We have growth in our buffers if they sit out for any time. So we pitch it every week. We would like to find some sort of additive that we might add that would not effect the chromatography.

Thank you in advance.

Sodium azide (100-200 ppm) is the classic antibacterial additive. Nasty stuff. Read up on it and how to dispose of wastes before considering using it.

Isn't there something a little safer that is typically used?

Try 20% MeCN, MeOH or 10% THF.

Proper microfiltration and subsequent cleanliness can also improve shelf life dramatically. There was an earlier discussion on this.

jclark the addition of that much organic might have an effect on my chromatography.

Everything is filtered but we still have growth after one week.

A suggestion was to use chloroform. What do you all think of that?

I have reviewed earlier discussions and have not seen anything on this topic.

There was an earlier thread on this subject. However, I agree with jclark. Just turn your buffer into mobile phase, add the organic fraction. I read somewhere that 2% ACN, or 10% MeOH were biocidic, I haven't confirmed this.

Some buffers (phosphate, diff. conc.) had been forgotten, sitting somewhere in the lab for nearly two years: no bacterial growth. Similar buffers have had the bugs after a week or even less (without an effort to keep it clean). Itīs a matter of cleanliness. As an example, if you filter and donīt throw away the initial filtrate you might as well forget it.
Also I have never seen microbes in commercial, sterilized buffers even after years in closed bottles, furthermore, I cannot recall bugs in mobile phase containig any organics. Many column manufacturers recommend azide or 20% MeOH for "sterile" column storage.

We are primarily using gradients. We have found with past experince that the premixing of mobile phase can change the chromatography. Also, for many methods, we start out with a lower amount of organic than has been suggested.

What again about chloroform? Will it work?

How does the keeping of the initial filtrate cause more bugs? I don't understand? If it is filtered isn't it filtered? What is different about the initial filtrate and the last filtrate.

This is a question, rather than a solution. I know that sonication has been used in the biological sciences to disrupt cells. I wonder whether sonication of mobile phases might be a non-chemical means of killing the bugs? Does anyone have any relevant experience?

The primary source of microbes is dust. If the bottom of the filter has dust on it, or any part of your equipment downstream from the filter, or the container in which you store it, you will be in trouble. The initial filtrate is used to wash the filtration apparatus, some of the later filtrate to wash the storage container, etc.
(This assumes that you are not letting anything stand around open, coughing into your solution, sticking you fingers in, etc. etc.)

It is extremely easy to microbially contaminate things. Unless you sterilized all equipment and chemicals (including water)used to make the mobile phases, and use aseptic techniques for their preparation, chances are that you will end up with some level of contamination. Filtration will not account for organisms in the mobile phase containers, lines, etc. As annoying as it may seem, it is probably best to make new buffers. If you want to add antibacterials (are you sure it is a bacteria and not a yeast or mold?) there are lots available. Included in these are sodium azide, benzalkonium or benzethonium chloride, parabens, etc. The use of these would be dependant upon your particular analysis.

Good Luck

If you do not mind using a salt-based buffer in your mobile phase, consider lithium (e.g. lithium acetate, lithium formate, etc.).

Lithium based salts result in a bacteriostatic environment.

<What again about chloroform? Will it work?
Of course adding chloroform will work, but it has a fairly high uv cutoff of about 245nm. Also, adding enough chloroform to be biocidic will also likely change the selectivity of your method(s).

I don't really understand the online vs offline mixing issue. Modern quaternary HPLCs (Agilent 1100s, etc.) have online mixing specs of about 1.5%. Assuming you have more than one and one could be high and one low - you need to validate (or think about) a robustness of about 3%. A 1000mL graduated cylinder has a tolerance of 2.5 mL or 0.5% when used to measure 500mL.

Thanks for the response Tom M. I was told that just a couple of drops would do it.

The second part of your message I think was for another discussion.

Regarding the use of chloroform as a preservative, I have investigated several options, primarily for preserving standards containing organic acids which are prone to attack by organisms even upon refrigeration. Although azide is often used, it's really not a very stable compound. In many cases there's not much left after a few weeks. I know that because in my method I can see the azide and its breakdown products. Chromate is incredibly effective, even at 10 ppm. It works for stabilizing standards for months to years even when stored at room temperature. Of course chromate's is a decent chromaphore so it's probably not a good choice for buffer stabilization. But sometimes I found chromate tended to oxidize my analytes. For the last few years I've been using chloroform instead. I just add a drop or two to my standard and this pretty much minimizes the problem. It should work well in the buffer stabilization case although I can't speak to whether it will introduce gradient artifacts (chloroform generally has a stabilizer added which would seem to imply other compounds may be present in old bottles).

An alternative procedure is to prepare a concentrate stock buffer (0.5M - 1M) and dilute amounts of this stock as you need. We store it at 4°C and prepare a fresh dilution (to 20 mM)every day. We have a daily routine of four isocratic methods, all of them with the same buffer but diff amounts of acetonitrile, changing the % of ACN during the sequence of analysis automatically as required by our sample schedule.
Therefore, we don't need to add any additive to the stock solution.
Maybe this can also work for you.
Regards

we use about the same procedure for our gradient analysis.
we make a stock solution ( 1M)filter it and store it in the vreezer (-19 °C)in portions of 50 ml. when needed we dilute the buffer with HPLC-water ( which comes filtered out of the machine) and its ready. remember that the pH of the stock solution is different from the diluted buffer.

Thank you Chris Pohl. I will give the chloroform a shot. Thanks to everyone else for their advice. It is nice to know how others are dealing with the problem

The suggestion to add a drop or two of chloroform is into what volume of mobile hase?

What about Diethyl pyrocarbonate? It's an effective antimicrobial agent when first added, but within a day or two has converted to CO2 and ethanol, thereby leaving the solution free of toxins.

Membrane filtering (Nucleopore for example) worked
in our lab for decades (using the standard lab
practice described by Muller,above). Personally I
like this better than introducing a compound at a
trace level: it will never reach equilibrium w the
stationary phase.

Until you solve your problem a word of warning-
-under GLP all solutions must have expiration
dates. Yours should reflect the onset of visible
contamination.

You might consider adding ProClin to your mobile phase. This is available from Supelco (Sigma Aldrich is the parent). This can be used in a variety of applications. Follow the link and do a search for the material. There are also links for the tech service people to find out which one works best for your application.

http://www.sigmaaldrich.com/Brands/Supelco_Home.html