Use of Infrared Radiation for Detection of Colorless Substances on Paper Chromatograms DONALD R. KALKWARF' and ARTHUR A.
FROST
Department o f Chemistry, Northwestern University, Evanston,
111.
It is desirable to have a general physical method for detecting colorless substances on paper chromatograms. For this purpose an apparatus has been developed to record infrared emission and transmission properties of paper strips. It has been found possible to detect a variety of substances in milligram quantities without the aid of a spectrophotometer
0
strip of plain filter paper which had been cut successively from the same sheet of paper could be mounted in the two steel guides placed in front of an electric heating mantle. Each strip could be pulled through the guide a t a constant rate by a small motor, as shown in Figure 2. Along each guide are two concentric rectangular apertures, 0.50 X 1.60 em., one in each wall. As a section of p:tper strip passes between these apertures, it is exposed on one side to the heating mantle, and radiation transmitted or emitted by the paper is allowed to pass out the aperture on the other side toward the detector.
NE of the important problems in the field of paper chromatography is the derelopnient of general techniques for detecting small amounts of colorless substances upon the completed chromatogram. The incorporation of radioisotopes into the substances to be separated resolves the problem of detection into that of locating the radioactive zones on the completed chromatogram for which several methods are available (6,10,14). Absorption of ultraviolet radiation has also been used to locate colorless zones either by the fluorescence imparted (3, 3, 12) or by direct transmission measurements of the completed chromatograms (4, 11, 16). Still, the paper chromatographic methods are largely limited to the separation of colored compounds or those which can be converted to colored compounds by means of some chemical reaction on the paper.
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Figure 2. Schematic Diagram of Apparatus As seen from bolometer housing
PREAMPLIFIER.
Figure 1.
A motor-driven chopping wheel was interposed between the guides and the detector in such a manner that radiation was allowed to pass through from alternately one and then the other paper strip a t a rate of 20 cycles per second. This pulsating radiation beam then had an intensity amplitude equal to the difference in the intensities of radiation coming from the two paper strips. The detector consisted of a thermistor-type bolometer enclosed in a metal jacket and fitted with a rock salt window. The bolometer and preamplifier were further enclosed in a metal housing fitted with a silver chloride window coated with silver sulfide to exclude visible radiation. The alternating intensity of the radiation striking the active thermistor flake gave rise to an alternating electric signal which was then amplified, rectified, and recorded.
,
Schematic Diagram of Apparatus Center section
The ability of infrared rctdiation to interact with practically all types of matter suggested that it could be utilized in an extremely general method for locating substances which were not visible in ordinary light. In addition, its great specificity of inheraction suggested the possibility of not only finding the separated zones but also distinguishing the chemical composition of one from another. Although spectrophotometry would be most advantageous in dealing with the question of specificity, there is also the possibility of using a negative or positive filter technique as used in infrared gas analyzers (6, 7 , 9 ) .
INSTRUMENTAL DETAILS
The heating mantle used was a commercial Nichrome wire heater mounted on a ceramic base and operated through a Variac. The chopping wheel is shown in Figure 3 and was conFigure 3. Chopping structed out of a l/lrinch sheet Wheel aluminum and rotated a t a speed of 300 r.p.m. b a small motor. The surface gcing the bolometer was polished to a mirror finish in order to locr.er its emissivity. The radiation detector used was a thermistor bolometer, Type BG-1, which, together with the preamplifier, was manufactured by the Servo Corp. of America. Operating a t a bias vole age of 150 volts and a radiation interruption rate of 20 cycles per
GENERAL METHOD
The general plan was to compare the emission or transmission of infrared radiation by a developed paper chroamtogram with the same properties of plain filter paper. The instrument developed for this purpose is shown in Figure 1. The developed chromatogram in the form of a narrow paper strip 2 X 30 em. and a similar 1
Present address, General Electric Co., Richland. Wash.
191
192
ANALYTICAL CHEMISTRY
second, the detector should have a sensitivity of approximately 470 volts per watt. After passing through the preamplifier with a listed gain of 330, the signal was sent to a Type 1231A am lifiernull detector manufactured by the General Radio Co. T i e null detector circuit rectified the alternate current signal and operated a direct current milliammeter which acted as a null detector. An Esterline-Angus recording milliammeter, Type AW, was placed in series with the null meter and was run a t a chart speed of 2 scale divisions (1.5 inches) per minute. The combination amplifier-null detector-recorder wae calibrated with a standardized 20-cycle-per-second test signal over the range of both amplifier gain settings and null point settings since the null meter scale is logarithmic.
acid, oxalic acid, phthalic acid, potassium chloride, succinic acid, sulfanilamide, tartaric acid, and urea. They were applied as solutions and after they had dried a sample strip and its reference strip of plain filter paper were placed in the strip guides and compared. AI1 of the substances investigated were easily detectable when 8 mg. of sample were between the apertures. For several compounds it was of interest to find the limiting amount which could be detected using this method of comparing dry paper strips. In the case of oxalic acid, amounta as little as 0.5 to 0.7 mg. uniformly spread over the total area of the aperture could consistently be detected. On the other hand, the detection limit for dextrose was approximately five times higher. A typical recording of a prepared strip is shown in Figure 4,B. Figure 4,A , shows a recording from the same regions viewed in Figure 4, B, before the samples were applied, showing that the instrument is also capable of detecting differences in plain paper strips, even though they be cut successively from the same sheet of paper. Attempts to eliminate such nonuniformities of the paper by washing were unsuccessful; however, they may be due to simply a varying thickness of the paper strip. Recordings of this type are reproducible to the extent shown in Figure 5 , provided the same regions of each strip are matched against each other during the runs. This can be done a t the beginning of a series by marking a line on each paper strip a t the place where it emerges from the guide. The strips can then be reset to these positions a t the beginning of the successive run. The instrument measures only the magnitude of the difference in intensities of radiation coming through the apertures in front of the sample and standard strips. A supplementary technique must be used to determine which radiation beam is more intense. A change in the size of the aperture in front of the reference strip gives this information conveniently. A11 the compounds listed caused a decrease in the intensity of radiation. Another technique tried in order to detect substancw on filter paper was to apply heavy mineral oil to the paper strips after the
Figure 4. Comparison of Paper C h r o m a t o g r a m s A . Comparison of t w o blank paper strips B . Comparison of s a m e dry strip after samples were applied
The noise level of the apparatus viewed a t the output of the preamplifier was found to be approximately 5 mv. (root mean square). Using the listed sensitivities for the bolometer and preamplifier, the minimum detectable power by the apparatus can be estimated to be about 3 X 10-8 watt. A recorded signal of zero could be obtained when sections of plain filter paper were being viewed in both guides by adjusting either the width of the apertures in front of the paper strips or the null point scale setting. Differences in emission or transmission of other sections of a developed chromatogram would then be recorded as deviations from this null point. As a means for estimating the temperature of the strip as it passed into the radiation beam, various types of wax were rubbed on the paper surface toward the source. As a section of paper containing a wax having a melting point lower than the temperature acquired by the paper in the radiation beam, the wax would melt, changing the translucence of the strip markedly. By adjusting the water temperature in the source housing and the voltage at the heating mantle, the temperature of the strip was kept between 50" and 60" C. when the strips were pulled through a t the normal rate of 1 inch per minute. EXPERIMENTAL
The paper used throughout the investigation was Whatman No. 1 filter paper. The transmission of this paper versus air, as measured with a Beckman Model IR-2 spectrophotometer, was found to vary from 0.2 to 0.3% in the wave-length range of 1 to 10 microns, much of the radiation, of course, being scattered. Strips of this paper were prepared with samples of various colorless compounds deposited upon them. These compounds included citric acid, dextrose, malic acid, maltose, naphthalene-2-sulfonic
Figure 5 .
Duplicates of Dry S t r i p Comparisons
193
V O L U M E 26, NO. 1, J A N U A R Y 1 9 5 4
gram was sufficiently developed so that the bands of oxalic acid and dextrose were clearly separated. The bands could be detected either by using the apparatus or by painting a solution of bromothymol blue in the area suspected of containing the oxctlic acid and a solution of ammoniacal silver nitrate in the region suspected of containing the dextrose. Using the oil technique, Figure 6 shows the recordings of such a separation and tbe reproducibility attainable.
Figure 6. Recordings of Developed Chromatogram Containing Oxalic Acid and Dextrose CA is maximum amount of a material between the apertures at any instant
eamples had been deposited. The oil was painted on the paper with a soft camel's-hair brush and any excess which remained on the surface was blotted off until no oily gloss remained. The transmission of the paper in this condition as measured with a Beckman Model IR-2 spectrophotometer had risen a t some wave lengths in the range 1 to 10 microns to as high as three times the transmission of the unoiled paper. Apparently a t these wave lengths, a decrease in the radiation reflected from the surface had more than offset any increase in radiation absorbed by the paper due to the oil present. Strips of oiled paper containing samples distributed at various spots were compared with plain oiled paper in the same manner and conditions used with the unoiled paper strips. As in the previous technique, the presence of any of the compounds listed caused a decrease in the intensity of the radiation. Recordings of these comparisons were found to be very reproducible, and reoiling of the paper had little effect on the chart obtained. I n general, this method was much more sensitive, an oxalic acid sample giving six times as great a response by this method as in the comparison of dry strips. Using either of the described techniques, the deflections obtained for oxalic acid, evaluated in terms of millivolts, were not found to follow a linear relationship with respect to amount. From the data available it was not possible to decide whether this behavior was a property of the system measured or whether the fault lay with the manner in which the deflections were evaluated. APPLICATION TO DEVELOPED CHROMATOGRAMS
Operating the apparatus under the conditions described in the previous section, developed chromatograms were examined by both the comparison of dry strips and oiled strips. One of the systems separated and detected was a mixture of oxalic acid and dextrose. A sample of a solution containing approximately equal quantities of these two substances was applied 2 inches from one end of a strip of filter paper. After drying, the strip was suspended in an enclosed glass cylinder with the end nearest the sample immersed about 1 em. in a reservoir of n-butyl alcohol saturated with water. I n approximately 4 hours, the chromato-
Figure 7. Recording of Developed Chromatogram Containing Malic and Tartaric Acids
Chromatograms of oxalic acid and dextrose were also examined by comparing dry strips of filter paper; however, the procedure required was more tedious. First, two plain strips of filter paper cut successively from the mme sheet of paper were compared in the apparatus. A sample of the mixture was then applied to one of the strips and it was developed as described previously. The developing solvent was also allowed to rise in the other strip in case the solvent would have any effect on the paper. After drying, the strips were again compared a t the same gain setting on the amplifier. The solvent appears to have had very little effect on the strips, since the deflections which showed up in the comparison of the plain strips also showed up in the comparison of the developea strips. Another system which was investigated was that of a mixture of tartaric and malic acids. The procedure used for separation was the same as that described previously, except that the developing solvent consisted of n-butyl alcohol saturated with an aqueous solution of 1N acetic acid (8). After development, the strip was placed in a drying oven at 60" C. to remove any acetic acid, and then compared with its reference strip of plain paper in the apparatus using the oil technique. Figure 7 shows a recording of such a chromatogram. In this case, a third component appeared in the developed chromatogram right a t the solvent front. This material was yellow in color and was due presumably to some impurity in the n-butyl alcohol used in this case. U S E OF NEGATIVE FILTER
In order to test the possibilities of identifying the separated components on a chromatogram, a negative filter was prepared for
A N A L Y T I C A L CHEMISTRY
194 use in the identification of the components of the dextrose-oxalic acid separation. A saturated solution of oxalic acid in ether was added to a stock solution of collodion and a sample of this mixture was placed on a glass plate and allowed to dry. The film thus formed was 0.0025 inch thick and was found to contain 30% oxalic acid by weight. When examined with a spectrophotometer, the film was found to have the absorbance spectrum versus air shown in Figure 8, A . T o examine the extent to which this film is characteristic of the oxalic acid it would be desirable for comparison to have an exactly similar film with the oxalic acid removed. Difficulties in standardizing the preparation of such films, and, in particular, the uncertainty as to what the oxalic acid film would k i p like if the oxalic acid were removed, prevented this direct comparison. The absorbance spectrum of a plain collodion film is shown in Figure 8,B, in order to gain some idea of what the oxalic acid film would transmit if the oxalic acid were not there. Considering the thickness of the films and their densities, i t is frlt that approuimately equal amounts of material are compared in these two spectra; hoivever, in the case of the oxalic acid film, only two thirds of the material is collodion. It would thus seem that the absorbance of the collodion portion of the oxalic acid film would be, in general, about two thirds of the values shown for the plain collodion film in Figure 8, B.
A
E
Figure 9. Recordings of Developed Chromatogram Containing Oxalic Acid and Dextrose A . Before interposing filter B . After interposing filter
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WAVELENGTH.
Figure 8 Infrared Absorption Spectra A. B.
Oxalic acid filter Collodion film
Although similar, these spectra show decided differences between l and 2.5 microns and again between 4.5 and 6.0 microns. Spectra of collodion (15) and oxalic acid crystals (IS) found in the literature show that oxalic acid has a broad absorption band betxeen 5 and 6 microns with sharp bands a t 2.8, 7.4, and 8.0 microns, while collodion has sharp bands at 3.0, 7.1, and 8.0 microns. The absorption spectrum of dextrose crystals has been published only for the range 1.6 to 4.6 microns (1). These data were felt to be insufficient to predict whether the filter prepared would actually act selectively as a negative filter; however, it was felt that the structural differences between oxalic acid and dextrose might be great enough so that some effect might be observed with even such a crude filter. This guess appears to be correct as shown in Figure 9 Chart B corresponds to the comparison of a developed chromatogram of oxalic acid and dextrose with its reference strip of plain paper, using the oil technique. Chart A corresponds to the comparison
of the same strips after interposiiig the oxalic acid film in front of both apertures. The film was mounted in a cardboard frame and was placed in a prepared holder on the strip guide assembly so that it was between the paper strip and the chopping wheel. The film attenuated the signal strongly so that the gain used for chart A was raised till the deflection given in the dextrose region WM approximately the same acl that shown in chart B. The deflection measured in millivolts given in the oxalic acid region is found to be attenuated to a much greater extent and shows a relative decrease of 91%, whereas the deflection decrease in the dextrose region is found to be 81% This effect can be interpreted in two ways. First, if transmitted radiation is being measured the film acts in the following way: Radiation characteristic of oxalic acid is absorbed from the radiation beam of the heating mantle by the oxalic acid on one paper strip. However, radiation characteristic of oxalic acid is being absorbed in both beams by the film so that the difference in intensities should be less than if the film were absent. On the other hand, radiation characteristic of dextrose will be absorbed by that substance on the sample strip, but the film will not absorb this radiation to as large an extent a8 it did radiation characteristic of oxalic acid. The signal from the dextrose region should then be less than it was without the filter, since all things absorb somewhat at all wave lengths, but the signal decrease should be proportionately smaller than the decrease of the oxalic acid signal. This effect can also be interpreted in terms of selective emission from the oxalic acid on the paper strip followed by selective absorption by the film. Radiation characteristic of oxalic acid is emitted from that region on the paper strip and is selectively absorbed by the film. Also, however, radiation a t these characteristic wave lengths is absorbed by the film in front of the plain
V O L U M E 26, NO. 1, J A N U A R Y 1 9 5 4 paper strip, so that the signal from the oxalic acid region should be less than in the absence of the film. By a similar argument, the signal from the dextrose region would not be affected to such an extent. DISCUSSION
The procedures described in the previous section have been shown to be capable of detecting a large assortment of chemical compounds and to be of some use in detecting the separated components on paper chromatograms. I n the procedure involving the comparison of dry paper strips, the measured signal indicating the presence of a sample on the strip appears to be due largely to the lower temperature of the surface containing the sample. A sizable contribution to the signal, however, is due to either a decrease in the transmission of the paper in the sample region or a lowering of the emissivity of the surface containing the a m p l e . No good method was found to distinguish between these latter possibilities. The relative insensitivity of the procedure in detecting sugars such as dextrose and maltose suggests that the chemical similarity of these substances with cellulose may give rise to similarities in the infrared emission and transmission of these substances. The measured signal in the comparison of oiled paper strips appears to be almost entirely due to the transmission of radiation from the heating mantle. Heat capacity effects were shown to be small and there is no reason to expect that the large increase in signal after application of the oil is due to larger differences in emissivities. This procedure is also relatively insensitive to sugars, again suggesting the influence of similarity in chemical structure between the sugars and cellulose. The greater sensitivity of this procedure together with the greater selectivity of transmission measurements over heat capacity measurements in identifying chemical compounds make this the preferred method. Chromatographic separations on paper usually involve material quantities in the order of several micrograms, whereas the quantities which can be successfully detected by this method are in the order of several milligrams. Now, as has been shown, chromatographic separations can be made with such large amounts on filter paper, but the relative movement of the bands must be widely different The major usefulness of paper chromatography on the other hand is its ability to deal with small samples and the trend of development for this technique appears to be in continually decreasing the sample size as soon as suitable detection methods become available. Thus, the present apparatus and technique cannot be considered as suitable for the detection of the majority of paper chromatograms.
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By changing the arrangement of the apparatus 80 that the conditions for making transmission measurements are at an optimum, the detection limits of this technique might be extended down into a more useful range. Improvement in transmission measurements could be accomplished by increasing the infrared intensity emitted by the source after properly shielding the strips in some thermostated enclosure. It is possible that the application of other inert nonvolatile liquids to the strips would also improve their transmission properties. Because of the proximity of the noise level, a narrow band pass filter in the amplifier circuit would also be very desirable. Some indication has been shown that transmittance measurements coupled with the use of a negative filter can be used not only to detect a substance but also to identify it. Here again, however, more quantitative work would be necessary e0 establish this technique firmly. ACKNOWLEDGMENT
This research has been supported in part by the Abbott Fund of Northwestern University and in part by research grant RG2749 of the National Institutes of Health, PLiblic Health Service. LITERATURE CITED
Barr, E. S., itnd Chrisman, C. H., J . Chem. Phys., 8,51 (1940). ( 2 ) Carter, C. E., J . Am. Chem. Soc., 72,5612 (1950). (3) Holiday, E. R., and Johnson, E. A,, Nature, 163,216 (1949). ( 4 ) Hotchkiss, R. D., J . B i d . Chem., 175,315 (1948). (5) Jamison, N. C., Kohler, T. R., andKoppius, 0. G . ,ANAL.CHEM., (1)
23, 553 (1951).
Jones, A. R., Ibid., 24, IO55 (1952). Kivenson, G. J., J . Opt. SOC.Amer., 40, 112 (1949). Lugg, J. W., and Overell, B. T., Nature, 160,87 (1947). Martin, G. A., Instruments, 22, 1102 (1949). hliiller, R. H., and Wise, E. N., ANAL.CHEM.,23,207 (1951). Paladine, A. C., and Leloir, L. F., Ibid., 24, 1024 (1952). (12) Pereira, A , , and Serra, J. rl., Science, 113,387 (1951). (13) Randall, H. hl., Fowler, R. G., Fuson, N.,and Dangle, J. R., “Infrared Determination of Organic Structures,” p. 105. New York, D. Van Nostrand Co., 1949. (14) Rockland, L. B., Lieberman, J., and Dunn, M. S., ANAL.CHEM., (6) (7) (8) (9) (IO) (11)
24,778 (1952). (15) Rowen, J. W., and Plyler, E. K., J . Research Natl. Bur. Standards, 44, 313 (1950). (16) Tennent, D. M., Whitla, J. B., and Florey, K., ANAL.CHEM., 23,1748 (1951). RECEIVED for review February 9, 1953. Accepted October 1, 1953. Abstracted from the Ph.D. thesis of Donald R . Kdkwarf, Northwestern University, 1952.
Spect rophotometric Tit rations with Ethylenediaminetetraacetic Acid (II) Determination of Magnesium, Calcium, Zinc, Cadmium, Titanium, and Zirconium PHILIP B. SWEETSER’ and C L A R K E. BRICKER Department o f Chemistry, Princeton University, Princeton,
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I D E use of ethylenediaminetetraacetic acid or its sodium salts (EDTA, Versenate, Sequestrene, or Complexone 111) as a volumetric agent has been possible because of the broad chelating power and stability of the Versenate chelates. Versenate forms a 1 to I chelate with B large number of di-, tri-, and, in some cases, tetravalent cations. By the proper use of buffers and additional complexing agents, the chelating power of the Versenate can be made very selective. This has been illustrated by Kinnunen and Merikanto in their procedure for the deter-
* Present addreese, Chemical Department, Experimental Station, E. I. du Pout de Nemoura L Co., h a . , Wilmington, Del.
N.1. mination of zinc in the presence of copper by the addition of cyanide to an ammonia-ammonium chloride solution of the metals, using Erichrome Black T as an indicator ( 2 ) . Cheng et al. have been able to determine calcium, magnesium, and iron in limestone with Versenate by varying the buffer conditions (1). The over-all versatility, sensitivity, and general convenience of Versenate as a volumetric reagent are, however, dependent upon the means of end-point detection used for the various titrations. Pribil has described a potentiometric and amperometric procedure for the determination of several cations ( 3 , 4). The use of a spectrophotometric end point for the determination of iron( 111), copper(II), and nickel(I1) with Versenate has been dwcribed*(6).
