500
VOLUME
19,
NO. 7
L I T E R A T U R E CITED
centrations of 200 mg. per 100 ml. of methanol-water to give a satisfactory end point. $-vinyl cyclohexene (butadiene dimer) gives low results. Oleic acid can be titrated, but reacts somewhat s l o ~ l ya t the end point. The method should be well suited to the determination of the enol content of substances such as 0-keto aldehydes, acetyl acetone, acetoacetic ester, etc., by the Meyer method, since with these compounds bromination is very rapid even a t the low temperatures necessary to retard tautomcsiization to the keto form. The first excess of bromine is at onm’ indicated when the amperomrtric techhique is employed.
Bartlett, P., and Tarbell, D., J..4m. Chem. Soc.. 58, 466 11936). ( 2 ) Kolthoff, I. M.,and Harris. W. E.. IND.ENG.CHERT., ~ A L ED., . 18, 161 (1946). ( 3 ) Laitinen, H. .1..and Kolthoff. I . 11..J . Ph,ys. Chem.. 45, 1079 (1941). (4) Morris, H. E., Stiles, K.B., and Lalie, If-. H., IKD. EXG.CHEM., A1x.kx.. ED.,18, 294 (1946); Lane, W.H., Ibid., 18, 295 (1946). ( 5 ) Nernst, W., arid Merriain. E., Z. ghysik. C‘hem..5 3 , 235 (1905). (1)
THISinvestigation was carried out unilrr t h e sponsorship of t h e Office of Rubber Reserve, Heconstruction Finance Corporation. in connection with thr Gowrninent Synthetic, Hilhhrr. Proarain.
Application of Reflectance Spectrophotometry to Quantitative Microanalysis Department
of
JULIUS SENDROY, JR. Experimental ,Medicine, Loyola University School of Medicine, and Mercy Hospital, Chicago, I l l . AND
WALTER C. GRANVILLE’ Research Laboratories, Interchemical Corporation, New York, N. Y . Reflectance spectrophotometry may be used in quantitative microanalysis by measuring changes in spectral refiectance corresponding to changes in color of a solid highly reactive chemical reagent in the form of an opaque-type surface coating. Limited application to cyto- and histochemistry is indicated.
A
LTHOUGH reflectometry has frequently been employed in industry for the descriptive evaluation of colorants and pigments in both wet and dry states (3, 5 ) , its greater use in quantitative chemistry has been long overdue, Chemical reagents have long been used t o bring about reactions resulting in color, the intensity of which serves as a measure of the amount of one of the reactants, and hence, directly or indirectly, of the amount of the substance to be determined. However, in such systems of quantitative analysis, the intensity of color has been determined in solutions or a liquid medium by light transmittance measurements. Although the intensity of the color of a solid can similarly be evaluated by light reflectance measurements, analytical chemists have paid little attention to the development of methods of analysis in which the color produced by, and hence indicative of the extent of, a reaction, is ultimately confined to a solid medium or surface. Steps in this direction have recently been taken, a i appears from the work of two laboratories. Compton, Granville, Boynton, and Phillips (1)used spectral reflectance measurements to evolve the quantitative relationship between variation in the green color of untreated, native apple leaves and their nitrogen content. Kienle, Steams, and Van der Meulen ( 2 ) determined the Hammett reaction constant for a photochemical reaction in which the azo bond of certain dyes was destroyed by light. This reaction was followed by quantitative spectral reflectance measurements for the compounds involved. The authors wish to call attention to the possibility of such B type of quantitative analysis by measurements of changes in spectral reflectance corresponding t o changes in color of a solid, highly reactive chemical reagent in the form. of an opaque-type surface coating. This interesting application occurred in the development by the senior author ( 4 ) of a simplified, smsitivr, Present address, Color Laboratorles Division, Container Corp America, Chicago, I11 1
uf
visual method for the detection and quantitative determination of carbon monoxide in air, based on its well-known reaction with palladium chloride. (In part because of wartime restrictions, publication of the method and of the spectral reflectance data presented here has necessarily bcen delayed.) The nature of the colorations involved made it necessary to identify and measure the changes produced, by an objective method, the results of which could be applied to the accurate reproduction of a standard color scale.
A
B
7 0 - REAGENT 66/50
C
D w
K
E
P
20
-
I
400
I
1
500 600 WAVELENGTH mu
1
700
Figure 1. Spectrophotometric Reflectance Curves forAir Samples Containing Carbon Monoxide A , hlank; B, 0.0012fh; C, 0.0031%; D , 0.0044%; E, 0.010%
ULY
1947
501
REAGENT 66/50
0 Q
Figure 1 shoNs the spectral reflectance curves obtained in one experiment ,with samples of air containing known amounts of carbon monoxide. Figure 2 shows the relationship of carbon monoxide concentration from 0 to 0.017,, to per cent reflectance from 550 t o 650 mp in steps of 25 mp. Figures 3 a n 2 4 are illustrative of the results obtained with a less sensitive reagent operating through a similar color range, from 0 to 0.1% carbon mono-dtl
650 mp 625 “
60 w
g
55
Q I-
? J
50
2
650 rnp 0 625 81 6 0 0 18 x 575 n 550 11 0
651iREAGENT 113
LL
45
I-
50
40
g
35
a
30tI YI I I I I I I I I I I 0
2 4 6 PER CENT
6
co
1
0
x 10-3
Figure 2. Relationship of Carbon RIonoxide Concentration in Air to Per Cent Reflectance The rengent consisted of a fine powder containing palladium chloride as t’he active ingredient, uniformly spread in a very thin layer to cover an area 1 inch square on a white paper strip, to whidh it was made to adhere by the application of pressure. The surface thus obtained was smooth and uniform in texture and color. Upon exposure for 1 minute to an atmosphere containing carbon monoxide, in a room or any smaller enclosed space or container, such reagent patches showed a graded color range from very pale orange to medium gray (1.S.C.C.-K.B.S. method of color designation), dependent on the concentration of carbon monoxide. Immediately following exposure to the test samples of air, spectral reflectance measurements of the color patches were made with the General Electric (Hardy) recording spectrophotometer, with a viewing geometry which completely included any specular component reflected from the sample. The standard of reflectance was a freshly prepared layer, 0.06 inch thick, of pure magnesium oxide.
0
2
4
6
PER CENT
6
1
0
GO x 10-2
Figure 4. Relationship of Carbon Monoxide Concentration in -4ir to Per Cent Reflectance
The spectral reflectance curves shown were obtained for. the prime purpose of furnishing a basis for color specification, for which they are sufficiently accurate. Although repetition of the work with an increased number of points would fix the position of the curves more exactly, the results as they stand indicate that in the lower range (0 to 0.017,) carbon monoxide may be determined by this method of measurement to within +0.0002%,, and in the upper range (0 to 0.1%) to within +0.002%. The particular reagents used have largely been supplemented by others of a series, varying in formulation, to be reported (4); hence, these data are not offered as supportive of a described method of carbon monoxide analysis but are presented rather to illustrate the possibilities of reflectometry in optical microchemistry, especially under conditionq where transmittance measurements are precluded owing to the opacity of the colored materials involved. Limited application t o cyto- and histochcmistry is likewise indicated. Obviously, less elaborate typcab of reflectometer than the one employed in this work may he and have bec.n, used (4). LITERATURE CITED
I 400
I
I
500 600 WAVELENGTH mp
I
700
Figure 3. Spectrophotometric Keflectance CurFes f o r Air Samples Containing Carbon Monoxide blank; B , 0.01%; C. 0.03%: I ) , 0.05%; E , 0.10%
(1) Conipton, C . C., Granville, W. C., Boynton, D., and Phillips:, E. S., Cornel1 Univ. Agr. Expt. Sta., Bull. 824 (1946). (2) Kienle, R. H., Stearns, E. I., and Van der Meulen, P. A . , J . P h y s . Chem., 50,363 (1946). (3) Mellon, M. G., “Colorimetry for Chemists,” Columbus, Ohio, G. Frederick Smith Chemical Co., 1945. (4) Sendroy, Julius, Jr., unpublished manuscript. ( 5 ) Wright, W. D., “Measurement of Color,” London, Adam Elilger,
.
1944.
