Use of Apparent Dissociation Constants in Qualitative Organic Analysis 1. V. PARKE and W. W. DAVIS Lilly Research Laboratories, Indianapolis 6, Ind.
Procedures are described for obtaining and interpreting potentiometric titration data in the form of hydrogen ion binding versus pH curves. Advantages and utility of data in this form are discussed.
relatively small. Simms ( 4 ) has determined the change in pKd with changing ionic strength for several typical acidic and basicgroups. His data indicate the deviation between pKL and pKa corresponding to the ionic strength occurring in the present experiments to be less than 0.1 p H unit. In general, obserred pK: ic: lo~verthan pk'a for acids nnd higher thnn p T c ~lor bases.
T
HE application of physical methods to qualitative organic analysis during recent years has led to widespread use of ultraviolet and infrared absorption spectrophotometry and, to n lesser extent, polarography in groupwise identification of organic structures. Potentiometric determination of dissociation constants of acidic and basic groups has been exploited far less, in spite of its great utility in such structure studies. Part of this neglect has been due to the lack of a relatively simple means for obtaining and interpreting potentiometric titration curvepes of acidic and basic groups. By pel forming titrations a t several temperature., it is possible to calculate the heat of dissociation for a given gioup. The heat of dissociation for a inonorarbovylic acid is generally less than 2000 calories per mole and is about 6000 calories per rnole for phenolic groups, while the AH for basic groups is generally greater than 5000 calories per mole ( 1 ) . Thiq technique has been used to great advantage in the qtudy of the dissociating groups in proteins. The second method involves titration in a nonaqueous solveiit or a qolvent-water mixtui e. Apparent dissociation constants of 27 carbo\\-lic acid. in varving ethyl alcohol-water mixtures have been studied by 1Iichaelis aiid Mizutani ( 2 ) . Progreqsive in-
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Figure 5 . Theoretical Curves from Transparent Mask Used Curves
for Graphic Analysis of Hydrogen Ion-Binding
A transparent inask shown in Figure 5 bearing this theoretical curve has been prepared for convenience in interpreting bound hydrogen ion versus p H curves. I n addition to the curve for exactly one equivalent hydrogen ion bound per mole of sample, other curves for 1.10, 0.90, 0.75, 0.50, and 0.25 equivalent per mole have been incorporated for upe with samples of unknown purity or molecular weight. An index mark a t the midpoint of the theoretical curve allows immediate determination of the p K i values with an accuracy of about f 0 . 0 5 p H unit. Use of this mask permits estimation of pKd a t the extremes of thc p H
ANALYTICAL CHEMISTRY
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AUREOMYCIN
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Figure 6. Hydrogen Ion-Binding Curve for Adipic Acid PH
Figure 8.
scale where only a portion of the complete theoretical titration curve may be observed. I n Figure 2 the midpoint of the inflecoccurs a t about the lowest tion for the carboxyl group- of glvcine __ p H a t which trustworthy values can be obtained, but the pKd can be estimated by fitting the observed points to the theoretical curve. Overlapping groups may be readily resolved and their pKd values determined accurately by use of the mask. Figure 6
pKAS OF DISSOCIATING GROUPS I
MONO- C O O H
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ORG A R S E N I C A C I D S ORG B O R I C A C I D S ORG P H O S A C I D S CARBOHYDRATES OFlGAlulC B A S E S ALlPH AMINES 8 - U N S A T D OR P O L A R S U B E - U N S A T D OR P O L A R S U E a - A M I N O ACIDS PEPTIDES AROM N HETERO N NON AROM AROM GUANiD NO
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Range of pK', of Dissociating Groups from Literature Data
Hydrogen Ion-Binding Curve for Aureomycin
illustrates use of the mask in resolution of the pKd values of adipic acid which are separated by approximately one pH unit ( p K i 4.23 and 5.24). Use of this mask is limited to aqueous titrations because the shaDe of inflections in nonaqueous titration curves would be expected to vary. Having determined the pKd of the dissociating group and having obtained some information as to its acidic or basic nature, it is frequently possible to make tentative assignments of structure of the dissociating group. The chart in Figure 7 has been drawn from aqueous titration data on 573 compounds taken from the literature. The ranges observed for typical dissociating groups are given and in several cases the effect of neighboring groups or substituents is noted. I t has been found helpful to employ a simple Keysort punched card system for indexing these titration data. While quantitative titrimetry ie not within the scope of this paper, some important quantitative information may be obtained from these titration curves. If the purity of the sample is known, the molecular weight can be determined within the limits of accuracy of the method, which is about 5%. Since use of the mask with the theoretical curve distinguishes partially overlapping inflections, a true molecular weight is determined in contrast to a neutral equivalent determined by an indicator titration. An exception would be a compound with two or more titratable groups having pKd values separated by less than about 0.4 p H unit. These would appear as a single titratable group and the apparent molecular weight would be the equivalent weight. Many substances exhibit a marked difference in other physical properties according t o the electrical state of their titratable groups. This difference is particularly important in their ultraviolet and visible absorption and in their behavior a t the dropping mercury cathode. Figure 8 shows the titrat@ curve for an antibiotic substance, aureomycin ( p K , 3.14, 7.33, and 9.24), and Table I shows the ultraviolet absorption data for the substance in each of its four electrical states. It can be seen that the effect of electrical state of ultraviolet absorption is profound in the case of this substance I n the investigation of the unknown structure of a new substance, potentiometric titration must precede determination of ultraviolet absorption or
V O L U M E 26, NO. 4, A P R I L 1 9 5 4 Table I.
Ultraviolet Absorption Maxima for Aureomycin pH 5.4
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polarographic behavior if these characteristics are to be obtained for each electrical state of the substance. The electrodes and electrometer used for titrations impose a limitation on the range of dependable p H values. This range may be extended by resort to more accurate but less convenient potentiometer circuits and by use of alternative but likewise less convenient electrode syqtenis. The range of valid pH values is further extended by u-e of smaller sample volumes and higher concentrations of acid or alkali used as titrant. Excellent data
on dissociation beyond this valid p H range can occasionally be obtained by measuring change in ultraviolet or visible light absorption or change in some other physical property as the niolecule dissoriates. Many organic substances exhibit inadequate solubility in n-ater to permit satisfactory titration. I n such cases it may be necessary to titrate the sample in a water-organic solvent mixture. Alcohols, dioxane. and diniethylformamide have proved useful for this purpose. Since pRd values will vary with different solvents, the data jn Figure 7 cannot be used for correlation with structure. LITERATURE CITED
(1) Cohn, E. J., and Edsall, .J. T., "Proteins, Peptides a n d .Imino Acids," p. 82, S e w Tork, Reinhold Publishing Corp., 1943. (2) Michaelis, L., and l I i e u t a n i , AI., 2. p h y s i b . Chem., 116, 185 (1925). (3) Mizutani. H., Ihid., 116, 350 (1925). (4) Simms, H. S., J . Phus. C'hem., 32, 1121 (1928). RECEIVED for review July 10, 195.3.
Accepted October 9 , I053
Pharmaceutical Control laboratory Record System GEORGE M. NAIMARK and ROBERT F. PRINDLE Strong
Cobb & Co., Inc., Cleveland 4, Ohio
A rapid, efficient pharmaceutical control laboratory record system is described. The system is based on two forms: an analytical "work sheet" and a marginally punched permanent record card, both of which contain the complete product formulation and job-identifying information. The marginal punching permits the coding of analytical and production information which aids in the critical evaluation of production and of laboratory operation.
T
HE authors have developed a record-keeping system for a
pharmaceutical control laboratory Yhich has proved extremely efficient. It evolved from a desire to create a system which xould emphasize ease of recording of analytical control data and simplification of the study of such accumulated data. The system makes use of two forms: a "work sheet" which has the dual function of indicating to the analyst that a specific assay is to be done and serving as the report form after the analysis has been completed; and a marginally punched card which acts as the permanent record of all analytical control data. An industrial analytical laboratory record system also based on a marginally punched card has recently been described ( 2 ) .
of a pharmaceutical product i? initiated by the creation of a manufacturing card which indicates product constituents, concentrations, manufacturing instructions, warnings of dangerous materials, customer's specifications, and all requisite jobidentifying data, such as customer, type of product, and code and lot number. At the time the manufacturing card is made, a duplicate copy is Ditto reproduced on the unprinted side of the control laboratory punched card (Figure 2). Having this complete and accurate copy of each manufacturing card in the control laboratory minimizes routine reference to the master files for the verification of product specifications. In addition, having the complete formula of each product aids the ag-
PUNCHED RECORD CARD
A marginally punched 6 X 8 inch Keysort card manufactured by the McBee Co., Athens, Ohio, serves as the permanent record of analytical control data for each product manufactured by the company. The card, which is printed on one side to minimize clerical preparation time (Figure l ) , is made of card stock which permits duplication on the blank side with a Ditto duplicator. I n this company, production
Figure 1. Punched Card for Permanent Record of Analytical Control Data
