Comparison of Transmittance and Reflection Spectra of the 2,4-Dinitrophenylhydrazones of Acetone and 4-Met hy l-2-penta none HARRY ZElTLlN and ALICE NllMOTO Chemistry Department, University o f HawaiE, Honolulu 7 4, Hawaii
b The reflection spectra of the 2,4dinitrophenylhydrazones of acetone and 4-methyl-2-pentanone in the powdered state were measured and compared with the transmittance spectra in ethyl alcohol. Similar comparisons were made with these compounds adsorbed as solids and in solution on various types and grades of aluminum oxide, silica gel, and filter paper. The spectral characteristics are retained, in most cases, in reflectance, although relatively constant bathochromic displacements take place in going from transmittance to reflectance. The emergence of characteristic spectra of the hydrazones in situ on the adsorbents studied i s attributed primarily to the fine state of subdivision of the adsorbate. The spectral displacements observed are not inherent in reflectance but are due to a state of subdivision.
L
on the use of absorbance (or transmittance) spectrophotometry for qualitative and quantitative purposes is voluminous. I n contrast, there are few accounts of simiIar applications of reflectance spectrophotometry because of the lack of detailed knowledge regarding the mutual relationship of transmittance and corresponding reflection spectra. Comparative studies of the reflection and corresponding transmittance spectra of various classes of compounds have been reported ( 1 , 9, 12, 17, 19, 21, 22, 26). I n most cases characteristic reflection spectra were obtained in which the optical peaks were displaced bathochroniically when compared n ith the transmittance spectra of the same compounds in solution. On the other hand, Fischer and Vratny (a),Yamaguchi et al. (24) n-orking with dyes on filter paper, and SaughLon, Frodyma, and leitlin (15) in a study of the heme pigmmts in tuna meat obtained reflection speclra in which no spectral displacements of the optical peaks were noted. This study has steinnied from conflicting and limited data concerning spectral displacenients in going from transmittance to reflectance, lack of ITERATURC
knowledge concerning the factors influencing them, and the need for an evaluation of the scope and limitations of spectral reflectance as an analytic tool. Shibata (19) has recently presented theories on the spectrophotometry of translucent and opaque materials. This paper compares the transmittance and reflection spectra of the 2,4dinitrophenylhydrazones of acetone and 4-methyl-2-pentanone. These are members of a well known and readily available series of compounds which possess characteristic spectra (4, 1 1 , 23) ; many methods for their separation by column and paper chromatography have been developed ( 5 , 7 , 14, 16). An early reflectance study by Yamaguchi and associates (25) of various members of this series was confined to the visible portion of the spectrum which is virtually featureless. This study is concerned with the ultraviolet range and is limited t o compounds reported to exist in one crystalline modification (6) , thus eliminating uncertainties that might arise from polymorphism. The effect of the particle size of the hydrazones and of various adsorbents is assessed by a comparison of the reflection spectra of the powdered hydrazones with the reflection spectra of the same compounds spotted on filter paper and mixed as solids and in solution with these adsorbents. EXPERIMENTAL
EQUIPMENT AND REAGENTS.All spectral measurements were made with a Beckman DU spectrophotometer equipped with a multiplier phototube and standard reflectance attachment. One-centimeter silica absorption cells were employed for transmittance studies. All chemicals mere C.P. or reagent grade. 2,4 - DINITROPHENY L H Y D R A zo N E s (DNP). The hydrazones mere prepared according to the method of Brady (3) and recrystallized several times from 95% ethvl alcohol. Acetone-2.4-DKP: orange geedles, melting point 125-6' (uncorr.), lit. 122-4' (6); A, ethyl alcohol 230. 260, 360 mu. lit. 230. 255. 362 mp (4). 4-Methyl-2-pentanon& D N P : orange-red needles, melting point 91-2.5' (uncorr.), lit. 92-4'
(6); , , ,A ethyl alcohol 230, 265, 360 mP ADSORBENTS.Three categories were chosen because of their use in the chromatographic separation of hydrazones. All except the filter paper were activated by heat prior to use. The acid adsorbent was silica gel, 40 to 60 mesh, > 140 mesh (Braun-KnechtHeimann Co.) ; the basic adsorbent, aluminum oxide, was Alundum, GO mesh (General Chemical Division), 90 mesh (illlied Chemical & Dye Corp.), and acid-washed, basic, and neutral chromatographic alumina (Woelni, activity grade 1). Whatman XOS.1, 3 MM., and 4 filter papers were u s d . PREPARATION OF POVDERCD S-411PLES. Copper screens of known mesh were used for screening samples. Finely powdcred samples were prepared by grinding with an agate mortar and pestle. MEASUREMEKT OF REFLECTION SmcTRA. Samples were placed on unglazed porcelain plates or packed into aluminum planchets and the spectra measured, where possible, from 220 to 600 mp. Samples and reference standards were taped to the stage of the reflecb ance attachment to minimize errors introduced by the movement of the samples. Samples of powdered hydrazones were placed on porcelain plates completely covering the area to be exposed to the light beam to minimize the reflection of light from the surface and the spectra were measured using a similar porcelain plate as reference. Hydrazones in the powdered state were mixed with different adsorbents and the spectra measured against pure adsorbent as reference. Solutions of the hydrazones in ethyl alcohol w r e added to the adsorbents in amounts just sufficient to wet them and the spectra measured against pure adsorbent. Reflrctiou spectra of the hydrazones on filter paper were measured 3s described by Zeitlin and Xiimoto (26). Q
Absorbance, the logarithm t o the base 10 of the reciprocal of reflectance was plotted against mave length in all reflectance work to enable direct comparison of reflection with transmittance spectra measured in absolute ethyl alcohol.
TRANSMITTANCE SPECTRA. The transmittance spectra of the hydrazones in VOL. 31, NO. 7, JULY 1959
1167
Table I.
Transmittance and Reflectance W a v e Length Maxima and Minima of the 2,4-Dinitrophenylhydrazones (DNP) of Acetone- and 4-Methyl-2-pentanone
Acetone Amax mp
Transmittance of DNP in ethyl alcohol Observed Braude and Jones (4) Reflectance of DNP Powdered on 40-60 mesh silica gel >140 mesh silica gel In ethyl alcohol on 40-60
> 140
4Methyl-%pentanone Xmsx mp
mp
Xmin
mp
230 228
260 255
360 362
295
230
265
...
360 ...
295
... ...
265-270 283
365-370 373-377
295 295
... ...
278 275-277
375-382 373-377
295 300
..,
272-275 270
362-365 362-365
295 295
... ...
275-277 273-275
362-365 36&368
293 292
268 268 273-275
368-373 370-373 383-387
300 300 300
233 233 233
267-270 270-273 268
373-376 371-374 368-373
303 300 300
266-268
368-372
298
230
268-270
373-376
298
275-276 270-280 265-266 265-266 263-268
374-376 374-377 365-367 365-367 365-367
295 295 297 293 295
232 227-232 227-232 227-232
278(?) 278 265-268 265-268 265-270
373-378 378 367 367 367
295 290 295 295 295
265-272 267-269 ... 266-273 266-273 266-273
368-372 368-372 ... 368-372 368-372 368-372
298 300 ...
233-243 233-243 233-238 228 230
263-268 263-268 263-268 268-272 265-268
364-367 364-367 364367 363-370 365
295-300 295 298 295 295
267 268-270 268-270 266-268
368-369 368-369 368-370 368-370
233 230-234 230 232-234
268 268 268 266-268
363 364 362-363 363-365
295 298 295 295
...
Powdered On acid-washed alumina 230-233 Basic 230 Neutral 230-232 In petroleum ether on acid-washed alumina 230 In ethyl alcohol on 60 mesh Alundum 232 90 222-228 .4cid-washed alumina 230-233 Basic 228-230 Neutral 223-230 Reflectance of DNP on filter paper Whatman No. 1 with chloroform 232 4 232 3 MM. ... 1 MM. nitrobenzene 232 232 4 230 3 MM. Transmittance of DNP on filter paper Whatman No. 1 with chloroform 233 4 233 1 with nitrobenzene 233 4 233
absolute ethyl alcohol were measured before the reflectance studies and throughout the investigation to establish their purity, and to furnish a basis for comparison with the reflection spectra. The hydrazones mixed with the powdered adsorbents and adsorbed on filter paper were eluted with absolute ethyl alcohol and the transmittance spectra obtained to determine whether chemical alteration of the hydrazones had occurred. The transmittance spectra of the hydrazones on filter paper were measured directly after insertion of the paper sample and reference blank
Amin
...
300
300 300 300
300 303 300
...
?
...
into the cell carrier of the spectrophotometer according to Bradfield and Flood (2). DISCUSSION AND RESULTS
The wave length maxima and minima of the reflection spectra of the two in Table I. hydrazones are T~~~~~~ plots representative of both compounds are given in Figures through 3. Pure Hydrazones. The reflection spectra of the pure hydrazones were
...
...
...
...
markedly different from the transmittance spectra in solution (Figure 1). They were characterized by one broad and generally flat absorption plateau extending over a range of 200 mP from the near ultraviolet to the visible. A very weak maximum displaced some 30 mp toward the red from the principal absorption peak of the hydrazones in solution seemed to be present on the plateau. As the particle size Of the hydrazone the broad absorption hand became narrower' The tendency toward more characteristic spectra with the finely ground powders is considered a particle size effect and is in agreement with the findings of Shibata (18), who compared the transmittance spectra of a- and pcarotene crystals suspended in water
Figure 1. Comparison of reflection spectra of powdered acetone and 4-methyl-2-pentanone-2,4dinitrophenylhydrazones of varying particle size with transmittance spectra in ethyl alcohol a, Acetone-DNP
< 4 0 mesh
b. Acetone-DNP > 1 4 0 mesh c. 4-Methyl-2-pentanone-DNP d. 4-Methyl-2-pentanone-DNP e.
f.
250
300
350 WAVE
1168
ANALYTICAL CHEMISTRY
400
LENGTH
450
my
500
550
< 4 0 mesh > 1 4 0 mesh
Acetone-DNP in ethyl alcohol (transmittance) 4-Methyl-2-pentanone in ethyl alcohol (transmittance)
with the transmittance spectra of these substances in solution and observed a flattening effect caused by an increase in particle size of the carotenes. Solid Hydrazones on Adsorbent Powders. The reflection spectra of the solid hydrazones mixed with a n inactive adsorbent such as Alundum were of the same type exhibited by t h e pure hydrazones. Characteristic reflection spectra were obtained when the powdered hydrazones were mixed with chromatographic alumina (Figure 2). The spectra exhibited three distinct maxima which corresponded with the three found in the transmittance spectra of the hydrazones in solution. The two peaks nearest the visible were shifted 10 to 15 mp toward the red while the peak in the far ultraviolet remained virtually undisplaced. The emergence of the characteristic spectra of the po\-idered hydrazones when mixed with an active adsorbent is striking and is attributed to the ability of the absorbent to reduce, in effect, the hydrazone crystals to a fine state of subdivision and to adsorb them by physical processes on its surface. An additional factor that may have been involved in the emergence of the characteristic spectra is multiple reflection between hydrazone and white adsorbent through the sharpening and intensification of the absorption bands by an increase in effective light path length as suggested by Shibata (20). However, this effect was not observed when a relatively inactive adsorbent such as Alundum was mixed n-ith
powdered hydrazone on alumina. However, the magnitude of these shifts was less on alumina by about 10 mfi. The reflection spectra of ethyl alcohol solutions of the hydrazones on silica gel were similar to those of the powdered hydrazones on this adsorbent but the maxima were shifted back to the ultraviolet. Thus, the principal absorption band appeared a t practically the same wave length as in transmittance and the weaker band was apparently shifted about 10 mp toward the red. The spectral characteristics of the hydrazones on silica gel and alumina appear similar in nature although on silica gel the absorption maximum in the ultraviolet was lost in all cases. Hydrazone Solutions on Filter Paper. The reflection spectra of the hydrazones on filter paper were similar to the reflection spectra of ethyl alcohol solutions of these compounds added t o alumina. The bathochromic shifts of the maxima in reflectance compared t o the maxima in solution ranged from virtually no displacement in the far ultraviolet t o about 10 mp for the other two maxima. As reported by Zeitlin and Niimoto (W6), the transmittance and reflection spectra of the filter paper samples were identical although the E,,, in transmittance was higher. Comparative transmittance studies of the solutions of the hydrazones which were recovered from all adsorbents by
powdered hydrazones. The spectra of the solid hydrazones were afiected similarly by acid-washed, basic, and neutral chromatographic alumina. Characteristic spectra were obtained in which the maxima were virtually identical on the three types of alumina (Figure 2). This appears to eliminate an acid-base type of interaction similar to that which occurs when 2,4dinitrophenylhydrazones in solution are treated with a base and the maxima in the transmittance spectra are shifted into the visible (11, 23). The reflection spectra of the solid hydrazones on silica gel displayed two maxima which appear to correspond with those found nearest the visible on alumina. The third band in the ultraviolet wm lost. The spectral characteristics of thc hydrazones were similar on silica gel of varying particle size. The magnitude of the bathochromic displacements on this acid adsorbent was approximately the same as those on alumina (Figure 2). Hydrazone Solutions on Adsorbent Powders. The reflection spectra of ethyl alcohol solutions of the hydrazones on all grades of alumina were characteristic and contained fairly sharp absorption bands corresponding with the transmittance spectra in solution (Figure 3). The bathochromic shifts of the maxima on Alundum were about the same as those observed in the spectra of the
9 ,-\,
j
7
i
i
i
.'
.' \
.-.
b
6
8 . '\
'\A
7 -
6 -
5 W
0 z a m a
0
v)
5 -
4
Y 0
z a
m
3
K
4 -
0
m
8
a
2
I
250
300 WAVE
350
400
LENGTH
450
500
mfl
250
Figure 2. Reflection spectra of powdered acetone-2,4dinitrophenylhydrazone on various grades of alumina and silica gel a.
Acid-washed alumina
b. Basic alumina e.
Neutral alumina
d. e.
Silica gel 60 mesh In ethyl alcohol (transmittance)
300 WAVE
350
400
450
500
my
LENGTH
Figure 3. Reflection spectra of ethyl alcohol solution of acetone-2,4-dinitrophenylhydrazoneon various grades of aluminum oxide and silica gel a.
b.
Basic alumina 60-mesh Alundum
c.
d.
60-mesh silica gel Transmittance
VOL. 31, NO. 7, JULY 1959
1169
elution with absolute ethyl alcohol demonstrate that spectral differences and displacements were not due to chemical alteration of the hydrazones by the adsorbents. Three studies reveal that bathochromic displacements in going from transmittance to reflectance do take place and are more pronounced, in the case of the two hydrazones studied, near the visible region of the spectrum. The displacements are fairly constant, attesting to the reproducibility of the reflectance technique and presumably to the similar nature of the factor(s) responsible. The effect of particle size in spectral reflectance and its role in causing spectral displacements have been considered briefly by Johnson and Studer (IO) and Lermond and Rogers ( I S ) . Accessory evidence supports the view that the spectral displacements are not inherent in the reflectance technique but are primarily due to or arise from a state of subdivision. Shibata (18) in his work on suspensions of carotene crystals in water found that the three absorption bands in the transmittance spectra of these suspensions were shifted toward shorter wave lengths when compared with the transmittance spectra of the benzene extracts of the carotenes. The displacements detected in the transmittance spectra of the suspensions are undoubtedly due to the state of subdivision of the carotene crystals in the suspensions relative to those in true solution. The findings of Naughton and
coworkers (15) can be explained similarly because no displacements were noted when the reflection spectra of the heme pigments in tuna meat were compared with the corresponding transmittance spectra in solution. These n-orkers may have been comparing substances of approximately similar particle size if the heme pigments, mainly oxy- and metmyoglobin, being macromolecules, were not in true solution but were present in both instances in the colloidal state. Finally, the most direct evidence is given by the results of the comparison of the maxima of the transmittance and reflection spectra of the two hydrazones measured directly on filter paper under a variety of conditions which show clearly that they are similar (Table I). These findings are considered significant because direct comparison of the transmittance and reflection spectra of substances of identical particle size revealed no displacements. LITERATURE CITED
(1) Borisov, M. D., Izvest. Akad. Nauk, S.S.S.R. Ser. Fiz. 17, 689 (1953). (2) Bradfield, A. E., Flood, A. E., J. Chem. Soc. 1952, 4740. 3) Brady, 0. L., Ibid., 1931, 756. 4) Braude, E. A., Jones, E. R. H., Ibid., 1945, 498. (5) Buyske, D. A, Owen, L. H., Wilder, P.. Hobbes. AI. E.. ANAL.CHEM.28. 910 (1956).' (6) Dirscherl, W.,Nahm, H., Ber. 73B, 448 (1940). (7) Elvidge, J. A,, Whalley, % Chem. I., & Ind. (London) 1955, 589.
I
(8) Fisher, R. B., Vratny, F., Anal. Chim. Acta 13, 588 (1955). (9) Guilmart, T., Bull. SOC. chim. [5], 5, 1209 (1938). (10) Johnson, P. D., Studer, F. J., J. Opt. SOC.Am. 40, 121 (1950). (11) Jones, L. A., Holmes, J. C., Seligman, R. B., ANAL.CHEW28, 190 (1956). (12) Lautsch, V. W., Kurth, G., Broser, W. J.. 2.Naturforsch. 8B. 640 (1953). (13) Lekmond, A . , Rbgers,' L. 'B., ANAL.CHEM.27,340 (1955). (14) Lynn, W. S., Steele, L. A., Staple, E.,Zbid.,28,132(1956). (15) >aughton, J. J., Frodyma, M. M., Zeitlin. H.. Science 125. 121 (1957). (16) Pippen,'E. L., Eyring, E. J,, Nonaka, Masahide, ASAL. CHEM. 29, 1305 (1957). (17) Pruckner, F., Schuienburn, hf., Schwuttke. G.. Naturwissenschaften38, 45 (1951).' ' (18) Shibata, K. , Biochim. Biophys. Acta 22,398 (1956). (19) Shibata, K., J. Biochem. (Japan) 45, 599 (1958). (20) Shibata, K., private communication. (21) Shihata, K., Benson, A. A., Calvin, M.. Biochim. BioDhvs. Acta 15. 461 (1954). (22) Smith, J. H. C., Shibata, K., Hart, R. W.,Arch. Biochem. Biophys. 72, 457 (1957). (23) Timmons, C. J., J . Chem. SOC.1957, 2613. (24) Yamaguchi, K., Fujii, S., Tahata, T., Kato, S., Yakugaku Zasshi. 74, 1322 (1954). (25) Ibid., p. 1327. (26) Zeitlin, H., Niimoto, A., Nature 181, 1616 (1958).
e.
I
"
RECEIVED for review August 11, 1958. Accepted hlarch 16, 1959. Work supported by a Frederick Gardner Cottrell grant from The Research Corp. of New York.
Spectrofluorometric Estimation of Adrenochrome in Human Plasma A. N. PAYZA and M. E. MAHON Psychiatric Research Unit, University Hospital, Saskatoon, Canada
b A determination of adrenochrome (2,3 -dihydro - 3 -hydroxy- N-methylindole-5,6-quinone) and similar compounds in plasma is based on the formation of a fluorescent compound in the presence of zinc acetate. The Farrand spectrofluorometer is used to measure fluorescence. Adrenochrome concentrations of 0.1 to 1 y yield a linear fluorescent curve. Adrenolutin (3,5,6-trihydroxy-N-methylindole) is the end product of the reaction. Other compounds tested-adrenaline, noradrenaline, tryptophan, tryptamine, 5-hydroxytryptophan, serotonin, 3-indofeacetic acid, 5,6-dihydroxy-N methylindole, 3,4-dihydroxyphenylalanine, dopachrome, epinine, epinochrome, and adrenoxyl-do not give 1170
ANALYTICAL CHEMISTRY
increased fluorescence after addition of zinc salts in acetone. 2-lodoadrenochrome, 5,6-dihydroxynorephedrinechrome, and noradrenochrome yield fluorescent substances with zinc salts to a lesser degree than adrenochrome,
follow the excretion of adrenochrome into urine in laboratory animals (2). It has also been used to develop an assay for small quantities of adrenochrome in plasma and cerebrospinal fluid.
A
Crystalline adrenochrome was prepared as described (6). Other compounds tested (reagent grade) were: DLtryptophan, serotonin, 3,4dihydroxyphenylalanine (Nutritional Biochemicals Corp.), noradrenaline hydrochloride (Delta Chemical Works, Inc.), 3,4dihydroxynorephedrine hydrochloride (Mann Research Laboratories Inc.), 3-indoleacetic acid, epinephrine, tryptamine hydrochloride (Eastman Kodak Co.), (epinine) 3,4-dihydroxyphenyl-
MATERIALS A N D METHOD
was an intermediate stage in the oxidation of epinephrine by a mammalian catechol oxidase in human plasma (6, 7 ) . Although this indicates adrenochrome may be present in human plasma, no assay method has been developed to test this possibility. Zinc and aluminum salts change adrenochrome to a fluorescent compound ( I , 4). This reaction was used to DRENOCHROME
