I am using an anion exchange column for my experiment and store it in 20% Ethanol as recommended. When the column is put to use weekly it gives a proper baseline with minimum stabilization.
However, when used after a period of 15 days or more, it gives a very wavy baseline and low resolution.
Any suggestions on alternative storage solutions that can be used or storage techniques that could be implemented?
Did you degass the 20% EtOH/water mixture? If not, possibly slow degassing of the solvent might create tiny air bubles in the pores, destroying the resolution and giving a wavy baseline. Flushing the column with a properly degassed eluent should restore its separation capabilities.
If you don't mention the column name or its chemistry, how can one provide a suggestion? Which anions are you separating? There are plenty of anion exchangers in the market.
I have never stored a ion exchange column in anything other than the eluent I was running it with.
Professor Omar, I'll try using sodium azide as suggested by you. Thank you.
Dr. Olieman, I degass all the buffers and cleaning solutions before passing it through the column. As a general policy we do it for five minutes and then the buffers pass through an in-line degasser before entering the column/system. Should I increase the degassing time? What do you suggest?
Dr. Wahab,
I am using the SOURCE 15Q 4.6/100 PE anion exchange column from GE Healthcare. It's a Polystyrene/ divinyl benzene matrix and is being used for separating proteins (transferrin isoforms) from plasma samples. Recommended storage solution for the column is 20% ethanol.
Alternately we have also tried Proteomix SAX NP-10 column from Sepax for the same application given that it had similar chemistry.
With both these columns, the issue of wavy baseline arises when column it put to use after a long haul.
Mihika, All stationary phases leach small amount of material, especially if you are using them after a long time. As they say nothing is permanent but change. No surface chemistry is ultra-stable. You will see some "disturbance" after using the column stored for a while. On the other hand, if the wavy baseline persists, and it has a definite low frequency (ripples), chances are it is coming from the pump.
Dr. Wahab,
In that case, should I clean the column every time I perform an assay after long term storage? Column insert mentions "rigorous cleaning" in the case of wavy baseline and low resolution, it involves using 1M HCl, 1M NaOH, and 1M NaCl.
Can this protocol be used frequently? Are there chances of hampering the column performance by using such strong chemicals?
Also, the baseline disturbance I mention is seen only with this particular column, for other applications, the baseline is quite stable and does not interfere with separation. I believe I can rule out pump malfunctioning then.
Michael, I use 2M NaCl as eluent, would you recommend storing column in this? We haven't stored column in 2M NaCl thinking NaCl would precipitate over a period of time. Any experience with high molarity storage solution?
What eluent do you use for your application?
Mihika I read the answers of other scientists and found it very interesting, but I would like to add one more thing here, to increase the life time of a chromatographic column it is strongly recommended to wash it with methanol or 2-propanole after every run to remove the analytes completely. After use, don't store the column full of buffers because the precipitation of buffer over the stationary phase will decrease the column performance. I read in one of your question that the column company recommended to store it in 20 % methanol, can you explain little more how you store in 20 % methanol, you just flow 20 % methanol through HPLC pump across the column and then remove it and store or something else? I will recommend to store your column in this way. In our laboratory we are making a variety of columns C18, mixed-mode etc, and effect of long term use on column performance. We use this method and our columns efficiency don't drop considerably and we are able to maintain it for one year or more. Washing column with strong acids or alkalis are usually not recommended because it damage the stationary phase. It can be use as a last option if the column is overloaded so much with sample and can't be clean with methanol/2-propanol etc.
Ashraf Ali, you are correct in saying that harsh acids/alkalis damage the column and hence I am skeptical in using the rigorous cleaning protocol.
We use 20% Ethanol for storing the column, it is allowed to run through the column at 1ml/minute flow rate until 20ml has passed and then the column is stored directly at room temperature.
"In that case, should I clean the column every time I perform an assay after long term storage? Column insert mentions "rigorous cleaning" in the case of wavy baseline and low resolution, it involves using 1M HCl, 1M NaOH, and 1M NaCl. Can this protocol be used frequently? Are there chances of hampering the column performance by using such strong chemicals?"
In general, ion-exchange resins can easily be exposed to acids and bases, unlike reversed or silica based phases. If the manufacturer has suggested these solutions, it means they must have tested this procedure, not once, but probably hundred of times. Do not worry about the initial baseline change after storage. First of all it will take a while to remove EtOH when a purely aqueous mobile phase is used. Do not use 2M NaCl as storage. Once it crystallizes, the column will be damaged. Without worrying too much, a classic sign of column fouling is a change in retention time. If you see your retention times have changed, it is time to clean it. otherwise simple wait for the column to equilibrate. You can also wash the column initially "offline" i.e. detector is disconnected. After 10-15 min, you can connect it back.
Dr. Wahab,
Thank you for the suggestions. I'll allow the column to equilibrate before starting the assay.
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