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calomel pH-cell in aqueous solutions containing dimethylsulphoxide at 25 and −12 °C
CRYOBIOLOGY 15, 340344 ( 1978)
The
Response of the Glass/Calomel pH-Cell in Aqueous Solutions Containing Dimethylsulphoxide at 25 and -12°C M. J. TAYLOR Ditiision of Cryoldology,
Clinical Research
,
Cl1
where X denotes the solution of unknown pH and S the standard reference buffer solution of known or assigned pH, while E is the electromotive force of a suitable Received June 28, 1977; accepted September 12, 1977.
OOII-2240/78/0153-0340.$02.00/O
HAI
3Uj,
England
A universal pH scale for all mixed solvents relating proton activity uniformly to the aqueous standard reference state is not yet a practical possibility (3), but separate scales for each mixed solvent medium can be achieved. Procedures analagous to those on which a practical aqueous pH scale is based have been used to establish operational acidity
340
Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved,
Harrow
galvanic cell consisting of an electrode reversible to hydrogen ions (usually a glass electrode) coupled with a suitable reference electrode (commonly a calomel electrode). A salt bridge consisting of a concentrated solution of potassium chloride may be used to connect the reference electrode with solution X or S. R, T, and F represent the gas constant, the absolute temperature, and the Faraday, respectively. The addition of DMSO to an aqueous solution gives rise to an apparent increase in pH when measured with a glass/ calomel pH-cell at ambient temperatures (6). Although the glass eIectrode has been used in DMSO solutions (9-11) it is uncertain whether this rise in pH represents a true change in hydrogen ion activity or whether the organic additive was in%uencing certain characteristics of the measuring electrodes, such as the liquid-junction potential or the asymmetry constant of the glass electrode.
Despite the many theories of the ways in which freezing injures biological materials, the precise mechanism is still not fully understood. The major difficulty is that during exposure to low temperature cells are subjected to a variety of concurrent phenomena all or any of which could lead to irreversible injury. A change in pH of the intra- and extracellular media ( 14 > is one of these phenomena and is one which has received relatively little attention (15, 16). This is undoubtedly due to the fact that the conventional methods of measuring pH values are unsuitable for measurements at Iow temperatures. Nevertheless, there is an increasing need for reliable pH measurements in aqueous solutions containing a cryoprotectant such as dimethylsulphoxide ( DMSO ) at physiological and subzero temperatures (6, 14). The widely used and accepted procedure for making an electrometric pH measurement is dependent upon an operational definition of pH ( 1). This is formulated as: pH (X) = pH (S) + (E;T;nE;O)F
Centre,
scales in DMSO-HZ0
mixed
soIvent
media
pH
MEASUREMENTS
AT
(13). Interpretation of the potential of an electrochemical cell, reversible to hydrogen ions, in terms of pH requires a knowledge of the standard electromotive force (E” ) of the cells. Standard molal emfs of the ceI1:
Pt; H~~p,~nw, HCl,m, &Cl; Ag L.21 for solvent compositions ranging from water to 30% (w/w ) DMSO-Ha0 at temperatures between 25 and -lZ.“C have been determined in this laboratory, and these E” vahres have in turn been used to assign standard pH values to selected buffer solutions under the same conditions. Standard pH values in mixed solvents are designated, pH* (S), where the asterisk signifies a quantity referred to the standard state in a solvent other than pure water. A description of the procedures used for deriving standard emfs and for assigning standard pH values is not the purpose of this comsmunication. However, detaiIs have been outlined eIsewhere (12, 13), and full communications are in preparation. Those resuhs of electrochemical measurements with the hydrogen-silver, silver chIoride primary standardising cell which have relevance to this assessment of the gIass/calomel pH-cell response in DMSOHz0 mixtures at 25 and -12°C are summarised in this preliminary communication. METHODS
Standard potentials (E” ) of cell 2 were derived by plotting a function involving the experimental cell potential corrected to 1 atm pressure, the molal concentration of electrolyte and the extended DebyeHiickel expression for the activity coefficient, against moIality (m) for a series of concentrations between 0.1 and 0.005 m. Extrapolation of this relationship to infinite dilution (m = 0) yields the standard molal emf of the cell (8). E” values obtained for binary solvent mixtures of DMSO and water permitted the assignment of standard pH” values to buffer compounds in
LOW
TEMPERATURES
341
these soIvent mixtures at temperatures between 25 and -12°C. The procedures and conventions used to assign pH* (S) values in this work were consistent with those adopted by standardising groups in many countries and recommended by the International Union of Pure and Applied Chemistry (2). pH”(S) values assigned to potassium hydrogen phthalate (KHphthaIate) and sodium N-Tris (hydroxymethyl) methyl-2-aminoethane sulphonate (TES Na) buffer solutions were used to assess the response of a combined glass and calomel electrode under the stated conditions of solvent compositions and temperature. A radiometer pH meter (TTT 2) was used in conjunction with a “low temperature” combined glass and calomeI electrode (Metrohm, temperature range +6O to -15OC) which incorporated a 3.0 hf KC1 reference electrolyte. The measurements were carried out in a “jacketed” titration vessel (Metrohm) immersed in a refrigerated bath containing a methanoIwater mixture controlled at either 25 or -12 * O.l”C. The electrode was standardised in a 0.05 m solution of potassium hydrogen phthalate ( KHphthalate) of known pH” (S) in 30% (w/w) DMSOHz0 (13), and its response in this solvent mixture at 25 and -12°C was assessed by measuring the known standard pH values of a sodium N-Tris (hydroxymethyl) methyl-Z-aminoethane sulphonate (TES Na) buffer. The TES Na buffer standards were prepared by converting 30 mm01 of a 100 mm solution of N-Tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (TES) to its sodium salt (13). The pH-cell response in 3070 (w/w) DMSOHZ0 was compared with its response in a purely aqueous solution by standardising the same cell in a 0.05 m KHphthaIate buffer of pH(S)=4.00 at 20°C (5, 7) and then taking a measurement when the electrode was equilibrated in a standard aque-
342
M.
J. TAYLOR TABLE
Standard
1
Cell Potentials (E”) and Standard Assess the Response of the Glms
pH Values for Buffer Electrode at 25 and
Solutions -12%
Used
PWS);;~PH*(S
to
PHW
K H phthalate (0.05 m)
Water 307~ (w/w) 30% (w/a)
23 26 -12
DhfSO-Hz0 DMSO-He0
0.22227 0.21839 0.23888
ous phosphate buffer [pH( S) = 7.00 at 2O”C]. Temperature cmpensation. The temperature compensator on the TTT2 pH meter is a manual device calibrated for adjustment between 0 and 100°C. For standardisation and measurements at temperatures below 0°C the “compensator” was arbitrarily set at “25GC” and corrections were appIied to the meter readings by calculating the necessary adjustments, in pH units, from the change in slope factor for the temperature difference between 25°C and the subzero temperature. The meter was calibrated in the usual way by making it read the known pH*( S) of a standard buffer at the temperature of measurement of the unknown, The correct pH* value, pH*( X), of the unknown was
pH’(X) +
Cell
TES
Na
(70 mm) TES
Na
solution
(30 mm) in 30% (30 mm)
pH*
pH* (S)],
[33
pH*(S)
[pB’(X)’
y
-
AND
DISCUSSION
Standard cell potentials and standard pH values pertinent to this assessment of the glass electrode response at 25 and -12°C are given in Table 1. The conditions of standardisation and measurement are summarised in Table 2 together with the meter readings and the corresponding pH values compensated for temperature, 2 pH-Cell w a Test at 25 and - 12’C
of Itu Retiponse
PH@)
Tern
in 30% DMSO-Hz0 in 30% DMSO-Hz0 (aqueous)
4.76 4.87 4.00 Tern
mature SbC)
Meter reading pH(X)’
erature lb
p&)
Assigned pH(a) or pH* (a)
reading,
25 -12 20 Correct PH(XI
PH
APH
+ TES DMSO-HB
7.13
25
7.09
7.00
-0.04
7.77
8.18
-0.03
6.96
6.96
-0.04
+ TES
(70 mm) in 3070 DMSO-HI0 BDH st.andard phosphate buffer
(aqueous)
=
RESULTS
Conditions of atsndardisation Standardking buffer
Measumements
7.026 7.128 8.210
where pH* (S) is the pH* value of the standard buffer solution at the temperature of measurement, T (“K).
with a Combined Glass/Calomel in 30% DMSO-Hz0 Solutions
0.05 M K H phthalate 0.05 M K H phthalate 0.05 M K H phthalate
4.007 4.761 4.870
caIculated from the meter (X)‘, using the reIationship:
TABLE Measurements
f 0.00002 f 0.00002 f 0.00001
;;r~H*(S) TES Ns (0.03 m)
8.21 7.00
-12 20
pH
MEASUREMENTS
AT
The most important observation from these results is that the response of the glass/calomel cell in 30% (w/w) DMSOHz0 at 25 and -12°C follows that of the hydrogen electrode within acceptable limits, The observed pH difference, ApH, between the measured pH value and the standard value assigned using the hydrogen-silver, silver chloride electrochemical cell was 0.04 units at 25°C and 0.03 units at -12°C. These ApH values were of the same order as those observed when the same gIass/calomel electrode was calibrated with a purely aqueous KHphthalate standard [pH(S)= 4.00 at 2O”C] and used to measure the pH value of a standard aqueous phosphate buffer [pH( S)= 7.00 at 2O”C]; ApH in this case was 0.04 units. The theoretical pH response as shown by the hydrogen gas electrode is never followed exactly by a glass electrode; consequently an error in its potential, with respect to the activity of hydrogen ions, between two standard points on the pH scale is to be expected. This error is minimised by calibrating the electrode at a point on the pH scale near to that of measurement. Bates (4) suggested that an acceptable degree of error for the response of a glass electrode between two standard points is 0.02 pH units. These results show that although the errors are 0.01-0.02 pH units Iarger than those recommended, possibIy due to insufficient preconditioning of the new electrode, the response of the glass/calomel pH-cell in 30% (w/w) DMSO-Hz0 at 25 and -12°C is acceptable for most purposes and certainly as good as its response in the standard aqueous system at room temperature. SUMMARY
The pH response of the glass/calomel electrochemical cell in aqueous solutions containing dimethylsulphoxide at 25 and -12°C has been assessed using buffer solutions whose standard pH values were
LOW
TEMPERATURES
343
assigned using the hydrogen-silver, silver chloride primary standardising cell. The glass/calomel pH cell was shown to function normally in 30% DMSO-Hz0 at 25 and -12°C and its response, compared to the hydrogen electrode, was as good as its response in the standard aqueous system at room temperature. REFERENCES 1. Bates, R. G., and Guggenheim, E. A. Report on the standardization of pH and related terminology. Pure A&. Chem. 1, 103-188 (1980). 2. Bates, R. G. Practical measurement of pH in amphiprotic and mixed solvents. Pure A&. Chem. 18, 421-425 ( 1969). 3. Bates, R. G. In “Determination of pH. Theory and Practice,” 2nd ed., Chap. 8. John Wiley, New York, 1973. 4. Bates, R. G. In “Determination of pH. Theory and Practice,” 2nd ed., Chap. I2. John Wiley, New York, 1973. 5. British Standards Institution. Specification for pH scale. British Standard 1047, London (1961). 6. Elford, B. C., and Walter, C. A. Effects of electrolyte composition and pH on the structure and function of smooth muscle cooled to -79°C in unfrozen media. CTYOhidugy 9, 82-100 ( 1972). 7. Hamer, W. J., Pinching, G. D., and Acree, S. F. pH standards at various temperatures in aqueous solutions of acid potassium phthalatc. .l. Res. Nat. Bur. Stand. 36, 4762 (1946). 8. Harned, H. S., and Owen, B. B. Ira “The Physical Chemistry of Electrolyte Solutions,” 3rd ed., Chap. 11. Reinhold, New York, 1958. 9. Kolthoff, I. M., and Reddy, T. B. Acid-base strength in dimethylsulphoxide. Inorg. Chom. 1, 189-194 (1962). 10. Morel, J. I’. Conductivitirs de quelques Blectrolytes, dissociation de l’acid acCtique et potentielsnormaux de l’electrode Ag-A&‘1 dans les melanges eau-dimethylsulpho;:ide. Bull. Sot. Chim. Fmnce 1405-1411 (1967). II. Ritchie, C. D., and Uschold, R. E. Acidity in non aqueous solvents. XIV. Hydrocarbon acids in dimethylsulphoxide. I. Amer. Chem. sot. 89, 1721-1725. (1967). 12. TayIor, M. J. An approach to the problem of pH measurement at low temperatures: The standard e.m.f. of the cell Pt/H2 (latm)
M.
J. TAYLOR
HCi(m)/AgCI-Ag in 20% (w/w) DMSOHa0 between 25” and -5S”C. In vitro v CSSR 3, 4%54 (1974). 13. TayIor, M. J. pH measurements in dimethylsulphoxidewater mixtures: Relevance to the integrity of smooth muscle exposed to low temperatures. Ph.D. Thesis, C.N.A.A., London (1977). 14. Taylor, M. J., Walter, C. A., and Elford, B. C. The pH-dependent recovery of smooth muscle from storage at -13°C in unfrozen
media. Cryohiolngy ( in press ). 15. Van den Berg, L. The effect of addition of sodium and potassium chloride to the reciprocal system: KHyPO,-NagHPO,HgO on pH and composition during freezing. Arch. Biochem. Biophys. 84, 305-315,
(1959). 16. Van den Berg, L. pH measurements at low temperatures using modified calomel and glass electrodes. And. Chem. 32, 62%631, ( 1960).
The
Response of the Glass/Calomel pH-Cell in Aqueous Solutions Containing Dimethylsulphoxide at 25 and -12°C M. J. TAYLOR Ditiision of Cryoldology,
Clinical Research
,
Cl1
where X denotes the solution of unknown pH and S the standard reference buffer solution of known or assigned pH, while E is the electromotive force of a suitable Received June 28, 1977; accepted September 12, 1977.
OOII-2240/78/0153-0340.$02.00/O
HAI
3Uj,
England
A universal pH scale for all mixed solvents relating proton activity uniformly to the aqueous standard reference state is not yet a practical possibility (3), but separate scales for each mixed solvent medium can be achieved. Procedures analagous to those on which a practical aqueous pH scale is based have been used to establish operational acidity
340
Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved,
Harrow
galvanic cell consisting of an electrode reversible to hydrogen ions (usually a glass electrode) coupled with a suitable reference electrode (commonly a calomel electrode). A salt bridge consisting of a concentrated solution of potassium chloride may be used to connect the reference electrode with solution X or S. R, T, and F represent the gas constant, the absolute temperature, and the Faraday, respectively. The addition of DMSO to an aqueous solution gives rise to an apparent increase in pH when measured with a glass/ calomel pH-cell at ambient temperatures (6). Although the glass eIectrode has been used in DMSO solutions (9-11) it is uncertain whether this rise in pH represents a true change in hydrogen ion activity or whether the organic additive was in%uencing certain characteristics of the measuring electrodes, such as the liquid-junction potential or the asymmetry constant of the glass electrode.
Despite the many theories of the ways in which freezing injures biological materials, the precise mechanism is still not fully understood. The major difficulty is that during exposure to low temperature cells are subjected to a variety of concurrent phenomena all or any of which could lead to irreversible injury. A change in pH of the intra- and extracellular media ( 14 > is one of these phenomena and is one which has received relatively little attention (15, 16). This is undoubtedly due to the fact that the conventional methods of measuring pH values are unsuitable for measurements at Iow temperatures. Nevertheless, there is an increasing need for reliable pH measurements in aqueous solutions containing a cryoprotectant such as dimethylsulphoxide ( DMSO ) at physiological and subzero temperatures (6, 14). The widely used and accepted procedure for making an electrometric pH measurement is dependent upon an operational definition of pH ( 1). This is formulated as: pH (X) = pH (S) + (E;T;nE;O)F
Centre,
scales in DMSO-HZ0
mixed
soIvent
media
pH
MEASUREMENTS
AT
(13). Interpretation of the potential of an electrochemical cell, reversible to hydrogen ions, in terms of pH requires a knowledge of the standard electromotive force (E” ) of the cells. Standard molal emfs of the ceI1:
Pt; H~~p,~nw, HCl,m, &Cl; Ag L.21 for solvent compositions ranging from water to 30% (w/w ) DMSO-Ha0 at temperatures between 25 and -lZ.“C have been determined in this laboratory, and these E” vahres have in turn been used to assign standard pH values to selected buffer solutions under the same conditions. Standard pH values in mixed solvents are designated, pH* (S), where the asterisk signifies a quantity referred to the standard state in a solvent other than pure water. A description of the procedures used for deriving standard emfs and for assigning standard pH values is not the purpose of this comsmunication. However, detaiIs have been outlined eIsewhere (12, 13), and full communications are in preparation. Those resuhs of electrochemical measurements with the hydrogen-silver, silver chIoride primary standardising cell which have relevance to this assessment of the gIass/calomel pH-cell response in DMSOHz0 mixtures at 25 and -12°C are summarised in this preliminary communication. METHODS
Standard potentials (E” ) of cell 2 were derived by plotting a function involving the experimental cell potential corrected to 1 atm pressure, the molal concentration of electrolyte and the extended DebyeHiickel expression for the activity coefficient, against moIality (m) for a series of concentrations between 0.1 and 0.005 m. Extrapolation of this relationship to infinite dilution (m = 0) yields the standard molal emf of the cell (8). E” values obtained for binary solvent mixtures of DMSO and water permitted the assignment of standard pH” values to buffer compounds in
LOW
TEMPERATURES
341
these soIvent mixtures at temperatures between 25 and -12°C. The procedures and conventions used to assign pH* (S) values in this work were consistent with those adopted by standardising groups in many countries and recommended by the International Union of Pure and Applied Chemistry (2). pH”(S) values assigned to potassium hydrogen phthalate (KHphthaIate) and sodium N-Tris (hydroxymethyl) methyl-2-aminoethane sulphonate (TES Na) buffer solutions were used to assess the response of a combined glass and calomel electrode under the stated conditions of solvent compositions and temperature. A radiometer pH meter (TTT 2) was used in conjunction with a “low temperature” combined glass and calomeI electrode (Metrohm, temperature range +6O to -15OC) which incorporated a 3.0 hf KC1 reference electrolyte. The measurements were carried out in a “jacketed” titration vessel (Metrohm) immersed in a refrigerated bath containing a methanoIwater mixture controlled at either 25 or -12 * O.l”C. The electrode was standardised in a 0.05 m solution of potassium hydrogen phthalate ( KHphthalate) of known pH” (S) in 30% (w/w) DMSOHz0 (13), and its response in this solvent mixture at 25 and -12°C was assessed by measuring the known standard pH values of a sodium N-Tris (hydroxymethyl) methyl-Z-aminoethane sulphonate (TES Na) buffer. The TES Na buffer standards were prepared by converting 30 mm01 of a 100 mm solution of N-Tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (TES) to its sodium salt (13). The pH-cell response in 3070 (w/w) DMSOHZ0 was compared with its response in a purely aqueous solution by standardising the same cell in a 0.05 m KHphthaIate buffer of pH(S)=4.00 at 20°C (5, 7) and then taking a measurement when the electrode was equilibrated in a standard aque-
342
M.
J. TAYLOR TABLE
Standard
1
Cell Potentials (E”) and Standard Assess the Response of the Glms
pH Values for Buffer Electrode at 25 and
Solutions -12%
Used
PWS);;~PH*(S
to
PHW
K H phthalate (0.05 m)
Water 307~ (w/w) 30% (w/a)
23 26 -12
DhfSO-Hz0 DMSO-He0
0.22227 0.21839 0.23888
ous phosphate buffer [pH( S) = 7.00 at 2O”C]. Temperature cmpensation. The temperature compensator on the TTT2 pH meter is a manual device calibrated for adjustment between 0 and 100°C. For standardisation and measurements at temperatures below 0°C the “compensator” was arbitrarily set at “25GC” and corrections were appIied to the meter readings by calculating the necessary adjustments, in pH units, from the change in slope factor for the temperature difference between 25°C and the subzero temperature. The meter was calibrated in the usual way by making it read the known pH*( S) of a standard buffer at the temperature of measurement of the unknown, The correct pH* value, pH*( X), of the unknown was
pH’(X) +
Cell
TES
Na
(70 mm) TES
Na
solution
(30 mm) in 30% (30 mm)
pH*
pH* (S)],
[33
pH*(S)
[pB’(X)’
y
-
AND
DISCUSSION
Standard cell potentials and standard pH values pertinent to this assessment of the glass electrode response at 25 and -12°C are given in Table 1. The conditions of standardisation and measurement are summarised in Table 2 together with the meter readings and the corresponding pH values compensated for temperature, 2 pH-Cell w a Test at 25 and - 12’C
of Itu Retiponse
PH@)
Tern
in 30% DMSO-Hz0 in 30% DMSO-Hz0 (aqueous)
4.76 4.87 4.00 Tern
mature SbC)
Meter reading pH(X)’
erature lb
p&)
Assigned pH(a) or pH* (a)
reading,
25 -12 20 Correct PH(XI
PH
APH
+ TES DMSO-HB
7.13
25
7.09
7.00
-0.04
7.77
8.18
-0.03
6.96
6.96
-0.04
+ TES
(70 mm) in 3070 DMSO-HI0 BDH st.andard phosphate buffer
(aqueous)
=
RESULTS
Conditions of atsndardisation Standardking buffer
Measumements
7.026 7.128 8.210
where pH* (S) is the pH* value of the standard buffer solution at the temperature of measurement, T (“K).
with a Combined Glass/Calomel in 30% DMSO-Hz0 Solutions
0.05 M K H phthalate 0.05 M K H phthalate 0.05 M K H phthalate
4.007 4.761 4.870
caIculated from the meter (X)‘, using the reIationship:
TABLE Measurements
f 0.00002 f 0.00002 f 0.00001
;;r~H*(S) TES Ns (0.03 m)
8.21 7.00
-12 20
pH
MEASUREMENTS
AT
The most important observation from these results is that the response of the glass/calomel cell in 30% (w/w) DMSOHz0 at 25 and -12°C follows that of the hydrogen electrode within acceptable limits, The observed pH difference, ApH, between the measured pH value and the standard value assigned using the hydrogen-silver, silver chloride electrochemical cell was 0.04 units at 25°C and 0.03 units at -12°C. These ApH values were of the same order as those observed when the same gIass/calomel electrode was calibrated with a purely aqueous KHphthalate standard [pH(S)= 4.00 at 2O”C] and used to measure the pH value of a standard aqueous phosphate buffer [pH( S)= 7.00 at 2O”C]; ApH in this case was 0.04 units. The theoretical pH response as shown by the hydrogen gas electrode is never followed exactly by a glass electrode; consequently an error in its potential, with respect to the activity of hydrogen ions, between two standard points on the pH scale is to be expected. This error is minimised by calibrating the electrode at a point on the pH scale near to that of measurement. Bates (4) suggested that an acceptable degree of error for the response of a glass electrode between two standard points is 0.02 pH units. These results show that although the errors are 0.01-0.02 pH units Iarger than those recommended, possibIy due to insufficient preconditioning of the new electrode, the response of the glass/calomel pH-cell in 30% (w/w) DMSO-Hz0 at 25 and -12°C is acceptable for most purposes and certainly as good as its response in the standard aqueous system at room temperature. SUMMARY
The pH response of the glass/calomel electrochemical cell in aqueous solutions containing dimethylsulphoxide at 25 and -12°C has been assessed using buffer solutions whose standard pH values were
LOW
TEMPERATURES
343
assigned using the hydrogen-silver, silver chloride primary standardising cell. The glass/calomel pH cell was shown to function normally in 30% DMSO-Hz0 at 25 and -12°C and its response, compared to the hydrogen electrode, was as good as its response in the standard aqueous system at room temperature. REFERENCES 1. Bates, R. G., and Guggenheim, E. A. Report on the standardization of pH and related terminology. Pure A&. Chem. 1, 103-188 (1980). 2. Bates, R. G. Practical measurement of pH in amphiprotic and mixed solvents. Pure A&. Chem. 18, 421-425 ( 1969). 3. Bates, R. G. In “Determination of pH. Theory and Practice,” 2nd ed., Chap. 8. John Wiley, New York, 1973. 4. Bates, R. G. In “Determination of pH. Theory and Practice,” 2nd ed., Chap. I2. John Wiley, New York, 1973. 5. British Standards Institution. Specification for pH scale. British Standard 1047, London (1961). 6. Elford, B. C., and Walter, C. A. Effects of electrolyte composition and pH on the structure and function of smooth muscle cooled to -79°C in unfrozen media. CTYOhidugy 9, 82-100 ( 1972). 7. Hamer, W. J., Pinching, G. D., and Acree, S. F. pH standards at various temperatures in aqueous solutions of acid potassium phthalatc. .l. Res. Nat. Bur. Stand. 36, 4762 (1946). 8. Harned, H. S., and Owen, B. B. Ira “The Physical Chemistry of Electrolyte Solutions,” 3rd ed., Chap. 11. Reinhold, New York, 1958. 9. Kolthoff, I. M., and Reddy, T. B. Acid-base strength in dimethylsulphoxide. Inorg. Chom. 1, 189-194 (1962). 10. Morel, J. I’. Conductivitirs de quelques Blectrolytes, dissociation de l’acid acCtique et potentielsnormaux de l’electrode Ag-A&‘1 dans les melanges eau-dimethylsulpho;:ide. Bull. Sot. Chim. Fmnce 1405-1411 (1967). II. Ritchie, C. D., and Uschold, R. E. Acidity in non aqueous solvents. XIV. Hydrocarbon acids in dimethylsulphoxide. I. Amer. Chem. sot. 89, 1721-1725. (1967). 12. TayIor, M. J. An approach to the problem of pH measurement at low temperatures: The standard e.m.f. of the cell Pt/H2 (latm)
M.
J. TAYLOR
HCi(m)/AgCI-Ag in 20% (w/w) DMSOHa0 between 25” and -5S”C. In vitro v CSSR 3, 4%54 (1974). 13. TayIor, M. J. pH measurements in dimethylsulphoxidewater mixtures: Relevance to the integrity of smooth muscle exposed to low temperatures. Ph.D. Thesis, C.N.A.A., London (1977). 14. Taylor, M. J., Walter, C. A., and Elford, B. C. The pH-dependent recovery of smooth muscle from storage at -13°C in unfrozen
media. Cryohiolngy ( in press ). 15. Van den Berg, L. The effect of addition of sodium and potassium chloride to the reciprocal system: KHyPO,-NagHPO,HgO on pH and composition during freezing. Arch. Biochem. Biophys. 84, 305-315,
(1959). 16. Van den Berg, L. pH measurements at low temperatures using modified calomel and glass electrodes. And. Chem. 32, 62%631, ( 1960).