Hi Sreetama, For most buffers, I use Katherine's method, but in the case of citrate I'd recommend just titrating citric acid with NaOH. I've made a pH series from citric acid and Na3Cit before, and it's a huge pain. It's very difficult to calculate how much of each you'll need, because citrate is triprotic and the pKas overlap. When I made a pH series this way, I ended up using much more stock than I anticipated, and just had an overall unpleasant afternoon.
One other point - high citrate concentrations can cause your pH meter to drift, so don't leave the probe in the citrate solution any longer than needed, and calibrate it frequently. I'm told this is because citrate chelates metals and that throws off the electrode, though admittedly I don't know the mechanism and can't find any references to back me up - it's just been lab folklore for me. Either way it might be worth testing the stability of your electrode over time to get an idea. Shane Caldwell McGill University On Thu, Jan 30, 2014 at 10:40 AM, Katherine Sippel < [email protected]> wrote: > Alternatively you could make a stock solution of citric acid (say 1 M for > example) and stock solution of sodium citrate (also 1 M). Mix them in the > appropriate ratio to ballpark the right pH and just adjust up or down with > the stock solution. The concentration of citrate will be the same no matter > the final volume. You can then dilute that down to whatever your final > concentration of citrate needs to be. > > If you are looking for the actual method to do the calculations I would > suggest finding a chemistry textbook and looking at the chapter on > buffering and the Henderson-Hasselbalch equation. > > Cheers, > Katherine > > > On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot <[email protected]>wrote: > >> But you have to be aware that pH depends on the concentration of the >> buffer. This is especially the case for phosphate and citrate buffer. >> Daniel >> >> Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit : >> >> It's a pain but I usually just make each pH of whatever buffer I'm using >> (if you make it concentrated then you'll only have to do it once). Also, >> if you haven't already found it, Hampton has a nice link to calculate >> volume of components while designing a tray as long as you tell it the >> concentrations. >> >> http://hamptonresearch.com/make_tray.aspx >> >> Nick >> >> From: Roger Rowlett <[email protected]> >> Reply-To: Roger Rowlett <[email protected]> >> Date: Thursday, January 30, 2014 at 7:23 AM >> To: "[email protected]" <[email protected]> >> Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5 >> >> The easiest way to produce repeatable conditions is to titrate a stock >> solution (say 1M) of citric acid with NaOH to the desired pH and use that >> to mix your screen. That's what Hampton does anyway. >> >> If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. >> The pH won't be linear with mixing ratio, but will be easily repeatable. >> The actual pH of the final, magic solution can be directly measured if >> desired. Calculations will never be exactly right; pKa values are ionic >> strength dependent. Better to measure. >> >> Roger Rowlett >> On Jan 30, 2014 2:37 AM, "sreetama das" <[email protected]> wrote: >> >>> Dear All, >>> We have obtained many tiny protein crystals in a condition >>> containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are >>> too small for mounting in loops. >>> >>> We intend to vary the salt concentration & pH to obtain >>> larger crystals. >>> >>> Could anyone direct us to some links, or provide us with a >>> method (with calculations) to calculate the amounts of citric acid & >>> trisodium citrate required to obtain buffers in a range of pH 3 - 6.5? >>> I have come across online buffer calculators and links where >>> the amounts of the components required are mentioned in grams, but none >>> explaining how those values were arrived at. >>> >>> Thanks & regards, >>> sreetama >>> >> >> > > > -- > "Nil illegitimo carborundum"* - *Didactylos >
