In article <[email protected]>, [email protected] says...
> I am using 10x PBS by Lonza withthis composition: > 1,440 mg/L of KH2PO4, 90,000 of mg/L NaCl, 7,950 mg/L of Na2HPO4. > I use it as 1x at pH 7.3, 8 and 9. > My problem is that, except for the sample at pH 7.3, in the cases at pH 8 and > 9 the value tends to low down. After a couple of days pH 9 solution has lowed > down to pH 8.3. > I changed the pH using high concentrated NaOH, as I found in literature. You probably should read up on how buffers work in a good book on general chemistry, or at http://www.rossu.edu/medical- school/students/documents/Basic_Chemistry.pdf. Briefly, buffers are mixtures of an acid and it's corresponding base, in the case of PBS those are H_2PO_4^- and HPO_4^2-, respectively. If both are present at near equal molar concentration, then the addition of either H^+ or OH^- leads to a change in the acid/base ratio rather than to a change of [H^+] and hence pH. The amount of H^+ or OH^- you can add to a buffer without noticeably changing the pH is called _buffering capacity_. This buffering capacity depends both on the buffer concentration and on the acid/base ratio. The capacity is highest when acid/base ~ 1 (based on molar concentration). Under that condition, the pH of the buffer will be near the pKa value of the acid, for H_2PO_4^- that is pH 6.86. The more the pH deviates from the pKa, the lower the buffering capacity. As a rule of thumb, buffering works reasonably well within +- 1 pH-unit of the pKa. When you added a lot of NaOH to your PBS, you in effect depleted the buffering capacity by converting most of the H_2PO_4^- present into HPO_4 ^2-. When you leave such a solution standing, it will over time take up CO_2 from the air, which gets converted into carbonic acid. This addition of acid will lower the pH, this lowering is not resisted by the buffer as you have no buffering capacity. Thus what you observed is exactly what would be predicted on theoretical grounds. If you want to work at higher pH than about 7.5, you should use a buffering substance with an appropriate pKa like Tris (pKa = 8.2), that is, use Tris-buffered saline (TBS) instead of phosphate buffered saline (PBS). The composition of TBS is 50 mM Tris (6.05 g/L), 150 mM NaCl (8.76 g/L) and HCl to the desired pH. Like PBS, it is ~ isotonic. Depending on your application, you may need to add K, Mg or Ca and perhaps some DTT or EDTA. It is possible to make a 10-fold concentrate, but upon dilution the pH may have to be re-adjusted (because the ion activity changes upon dilution). _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
