, I
I
1
80
10
20
30
40
50
Nickel, Micrograms
Figure 15.
Calibration for nickel on filter paper
(2) Felten, E. J., Fankuchen, I., Steigman, J., AKAL.CHEM.31, 1771 (1959). (3) Liebhafsky, H. A., Pfeiffer, H. G.,
a fixed quantity of cobalt t o serve as an internal standard, evaporating the acid solution onto a 13/*-inch circle of filter paper, and measuring in a n x-ray fluorescence spectrometer. The metals nickel, vanadium, and iron have been determined by the paper film technique. A typical calibration for nickel is illustrated in Figure 15. This curve illustrates the linearity and the precision of the calibration data. The measurement of peak intensity alone for the sought element provided
much less satisfactory results than the use of the internal standard. ACKNOWLEDGMENT
The permission of the Humble Oil & Refining Co. to publish this paper is acknowledged. LITERATURE CITED
(1) Birks, L. S., “X-Ray Spectrochemical Analysis,” Vol. 11, “Chemical Anal-
ysis,” Interscience, New York, 1959.
Winslow, E. H., Zemany, P. D., “X-Ray Absorption and Emission in Analytical Chemistry,” Wiley, New York, 1960. (4) Pfeiffer, H. G., Zemany, P. D., Nature 174,397 (1954). ( 5 ) Rhodin, T. N., Jr., ANAL. CHEW 27, 1857 (1955). (6) Winslow, E. H., Liefhafsky, H. A., Ibid., 21, 1338(1949). (7) JT‘yckoff, R. W. G., “Crystal Structures,” Vol. I, Interscience, New York, 1948. RECEIVEDfor review December 8, 1960. Accepted March 16, 1961. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa. February-March 1961.
Curvature Inversion Technique in Absorbance Compensation Spectrophotometry C. F. HISKEY Endo laboratories, Richmond Hill,
N. Y.
b The principles and technique of compensation spectrophotometry are discussed in relation to a curvature inversion technique for determining when the concentration of a substance being determined is identical with that in the reference standard. The method is illustrated with a few typical problems, and, in addition, various practical aspects of this technique are discussed.
T
SITUATION frequently occurs, where the analyst must determine spectrophotometrica~lysome particular absorbing constituent in the presence of other absorbing materials whose concentration and composition are not known and, therefore] their light absorbing characteristics are not known. It often happens that these unidentified materials vary from sample to sample, and therefore, for each assay it is necesHE
sary for the analyst to discover in the assay process itself, their light absorbing characteristics so that suitable compensation can be made in each case. Various partial solutions of this problem have been proposed from time t o time. For example, in the determination of vitamin A according to U.S. Pharmacopeia XVI, p. 938, various amounts of unsaponified absorbing material accompany the vitamin and compensation for its effects on the spectrum must be made. The partial solution is to assume that the impurities have an absorption spectrum that varies linearly with wave length in the vicinity of the vitamin absorption peak. B y making measurements a t three appropriately chosen wave lengths and knowing the spectrum of the pure vitamin, a suitable base line can be established which permits the calculation of the
background correction at the vitamin absorption peak. This technique] first employed by Morton and Stubbs (S), has been extremely useful in this and several other cases where it could be applied. It is particularly useful to the analyst who does not have a recording spectrophotometer available t o him and must, therefore] rely for his assay, on a limited number of measurements made after manually setting the wave length of the pass band. However, when a recording spectrophotometer is available, then by absorbance difference measurements over a suitable wave length interval, it becomes possible in principle t o discover in each case what the residual or background absorbance is and thus to obtain an exact solution. It is also possible to accomplish this objective with a manual instrument] but the extended period of VOL. 33, NO. 7, JUNE 1961
927
iii(~,isiir(~iii(~iit r r q i i i i u t is discaour:igiiig i i i i d c r sii(’1i c-ircuiiist:iiiccs the iiwtlrod docs not lciitt i t d f rendily to I”otluctiou control :LSS:L~S. ‘ I h purpose of tliis article is to dcvc.lol) tlic tlicw-y u n d d y i n g this type of :iss:ij’, to sIio\v soiiic of it5 :idvant:igcs :ind limit:Ltions, : i d to discuss pmcticd pro\)lims and tcchniqucs rchting to its :ippIiration in c8hcniic:il :iss:~y. 1Iic :ipprowh (~niplnycdlicrc is w r y sirni1:tr to tlic v:iri:ibIc rofcrcncc tcchniqric first dcsrribcd by Jones, C h k , and I1:irrow (a) and tlicii :tpplicd to vitainin :~ssnys by Schiaffino et al. (4). It tliffcrs from their approach in thnt tlic il1ustr:itions prcscntcd are tnkcn from :tctunl production snmples for which esnct compensation for all constituents is not possible. In addition, new opcr:iting techniques are dcscribed. This technique has been especi:illy useful in the assay of pharmaceutical forniulations. One often encounters light scattering when attempting to make :L direct spectrophotonietric determination of sonie particular constituent. In sirup this light Scattering is usu:illy caused by the liquid sugar used; in suppositories, it may result from the waxes or othcr high polymeric bases used in the formuhtion, and in tablets it is often c:tused by talc fines or traces of stearates which may remain in the filtrate when the granulation is extracted and filtered. At any rate, the problem is encountered often enough, but with the technique to be described, one may proceed with the assay without waiting to solve the problem of eliminating the iriterfcrence. :incl
I
?
PROBLEM O F BACKGROUND
The problem of resolving the spectrum of a pure substance from that of the background is but a specific instance of the more general case of resolving it from the combined spectra of a large number of pure substances whose identities are unknown to the analyst. It is equivalent to separating the spectra of two substances under conditions where there is overlap in their bands. I n this context, background is seen to be nothing more than the spectra of one or more unknown pure substances. It may also include as one of its components, the light scattering of suspended particles in the medium. Initially, i t is assumed that the Bouger-Beer relation applies to a solution containing a n absorbing species, X, which is a pure compound and another absorbing species, B, which is also a pure compound. Then the absorbance A , a t any \Trave length is given by the sum of the separate absorbances, vie. A = Ax
928
+ AB
ANALYTICAL CHEMISTRY
(1)
t,
h
t
A‘
Figure 1 . Residual spectra using different reference solutions
I n turn each of these individual absorbances varies with wave length in a way d i i c h is characteristic for each of the two substances, Le.,
+
could tliiis bc tl~~tc~i~niincd, but in p r x tic(., s r v ( w 1iinit:itioiis bccoinc evid(wt. Xltlioiigh iiiiti:illy the :issumption \vas niadc tlint thc, Ih1ngc~r-13cerrclatioilship applied to conq)ontmt Y , it is cvidcrit that a t tlic point of coniplcte conipcnsntion for it, this rcquiremcmt is no longtr ncccssaq.. Only in the cvcnt that X wcrc to iiitcr:~ct with some othcr constitucrit in the samplc for which no compensation can be ninde, will there be an error in this approach. Although the illustrations of this technique are given here only for the ultraviolct portion of the spectrum, ncvertheless the principle applies without modification to other portions of the spectrum and one may work in the visiblc and infrared with virtually no change in technique except as indicated bclow . ilt this point, it has been established that any solvent medium containing substances absorbing radiation from the incident beam and having different absorption functions as regards the wave length of light can be treated for assay purposes as a two-component system with a resolvable part and a residual part. The technique for accomplishing this resolution will nom be treated. METHOD FOR DETERMINING RESIDUAL SPECTRUM
To introduce the method and technique for acconiplishing the above, conThe total absorbance function, f(-i)x, sider the results which would be derived is the sum of two other absorbance from the following experiment. I n the functions and these individual functions sample cuvette of the recording spectroare composed of a wave lengthdependphotometer is placed a solution conent and a wave length-independent taining a known concentration of an part. The wave length-dependent part absorber and its spectrum is measured is ternied the absorptivity function and with the solvent in the reference beam. where the Bouger-Beer relation applies The spectrum obtained is similar to that it is not affected by the magnitude of shown in curve 1 of Figure 1. Now the concentration term. suppose that the reference solvent is If by some device, it is possible to replaced by solutions containing 50, discover ( a x ) ~ Cthen ~ , by simple sub90, 100, 110, 150, and 200% of the contraction of that function from the centration of absorber in the first measured total absorbance function solution. These curves are labeled to f(A)x, there results the spectruni due show the percentage of the original conto pure component B. Since ~ ( U B ) X C B centration which has been placed in the can be any general absorbance function, reference beam. An inspection of this it can also be an absorbance, due on family of curves reveals that the abthe one hand, to light scattering or to a sorption peak in the vicinity of A’’ mixture of any number of pure subdiminishes in proportion to the constances in such combinations of concentration contained in the reference centrations and absorptivity functions beam so that when the reference soluas to effectively produce ~ ( U B ) Xwhen tion has a concentration identical nith their individual absorbance functions that of the original solution a straight are summed. Thus according to the line results, i.e., the relative absorbance above is zero for all wave lengths of interest. If the concentration in the reference ~ ( U B ) A= CB ~(~D)ACD beam is increased above that of the ~ ( U E ) A C E. . .f(i(as)~C,v ( 3 ) sample then a negative absorbance function appears and in the A’ region Hence the resolving of the spectra of a the absorbance maximum previously compound for which an assay is sought observed is now replaced by an absorpis simply that of separating i t from a tion minimum. Similarly, on the right residual spectrum. I n principle this side of the other absorption peak, Le., suggests that all the individual species ~ ( A ) A= ~ ( U X ) A C X~ ( U B ) A C B (2)
++
1'4
I
\
I
280
I
1
I
I
3x1
300
WAVE LENGTH,
I
I
4k7
M p
Figure 2. Comparison of spectrum of pyrilamine maleate reference and sirup containing it
in the vicinity A' there is also an inversion of curvature as the reference standard is varied through the region of 100% compensation. This group of curves illustrates the technique of discovering when exact compensation had been achieved. In a practical way, if the sample concentration were unknown it would only be necessary t o measure the spectra of this unknown solution of curves such as are presented in Figure 1 . Then by eiamining this group of curves in the vicinity of an absorption peak and interpolating them to that end point where inversion occurs i t becomes possible to discover the concentration of reference standard which e ~ a c t l ycompensates for the absorber in the solution. \Then this is known, then by subtracting the absorption function of that reference standard from the unknown spectrum a t each wave length, it is possible in principle t o discover the residual. I n this hypothetical case that residual is zero but utility of this technique is for those cases where the residual is substantial. This is illustrated in the following elample. APPLICATION TO ROUTINE ASSAYS
I n the quality control of one particular cough preparation, pyrilamine maleate must be determined. This substance is present however, with other constituents such as esters of p-hydroxybenzoic acid, dye (used for coloring the formulation), haze (introduced by the liquid sugar used) and alkaloids which have weak, but nevertheless interfering, absorption peaks in the vicinity of the pyrilamine peak. Figure 2, curve A ,
I
shons the absorption spectrum of a st:md:ird amount of pyrilamine maleate dissolved in 0.1.V hj-drochloric acid. This is comliared with curve B 15 here an aliquot of the sirup in question has been dilutcd similarly into the exact same rangc as the reference standard. The other absorbers have increased the absorbance a t 315 mp and below 290 mp the absorbance which is due to the p-hydroxybenzoic esters is so great that the instrument goes off scale. Small variations in the specifications and concentrations of all of the constituents used in production of this item led to considerable variability in the appearance of the sirup spectrum from batch to batch. Consequently no single correction can be applied in practice: instead, this correction must be discovered as part of the assay procedure itself. I n Figure 3 a family of curves are presented in which successively stronger reference standards have been placed in the reference cell and the sirup spectrum is determined relative to those standards. I n the uppermost curve marked 80, the reference standard was 80% of label. The other numbers associated with the other curves indicate the percentage of label of those reference standards. In the 315-mp region, the absorption peak steadily falls with increasing strength of the reference standard until the absorption peak is replaced by an absorption minimum. By interpolation, the inversion of curvature occurs when the reference standard has a concentration of about 105% of label. This corresponds t o the anticipated result since a 594 overage was introduced. The absorbance a t 315 mp of 0.03 unit com-
ao
-
-02
I
1
em
1
,
300
320
I
3 0
WAVE LENGTH, M p
Figure 3. Residual spectra of pyrilamine maleate sirup
w
0 04
2
SE
8m a 02
00
2 53
2 70
2'0
WAVE LENGTH, M p
Figure 4. Spectra of dihydrohydroxymorphinone reference compared with assay sample
pares favorably with the difference observed in Figure 2 of 0.027 unit. The introduction of high absorbance reference standards necessitates a larger slit width and this in turn means t h a t resolution is lost ( 1 ) . This does not affect the assay, but it does mean that the residual spectrum obtained a t 100% compensation may look different from the way it would if the spectrum were determined in the absence of the component which was compensated for. From a more careful scrutiny of Figure 3, it will be observed that although one can readily interpolate to the vicinity of curvature inversion there is probably some uncertainty as to the precise concentration in the reference standard, which would exactly compensate for the pyrilamine present. I n the next instance shown in Figure 4, this difficulty is even greater. The curve labeled A is t h a t of dihydrohydroxymorphinone reference standard dissolved in 0 . l N hydrochloric acid. I n the case of this particular assay procedure a variable amount of contamination came with the alkaloid as revealed by the new spectrum. A series of reference standards a t the 90, 100, 110, and 120% of label were used and the difference spectra so obtained are presented. Although the inversion can be placed with certainty in the 100- to 120-91, interval and probably in the 100- t o llO-yointerval, nevertheless it would be better if the decision could be made with greater precision. This may be achieved in part by compressing the n-ave length scale so that the absorption peaks are given a greater apparent curvature. To do this, a set of substitute gearing described below, was installed in the DK-2, which compressed the wave length scale seven and a half times and proVOL. 33, NO. 7, JUNE 1961
0
929
Ob
W
0
5 04 m
(r
105
s1 m a
I15 02
Figure 6.
IC0
1
WAVE LENGTH
Figure 5. Residual spectra of dihydrohydroxymorphinone
duced the family of curves shown in Figure 5. The peak absorbance of the main spectral curve should be examined in relation t o curve B, of Figure 4, because they are made with the same solution in the sample beam of the instrument. Thus an indication of the wave length compression may be had. It is first necessary t o have the alkaloid spectrum superimposed upon the family of absorbance spectra as is done in the figure t o fix the position of the maximum relative to the abscissa. Operating with the new gearing, the wave length numerals may be made to coincide with the wave length of the pass band a t only one point on the chart. Consequently, the analyst a t first needs this marking of the absorption peak to calibrate the paper but with experience even this requirement can be dropped. The family of difference curves cover reference standards of 90, 95, 100, 105, 110, 115, and 120% of label and from a study of them, it is easy to decide that the inversion is close t o the 105% curve which in this instance was the concentration a t which the parenteral solution was made. I t is important to indicate one spurious aspect of the family of curves in Figure 5. These data were obtained by running the wave length drive at the highest possible speed so that the wave length interval represented by this figure was covered in about 11/4 minutes. When the 260-mp region is reached, the absorption curve in the standard rises so steeply that the pen is sent off the paper. At these rates of scanning the slit mechanism was not able to keep the reference power a t the constant value intended by the manufac930
Schematic outline of gearing in Beckman
DK-2
ANALYTICAL CHEMISTRY
turer. Thus the reference standard in such a region appears t o be less concentrated than it really is and, therefore, the data below 260 mp may not, in this instance, be used for detecting the inversion. If one wishes t o use the region below 260 mp in this assay, it would be necessary to reduce the scanning rate to a speed which the slit mechanism could follow. In extreme cases this may even be done manually. PRACTICAL TECHNIQUES
There are a number of ways of varying the reference standard. The most obvious is to prepare a series of standards and mechanically substitute one after another in the reference beam. A somewhat easier method is to use a variable thickness cell which then requires only a single standard and adjustment of the variable thickness cell for each scanning. Alternatively one may use a single reference standard whose concentration is somewhat in excess of the anticipated concentration in the sample and then reduce the path length through the cell by a series of wedges so chosen with respect to the thickness that a series of nicely spaced curves result. Clark, Jones, and Harrow, have introduced a flow cell technique which is useful in this type of assay. The unit consists of a flow cell (the flow cell kit developed by the Pyrocell Manufacturing Co., New York 28, N. Y. is suitable for the DK-2) adapted for the particular instrument one may have which is connected in a closed circuit with a reservoir and a pump. Then one starts with a known volume of solvent in the reference cell and reservoir and makes additions of known volumes of a concentrated standard solution to the reservoir, with scanning between each addition of standard. In this way a very large number of spectra may be generated with very little effort. Alternatively, the analyst can start with a known volume of standard
solution in the reservoir whose concentration is about 10% or more above that in the sample and then generate absorbance difference functions by scanning after adding known increments of pure solvent. I t is also possible to do this type of assay using an analog computer. The assay is accomplished with only two sets of data-the spectrum of the sample and of the pure substance. Each of these spectra is simulated by separate electronic function generators which are exactly phased with each other on the wave length term but opposed to each other, Le., in a network where the pure component function is subtracted from the sample function. The difference function which thus results is displayed on an oscilloscope screen. Now by varying the amplifier gain on the pure component function while keeping the gain on the sample function constant, one can generate all of the difference spectra that could be generated by the techniques previously described. ADAPTATION OF SPECTROPHOTOMETER
The method whereby the Beckman DK-2 recording spectrophotometer can have its present carriage movement, relative to the wave length drive altered, is illustrated by the schematic outline of the gears shown in Figure 6. The drive motor gear with 100 teeth turns a 32-tooth gear which rotates the shaft and drives the larger gear, -4. Gear A communicates its motion t o both the wave length drive gear and t o B. B in turn rotates the shaft arid the 48-tooth spur gear which in turn drives the 96-tooth pen drive gear. The pen drive shaft has a rate of rotation equal to 100/64 times or about one and one half times that of the drive shaft. To reduce that drive ratio substantially, the 48-tooth gear is lifted off its shaft and a direct gear linkage is installed between the drive motor and the pen drive gears. When this is done the wave length drive rat2 relative to the
drive motor nil1 not be altered. It was convenient to install a 20-tooth gear above the drive motor gear and a 96tooth gear above the 96-tooth pen drive gear. This is accomplished by first installing two 1/4-inch sleeve couplings on the shafts protruding above the pen drive and motor drive gears, then inserting approyimately 3 inches of inch shafting into each of these two sleeve couplings and finally mounting a 96-tooth spur gear on the pen drive shaft about 3 inches above the other gear and a t a similar position on the drive motor extension mounting a 20-tooth spur gear. It is necessary to work at this elevation in this instrument to obtain the necessary clearances. I n making this installation some slight adjustment of the gear support will be required. With this installation, the drive motor shaft will now turn 4.8 times faster
tlinn the pen drive shaft, giving a n ovcr-all compression of the spectra equal to 7.5. It was convenient to loosen the setscrew on the sleeve coupling of the auxiliary gear and thus remove i t whenever the instrument is to be operatcd normally. This conversion from one gearing to another can be effected in seconds. If both the 48- and the 32-tooth gears are simultaneously removed and the auxiliary gear installed, the wave length drive is inoperative and the abscissa on the recording paper becomes a time scale. With the scanning time dial set a t the number one position, i t takes about 15 minutes for the present carriage to traverse the 14 inches of the recorder paper. The other time scale will double and quintuple the time duration for traverse. The entire installation may be made for less than $20. The necessary ma-
terial was purchased from stock from the P I C Design Corp., 477 Atlantic Ave., East Rockaway, L. I., N. Y. It included the following items; one G41-96 spur gear; one G41-20 spur gear; two D1-3 sleeve couplings and two A3-35 shafting. I ITERATURE CITED
(1) Hiskey, C. F., Young, I. G., -4NAL. CHEM.23, 1196 (1951). (2) Jones, J. H., Clark, G. R., Harrow, L. S., J . Assoc. Ob%. Agr. Chemists 34,135-79 (1951). (3) Morton, A., Stubbs, M., Analyst 71, 348 (1946). (4)Schiaffino, S. S., L o p , H. W., Kline, 0. L., Harrow, L. S., Ibid., 39, 180 (1956). Received for review December 27, 1960. Accepted March 17, 1961. Presented in part Gordon Research Conference on Analytical Chemistry, New Hanipton, N. H., August 1959 and the Eastern Analytical Symposium, New York, N. Y., November 5, 1959.
Infrared Studies of Some New Polycyclic Hydrocarbons and Their Derivatives Containing the CycIopropyI Ring S. A. LIEBMAN and B. J. GUDZINOWICZ Special P rojecfs Department, Research and Engineering Division, Monsanto Chemical Co., Everetf, Mass.
b Data are presented to illustrate the usefulness of previously reported frequency assignments for the cyclopropyl ring structure in some new polycyclic hydrocarbons and their derivatives in the regions of C--H stretching (-3050 cm.-l) and ring deformation (-1 020 cm.-'). From these investigations, it is noted that deviations do occur if one attempts to use the 860-cm.-' as well as these other spectral regions for the identification of the cyclopropyl ring without considering the type of compound in question, the presence of interfering groups, and the resolving power of the instrument.
P
INVESTIGATORS have noted that the cyclopropyl ring structure in organic compounds can be identified by characteristic absorptions in specific infrared spectral regions. Bartleson, Burk, and Lankelma ( 2 ) cited two absorption bands in alkyl cyclopropanes near 1020 em.-' and 860 em.-' as indicative of the presence of the ring system. I n 1952, Wiberley and Bunce ( 1 9 ) ,using lithium fluoride optics to study the absorption of nine cyclopropyl derivatives, showed two characteristic cyclopropyl CH2 absorption frequencies a t 3100 cm.-l and 3012 cni.-l resulting from asymmetric and symmetric CH2 stretching vibrations, respectively. It was concluded that,
REVIOUS
when used with the 1 0 2 0 - ~ m . - ~and 860-cm.-l bands, the CH2 stretching region could provide additional evidence t o substantiate the presence of the cyclopropyl ring. Recently, Wiberley, Bunce, and Bauer (90) extended the range of the two CHz stretching vibrations to 2995 t o 3033 cm.-' and 3072 to 3099 em.-' in a comprehensive study of more than 60 monosubstituted cyclopropanes, some of which had functional groups in their structures. However, all three absorption regions near 3050, 1020, and 860 em.-' have been questioned by investigators as to their usefulness for identification purposes. Derfer, Pickett, and Boord (7) presented data for 14 variously substituted cyclopropane hydrocarbons, utilizing a band centered a t 1010 cm.-l as characteristic of the cyclopropyl ring. The 860-cm.-l region failed to be useful because of its inconsistency. From qualitative studies of 34 cyclopropyl derivatives, Slabey (18) evaluated these three spectral regions and concluded that the 1050- t o lOOO-cm.-l region was most suitable for determining the presence of the cyclopropyl ring. Furthermore, he noted that, when instrumentation with higher resolution is available, the C-H stretching region could offer additional confirmation of the ring's presence. I n 1957 Allen et al. ( I ) , studying heavily substituted
cyclopropyl derivatives with functional groups adjacent to the ring, stated that neither the C-H stretching nor the ring deformation region near 1020 cm.-l was adequate t o characterize the cyclopropyl ring clearly. In more complex polycyclics, Josien, Fuson, and Cary ( I O ) , Barton (S), and Cole (6) utilized either individual or multiple absorption bands at frequencies of 3024 to 3058, 1010, 860, and 800 cm.-' to establish the presence of modified cyclopropyl structures. For nortricyclene compounds, Kaplan, Kwart, and von Schleyer (11) cited the 860-cm.-' frequency to substantiate the presence of substituted cyclopropanes with special reference made to the investigations of Hart and Martin (9) and von Schleyer and O'Connor (16). I n a series of nine monosubstituted nortricyclenes, Hart and coworkers observed that bands mere consistently present a t 860 and 790 cni.-l The failure of the usefulness of the 1020-cm.-l region was reported in 1958 by Paasivirta (15). Nevertheless, he found characteristic 850 to 860-cm.-l and 3050 to 3090-cm.-' absorptions in 10 compounds having the skeletal nortricyclene structure. A literature survey has revealed few recorded spectra of spiranes containing the cyclopropyl ring. Cleveland, Murray, and Gallaway (5) presented the spiropentane spectrum showing strong VOL. 33, NO. 7, JUNE 1961
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