cence." M. J. Cormier, D. M. Hercules, and J. Lee, Ed., Plenum, New York, N.Y., 1973, pp 427-449. (9) K. Gleu and K. Pfannsteil, J. Pract. Chem., 146, 137 (1936). (10) W. Langenbeck and U.Ruge, 5erDeut. Chem. Ges. 5, 70,367 (1937). (11) L. I. Dubovenko. M. S. Rigun, and V. 0. Bilochenko, Vim. Kiiv. Univ., Ser. Khim., 13, 25 (1972). (12) A. K. Babko and N. M. Lukovskaya, Ukr., Khem. Zhr.. 27, 519 (1961). (13) A. K. Babko and N. M. Lukovskaya, Zh. Anal. Khim., 17, 50 (1962). (14) W. A. Armstrong and W. G. Humphreys, Can. J. Chem., 43, 2576 (1965). (15) A. A. Ponomarenko. Tr. Mask. Obshchest. lspyt. Prir., Otd. Biol., 21, 165 (1965). (16) N. M. Lukovskaya, A. V. Terletskaya, and N. I. Isaenko, Zavod. Lab., 37, 897 (1971). (17) A. Steigmann, J. SOC.Chem. lnd., 61, 36 (1942). (18) J. Kubal, Chem. Listy, 62, 1478 (1968). (19) H. Ojima and R. Iwaki, NipponKagakuZasshi, 78, 1632 (1957). (20) A. K. Babko and I. E. Kalinichenko, Ukr. Chem. Zh., 31, 1092 (1957). (21) A. Dorabialska and A. Kalinowska, Rocz. Chem., 42, 1905 (1965). (22) A. G. Stepanova and E. A. Bozhevol'nov, Tr. Vses. Nauch.-lssled, lnst. Khim. Reactiv. Osobo,Chist. Khim. Veshchestv, 32, 384 (1970). (23) V. K. Zinchuk and A. I. Komlev, Zh. Anal. Khim., 28,616(1973). (24) D. T. Bostick and D. M. Hercules, Anal. Letts., 7, 347 (1974). (25) J. P. Auses and J. T. Maloy, Paper No. 89, presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 4, 1974. (26) E. H. Huntress, L. N. Stanley, and A. S. Parker, J. Chem. Educ., 11, 142 (1934).
(27) G. H. Ayers, "Quantitative Chemical Analysis," 2nd ed., Harper and Row, New York, N.Y.. 1968, p. 621. (28) W. L. Schumb, C. N. Satterfield, and R. L. Wentworth, "Hydrogen Peroxide." Reinhold, New York, MY.. 1955. (29) H. J. Bright and M. Appleby, J. Bid. Chem., 244, 3625 (1969). (30) H. A. Sober, Ed., "CRC Handbook of Biochemistry," 2nd ed., Chemical Rubber Co., Cleveland, Ohio, 1970, pp J195-197. (31) S. A. Levinson and R. P. MacFate, "Clinical Diagnosis," 7th ed., Lea and Fibiger, Philadelphia, Pa., 1969, pp 4-7. (32) W. R. Seitz. W. W. Suydam, and D. M. Hercules, Anal. Chem., 44, 957 (1972). (33) D. Keilen and E . F. Hartree, Biochem. J., 42, 221 (1948). (34) J. A. Johnson and R. M. Fusaro, Anal. Biochem., 13, 412 (1965). (35) E. C. Adams. Jr., R. L. Mast, and A. H. Free, Arch. Biochem. Biophys., 91, 230 (1969). (36) K. Diem and C. Lenter, Ed., "Scientific Tables,: CIBA-Geigy Ltd., Basle, Switzerland, 1973, p 673. (37) N. Matsaniotis, C. Daneltou-Athanassiadou. C. Katerlos, P. Hartokalis, and E. Apostolopouiou, J. Pediat., 78, 85 (1971). (38) W. T. Carraway and C. W. Kammeyer, Clin. Chem. Acta, 41, 395 (1972).
RECEIVEDfor review August 26,1974. Accepted November 4, 1974. This work was supported by the National Institutes of General Medical Sciences under grant GM-17913.
Comparative Study of Colorimetric and Fluorometric Determination of Thiamine (Vitamin B,) by an Automated Discrete-Sampling Technique John Y. Park Beckman Instruments, Iflc., P.O. Box 0 - W , Irvifle, Calif. 92664
Thiamine content In vitamin Preparations has been analyzed by employing a newly developed automated materials analyzer. In this study, two analytical methods were used in determining thiamine content under controlled condltions: the fluorometric method and the colorimetric method. Based on experimental results of the above two methods, discussions and comparative recommendations are given for rapid quality control work and general laboratory practlces. The fluorometric method is preferred in a case of low thiamine content and the acid-dye method preferred when there is high thiamine content. This is due, mainly, to the limitations of their inherent specificity and errors introduced by dilution to their analytical concentrations.
Because of the ever increasing number of analyses for drugs, vitamins, and nutritional products ( I ) , rapid and reliable analytical methods are becoming more essential in pharmaceutical, biological, and food industries. The assay procedure for thiamine determination described in the U S . Pharmacopeia XVIII (1970) ( 2 ) ,and the National Formulary XI11 ( 3 ) requires a great deal of analysis time for manual operation because of the chemical nature of the analysis involved. This makes tablet-by-tablet analysis a difficult task for chemical analysts, especially in cases of rapid control work. With a highly mechanized automatic instrument, coupled with various detection systems, the standard operations of analytical chemistry can be facilitated with precision and accuracy. Such an instrument has been developed ( 4 , 5 ) . 452
ANALYTICAL CHEMISTRY, VOL. 47,
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The assay method of thiamine recommended by the committees of the U S . Pharmacopeia and National Formulary is based on the thiochrome reaction-the so-called fluorometric method. This method is most widely used for thiamine determination in pharmaceutical quality control laboratories (6). Although the fluorometric method is the most specific of all the methods in general use for thiamine determination, the need to give close attention to the details of the procedure detracts from its use for rapid control work (7). On the other hand, while colorimetric inethods receive very little attention, a promising colorimetric method utilizing the basicity of thiamine has been reported recently by Das Gupta and Cadwallader ( 7 ) . The nature of the colorimetric method-namely, aciddye method, appears to be based on a simple acid-base reaction between the thiamine and bromothymol blue in a pH 6.6 buffer medium. When considering the most basic property of thiamine among the vitamins, the reaction could be ideally used for a determination of thiamine in vitamin preparations. Furthermore, the method seems to be convenient and simple to adapt for rapid analysis, especially for an automatic operation; as it gains merit from the simple acid-base reaction which produces the stable colored salt (the acid-dye salt). These two analytical schemes are shown on page 453. Therefore, the present study was carried out with the automatic instrumentation to develop automated procedures for both the fluorometric and the colorimetric methods, and to test their applicabilities to the thiamine assay in vitamin preparations especially intended for general practices. Included are discussions on the limitations of both
MARCH 1975
7171 Pipetter
Disruptor
>
Chemical Table
-+
-
Detector
*
c
Teletype
zj Computer I
I
A
Figure 1. Block diagram for the modular design of the automated materials analyzer system
70
60
50
40
ACID DYE SALTS
30
(1 1 Ratio) 20
m e t h o d s and recornmendations for practical a p p l i c a t i o n s t o v i t a m i n quality c o n t r o l work.
EXPERIMENTAL Instrumentation. T h e Beckman Automated Materials Analyzer (AMA 40) was used for this study. Shown in Figure 1 is the block diagram of the instrument. The general descriptions and the automatic characteristics of the instrument have been described in detail elsewhere ( 4 , 5). A Beckman DB-GT Spectrophotometer for absorption measurements and a Ratio Fluorometer for fluorescence measurements were used as a read-out module. which was equipped with a 10inch Potentiometric Recorder. In addition, a newly designed semimicro flow cell was used for fluorescence measurements. Although the characteristics and advantages of the new flow cell over the conventional fluorometer flow cell were described in detail ( 8 ) ,the main advantages are as follows: Versatile adaptability for automatic sampling systems; small cell volume (20.4 ml), with minimum source light scattering effects, allowing semi-micro sample analysis and less solvent for cleaning; and non-breakable features. It is well known that cross contaminations from one sample to the next are important. factors in physicochemical or chemico-analytical treatment of samples, which can be solid, liquid, or gas. This phenomenon appears in various forms depending on the chemical and physical nature of the samples and physical design of the instrument. Realizing this natural phenomenon, it has been a common laboratory practice to flush out the system (especially the flow-cell) with a sample to be analyzed in order to minimize the undesirable effect whenever it was found to be interfering with the final results. Consequences of such an inherent effect may be noticeable when handling a highly viscous solution with a continuous automatic mode. For example, a high density, viscous sample, isobutyl alcohol solution used in the thiamine determinations gives a higher effect than the aqueous quinine sulfate solutions. Figure 2 shows the observed cross-contamination effect which is much less than 1.0% with quinine sulfate solution. As a first approximation, the following equation can be written for such an effect:
10
0
.
~ - _
Figure 2. Performance test with quinine sulfate
The actual strip-chart recording for 2 blanks. 10 samples, 2 blanks. The arrow signs are placed where cross-contamination is most effective in the analytical automated system (for details, refer to text)
where p is the final cross-contamination effect, I C is a concentration difference between the experimentally measured and the expected concentration for a given sample, and C is the expected concentration of the sample (or standardj taken as reference for the calculation of p. T o minimize the effect of JC, the analyzer is designed on the basis of discrete-sampling technique. Furthermore, as shown in Equation 1, p is the function of AC: thus the term p becomes negligible between either samples or blanks. On the other hand, a pronounced increase of the term p will be apparent where a large concentration difference exists between two consecutive solutions (e.g., between a blank and a sample, or vice versa), if any contamination effect is present in an automated system. Therefore, it is convenient to add an extra standard a t the beginning and an ext,ra blank a t the end to compensate for the first standard reading and the last blank reading. p is expressed as %. As described in previous reports (5, 9), data reductions for the content uniformity test, the assay test, and the calibration curve were done by computer. Printed data and punched tapes were obtained from a teletype. The teletype was connected to the detector by means of an intercoupler which is capable of monitoring both time intervals between readings and the number of readings for an individual sample. Finally, the punched data were fed into an IBM 360 electronic computer by means of a time-shared terminal (CAILOS-FORTRAN). The results are shown in Figures 3 , 4 and 5 . ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
453
POTENCY
POTENCY
.................................................... . . . . . . . . . .
IO
80
90
100
................................................... . . . . . . . . . . . .
70
I20
110
1
99.4
I
2
98.3
2
103.1
3
101.7
3
103.9
4
98.9
4
98.7
5
98.9
5
102.5
6
99.4
6
98.7
103.4
7
100.7
8
100.1
8
101.9
9
98.3
9
97.2
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94.9
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99.2
................................................... 90
80
95.0
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115.0
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100
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W O E AV E
....................... . . . .
80
90
t
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120
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.o
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T h i a m i n e hydrochloride o r t
*
mononitrate tablet
1) Crush and dissolve in 2? HC1 solvent
2 ) Dilute i t with 2? HC1 solution 3) Addition of oxidizing agent
.*
.................................................... . . . . . . . . . .
0
5
LO
15
20
Thiochrome
25
Thiochrome
PRI ITHI AM I N E)
1) Extract with ethanolic isobutyl alcohol
Figure 4. Calibration curve (at Xabs = 420 nm) obtained by acid-dye method (the curve plot was prepared by computer-this is a photographic reproduction of computer output)
Sample tablets were crushed and dissolved simultaneously on a tablet-by-tablet basis. For fluorometric measurements, the Ratio Fluorometer was calibrated by using quinine sulfate standard solution; that is, the fluorescence reading was set a t 100%with the quinine standard solution, and 0% with 0.28% (v/v) sulfuric acid solution. After the fluorometer is calibrated, a concentration of quinine sulfate solution is taken and divided into 10 different T-cups (sample cups) for analyzing in an automatic mode, (e.g., successive dilutions and sampling into the fluorometer). The result is shown in Figure 2. Reagents. All chemicals used were ACS certified reagent grade. Necessary reagents for the colorimetric method were prepared according to the procedure given by Das Gupta and Cadwallader (7). Detailed procedures for reagent preparations described in U.S. Pharmacopeia XVIII were followed for the thiochrome method. Note that the oxidizing reagents (K3 [Fe(CN)e]in NaOH solution) should be used within 4 hours after preparation. The thiamine reference standard was obtained from U.S. Pharmacopeia, Bethesda, Md. Commercially-available thiamine tablets were used as samples. 454
110
Automated Procedure. Most manual analytical procedures can be easily converted into automated procedures, (working procedures). I t is imperative to point out that the principle of the assay method (in some specific cases, each analytical step) should be well understood prior to establishing automated procedures. This is necessary because of the fact that most automated analytical instruments are based on a semi-micro scale. The analytical principles of both fluorometric and colorimetric methods are summarized below. Fluorometric Analysis
i E 5 0 RBAV C E
22
100.6
100
Figure 5. Potency and assay plot results for thiamine determination by acid-dye method. (The plots were prepared by computer-this is a photographic reproduction of computer output)
Figure 3. Potency and assay plot results for thiamine determination by thiochrome method (the plots were prepared by computer-this is a photographic reproduction of computer output)
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an
ANALYTICAL CHEMISTRY, VOL. 47,
NO. 3,
Sampling into f l u o r o m e t e r In the development of an automated procedure, the number of dilution operations, stability of the oxidizing agent, and the isobutanol extraction process were considered to be of special importance. In addition, it is known that addition of excess amounts of an oxidizing agent destroys the final product, thiochrome. These analytically important factors lead to a considerable reproducibility-error (*5 10%) (IO),in fluorescence analysis. On the other hand, the linear analytical dynamic range was observed to be an order of magnitude greater than the colorimetric. Figure 6 gives an experimental setup diagram for fluorometric analysis based on 25-mg thiamine tablets. NOTE: In this analysis, small quantities of ethanol addition act as an effective agent for defoaming and clean phase separation during and after the extraction process. Colorimetric Analysis
-
T h i a m i n e hydrochloride ( o r mononitrate)
MARCH 1975
1) Crush and dissolve in pH 6 . 6 buffer
2 ) Dilute with pH 6.6 bmffer 3) Addition of dye reagent
Acid-dye salts
RP:
Reagent
Pump
FIL: F i l t e r MIX: M i x e r
SAM: S a m p l e P r o b e
TC: T-Cup TT: T-Tube
Figure 6. Automated analytical setup for thiamine determination by thiochrome method
Acid-dye salts
Sampling into spectrophotometer, read at 420 nm
The experimental setup diagram for the colorimetric method is shown in Figure 7, which is based on 50-mg thiamine hydrochloride tablets.
Since the dye reagent is prepared in CHC13 solvent, the extraction process requires no additional extracting solvent. However, special attention was given to the extraction process because the acid-base reaction is taking place in the solvent media (aqueous and organic phases) a t the same time to form the salts during the extraction process. This means that the amount of time for effective mixing of the two phases is the critical factor. Even though the efficiency of the mixer (mechanical stirrer) was excellent (5),a series of mixer probes were used as shown in Figure 7, t o give sufficient reaction time. The use of the increased number of mixer probes to meet the necessary reaction time is mainly due to the time of one full automatic cycle, because the time for a complete crushing and dissolution of the sample tablet is predetermined.
Fluorometric Method. The result shown in Figure 2 reveals two important analytical factors in automated fluorometric instrumentation; first, performance of the newly designed semi-micro flow cell for fluorescence analysis concerning cross-contamination from one sample to the next. The largest contamination expected is indicated hy an arrow in this figure. The observed effect is less than 1.0%in the case of quinine sulfate solution. Second, the repeatability of fluorescence measurements in the automated replicate analyses of ten identical samples (quinine sulfate solu-
1) Estraction of x i d - d y e salts into CHC13
RESULTS AND DISCUSSION
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
455
P-D: FIL:
Pipetter-Diluter
Filter
MIX: M i x e r
TC:
T-Cup
TT:
T-Tube
Figure 7. Automated analytical setup for thiamine determination by acid-dye method
Table I. Relative Fluorescence Intensity from Standards and Commercial Tabletsa Standards a 1 0 . a y l e t .
Fluorescence reading a
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88.5
88.5
88.0
87.0
90.0
87.5
87.5
88.0
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88.6
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84.0
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1
Fluorescence reading
88.2
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88.2
88.2
88.0
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456
ANALYTICAL CHEMISTRY, VOL. 47,
NO. 3,
MARCH 1975
88.3
6
7
5
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87.2
87.2
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10
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tion). Figure 2 reveals an excellent repeatable result [Relative Standard Deviation (RSD) < 1.0%], possibly due to the discrete sampling technique adopted for the automated analytical instrumentation used in this experiment. Following the automated fluorometric procedure (see Figure 6), commercial vitamin preparations containing 25 mg of thiamine hydrochloride were analyzed (see Table I). U.S.P. reference standards were analyzed in the same run, under the same conditions for the purpose of comparison and potency calculation of each tablet (see Table I). Based on these data, calculations of potency and assay tests were processed by computer. Figure 3 shows the results of these tests. Although tablet potency (94.9-103.4%) and assay (99.3%) test results are satisfactory, the experimentally determined potency spread of 8.5% is of particular interest. T o justify that the observed spread is due to the potency of each tablet, the remaining sample solutions were uniformly mixed in a large beaker and divided into ten different T cups (sample cups), and analyzed again automatically under the same conditions. The results are shown in Table 11. All ten samples gave the same fluorescence intensity with a mean deviation of f0.5%. This clearly presents strong evidence for the experimental result that the observed potency spread is due to the amount of thiamine hydrochloride contained in each tablet. Furthermore, this result also indicates reliable repeatability and precision of the instrumental functions. Colorimetric Method. Detailed principles for the colorimetric method have been given in the literature (7), which takes advantage of the most basic property of thiamine in vitamins. Thus, it is expected that the interference effect from the other vitamins may not be highly probable. Of particular interest in this method is the separation of the final product (the acid-dye salt which is formed from the acid-base reaction in the two different solvent media), by utilizing its solubility in the organic phase rather than in the aqueous phase in the same reaction tube. Even though the acid-dye salt has an ionic character, it is more soluble in the chloroform phase than in the aqueous phase because of the large molecular weight of the salt (7). In automating such an extraction, the discrete-sampling technique as described fully in previous reports (4, 5, 9) would be expected to be superior over other types of automatic techniques (e.g., continuous flow type, etc.). Furthermore, the successful use of a series of many mixer probes for achieving simultaneous extraction and completion of chemical reactions in the same reaction tube, demonstrates not only the excellent efficiency of mixing but also the versatility of the mixer probe (e.g., back extractions and multiple extractions are possible). With the automated procedure described in the previous section, the calibration curve for thiamine hydrochloride was obtained and is presented in Figure 4. The linear response between 2 pg/ml to 12 pg/ml is evident from the calibration curve. Although the absorbance values between standards were observed to be identical within the experimental error (RSD < 1.0%),some variations (potency spread 6.6%) in ten individual tablet analyses of commercial thiamine preparation can be seen in Figure 8. Based on these values, calculations and plots of content uniformity test and assay test were processed by computer. The results are shown in Figure 5 . In general, reproducibility and precision are observed to be quite satisfactory (in most instances, errors of repetitive determinations were less than 2.0% (RSD). Studies on thiamine determination have been extended to various multi-vitamin preparations. I t was found that two types of interference had occurred; increase or decrease of the absorption intensity. This may well be due to the na-
4 300 A
Figure 8.
The actual strip-chart recording for thiamine determination
by acid-dye method; 2 blanks, 1 standard, 10 tablets, 3 blanks
ture of the compositional constituents in multi-vitamin preparations (note that, in general, most multi-vitamin preparations contain 10 mg or 25 mg of thiamine hydrochloride). Furthermore, the amounts of the analyte (thiamine) that were of interest compared t o the other components in the sample would make the final result vary from one sample to the other. This observation indicates that the colorimetric method is not highly specific, even though it gave reproducible absorption responses (increased or decreased) for a given sample. Therefore, in adapting this method for an analysis of multi-vitamin preparations, background corrections are necessary. This is possible when using a placebo as a blank reference, if the nature of the chemical interferences with this method is known for a given multi-vitamin preparation. (The term “placebo” in Pharmaceutical Analysis Laboratories refers to a blank reference material which is a material containing every component except the component to be determined.) CONCLUSIONS Both fluorometric and colorimetric assay methods work equally well with the automated procedures described in this report, particularly for single thiamine tablet analyses. However, because of the highly sensitive nature of the fluorescence method compared to the absorption, erors introduced by successive dilutions to a working concentration (pg range) may make the final results vary more significantly in fluorescence measurements than in absorption from one sample to the other. This effect becomes obvious when analyzing high thiamine content tablets. In addition, the fluorometric method required double analysis timethat is, blank readings and sample readings; whereas the colorimetric method appears to be simple and rapid requiring single analysis time. Although detailed advantages and disadvantages for both methods are not given here, the colorimetric method is preferred to the fluorometric in the case of high thiamine content analyses (concentrations above 50 mg of thiamine), because of the relatively less sensitive nature of the absorption method to the fluorescence method. Furthermore, the analysis rates have been observed to be forty readings per hour for both methods. This means that forty samples per hour with the colorimetric method are possible. With the fluorometric method, it would take two hours for forty samples. Therefore, this colorimetric method should be used in applications whenever rapid analysis of high potency thiamine (above 50 mg/tablet) is desirable. For multi-vitamin analysis, the fluorometric method is advantageous over the colorimetric method because the blank measurement is, in essence, like using a placebo, while the colorimetric method may need the use of a placebo to improve the final results. Finally, special applications with the automated materials analyzer utilizing a discrete-sampling technique would provide not only an effective and efficient inter-laboratory collaboration work for food and drug analyses, but would
A N A L Y T I C A L CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
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eliminate the possibility of human errors and also facilitate manual analytical processes much more effectively.
J. Ramirez-Munoz, Anal. Chim. Acta, 71,321-331 (1974). T. Higuchi and E. Brochmann-Hanssen, "Pharmaceutical Analysis," Interscience, New York, N.Y., 1961. V. Das Gupta and D. E. Cadwallader, J. fharm. Sci., 57, 112 (1968). J. Y. Park, paper presented at the Pitisburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 6, 1974, Cleveland, Ohio. (9) J. Y. Park, submitted to J. Pharm. Sci., "Determinations of vitamin A and vitamin E in vitamin tablets or capsules by discrete sample automatic analysis." (10) Rolf Strobecker and Heinz M. Henning, "Vitamin Assay Tested Methods," Verlag Chemie, Weinkeim, Germany, 1966, pp 65-97.
ACKNOWLEDGMENT The author thanks Juan Ramirez-Muiioz and W. F. U1rich for productive discussion and suggestions. LITERATURE CITED (1) H L. Reynolds, H. P. Eiduson, J. R. Weatherwax, and D. D. Dechert, Anal. Chern., 44, (13), 22A-34A (1972). (2) U S. Pharmacopeia XVIIi, U S . Pharmacopeia Convention, Inc., Bethesda, Md, 1970. (3) National Formulary XIII, American Pharmaceutical Association, Washington, D.C., 1970. (4) D. G. Rohrbaugh and J. Ramirez-Munoz, Anal. Chim. Acta, 71, 31 1-320 (1974).
RECEIVEDfor review June 3, 1974. Accepted October 25, 1974. Presented in part a t the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1974.
Systematic Identification of Unknown Drugs in Powder Form by Means of Ultraviolet Spectrophotometry in Forensic Toxicology Mark Martens, Frank Martens, Patrick Maenhout, and Aubin Heyndrickx Department of Toxicology, State University of Ghent, Hospitaalstraat 13, 8-9000 Ghent, Belgium
Since rapid and safe determination of unknown pharmaceutical powders has become a need, several laboratories have designed identification systems by means of punched card systems. It is our aim to attempt identification by using only the electronic properties of the unknown molecule. Using buffers of pH 2, 7, and 12 and solvents of different polarity as ethanol and n-heptane, it is possible to build up a pharmaceutical Keydex punched card system, which offers the possibility of determining unknown powders by 125 UV criteria. By optical coincidence of the punched cards, carrying the UV information, one molecule can be selected out of hundreds. Afterwards, additional GLC and TLC analyses may affirm Its identity.
The increasing amount of drugs, being seized by the police daily, has made a fast, accurate, and simple method indispensable for the determination of narcotics and psychoanaleptic pharmaceuticals. Usually these samples are delivered as crystalline or amorphous powders. In such cases, it is a nearly impossible task to identify unambiguously the unknown powder by means of a common spot test and a single UV spectrum, taking into consideration the enormous variety of pharmaceuticals. Lowell W. Bradford and James W. Brackett ( I ) built up an identification system based on the comparison of UV spectra and the computation of the specific absorptivities. By taking advantage of the electronic properties of a series of chemical compounds in alkaline and acid solutions, further differentiation of unknown substances is described. But many questions may arise about the system: To what extent do the unknown molecules change their electronic behavior as a function of the polarity of the solvent used? What is the reproducibility of the UV spectra of pharmaceutical compounds extracted by a water-solvent equilibration system, when putrefied human forensic samples are considered? To this last question, one can only answer that UV spectrophotometry is an inadequate tool in the analysis of biological samples. TLC, GLC, and GC-MS are undoubtedly 458
the only means to obtain plausible solutions to this particular problem. Furthermore, if a classification has to be set up, one must consider the possibility of extending the system with new compounds, without disturbing the uniformity. Charles McArdle (2) designed an efficient identification system for tablets by means of a punched card system. He chose color, size, marking, and taste as identification criteria for tablets. Later on, the Anti-poison Center of Nancy, France, developed a more complete system, also based on marginal perforation. This laboratory switched over to the optical coincidence punched card system recently ( 3 ) . At the Metropolitan Police Laboratories in London, optical coincidence systems have been in use for a long time. Not only the morphology of tablets is registered, but also the electronic spectra of the active substances in alkaline and acid media ( 4 ) . On the basis of the systems mentioned, we set up a method to determine unknown powders by using the electronic properties of the compound only. The Frank-Condon principle states that during the electronic transition, atoms do not move. Electrons, including those of the solvent molecules, may reorganize. Most transitions result in an excited state, more polar than the ground state; the dipole-dipole interactions with solvent molecules will, therefore, lower the energy of the excited state more than that of the ground state. Thus, it is usually observed that ethanol solutions give longer wavelength maxima than n- heptane solutions do. The weak transition of the oxygen lone pair in ketones, the n T * transition shows a solvent effect in the opposite direction. The solvent effect is now due to the lesser extent to which solvents can hydrogen-bond to the carbonyl group in the excited state. In case of potentially tautomeric molecules, the change in the absorption maxima with the change of the p H is due sometimes to a change in the chromophore as a result of the tautomerism and sometimes to simple protonation or deprotonation. Therefore it was necessary not only to consider acid-base shifts in aqueous media but also the change of the curve shape ii; solvents of different polarity. Furthermore, the information, obtained from the interpretation of the UV
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
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