in the Oasis Applications Booklet at Waters.com. If you tell me the nature of your cryoprotectants, I may be able to give you more concrete advise.

Hi Uwe

This is the complete story,

I used this column in the beginning for nucleotides analysis. mobile phase 100mM KH2PO4, 5mM TBAHS, pH 7.4 and 5% Methanol. Nucleotides were extracted from homogenization, acid precipitation, neutralization, and filtration with 0.2 micron.

After several injections (~ 50 injections) the column backpressure was abnormally high but I fixed it with reverse flushing. When I did the column validity by running some standards stated on column certification sheet, to my surprise the standards analytes Benzene and benzophenone were eluted together and earlier. originally they were separated within 1 minute and eluted later (0.8 minutes later).

I changed the column guard cartridge but still the same thing.

I am really thinking this is because of some lipids, hydrophobic proteins/metabolites that got adsorped on the column packing material.

How to prevent this problem? I thought the guard cartridge should be able to prevent this. and How to regenerate my C-18 column...any good cleaning solutions?

For my cryoprotectant analysis...the cryoprotectants are DMSO, Formamide, Ethlyne Glycol, and glycerol, and etc....I used REZEX column. In this experiment, I don't want to see the protein at all...I want to remove them completely.

I think SEP is the way to go...

If you washed with MeCN, THF, MeOH etc., the column is rather clean of any small molecules. Phospholipids will have been cleaned out, as will have any hydrolyzed C18 that may have developed during your run at pH 7.4.

The cryoprotectants that you mention are small rather polar molecules. I would consider loading the sample on a silica column under some controlled conditions, maybe pH 7 phosphate. Proteins will stick to the silica, peptides may still elute, as do salts. I don't know the Rezek column, so I don't know what it can tolerate.

There are quite a few examples of cleaning out proteins in this forum, for instance a method I have used as a last resort is a mixture of Li-dodecylsulfate and dithiothreitol. Now, pure organics like MeOH and ACN are common precipitants of proteins, you may cement proteins to the column if you wash with those solvents.

Hi all..

thanks for the reply...

is 100% Hexane safe for C-18 column? I saw some protocols used hexanes for column cleaning.
But as far as I know...hexane could dissolve the C-18 linkage...just like methanol to normal silica....


Also on many papers..they used 100% water to clean the C-18 column...is this also safe? I thought 100% water will collapse the c-18 chains?


Thank you for your input...

Hexane is safe, but I don't think it will give you any better results than the wash protocol mentioned in your first post. Also water is safe - the hydrophobic collapse is a reversible phenomenon. (I prefer to call it dewetting.)