BioUltra Reagents

Biological Buffers

To view a complete list of buffers, please visit the Buffer Explorer.

To meet highest demands for quality, we offer a selection of BioUltra Biological Buffers.

 

 Introduction

A buffer, as defined by Van Slyke [1], is "a substance which by its presence in solution increases the amount of acid or alkali that must be added to cause unit change in pH". Buffers are thus very important components in experiments designed to study biological reactions by maintaining a constant concentration of hydrogen ions within the physiological range. The pH of mammalian blood is maintained close to 7.38 by buffer systems such as

H2PO4- <=> HPO42-, CO2 <=> H2CO3,

H2CO3 <=> HCO3-,

In living plants, the normal range of pH in tissues is about 4.0-6.2. It is not as narrowly defined as in mammalian tissues.

Universally applicable buffers for biochemistry must display:

  • water solubility
  • no interference with biological processes
  • known complex-forming tendency with metal ions
  • non-toxicity
  • no interference with biological membranes (penetration, solubilisation, adsorption on surface etc.)
  • very low U.V. absorption at wavelength >260 nm

"BioUltra" Zwitterlonic (Good's) Buffers

The use of buffers based on inorganic or organic salts is limited because of the interference of buffer cations and anions with the biological reaction under study. The development and introduction of the Zwitterionic Biological Buffers by Good [2] did much to change this situation. This type of buffer displays the desired characteristics: Low interference with biological processes is due to the fact that anionic and cationic sites are present as non-interacting carboxylate or sulfonate and cationic ammonium groups. The pK and the buffer range of the zwitterionic substances lie within the physiological limits (pKa 6.15-9.55). Moreover the zwitterionic nature of these buffers makes them very water soluble, normally above the one-molar range. Physical constants of the buffer substance (pKa, D pKa/°C, solubility, pH- and UV-range) are included under the product entry in the alphabetical list.

"BioUltra" Buffer Salts and other Buffer Components

Buffers based on organic and inorganic salts, acids and bases are widely used in biochemical and biological research. The statements regarding the effect of anions and cations on biological systems also apply here.

 

 Primary Standards

N.B.S.* Standard Buffer Substances [4]
Primary Standards

Composition and properties of the five primary standard buffers at 25°C (see notes on preparation, below).

Buffer Solution

  Tartrate Phthalate Phosphate D Phosphate E Borate
Buffer substance KHC4H4O6 KHC8H4O4 KH2PO4 +
Na2HPO4

KH2PO4 +
Na2HPO4
Na2B4O7
10 H2O
g/l soln. at 25 °C Saturated
at 25°C
10.12 3.39 [b]
3.53 [c]
1.179 [b]
4.30 [c]
3.80
Molality (m) 0.0341 0.05 0.025 [a] 0.008695 [b]
0.03043 [c]
0.01
Molarity (M) 0.034 0.04958 0.02490 [a] 0.008665 [b]
0.03032 [c]
0.009971
Density (g/ml) 1.0036 1.0017 1.0028 1.0020 0.9996
pH at 25°C 3.557 4.008 6.865 7.413 9.180
Dilution value,
D pH½
+0.049 +0.052 +0.080 +0.07 [d] +0.01
Buffer value, b ,
equiv./pH
0.027 0.016 0.029 0.016 0.020
Temp. coeff., dpH(S)/dt, units/°C -0.0014 +0.0012 -0.0028 -0.0028 -0.0082

[a] Concentration of each phosphate salt. [b] KH2PO4. [c]
Na2HPO4. [d] Calculated value.

Recommended standard values of pH(S) for primary standard buffers (+/- 0.005 at 0 - 60°C and +/-0.008 from 60-90°C).

Buffer pH

Temp. (°C) Tartrate Phthalate Phosphate D Phosphate E Borate
0   4.003 6.984 7.534 9.464
5   3.999 6.951 7.500 9.395
10   3.998 6.923 7.472 9.332
15   3.999 6.900 7.448 9.276
20   4.002 6.881 7.429 9.225
25 3.557 4.008 6.865 7.413 9.180
30 3.552 4.015 6.853 7.400 9.139
35 3.549 4.024 6.844 7.389 9.102
38 3.548 4.030 6.840 7.384 9.081
40 3.547 4.035 6.838 7.380 9.068
45 3.547 4.047 6.834 7.373 9.038
50 3.549 4.060 6.833 7.367 9.011
55 3.554 40.75 6.834   8.985
60 3.560 4.091 6.836   8.962
70 3.580 4.126 6.845   8.921
80 3.609 4.164 6.859   8.885
90 3.650 4.205 6.877   8.8850
95 3.674 4.227 6.886   8.833

*N.B.S. National Bureau of Standards

 Secondary Standards

Composition and properties of the two secondary standard buffers at 25°C.

Buffer Solution

  Tetraoxalate Calcium hydroxide
Buffer substance KH3(C2O4)2. 2H2O Ca(OH)2
g/l of soln. at 25°C 12.61 Saturated at 25°C
Molality (m) 0.05 0.0203
Molarity (M) 0.04962 0.02025
Density (g/ml) 1.0032 0.9991
pH at 25°C 1.0032 12.454
Dilution value,D pH1/2 1.679 - 0.28
Buffer value,b, equiv./pH + 0.186 0 09
Temp. coeff.,
dpH (S)/dt, units/°C
0.070 - 0.033

Recommended standard values of pH(S) for the secondary buffer standards.

Buffer pH

Temp (°C) Tetraoxalate Calcium hydroxide
0 1.666 13.423
5 1.668 13.207
10 1.670 13.003
15 1.672 12.810
20 1.675 12.627
25 1.679 12.454
30 1.683 12.289
35 1.688 12.133
38 1.691 12.043
40 1.694 11.984
45 1.700 11.841
50 1.707 11.705
55 1.715 11.574
60 1.723 11.449
70 1.743  
80 1.766  
90 1.792  
95 1.806  

Note. See below for remarks on drying potassium tetraoxalate dihydrate.

Primary N.B.S.* Standard Buffer Substances
Product No. Description
60219 Potassium dihydrogen phospate
60359 Potassium hydrogen phthalate
60366 Potassium hydrogen D-tartrate
71639 di-Sodium hydrogen phosphate anhydrous
71999 Sodium tetraborate Decahydrate
 
Secondary N.B.S.* Standard Buffer Substances
Product No. Description
21181 Calcium hydroxide
60589 Potassium tetraoxalate Dihydrate

 

 Notes

Notes on the Preparation

The primary and secondary standard buffer solutions are prepared from the standard buffer substances as indicated in the table. The standard pH [pH(S)] of the buffer solution in the temperature range 0-95°C are indicated and can be used for calibration purposes. Buffer compositions are on the molal scale. Only freshly prepared solutions should be used, as the initial buffer composition may change very rapidly. Tartrate, phthalate, and phosphates may be dried at 110°C for 1-2 hours prior to use. Potassium tetraoxalate should not be dried above 60°C, and borax should not be dried at all.
 

Molality (m) dimension: mole per kilogram of solvent

Molarity (M) dimension: mole per litre of solution

Dilution value (DpH1/2) change of pH value observed by dilution of a buffer solution with an equal volume of pure water. It is positive when pH increases and negative when pH decreases with increasing dilution

Buffer value b (equiv./pH) (also Buffer capacity, Van Slyke Buffer value) b = d[B]/dpH, where d[B] is the increment (in equivalents) of a strong base required to produce a certain pH change of the buffer solution. Strong acids effect negative (-d[B]) increment and thus lower the pH

Temperature coefficient (pH unit/°C) dpH(S)/dT standard change of pH value per degree centigrade. It can be positive or negative

 

General Aspects regarding Buffer Applications

With few exceptions, studies of biochemical systems require the use of a buffer in order to control the pH value. Therefore the action of the buffer is of prime importance. Factors influencing the action of buffer solutions and pH are [5, 6]:

  • activity effects: concentration and electrical charge of the species involved
  • salt effects: added "indifferent" electrolytes
  • dilution effect: pH-variation on dilution of buffer solutions buffer capacity: added base or acid
  • temperature dependence

The choice of the correct buffer for a particular biochemical system or technique depends on a number of additional factors. For example: undesired interaction of the buffer with the biopolymer, redox stability, metal ion complexing properties and purity. One way to solve the difficult problem of selecting the right buffer is to evaluate as many buffers as possible. Reviews on the use of buffers in various areas are available [7-9]. However they do not provide detailed information and a comprehensive treatise on the subject should be consulted.

Practical Aspects of Buffer Application

  • Activity and salt effects have a marked influence on the pH value of a solution according to the equation

pH = pKa' + log[B]/[BH] (1)

where

pKa' = pKa + correction factor

The factors for different ionic strengths are tabulated in [5] and range from 0.015 for ionic strength I = 0.001 to 0.159 for I = 0.5.

  • lonic strength is defined as in   

where ci is the concentration of species i, and z is the corresponding charge. I can be calculated very easily from the experimental parameters.

Buffer capacity. The maximum buffer capacity bmax of a monovalent species is found to be at pH = pKa', the practical pK-value. bmax in the pH range 3-11 is calculated according to equation (3)

bmax = 0.576 c (3)

where c is the total concentration of the buffer substance. Thus the useful buffer capacity lies within a pH range of pKa± 1 unit. If more than 50% of the maximum buffer capacity must be realized the corresponding range is only pKa' + 0.75 units.

The Practical Buffer Range

b, the buffer capacity, is defined as given in (4)

b =d [B] (4)
      dpH

where [B] is the amount of base added to the buffer component BH. The buffer capacity of a mixed weak acid-base buffer system is greater, the closer the individual pKa values lie. b values of a mixture of buffers are additive.

From equation (5) it is possible to calculate the molar ratio [basic species]/[acidic species] which leads to a desired pH within the practical buffer range, pKa± 1 unit.

pH = pK + log[basic species] (5)
                     [acid species]

From the diagram on page ...
[B]/[BH] - pH - % buffer capacity can quickly be estimated.

Temperature effects on the pH of a given solution may be considerable. TRIS has a pKa of 8.55 at 0°, 8.06 at 25° and 7.22 at 37° (mean dpH/dT-0.03 pH units/°C). Salt buffers, such as the Primary Standards show dpH/dT of about 0.002 pH-units/°C. The change can be either positive or negative.

Dilution effects depend mainly on the charge of the buffer species; dilution of a 0.1 M HA/A- buffer system (total concentration) with an equal volume of water results in a pH-value change of 0.024 units, whereby the pH is lowered in the case of basic buffers and increased when acidic ones are diluted. The pH variation of HA-/A2- buffer systems are increasod by a factor of approximately three.

Use of diagram: Determine from experimental parameters the molar concentration ratio of basic and acidic speries in the buffer system.

(1 to 10)
10     1

Read the pH of the Solution from the upper diagram of pH deviation and from the lower diagram the % of maximum buffer capacity (% bmax).

 pKa Value and Buffer Range of important Biological Buffers

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Effective pH range pKa 25°C Buffer
1.2-2.6 1.97 maleate (pK1)
1.7-2.9 2.15 phosphate (pK1)
2.2-3.6 2.35 glycine (pK1)
2.2-6.5 3.13 citrate (pK1)
2.5-3.8 3.14 glycylglycine (pK1)
2.7-4.2 3.40 malate (pK1)
3.0-4.5 3.75 formate
3.0-6.2 4.76 citrate (pK2)
3.2-5.2 4.21 succinate (pK1)
3.6-5.6 4.76 acetate
3.8-5.6 4.87 propionate
4.0-6.0 5.13 malate (pK2)
4.9-5.9 5.23 pyridine
5.0-6.0 5.33 piperazine (pK1)
5.0-7.4 6.27 cacodylate
5.5-6.5 5.64 succinate (pK2)
5.5-6.7 6.10 MES
5.5-7.2 6.40 citrate (pK3)
5.5-7.2 6.24 maleate (pK2)
5.5-7.4 1.70, 6.04, 9.09 histidine
5.8-7.2 6.46 bis-tris
5.8-8.0 7.20 phosphate (pK2)
6.0-12.0 9.50 ethanolamine
6.0-7.2 6.59 ADA
6.0-8.0 6.35 carbonate (pK1)
6.1-7.5 6.78 ACES
6.1-7.5 6.76 PIPES
6.2-7.6 6.87 MOPSO
6.2-7.8 6.95 imidazole
6.3-9.5 6.80, 9.00 BIS-TRIS propane
6.4-7.8 7.09 BES
6.5-7.9 7.14 MOPS
6.8-8.2 7.48 HEPES
6.8-8.2 7.40 TES
6.9-8.3 7.60 MOBS
7.0-8.2 7.52 DIPSO
7.0-8.2 7.61 TAPSO
7.0-8.3 7.76 triethanolamine (TEA)
7.0-9.0 0.91, 2.10, 6.70, 9.32 pyrophosphate
7.1-8.5 7.85 HEPPSO
7.2-8.5 7.78 POPSO
7.4-8.8 8.05 tricine
7.5-10.0 8.10 hydrazine
7.5-8.9 8.25 glycylglycine (pK2)
7.5-9.0 8.06 Trizma (tris)
7.6-8.6 8.00 EPPS, HEPPS
7.6-9.0 8.26 BICINE
7.6-9.0 8.30 HEPBS
7.7-9.1 8.40 TAPS
7.8-9.7 8.80 2-amino-2-methyl-1,3-propanediol (AMPD)
8.2-9.6 8.90 TABS
8.3-9.7 9.00 AMPSO
8.4-9.6 9.06 taurine (AES)
8.5-10.2 9.23, 12.74, 13.80 borate
8.6-10.0 9.50 CHES
8.7-10.4 9.69 2-amino-2-methyl-1-propanol (AMP)
8.8-10.6 9.78 glycine (pK2)
8.8-9.9 9.25 ammonium hydroxide
8.9-10.3 9.60 CAPSO
9.5-11.1 10.33 carbonate (pK2)
9.5-11.5 10.66 methylamine
9.5-9.8 9.73 piperazine (pK2)
9.7-11.1 10.40 CAPS
10.0-11.4 10.70 CABS
10.5-12.0 11.12 piperidine
  12.33 phosphate (pK3)

References

  1. Van Slyke, J. Biol. Chem. 52, 525 (1922).
  2. N.E.Good, G.D.Winget, W.Winter, TN.Conolly, S.lzawa and R.M.M.Singh, Biochemistry 5, 467 (1966); N.E. Good, S.lzawa, Methods Enzymol. 24, 62 (1972).
  3. G. Gomori, Methods Enzymol. 1, 138 (1955)
  4. Bates, J. Res. Natn. Bur. Stand. 66A, 179 (1962), see also R.M.C. Dawson et al., 3rd ed., p. 421, Clarendon Press,Oxford (1986).
  5. D.D. Perrin, B. Dempsey, Buffers for pH and Metal lon Control, Chapman and Hall Laboratory Manuals, London (1974).
  6. R.M.C. Dawson, D.C. Elliot, W.H. Elliot, K.M.Jones, Data for Biochemical Research, 3rd ed., Oxford Science Publ.(1986).
  7. J.S. Blanchard, Methods Enzymol. 104, 404 (1984); V.S. Stoll, J.S. Blanchard, ibid. 182, 24(1990).
  8. K.J. Ellis and J.F. Morrison, Methods Enzymol. 87, 405 (1982).
  9. f McLellan, Anal. Biochem. 126, 94 (1982).

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