Tris (USP, BP, Ph. Eur.) pure, pharma grade

Comments

Tris(hydroxymethyl)aminomethane (abbreviated TRIS or THAM), also known as tromethamine, trometamol (INN, International Nonproprietary Name) and TRIS buffer. Chemically, it is a primary amine with three alcoholic hydroxyl groups.

Synthesis
First, three moles of formaldehyde are added to nitromethane in an aldol reaction to form tris(hydroxymethyl)nitromethane. Subsequently, the nitro group is reduced to the amino group.

Chemical properties
Tris(hydroxymethyl)aminomethane is used for biochemical, molecular biological, microbiological and pharmaceutical purposes as a buffer substance. At a pKa 8.2 (at 20 °C), TRIS has a good buffering capacity between pH 7.2-9.0, but it shows a relatively strong temperature dependence of the acid constant (ΔpKa = -0.031 K-1).The pH increases when the solution is cooled or decreases when the solution is heated.

Content determination
The content of tris(hydroxymethyl)aminomethane can be determined as a weak base in aqueous medium using methyl red as an indicator against hydrochloric acid. As an ethanolamine derivative, TRIS can be detected by the Chen-Kao reaction. In this reaction, the substance is mixed with sodium hydroxide solution and copper(II) sulfate (CuSO4) (blue-violet coloration).

Use

Buffer substance in biochemistry
Tris is commonly used as a buffer substance in biochemistry, e.g. in the TE buffer for DNA purification or in electrophoresis buffers such as for the separation of proteins by SDS-PAGE or the separation of nucleic acids by agarose gel electrophoresis (TAE buffer, TBE buffer). Furthermore, tris(hydroxymethyl)aminomethane is used for buffers to solubilize proteins, e.g. in TBS buffer and TBST buffer. Since the compound has a reactive primary amino group, the buffer is not suitable for some chemical applications, which is why Good buffers or inorganic buffers are then used.

Excipient in pharmaceuticals
Trometamol is used in various pharmaceutical dosage forms such as injection and infusion solutions, eye drops, creams and gels as an excipient for stabilization. It has an alkalizing and buffering effect.

Drug substance/buffer substance in medicine
In the form of its hydrochloride, tris(hydroxymethyl)aminomethane is used as a drug for the treatment of metabolic acidosis, and also for the alkalinization of urine in cases of poisoning with weakly acidic substances such as barbiturates. It is administered intravenously. Since Tris buffer has a respiratory depressant effect, its use is contraindicated in patients with respiratory insufficiency who are still breathing spontaneously. As an organic base, tris(hydroxymethyl)aminomethane forms salts with mineral acids. Carbon dioxide (CO2) dissolved in the blood can be neutralized in this way. At the physiological pH of 7.4, approx. 70 % of the tris(hydroxymethyl)aminomethane in the blood plasma is in the ionized (protonated) form. Non-ionized trometamol penetrates cell membranes and is also effective intracellularly as a buffer; this causes potassium shifts from the intra- to the extracellular space. Hyperkalemia and secondary hypokalemia may occur initially. The hydrochloride of the compound is available as an infusion solution or infusion solution concentrate.

Tris is the most commonly used buffer in biological research. One of the most important applications is the use as electrophoresis buffer such as TBE (see A1417 and A0972) or TAE (see A1691) for polyacrylamide and agarose gel electrophoresis, respectively. Tris should not be used at pH values below pH 7.2 or above pH 9.0. The pH value of a Tris buffer strongly depends on the temperature. Therefore, Tris buffers should be prepared at the temperature where it is used. Besides, dilution of concentrated Tris buffers will result in a decrease in the pH value of 0.1 pH units per tenfold dilution. Please take into account when preparing RNase-free reagents by DEPC-treatment that Tris will inactivate DEPC. In addition, Tris may form Schiff bases with aldehydes and ketones.

Literature

(1) Good, N.E. et al. (1966) Biochemistry 5, 467-477. Synthesis and characterization of various 'H+ buffers' for biological research. (2) Good, N.E. & Izawa, S. (1972) Methods Enzymol. 24, 53-68. Further discussion on the selection of buffers in biological research, here with emphasis on their suitability for the study of photosynthetic processes. (3) Ogden, R.C. & Adams, D.A. (1987) Methods Enzymol. 152, 61-87. Overview of gel electrophoresis of DNA and RNA with recipes for buffers and solutions and performance of gel electrophoresis.