V O L U M E 2 5 , NO. 12, D E C E M B E R 1 9 5 3 by the method already described with an average error of &2.5%. The use of Versene forthe separation of vanadium from chromium, iron, and other elements will be reported later. APPARATUS 4ND MATERIALS
Copper sulfate solution. A solution of copper sulfate was prepared containing 0.63 mg. of copper per ml. Ethylenediaminetetraacetic acid, tetrasodium salt (Versene). A 10% solution prepared from a 34y0 solution was used in these determinations. The Versene was obtained from the Bersworth Chemical Co.
1865
precipitation of the copper was obtained by the addition of 10 ml. of the 6% cupferron solution, even when the pH was less than 0.1. Precipitation of Vanadium in Presence of Copper. In solutions containing varying amounts of vanadium and co per, the precipitation of the vanadium was carried out at a pH Ess than 1.0, and the vanadium cupferrate precipitate was 6rst washed with a 2% Versene solution, adjusted to a pH of 1 to 1.5, and finally washed with 10% sulfuric acid. The results obtained for the vanadium checked well with the work on solutions which contained vanadium alone (Table 11). The acetone solution of vanadium c u p ferrate was checked for the presence of copper by the dithizone method as described by Sandell ( 2 ) and no copper was found. PROCEDURE
EXPERIMENTAL
Effect of Concentration of Versene Solution on Precipitation of Vanadium. I t has been reported that vanadium precipitates in solutions with a pH of 3 to less than 0 ( I , 3, 4). In the presence of Versene there was no precipitation of the vanadium cupferrate until the pH was below 1.5, and a t a pH of 0.5 precipitation was complete. From 2 to 20 ml. of 10% Versene solution was added to solutions containing 0.0255 mg. of vanadium(V) per ml. The vanadium(l7) precipitated quantitatively with cupferron even in the presence of a large excess of Versene, and the excess could be washed readily from the precipitate. Effect of Versene Solution on Precipitation of Copper with Cupferron. In solutions rontaining 5 ml. of the standard copper solution and from 2 to 10 ml. of the 1 0 7 Versene solution, no
To a solution containing the vanadium and copper salts was added 10 ml. of 10% Versene solution, and the procedure described \vas followed. LITERATURE CITED
(1) Bertrand, D., BzdL
SOC. chim. France, 9, 122 (1942). (2) Sandell, E. R., “Colorimetric Determination of Traces of Metals,” 2nd ed., p. 296, Kew York, Interscience Publishers, 1950. (3) Smith, G. F., “Cupferron and Keo-Cupferron,” G. Frederick
Smith Chemical Co., Columbus, Ohio. Drexler, S. J., ,J. Opt.
(4) Strock, L. R., and (1941).
Soe. Amer., 31, 167
R E C E I V Efor D review June 10, 1953. Accepted October 23, 1953.
Automatic Measurement of light Absorption and Fluorescence on Paper Chromatograms J44IES A. BROWN1AND MAX 31. ,MARSH Eli Lilly and Co., Indianapolis, Ind. Previous automatic devices for scanning paper strip chromatograms have either been of the intermittent, nonrecording type or have required the use of an automatic recording spectrophotometer. The attachment described is designed for use with a commercially available monochromator employing a stabilized light source such as the Beckman Model DU spectrophotometer. An innovation in the assembly described is the use of interference filters to
T
HE application of the paper chromatographic technique to the quantitative as well as qualitative evaluation of complex mixtures continues to receive considerable attention. Recent studies have resulted in the construction of several devices for the scanning of paper strips from the standpoint of light absorption measurements (3-6). The present work represents a continuation of some preliminary observations reported earlier ( 2 )and is an attempt to broaden the utility of the automatic technique, by providing for the measurement of fluorescence on paper strips and by incorporating components which are more accessible to the average laboratory. DESCRIFTIQN OF APPARATUS
The attachment for automatic scanning consists of five major parts-a spacer, a scanning chamber, a strip transporting mechanism, a photomultiplier tube detector with amplifier, and a strip chart recorder. Since paper strip chromatograms can be prepared most conveniently on strips 0.5 inch wide ( 1 ) . the first requirement in designing a scanner was to provide a beam of light a t least that 1
Present addrese, Truesdail Laboratories, Inc., Los Angeles. Calif
permit the measurement of fluorescence on paper strips. Strip chart records, both of absorption us. strip length and of fluorescence us. strip length, have been obtained. The reproducibility of results appears to be better than that obtained with other scanningdevices. Byplottingper cent transmittance rather than absorbancy, greater sensitivity is observed in evaluating low concentrations of absorbing materials on the strips.
wide through which the strip could be transported. Examination of the characteristics of the light beam emitted by the Beckman DU monochromator used in this work showed that in the vicinity of the absorption cells as normally placed in the cell compartment the beam is a vertical rectangle about 1/8 X 3/g inch. Kith the hydrogen lamp as a source, this beam diverges in the horizontal dimension and converges in the vertical dimension as the distance from the exit slit increases, and at a distance of about 8 inches it forms a slightly diffuse horizontal rectangle about 3,’~8 X 5 / / ~ inch. With the tungsten lamp as the source, the image at the %inch distance is a vertical ellipse about )/8 inch wide and 1 inch high but with the brightest portion concentrated X 5 / / ~ inch. Thus, it in a diffuse horizontal rectangle about appeared feasible to transport the strip vertically through the beam at a distance of 8 inches from the block supporting the filter slide and exit slit. An alternate method would be to place the scanner closer to the block and alter the beam with lenses but this appeared to be an unnecessary complication. The use of the beam at the 8-inch point has at least two advantages. First, the chamber and scanner are located where there is no interference with the light source housing; secondly, it allows for the transporting of thestripverticallythroughthe beam. Thus, provision is
1866
ANALYTICAL CHEMISTRY
easilv made for shlftine the stnn out of . .
.
on a sliding top to the light chamber.
ing of the strip would present a more difficult mechanical problem.
by 1.5 inch in outside diameter in such a manner that the over-all length of the spacer was 6"/,, inches. Ti& plates
Figure 1. General View of Complete Instrument
convenienoe, both plates were drilled fdentichy. The plates were machined after the brazing so that the faces were psritllel t o each other, perpendicular to the pipe, and fitted precisely to the filter slide block and scanner ohemher. The chamber was constructed of an easily workable and extremely light weight magnesium allay. The two identical sides are 6.75 inches high, 7 inches wide, and 0.25 inch t,hiek, with a notch 1"/1a inches wide and 1.75 inches high cut out of the lower corner to nrovide room for the cables so that a standard Beckman photdmultiplier attachment could be mounted if desired. The ends and bottom of the chamber were made from
TG parts were &embled with 38 countkrsuKk michine screws. The sides were drilled, one to fit the spacer plate and the other to aocommodate either the Beckman or the-Aminco (Amerioitn Instrument Go., photomultiplier microphotometer) phototube housine. The lid 1s a niece of the mme allav 8 inches lone. 2 inches kide, and 8 / 8 inch thick, with 0.25 X 0.25 inch rabbitson eaob side. The sides of the chamher extend 0.25 inoh above the ends, the lid fitting between these extensions of the sides and oveilapping the top to make a light-tight fit. The lid is free to slide. The insides of both the spacer and the chamber were painted with a flat black p i n t to minimiee reflections. The scanner is similar to one described bv Pilrke and Davis (5) .. modified by Brown (1). It incorporates one solid brass roller, roughened to eliminate slippage of the strin. which is e a r e d to a synchronous motor. The -br& frame holding thegears and roller is mounted on a brass block a/a inch thick which is in turn mounted an the underside of the lid. The brass block contains the aperture which is a hole 0.5 inch in diameter; over this me mounted two movable plates t o provide an adjustable slit. The brass block also contains an adjustable track to guide and retain the paper strip and a spring-loaded companion roller t o the driving roller so that a slight pressure is exerted on the paper strip as i t is being driven. Provision is made for mounting stationary reference strip of paper with an identical aperture and adjustable slit The dimensions of the scanner and its placement m e such that when one end of the lid is flush with the end of the chamher the reference specimen aperture is located in the beam and when the other end is flush with the other end of the chamber the main aperture is in the beam. Adjustable stops were placed on the ends of the chamber to limit the travel of the sliding top. All of the parts vere constructed so that the apertures were in a line centered at the exit slit and perpendicular to the filter slide block on the monochromator. The position of the light beam can be shifted somewhat with the focusing adjustments provided on the monochromator; the final aligriment of the beam to cover the aperture was accomplished in this manner. The depth of the chamber was made large as possible t o provide space for the strip to collect after it had passed the aperture. The synchronous motor is commercially available (R. W. Cramer Co., Centerbrook, Conn.). It is of a high torque design with an electric a8
clutch which disengages when the power is off so that the strip can be moved by hand. Its output shaft speed is 4 r.p.m. and the strip travels about 7 inches per minute. As mentioned bv Parke and Davis (6). . .. the lieht. after nassina through the paper; is essentially d l scattered. -This light pass& through the 8J8-inch thick hole in the body of the scanner and this may result in some collimation. The scanner was placed as close as nossible to the exit side of the ohamher and when the Beckman phototube housing is mounted on the chamber the phototube is located close enough to the scanner so that an adequate amount of light strikes it. This is not true when the Aminco photomultiplier tube housing is used since there is a sizable chamber built into the photomultiplier tube housing t o accommodate filters; therefore, an inadequate amount of light strikes the phototube unless the beam is further collimated. This wm done by mounting a piece of stainless steel tubing of 0.5-inch inside diameter and 1.5-inch leneth inside the ninde .. oonnecting the phototube housing to the scanner chamber, using a suitable brass bushing, so that one end was aa close as possible t o the scanner. The interior surface of the tube was highly polished. All of the lieht from the strin . D.~ S S B Sinto this tube and nraetioallv all i s collimated into a beam which strikes the phototube. Only enough space was left between the collimating tube and the phototube to insert the thin interference filter which is necessary when fluorescence measurements are being made. The detecting element is an RCA lPS8 photomultiplier tube. The assembly shown in Figure 1 utiliaes the Aminco phatamultiplier tube housing and lineoperated direct current amplifier and power supply (American Instrument Co.). It has also been found possible to install the photomultiplier tube in the standard photocell housing of the Beckman spectrophotometer and t o employ the internal amplification system of the instrument itself, the take-off to the recorder being in the null meter circuit. In the latter case. an external batter""nower . suonlv .." for the nhotomultiplier tube is necessary. The Beckman photomultiplier attachment includes a power supply and operates satisfactorily far this application (2). The recorder selected for use in these studies was a Brown Instrument Co., fast-acting strip-chart recorder. The pen speed is one second far full-scale travel and the voltage range i s 0 to 1 mv. For most applications a recorder with a 0- to IO-mv. span would be satisfactory but a high-speed pen travel is mandatory. In order to secure the proper input potential, the output of the amplifier wm supplied to a suitable voltage dividing resistor wired in parallel with the recorder. Figure 1 shows a photograph of the complete apparatus. Figure 2 is a close-up exploded view which shows the various parts and their approximate relationship to each other. ~
~
OPERATION
Absorption Measurements. The proper operating range of the instrument is determined by choice of slit width, load resistor, and
V O L U M E 25, NO. 12, D E C E M B E R 1 9 5 3
1867
high-voltage setting. The general specifioations for photomultiplier tubes indicate that maximum stability and reproducibility of output is obtained when the high voltage is kept as low as possible. It has seldom, if ever, been necessary to increase the high voltage above the 680-volt minimum of the Aminco ilmplifier. To reduce the noise output, the laad resistor should be as low as possible. It is necessary to fix the 0 and 100% ends of the recorder pen travel corresponding to zero and maximum amplifier output, respectively. The zero end is fixed first, with the scanner in a mid-position in the chamber so that no light falls on the phototube, by adjusting the zero adjust (dark current compensating) knob an the amplifier. The 100% end is fixed by shifting the reference section of the scanner which contains a typical specimen of the paper being used into the beam and adjusting the slit on the optical bench. It is often desirable to fix the 100% paint at a small distance from the end of the chart so that the pen may travel above 100% because of normal fluctuation of the background absorption. The sample strip is backed out of the beam by rotating the clutch mechanism of the synchronous motor: then, after setting the transport mechanism in the sample pasition, the motor is started and a t the first deflection of the pen, indicating entrance of the strip into the beam, the chart motor is started, thus synchroniaing the chart record with strip position.
Settings a t both ends of the recorder scale are checked and readjusted if necessary before each strip is run through or whenever the wave-length setting is changed. The prinoipal precaution in making these settings is to make them approximately the same for all strips of a given series since the height and thus the area of a peak is dependent on the total span of the pen Fluorescence Measurements. Far the measurement of fluorescent mnes on paper Strips, a mercury vapor lamp (available from Beckman Instruments, Inc. and recommended by them for calibration of the wavelength scale of the DU spectrophotometer) is placed in the normal position for the light source. The wavelength dial is set a t 365 mp which effectively isolates the fluorophor-activating line. An interference filter baviug a peak transmittance a t a waive length near the wave length maximum of the emission spectrum of the fluorophor is placed in the light beam between the paper strip and the photomultiplier tube. Suitable filters having essentially complete cutoff a t 365 mp and 25 to 35% transmittance st 450 mp (blue fluorescence) or 580 mp (yellow fluoretrcence) have been purchased from Baird Associates and Farrand Optical Co. With this sssembly, light strikes the photocell only when a fluorescent zone is passing through the beam. This provides s recording of emission ver8us strip length. It is necessary to employ a standard fluorescence for reference with each sample series studied sinceareferencelightintensityisnototherwise obtainable. This can be conveniently done by placing a spot of the material being tested or of any stable fluorescent substance having a similar emission spectrum, on zt piece of paper strip and placing i t in the reference aperture of the scanner. Then, before eaxh strip is scanned, the pen is adjusted to a fixed reference point on the chart with the reference specimen in the beam by adjusting the slit. The only other adjustment necessary is that to compensate for zero drift. EXPERIMENTAL
Paper strips 0.5 X 22.5 inches were used in developing chromatograms according to the method described previously (a). After drying, the strips were scanned a t the appropriate wave lengths using the described apparatus. n
RESULTS
1
Figure 3 shows a typical graph of relative per cent transmittance of a pyridoxine hydrochloride band versus a linear function of strip length. Figure 4 shows a typical waph of relative fluorescence intensity of a riboflavin hand versus a linear function of strip length. Table I and Figure 5 show the quantibtive results of measuring the areas of the percent transmittance peaks from a series of concentrations of ovridoxine hydrochloride, and Table I1 and Figure 6 show similar data from the fluorescence peaks from a series of concentrations of
."
I
E P E L A T I V L P O S I T I O N ALONG PAPER STRIP
Figure 3.
-0
Per Cent Transmittance at 297 mp ex. Strip Length of a Filter Paper Strip Containing Pyridoxine Hydrochloride A.
B.
e.
D. E.
Bass line (soan of the stdp before applying the sample) Scan 01the strip *Iter development Point of sample application Pyridoxine hydrochloride band Solvent front
DISCUSSION
I n Fimre 3 the typical background absorption of a paper
ANALYTICAL CHEMISTRY
1868 Table I.
Concentration-AreaData for Various Concentrations of Pyridoxine Hydrochloride
(Four strips a t each concentration. Wave length = 297 mfi) Concentration.. Y. .Der 0.625 1.25 1.875 2.5 3.126 strip Area of absorption zone, 0.83 1.46 2.23 2.94 3.69 sq. inch 0.81 1.53 2.24 3.00 3.52 0.77 1.65 2.23 2.94 3.64 0.81 1.52 2.16 2.86 3.83 Avera e Max. i e v . from average, 70
0.80 +3.7 -3.7
1.51 +2.6 -3.3
2.21 $1.4 -2.3
2.93 $2.4 -2.4
3.67 +4.4
-4.1
Table 11. Concentration-Area Data for Various Concentrations of Riboflavin ( F o u r strips Concentration, y per strip Areaof fluorescent zone, sq. inch
@4T."Zv. from average, 70
a t each concentration) 2.6 5.2 7.8 3.30 5.24 7.02 5.30 6.64 3.46 6.72 3.34 5.41 6.73 5.30 3.44
3.39 +2.1 -2.7
5.31 +1.9 -1.3
6.78 +3.5 -2.1
10 4 7.40
7.66 7.75 7 32
13.0 8 16 8.43 8.22 8 24
7.53 +2.9 -2.8
8 26 +2.1 -1.2
strip is shown and it is evident that the variation in absorption is considerably more than is shown in the previous work ( Z ) , where absorbance versus strip length was plotted instead of per cent transmittance versus strip length as in the present study. This is to be expected since, in the plotting of per cent transmittance, small variations in transmittance are magnified in relation to large variations as compared to an absorbance (logarithmic) plot. The plot of per cent transmittance is on a highly expanded scale for low transmittance values as compared to the absorbance scale on the Cary Recording spectrophotometer used in ( 2 ) . The disadvantage of having a more variable base line is offset by the greatly increased sensitivity of the present instrument which allows, for example, the determination of as little as 0.6257 of pyridoxine hydrochloride on a strip as shown in Table I. The ability of the instrument to reproduce a trace, including all of the background peaks, is well demonstrated in Figure 3.
I
-0
The zero setting was checked before and after each strip was scanned and occasionally small discrepancies were noted, but the only changes which would have significant effect are those which might occur while an actual band was passing the beam, since gradual range changes are compensated for when superimposing the base line. In Figure 4 it is evident that the background variation is no problem Kith fluorescence measurements eince it represents an infinite value in terms of absorption. Thus, no prescanning of the strips is necessary when only fluorescence measurements are to be made. Any background fluctuation which does occur can be attributed to instrument noise and is not reproducible.
Figure 5. Plot of Areas of Absorption Peaks of Pyridoxine Hydrochloride us. Concentration
The precision obtained in quantitative measurement of fluorescence as shown in Table I1 is somewhat better than that from absorption measurements. This might be attributed to the absence of the variable background absorption and perhaps the comparision shows how much the precision of absorption measurement is affected by the variable background. Measurements of both absorption and fluorescence of bands on strips are subject to some error and variability, because of the imperfect distribution of the material in the band, the finite dimensions of the beam of light passing through the strip, the fact that only approximately 94% of the width of the strip is covered by the beam, etc. These factors affect both fluorescence and absorption measurements. The forementioned factors also affect the form of the concentration-area relationship curves as do such things as amplifier and phototube linearity, etc. Figure 5 indicates that the concentration-area curve for per cent trancmittance measurements is
I
R E L A T I V E POJlT'lON A L O N G PAPER S T R I P
Figure 4.
Fluorescence vs. Strip Length of a Developed Filter Paper Containing Riboflavin
A 580-mrinterference filter was used in front of the phototube .4. Point of sample application B . Riboflavin band
It might be expected that with the more variable base line, less precision would be attained in quantitative work. This apparently is not necessarily the case, as shown in Table I. One reason for this may be the faster pen response of the recorder as compared to that of the Cary instrument used in the previous work ( 1 , 2). While the present instrument is subject to zero drift, light fluctuations, etc., these apparently are of little consequence.
N
2
i
a
5
6
T B I i O
I 15
CONCENTRATIOW-MCG.PCR STRIP
Figure 6. Semilog Plot of Areas of Fluorescence Peaks of Riboflavin us. Concentration
V O L U M E 2 5 , NO. 1 2 , D E C E M B E R 1 9 5 3 essentially a straight line over the range used, which is contrary to Beer's lam and the usual experience with solutions. There is no ready explanation for this except to say that it is probably due to compensating effects of the various factors mentioned. The concentrations used in this series were chosen so that the maximum deflection of the pen was not over about 50% on the transmittance scale. Obviously, the closer to 0% transmittance the deflection becomes, the less deflection there is per unit of concentration increase and the sensitivity decreases rapidly. Therefore. the linear relationship shown in Figure 5 could not hold for larger concentrations. I t appears to be best, for quantitative work, to limit the maximum deflection to around 50%. The semilogarithmic relationship indicated in Figure 6 for fluorescence measurements again is the result of all of the various facators. Since. in quantitative work, the analytical results are always based on results from standards run under the same conditions, the form of the calibration curve is not particularly important. If the Beckman photomultiplier attachment is used, and the output to the null-point meter is supplied to the recorder, the instrument is operated in a state of unhalance (a d a t e for nhich it
1869 was not designed) and the nonlinearity of the amplifier under these conditions becomes a factor. For qualitative work this is not important and for quantitative work it appears that the only effect would be on the shape of the concentration-area calibration curve. 4CKNOWLEDGMENT
The authors gratefully acknowledge the contributions of Elmer Lee to the construction of the apparatus described and to Frieda Lillis for the layout of some of the figures. LITERATURE CITED
Brown, J. A , , . ~ u L . CHEX.,25, 774-7 (1953). Brown, J. A , , and l l a r s h , 11. lI., Ihid.,24, 1952-6 (1952). ( 3 ) Hashimoto, Y . , a n d lIori, I., S a t u w , 170, 1024 (1952). (4) Paladini, A. C',, and Leloir, L. F., .~s.\I.. CHEM.,24, 1024-5
(I) (2)
(1952). ( 5 ) Parke, T. V.,and Dai-is, W.W., Ibid., 24, 2019 (1952). ( 6 ) Redfield, R. R., and Cuznian, Barron, E. S., Arch. Biochem. and Biophys., 35, 443-61 (1952).
RECEIVED for review July 2 5 , 1953. Accepted September 21, 1953.
Dichromate Oxidation of Diethylene Glycol MARIO J . CARDOXE AND JOHN W. COMPTOI\J Research and Development Division, Wyandotte Chemicals Corp., Wyandote, IWich. .4n oxidation method for resolving organic mixtures containing nonvicinal hydroxyl and/or ethereal oxygen,asin glycolmixtures,requires astrong oxidant acting differentially. The behavior of dichromate as a differential oxidant is indicated by a study of the literature. Oxidation of diethylene glycol by dichromate in sulfuric acid solution at 100" C. results in three well-defined stages or levels of oxidation indicative of definite stoichiometric reaction mechanisms. These levels are attained in solutions which are 50,25, and 12.5f7"by volume in sulfuric acid and are independent of the glycol and dichromate concentrations, provided that a moderate excess of dichromate over that stoichiometrically required for
I
N THE analysis of either pure compounds or organic mixtures the use of dichromate as an oxidant is not limited to vicinal hydroxyl compounds as is periodic acid in the Malaprade reaction (1). Dichromate oxidation has been employed as a means of analytically resolving organic mixtures by one of three general approaches: (1) total oxidation by dichromate and subsequent correction for the oxidizable components ( 2 ) which are determined separately by independent methods, such as the Malaprade reaction, (2) removal or destruction of the more easily oxidizable components by a prior treatment (10) such as the Malaprade reaction and subsequent total oxidation of the remaining components with dichromate, and (3) differential dichromate oxidation (9, eo), which embodies independent oxidations under separate and different oxidizing conditions. I n the latter case only the component in question varies in behavior. Selection of the conditions necessary to effect complete oxidation of the organic compound or compounds by dichromate in the first two methods is of fundamental importance, but an examination of the literature discloses considerable latitude in the conditions used (2, 4, 9, 10, 12, 2 0 ) . In the the third method usually one of the oxidation conditions selected is the highest level of oxidation-Le., Oxidation to carbon dioxide and water. Since only the differrntial in oxidixability is required. assurance
the oxidation of the glycol to the respective level is provided. Application of the differential oxidation method for the resolution of the binary mixture, diethylene glycol and ethylene glycol, was successful over a limited composition range with an accuracy within &5% of the glycol content. The accuracy limitation was due to the lack of differential oxidation behavior of ethylene glycol under the conditions, coupled with the relatively small oxidation level change of the diethylene glycol. Further study providing additional data on the dichromate oxidation characteristics of these and other organic compounds should result in considerable improvement of the differential oxidation method.
of complete oxidation is not so critical as in the first two methods. The chief objection to the differential method lies in the use of different oxidizability. This has proved to be satisfactory for the diethylene glycol but represents only an arbitrary experimental behavior under the prescribed conditions. Under these circumstances, the method is open to the criticism of being empirical. In order to circumvent this objection, this investigation had as its purpose the determination of the dichromate oxidation characteristics of organic compounds (specifically, diethylene glycol) which are not empirical levels of oxidation, but which are represented by stoichiometric oxidation mechanisms. CHEMICALS AND SOLUTIONS
All chemicals used were the highest quality reagent grade material available, and no attempt was made to purify any of these materials further. Glycolic Acid, Matheson Co., No. 5770, assay 9 9 k yo and containing only water as impurity. Sodium Oxalate, Bureau of Standards No. 40e. All other chemicals have been described (5). OXIDATION AND DETERMINATION PROCEDURE
The oxidation procedure and the spectrophotometric method for following the oxidation have been described in detail pre-
