I don't think it will damage the column but I have never used this particular column.

I would suggest to use Milli-Q water instead (doubly deionized water) because of its ultrapurity, the water is slightly acidic.

I hope this help.

Dear Pat, Nickel Odon,

In the past (hoaw ! 20 years, I am a senior I realize it :-)) , I performed HPLC essentially for hydrophobic compounds (eicosanoids) and used coresponding colums (C18 graft colums).

For proteins, I think that are hydrophilic ones , and you can used an elution solution with pH at a mean range compared to proteinscaracteristics; I suppose with these column pH 7.4 (proteins stick , then unstick at a differential time at this mean pH).

So, you have to distinguish a pH for the best separation of proteins in your mixture, from a pH of a solution used between your experimentations to wash your column, and unstick all "contaminant" before a new use.

In this case you can use another a solution (than elutionone) , but with a more basic pH (8.4). I suppose that the column is not altered with this pH (groups used by it to stick your proteins) , and you have to use the one recommanded by manufacturer.

To my mind, you had not to change this pH; if not, it wouldn't be more a washing solution.

I hope the above will help you.

Best regards

Didier JAMBOU

PS For my HPLC solution, I used, distillized and unionized water (millipore).

You don't HAVE to adjust the pH or ionic strength of the mobile phase, but if you want your separation to be reproducible, and you want your columns to perform effectively over a reasonable lifetime, you will. The TSK SW series are silica based and shouldn't be used above pH 7.5 or the base silica dissolves - you slowly destroy your column. And even if you're using the PW or Alpha material, with a pH range of 2-12, relying on the distilled water to be perfectly constant is asking for trouble. Proteins are polyelectrolytes - their chemistry changes with pH. To get consistent separations, you want all your samples to be treated the same.

Why don't you follow the suggested protocol and use a modest amount of phosphate buffer? If phosphate is a problem downsteam, we've used acetate, citrate, and triethanolamine hydrochloride buffers. The last is nice in that triethanolamine is a liquid, so no weighing is necessary.

Flush the instrument (without column) with hot water for 30 mins at 1ml/min flow. Do this before every time you want to use the instrument.

For column cleaning always use Milli-Q water (20% ethanol).

Please check the pH of water with calibrated pH meter Ideally pH of HPLC grade/purified water should be 5 to 8

Hi,

In full agreement with John Ford suggestion:

-HPLC and LC-MS grade Water are slightly acidic (Storage),

-silica in silica-based column (If you are using column such as the TSKgel G3000SWxl) begin to dissolves at pH higher than 8 and

-by working with proteins is always advisable to use a buffer,

As recommended by the manufacturer (http://www.separations.us.tosohbioscience.com/Products/HPLCColumns/SizeExclusion/ProteinsBiopolymers/SWxl/TSKgelG3000SWxl.htm), I would suggest to use a low concentrated phosphate buffer (http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html)

and

please check the calibration of your pH-meter using fresh buffer solution for calibration (contamination or CO2 dissolution).

My two cents

Thanks for all comments

maybe I'm not clear about my question ... what i curious is that

1. Can I use commercial HPLC grade water which the pH 8.39 to flush the column that has allowable range pH of 2.5-7.5

and yes i used a calibrated pH meter (my lab is a GLP one that use freshly calibrating solution also)

2. some of the answers talk about Millipore/ milliQ water ..today i do measure the pH of milliQ and it turn out to be 8.5

Do we really have to concern about pH of water ? i mean just water that use in flushing step....?

If yes please suggest me the water for HPLC that have pH below 7.5

best,

Pat

I am working on pesticides and the pH of buffer is always 2 . even when i used 0.3% formic acid i m very tensed how can i solve this problem?

If I have now well understood, puified water used to make solutions is evidently basic (milliQ one ie), but to have your eluent solution for HPLC you mix this basic water with compound (ions, nitril ...) ; so, the pH change, and is no more > 8 that could alter the column.

And it's the role of ie a phosphate buffererd saline solution used in case of protein separation.

When we begin an HPLC experimentation, firstt, we equilibrate the column wiht th solution used (ionic or hydrophobic) until stable baseline, then the solution to analyze.

If we think that somme compond after anbalysis are yet sticked to the column, able to "contaminate " further esperient, we can try to "unstick" with column tretament (and suggestion of hot , neutral pH water is one possibility, as suggested) with a risk to alter the column.

Best regards

Didier J