Automated, Stepping Differential Calorimeter for the Analysis of Purity J. Zynger Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, IN 46206
An automated, stepping differential calorimeter is described for quantitative analysis on an absolute basis. The lnstrument consists of an electronic sequencer which controls both a Perkin-Elmer DSC-1B Calorimeter and a digital integrator/printer. A computer program which facilitates the data analysis is also described. When compared to conventional differential scanning calorimetry, the instrument is shown to have Increased sensitivity, and applicability to a much wider purity range. The instrument has been evaluated using mixtures of phenacetin containing various amounts of benzamide as an impurity. Over the 92-100% purity range, the average precision for quantitative analysis is 0.5 %; the average precision for determining the heat of fusion is 4%.
The colligative properties of a compound have long been recognized as a sensitive and accurate measure of purity. Recently, Watson and coworkers (1, 2) described a procedure for determining purity that is based on the information contained in a thermogram, generated by melting a sample in a differential scanning calorimeter (DSC). The procedure involved manually segmenting the melting curve so that fractions of the sample melted a t various temperatures can be measured. Assuming ideal behavior between the components, the inverse of the fraction melted when plotted vs. the temperature should yield a straight line that is defined by the van’t Hoff equation (3):
where T,= instantaneous sample temperature, To = melting point of the pure compound, R = gas constant, AHf = heat of fusion, X = mole fraction of impurity, and F = fraction melted a t T,. This technique has been critically evaluated (4-12) in view of both its theoretical and instrumental limitations. One major criticism arising from these studies has been that the DSC analysis is limited to concentrations of eutectic impurities not exceeding about 1 mole %. Hence, the technique exhibits only a 1%total dynamic range. Various methods have been described which attempt to extend this working region. DeAngelis and Papariello (5) and Schumacher and Felder (13) have suggested that one should dilute the impurity in an impure sample by adding “pure” main component, until the sample is purer than 99%. This procedure is not only time consuming, but also it requires that one have access to “pure” main component. Rossini (3),using adiabatic calorimetry, has shown that accurate analysis can be performed directly on the sample even a t impurity levels exceeding 5%. Also, Staub and Perron ( 1 4 ) have recently demonstrated that a stepped heating technique dramatically extends the working region for purity determinations. However, some controversy exists as t o the rationale responsible for the extension (15). Nonetheless, it appears that the 1%impurity limitation observed for the DSC technique is instrumental rather than theoretical in nature. 1380
ANALYTICAL CHEMISTRY, VOL. 47, NO., 8, JULY 1975
Using a temperature stepping approach, an automated stepping differential calorimeter, based upon a PerkinElmer DSC-lB, is described for the absolute analysis of purity. A computer program is discussed which facilitates the data reduction and which makes maximum use of the theoretical and instrumental advantages obtained by this procedure. Data obtained for a variety of compounds are presented which illustrates the precision and accuracy of the new instrument and which demonstrate that a purity of as low as 92% can be accurately determined.
EXPERIMENTAL Instrumentation. A block diagram of the instrumentation used is shown in Figure 1. A Perkin-Elmer Model DSC-1B calorimeter was modified by replacing the eight-position scan-speed selector knob with a nine-position selector switch. The first eight positions serve the identical functions as in the original instrument. The ninth position (REMOTE) disengages the timing circuitry of the instrument and places the stepping motor of the temperature programmer under the control of the external electronic sequencer. For all the data to be presented, the DSC is, operated at its most sensitive range setting of 1 mcal/sec (10 mV full scale). The output from the calorimeter is connected to a Honeywell Model 194 potentiometric recorder and to an Infotronics Model CRS-100 digital integrator, equipped with a printer. The integrator is used to integrate and record the energy absorbed by the sample, due to each temperature increment. The integrator is operated in its “manual” mode and as such serves to continually digitize the analog signal from the DSC-1B. For all the data that will be presented, the count rate of the integrator is set a 4 kHz/mV; the base-line tracking rate is held at 6 mV/min; and the voltage threshold level is set at 25 pV. To facilitate the data logging, an analog representation of the temperature, available from the DSC-lB, is converted into the digital domain with a Hybrid Systems Model 535-3-BCD analog to digital converter (ADC). The ADC output replaces the original “elapsed time counter” of the integrator and, therefore, both the energy absorbed together with the temperature are automatically recorded. The points in time at which integration begins and ceases are under the control of the external sequencer. A circuit diagram of the logic system which controls both the integrator and the calorimeter is shown in Figure 2. A system clock (Heath Co., Model EU-800-KC) provides the requisite frequencies to the logic. The circuit elements are mounted on two breadboard cards (Heath EU-
RECORDER
Step
Senlng
t
.f
PRINTER/ COMPUTER U Area
Figure 1. Block diagram of
I
the instrumentation
Figure 2. Circuit diagram of the digital sequencer and controller
50-MG) which are housed, together with the clock, in a Heath EU800-D Instrumentation ADD. The ADD provides the push button (PB) by which the analyst controls the system. Basically, the logic controller functions to connect the 50 Hz output of flip flop 4 (FF4) to the stepping motor of the calorimeter for a length of stepping time that the analyst has set into counter 1 (Cl) using the ABCD push button preset. Step times can vary from 0.1 to 1.5 seconds, which give rise to temperature increases of from 0.056 to 0.84 K respectively, in intervals of 0.056 K. Initial turn-on is accomplished through PB1. At the start of a run, the sequence of operations is manually initiated using PB2. This causes the “integrate” line to go to the low state which forces the integrator to establish a base line for the measurement by tracking the calorimeter output for 16 seconds. The temperature is then incremented by the amount corresponding to the step setting. Simultaneously, the “integrate” line is released, which allows integration to begin, and the voltage representing the temperature is strobed into the ADC. Following a 16-second delay, during which integration continues, the logic monitors the frequency of the integrator’s voltage-to-frequency converter on the “count” input line. When this frequency drops below 10 Hz (27.5 fiV above base line), the digitize line goes to the low state and integration is halted. The integral and the temperature are printed and the entire process is repeated by establishing a new base line in the next 16 seconds. The system continues to run unattended through the sample’s melting temperature until PB1 is again depressed. Procedure. Samples to be analyzed are normally weighed, between 2 and 4 mg, to an accuracy of 10 fig and are then sealed in aluminum sample pans using a Perkin-Elmer volatile sample sealer. Both the temperature axis and the energy axis (integrator counts) are calibrated using a known weight of indium wire (99.999%, K&K Laboratories). Before beginning the step operation, the sample is scanned through its melting transition a t 2.5 K/min to establish the melting range. The range is then used to compute the step setting necessary to obtain about 20 temperature increments within the melting region. Depending on the purity of the sample, the stepping experiment requires between one-half and one hour of instrument time. After completing the experiment, the analyst enters the logged data to a computer program that is described in the next section.
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TIME IMINUTESI 16 20 24
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28
4000
32
4050
.
-
36
40
4100
TEMPERATURE (OEG K I
Figure 3. Thermogram obtained by continuous melting of a 96.04
mole YO phenacetin sample
RESULTS AND DISCUSSION Figure 3 shows a thermogram obtained by scanning through the melting region of a 2.75-mg sample of phenacetin, containing an impurity of 3.96 mole % benzamide, a t a rate of 0.625 K/min. From the curve, it appears that melting begins a t 395.0 K and ends a t 405.3 K. Figure 4 was generated by stepping the identical sample through its melting transition a t a rate of 6 steps/5 K. The integral of each peak is listed in Figure 4. Integration begins when the rising portion of the signal reaches the previously determined base line. Integration is halted when the falling portion returns to base line, and a base line for the next peak is then established. The initial 11 peaks and the last 3 peaks arise from the difference in the heat capacities (AC) of the sample and reference pans. The integral of each of those 14 peaks is, therefore, merely the differential heat capacity energy (AQ) that is proportional to the temperature increment ( A T ) . Since the temperature step is fixed, AQ will remain essentially constant over the melting ANALYTICALCHEMISTRY, VOL. 47, NO.
8 , JULY 1975
1381
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Figure 4. Thermogram obtained by stepwise melting of the 96.04 mole % phenacetin sample (note time is not linear with temperature). Peak areas are expressed in thousands
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Figure 5. Computer generated fit for the data represented in Figure 4
region. Applying the calibration constant obtained for indium (27.6 mcal/million counts), the data in Figure 4 shows that AQ equals 1700 f 100 Gcal. From the integrals shown in Figure 4, it is apparent that melting begins a t 385.0 K and ceases a t a temperature not higher than 405.0 K. With temperature stepping, melting is detected at a lower temperature. In fact, the energy not observed in Figure 3 by temperature scanning (the initial 13 melting peaks in Figure 4, peaks 12 through 24) corresponds to about 8% of the total fusion energy. Not only is the stepping technique more sensitive but also, since complete thermal equilibrium is established after each peak, the analyst need not be concerned with the thermal resistance (Ro) of the system. In the scanning 1382
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
method, Ro is of major concern and calibrations and corrections must be made to account for the thermal lag in the system (16) which causes the sample temperature to lag behind the observed temperature. This is indicated by comparing the final melting temperatures in Figures 3 and 4. Since the melting process is an isothermal transition, the thermal resistance and the thermal lag of the sample change drastically as melting proceeds. A knowledge of Ro is required in the scanning technique and since it can only be determined by inference ( I , Z ) , the scanning technique mandates that only the initial 10 to 50% of the melting peak be used for calculation. On the other hand, an analysis using the stepping technique utilizes all the data, especially the latter 50% of the melt where most of the purity information is concentrated, and where sensitivity limitations are the lowest. Even though a larger percentage of the fusion energy is observed by temperature stepping, a fraction of the fusion energy still remains unrecorded due to the 40 wcal/sec threshold level of the DSC-1B (2). T o account for this unrecorded energy, a computerized linearization procedure is used. The logged data are entered to a computer via a teletypewriter. The program which is written in Extended Basic for a PDP-10 time-share facility, subtracts the average heat capacity energy from each melting integral. It then uses a least-squares procedure to fit the data to Equation 1. The program corrects the total heat of fusion obtained by 0.5% increments to a 50% maximum correction and recalculates the impurity concentration. The best value for AHf and X is obtained when the F test (17) of the least-squares fit is a maximum. Since the fusion energies of the initial peaks are relatively small, they contribute the greatest amount of uncertainty to the fit. The program, therefore, iterates through the correction and fitting routine by successively disregarding each initial peak in the linearization procedure. If the initial data are good, the F value should decrease; conversely, if the data are bad, the F value should dramatically increase. Therefore, the upper and lower limits for the fraction melted are not chosen arbitrarily as in the scanning technique (2, 5-10), but rather they are functions of the data itself. The slope of the line obtained with the maximum F value is used to determine the heat of fusion and the purity of the sample, according to Equation 1. Figure 5 shows the computer output for the data represented by Figure 4. An 8.5% correction was necessary to produce the results shown. The “X” shown at 406.0 K repre-
/
~
Table I. Results for Phenacetin Samples Spiked with Benzamide Impurity Prepared p u i t y , %
Found,
%
99.61 99.6 99.20 99.2 98.45 98.4 98.6 98 -21 97.75 98 .O 97.60 97.9 97.14 97.0 96.51 96.5 96.04 96.3 95.90 96.5 95.39 95.5 95.10 96.0 94 -64 95.5 93.94 94.5 93.18 92.9 92.70 93.0 92.04 92.6 91.54 94 .O 90.66 92.4 84.70 90.0 Relative standard deviation for three determinations.
R S D , %a
Error, %
0.1 0.1 0.3 0.1 0.3 0.7 0.6 0.7 0.2 1.1 0.6 0.4 0.7 0.9 0.6 0.9 0.9 1.0 2.7 1.o
0.0 0 .o 0.1 0.4 0.3 0.3 0.1 0.0 0.3 0.6 0.1 0.9 0.9 0.6 0.3 0.3 0.6 2.7 1.9 6.2
A H f found
(callmole)
A H f - ~ s % ~ ,
7630 7600 7260 7210 7210 7080 7370 7790 7480 7490 7120 7320 7600 7550 7400 7460 7200 6820 7410 6750
5.2 5.8 5.9 0.8 4.9 3.1 1.9 3.5 3.8 4.1 3.4 3.2 1.9 3.8 3.3 3.8 2.8 7.9 13.9 4.1
-Table 11. Comparative Purity Results on an Investigational Herbicide Lot so,
DSC
1 99.7 2 98.7 3 98.2 4 96.3 5 94.4 Gas-liquid chromatography.
PREPARED PURITY ( M O L E %) Figure 6. Comparison of purity obtained by iterating to find and by using AH, = 7 6 1 0 ( 0 )
LYf(0)
sents To in Equation 1. The calculated purity is 96.35 mole the calculated A H f is 7645 cal/mole. A binary mixture consisting of phenacetin “spiked” with various percentages of benzamide was chosen to evaluate the instrument with regard to precision and accuracy. Benzamide was weighed with an accuracy of 0.2 wg on a microbalance (Cahn-Ventron, Model G-2), directly into preweighed phenacetin containing pans. Three determinations were performed on each prepared sample, and the results of these experiments are listed in Table I. The data indicate that the precision and the accuracy of the impurity determination remains essentially constant over the 92-100% purity range. Over this range, the average accuracy is about 0.3% with an average precision of about 0.5% in the main component. From the data, it can be seen that the method becomes unacceptable at impurity levels exceeding 8%. This results from the breakdown of the simplifying assumptions used to obtain Equation 1. % and
‘5 H t
CLCO
6430 6160 6170 6370 6370
99.8 98.6 98.5 96.7 93.7
The average value of the 3 determinations for AHf and its precision, a t each purity level, are also listed in Table I. These data indicate an average precision and accuracy of about 4% in the determination of AHf over the useful range of this procedure. T o determine if the prior knowledge of AHf would improve the accuracy of the analysis, the computer program was also instructed to calculate the purity of each test mixture, based on AHf equal to 7610 cal/mole ( 1 5 ) . Figure 6 illustrates the graphical comparison of the results of this experiment with the data tabulated in Table I. The data indicate that only a slight improvement in accuracy is obtained when one has prior knowledge of Hf. Also, note that the average calculated purity was within 1% of the prepared purity in all cases. The instrument has now been routinely used to perform over 200 purity determinations on some 40 different compounds. Table I1 lists the results obtained using the present system for the analysis of various lots of an investigational herbicide. Table I1 also lists comparative data obtained by gas chromatographic analysis. The good agreement between the analytical techniques is apparent from the data.
ACKNOWLEDGMENT The author thanks Marc Anderson for obtaining some of the DSC data and Charles Doty for obtaining the gas chromatographic data.
LITERATURE CITED (1) E. S.Watson, M. J. O’Neill, J. Justin and N. Brennen, Anal. Chem., 36, 1233 (1964). (2) Perkin-Elmer Corp., Thermal Analysis Newsletter, No. 5 and No. 6.
ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
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(3) F. D. Rossini. "Chemical Thermodynamics, "University of Notre Dame, Notre Dame, IN, 1949, pp 89-1 11. (4) R. Reubke and J. A. Mollica, Jr., J. Pharm. Sci., 56, 822 (1967). (5) N. J. DeAngelis and G. J. Papariello, J. Pharm. Sci., 51, 1868 (1968). (6) C. Plato and A. R. Glasgow, Jr., Anal. Chem.. 41, 330 (1969). (7) G. L. Driscoll, I. N. Duling. and F. Magnotta in "Analytical Calorimetry". Vol. I, R. S. Porter and J. F. Johnson, Ed.. Plenum Press, New York, 1968, pp 271-278. (8) G. J. Davis and R. S . Porter, J. Therm. Anal., 1, 449 (1969). (9) E. M. Barrall II and R. D. Diller, Thermochim. Acta, 1, 509 (1970). (IO) E. F. Joy, J. D. Bonn and A. J. Barnard, Jr., Thermochim. Acta, 2, 57 (1971). (11) D. L. Sondack, Anal. Chem., 44, 888 (1972)
(12) E. E. Marti, Thermochim. Acta, 4, 173 (1972). (13) R. Schumacher and B. Felder, Fresenius' 2. Anal. Chem., 254, 265 (1971). (14) H. Staub and W. Perron, Anal. Chem., 46, 128 (1974). (15) Perkin-Elmer Corp. Thermal Analysis Application Study, No. 3 and No. 10. (16) H. M. Heuvel and K . C. J. B. Lind, Anal. Chem., 42, 1044 (1970). (17) H. A. Laitinen, "Chemical Analysis," McGraw-Hill, New York, 1960, pp 537-578.
RECEIVEDfor review December 13, 1974. Accepted March 25, 1975.
Determination of Sulfonamides of Pharmaceutical Importance by Catalytic Thermometric Titration Edward J. Greenhow and Leslie E. Spencer Depafiment
of Chemistry, University of London, Manresa Road, London, S W3 6LX, England
Tetra-n-butylammonium hydroxide, sodium methoxide, potassium methoxide, and potassium hydroxide, in nonaqueous solution, and pyridine, dimethylformamide dimethylsulfoxide, 1,1,3,34etramethylurea, and N,N,N',N'-tetramethyll,2-dlaminoethane, have been evaluated as titrants and sample solvents, respectively, in the determination of the acidic functions of sulfanilamide derivatives and sulfonamide formulations of pharmaceutical importance by catalytic thermometric tltration. When acrylonltrlle Is used as the thermometric indicator, both the end-point sharpness and the reaction stoichiometry corresponding to this end point are influenced by the nature of the titrant and the sample solvent, particularly in determinations of sulfanilamide and sulfaguanidine. Satisfactory assay values are obtained with potassium hydroxide in propan-2-01 as the titrant, acrylonitrile as the solvent for sulfanilamide, dimethylsulfoxide as the solvent for sulfaguanidine, and dimethylformamlde or dlmethylsulfoxlde as the solvent for other sulfonamides. Preclslons of better than 1 and 2 % have been obtained with 0.01 and 0.001 mequlv samples, respectively.
Various titrimetric methods are, or have been, recommended for the routine assay of the sulfonamides of pharmaceutical importance and the sulfonamide content of pharmaceutical formulations. Titration with sodium nitrite solution, to determine the aromatic amine function, is now the most widely used assay procedure (1-3), but titration of acidic hydrogen in the sulfonamide group is an acceptable alternative method (4-6). According to Garratt (7), sulfisoxazole (sulfafurazole) cannot be determined satisfactorily by the nitrite method, and this compound and the related sulfamethoxazole are currently assayed (1, 2 ) by nonaqueous acid-base titration using a visual indicator to locate the end point. The latter procedure was previously recommended for the determination of sulfamethoxypyridazine ( 2 ) . Conventional thermometric titration has been used for the determination of sulfonamides in aqueous solution. The reactions employed include diazotization with 0.1M sodium nitrite ( 8 ) , oxidation with 0.5M sodium hypochlorite ( 9 ) , and the formation of silver derivatives with 0.3M 1384
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
silver nitrate (10). Catalytic thermometric titrimetry, in which the heat evolved during the alkali-catalyzed anionic polymerization of acrylonitrile is used to establish the end point, has been shown to be suitable for the determination of carboxylic acids, phenols, and other weak acids in nonaqueous solution (11).The polymerization is initiated by a small excess of the alkali titrant, after the acid sample has been neutralized:
+ CH2=CHCN HOCH2CH-CN HOCH2CH-CN + nCHz=CHCN OH-
+
+
HO(CHzCHCN),CH&H-CN AHp = -18.3 kcal mol-1 (of monomer) An evaluation of the use of this technique for the assay of sulfonamides, the subject of the present study is, in addition to its practical relevance, of theoretical interest because the sulfonamides represent a convenient series of weak acids suitable for a systematic investigation of the factors influencing the titrimetric process. In the earlier investigation, it was observed that, with some compounds, the end points corresponded to sub-stoichiometric neutralization reactions. For example, the titrant:acid molar ratios a t the thermometric end point were 0.17 and 0.69 when solutions of succinimide in dimethylformamide were titrated with 0.1M tetra-n-butylammonium hydroxide in toluenemethanol and 0.1M potassium hydroxide in propan-2-01, respectively. In the titration of 4-methyl-2,6-di-tert-butylphenol, in which the acidic function is sterically hindered, the corresponding reaction stoichiometries were 0.5 and 0.73. In the present study, pyridine, dimethylformamide, dimethylsulfoxide, 1,1,3,3-tetramethylurea and N,N,N',N'tetramethyl-1,2-diaminoethanehave been evaluated as solvents in combination with four titrants, namely, tetran-butylammonium hydroxide, sodium methoxide, potassium methoxide, and potassium hydroxide. The first three solvents are widely used in nonaqueous potentiometric and indicator titrations of weak acids, while 1,1,3,3-tetramethylurea has been evaluated (12) as a solvent in the determination of sulfonamides by these techniques. N,N,N',N'-tetramethyl-1,2-diaminoethanewas investigated as a repre-
