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^- <=> HPO₄^2-, 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

[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

*N.B.S. National Bureau of Standards

 Secondary Standards

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

Buffer Solution

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

Buffer pH

Note. See below for remarks on drying 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.
 

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 c_i 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 b_max of a monovalent species is found to be at pH = pKa', the practical pK-value. b_max in the pH range 3-11 is calculated according to equation (3)

b_max = 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 (% b_max).

 pK_a Value and Buffer Range of important Biological Buffers

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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