Spectroscopic Analysis of Isomeric Xylene Mixtures Method for Analysis of Mixtures with Overlapping Absorption Bands R i Y W O N D T. \-.AUGHY1 AND ALLEY E. STE.ARY I-niversity of Missouri, Columbia, .Mo. A n ultratiolet spectroscopic method is described for the analysis of mixtures with oterlapping absorption bands, and is applied to isomeric xjlene mixtures. This method involves the preparation of a working chart from the difference in measured optical densities a t selected wave lengths and the known composition of the samples. From ultra\ iolet absorption measurements a t four wa\e lengths. the composition of isomeric xylene mixtures can be read
from the working chart. The mean deviation between the known composition of synthetic isomeric xylene samples and their composition as obtained Application from the \c-orlcingchart is less than 1%. of the method is rapid and does not require measurement of extinction coefficients or involve solvent and cell corrections, and eliminates extraneous errors due to “fingerprints,” etc. Departure from Beer’s law- does not affect accuracy of results.
T
HE uaual method for the spectroscopic analy+ of niulticomponent mixtures with overlapping ahsorption bands requires the determination of the extinction coefficient of each component a t wave lengths where absorption is to be measured. For a mixture of n components, absorption measurements a t n wave lengths, followed by the solution of the n siniultaneous equations, gives the concentration of the various c,oniponents. Variations of this method have been used by preparing lvorking curves for the binary mixtures and making corrections for the component having the least absorption ( 2 , 5 ) . Robertson, Ginsburg. and Matsen (6) were able to analyze a mixture of isomeric cresols with an overlapping absorption band by making correction3 for the absorption of two of the components, the concentration of u-hich had been determined from absorption measurements using independent bands.
2500
2300
2700
Figure 2.
Ultraviolet Absorption Spectrogram of 1,3-Dimethylbenzene (m-Xylene)
IO0
2300 2500 2700 WAVE LENGTH IN ANGSTROM UNITS
Figure 3.
WAVE LENGTH IN ANGSTROM UNITS
Figure 1. Ultraviolet Absorption Spectrogram of 1.2-Dimethylbenzene (0-Xylene)
Inspection of the spectrograms of the isomeric x~.lenesas s h o w in Figures 1, 2, and 3 shows that no relatively independent bands exist and that the extinction coefficients of the isomers a t the wave lengths practicable for analysis are of the same order of magnitude. Because the accuracy obtained by the solution of n simultaneous equations is determined by the accuracy of determination of the extinction coefficients and their order of magnitude, as well as that of the absorption measurements a t n wave lengths, any error in the determination of extinction coefficients or the measurement of total absorption may affect the results considerably. The preparation of a working chart from the difference in measured optical densities of synthetic mixtures, a t selected wave lenths. tends to reduce the precnutions required for the securing of accurate values, inasmuch as extinction coefficients 1
2900
WAVE LENGTH IN ANGSTROM UNITS
2900
Ultraviolet Absorption Spectrogram of 1’4-Dimethylbenzene (p-Xylene)
are not required and any extraneous absorption, which is constant a t the two wave lengths used, cancels. Corrections for any deviation from Beer’s law are eliminated, because the composition of the synthetic mixtures covers the entire composition range of the analytical samples and any deviation from Beer’s law will appear in the working chart. THEORETICAL
For a single component, the analJ tical procedure is reduced to the preparation of a working curve by plotting log Ic,/I, the optical density, against concentration. If the absorbing substance obeys Beer’s law, the working curve is a straight line according to the relationship log Io/I
ECd
where E is the extinction coeficient, c is the concentration, and d is the thickness of the absorbing material. A mixture of independently absorbing substances absorbs light according to the equation
Present address, Chemistry Department, Southwestern-at-Jlemphis,
log Io/I
Memphis, Tenn.
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= (€ICI
+
E*C*
+ . . . .)d
ANALYTICAL CHEMISTRY
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coincide with regions of minimum absorption of the other components, and the wave lengths selected should provide a maximum difference in absorption for a single component. From inspectiOD of the spectrograms of o-, m-,and p-xylene shown in Figures 1,2, and 3, it is noted that p-xylene hae maximum absorption bands a t 2685 and 2745 A with relatively ;Teak absorption a t wave lengths 2620 and 2720 A. Strong bands for the meta isomer are found a t 2650 and 2725 with minimum absorption a t 2675 and 2705 A. Th: ortho component has maxima a t 2630 and 2710 A. with a minimum a t 2690 A. A4pplying the criteria stated above, wave lengths 2685 and 2625 A. nere selected for measuremeont of the para content. A maximum at NGTE 0 - i Percent 2725 A. and minimum a t 2705 gave a measurement of the meta content equal in sensitivity to about two thirds of that of tbe para isomer. The Figure 1. Working C h a r t for Analisia of Xylene hIixtures positions, 2705 and 2625 A , , corresponding to minimum absorption for the meta and para iscmers, respectively, coincide with positions of where el, ea . . are the extinction coefficients of noninteracting maximum absorption for the :ortho component. The ortho substances having concentrations CI,CZ . ., respectively. The difisomer has a minimum at 2685 A. which coinrides with a maxiference in density between bands from two different compounds in mum of the para; thus the wave lengths selected serve equally a single mixture is a more sensitive function of concentration than well for the measurement of the ortho content. the density of either band alone, and those regions of maximum As Figure 4 shows, the difference in optical density, 3 2 6 8 5 A. and minimum ahsorption give the grratest sensitivity. By the E2625 A , a t wave lengths 2685 and 2625 d. was plotted against method of differences, the optical densities a t two wave lengths are the difference in optical density, 3 2 7 2 8 A. - E2705 A , a t wave required for a single measurement. With constant total concenlengths 2725 and 2705 8. tration, two distinct measurements are sufficientto determine the APPARATUS AND REAGENTS composition of a three-component system, thus requiring optical The instrument used in this work was a Beckman quartz specdensity measurements a t three wave lengths. Measurements a t trophotometer (1 ), model DU, equipped for ultraviolet spectrosfour wave lengths mag provide a greater sensitivity, as the wave copy. The absorption cells were uartz, 1 em. in length. The lengths selected for the measurement of one component may not solvent, 2,2,4-trimethylpentane ?so-octane) was purified by selective adsorption on silica gel (3, 4 ) . Pure grade iso-octane, 99 be a sensitive function of concentration for the second. mole % purity, obtained from the Phillips Petroleum Company, Absorption measurements for a three-component mixture are: was passed through the silica gel column until its transmittancy equaled or excelled that of a standard sample obtained from the (1) E? = &cI E~:CZ E,!,:’c~ Sational Bureau of Standards. The solvent from the analyzed samples was reclaimed bv passage through the silica gel column E;, = ti,^, E;:CS c,!,:’c~ (2 1 The activated silica gel, ko. 22-08-08-226, was obtained from the E!: - Et: = (& &) ~1 (E:: E:!,) CP (&’ - & ‘ ) ~ 3 (3) Davison Chemical Corporation. where Eto$is the optical drnsity of the mixture. -4similar relaPREPARATION OF T H E WORKING CHART tionship can be written for the difference in optical densities a t Synthetic samples were prepared from “standard samples” of wave lengths XI and X I . By fixing the concentration of one comhvdrocarbons obtained from the National Bureau of Standards ponent with the total concentration constant, and varying the The purity of the standard samples was as follorrs: m-xylene concentration of the two remaining, a straight-line relationship is 99.91 * 0.04%, p-xylene 99.93 * 0.03%, o-xylene 99.99 * 0.007%. expected between the difference in optical densities a t two wave In preparing the synthetic samples, 0.65 ml. of the standard lengths, X I and AB, and the composition of the mixture. All lines sample, measured with a pipet, was introduced into 25 ml. of representing fixed quantities of one component are parallel. solvent (pipet) which had been weighed previously on an analytiSimilarly, lines representing constant concentrations of the second cal balance. From the weight of the resulting solution, the weight of the isomeric xylene was obtained. One miUiliter of this component will be parallel and have a slope different from that of solution (pipet) was added to 75 ml. of solvent measured from the first if their extinction coefficients a t the two wave lengths calibrated pipets. Samples of each isomer were prepared in an differ. A difference of optical densities a t wave lengths A3 and X4 analogous manner. The ratio of the weights of 0.65 ml. of the fixes the points of intersection of these b o groups of parallel standard samples checked the ratio of the densities of the isomers to within 0.04%. All measurements were made at 25” * 0.1 O C. lines, thus giving the composition of the mixture. The above refrom a constant-temperature bath. By means of 1-ml. and 5-ml. lationships are shown graphically in Figure 4 for the ternary syspipets calibrated and found to deliver volumes in the ratio of 1 to tem G-, m-, and p-xylene, which served as a working chart for the 5 to within 0.137,, 10-ml. samples were prepared, varying in comanalysis of isomeric xylene mixtures. position by 10%. Any extraneous absorption due to solvent, cells, fingerprints, The absorption of the samples n as measured a t the four wave etc., appearing in Equations 1 and 2 is eliminated in Equation 3, lengths designated above and plotted as shown in Figure 4. This if constant for the n-ave lengths selected. graph, covering the entire composition range on the ternary cornThe curvature of the constant percentage lines in those regions position axes, served as a working chart for the analysis of experiof high concentration of the para component, as shown in Figure mental samples. The same pipets and quantities were used in 4, indicates a deviation from Beer’s law. preparing the experimental samples for analysis.
h.
a.
.
.
+
-
+
+
-
+ +
+
ANALYSIS OF SPECTROGRAMS
ACCURACY OF T H E METHOD
For maximum sensitivity from a working chart as described above, regions of maximum absorption of one component must
The triangular coordinates of Figure 4, constructed from the measured absorption of the synthetic samples, indicate the
V O L U M E 2 1 , NO. 11, N O V E M B E R 1 9 4 9 Table I. %mple
Analyses of Isomeric Xylene Mixtures Actiial Composjtion, Volume Rleta Para
Ortho
Observed Composition. Volume yo Meta Para
Ortho
1363 The two analyses of sample I11 indicate a possible error in the preparation of the synthetic hydrocarbon mixture. A decrease in concentration of total xylenes would probably eliminate the curvature of the constant percentage lines in regions of high concentration of one component, thus giving a triangular coordinate system. ACKNOWLEDGMENT
accuracy of the method. The variation in distance between lines
of constant percentage does not exceed *0.5% except in the regions of high concentration of one component; also, the experimental points do not vary by more than *0.5% from straight constant percentage lines except in those regions near the vertexes and a few points along the 0% para coordinate. The intersections of the constant percentage lines at the experimentally determined points, also, indicate the accuracy of construction of the absorption diagram. As the preparation of the 10-ml. samples used for calibration purposes is omitted in the analysis of isomeric xylene mixtures, the analysis of a mixture would be expected to be more accurate than the location of any point used in the preparation of the working chart. To test further the accuracy of the method, synthetic samples, prepared from synthetic mixtures of the pure hydrocarbons, were analyzed (Table I).
The authors are indebted t o the American Petroleum Institute Research Project 44 a t the Kational Bureau of Standards for supplying samples of pure isomeric xylenes for spectroscopic calibration purposes. LITERATURE CITED
(1) Cary, H. H., and Beckman, A. O., J . Optical Soc. Am., 31, 682 (1941).
(2) Fry, D. L., Nusbaum, R. E., and Randall, H. M., J. AppZied P h y s . , 17, 150 (1946). (3) Graff, M. M., O’Connor, R. T., and Skau, E. L., IND. ENO. CHEM.,ANAL.ED., 16, 556 (1944). (4) Mair, B. J., J . Research Natl. Bur. Standards, 34, 435 (1945). ( 5 ) Nielsen, J. R., and Smith, D. C., IND.ENQ.CHEM.,ANAL.ED., 15, 609 (1943). (6) Robertson, W. W., Ginsburg, N., and Matsen, F. 8 . ,Ibid., 18, 746 (1946). RECEIVED July 30, 1948. Part of a thesis submitted by Raymond T. Vaughn t o the Graduate Faculty, University of RIissouri, in partial fulfillment of thi requirements for the degree of doctor of philosophy.
Determination of Sugar in Forage Plants J. W. THORI..IS, C. G . MELIN, AND L. A. MOORE Bureau of Dairy I n d u s t r y , U . S. D e p a r t m e n t of Agriculture, Washington, D . C . The time required to make sugar determinations on forage plants has been considerably shortened. Extracting either green or dried material with an alcoholic solution in the Waring Blendor was as complete as with the longer A.O.A.C. method employing a Soxhlet extraction. Carotene and sugar were extracted simultaneously from green material by the “foaming mixture” in the blender. Clarification of the extract with lead did not alter the amount of reducing substances found in the plants studied.
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HE soluble carbohydrate fraction of forage crops may account for as much as 20% of the feeding value of the crop.
During the harvesting procedures a large proportion of these labile constituents may be lost, especially under adverse harvesting conditions ( 1 , 8). Recently there has been an increase in the use of artificial methods of drying forages, and many studies are being conducted to determine the effect of the various drying procedures on the amount of nutrients preserved ( 1 , 8). It is also desirable to study the relative loss of sugars when the crop is cured by various processes, but such a study is handicapped by the lengthy procedure used to perform sugar analyses on the forage. The object of the present study was to determine whether the usual procedure for sugar analysis could be shortened by using a more rapid method of extraction and by eliminating the lead clarification of the extracts. Because the carotene content is also of interest when the forages are preserved by different methods, an attempt was made to determine both the sugar and the carotene content by one extraction of the same sample of green material. EXPER1,MENTAL
Two methods of extracting dry forage plants were compared. The dry forage material was ground in a Wiley mill, using the
screen with 1.0-mm. holes, and 2.0-gram samples were used for all extractions. In one method of extraction, the procedure described for plant tissues by the Association of Official Agricultural Chemists ( 2 ) was followed, except that a 50 to 60% solution of ethyl alcohol as used for feeds was used in place of 80% alcohol. Others (3, 5 ) also have found this substitution satisfactory. In this procedure, preliminary heating of the forage material in alcohol on a steam bath was found to be necessary in order to extract all the sugar from the tissue. The second or experimental extraction procedure used was to extract the dry material with 200 nil. of 75% alcohol in a Waring Blendor for 5 to 7 minutes. The alcohol solution was then filtered by using a Buchner funnel or fritted filter with vacuum and the residue was washed five to six times with 50% alcohol. During the investigations it was found that the filtration process was hastened by layering Hyflo Super-cel suspended in 70% alcohol over the filter paper on the Bdchner or over the fritted filter. Green forage material was extracted by three procedures. In each case 5.0 grams of material were used. Procedure 1 was the method described by the Association of Official Bgricultural Chemists ( 2 ) ,as modified for use Lyith the dry material. Procedure 2 was the method using the Tl-aring Blendor, with 75% alcohol, for extraction purposes. The third procedure combined the extraction of carotene and sugar from the same sample by employing the “foaming mixture” ( 6 ) . This consisted of using an extraction mixture of 100 ml. of absolute alcohol and 70 ml. of Skellysolve B. The water content of the green material was usually sufficient to give the necessary proportions of alcohol to Skellysolve to produce a desirable foaming mixture. For use in the Waring Blendor the sample was cut as fine as possible (1.25-em. lengths or leas)
