V O L U M E 2 2 , NO. 12, D E C E M B E R 19.50
*
di,o\itie sulution in the presenre of 3 nig. of chloride ion as sodium chloride and 4 mg. of bromide ion as sodium bromide. The results obtained, given in Table I, demonst,rate t h a t chloride and hromide ions do not interfere in the titration. T o test the interference of metal ions typically found in used, doped oils ( d ) , duplicate 1-gram samples of two different, oils containing it variety of metals n - e i ~ashed in air and known amounts of silver nitrate solution were added to the ignited residues. After the resiclues had been taken to a paste by heating with nitric acid, t8heywere lixiviated wit,h ammonium hydroxide and titrated without filtering by the aniperometric method. Very satisfactory recovery of silver \vas obtained as shown in Table 11. T h e method was furthrr tested by adding known amounts of pure silver metal to thrx oil before ignition. I n this aeries, the solutions were filtered before titration, as aliquots were also analyzed by the giavimrtric: method. A s shown in Table 111, satisfactory recovery of silvcr K:M oht:tined t)>- both methods. The maximum tmnipcraturr of ignition was found to be rather ci.itical. When samples of known silver (tontent were ignited in porcclain or platinum dishes to a temperature of over 600" C., rrsults were 25 t o 50Y0 low by both the amperometric and gravimetric methods. No difficulty was experienced when the temperature of ignition was held to 500" =t50' C. In t h r iimperometric titration it \vas found convenient to use a
1507 sctturated calomel reference electrode and to apply -0.23 volt on the electrode. This eliminated the need for a special reference electrode of this potential described by Kolthoff and Harris ( 2 ) * The latter electrode would be necessary, however, if no provision were available for applying a potential The choice between the two methods for silver will depend somewhat on the sensitivity required and the equipment available. Either gives good results in the concentration range of silver normally expected in used lubricating oils. The amperometric method is much more rapid than the gravimetric method and is especially applicable to 1015 amounts of the metal, less than 0.00170being easily determined by using 0.001 A47standard iodide solution. Elements expected in new or u3ed additive-containing oils do not interfere with either method. LITERATURE CITED
W. F., and Lundell, G. E. F., "Applied Inorganic Analysis," p. 591, New Tork, .John Wiley & Sons, 1929. ( 2 ) Kolthoff, I. M., and Harris, W ,E., IND.ESG. CHEM.,A x . 4 ~ED., . 18, 161-2 (1936). (3) Laitinen, H. d.,Jennings, JY.P., :ind Parks, T. D., I h i d . , 18,358-9 (1946). (4) Lykken, L., Fitesimmons, K . K., Tibbetts, 8. A , and \Tyld. G., Petroleum Refiner, 24, S o . 10, 405--14 (1935). ( 1 ) Hillebrand,
RECEITEDFebruary 6 , 1950.
Chromatographic Separation Employing Ultraviolet Absorbancy Ratios IIORTOR' BEKOZA Bureau oj Entomology arid Plant Quarantine, United States Department of Agriculture, Reltscille, M d .
A method for detecting impurities in chromatographic zones employing ultraviolet absorbancy ratios has been applied to the separation of alkaloids from a mixture. Its advantages, limitations, and sources of error are presented.
A
STSTElIATIC c~ollectionof the effluent from a chromato-
giitphic column, cwmbined n i t h ultraviolet spectrophotometiir analysis. has been used to isolate two alkaloids in a state c 1 0 ~to puiity. C n d ~ ithe conditions used, no additional time
O
,
I2
l
I6
20 0
N U M B E R O f TUBE
Figure 1. Countercurrent Distribution Pattern of Wilfordine Benzene+=%HCl
\vas required to develop the c~hromatoyram, because spectrophotometric measurements a e r e made a.hile the chromatogram was being developed. The method reveals much valuable information in a minimum of time. The insecticidal alkaloid, nilfordine, whose isolation has been recently reported by Acree and Haller ( 1 ) has been shown by count,ercurrent distribution ( 2 ) to be a mist'ure (see Figure 1 ) . Because separation of the components hy countercurrent distribution on a large scale is laborious, partition chromatography ( 7 ) was employed. The method of chromatographing involves the collection of fractions of convenient volume, \vhose absorbancies are read in the spectrophotometer at two or three wave lengths. The wave lengths used may correspond to a peak and a n adjoining minimum, two peaks, or any other caivenient arritngement. For any pure ronipound the absorbancy r:ttio between these two wave lengths should remain constant, provided the concentration is proportional to the absorbanry. Thus, if a zone is collected in 15 frac.tions and the ratio of the absorkiancies remains constant for all these fracxtions, i t is a good indication that the r*ompound is pure. Although absorbancy ratios have been used to estimate the amount of each constituent of a binary mixture in a chromato~ graphic zone where the individual absorption spectra of each constituent are known (3-5), in research work on new cwmpounds or compounds of unknown purity t,he spectra of the individual constituents are not known; in such cases, the method described below is applicable. The method should be of general use
ANALYTICAL CHEMISTRY
1SO8 in the chromatographic separation of compounds, spectrally vharacterized or not, which absorb in the ultraviolet. EXPERIMENTAL
All spectrophotometric measurements were made \\ ith a Beckman quartz spectrophotometer, Model DU, Serial S o . 757.
0
100
200
300 MlLLlLlTERS
Figufe 2.
400
500
600
3olvent a r r ShoFvn in Figures 5 and 6, respectively. The higher corioentration of hydrochloric x i d causes slower movement of the alkaloid and greater resolution. This higher conrentration of hydrochloric acid cxould not be used on the first chromatographing, as an excessive quantity of mobile solvent would be required to remove compound C-2. The ratios in Figure 5 indicate that C-1 seems to be pure hetxveen 750 and 1300 ml. Likewise, the ratios indicate just Jvhich fractions are pure iii Figure 6 and permit the best separation of the impurity to be realized-that is, the pure compound ran be separated (175 to 237.5 ml.) and, because the amount of impurity in the remaining frartions can be readily estimated, only those fractions which 1r:ive :i sufficient amount of the draired compound are combined
700
EFFLUENT
Partition Chromatography of Wilfordine Ether-1% HCI
Figure 2 shows the first iesult obtained n hen wiltoidiiie \vas subjected to partition c-hromatography on silica gel, 1% h] drochloric acid being used as the immobile solvent and ether as the mobile solvent. The two main constituents are widely separated. 1Xaterial C-I seems to be pure, but the latter fractions of C-2 obviously are c~ontaniinated. Because C-1 appeared to be pure, it was subjwted to countercurrent distribution. Figure 3 shows that the ompound is impure. Figure 3. Countercurrent Distribution Pattern of Compound C-1 One-half gram of milfordine \I as then chromatographed, and the absorbancy v a s measured a t 270 and 255 mp. I n this rase Benzene-12 7~HCI SO-ml. fractions \\ere collected, the last 3 nil. of each fraction directly in the cuvette. For the sake of brevlty only compouiitl C-1 is considered here. The lower graph of Figure 4 s h o w the absorbancy a t 270 m p versus milliliters of effluent; t h e upper graph shows the ratio of absorbanvies a t 270 and 255 mp versus milliI=== liters of effluent. The ratio is relatively constant between 400 and 600 nil., then decreases, and then levels off again. The constant ratio is strong evidence for the presence gm 21.5 .0 of a pure compound; the decreasing ratio indicates that increasing amounts of an impurity or impurities are appearing. The relative position of the absorbancy ratio of any fraction between t h a t of the 1.0 pure compound and that of the impurity isameasure of the absorbancy due to the impurity in that fraction. 0.5 The shaded area shows the absorbancy estimated t o be due to the im500 1000 I600 LOO purity. 0 0 0 MILLILITERS EFFLUENT The results of rechromatographMILLILITERS EFFLUENT i n g -4 (350 to 650 ml.) and B (650 Figure 4. Partition Chromatography Figure 5 . Partition Chromatography t o 750 ml.) of Figure 4 with 2% of Wilfordine Using Absorbancy Ratios of '4 (Figure 4) Using Absorbancy Ratios hydrochloric acid as the immobile c - 1 only
i2'l
*
V O L U M E 22, NO. 12, D E C E M B E R 1 9 5 0
1so9 order to determine the distribution of the material that was not eluted. By this means it is usually possible to predict, whether thp separation will be successful. The results ma:. point to a stronger or weaker mobile or immobile solvent. Figure 9 shows thr. preliminary trial on 2 grams of adsorbarit withl.8 mg. of wilfordine that led to the SUP cessful chromatogram obtained in Figure 2. DISCUSSION
To use the foregoing method most effectively, the advantages, limitations, anti sources of error must be recognized. If the desiredcompound orrompoundsal)sorbinthe ultraviolet, the solvent should be sufficiently 100 200 100 400 MILLILITERS EFFLUENT transparent so that measurements may t w made a t the desired wave lengths. If it is Figure 6. Partition Chroniatography of B (Figure 4) Using impossible or inconvenient to use a transAbsorbanc? Ratios parent solvent, a nontransparent solvent may be used, aliquots being evaporated conipletely and then taken up in a transpareIit solvent for absorbancy measurements. In such cases it may be advisable to evaporate Figure 7 . Countercurrent Distrioff every third fraction escept at those point3 \,.here data are desired. hution Pattern of Compound C-1 Benzene-10 70 HCl fksause ultraviolet measurements ma!' I)(> used to detect minute quantities, fairly arcurate data may be obtained with a fen. 1.4 milligrams of material. This extreme sensitivity of measurement in the ultraviolet i:, uset'ul in detecting the exact beginning and 1.2 end of a chromatographic zone. When i t rolored material is chromatographed, separation by eye often may be unreliable hecausr, 1.0 of the paleness of the fractions. 3Iucah i l to 1)e gained 1-, following the compound in the ultraviolet, where its absorbancy is usually many times greater and i t can be definitely shown that one compound does or I does not tail into the other. ae * The elution pattern of the chromato0 graphic zones, as determined by the ultras 0.4 violet absorhancy, is an index of purity an(i also shows 1 1 0 ~well ~ a column is operating. U I n one run in whic.h a poorly formed zonrs NUYBER OF TUBE came off the column, careful esamination Figure 8. Countercurrent Distriof the column disclosed a crack in the gc.1. hution Pattern of Compound C-2 Martin (6) has described the type of zon+ B e n z e n e 4 70HCI to be expected, and Moore and Stein (81 1 2 1 4 5 I L 1 4 5 8 7 I 9 I O I l K V E N ml. FRACTIONS COLUMN SECTIONS have published some typical curves obtaintd in work on the separation of amino acid,.. ~ i t the h nest crude Iiatch to be iesolved. Figure 9. Preliminary Selecting the proper wave lengths to m e w Thcs f i actions composed mostly of imChromatography of Wilfordine Ether-1 70HCI ure the al)sorbancies is important. Gencsrpinit\ may be discarded or saved as ally, the best procedure is t,o determine tliv desired. spectrum of the starting material. If the, Figure 7shows the countercurrent distrihution of compound C-1 after the second chromatographing:' spectrum has a maximurn and a minimum, the n-ave lengths corresponding to these points may be used. .Uter preliniiriai,?. ('-1 appears t o be close to purity (9). Figure 8 shows the counterr n i w n t distribution of compound C - 2 treated in the same manner: separation it may be desirable to determine the spectrum of tlic C-2 is also close t o pui.ity. By utilization of these general prinpurest fractions and choose new wave lengths which correspond ciples, the most effective separation can be achieved with a t o its maximum and minimum. Sometimes it is desirable to dcniiriimum loss of material in a minimum time. termine the spectrum of the fore and rear of a chromatographic Preliminary Procedure. h s a general rule it is recommended zone and use the two wave lengths that show the greatest at)that 1 or 2 mg. of material tie tried on 1 or 2 grams of adsorbant sorbancy ratio difference. If no niasimum or minimum is presI d o r e a large column is used. In these preliminary trials results pnt, two points about 20 to 50 m p apart may be arbitrarily taken. m a y be obtained most rapidly if the height of t h e column is about If three wave lengths are taken, another set of ratios may be calequal t o the diameter. After development of the chromatogram, culated. Should both sets of ratios remain constant for a Z O I I P , the column should be sectioned. and the sections extracted in it would he even stronger evidence that the zone is pure. v)
ANALYTICAL CHEMISTRY
1510 In the application of al)sorl)ancy ratios t o deteniiine purity it is assumed that the absorbancy of the compounds obeys Beer's 1x1~. This point may be readily checked by taking the eluate corresponding to the purest sertion of a zone (generally the peak) and making several dilutions thereof. If the ahsor1)ancy doer not obey Beer's law a graph may he plotted for any oiie (>ompound, from which the adjustrd al)sor\)anryratio for a n y coiirc~i!tration may he read. At)sorbanc.y ratios m a y lie applied n-hen R pure (bornpound or o i i (.lose ~ to purity may I)e oI>tained. They n-ould probably be u.ir~lesson a (.rude mixture. The method does not detect ztn impurity that has an :ihsorlxinc.~. i,:rtio ideiitical with that of the desired compound, urilrsa t h e impurit!. separates enough to distort the shape of th(1 chromutographic z o n e , nor docs it detect rantaminants that, do not ahsoi,l) :it the I\ ave lengths measured. Sources of Error. I t is necessary t o correct for differences in absorbanc,ies of the cells themselves. Temperature effects should tie hrltl to a minimum. T h e column should be developed with :t uniforni tior$- rate of solvent, as stoppages for an extended tinw in the middle of a zone may cause distortion of the elution pattern. Tf neccssary, the c.olumn may t)r interrupted or speeded up het w e n zones. I n adsorptive chi~omatographyit is a good idea to equilibrate the solvent and adsorbant prior to column formation. Scrupulous care must be eserrised to avoid contamination. Glass-stoppered glassware is preferable, although corks or rubber stoppers may be used if they arc covered with a metal foil t h a t does not react with the mohile solvent. T h e column should he connected to the source of pressure lvith a ground-glass joint, and if it is t o be plugged, glass wool rinsed with the mobile solvent is preferred. Cotton should not be used, as it contains materials that absorb in the ultraviolet. Finally, a parallel t)lank drtci~ninationis necaessary to show that all iiitert'eiwic.es h:tve 1)et.n c.liminated. OTHER APPLlCATlOY S
.4bsorbancty ratios are b y no means limited to partition chromatography, b u t may be applied to straight chromatographic separations as well as t o countercwrent distribution.
In instances in \vhicah c ~ h r o ~ n s t o g ~ ~ or s p 1countercurrent ~~distribution is used to determine purity, a constant absorbancy ratio of the fracntions tvould make the proof of purity more rigid. Sensitive compounds may he sqxtrated ir! pure form I)>- several ratios heing used t o deterquick passes on a column, a1isoi~l~aiic.y mine which is the desired matt>ri:iI, .\l)wrh:tncy ratios should ;tlso prove useful in control ivork, ljec*:tuseit is possihle to detect rontamiiiation in a zone that appears t o he homogeneous. The use of al~sorl~ancy ratios i i i quantitative anal> OF the constituents are k n o ~ v nhas Iwen men Ahsorhancy ratios of c.hroni:rtogial,hi(, frnctiona in the v i ~ i h l c or infrared may also he uic3ful. SL.11 hI Z R Y
Uy use of a systematic. cdlec.tion of c.hromatographic fi~actious from n-hich ultraviolet ahsorhancy ratios are determined, it has been possihle t o detect impurities in cahromatographic zones. The method has been used to isolate the two main constituents of the cbrystalline alkaloid ir-ilfordine. T h e method may he generdl>- applicable t o the vhromatographic or countercurrent separation of compounds Lvhich :~IIsoI,I) in the ultraviolet and possibly to compounds which ahso~,l)in other parts of the spectrum. LITERATPRE CITED
(1j Acree, F.,and Hailer. H. L.. J . A m . Chenz. S o c . , 72, 1608 (1950). (2) Craig. L. C., and Post, O., .IX.%L. CHEM.,21, 500 (1949). (3) Gibson. 51. R., and Bchwarting. A. E., J . A 7 n . P h a r m . Assoc., Sci. Ed., 37, 206 (1948). (4) Haskins. .L L., Shermali, A . I., and Allen, W. lI,,6.Bi'ol. C h e m . , 182,429 (1950). (5) Hotchkiss, R. D., I b i d . , 175, 315 (1948). (6) Martin, -I.J. P., Endearour, 6, 21 (1947). (7) Martin, A. J. P., and Synge. R. L. M., Biocheni. J . , 35, 1358 (1941). (8) ,\1oore, S.,and Stein, If-. H., A m . S. I'. A c a d . Sci., 49, 265 (1948). (9) IVilliamsoii, B., and Craig, L. C., J . B i d . Chem., 168, 687 (1947). RECEIVED April 28, 1950. P a r t of Ph.D. thesis submitted by hiorton Beroza to Graduate School of Georgetown University. Report of a study made under the Research and 3larketing Act of 1946.
Determination of Traces of. Silicon in Vanadium and Uranium Spectrophotometric Method RUTH GUENTHER ~ N D RICHARD H . GALE Knolls .4tomic Power Laboratory, General Electric Company, Schenectady,
1 9 ' .
Y.
-4 spectrophotometric method for the determination of silicon in vanadium
and uranium metals is described. The metal is removed by chloroform extraction of its cupferrate, the silicomolgbdate color is developed in the residual solution, and the transmittancy is determined at 390 m p . The system shows an adherence to Beer's law over the range of 0.02 to 0.28 mg. of silicon per 100 ml. with a reproducibility of 10.004 mg.
T
HE procedure described herein was developed during a research program t h a t necessitated a precise analysis of microgram quantities of silicon in high purity vanadium and uranium. Silicon is present in these metals as a silicide capable of being transformed into a soluble silicate. For trace quantities of silica, a microtechnique is available if a gravimetric method is desired (2); however, the generally applicable procedures for traces of
silicba utilize either the yellow silicomolybdate color developed in dilute acid (3, 4)or the molybdenum blue color developed in a reducing medium ( I ) . There appears to be nothing gained through u,ye of the molybdenum blue complex, for accuracy and precision in the two processes are comparable. A separation of the silica M as necessary, because both uranium and vanadium interfere in the above methods.
,
