Biological

Buffer Substances
– in high purity

Advancing Your Life Sciences –

From Discovery to LaunchTM
 Contents

Introduction 3

Biological buffers substances 4 - 15

Ordering information 16 -19

Helpful hints 20

pK α-values of selected 21
biological buffers,
Buffering ranges

Literature 22

2
Introduction
The idea behind this brochure is to
give you an overview of the wide
range of buffers available from Merck
KGaA that are indispensible when
working with biological molecules.
In addition, we have tried to compile
what will hopefully be useful in-
formation on the individual substan-
ces so that you can select the most
suitable for your own applications.
The tabular information included as
appendices should make the brochure
a valuable work of reference for your
future activities.
Work involving biological molecules
invariably involves the use of buffer
solutions. The task of the buffer is
to create a defined environment
for the biomolecule so that it can be
kept in a stable and native form
and its activity is not influenced. The
buffer itself, however, should not
exert any influence on the biological
compound.
Equally important, the buffer should
not contain any impurities that may
have a negative effect on the activity
or stability of the biomolecule. Hence,
only highly purified substances of
defined specification should be used
which guarantee reproducible pro-
cesses on a lab or production scale.
Your Benefits:
• Setting standards for high product quality
• Broad range of product specifications and packages
• Complete documentation for regulatory issues
Merck supplies buffers in various
grades. The differences between them
rarely involve purity itself, but more
the additional parameters that are
specified and tested prior to delivery.
For example, the content of nucleases
may be an important quality criterion
for biotechnological applications
and fulfilling such an additional re-
quirement gives security for the user
working in production of pharma-
ceutical components.
However, for certain applications, it
may be desirable to include additional
quality parameters not included
in our Certificate of Analysis in the
testing programme. Normally, it
is possible for this to be done so that
a customised product results. Should
you require such products, please
contact us.
The same applies to individual
package sizes. You may wish to have
a pack containing e.g. precisely 11.4
kg of glycine for frequent routine
use. We could supply this as a special
pack. Alternatively, you may require
a special mixture of buffers; also
in such cases we can oblige. We can
produce mixtures, ready-to-use
solutions and concentrates according
to your wishes or using your
procedures - all of course in strict
confidentiality.
3
 Biological buffer substances
Zwitterionic buffers according to Good and co-workers [3]
have proven suitable for work involving biological molecules.
They have several advantages compared with conventional
buffers such as phosphate-, tris-, borate- and carbonate buffer
solutions. "Good" buffers approach the "ideal" buffer state:
pKα values are within the physiologically interesting range of
pH 6.0 and pH 9.0; and the pKα value is mostly independent
of temperature and concentration.

All the compounds are chemically stable, non-toxic and do not

interfere with biochemical reactions. They are eminently suitable
for cell cultivation as they are not absorbed through the cell
membranes. Their inherent absorption within the wavelength
range 240 - 700 nm is also minimal.

ACES Merck Cat. No. 1.15226

CAS-No. 7365-82-4
N-(2-Acetamido)-2-aminoethanesulfonic acid
EINECS-No. 230-908-4
Summation formula C4H10N2O4S
Molar mass 182.20 g/mol
Solubility soluble in water; insoluble in toluene and ether
pKα 6.90 (20 °C)
dpKα /dT –0.02/°C
Application notes ACES belongs to the zwitterionic "Good" buffers [3–5]. Its buffering range
covers the physiological pH range. It has a low UV absorbance at 260
and 280 nm. ACES is useful as elution buffer in the chromatographic
purification of proteins. It binds Cu2+, but not Mg2+, Ca2+, or Mn2+.

Buffering range pH 6.1–7.6

Working range 20–100 mM

Specification:

Assay (alkalimetric) ≥ 99 %
Identity (IR-spectrum) passes test
UV absorption (250 nm, 2 % in water, 1cm) ≤ 0.05
Chloride (Cl) ≤ 0.05 %
Appearance white, crystalline powder
Test solution Dissolve 2 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.

4
ADA Merck Cat. No. 1.15227
CAS-No. 26239-55-4
N-(2-Acetamido)iminodiacetic acid
EINECS-No. 247-530-0
Summation formula C6H10N2O5 [(Carbamoylmethyl)imino] diacetic acid
Molar mass 190.16 g/mol
Solubility soluble in water; insoluble in toluene and ether
pK α 6.60 (20 °C)
dpKα /dT –0.011/°C
Application notes ADA belongs to the zwitterionic "Good" buffers [3]. It shows minimal
interferences in biological applications, but it has some ability to bind
metal ions.
Buffering range pH 5.9–7.3
Working range 20 –100 mM

Specification:

Assay (acidimetric) ≥ 99 %
Identity (IR-spectrum) passes test
UV absorption (250 nm, 5 g/l, 1 cm) ≤ 0.25
Chloride (Cl) ≤ 0.02 %
Sulfate (SO4 ) ≤ 0.005 %
Sodium (Na) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105 °C) ≤ 0.5 %
Appearance white, crystalline powder
Test solution Dissolve 0.5 g of substance, exactly
weighed, in 100 ml redistilled water.
The solution is clear and colourless.

BES Merck Cat. No. 1.15228

CAS-No. 10191-18-1
N,N-Bis(2-hydroxyethyl)-2-aminoethane-
EINECS-No. 233- 465-5
sulfonic acid
Summation formula C6H15NO5S
Molar mass 213.25 g/mol
Solubility soluble in water; insoluble in toluene and ether
pKα 7.15 (20 °C)
dpKα /dT –0.016/°C
Application notes BES shows low absorption at 260 and 280 nm. It binds only Cu2+
to some extent, not Ca2+, Mg2+ or Mn2+.
Buffering range pH 6.4–7.8
Working range 20 –100 mM

Specification:

Assay (alkalimetric) ≥ 99 %
Identity (IR-spectrum) passes test
UV absorption (250 nm, 5 % in water, 1cm) ≤ 0.05
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4 ) ≤ 0.005 %
Sodium (Na) ≤ 0.01 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105°C) ≤ 0.5 %
Appearance white, crystalline powder
Test solution Dissolve 2.5 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.
5
 BICINE Merck Cat. No. 1. 01910
CAS-No. 150-25-4
N,N-Bis(2-hydroxyethyl)glycine
EINECS-No. 205-755-1
Summation formula C6H13NO4 N,N-bis(hydroxyethyl)aminoacetic acid
Molar mass 163.17 g/mol
Solubility soluble in water; hardly soluble in ethanol, ether and chloroform
pKα 8.35 (20°C)
pKα /dT –0.018 /°C
Application notes BICINE is a zwitterionic buffer substance belonging to the "Good"
buffers. It is often used for biochemical applications at low temperatures
[3,8,9]. BICINE acts as a chelating agent for divalent metal ions
(Cu2+ > Ca2+ >> Mg2+). Interferences with the Lowry protein test have
been reported.

Buffering range pH 7.6–9.0

Working range 20–200 mM

Specification:

Assay (by perchloric acid titration) ≥ 99 %

Identity (IR-spectrum) passes test
UV absorption (250 nm, 5 % in water, 1cm) ≤ 0.05
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.005 %
Sodium (Na) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105°C) ≤ 0.2 %
Appearance white, crystalline powder
Test solution Dissolve 2.5 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.

BIS-TRIS Merck Cat. No. 1. 03252

CAS-No. 6976-37-0 2,2-Bis-(hydroxyethyl)-
EINECS-No. 230-237-7 iminotris(hydroxymethyl)methane

Summation formula C8H19NO5 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-

Molar mass 209.24 g/mol 1,3-propanediol
Solubility soluble in water 2,2-Bis(hydroxymethyl)-2,2',2''-nitrilotriethanol
pKα 6.46 (25°C)
dpKα /dT –0.02 /°C
Application notes Like Tris buffers, Bis-Tris shows a significant temperature dependency.
It is used as cathodic buffer in IEF and 2-D electrophoresis [10–12].
Buffering range pH 5.8–7.3
Working range 20–200 mM

Specification:

Assay (by perchloric acid titration) ≥ 99 %

Identity (IR-spectrum) passes test
UV absorption (280 nm, 10 % in water, 1cm) ≤ 0.1
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.01 %
page 7

6
Iron (Fe) ≤ 0.001 %
Lead (Pb) ≤ 0.001 %
pH value (2 % in water) 9.4–9.8
Sulfated ash ≤ 0.05 %
Loss on drying (60°C, vacuum) ≤ 1.0 %
Appearance almost white, crystalline powder
Test solution Dissolve 10 g of substance, exactly
weighed, in 100 ml carbon dioxide-free
water. The solution is almost clear and
colourless.

CHES Merck Cat. No. 1.15229

CAS-No. 103-47-9 2-(Cyclohexylamino)ethane-sulfonic acid
EINECS-No. 203-115-6
Summation formula C8H17NO3S
Molar mass 207.29 g/mol
Solubility soluble in water; insoluble in toluene and ether
pKα 9.55 (20°C)
dpKα /dT –0.018 /°C
Application notes CHES shows low UV absorption at 260–280 nm.
Buffering range pH 8.6–10.0
Working range 10–100 mM

Specification:

Assay (alkalimetric; on dried substance) ≥ 99 %

Identity (IR-spectrum) passes test
UV absorption (250 nm, 5 % in water, 1cm) ≤ 0.05
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.025 %
Sodium (Na) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105°C) ≤ 1.0 %
Appearance white, crystalline powder
Test solution Dissolve 2.5 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.

7
 Glycylglycine Merck Cat. No. 1. 04233
CAS-No. 556-50-3
N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid
EINECS-No. 209-127-8
Summation formula C4H8N2O3
Molar mass 132.12 g/mol
Solubility soluble in water, slightly soluble in ethanol, insoluble in ether
pK α1 3.14 (25 °C)
pK α2 8.4 (20 °C)
dpK α /dT –0.028
Application notes This buffer substance shows a temperature dependence similar to Tris.
Buffering range pH 7.5–8.9
Working range 20–200 mM

Specification: Merck Cat. No. 1.04233

Assay ≥ 99 %
(by perchloric acid titration; on anhydrous substance) *
Identity (IR-spectrum) passes test
UV absorption (250 nm, 2 % in water, 1cm) ≤ 0.05
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.005 %
Sodium (Na) ≤ 0.01 %
Glycine ≤ 0.15 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105 °C) ≤ 1.0 %
Appearance white, crystalline powder
Test solution Dissolve 2.5 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.

HEPES Merck Cat. No.: 1.10110 (buffer substance), 1.17839 (for biotechnology)
CAS-No. 7365-45-9
2-[4-(2-Hydroxyethyl)-1-piperazine]
EINECS-No. 230-907-9 ethanesulfonic acid
Summation formula C8H18N2O4S
Molar mass 238.30 g/moll
Solubility soluble in water; insoluble in toluene and ether
pKα 7.55 (20 °C)
dpKα /dT –0.014/°C
Application notes HEPES buffer belongs to the zwitterionic biological buffers described by Good
et al.[3,4]. HEPES buffer systems are general purpose buffers that can be used
for many applications [6]. They can be recommended for freezing of protein
solutions. The increase in acidity induced by freezing, that can be observed
with many buffers, is negligible with HEPES. It prevents enzymes and
coenzymes (e.g. NADPH) from denaturation and inactivation during freeze-
drying and subsequent storage. HEPES is widely used in cell culture media,
but it was reported, that it stimulates the growth of certain cell lines [5]. In
comparison to Tris buffers the temperature dependence of HEPES is much
less. This buffer does not form complexes with divalent metal ions, sometimes
acting as cofactors or activators for certain enzymes e.g. Mg2+ for Benzonase®.
HEPES shows low absorbance in the UV range, but it does interfere with the
Lowry protein assay giving false positive results [7]. HEPES buffers can be
prepared by mixing equimolar solutions of HEPES and HEPES-Na.
Buffering range pH 6.5–8.5
Working range 20–100 mM page 9

8
Specification: Merck Cat. No. 1. 10110 (buffer substance) Merck Cat. No. 1. 17839 (for biotechnology)

Assay (alkalimetric) ≥ 99 % ≥ 99 %
Identity (IR-Spectrum) passes test passes test
Appearance of solution (5 %; water) Clear and colourless
UV-Absorption:
260 nm; 1mol/l; 1 cm; water ≤ 0.1 ≤ 0.1
280 nm; 1mol/l; 1 cm; water ≤ 0.08 ≤ 0.08
330 nm; 50 %; 4 cm; water ≤ 0.4 -
440 nm; 50 %; 4 cm; water ≤ 0.2 ≤ 0.2
Chloride (Cl) ≤ 0.005 %
Sulphate (SO4) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0001 % ≤ 0.0001 %
Cu (Copper) ≤ 0.0002 %
Fe (Iron) ≤ 0.0002 %
Pb (lead) ≤ 0.0001 %
Sodium (Na) ≤ 0.005 %
Sulphated ash ≤ 0.1 %
Loss on drying (105°C) ≤ 0.2 % ≤ 0.2 %
Zwitterionic buffer, pK α at 20°C 7.55
DNAses (Exo- and Endonucleases): not detectable
RNAses not detectable
Proteases not detectable
Endotoxins ≤ 20 U/g
Suitable for tissue culture work suitable
Appearance white to almost white, crystalline powder
Appearance of test solution Dissolve 2.5 g substance, exactly weighed, Dissolve 2.5 g substance, exactly weighed,
in 50 ml carbon dioxide-free water. in 50 ml carbon dioxide-free water.
The solution is clear and colourless. The solution is clear and colourless.

HEPES-Na Merck Cat. No. 1.15231

CAS-No. 75277-39-3
2-[4-(2-Hydroxyethyl)-1-piperazine]ethane-
EINECS-No. 278-169-7
sulfonic acid sodium salt
Summation formula C8H17N2NaO4S
Molar mass 260.28 g /mol
Solubility soluble in water; insoluble in toluene and ether
pKα 7.55 (20°C)
dpKα /dT –0.014/°C
Application notes see HEPES
Buffering range pH 6.5–8.5
Working range 20–100 mM

Specification:

Assay (perchloric acid tibration; on anhydrous substance) ≥ 99 %

Identity (IR-spectrum) passes test
UV absorption (250 nm, 2 % in water, 1cm) ≤ 0.06 %
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105°C) ≤6%
Appearance white, crystalline powder
Test solution Dissolve 1.0 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.
9
 HEPPS Merck Cat. No. 1.15230
CAS-No. 16052-06-5
3-[4-(2-Hydroxyethyl)-1-piperazinyl]-propane-
EINECS-No. 240-198-8
sulfonic acid
Summation formula C9H20N2O4S
N-2-Hydroxyethylpiperazine-N'-3-propanesulfonic acid
Molar mass 252.33 g/mol
Solubility soluble in water; insoluble in toluene and ether
pK α 8.00 (20 °C)
dpK α /dT – 0.014/°C
Application notes HEPPS is similar to HEPES. HEPPS does not bind divalent cations.
Interferences with the Folin protein assay may occur.
Buffering range pH 7.3–8.7
Working range 20–200 mM

Specification:

Assay (acidimetric) ≥ 99 %
Identity (IR-spectrum) passes test
UV absorption (250 nm, 2 % in water, 1 cm) ≤ 0.05
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.005 %
Sodium (Na) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105°C) ≤ 0.5 %
Appearance white, crystalline powder
Test solution Dissolve 1 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.

MES Merck Cat. No. 1. 061266

CAS-No. 145224-94-8
2-Morpholinoethanesulfonic acid
EINECS-No. 224-632-3
monohydrate
Summation formula C6H13NO4S · H2O
Molar mass 213.25 g/mol
Solubility soluble in water; insoluble in toluene and ether
pKα 6.15 (20°C)
dpKα/dT –0.011/°C
Application notes MES is a zwitterionic buffer described by Good et al. [3]. This buffer is often
used in the preparation of media for the culture of microorganisms, plant
cells and algae [13–15]. MES is a very convenient buffer substance in the
given pH-range, due to its biological inertness. In comparison to other "Good"
buffers, MES has a minimal chelating ability. MES may undergo
partly decomposition when it is autoclaved in presence of glucose.
Buffering range pH 5.6–6.7
Working range 10–100 mM

Specification:

Assay (alkalimetric; on anhydrous substance) ≥ 99 %

Identity (IR-spectrum) passes test
UV absorption (250 nm, 5 % in water, 1 cm) ≤ 0.05
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.005 % page 11

10
Sodium (Na) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Water (acc. to Karl Fischer) 8.0 – 9.0 %
Appearance white, crystalline powder
Test solution Dissolve 2.5 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.

MOPS Merck Cat. No. 1. 06129

CAS-No. 1132-61-2 3-Morpholinopropanesulfonic acid
EINECS-No. 214-478-5
MPS, 3-(N-Morpholino)propane sulfonic acid,
Summation formula C7H15NO4S N-(3-sulfopropyl)morpholine
Molar mass 209.26 g/mol
Solubility soluble in water; insoluble in toluene and ether
pKα 7.20 (20°C)
dpKα/dT –0.013/°C [20]
Application notes MOPS belongs to the zwitterionic "Good's" buffers [3]. It can be used in
many bioanalytical methods (e.g. IEF, protein assays) and the preparation of
fermentation media [18-22]. Its metal binding capacity is negligble.
Buffering range pH 6.5–7.9
Working range 20–100 mM

Specification:

Assay (alkalimetric) ≥ 99 %
Identity (IR-spectrum) passes test
UV absorption (250 nm, 5 % in water, 1 cm) ≤ 0.05
Sodium (Na) ≤ 0.005 %
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105°C) ≤ 0.2 %
Appearance white, crystalline powder
Test solution Dissolve 2,5 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.

11
 PIPES Merck Cat. No. 1.10220
CAS-No. 5625-37-6
Piperazine-1,4-bis(2-ethanesulfonic acid)
EINECS-No. 227-05-76 1,4-Piperazinediethanesulfonic acid
Summation formula C8H18N2O6S2
Molar mass 302.36 g/mol
Solubility soluble in alkalis, slightly soluble in water; insoluble in toluene and ether
pKα 6.80 (20 °C)
dpKα /dT –0.0085/°C
Application notes PIPES is a zwitterionic buffer described by Good et al. [3,4].
It interferes in the Lowry protein assay [7].
Buffering range pH 6.1–7.5
Working range 20–100 mM

Specification:

Assay (acidimetry) ≥ 99 %
Identity (IR-spectrum) passes test
UV absorption ≤ 0.05
250 nm, 2 % in sodium hydroxide solution 1mol/l, 1cm
Chloride (Cl) ≤ 0.2 %
Sulfate (SO4) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Sodium ≤ 0.2 %
Loss on drying (105°C) ≤ 0.5 %
Appearance white to slightly yellowish,
crystalline powder
Test solution Dissolve 0.2 g of substance, exactly
weighed, in 10 ml NaOH 0.1 mol/l.
The solution is clear and colourless.

TES Merck Cat. No. 1.10695

CAS-No. 7365-44-8
EINECS-No. 230-906-3 N-[Tris(hydroxymethyl)-methyl]-2-
aminoethane sulfonic acid
Summation formula C6H15NO6S
Molar mass 229.25 g/mol 3-{[tris-(hydroxymethyl)methyl]amino}
ethanesulfonic acid
Solubility soluble in water; insoluble in toluene and ether
pKα 7.50 (20 °C)
dpKα/dT –0.02/°C
Application notes TES belongs to the zwitterionic buffers acc. to Good et al. [3-5]
Buffering range 6.8–8.2
Working range 20–100 mM

Specification:

Assay (alkalimetric) ≥ 99 %
Identity (IR-spectrum) passes test
UV absorption (250 nm, 5% in water, 1cm) ≤ 0.05
Chloride (Cl) ≤ 0.005 %
Sulfate (SO4) ≤ 0.005 %
Sodium (Na) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105 °C) ≤ 0.2 %
Appearance white, crystalline powder, partly clogged
Test solution Dissolve 2.5 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.
12
TRICINE Merck Cat. No. 1. 08602
CAS-No. 5704-04-1
N-[Tris(hydroxymethyl)-methyl]-glycine
EINECS-No. 227-193-6
Summation formula C6H13NO5 N-[2-(tris(hydroxymethyl)methyl]glycine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)-
Molar mass 179.17 g/mol ethyl]glycine
Solubility soluble in water; insoluble in toluene and ether.
pKα 8.15 (20°C)
dpKα /dT –0.021/°C
Application notes One of the zwitterionic buffer substances known as "Good" buffers with
similar properties as BICINE [3,4]. It can replace TRIS in many systems. It is
mainly used as buffer in capillary, SDS gel electrophoresis and IEF [23–25].
Buffering range 7.2–9.2
Working range 20–200 mM

Specification:

Assay (perchloric acid titration) ≥ 99 %

Identity (IR-spectrum) passes test
UV absorption (250 nm, 5 % in water, 1cm) ≤ 0.075
Chloride (Cl) ≤ 0.05 %
Sulfate (SO4) ≤ 0.005 %
Sodium (Na) ≤ 0.005 %
Heavy metals (as Pb) ≤ 0.0005 %
Loss on drying (105°C) ≤ 0.2 %
Appearance white, crystalline powder,
partly clogged
Test solution Dissolve 2.5 g of substance, exactly
weighed, in 50 ml redistilled water.
The solution is clear and colourless.

TRIS Merck Cat. No.: 1. 08382 (GR f. a., ACS, Reag. PhEur), 1.08386 (PhEur, BP, USP ), 1.08387 (LAB)
CAS-No. 77-86-1
Tris(hydroxymethyl)aminomethane
EINECS-No. 201-06-44
THAM, Trometamol, Tromethamin
Summation formula C4H11No3
Molar mass 121.14 g/mol
Solubility readily soluble in water, very slightly soluble in methanol and ethanol
pK α 8.30 (20°C)
dpK α /dT –0.028/°C
Application notes Tris-HCl (see below) and Tris base are inexpensive, biological standard buffers
showing a buffering capacity within the physiological range [1]. For this and
because of its low UV absorbance, Tris is often used as buffer system for
diagnostic assays. NADH and NADPH show better stability at pH above
7.5 in Tris buffer than in phosphate buffers. Tris does not precipitate calcium
salts and maintains solubility of manganese salts. However, it should be
noted, that Tris buffers, although ubiqitious, have some obvious disadvan-
tages. These buffers show for instance a strong temperature sensitivity (see
Chapter 4) . As a primary amine, Tris is a reactive compound and may
therefore interfere in certain reactions e.g. immobilisation of proteins. It may
also penetrate biological membranes. Please note as well, that Tris buffers
absorb CO2 from air. With the Bradford protein assay [29] interferences occur,
because of the primary amino group of Tris.
Buffering range pH 7.2 - 9.0
Working range 20 - 200 mM page 14

13
 TRIS
Merck Cat. No.: 1. 08382 1. 08386 1. 08387
Specification: (GR f.a., ACS, Reag. PhEur) (PhEur, BP, USP) (LAB)

Assay (acidimetric, calc. on anhydrous substance) 99.8 – 100.1% 99 – 100.5 % 99.8 – 100.1%
Identity (IR-Spectrum) passes test passes test passes test
In water insoluble matter max. 0.003 % ≤ 0.005
Melting point 169 – 172˚C 168 – 172˚C
Appearance of solution (5 %; water) conforms conforms
pH-value (1%; water) - 10.3 – 10.7
pH-value (5 %; water) 10.3 – 10.9 10.0 – 11.5
UV-Absorbance:
260 nm; 10 %; 1cm; water max. 0.02
280 nm; 10 %; 1cm; water max. 0.02
300 nm; 10 %; 1cm; water max. 0.02
405 nm; 10 %; 1cm; water max. 0.004
430 nm; 10 %; 1cm; water max. 0.004
290 nm; 40 %; 1cm; water max. 0.1 ≤ 0.2
Chloride (Cl) max. 0.0005 % ≤ 0.001 % ≤ 0.001 %
Sulphate (SO4) max. 0.0005 % ≤ 0.005 % ≤ 0.005 %
Heavy metals (as Pb) max. 0.0005 % ≤ 0.0010 % ≤ 0.0005 %
As (Arsenic) max. 0.00002 % ≤ 0.00002 %
Ca (Calcium) max. 0.0001 %
Cd (Cadmium) max. 0.00001 %
Cu (Copper) max. 0.00001 %
Fe (Iron) max. 0.00002 % ≤ 0.0010 % ≤ 0.0005 %
K (Potassium) max. 0.00003 %
Mg (Magnesium) max. 0.00003 %
Na (Sodium max. 0.0001 %
Pb (lead) max. 0.00001 %
Zn (Zinc) max. 0.00001 %
Sulphated ash max. 0.01 % ≤ 0.1 % ≤ 0.05 %
Loss on drying (105˚C) max. 0.5 % ≤ 0.5 %
Water max. 0.1 % ≤ 0.5 %
Filter sample (0.45 mm) conforms conforms
Related substances (TLC) conforms conforms
Residual solvents class 3 (PhEur / ICH) ≤ 0.5 %
Other residual solvents (PhEur / ICH) excluded by
production process
Organic volatile impurities (USP/ NF) conforms
Endotoxins ≤ 0.03 I.U./mg
Colony acounts
aerobic bacteria ≤ 100 CFU/g
Yeast and moulds ≤ 100 CFU/g
Salmonella species absent in 10 g
E.coli absent in 10 g
Pseudomonas aeruginosa absent in 10 g
Staphylococcus aureus absent in 10 g
Pharmacopeia Correspond: PhEur, BP, USP
Appearance white to almost white, white to almost white, white to almost white,
crystalline powder crystalline powder crystalline powder
Appearance of test solution Dissolve 5.0 g of Dissolve 2.5 g of Dissolve 5.0 g of
sustance, exactly sustance, exactly substance, exactly
weighed, in carbon weighed, in carbon weighed, in carbon
dioxide-free water to dioxide-free water to dioxide-free water to
make 100 ml. The make 50 ml. The make 100 ml. The
solution is clear and solution is clear and solution is clear and
14 colourless. colourless. colourless.
TRIS HCI Merck Cat. No. 1. 08219
CAS-No. 1185-53-1
Tris(hydroxymethyl)aminomethane hydrochloride
EINECS-No. 214-684-5
THAM-HCl, Trometamol hydrochloride,
Summation formula C4H11NO3 · HCl 2-Amino-2-hydroxymethyl-1,3-propanediol
Molar mass 157.60 g/mol hydrochloride
Solubility readily soluble in water CIH
pK α 8.3 (20°C)
dpK α /dT –0.028/°C
Application notes see TRIS
Buffering range pH 7.2–9.0
Working range 20–200 mM

Specification:

Assay (perchloric acid titration; on anhydrous substance) min. 99 %

Identity (IR-spectrum) passes test
UV absorption (10 % in water, 1cm)
230 nm max. 0.1
260 nm –
280 nm –
300 nm max. 0.02
405 nm max. 0.004
Melting range 145 – 155°C
Sulfated ash < 0.03 %
Water (acc. to Karl-Fischer) < 0.5 %
Cadmium (Cd) max. 0.0001 %
Copper (Cu) max. 0.0001 %
Iron (Fe) max. 0.0003 %
Lead (Pb) max. 0.0002 %
Zinc (Zn) max. 0.0002 %
Appearance off white to slightly yellowish,
crystalline powder
Test solution Dissolve 5 g of the substance, exactly
weighed in 50 ml of redistilled water.
The solution is almost clear and
colourless.

15
A-H
Ordering information
Biological Buffer Substances and other Buffer Components

Product Merck Cat. No. Pack size

A
ACES 1.15226.0025 25 g
N-(2-Acetamido)-2-aminoethanesulfonic acid buffer substance 1.15226.0250 250 g
1.15226.9010 10 kg
Acetic acid 100 % extra pure DAB, BP, USP 1.00056.2500 5l
1.00056.9025 25 l
ADA 1.15227.0025 25 g
N-(2-Acetamido)iminodiacetic acid buffer substance 1.15227.0250 250 g
1.15227.9010 10 kg
ß-Alanine for biochemistry 1.01008.0250 250 g
1.01008.1000 1 kg
1.01008.9025 25 kg
Ammonium acetate extra pure 1.01115.1000 1 kg
1.01115.5000 5 kg
1.01115.9050 50 kg
Ammonium hydrogen carbonate extra pure, Ph Eur, BP 1.01131.5000 5 kg
1.01131.9029 50 kg

B
BES 1.15228.0025 25 g
N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer substance 1.15228.0250 250 g
1.15228.9010 10 kg
BICINE 1.01910.0025 25 g
N,N-Bis(2-hydroxyethyl)glycine buffer substance 1.01910.0250 250 g
1.01910.9010 10 kg
BIS-TRIS 1.03252.0025 25 g
2,2-Bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)-methane 1.03252.0250 250 g
Boric acid cryst. extra pure Ph Eur, BP, NF 1.00160.5000 5 kg
1.00160.9050 50 kg
Boric acid GR for analysis, ACS, ISO 1.00165.0100 100 g
1.00165.0500 500 g
1.00165.1000 1 kg
1.00165.5000 5 kg
1.00165.9050 50 kg

C-F
Calcium Triplex® dihydrate Ph Eur, BP, USP 1.08439.0500 500 g
1.08439.2500 2.5 kg
1.08439.9029 25 kg
CHES 1.15229.0025 25 g
2-(Cyclohexylamino)ethanesulfonic acid buffer substance 1.15229.0250 250 g
Citric acid monohydrate cryst. extra pure Ph Eur, BP, USP 1.00242.5000 5 kg
1.00242.9025 /9029 25 kg
Citric acid monohydrate GR for analysis, ACS, ISO 1.00244.0500 500 g
1.00244.1000 1 kg
1.00244.5000 5 kg

G
Glycine cryst. Ph Eur, BP, USP 5.00190.1000 1 kg
5.00190.5000 5 kg
5.00190.9025 25 kg
Glycine GR for analysis 1.04201.0100 100 g
1.04201.0250 250 g
1.04201.1000 1 kg
1.04201.5000 5 kg
1.04201.9025 25 kg
Glycylglycine buffer substance 1.04233.0010 10 g
1.04233.0100 100 g
1.04233.1000 1 kg
1.04233.9010 10 kg

H
HEPES 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid 1.10110.0025 25 g
buffer substance 1.10110.0250 250 g
1.10110.1000 1 kg
1.10110.9025 25 kg
HEPES 1.17839.0100 100 g
2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acidfor biotechnology 1.17839.1000 1 kg
1.17839.9010 10 kg
16
These products are not intended for use as in-vitro-diagnostics in terms of European Directive 98/79EC.
They are for research purposes only, for investigating in-vitro samples without any medical objective.
Product Merck Cat. No. Pack size
I-S
HEPES Sodium 1.15231.0025 25 g
2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid sodium 1.15231.0250 250 g
salt buffer substance 1.15231.2500 2.5 kg
1.15231.9010 10 kg
HEPPS 1.15230.0025 25 g
3-[4-(2-Hydroxyethyl)-1-piperazinyl]-propanesulfonic acid buffer substance 1.15230.0250 250 g
1.15230.2500 2.5 kg
1.15230.9010 10 kg
L-Histidine monohydrochloride monohydrate extra pure Ph Eur, BP 1.04354.0500 500 g
1.04354.5000 5 kg
L-Histidine monohydrochloride monohydrate for biochemistry 1.04350.0025 25 g
1.04350.0100 100 g
1.04350.0500 500 g
1.04350.9010 10 kg
Hydrochloric acid 1 mol/l, 1N solution 1.09057.1000 1l
1.09057.2500 2.5 l
1.09057.5000 5l
1.09057.9010 10 l
Hydrochloric acid fuming 37 % extra pure, Ph Eur, BP, JP, NF 1.00314.1000 1l
1.00314.2500 2.5 l
1.00314.9025 25 l

I-L
Imidazol buffer substance 1.04716.0050 50 g
1.04716.0250 250 g
1.04716.1000 1 kg

M-N
MES 1.06126.0025 25 g
2-Morpholinoethanesulfonic acid monohydrate buffer substance 1.06126.0250 250 g
1.06126.1000 1 kg
1.06126.9010 10 kg
MOPS 1.06129.0025 25 g
2-Morpholinopropane sulfonic acid buffer substance 1.06129.0250 250 g
1.06129.5000 5 kg
1.06129.9010 10 kg

O
ortho-Phosphoric acid 85 % extra pure, Ph Eur, BP, NF 1.00563.1000 1l
1.00563.2500 2.5 l
1.00563.9025 25 l

P
PIPES 1.10220.0025 25 g
Piperazine-1,4-bis(ethanesulfonic acid) buffer substance 1.10220.0250 250 g
1.10220.1000 1 kg
1.10220.9010 10 kg
Potassium acetate extra pure Ph Eur, BP 1.04820.1000 1 kg
1.04820.5000 5 kg
1.04820.9050 50 kg
Potassium dihydrogen phosphate cryst. extra pure, Ph Eur, BP, NF 1.04871.5000 5 kg
1.04871.9050 50 kg
Potassium dihydrogen phosphate GR for analysis, ISO 1.04873.0250 250 g
1.04873.1000 1 kg
1.04873.5000 5 kg
di-Potassium hydrogen phosphate anhydrous, extra pure, Ph Eur, BP 1.05101.1000 1 kg
1.05101.5000 5 kg
1.05101.9029 25 kg
di-Potassium hydrogen phosphate trihydrate, GR for analysis 1.05099.0250 250 g
1.05099.1000 1 kg
1.05099.5000 5 kg
Potassium hydroxide pellets, extra pure, Ph Eur, BP, JP, NF 1.05032.1000 1 kg
1.05032.5000 5 kg
1.05032.9025 25 kg
Potassium hydroxide pellets, GR for analysis 1.05033.0500 500 g
1.05033.1000 1 kg
1.05033.5000 5 kg

S
Sodium acetate trihydrate extra pure, Ph Eur, BP, JP, USP 1.06265.5000 5 kg
1.06265.9029 25 kg
Sodium carbonate anhydrous, extra pure, Ph Eur, BP, NF 1.06398.5000 5 kg
1.06398.9029 25 kg
Sodium carbonate GR for analysis, ISO 1.06392.0500 500 g
1.06392.1000 1 kg
1.06392.5000 5 kg
17
These products are not intended for use as in-vitro-diagnostics in terms of European Directive 98/79EC.
They are for research purposes only, for investigating in-vitro samples without any medical objective.
 Product Merck Cat. No. Pack size
T-Z
tri-Sodium citrate dihydrate cryst, extra pure Ph Eur, BP, USP 1.06432.5000 5 kg
1.06432.9025 /9029 25 kg
tri-Sodium citrate dihydrate GR for analysis, ACS, ISO 1.06448.0500 500 g
1.06448.1000 1 kg
1.06448.5000 5 kg
Sodium dihydrogen phosphate dihydrate extra pure Ph Eur, USP 1.06435.1000 1 kg
1.06435.5000 5 kg
1.06435.9025 25 kg
Sodium hydrogen carbonate extra pure, Ph Eur, BP, USP 1.06323.2500 2.5 kg
1.06323.9029 25 kg
Sodium hydrogen carbonate GR for analysis, ACS, ISO 1.06329.0500 500 g
1.06329.1000 1 kg
1.06329.5000 5 kg
di-Sodium hydrogen phosphate anhydrous extra pure Ph Eur, BP, USP 1.06585.5000 5 kg
1.06585.9029 25 kg
di-Sodium hydrogen phosphate anhydrous GR for analysis, ACS 1.06586.0500 500 g
1.06586.2500 2.5 kg
di-Sodium hydrogen phosphate dihydrate extra pure Ph Eur, BP, USP 1.06576.5000 5 kg
1.06576.9025 /9029 25 kg
di-Sodium hydrogen phosphate dihydrate GR for analysis 1.06580.0500 500 g
1.06580.1000 1 kg
1.06580.5000 5 kg
di-Sodium hydrogen phosphate dodecahydrat cryst. extra pure Ph Eur, BP, USP 1.06573.5000 5 kg
1.06573.9025 /9029 25 kg
di-Sodium hydrogen phosphate dodecahydrat GR for analysis, ISO 1.06579.0500 500 g
1.06579.1000 1 kg
1.06579.5000 5 kg
Sodium hydroxide pellets extra pure, Ph Eur, BP, JP, NF 1.06482.1000 1 kg
1.06482.5000 5 kg
1.06482.9025 25 kg
Sodium hydroxide solution 4.0 mol/l, 4 N solution 1.11584.5000 5l
di-Sodium tetraborate decahydrate extra pure, Ph Eur, JP, NF 1.06303.1000 1 kg
1.06303.9025 25 kg

T-Z
TES 1.10695.0025 25 g
N-[Tris(hydroxymethyl)-methyl]-2-aminoethanesulfonic acid buffer substance 1.10695.0250 250 g
1.10695.1000 1 kg
1.10965.9010 10 kg
Titriplex® III Ph Eur, BP, JP, USP 1.08421.1000 1 kg
Ethylenedinitrilotetraacetic acid disodium salt dihydrate 1.08421.9025 25 kg
TRICINE 1.08602.0025 25 g
N-[Tris-(hydroxymethyl)methyl]glycine buffer substance 1.08602.0250 250 g
1.08602.1000 1 kg
1.08602.9010 10 kg
Triethanolamine GR for analysis 1.08379.0250 250 ml
1.08379.1000 1l
Triethanolamine Ph Eur 1.08372.5000 5l
1.08372.9025 25 l
Triethanolamine hydrochloride GR for analysis 1.08357.0100 100 g
1.08357.1000 1 kg
TRIS Tris(hydroxymethyl)aminomethane GR for analysis, ACS, Reag. Ph Eur 1.08382.0100 100 g
1.08382.0500 500 g
1.08382.1000 1 kg
1.08382.2500 2.5 kg
1.08382.5000 5 kg
1.08382.9025 25 kg
TRIS Tris(hydroxymethyl)aminomethane Ph Eur, BP, USP 1.08386.1000 1 kg
1.08386.5000 5 kg
1.08386.9025 25 kg
TRIS Tris(hydroxymethyl)aminomethane LAB 1.08387.0500 500 g
1.08387.2500 2.5 kg
1.08387.9025 25 kg
TRIS HCl Tris(hydroxymethyl)aminomethane hydrochloride GR for analysis 1.08219.0100 100 g
1.08219.1000 1 kg
1.08219.9010 10 kg
1.08219.9025 25 kg

These products are not intended for use as in-vitro-diagnostics in terms of European Directive 98/79EC.
They are for research purposes only, for investigating in-vitro samples without any medical objective.

18
Supplements A-Z
Product Merck Cat. No. Pack size
A-B
Ammonium chloride extra pure Ph Eur, BP, USP 1.00924.5000 5 kg
1.00924.9029 25 kg
Ammonium chloride GR for analysis, ACS, ISO 1.01145.0500 500 g
1.01145.1000 1 kg
1.01145.5000 5 kg
Ammonium sulfate for biochemistry 1.01211.0250 250 g
1.01211.1000 1 kg
1.01211.5000 5 kg
1.01211.9050 50 kg

C-D
Calcium chloride dihydrate GR for analysis, ACS 1.02382.0250 250 g
1.02382.0500 500 g
1.02382.1000 1 kg
1.02382.5000 5 kg
Ethylenedinitrilotetra acetic acid disodium salt dihydrate 1.08421.1000 1 kg
(Titriplex® III) Ph Eur, BP, USP 1.08421.9025 25 kg
Ethylenedinitrilotetra acetic acid disodium salt dihydrate 1.08418.0100 100 g
(Titriplex® III) GR for analysis 1.08418.0250 250 g
1.08418.1000 1 kg
1.08418.5000 5 kg
1.08418.9010 10 kg

G-L
Glycerol about 87 % extra pure, Ph Eur, BP 1.04091.1000 1l
1.04091.2500 2,5 l
1.04091.9025 25 l
Glycerol anhydrous pure, Ph Eur, BP, JP, USP 1.04093.1000 1l
1.04093.2500 2.5 l
1.04093.9025 25 l

M-O
Magnesium chloride hexahydrate extra pure, Ph Eur, BP, USP 1.05832.5000 5 kg
1.05832.9029 25 kg
Magnesium chloride hexahydrate GR for analysis, ACS, ISO 1.05833.0250 250 g
1.05833.1000 1 kg
1.05833.5000 5 kg

P-R
Potassium chloride extra pure, Ph Eur, BP, USP 1.04935.5000 5 kg
1.04935.9029 25 kg
Potassium chloride GR for analysis 1.04936.0250 250 g
1.04936.0500 500 g
1.04936.1000 1 kg
1.04936.5000 5 kg

S-Z
Sodium azide extra pure 1.06688.0100 100 g
1.06688.0250 250 g
1.06688.1000 1 kg
1.06688.9010 10 kg
Sodium chloride extra pure, low in endotoxins, Ph Eur, BP, JP, USP 1.16224.5000 5 kg
1.16224.9029 25 kg
Sodium chloride extra pure, Ph Eur, BP, JP, USP 1.06400.5000 5 kg
1.06400.9029 25 kg
Sodium chloride GR for analysis, ACS, ISO 1.06404.0500 500 g
1.06404.1000 1 kg
1.06404.5000 5 kg
Sodium sulfate anhydrous extra pure, Ph Eur, BP, USP 1.06643.2500 2.5 kg
1.06643.9025 25 kg
Sodium sulfate decahydrate extra pure, Ph Eur, BP, USP 1.06642.2500 2.5 kg
1.06642.9025 25 kg

These products are not intended for use as in-vitro-diagnostics in terms of European Directive 98/79EC.
They are for research purposes only, for investigating in-vitro samples without any medical objective.

For more information, products and qualities please ask for the Merck catalogue for Chemicals & Reagents
or the Merck ChemDAT ®. We also welcome you on our homepage http: // LSA .merck.de

19
 Helpful hints
99 % water! Buffers consist mostly of water, a
fact that is largely ignored. If the quality of the
water used is inferior, the purest of buffer sub-
stances won't help. Very often, badly serviced
water-processing plants are responsible for many
of the “unexplainable phenomena” frequently
encountered in the lab. Have you ever wondered
e.g. why so many experiments seem to go wrong
on a Monday? Perhaps the water is responsible;
it may have been standing in the pipes over the
weekend and you have then used it to make up
your "fresh" buffer solutions. Even if it means a
lot more work, you should always use redistilled
water and even go to the extent of ultrafiltering
it before use.
Always try to prepare buffers at the temperature
and concentration that you plan to use during
your experiment. When working with stock
solutions, always adjust the pH after dilution and
temperature adjustment.
Should you have to keep larger quantities of
buffer solutions as stock solutions, make sure
that there is no turbidity visible before using.
Turbidity usually occurs as a result of microbial
contamination or precipitated salts. Do not use
such solutions, even in cases of doubt. Keep
all solutions in a cool place and tightly closed.
And use bactericides such as sodium azide or
thiomerosal only if you are certain that they
will not have any effect on your proposed bio-
logical system.
Phosphate buffers
Although phosphate is far from an ideal buffer
substance, it is still widely used. The problem
with phosphate buffers is that it acts as an active
compound i.e. as metabolite, activator or in-
hibitor in many biological reactions. Phosphate
can bind or precipitate polyvalent cations. Below
pH 7.5 their buffering capacity is fairly poor
although ranges from pH 5.8 to 8.0 are listed in
various tables in the literature [26].
20
Solid buffer substances are stable for at least five
years when stored in tightly closed containers
at ambient temperatures, dry and protected from
light.
When selecting an appropriate buffer substan-
ce, always choose one with a pKα close to the
desired working pH. If you expect the pH to drop
during the experiment, choose a buffer with a
pKα slightly lower than the working pH. If the pH
is expected to increase, use a buffer with a
slightly higher pKα.
Sometimes it is desireable to prepare buffers
without mineral cations e.g. in order to check
the influence of sodium or potassium ions
on a given biological system. In this case, an
aqueous solution of the quaternary organic
amine tetramethylammonium hydroxide can be
used to adjust the pH instead of NaOH or KOH.
Buffers prepared with this base can be supple-
mented by inorganic salts if you wish to evaluate
the impact of the cations.
In certain cases volatile buffers may be desired
in order to remove a buffer quickly and com-
pletely. A compilation of such volatile buffers
can be found e.g. in [27]. For other studies,
buffers with a very broad buffering range should
be used. This can be achieved by mixing buffer
substances e.g. “Good” buffers [28].
pKα-values of Buffer pKα (4 °C) pKα (20 °C) pKα (25 °C) pKα (37 °C) ∆pKα/°C
selected ACES 7.22 6.90 6.80 6.56 –0.020
biological buffers ADA
BES
6.80
7.41
6.62
7.15
6.56
7.07
6.43
6.88
–0.011
–0.016
BICINE 8.64 8.35 8.26 8.04 –0.018
BIS-TRIS 6.88 6.56 6.46 6.22 –0.020
CHES 9.73 9.55 9.50 9.36 –0.011
Citrate pKα2 4.79 4.77 4.76 4.74 –0.0016
Glycine pKα2 10.32 9.91 9.78 9.47 –0.026
Gly-Gly 8.85 8.40 8.26 7.92 –0.028
HEPES 7.77 7.55 7.48 7.3 –0.014
HEPPS 8.18 8.00 7.95 7.8 –0.011
Imidazole 7.37 7.05 6.95 6.71 –0.020
MES 6.33 6.15 6.10 5.97 –0.011
MOPS 7.41 7.20 7.14 6.98 –0.013
PIPES 6.94 6.80 6.76 6.66 –0.0085
Phosphate pKα2 7.26 7.21 7.20 7.17 –0.0028
TAPS 8.02 8.31 8.40 8.62 +0.018
TES 7.82 7.50 7.40 7.16 –0.020
TRICINE 8.49 8.15 8.05 7.79 –0.021
TRIS 8.75 8.30 8.08 7.82 –0.028

Buffering ranges Buffer

Glycine/HCl
Citric acid/Na-citrate
Acetic acid/Na-acetate
KH2PO4/Na2HPO4
MES
BIS-TRIS
ADA
ACES
PIPES
Imidazole/HCL
BES
MOPS
HEPES
TES
TRIS/HCl
HEPPS
TRICINE
GLY-GLY
BICINE
Na-borate/HCl
Glycine/NaOH
CHES
AMP/HCL
Na2CO3/NaHCO3
Na-borate/NaOH
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

21
 Literature
1. R.J. Beyon & J.S. Easterby: Buffer solutions The Basics, (C. Howe, ed.) 1 st ed. (1996), IRL Press, Oxford
2. I.H. Segel: Biochemical Calculations, 2nd ed. (1976), J. Wiley & Sons, New York
3. Good, N.E. et al: Biochemistry 5, 467 (1966)
4. Good, N.E. & Izawana, S.: Methods Enzymol., Part B, Vol. 24, p. 53 ff. (Pietro, ed.) (1972) Academic Press, New York
5. Ferguson; W.J. et al: Anal. Biochem. 104, 300 (1980)
6. Eagle, H.: Science 174, 500 (1971)
7. Himmel, H.M. & Heller, W.: J. Clin. Chem. Clin. Biochem. 25, 909 (1987)
8. Williams-Smith, D.L. et al: Biochem. J. 167, 593 (1977)
9. Soni M.L & R. & Kapoor, R.C.: Int. J. Quant. Chem. 20, 385 (1981)
10. Nealon, D.A., et al: Clin. Chem. 26 (10), 1516 (1980)
11. Schroeder, W.A. et al: J. Chromatogr. 118 (3), 925 (1976)
12. Wiltfang, J., et al: Electrophoresis 12 (5), 352 (1991)
13. Wehr, J.D., et al: J. Phycol. 22, 88 (1986)
14. Schwenke, J: Plant and Soil 137, 31 (1991)
15. Howiesson, J.G.: Plant and Soil 88, 367 (1985)
16. Hausfeld, A.D.: Anal. Biochem. 212, 237 (1993)
17. Sankar, M. & Bates, R.G.: Anal. Chem. 50, 1922 (1978)
18. Paques, E.P. et al: Eur. J. Biochem. 107, 447 (1980)
19. Sonna, A. et al: J. Biol. Chem. 263, 6625 (1988)
20. Aharonowitz, Y. & Demain, A.L.: Arch. Mircobiol. 115, 169 (1977)
21. Shawar, R.V. et al: J. Clin. Microbiol. 30, 1976 (1992)
22. MacKerrow, S.D. et al: J. Clin. Microbiol. 25, 85 (1987)
23. Gardner, R.S.: J. Cell Biol. 42, 320 (1969)
24. Spendlove, R.S. et al: Proc. Soc. Exp. Med. 137, 258 (1971)
25. Schaegger, H. & Von Jagow, G.: Anal. Biochem. 166, 368 (1987)
26. Sørensen, S.P.L.: Biochem Z. 21, 131 (1909) and Biochem. Z. 22, 352 (1909)
27. Perrin, D.D. & Dempsey, B.: Buffers for pH and Metal Ion Control, Chapman and Hall, London (1974)
28. Ellis, K.J. & Morrison, J.F.: Methods Enzymol. 87, 405 (1982)
29. Bradford, M.M.: Anal. Biochem. 22, 248 (1976)

Further reading
Gomori, G.: Methods Enzymol. Vol. 1, 138 (1955); Data on the preparation of phosphate amd acetate buffer
McIlvaine: J. Biol. Chem. 49, 183 (1921); Data on citrate-phosphate universal buffer
Stoll, V. & Blanchard J.S.: Methods Enzymol. 182, 24 (1990); Overview of common buffer systems, recipes for
preparation and detailed description of their properties

22
Abbreviations
BP: British Pharmacopoeia
CAS No.: Chemical Abstracts Service
Registry Number
DAB: German Pharmacopoeia
EINECS: European Inventory of
Existing Chemical Substances
Ph Eur: European Pharmacopoeia
Ph Franc: French Pharmacopoeia
Ph Helv: Swiss Pharmacopoeia
JP: The Pharmacopoeia of Japan
OEAB: Austrian Pharmacopoeia
USP: The United States Pharmacopoeia

23
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