There are so many answers to the question for brevities sake I will add mine
1. Phosphate determines the pH, not chloride salts added. These monovalent cation/chloride solutions should have all but no buffering capacity. If the sodium or potassium chlorides show a significant buffering capacity they may have been contaminated by other salts, replace. I've seen worse things happen. 2. Dibasic phosphate is basic and if left exposed to air at room temperature will absorb both moisture and carbon dioxide, this can drop the pH and make the salt more difficult to handle. It is, as basic salts go, pretty resilient to acidification, but conditions in the laboratory may vary (e.g. gas space heaters, storage around areas were flame is used). I seriously doubt this is the issue. The way to know "what's up with this" is keep a record of your buffer salts, for example a 0.01 M solution of dibasic should always be the same pH. So you can record this on the bottle. If the pH of the diluted salt changes over time and it may be time to discard and replace _or_ your pH meter may have a problem. This is a significant problem in the laboratory, DNA hydration buffers at pH8.3 tend to loose about 0.1 pH units per 6 mos, faster if they are frequently opened. Hydroxides are the worst, absorbing both water and carbon dioxide quickly. If the dibasic absorbs enough water it can! alter the pH because its molar contribution per weight will be less. This is unlikely to be the cause. For both of these, if you make the solution repeated and repeatedly you get the same deviant pH, then replace. 4. Two salts were confused at weighing, whoops. Seen this one a lot, I've done it and a number of my trainees have done it. The solution remade comes out perfect pH. As my TA used to say 'you make a cook-book buffer, if the pH is wrong, that's a good thing, don't adjust the pH, remake the buffer'. Here's the recipe for 10X PBS pH 7.2 NaCl 87.7 NaH2PO4 3.05 Na2HPO4 11.1 Initial ddH20 0.9 L (e.g twice distilled water is all that we use, house DI systems cannot be relied upon) ddH20 Water to 1L Dibasic will not dissolve quickly, requires about 2-4 hours to dissolve, so keep the vessel covered with parafilm or flat piece of glass. This is not something that you leave overnight on the bench stirring in open air. Make 100 ml of 1X and test the pH. pH should be 7.2 +/- 0.05 you can lower the monobasic and increase the dibasic to achieve pH 7.4 (See other post). NaOH can be added in small amounts to correct the pH, in most case pH adjustment is not needed. 5. Divalent and trivalent cations are easily trapped by higher ionization states of phosphate, thus heating phosphate can trap calcium and potentially drop the pH. Divalent cation solutions should be added at STP and in diluted form. In addition, one expects as the pH to go up, the stability of calcium phosphate in solution to go down. Sometimes we heat buffers to get them in solutions, but this may not work well with some buffers, or may cause problem with sterilizing solutions. In this case calcium chloride and phosphate buffers need to be sterilized separately, and then combined after they cool down to room temperature. Unless your water is made from Antarctic ice, it probably has a significant amount of contaminants in it, in our area water needs to be twice distilled (deionized and then steam distilled). Deionization removes hardening solutes, whereas distillation removes volatiles such as gases in the water (such as ammonia, which occurs in areas where chloramines is added to the water supply). All chemistry requires near 18 mega-ohm water, whereas glassware washing, soap solutions, etc, should be done in deionized water. Residue on bottles are frequently carbonates (calcium), at high temperatures (such as in a drying oven) the carbonate can be converted to oxides and hydroxides, which will trap phosphate. A common reason for scale is that someone made a reagent, left it to soak in tap water but forgot about it. The water evaporated leaving a thin coating of calcium. Soaps will not remove this. Bottles that have hard water scale, rust deposits, on them can be cleaned by soaking with 0.01! M hydrochloric acid or dilute phosphoric acid. I can go into almost any lab, and if I look at the glassware shelf long enough I will find bottles with scale on the inside of the bottle. 6. Another tip, something people overlook, don't assume that glassware on the shelf is rinsed properly. Frequently, soap residue is found in the glassware. Add some deI and shake, if you see lots of bubbles, it was not clean. This happens much and in almost every lab I have been in. In managing an animal facility for the last 15 years, I found that soaps are not at neutral pH, some soaps are acidic or alkaline enough to damage plastics in the autoclave (yellowing, fissuring and opaqueness), this can lead to trapping of soap and debris. The solution to the problem is that we made our own soap from technical grade SDS (near saturation) that was pHed to neutral with Tris-HCL. In addition use only enough soap to get the job done, and be wary of soap precipitates (premaking soap solution virtually eliminates precipitation of soap). In many cases, you are using an inert aqueous buffer, simply deI rinse the container when done, put upside down on the bench and let set, reuse. How m! any folks get tenacious tiny bubbles in the melted agarose the first time they microwave in a fresh glass? That's due to glass-ware washing residue on the glass that cannot be rinsed away. For many buffers, simply deI rinse the vessel, invert glass on bench to dry, reuse. 7. Check your pH calibration solutions occasionally. The pH 10 buffer is extremely susceptible to carbonate neutralization. For this reason I calibrate the meter for all pHs below pH 8.5 with the pH 7.0 and pH 4.0 buffers. Before I use pH 10 calibration I first calibrate at pH 4 and 7 and the pH 10 reading should be 'ballpark-ish', if not open an new bottle and compare pHs. To get the best calibrations I rinse the electrode once in each buffer (usually the buffer from the last calibration, sealed in a 20 ml scintillation vial) and replace it with fresh buffer, then set the calibration point. Occasionally one has to deal with an old electrode, the surfaces are coated and equilibration is quite slow, particularly at pH7.0, that's not good at pH 10 if the electrode takes 15 minutes to equilibrate since pH of the buffer has already dropped. In this case you might purge cover electrode in the scint. vial with paraffin. This is not the most common cause of pH difficulties that I ! see with trainees, but is a reasonably common problem. 8. There are other issues, with specific buffers like Tris/EDTA in that pH can change markedly with dilution and temperature, thus one has to do some buffer tinkering to get the desired pH. Again, one way to know if you have a bad buffer (either because of age or mismaking) is to make a buffer from newly purchased material and determine the pH both concentrated and diluted so that one knows exactly what to expect. It goes without saying that the buffer needs to be pHed at the standard temperature. I hope this resolves your recurring problem. Philip -----Original Message----- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of sudheer sangeetham Sent: Tuesday, January 03, 2012 10:53 AM To: meth...@magpie.bio.indiana.edu Subject: 10X PBS Hi People I have few doubts regarding phosphte buffer saline (PBS). Recently I found one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4. Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but not KCl, am I right? I have prepared the buffer like this NaH2PO4 ( 0.038M) Na2HPO4.2H20 ( 0.162M) NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct? did I do any mistake.... Could any one please tell me did i do any mistake? Thank you in advance -- Sudheer Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary. _______________________________________________ Methods mailing list http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
