column cleaning

Record: 660

Category: Column Problem
Product: KW
Sample:

Question
I am using the KW-400 series columns and I see a loss of resolution if I run about 25 injections and let it sit for more than a week. Also, the pressure tends to creep up. I thought that I might have the pH wrong and the silica is decaying but I don’t see any large peaks coming off which is usually indicative of crushed or damaged silica. I store it in water as it came in water. I thought that micro growth might be the problem and I was wondering what you would recommend for cleaning. I read in your applications notes that the flow should be reversed and I will try that but I didn’t see anything that would effectively remove protein or micro stuck on the column. I have tried using a 20% ethanol solution and that helps a little but there is still a lot of gunk on my column. I thought maybe I could store it in triclosan or at least wash it using a very dilute solution of it.

I analyze protein (ranges from peptides to 10^7) using 4M NaSCN+25mM Trisma HCl at pH 7.5

Answer
Please let me ask you several questions in order to solve and prevent the problem.
Did you see gradual increase of column pressure as you inject more samples?
Would you be able to tell me the type of your protein sample, concentration, and injection volume?
Also, how were the sample prepared? – what solvent? Do you use filter/centrifuge?
Are you using guard column?

I realized the salt concentration you are using is very high.
Recommended salt range is 0.1 – 0.3M. Is there any reason that you have to use such a high salt?

Meanwhile, below is a recommended cleaning step for the KW series column/
When observing the high pressure, reverse the flow and introduce the eluent at slower flow rate.
When suspecting protein adsorption, use ~0.3M NaCl solution to clean the column.
*do not leave NaCl solution in the column for a long time period since it may cause rust to the column housing.

When suspecting organic substance adsorption, clean with DI water first, and then clean with organic solvent such as MeOH.
Storing the column with 20% MeOH is recommended over sodium azide solution, since NaN3 is a basic eluent and KW series is not recommended to use under basic conditions.

Also for the long term storage, using the following method is the best way.
After cleaning the column with DI water and 20 % MeOH.
1. Remove the exit end of the column from the HPLC system, attach a 1/16 inc 0.8 mm i.d., 50 cm Teflon tubing.
2. Start the pump and once observing eluent flow from the Teflon tubing, stop the pump and place the Teflon tubing in a 100mL solvent resorvoirs which contains 80 mL of the solvent.
3. Remove the entrance side of the column from the HPLC system, screw the end-cap and keep the column (with eluent) at a place with small temperature fluctuations. It is also recommended to change the reserving eluent one a month.

 
 
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