Modern Trends of Absorption Spectrophotometry In the Ultraviolet and Visual Kegions E. I. STEARNS .4pplication Research Department, Calco Chemical Division, American Cyanamid Co., Bound Brook, N. J . This review paper presents some of the modern trends toward newer methods of analysis, although the well-known standard methods still comprise the bulk of visual and ultraviolet analyses. A trend away from standard measurement proceduresis illustrated by use of an absorbing reference solution to get increased precision. A trend toward complexity of solution preparation is illustrated by the variable reference solution technique. A trend toward complexity in analys i s of data is illustrated by the determination of empirical formulas of complexes. Illustrations are given of reflectance and fluorescence emission analysis. As a result of this diversification of methods, the spectrophotometric method can be applied to a larger variety of analytical problems.
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T T H E present time, about 10,000 ultraviolet and visual
spectrophotometers are in use in various laboratories. By far the greatest amount of work with these instruments involves quantitative determination of one component or identification of single components. Extrnsion of these methods to niulticomponent systems is also coinmoil. The methods are all very well worked out and discussed in textbooks ( I O ) . Examples of applications for many materials can be found in the literature ( 2 ) . The purpose of this review article is not to discuss these wellestablished methods but rather to present the modern trends of spectrophotometric analysis toward neJv methods
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DEVIATIONS FROTI STAND4RD PROCEDURES
Absorption spectrophotometry has been so well standardized that there is a tendency to carry out analyses without thinking about them very much. In these so-called “standard” procedures most measurements are made by determining the transmittancy of a solution a t the wave length of the absorption maximum of the cornpound with a solvent-filled cell as the reference. Beer’s law is assumed. To analyze for two or three components, transmittancies are determined at two or three wave lengths. However, these widely accepted practices are not necessarily the best and should not be blindly accepted. All analyses need not be carried out a t the absorption maximums. The selection of the wave lengths for analysis should be based upon several considerations ( I O ) . For instance, Figure 1 shows the effect of irradiation on the absorption spectrum of Thio-Indigo Orange R (C.I. 1217, 13). The chemical explanation of the two forms of this compound which give the two curves shown in Figure 1 has been shown to be cis-trans isomerism (29). If an attempt is made to measure this compound a t the absorption maximum, it is necessary to control the temperatuie and the llluminatlon very exactly in order to obtain valid results. If, however, a wave length of 480 mp is chosen, a correct analysis of concentration may be made a-ithout exercising great care. Thus the absorption maximum is not always the best wave length for analysis. If two components are to Le determined, it is not necessary to take readings of transmittanc! at two wave lengths. Thus, in multicomponent analyses, measurements of absorbancy coefficients with different solutions of identical concentration may be jugt as useful as measurements of absorbancy coefficients a t different wave lengths The reference solution need not be the solvent at zero concentiation of the sample to be analyzed I t has been pointed out .(.vera1 times that increased accuracy can result from maawring :-I wlution aqainst an optical g h s i filter or .i reference cell n hich
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Figure 1. Effect of Irradiation on Absorption Spectrum of Thio-Indigo Orange R in Pyridine 4. B.
After dark storage After irradiation by 50-watt tungsten distant Copied from Mellon (10)
1 minute. 6 inches
contains a known concentration of one or more of the components being determined ( I O ) . K i t h this technique, it has.been reported ( 1 ) that a precision better than 0.1% can be obtained. This precision is of the same order of magnitude as many volumetric and gravimetric analytical chemical techniques. Thus, the reference solution should not be the solvent in many cases when greater precision is desired. If Beer’s law is found valid for individual compounds, it should not be assumed valid for mixtures of these compounds in carrying out multicomponent analyses. Beer’s law states that the absorptive capacity is directly proportional to the concentration of the solute. The additivity of absorbancies of mixed compounds is a corollary of Beer’s Ian. Hoxever, it iF perfectly possible for Beer’s law to be valid foi the individual components and yet have the corollary fail for the mixture. Figure 2 shows the curve of two dyes, a yellow (C.I. 365) and a blue (C.I. 518). Curve C is the predicted curve of a mixture of 25% blue and 75% yellow. Curve D is the measured curve of this same mixture. The predicted rurve deviates from the measured curve because of a failure of additivity of absorbancies of the t u o components. -4twocomponent analysis for these two dyes may not be carried out without special precautions. Thus, Beer’. law for single components, and its corollar~for mixtures. should be checked separately. 1004
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WAVE LENGTH, MILLIMICRONS
Figure 2. Effect of Component Interaction on ibsorption Spectrum of Mixture of Diamine Sky Blue FF and Chrj sophenitie Diamine Sky Blue FF (C.I. 518) Chrysophenine (C.I. 365) Predicted curve of 25% A and 75 % B Measured curve of 2570 A and 7570 B
A. B. C. D.
INCRE4SING COMPLEXITY IN SOLUTION PREPARATION
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Figure 3.
Absorption
C u r \ e of Fluorescein Dyes
in Dilute Ammonia Concentrations arbitrary Fluorescein Dibromofluorescein Tetrabromofluorescein (hpird from Jones e t o l . ( 8 )
1. 2. 3.
Copied from Mellon ( I O )
As an example of extended utility of spectrophotometric methods by greater complexity in solution preparation, the work of Jones et al. may be cited. One of the advantages of a doublebeam spectrophotometer is that it enables the operator to eliminate the absorption of one or more of the components of a mis by putting the same amount of the component in the reference beam ( 1 2 ) . This technique has been carried to the extreme hy Jones et al. ( 8 ) , who varied the reference solution as part of the determination. In this procedure, the 100% transmittancy line of the spectrophotometer is adjusted so that it fa115 near the center of the plotting paper. The solution 1 0 9 to be analyzed is placed in one beam of the spectrophotometer. The cell in the other beam is connected to a titration vessel by a circulating pump. The components which are in the sample cell are added separately and in measured amounts by a buret into the titration vessel. The spectrophotometric. curve is run after each addition. By an examination of the curve it is apparent, after a little practice, which materials must be added. The end point is reached when the instrument plots the 100% line. One of the advantages of this method is that the precision of the wave-length setting is not important. Figure 3 shows the curves of fluorescein, dibromofluorescein, and tetrabromofluorescein known aa D&C Yellow S o . i , D&C Orange KO.5 , and D&C Red S o . 21. These are not true transmittancy curves, because the setting of the instrument has been altered so that the 100% line plots in a part of the paper where the pen may record above as well as below the 100% line. If an attempt were made to select three wave lengths for the simultaneous equation determination of these three components, a t least t n o of the wave lengths would fall on a part of the absorpL tion curve of the unknonm solution where the 400 500 600 slope of the curve is very steep. A very small WAVE LENGTH, Mer change in the wave-length settings would result Figure 4. Analysis of Commercial in a large difference in the apparent transmittancy Sample of D&C Orange No. 3 and consequently the analysis would not be w r y Variable reference solution teohniqirr nccura te. Copied from Jones et ai. ( 8 )
The applications of spectrophotometry are being extended by increasing the complexity of the solution preparation. For example, the solution cell has been connected t o a very complicated apparatus for studying the exhaustion of dye from a solution to n fiber (14, 16). The solution cell has been connected to the blood vessels of a living dog to study the constituents of blood ( 5 ) . Special solution cells, such as those for measuring very small quantities of liquid (9),have been used.
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All concentrations 0.015 gram/liter total d>e , all solutions irradiated, solvent water
ANALYTICAL CHEMISTRY
1006 The variable reference solution technique is illustrated by Figure 4,which shows the analysis of a commercial sample of D&C Orange No. 5 , which is a mixture of the three components shown in Figure 3. Curve 1 is the initial sample. Inspection shows that it is primarily dibromofluorescein and consequently 3 mg. per liter of this compound are placed in the reference cell. When this combination of two solutions is measured, curve 2 results. Curve 2 represents the residual absorbing materials which are present in the sample cell in excess of what has been added to the reference cell. I t is evident that still more dibromofluorescein is present. Consequently, two further additions of dibromofluorescein are made, giving curves 3 and 4. Curve 4 has an evident absorption a t the wave length where the tetrabromofluorescein absorbs. Conse uently, a small amount of tetrabromofluorescein is added to %e reference cell and curve 5 is run. Successive small amounts of the three components are added to the reference solution, as an inspection of the absorbancy curve indicates necessary, until finally a line results which is a close approximation to the 100% line. As no transmittancy readings are taken on a steep part of the curve, no error is introduced from this source. Table I shows the composition of the reference solution for the different curves of Figure 4.
Table I. Fig 4 Curve
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Composition of Reference Solution D&C Orange ho. 5, Mg.
Calcd. compn. of unknown. %
D&C
Red Xo. 21, Mg.
0.00
0.00 3.00 3.50 3.95 3.95 3.95 3.95 4.02 4.08 82
0.00 0.00 0.00 0.29 0.37 0.37 0.40 0.40 8.0
Variable reference solution technique Copied from Jones et at. ( 8 )
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0.00 0.00 0.00 0.14 0.14 0.16 3.2
Mp
Figure 5. Analysis of Benzene and Toluene lMixture in a Solution of 95% Ethyl Alcohol
D&C Yellow No. 7, Volume, Mg. M1. 0.00 1000 0.00
Table 111. Composition of Reference Solution 1005
I t is very difficult to compare the accuracy of an analysis based on simultaneous equations to the accuracy of an analysis by the variable reference solution technique. I n some analyses of the compounds illustrated in Figure 3 by the method of simultaneous equations, the calculated values for the components present in small amounts were absurd-for instance, a negative value has been found occasionally for a component known to be present. For a mixture of 90% tetrabromofluorescein and 10% dibromofluorescein, the method of simultaneous equations gave a result for the dibromofluorescein dependable to =J=20% of the amount present. By the variable solution technique, the value obtained for the dibromofluorescein has been found dependable to i10%. While it is impossible to give a simple pair of comparative accuracies applicable generally to all mixtures, in individual cases the variable reference solution technique gives more accurate results. Another advantage of the method is that the precision of the slit width setting is unimportant, Figure 5 and Table I1 illustrate the analysis of a mixture of benzene and toluene in solution in 95% alcohol. It is likely that in a two-component analysis a t two wave lengths the absorption maximum of toluene at 269
Curve
FDB-C Yrliow KO.6
Calcd. conipn. of unknown, mg./l. Actual compn. of unknown, mg./l.
8.9
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mM would be used as one of the wave lengths. This peak, however, is very sharp, so that different slit widths will give a different indicated transmittancy and, therefore, a difference in the final analysis I n the variable reference solution technique, the sharp-
Table 11. Composition of Reference Solution Curve
4 5 6 Calcd. compn. of unknown, mg./l. Actual compn. of unknown, mg./l.
Benzene, hlg. 0.00 99.5 125.0 125.0 174.8 178.8 195.0
Toluene, Mg. 0.00 124.8 161.8 181.0 181.0 184.8 199.0
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915
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W A V E LENGTH, Mr Figure 6. Determination of FD&C Yellow No. 6 in Presence of Caramel Dye Variable reference solution teohnique Copied from Jones e t al. ( 8 )
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ness of this peak is an advantage rather than disadvantage. As toluene is added to the reference solution, its peak becomes smaller and smaller and finally appears as an inverted peak which is just barely evident in curve 6 of Figure 5. A third advantage of this technique is that it is possible to correct for a “reasonable” background. Figure 6 and Table I11 show the spectrophotometric titration of a mixture containing FD&C Yellow KO.6 and caramel.
. Figure 7.
Determination of Two Dyes Which Interact in Solution Variable reference solution technique Copied from Jones et al. ( 8 )
Given the series of seven curves in Figure 6, the problem is to select the most reasonable background. There is obviously an excess of dye in the solution cell for curve 2,because there is some absorption a t the wave length where the dye absorbs strongly. Curve 7 obviously represents a case where a deficiency of dye is pr-esent in the solution cell, because there is an unnatural deficiency in absorption at the region where the dye absorbs. With a little euperience, curve 5 may be chosen as the most reasonable background. If no correction is made for hackground, the single-component analysis for quantity of FD&C Yellow S o . 6 present gives the result of 10.35 mg. per liter. If a correction for linear background absorption in the region 420 to 580 mp is made, analysis of the curve gives a result of 8.5 nig. per liter of dye present. The reasonable background, a- determined by the method of variable reference solution, gives a result of 8.9mg. per liter. In this particular case, the sample was known to have 9.0 mg. per liter of dye added to the caramel. Hence, the variable reference solution technique gave the most accurate result. This analysis could not be handled by the usual two-component method because the curve of caramel is somm hat variable.
A fourth advantage of the method is that deviations from Beer’s law or deviations of additivity of absorbancy are unimportant. Figure 7 shows the titration of the two dyes shown in Figure 2 for which absorbancies are not additive. Curve Y is the curve of the yellow dye by itself (C.I. 365), curve B is the curve of the hlue dye (C.I. 518), and curve 1 is the curve of the miuture. The titration data are given in Table IV.
Table IV. Composition of Reference Solution Curve
Calcd. cornpn. of unknown, mg./l. -4ctual compn. of unknown. mg./l.
Blue,
Yellow,
Mg.
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Yolume. 111.
Curve 1 cannot be predicted on the assumption of additive absorbancies. This indicates that the two dyes react in solution. It is possible to analyze these curves, as is pointed out by hlellon (io),page 380. However, this conventional method is somewhat involved. The interpretation of data is much iimpler by the method of variable reference solution. I n Figure 7, curve 2 shows that enough blue dye has been added .to the reference cell to compensate for the complete absorption a t 580 mp. Curve 3 shows the plot when enough yellow dye has been added to the reference solution to compensate for most of the absorption a t 400 mp. Some of the yellow dye added has reacted with the blue dye, so it is now evident that not enough hlue dye has been added. Addition of more dye of each type finally gives the flat 100% line which is curve 4. Provided the reaction of the two dyes takes place very quick1 the same quantities of dye and dye complex are present in botx’the sample and reference solution cells and no correction for failure of the corollary of Beer’s law is necessary. Several precautions must be observed. I n a spectrophotometer operating on the principle of the Cary spectrophotometer, it is necessary to put the unknown solution in the reference beam, so that the slit width of the instrument will be constant during the entire determination. The two solution cells must be identical in thickness and transparency, The circulation of the solution in the reference cell must be stopped before the measurement to avoid any air bubbles from scattering light and introducing a false absorption. It is desirable to add the major component first to the reference cell in order to reduce the amount of solvent which must be added if i t is necessary to back-titrate. However, none of these precautions is prohibitive. The variable referencesolution technique is an example of complexityin solutionpreparation but simplicity in interpretation. The concentrations of the several components present are determined by the titration data in the reference solution preparation. There really is no interpretation of the spectrophotometric curve a t all except to select the curve closest to a flat 100% line which represents equality of the samples in reference and solution beams. 0 0.5 1.0
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RELATIVE PROPORTION OF TIRON
Figure 8.
Determina-
tion of Empirical Formula
of Iron-Tiron Complex Method of continuous variations Copied from Harvey e t al. (6)
INCREASING COMPLEXITY IN INTERPRETATION OF DATA
The field of spectropho-
,tometric application is being
extended also by obtaining data in the normal, simple way, but then using the data PO obtained in further calculations. This may be considered as getting more information from the available data. -4s an example of this extension, the determination of empirical formulas of complexes in solution has heen chosen. If two compounds react in solution, it is frequently possible to determine the empirical formula of the complex from the absorption curves of suitably prepared solutions. Suppose that a single compound of well determined molecular formula is formed and that the law of mass action is applicable. I n the reaction md nB 9 A,B, the problem is to determine the values of m and n. As a specific example, consider the t x o components ferric iron and 1,2-dihydroxybenzene-3,5-disulfonate (trade name Tiron). It is observed experimentally that a mixture of these two constituents gives a distinct red complex a t p H 9.4to 9.6. To determine the numerical values of m and n by the continuous variation method of Job ( 7 , 18), solutions of components d and B of the same molar concentration are mixed in various proportions and the absorbancy of the various mixtures is measured. The difference between the absorbancy found for each
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ANALYTICAL CHEMISTRY
1008 solution and the corresponding value of absorbancy calculated for each solution on the assumption of additive absorbancies of the individual components is plotted against the composition. This curve will have a maximum or a minimum in the usual case. The composition a t which the difference in absorbancy is a masimum or a minimum is the exact composition of the complex which has formed. This is illustrated by Figure 8. The difference in predicted and measured absorbancy is the actual absorbancy in this case because the individual constituents are nonabsorbing a t this wave length (480 mg). It is evident that the absorbancv maximum reaches a peak a t a ratio of 0.750 mole of l,2-dihidroxybenzene-3,5-disulfonateto 0.250 mole of ferric iron. This is evidence that the red complex has the molar ratio of 3 t o 1. The molar ratio method ( 8 ) depends upon the following observations. If a stable complex is formed, a plot of absorbancy against molar ratio of component B to component A , with A held constant, rises from the origin as a straight line and breaks sharply to constant absorbancy a t the molar ratio of the components in the complex. By increasing the ionic strength of the solution and thereby decreasing the dissociation of the complex, the usefulness of the method is extended ( 6 ) .
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Figure 10. Curves of Various Mixtures of Magnesium Ion and 2-Amino-4-chlorophenol-6-sulfonicAcid + 2-Naphthol in Ammoniacal Solutions Concentration of dye constant
Irl, 0 3 6 9 MOLES TIRON’MOLE Fe
Figure 9. Determination of Empirical Formula of Iron-Tiron Complex Molar ratio method. Ionic strength = 0.60 Copied from Harveg e t al. ( 6 )
Figure 9 shows data on the same iron-Tiion complex which was discussed in connection with Figure 8. The curves of Figure 9 show the analysis at two levels of concentration of iron, both of which show a “break” a t the same molar ratio. From these curves, which level off a t a molar ratio of 3 moles of Tiron to 1 ot iron, it is deduced that the empirical formula of the complex is 3 Tiron to 1 iron. The slope ratio method ( 6 )depends upon the following observations. If the concentration of constituent B is constant and in eicess.. the slope of the line of absorbancy of the mixture versus concentration of A is proportional to a constant divided by m. If A 1constant and in excess, the slope of the line of absorbancy of the mixture versus concentration of B will be proportional to the same constant divided by n. Hence, the ratio of the slopes of the ta o lines will give the ratio of m and n and, therefore, the empirical formula of the complex A,B,. One application of these methods of analysis of complexes is ni the determination of purity of a dye. The color of certain dyes in water solution is markedly changed by the presence of a metal ion. Figure 10 shows a set of spectrophotometric curves which have been measured after successive additions of a magnesium reagent of known purity to an ammoniacal solution of 2-amino-4chlorophenol-6-sulfonic acid + 2-naphthol. The isobestic points are an indication that the system contains two colored components, the dye, D, and the complex, DMg. By a conventional two-component analysis ( 1 5 ) ,it is possible to determine the percentage of the complex present in each solution. The assumption is made that with a large excess of magnesium all the dye i p present in the form of the complex.
When a plot is made of per cent complex us. moles of magnesium added, Figure 11 is obtained. The lower portion of the curve is essentially linear. This is to be expected because with excess dye any magnesium is essentially completely converted to the complex. For solutions containing higher concentrations of the complex the curve of Figure 11 departs from linearity. This is a result of dissociation of the complex. If the linear portion of the curve is extrapolated upward to intersect the horizontal line drawn a t 100% complex, the abscissa of this intersection represents the number of moles of magnesium equivalent to the moles of dye present for a 1 to 1 complex. The purity of the dye may then be calculated by the relation, %purity =
(moles of Mgequiv. to 100%DMg)(M.W. of dye) 100 (wt. of dye sample)
The dissociation constant of the magnesium dye complex can also be derived from the curve in Figure 11. The dissociation constant, K , is given by the relationship
The concentration of each of the components in the dissociation constant equation is available from the nonlinear portion of the plot in Figure 11. The concentration of the complex is given in Figure 11 by the distance .4B,the concentration of dye by the distance BC, and that of magnesium by the distance BD. The dissociation constant for the magnesium complex of 2-amino-4chlorophenol-6-sulfonic acid + 2-naphthol was found to be 2.5 x 10-6 by using the above-described method. DEVIATIONS FROM SOLUTION SPECTROPHOTOMETRY
One extension of the application of spectrophotometric mea
