V O L U M E 26, NO. 6, J U N E 1 9 5 4 Table
983
V. Final Results Composition by X-Ray b y Proportional Intensities,
19.85 65.94 13.98
Composition by X-Rag Csing Parameters, % 20.29 05.95 13 76
Error + 2 22 Nil -1.57
+
27.7 52.1 6.98
Error +39.5 -21.0 -50.0
Composition b y Chemical Analysis,
%
%
Alloy 1
Element Cr Fe Si
2
Cr Fe Xi
21.23 13.85 64.84
20 39 l5,04 64.57
-3.96 +8.60 -0.42
28.7 15.1 46.5
f35.2 + 9.0 -28.3
3
Cr Fe Ni
32.13 35.43 31.86
32.44 35.86 31.66
+0.97 +1.21 -0.63
40.4 27.3 18.6
4-25 8 -22.9 -41.5
4
Cr re
16 50 26 6
45 20
+3.10 $2.29 -4.70 -6.10
19 8 81 0 15.1 6.52
+20.4
55
16.96 51.35 25.54 6.15
18 66 12 2
43 26 99 32
19.63 64.93 13.23 2.22
+6.61 -2.01 +1.85 -4.31
24.9 51 5 6.79 2.42
+35.1 -22.3 -48.4 4.3
S I
110 J
Cr Fe N1
.\Io
76
80
%
+61.4
-43 6 0.5
-
+
direction of the surface. Gillam and Heal ( 1 ) gave, without derivation, an expression for the effect, which they called mutual fluorescence, and which was to be added to a term analogous to Equation 2. The inclusion of thls expression would prohibit the use of linear equations with empirical coefficients. The upe of empirically determined absorption parameters compensates for this omission. This can be seen from the values of the absorption parameters (Table 111). Thus A c ~ F A ~ c, ~ N ) , and A F ~ have N ~ values less than unity-Le., less than the selfabsorption value. Since the absorbing element in each case is a little heavier than the radiating element, the low absorption parameters can be due only to mutual fluorescencr. Hence, the empirically determined parameters give an automatic correction for mutual fluorescence. The three corresponding parameters with the subscripts reversed, on the other hand, are appreciably greatel than unity. Close examination shows that there is a correspondence in the departure from unity between the two sets of three parameters. This stems from the reciprocal relation: high excitation of A from B’s K a radiation means high absorption of B’Rradiation by A . That the absorption parameters with their automatic correction for mutual fluorescence are not strictly constant can be seen
in Table V. In Alloy 2, having a high nickel-low iron content, the x-ray reading of iron is 8.6% too high; whereas, for Alloy 5, in which the nickel-iron composition is reversed, the x-ray reading of iron is 2% too low. Thus it is well to have the binaries, from which the parameters are determined, near the 50-50 composition. The last two columns in Table V give the compositions and relative errors, assuming the relative compositions to be inversely proportional to the intensity ratios of Table IV. The effects of absorption and mutual fluorescence are readily observed, and the effectiveness of the absorption parameters in correcting for these effects is in evidence. Analyses based on this procedure offer the significant advantages of a physical method of analysis that is not restricted by the need for reference or standard samples with a composition similar to the sample being analyzed. CONCLUSIONS
Absorption-excitation effects can be expressed in terms of empirical parameters relating pairs of elements. These parameters and flourescent intensity data can be combined in theoretically simultaneous, linear equations. A solution of these equations ultimately gives the percentage by weight of each element in an alloy. This definite system for solving these overdetermined equations greatly simplifies the mathematical manipulation. The accuracy of results obtained on ternary and quaternary alloys compares favorably to that obtained by routine chemical procedures. This method is not restricted by need for reference samples. ACKNOWLEDGMENT
The authors are indebted to Sol Gootman for the careful chemical analyses of the alloyP. LITERATURE CITED (1)
Gillam, E., and Heal, H. T., Brit. J . A p p l . Phys.. 3, 363-8
(2)
Koh, P. K., and Caugherty, B., J . A p p l . P h y s . , 23, 427-33
(3)
Sherman, J., Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, l l a r c h 1953.
(1962).
(1 952).
RECEIVEDfor review September 14, 1953
.\ccepted Rlarch 29, 1954.
Spectrophotometric Determination of Mumulone Complex and Lupulone in Mops G O R D O N A L D E R T O N , G.
F. BAILEY, 1. C . LEWIS,
and
FRED STITT U. S. Department o f , .Jriculture,
Agricultural Research Service, Western Utilization Research Branch,
A rapid method for determination of lupulone and of humulone complex (humulone plus cohumulone) in petroleum ether extracts of hops is based on absorbance measurements at wave lengths of 275, 325, and 355 mp. The influence of solvent and of alkali in methanol on the spectrophotometric stability of lupulone and humulone complex has been studied. The background absorption of hop extracts due to components other than these two has been investigated by adsorption on ion exchange resin and silica gel, by molecular distillation, and by alkaline extraction of hop extracts. Conditions to w-hich a background absorption must con-
Albany 6, Calif.
form in a ternary spectrophotometric analysis, so that it makes no contribution to values for each of two real components, are pointed out. These conditions are shown to be approximated well by the variable background absorption of hop extracts due to components other than lupulone and humulone complex.
T
HERE is need for a rapid method for determination of the
resinous constituents of hops, which are important in brewing. These include humulone, lupulone, and cohumulone. Cohumulone was discovered recently by Rigby and Bethune (11), who isolated it by countercurrent distribution. It is
ANALYTICAL CHEMISTRY
984
precipitated with humulone by lead and o-phenylenediamine, and its absorption spectrum and optical rotation are nearly identical with those of humulone ( 9 ) . Since humulone, cohumulone, and possibly a third component, ( 1 1 ) are not distinguished by the ultraviolet spectrophotometric method described or by any other method, with the exception of countercurrent distribution ( I O ) , the term "humulone comples" is used for the mixture and "humulone" for the pure compound. A tentative proposal for spectrophotometric determination made by the authors in 1949 (6) was based on their observation that lupulone and humulone complex give rise t o the principal absorption of hop estracts in the spectral region approximately from 300 to 400 mH. -4lthough the tentative method proved very useful in following the isolation of lupulone and humulone complex, subsequent Tvork by Rigby and Bethune (10) and 11-ork in this laboratory has shown that the content of humdone complex is overestimated systematically because of interference by nonspecific absorption, which increases with decreasing wave length. The modified spectrophotometric method of Rigby and Bethune ( 1 0 ) was based on a preliminary purification of the hop extract by adsorption on silica gel, folloxing the procedure of Govaert and Verzelt, ( 4 ) . Investigations of various methods of fractionation of hop estracts have led t o the wholly spectrophotometric method reported here. The influence of the illdefined absorbing impurities is corrected by measuring the absorption at 275 niw, a wave length of minimum absorption for both lupulone and humulone complex. The method is rapid; results on prepared estracts can be obtained within 5 minutes. The nature of the interfering background absorption was investigated by fractionating the impurities, that absorb a t low wave lengths, away from lupulone and humulone complex by molecular distillation, by adsorption on ion eschange ,resin and silica gel, and by alkaline extraction. hlthough the background spectra thus obtained did not conform to varying concentrations of a single hypothetical compound, they did follow the i43?5:A2ij= 1:(0.93 O.lSX):X, which allows a solution for lupulone and for humulone complex hy ternary simultaneous equations. It is popsible that such a property of ternary equations, in allowing a certain variation in the spectrum of background absorption, has not been reported previously and niay be useful in other applications. For esample, backgrounds of constant spectra will rarely, if ever, be found for plant estracts. Lupulone and humulone complex, although relatively stable in hop extrarts, were found t o be much less stable in more highly purified form in various solvents, so that special precautions are necessary ill fractionation and standardization.
cell, I , the energy transmittcd hy sample cell, c, the snniple concrntration in grams per liter, d, the cell depth (path length) in centimeters, and 01, the specific absorption coefficient. Thus, where specific absorption coefficients are given, they are in units of liters per gram centimeter. Another symbol, A i , is useful in describing the attempts t o separate the ultraviolet-absorbing impurities from the lupulone and humulone complex in hop extracts by liquid-liquid extraction, by adsorption analysis, and by vacuum distillation. In such cases the fractional absorbance A ; = AhU, where -4X is the observed absorbance for an aliquot of any fraction, and D the dilution factor from the starting hop estract. It was found early in this investigation that both lupulone and humulone complex in methanol solution separately obey Beer's law, and that the absorbance is additive in mixtures. These conditions are, of course, essential to the development of convenient equations for the analytical procedure. 1
Lupulone, Aikaline
Spectrophotometric observations usually n-ere made rvith a Cary hlodel 11 recording spectrophotometer. Photometric accuracy was verified with a yellow and a blue reference filter (XBS-G-5145) and alkaline potassium chromate solutions preparcxd ncrording to Haupt (6). llercury and cadmium emission lines n'ere used as standards for wave-length calibration. I n general, absorption spectra were scanned between 400 and 220 mu. Although the Beckman Xodel DU spectrophotometer was used only occasionally, the instrument may be used to advantage in the analytical procedure where absorbance is t o be measured at only a few wave lengths. Concentrations should then be somewhat lower than shown here, in order t o fall within the best precision of this instrument. I n the follom-ing discussion the absorbance of a solution at a given wave length, A, is given by the expression:
/'
200
300
250
'\
'C 3
350
Wove length in Millimicrons Figure 1. ibsorption Spectra of Lupulone and Humulone Complex in Acidic (0.002 V) and Alkaline (0.002 V) 3Iethanol
Absorption spectra of lupulone and humulone complex in methanol are shoivn in Figure 1; details of the purification procedures are given in the next section. Spectra usually were xanned immediately after mixing 0.03 nil. of 0.2.Y sodium hydroside or 0.2s hydrochloric acid with the 3.0-ml. contcnts of the 1.00-em. fused-silica absorption cells. The solutions, 0.002S in acid or alkali, were found to give rrproducible spectra over rcztsonable periods of time, as shown in the section on stability. The alkaline spectra proved most useful, and undefined readings refer to this condition. Reagent-grade absolute methanol was used as a rcference solvent; the small changes in solvent absorption caused by the acid w x e vcry rrproducible. The alkali pro-
Table I. Concn. of Humulone Complex lig,/Ml: 10 5 1 1 0.054
0,054 0.054 0.040 0,040
where ZO is the energy transmitted by solvrnt-filled referenre
/-y
i c I
+
SPECTROPHOTO\IETRIC I'ROPERrIES O F LUPULONE AND HUIlULOZE COIIPLEX
1
I
Stability of Humulone Complex in Neutral RIethanol Storam Conditions ~ ~ Approx. temp., IlluniiC. Time nation
Increaae _ _ in Absorbance. "c
7 days
-24 1
23 5 2,5 25 25 25 25 25 25
28 days 90 days 24 hours 4 hours 2 hours 1 hour 4 hours 4 hours
Room Dark Room Room Room Room Room Room Dark
353 mfi -25 1 -47 0 5 -2 -1 0 5 -5 0.5
3 2 6 mp -44 0 5 -2 -1 0.5
-6 0.5
275 nip 67 27 83
... 1. ..
14 2
V O L U M E 2 6 , NO. 6, J U N E 1 9 5 4 Tahle 11. NaOH conrn.,
.v
0 02 0 01 0 02 0.002 0 02 0 012
0 0" 0.02 0 001
H20 concn.,
%
1 0.5 1 1 10 0 3
1 1 0 5
985 tant primarily in fractionation, purification, and standardization. Unlike similar solutions Absorbance Storage of the purified components, Change, % titne, min. 353 ,1111 3 2 5 n1p 275 n i p petroleum ether extracts of hops are unexpectedly stable. Y!i0 -51 -9 14 Absorption Coefficients of 1J
