What Tris Buffer Is
Tris, or tris(hydroxymethyl)aminomethane, is the buffering agent used in most biochemical and molecular biology protocols. Its formula is C4H11NO3, its molecular weight is 121.14, its CAS number is 77-86-1, and it is a white crystalline solid with a melting point of about 171 to 172 C. In solution it has a pKa of about 8.06 at 25 C, which places its useful buffering range between roughly pH 7.0 and 9.0 and makes it the standard choice for DNA, RNA and protein work. Turbidity in a working solution is almost never caused by the Tris itself, because the solid is freely soluble; it is caused by something else in the bottle, the water or the glassware.
Prevention and Correct Preparation
Prepare buffers with reagent-grade Tris and reagent water, dissolve at room temperature with vigorous stirring until the solution is completely clear, and adjust pH with a calibrated electrode using the method described in ASTM E70 for pH measurement with a glass electrode. Filter the finished buffer through a 0.22 micrometre membrane, store it in a clean, tightly closed container protected from light, and hold working solutions at 2 to 8 C for no longer than a few weeks. Solutions that will be kept longer should be filter sterilised rather than autoclaved, because autoclaving drives off carbon dioxide and shifts the pH. Never top up an old bottle with fresh buffer, and never mix Tris with phosphate containing solutions unless a precipitate-free combination has been verified.
Selection and Supply Considerations
Buyers comparing offers for this material should ask for the mill test certificate that accompanies the shipment and check that the heat number on the certificate matches the marking on the product. Certificate values are the controlling evidence of compliance; brochure data sheets are indicative only.
Tolerances, surface condition, temper or heat-treatment state and packing method should be written into the purchase order rather than agreed verbally, because these details determine whether the material can be processed without rework after arrival. For export shipments, confirm the inspection regime, the acceptable third-party inspector and the documentation set required for customs clearance before the order is released to production.
Frequently Asked Questions
Q: Why does my Tris buffer turn cloudy after a few days in the fridge?
A: Cold storage lowers the solubility of dissolved salts and shifts the pH because the pKa of Tris is strongly temperature dependent. Warm the solution to room temperature and re-check before use; if cloudiness persists, prepare a fresh batch with reagent water and filter it.
Q: Does Tris buffer itself cause turbidity?
A: Pure Tris is highly soluble and gives clear solutions. Turbidity indicates an impurity, undissolved powder, microbial contamination, poor quality water or an incompatible additive rather than the buffer substance itself.
Q: What water should be used to prepare Tris buffer?
A: Reagent water meeting ASTM D1193 Type II or ISO 3696 Grade 2 is appropriate. It limits calcium, magnesium, silicate and organic load, all of which can produce haze or precipitate at alkaline pH.
Q: Can turbid buffer be filtered and reused?
A: Filtering through a 0.22 micrometre membrane removes particles and microbes, but it does not undo the chemical change that caused precipitation, and it does not tell you whether the pH or ionic strength is still correct. For quantitative work, prepare a fresh solution.
Q: How should Tris buffer be sterilised?
A: Filter sterilisation is preferred. Autoclaving is possible but the pH drops because carbon dioxide is expelled, so the pH must be re-checked and adjusted after the solution has cooled to working temperature.
Q: Does the pH of Tris change with temperature?
A: Yes. The pKa decreases by about 0.028 units per degree Celsius, so a buffer set to pH 8.0 at 25 C reads higher when chilled. Always adjust and measure at the temperature at which the buffer will be used.
