An Electronic Image Converter and Its Use in Chromatography ZABOJ V. HARVALIK Institute of Science and Technology, University of Arkansas, Fayetteuille, Ark.
A n electronic image converter and its accessories, enabling observation of textures and color reactions of adsorption columns illuminated hy an infrared light source, are described. The converter employs an image tube, Type CV-147, which conyerts infrared into visible light. The color sensitivity of the tube also permits observation of ultraviolet light. The color range of this deviee is 300 to 1400 milli-
OLORLESS substances adsorbed in a chromatographic column are difficult to locate, Xarrer and sch6pp (8)
C
microns and its magnification power is 2.5X. It i s suggested that the converter he applied to medical, criminological, and industrial investigations as well as problems in analytical chemistry. The addition of this device to chromatographic teohniques necessitates. a new or broader definition of the term “ultrachromatography,” and the introduction of the terms “infra-” and “fluoroohromatography.”
verter (Figure 1, center), where an optical, infrared image is focused upon the photocathode of the image tube (also Figure 2, PI. The electrons released by the photocathode of tbeimagetube,P, fall a fluorescentscreen, W, covered with B phosphorof acomposition similar to that of cathode-ray tubes. Electrons activate the phosphor, S, to a greenish luminescence and the electron im-
and Winterstein and Schon (27) used filtered ultraviolet light for identification of some colorless substances because of their fluorescence. This method of identification, erroneously called ultraehromatogrsphy, is limited in its because not dl colorpiece, age formed EP. on The theimage screen, onS,the is observed screen canthroughamagnifgingeye be photographed on any 1es substances fluoresce in the region of visible light when actiereen.sensitive film, vated with ultraviolet radiation. Some colorless substances form fluorescent compounds after their adsorption in the chromatographic column when chemical reagents are added (9, 24-28). Strain (29) suggested forming fluorescent compounds of colorless samples before they are adsorbed in the chromatographic ~olumn,and identifying them after their adsorption. Some adsorbents interfere with the fluorescence of the compounds; they fluoresce and make identification of chromatographic layers difficult. The author investigated reflection, absorption, and fluores cenee in the infrmed region of the spectrum of some colorless compounds to study the possibility of chromatographic identification using infrared as B light Source. Infrared light is invisible to the human eye, and can he observed only by devices for converting infrared to visible light. Electronic image converters permit the observation of infrared absorption and reflections from specimensilluminated by various light sources. Electronic image converters use image tubes as elements for converting infrared to visible light. Aa early BS 1934, image tubes were used to study the infrared-reflecting and absorbing properties of materials. An image tube was attached to a EP microscope or telescope and objects illuminated with infrared light were observed (2,22, 18). The image tube WBS improved in ita physical and electronic performanee during World War I1 (9, 20). An electronic eyepiece attached to a spectroscope, permitting direct observation of the infrared and ultraviolet portion of the spectrum, was described by the author a t the Chicago meeting of the American Physical Society in 1947 (6, 7). The use of the image tube attached to a microscope (previously reported,, 28) was reported and applied to petrographic research in 1948 by Bailly ( 2 ) . The importance of electronic image converters in analytical chemistry, and possible uses in criminology, medical sciences, and industry were pointed out bv the author at the meetina of the AMERICANCHEMICAL S O C I ESeptember ~, 1948 (6). ..,rr.rl,,”...~ Yh.. .,.,,,””..”. .... tion, can tie huilt by any individual fsmiliar with rltvtronir rirThe transformation of infrared light into visible light by the ruirs. It (Vigiire 2 ) consists n i yhc itwig,: t u l w f-‘\’-i47 (fl),(t;iguse of the image tube is explained as follows: itre 2., CV-117 I. the ohirctive I m p 3 w t t . m . 0.and tlre magnifying -~ &&cc. EP. The &ape tube is bounted’in a brass shielding The chromatographic column (Figure 1, center), jlluminated by tkb’e, thk inside of whi& is lined with mica. An outer tube is an infrared light mume (15C-watt projector bulb mserted into a slipped over the shielding tube. As a focusing mount, FMI, for light-tight box and equipped with an infrared filter, Corning No. the objective lens system, 0,a tube of mmewhat smaller diameter 2540, Figure 1, left), reflects and absorbs a certain amount of inis attached to the outer tube. The objective lens system, 0,has frared light. Infrared light reaches the electronic image con-
,... .,.,... ~
~~~
1149
I,
1150
ANALYTICAL CHEMISTRY
a focal length of 5.0 cm., and is used to focus the object on the photocathode, P , of the image tube. To prevent visible light from reaching the photocathode, a filter, F , can be slipped over the objective lens system (15). The magnifying eyepiece, EP, Kellner, with a focal length of approximately 6.0 em., is placed in the focusing mount end of the image tube, FM,. The image tube is connected to the power supply by a socket, 2’. The power supply (Figure 1, right, and Figure 3) consists of a high voltage transformer (1800 to 2000 volts secondary), a filament transformer for the rectifier tube (2.5 volts secondary), a filter condenser of 0.25 microfarad having an’ insulation of 3000 volts, and a resistor of 5 megohms and one resistor of 10 megohms, both of 2-watt type. A resistor of 40 megohms shunts the 0.25microfarad condenser. This shunt resistor consists of four 10megohm resistors (2-watt type in series connection).
Figure 3
The electronic image converter enables the observation of ultraviolet from 300 to 400 mp, and infrared from 760 to 1400 mp, as well as the visible range of the spectrum (400 to 760 mp). The greatest sensitivity of the electronic image converter is in the ranges between 350 and 450 mp, and 560 and 900 mp. The phosphor of the screen has its maximum radiation around 525 mp, and coincides closely with the color range of the maximum sensitivity of the human eye; it has a resolution definition of about 350 lines per inch, using television standards, and a medium persistency. The magnification of the electronic image converter is 2 . 5 X , and can be increased or decreased, depending on the focal length of the objective lens system. The electronic image converter can be used in different ways to aid the discrimination of layers in the chromatographic column. OBSERVATION IN THE INFRARED REGION
The illumination of the chromatographic column with infrared light is achieved by placing a Corning No. 2540 infrared transmitting filter, polished, 2 mm. thick, in front of the light source. Observation is made in a dark room. If any infrared light is reflected, it will be ‘detected through the image converter. This type of identification of chromatographic zones is termed infrachromatography.
from reaching the image tube. The incandescent light source described above is used, and the Corning 2540 is replaced by a filter consisting of Corning Nos. 3389 and 4407, polished, standard thickness ( 4 ) . This filter combination transmits visible light only. Some substances require higher activation energies than the energy transmitted through the Corning No. 3389 and 4407 filter combination. When the former is omitted, a more bluish light will be transmitted, and this iricludes some ultraviolet. High activation energies can be obtained by using a Corning No. 5860 ultraviolet-transmitting filter in connection with a mercury vapor lamp. Light reaches the chromatographic column and activates the adsorbed substances to fluorescence. Because only infrared light is transmitted to the image tube, and no infrared was present in the light source, any infrared observed through the electronic, image converter originates in the adsorbed compound. This method of observation using a light source emitting shorter wave-length light than the light observed through the electronic magnifier is called fluorochromatography. This term is also applicable to the fluorescence produced in adsorbed layers when they are exposed to filtered ultraviolet light, and is synonymous with Karrer and Schopp’s definition of ultrachromatography (8). The above-mentioned observation methods suggest a more precise terminology: Ultrachromatography uses an ultraviolet light source, and the light observed is reflected from the chromatographic column, and is in the ultraviolet region of the spectrum. Infrachromatography uses an infrared light source, and the light* reflected from the chromatographic column is in the infrared region of the spectrum. Fluorochromatography uses any convenient light source, and the light emitted from the chromatographic column is of longer wave length than the longest wave length emitted by the light source. PREPARATION OF SAMPLES
Selected leaves of the mulberry tree, MOTUSrubra (200 grams)), were boiled in 2000 ml. of distilled water for 3 hours and stored a t 4‘ C. for several days. The extract was filtered, and a small portion of it was diluted with distilled water to ten times the original volume. This diluted extract, of brown-greenish color, was used in the adsorption tests. IDENTIFICATION OF ZONES USING CHROMATOGRAPHIC COLUMNS
Activated charcoal, silica gel, alumina, magnesium oxide, and fuller’s earth were used as adsorbants. Each adsorbant was placed in a borosilicate glass tubing 1.1 cm. in diameter and 35 cm. in length. Six columns were prepared for each type of adsorbant and the diluted extract was permitted to flow through the columns. Observations of the resulting chromatographic zones were made in visible light and by illumination of the chromatographic column with infrared light, using the electronic image converter (Figure 1). Table I shows the result of these observations.
OBSERVATION IN THE ULTRAVIOLET REGION
The illumination of the chromatographic column by filtered ultraviolet light is achieved by a mercury arc as light source in connection with a Corning No. 5860 ultraviolet transmitting filter, polished, 3 mm. thick. At the same time, a Corning No. 5860 ultraviolet transmitting filter, polished, 3 mm. thick, is attached to an electronic image converter to prevent any visible (fluorescent) light from reaching the photocathode of the image tube. The chromatographic column of borosilicate or quartz glass ensures maximum ultraviolet transmission. Individual chromatographic layers reflect ultraviolet light, and are observed by the electronic image converter. This method differs in principle from the method Karrer and Schopp described in 1934 (8). This type of identification of chromatographic zones is termed ultrachromatography.
Table I.
Number of Zones Separated in Adsorption Column
Adsorbant Charcoal Visible Infrared
Eluant Ethyl Before alco- Ethyl AceElution Water bo1 ether tone
Ben-
CCh reno
?
0
0
0
0
0
0
7
0
0
0
0
0
0
Silica el Visi6le Infrared
4
6
3 4
4 5
4 5
4 5
4 5
4 5
Alumina Visible Infrared
4 6
3 4
4 51
4 5
4 5
4 5
OBSERVATION OF FLUORESCENCE IN THE INFRARED REGION
Magnesia Visible Infrared
3 5
2 3
3 4
3 4
3
3
To observe fluorescence in the infrared region of the chromatoraphic column, the electronic image converter is covered with a borning No. 2540 infrared transmitting filter, polished, 2 mm thick (Figure 2, F ) , which prevents all ultraviolet and visible light
Fuller’s earth Visible Infrared
4 6
4
4
4 5
3
?
5
5
4
?
3 5
5
4
5
?
4 6
5
4
8
?
V O L U M E 2 2 , NO. 9, S E P T E M B E R 1 9 5 0
1151 LITERATURE CITED
Table 11. Number of Zones Separated by Paper Chromatogram Eluant Ethyl ~100-
Water Visible light Infrared light
3 7
bo1
Ethyl ether
4 6
3 6
Aoetone 2 5
CClr 2 6
Benzene 2 2
IDENTIFICATION OF ZONES USING PAPER CHROMATOGRAPHY
Filter paper strips, 6 X 200 mm., were cut and a small drop of the undiluted extract was placed on the strip 25 mm. from the lower end. The paper strip was immersed in the eluant. Six strips were prepared, one for each eluant (water, ethyl alcohol, ethyl ether, acetone, carbon tetrachloride, benzene). After 2 hours’ elution the chromatographic zones were observed in visible and infrared light. Table I1 gives the results of the observations. CONCLUSION
The experimental data indicate that the use of an infrared light source in connection with the electronic image converter increases discriminat,ion of chromatographic layers.
Bailly, R.,Science, 108, 143 (1948). Brilche, E.,and Scherser, O., “Geometrische Elektronenoptik,” Berlin, Julius Springer, 1934. Cholnoky, L. V.,Magyar Chem. FoEydirat, 39, 138 (1933). Corning Glass Works, Corning, N. Y . , “Glass Color Filters,” 1948.
Harvalik, Z. V., Abstracts of 114th Meeting, p. 13B,AM. C H ~ M . SOC.,St. Louis, Mo., 1948. Harvalik, Z. V., A m . J . Phys., 18, No.3,151-3 (1950). Harvalik, Z.V.,Rev. Sci. Instruments, 19, 254 (1948). Karrer, P.,and Schopp;K., Helv. Chim. Acta, 17, 693 (1934). Morton, G.A., and Flory, L. E., Electwnics, 19, 112 (1946). Morton, G.A,, and Flory, L. E., R C A Review, 7, 385 (1946). Pratt, T.H., J . Sci. Instruments, 24,312 (1947). Schaffernicht,B.,2.tech. Physik, 17, 12 (1936). Strain, H.H., J . A m . Chem. Soc., 57, 758 (1935). Tschesche, R.,and Offe, H. A . , Ber., 68, 1998 (1935). Ibid., 69, 2361 (1936).
Wieland, H., Hesse, G., and Huttel, R., Ann., 524,203 (1936). Winterstein, A,, and Schon, K., Z . physiol. Chem., 230, 139 (1934).
Zworykin, V. K., and Morton, G. A,, J . Optical SOC.Am., 26,181 (1936). RECEIVED November 1, 1948. sity of Arkansas.
Research Paper 928.Journal Series, Univer-
Reduction of Nitroammonocarbonic Acids with Titanous Ion in Acid Media ROBERT P. ZIMMERMAN AND EUGENE LIEBER Zllirwis I n s t i t u t e of Technology, Chicago, Ill. T h e behavior of a n u m b e r of compounds containing t h e -“NO2 group, when one a t t e m p t s t o reduce t h e m with titanous chloride, h a s been studied. Such nitroammonocarbonic acids as nitroguanidine, nitro aminoguanidine, a n d their alkyl a n d acyl derivatives do n o t give reproducible values i n reduction by titanous chjoride. In s u c h cases ferrous ions have a marked effect, i n t h a t t h e a m o u n t of titanous ion consumed :aries with t h e a m o u n t of ferrous ion added. By adjusting t h e concentration of ferrous ion t o a value dependent on t h e type of nitroammonocarbonic acid concerned, i t is possible to a t t a i n a reduction equivalent of 6 titanous ions per nitro group. Both nitroguanidine‘ a n d nitroaminoguanidine require about 0.75 t o 0.85 equivalent of ferrous
I
N T H E synthesis of compounds of high nitrogen content intermediates containing the “nitramide” group, --PI”SOI,
3re of frequent occurrence. Nitroguanidine, nitrourea, nitroaminoguanidine, and their alkyl and aryl derivatives are typical examples of compounds which are widely used in this field. As a group they may be termed “nitroammonocarbonic acids” and at present no rapid and accurate methods are available for their assay. I t is rational to approach this problem by utilizing the reactivity of the nitro group, which is the characteristic functional group common to them all. The present paper summarizes the results of an investigation of the reduction of nitroammonocarbonk acids by titration in acid media and the effect of ferrous ions on this reduction, together with suggestions as to the mechanism involved in the reduction. PREVIOUS WORK
The titanous ion, in a high concentration of hydrogen ion, has long been used for the estimation of the aromatic nitro group (IO):
ion for t h i s purpose; on t h e other h a n d , nitrourea, i n either t h e presence or absence of ferrous ions, copsumed only 2 equivalents. The use of suitably chosen concentrations of ferrous ion permits a n assay of nitroguanidine a n d nitroaminoguanidine between 1.3 a n d 2.20/,, comparable with t h a t often obtained i n assaying t h e m by t h e Dumas nitrogen method. However, t h e r e a c t i o n should ‘not be used as a quantitative procedure unless one h a s a n understanding of t h e pitfalls involved. Mechanisms are suggested to account for t h e consumption or 4 or more equivalents of titanous chloride i n t h e reduction of nitro- a n d nitroaminoguanidine. T h e stoichiometric mechanisms assume t h a t reductive cleavage may occur d u r i n g t h e reduction.
+ C6HsN02 + 6 H +
6Ti+++
-.
+
+
C6H5NH2 6 T i + + + + 2H20
An examination of the extensive literature indicates that this ieaction is characteristic of the nitro group, 6 equivalents of titanous ion being required for each nitro group. The first work of any importance was done by Knecht and Hibbert ( 9 ) , and has since been incorporated in their book (IO). They found that titanous chloride in acid media reduced aromatic nitro groups to the amine, consuming 6 equivalents of the titanous ion for each nitro group. Their method was to dissolve the sample in water, acid, or alcohol, add an excess of acidic titanous chloride, boil the solution, and then titrate the unrescted titanous chloride with standard ferric alum solution, using potassium thiocyanate as the indicator. Kolthoff (I I ) found that the reduction potential of titanous chloride increased with decreasing acid content. Thus, titanous chloride is a much more powerful reducing agent in basic solution than in acid solution. Van Duin ( 2 2 )had previously reported that the titer of titanous chloride solutions decreased aa acid concentration increased. Kolthoff and Robinson (12) sug-
