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ANGUS Chemical Company Technical Bulletin TRIS AMINO ® Tris(Hydroxymethyl)Aminomethane AS A BUFFER FOR pH CONTROL CAS Registry No. 77-86-1 EINECS No. 201-064-4 TB 69 TRIS AMINO brand of tris(hydroxymethyl)- aminomethane or 2-amino-2-hydroxymethyl-1,3- propanediol is highly useful as a pH buffer for numerous analytical, reagent, diagnostic and biological applications. TRIS AMINO is a registered trademark of ANGUS Chemical Company, the primary producer of tris(hydroxymethyl)aminomethane. This pr
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  ANGUS Chemical CompanyTechnical Bulletin TRISAMINO  ®  Tris(Hydroxymethyl)Aminomethane AS A BUFFER FOR pH CONTROLCASRegistry No.77-86-1EINECS No.201-064-4 TB 69 TRISAMINObrand of tris(hydroxymethyl)-aminomethane or 2-amino-2-hydroxymethyl-1,3-propanediol is highly useful as a pH buffer fornumerous analytical,reagent,diagnostic andbiological applications. TRISAMINO is a registered trademark of ANGUS Chemical Company,theprimary producer of tris(hydroxymethyl)aminomethane.This product is soldunder other product names:TRIS,TRISBuffer,TRISBase,THAM,Tromethamine,Trisaminol,Trometamol,etc. Typical Properties The following are typical properties of TRISAMINO.Theyare not to be considered product specifications.Molecular weight (calc.) . . . . . . . . . . . . . . . . . . . . .121.14Boiling point @ 10 mm Hg, °C . . . . . . . . . . . . . . .219-220Melting range °C . . . . . . . . . . . . . . . . . . . . . . . . . .165-172pKa @ 25°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.03pH of 0.1M aqueous solution @ 20°C . . . . . . . . . . . .10.4Solubility in water at 20°C, g/100 mL . . . . . . . . . . . . . . .80 CCH 2 OHCH 2 OHNH 2 HOCH 2 A subsidiary of The Dow Chemical Company ANGUS Chemical Company1500 E.Lake Cook RoadBuffalo Grove,IL 60089Phone:847-215-8600Toll Free:800-447-4369Fax:847-215-8626  Selecting a Buffer Most biochemical reactions must be carried out insolutions buffered to a pH range of 6 to 8.However, manycommonly used buffering systems have shortcomings inthis range.In a comprehensive study of buffer systems,Good, et al. identified nine criteria for a hypothetical “ ideal ” buffer. 1 1.pKa between 6 and 8.2.High solubility in aqueous systems.3.Exclusion by biological membranes.4.Minimal salt effects.5.Minimal effects on dissociation due to concentration,temperature, and ionic composition.6.Well defined or nonexistent interactions with mineralcations.7.Chemical stability.8.Insignificant light absorption between 240 and 700nanometers.9.Easily available in pure form.No single buffering agent meets all these criteria.Phosphate buffers, for instance, tend to precipitate or bindwith polyvalent cations, lose their buffer strength above apH of 7.5, and frequently act as participating or inhibitingmetabolites in biological reactions.Carbonates tend to bepoor buffers due to the low solubility of carbon dioxide.Other compounds, such as borates, can react to formcomplexes with certain respiratory metabolites and otherorganic compounds. 1 Good, N.E., G.D.Winget, W.Winter, T.N.Connolly, S.Izawa, and R.M.M.Singh, Biochem  ., 5:467 (1966) Performance of TRISAMINOas a Buffer TRISAMINOperforms well as a buffer in the pH range ofseven to nine (buffers generally perform well over a rangeof two pH units centered on the pKa).It is highly soluble inwater and has insignificant light adsorption between 240and 700 nanometers.TRISAMINOis readily available inits pure form and is quite stable under normal ambientconditions.In selecting a buffer, knowledge of the chemical reactivityof the buffer is required, so that unwanted reactions withchemical species present in the solution will not occur.TRISAMINO is a mildly-basic primary amino alcohol.Assuch, it enters into reactions typical of primary amines andalcohols.For instance, TRISAMINOreacts withformaldehyde at room temperature to form an oxazolidine.However, in most biochemical or diagnostic reactions, theinterference of TRISAMINO with other species or ionspresent is negligible.For a more complete discussion ofthe chemical properties of TRISAMINO, see ANGUSTechnical Data Sheet No.10, “ Primary Amino Alcohols. ” TRISAMINO is not excluded by biological membranes,but in most applications this is of little significance.Ifbiological membrane exclusion is essential, it isrecommended that zwitterionic buffers be employed.The dissociation constants of all buffers change with theirconcentration in solution, but some buffers are moresensitive to this ionic effect than are others.If the buffer ofchoice has a dissociation constant which changes littlewith buffer concentration, a concentrated buffer solutioncan be diluted as required for numerous test solutionswithout readjustment of pH.TRISAMINO solutions will drop roughly 0.1 pH unit perten-fold dilution.This is excellent performance incomparison to phosphate buffers, for example, whichincrease in pH from 6.6 to 6.9 when diluted from 0.5M to0.05M.The dissociation constants of most buffers are affecteddramatically by temperature, and TRISAMINOis noexception.When using TRISAMINOas a buffer, caremust be taken to prepare each buffer solution so that thedesired pH is obtained at the temperature at which thesolution is to be utilized.The table on page three presentsthe pH at several temperatures of 0.05M TRISAMINObuffer solutions prepared by dissolving various amounts ofTRISAMINO and TRISAMINOhydrochloride in water.2  The Effect of Temperature on the pH of 0.05MTRISAMINOBuffer SolutionsTRISAMINOTRISAMINOhydrochloridepHpHpHgrams/litergrams/liter@5°C@25°C@37°C 0.677.027.767.206.910.806.857.897.307.020.976.617.977.407.121.186.358.077.507.221.396.068.187.607.301.665.728.267.707.401.975.328.377.807.522.304.888.487.907.622.654.448.588.007.712.974.028.688.107.803.343.548.788.207.913.703.078.888.308.014.032.648.988.408.104.362.219.098.508.224.651.839.188.608.314.901.509.288.708.425.131.239.368.808.515.320.969.478.908.625.470.769.569.008.70When TRISAMINO is completely neutralized withhydrochloric acid, a solution pH of about 4.7 is obtained. TRISAMINO–Phosphate Mixed Buffers By using a 1:1 by weight combination of TRISAMINO andpotassium dihydrogen phosphate (KH 2 PO 4 ), the lowesteffective buffering pH level of a 0.10M solution is reducedto about 6.5.Moreover, this value is much lesstemperature-dependent for the TRISAMINO/KH 2 PO 4 combination than is the lower end of the effective pHbuffering range obtained from TRISAMINOalone.Thiscombination of buffers may be considered in lower pHapplications where phosphate interference does not affectthe reaction environment. Buffer4°C20°C37°C TRISAMINOeffective range8.1-9.67.5-9.07.2-8.7pKa8.828.267.92KH 2 PO 4 effective range6.4-7.86.3-7.86.3-7.7pKa7.107.066.981:1 Comb.effective range6.5-9.46.4-9.06.3-8.7pKa7.957.727.51 Applications for TRISAMINOBuffer TRISAMINO is ideally suited for use as a mild alkalinebuffering agent and acidimetric standard.Asdemonstrated in the table, the combination of TRISAMINOand TRISAMINOhydrochloride can generateany desired pH between seven and nine.The highequivalent weight, low order of toxicity, high purity, andsuperior chemical stability of TRISAMINOposition it asthe buffer of choice for many applications. Enzyme Production  TRISAMINOstabilizes the pH of solutions in the rangeof most favorable for the production of many enzymes.TRISAMINOdoes not interfere with enzyme activity;infact, it helps to prolong the life expectancy of the enzymeformers. Enzyme Enhancement  The use of TRISAMINOin conjunction with enzymessuch as proteases, lipases and amylases appears tonoticeably improve their digestion of soils and stains.This is due in part to the ability of TRISAMINOtoscavenge residual chlorine, often found in municipalwater supplies, which would otherwise substantiallydeactivate enzymes used in such water. Contact Lens Cleaner  TRISAMINOholds the pH of contact lens cleaningsolutions in the range most favorable for hydrolysis ofprotein films on lens surfaces.This effects a more rapidremoval of such films and improves the overall cleaningefficiency of lens cleaners.Also, the low toxicity and lackof eye irritancy of TRISAMINO are of primeconsideration in its choice for this application. Medical Diagnostics  TRISAMINOis an important buffer for the production,storage and use of enzymes for medical diagnosticassays.Ultra Pure TRISAMINO is a standard for use inbuffering alkaline phosphate and is commonly used inthe preparation of enzyme antibody conjugates.3  Air Washing  As a scavenger of CO 2 , TRISAMINOcan be combinedwith the enzyme carbonic anhydrase to form a highly-efficient agent for the removal of CO 2 from anatmosphere.The rapid release of the CO 2 from the TRISAMINOis achieved under mild vacuum or heating toregenerate the scavenging agent.This absorption systemis highly effective for reconditioning air in enclosed areassuch as submarines. Fish Transport  TRISAMINO acts to regulate the pH of the water, andscavenge CO 2 resulting from respiration, in the transportof live fish.It has been demonstrated that the use of TRISAMINOin the water will extend the survival rate of fish inclosed, non-aerated containers by as much as threefold.TRISAMINO will achieve the desired results in bothseawater and fresh water without precipitating dissolvedcalcium. Acidimetric Standard  TRISAMINOcan be used directly for the standardizationof strong acid solutions.It gives sharp inflections at theequivalence point of potentiometric titrations ofhydrochloric acid in water and perchloric acid in aceticacid.Standardizations may also be accomplished using asuitable indicator such as p-sulfo-o-methoxylbenzene-azodimethyl-1-naphthylamine.TRISAMINO buffers alsofind use as solutions for the standardization of electrodes,whether pH-sensitive or ion-specific. Protein Electrophoresis  TRISAMINOsalts find use in gel electrophoresis forprotein separations.As an example, for electrophoresis innon-dissociating buffer systems, TRISAMINO salts arethe buffers of choice above pH 7.0 (Tris-glycine or Tris-borate for pH 8.3-9.3;Tris-acetate for pH 7.2-8.5;Tris-citrate for pH 7.0-8.5). TRISAMINO is aRegistered Trademark of ANGUS Chemical Company. HFP/JHV –  Form No.319-00042 ©1999 by ANGUSChemical Company.All Rights Reserved.Printed in U.S.A. The information and data contained herein are believed to be correct.However, we do not warranteither expressly or by implication the accuracy thereof.In presenting uses for this product, noattempt has been made to investigate or discuss any patent situations which may be involved. A subsidiary of The Dow Chemical Company
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