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 product is sold
under other product names: TRIS, TRIS Buffer, TRIS Base, THAM,
Tromethamine, Trisaminol, Trometamol, etc.
Typical Properties
The following are typical properties of TRIS AMINO. They
are not to be considered product specifications.
Molecular weight (calc.) . . . . . . . . . . . . . . . . . . . . .121.14
Boiling point @ 10 mm Hg, C . . . . . . . . . . . . . . .219-220
Melting range C . . . . . . . . . . . . . . . . . . . . . . . . . .165-172
pKa @ 25C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.03
pH of 0.1M aqueous solution @ 20C . . . . . . . . . . . .10.4
Solubility in water at 20C, g/100 mL . . . . . . . . . . . . . . .80
C
CH2OH
CH2OH
NH2
HOCH2
A subsidiary of The Dow Chemical Company
ANGUS Chemical Company
1500 E. Lake Cook Road
Buffalo Grove, IL 60089
Phone: 847-215-8600
Toll Free: 800-447-4369
Fax: 847-215-8626
Selecting a Buffer
Most biochemical reactions must be carried out in
solutions buffered to a pH range of 6 to 8. However, many
commonly used buffering systems have shortcomings in
this 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 mineral
cations.
7. Chemical stability.
8. Insignificant light absorption between 240 and 700
nanometers.
9. Easily available in pure form.
No single buffering agent meets all these criteria.
Phosphate buffers, for instance, tend to precipitate or bind
with polyvalent cations, lose their buffer strength above a
pH of 7.5, and frequently act as participating or inhibiting
metabolites in biological reactions. Carbonates tend to be
poor buffers due to the low solubility of carbon dioxide.
Other compounds, such as borates, can react to form
complexes with certain respiratory metabolites and other
organic 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 TRIS AMINO as a Buffer
TRIS AMINO performs well as a buffer in the pH range of
seven to nine (buffers generally perform well over a range
of two pH units centered on the pKa). It is highly soluble in
water and has insignificant light adsorption between 240
and 700 nanometers. TRIS AMINO is readily available in
its pure form and is quite stable under normal ambient
conditions.
In selecting a buffer, knowledge of the chemical reactivity
of the buffer is required, so that unwanted reactions with
chemical species present in the solution will not occur.
TRIS AMINO is a mildly-basic primary amino alcohol. As
such, it enters into reactions typical of primary amines and
alcohols. For instance, TRIS AMINO reacts with
formaldehyde at room temperature to form an oxazolidine.
However, in most biochemical or diagnostic reactions, the
interference of TRIS AMINO with other species or ions
present is negligible. For a more complete discussion of
the chemical properties of TRIS AMINO, see ANGUS
Technical Data Sheet No. 10, Primary Amino Alcohols.
TRIS AMINO is not excluded by biological membranes,
but in most applications this is of little significance. If
biological membrane exclusion is essential, it is
recommended that zwitterionic buffers be employed.
The dissociation constants of all buffers change with their
concentration in solution, but some buffers are more
sensitive to this ionic effect than are others. If the buffer of
choice has a dissociation constant which changes little
with buffer concentration, a concentrated buffer solution
can be diluted as required for numerous test solutions
without readjustment of pH.
TRIS AMINO solutions will drop roughly 0.1 pH unit per
ten-fold dilution. This is excellent performance in
comparison to phosphate buffers, for example, which
increase in pH from 6.6 to 6.9 when diluted from 0.5M to
0.05M.
The dissociation constants of most buffers are affected
dramatically by temperature, and TRIS AMINO is no
exception. When using TRIS AMINO as a buffer, care
must be taken to prepare each buffer solution so that the
desired pH is obtained at the temperature at which the
solution is to be utilized. The table on page three presents
the pH at several temperatures of 0.05M TRIS AMINO
buffer solutions prepared by dissolving various amounts of
TRIS AMINO and TRIS AMINO hydrochloride in water.
2
The Effect of Temperature on the pH of 0.05M
TRIS AMINO Buffer Solutions
TRIS AMINO
TRIS AMINO hydrochloride pH pH pH
grams/liter grams/liter @5C @25C @37C
0.67 7.02 7.76 7.20 6.91
0.80 6.85 7.89 7.30 7.02
0.97 6.61 7.97 7.40 7.12
1.18 6.35 8.07 7.50 7.22
1.39 6.06 8.18 7.60 7.30
1.66 5.72 8.26 7.70 7.40
1.97 5.32 8.37 7.80 7.52
2.30 4.88 8.48 7.90 7.62
2.65 4.44 8.58 8.00 7.71
2.97 4.02 8.68 8.10 7.80
3.34 3.54 8.78 8.20 7.91
3.70 3.07 8.88 8.30 8.01
4.03 2.64 8.98 8.40 8.10
4.36 2.21 9.09 8.50 8.22
4.65 1.83 9.18 8.60 8.31
4.90 1.50 9.28 8.70 8.42
5.13 1.23 9.36 8.80 8.51
5.32 0.96 9.47 8.90 8.62
5.47 0.76 9.56 9.00 8.70
When TRIS AMINO is completely neutralized with
hydrochloric acid, a solution pH of about 4.7 is obtained.
TRIS AMINOPhosphate Mixed Buffers
By using a 1:1 by weight combination of TRIS AMINO and
potassium dihydrogen phosphate (KH
2
PO
4
), the lowest
effective buffering pH level of a 0.10M solution is reduced
to about 6.5. Moreover, this value is much less
temperature-dependent for the TRIS AMINO/KH
2
PO
4
combination than is the lower end of the effective pH
buffering range obtained from TRIS AMINO alone. This
combination of buffers may be considered in lower pH
applications where phosphate interference does not affect
the reaction environment.
Buffer 4C 20C 37C
TRIS AMINO effective range 8.1-9.6 7.5-9.0 7.2-8.7
pKa 8.82 8.26 7.92
KH
2
PO
4
effective range 6.4-7.8 6.3-7.8 6.3-7.7
pKa 7.10 7.06 6.98
1:1 Comb. effective range 6.5-9.4 6.4-9.0 6.3-8.7
pKa 7.95 7.72 7.51
Applications for TRIS AMINO Buffer
TRIS AMINO is ideally suited for use as a mild alkaline
buffering agent and acidimetric standard. As
demonstrated in the table, the combination of TRIS
AMINO and TRIS AMINO hydrochloride can generate
any desired pH between seven and nine. The high
equivalent weight, low order of toxicity, high purity, and
superior chemical stability of TRIS AMINO position it as
the buffer of choice for many applications.
Enzyme Production
TRIS AMINO stabilizes the pH of solutions in the range
of most favorable for the production of many enzymes.
TRIS AMINO does not interfere with enzyme activity; in
fact, it helps to prolong the life expectancy of the enzyme
formers.
Enzyme Enhancement
The use of TRIS AMINO in conjunction with enzymes
such as proteases, lipases and amylases appears to
noticeably improve their digestion of soils and stains.
This is due in part to the ability of TRIS AMINO to
scavenge residual chlorine, often found in municipal
water supplies, which would otherwise substantially
deactivate enzymes used in such water.
Contact Lens Cleaner
TRIS AMINO holds the pH of contact lens cleaning
solutions in the range most favorable for hydrolysis of
protein films on lens surfaces. This effects a more rapid
removal of such films and improves the overall cleaning
efficiency of lens cleaners. Also, the low toxicity and lack
of eye irritancy of TRIS AMINO are of prime
consideration in its choice for this application.
Medical Diagnostics
TRIS AMINO is an important buffer for the production,
storage and use of enzymes for medical diagnostic
assays. Ultra Pure TRIS AMINO is a standard for use in
buffering alkaline phosphate and is commonly used in
the preparation of enzyme antibody conjugates.
3
Air Washing
As a scavenger of CO
2
, TRIS AMINO can be combined
with the enzyme carbonic anhydrase to form a highly-
efficient agent for the removal of CO
2
from an
atmosphere. The rapid release of the CO
2
from the TRIS
AMINO is achieved under mild vacuum or heating to
regenerate the scavenging agent. This absorption system
is highly effective for reconditioning air in enclosed areas
such as submarines.
Fish Transport
TRIS AMINO acts to regulate the pH of the water, and
scavenge CO
2
resulting from respiration, in the transport
of live fish. It has been demonstrated that the use of TRIS
AMINO in the water will extend the survival rate of fish in
closed, non-aerated containers by as much as threefold.
TRIS AMINO will achieve the desired results in both
seawater and fresh water without precipitating dissolved
calcium.
Acidimetric Standard
TRIS AMINO can be used directly for the standardization
of strong acid solutions. It gives sharp inflections at the
equivalence point of potentiometric titrations of
hydrochloric acid in water and perchloric acid in acetic
acid. Standardizations may also be accomplished using a
suitable indicator such as p-sulfo-o-methoxylbenzene-
azodimethyl-1-naphthylamine. TRIS AMINO buffers also
find use as solutions for the standardization of electrodes,
whether pH-sensitive or ion-specific.
Protein Electrophoresis
TRIS AMINO salts find use in gel electrophoresis for
protein separations. As an example, for electrophoresis in
non-dissociating buffer systems, TRIS AMINO salts are
the 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).
TRIS AMINO is a
Registered Trademark of
ANGUS Chemical Company.
HFP/JHV Form No. 319-00042
1999 by ANGUS Chemical Company.
All Rights Reserved. Printed in U.S.A.
The information and data contained herein are believed to be correct. However, we do not warrant
either expressly or by implication the accuracy thereof. In presenting uses for this product, no
attempt has been made to investigate or discuss any patent situations which may be involved.
A subsidiary of The Dow Chemical Company