Amperometric Titration of Oxidized Glutathione in Presence of Cystine Polarographic Determination of Cystine and Oxidized Glutathione in a Mixture WALTER STRICKS ANI 1. &I. KOLTHOFF University of Minnesota, Minneapolis, Minn. From a polarographic study on the rate of alkaline fission of oxidized glutathione and of cystine it was inferred that reactivity in alkaline medium can be made the basis of a n amperometric mercurimetric titration of the peptide in the presence of large concentrations of cystine and of a polarographic determination of cystine in the presence of large concentrations of peptide. Conditions have been established under which the hydrolytic fission of oxidized glutathione according to the over-all equation 2GSSG 30" # 3GS' GSOOH Hz0 can be made to run to completion by removing the GS' with mercury, while cystine is not attacked. The GS- is determined by amperometric titration with mercuric chloride using the rotating platinum electrode as indicator electrode. In the polarographic determination of cystine, mercuric chloride is added to the alkaline mixture of peptide and amino acid. The mercury glutathionate and the excess mercury are
+
+
+
A
PPLICATIOK of reaction kinetics to analytical problems is still relatively rare (8, 9). The authors have found an interesting analytical application of different reactivity in the determination of oxidized glutathione (GSSG) in the presence of cystine (RSSR). I t is a simple matter to determine the sum of both by amperometric titration with cupric copper (6). The determination of the total cathodic diffusion current of oxidized glutathione plus cystine is not a direct measure of the sum of the concentration of oxidized glutathione and cystine, because the diffusion coefficient of both constituents is different. The rate of fission of oxidized glutathione by alkali is considerably greater than that of cystine (1, 11). The hydrolytic splitting of disulfide compounds is determined by the following reactions (1): 2GSSG 2 0 H - e 2GS2GSOH (1) 2GSOH OH- e GSGSOOH HzO (2) 2GSSG 3 0 H - e 3GSGSOOH H20' (3) From a polarographic study of the kinetics of the reactions of oxidized glutathione and cystine with alkali, to be reported in a subsequent paper, it was inferred that the reversible Reaction 3 can be made to run to completion for oxidized glutathione by removing reduced glutathione (GS-) with mercury or silver under conditions under which cystine is not attacked. This behavior has been made the basis of the determination of oxidized glutathione in the presence of cystine. At a given alkalinity the reduced glutathione formed is determined by amperometric titration with mercuric chloride, using the rotating platinum wire electrode as indicator electrode. I n previous work ( 7 ) silver nitrate has been used for the amperometric titration of sulfhydryl groups. However, under the experimental conditions used in the present work, silver does not give a well defined end point. It was found that mercuric chloride was very suitable as a reagent. A separate study on the reactions between mercuric chloride with reduced glutathione and cysteine be reported in a future communication. I t has been found possible to determine small amounts of oxidized glutathione in the presence of relatively large amounts of cystine.
+ + +
+ + +
precipitated with thionalide in dilute acetic acid. After removal of the excess thionalide from the aqueous solution with o-dichlorobenzene, the cathodic wave of cystine is determined a t the dropping mercury electrode. Another polarographic method for the determination of oxidized glutathione and cystine in a mixture of both is based on the fact that surface active substances a t the proper wncentration displace the cystine wave to more negative potentials, while the peptide wave is practically unaffected. With thymol as surface active agent in dilute acetic acid i t is possible to obtain a well defined glutathione and cystine wave in a mixture of both. The determination of disulfide-containing peptides and of cystine in solutions of partially hydrolyzed proteins is of great significance in rate studies of the hydrolysis and denaturation of proteins in biological materials like normal and pathological blood sera.
Csing also the amperometric titration method with copper, the cystine concentration is found by deducting the oxidized glutathione concentration from the sum of concentrations of both oxidized glutathione and cystine. A direct determination of cystine is often desirable, particularly in mixtures of small cystine, and large oxidized glutathione concentrations. For this purpose a polarographic determination of cystine in the presence of large concentrations of oxidized glutathione has been developed. Maliing use of Equation 3 mercuric chloride is added to the alkaline
+ +
1050
I
0
l
-1
l -2
1 -3
1 -4
1
-5
1
-e
Ed*,
1
-7
1
- 8
1 -9
I -10
l
.I1
l
42'
VOLT
Figure 1. Current-Voltage Curves Of 5 X 10-5 .M G S S G and of 1.25 X 10-5 ,M Hg(I1) in 0.1 J4 "3, 0.1 ,M Xah-03, 0.013 M S a O H a t rotating plati. num electrode. Volume of solution 40 ml A. B. C.
Supporting electrolyte 6 x 10-5 M G S S G 1.5 ml. 10-3 MHgClzadded t o B D. 0.5 mi. 10-3 M HgC12 added t o L E . 1.25 X 10-6 .M HOC12
V O L U M E 25, NO. 7, J U L Y 1 9 5 3 mixture of oxidized glutathione and cystine. The mercury glutathionate [Hg(GS)Z]and excess mercuric chloride are removed in acetic acid buffer with thionalide. After removal of the excess thionalide from the solution by treatment with 0-dichlorobenzene, the cathodic wave of cystine is determined a t the dropping mercury electrode.
1051
current. 911 potentials were measured against the saturated calomel electrode (S.C.E.). Oxygen was removed from the solutions in the cell with pure nitrogen. During an experiment an atmosphere of nitrogen was maintained over the solution. The characteristics of the capillary used were: m = 1.66 mg. s e - 3 ; t = 4.82 seconds (open circuit); m2I3tile = 1.748 mg.*I3 pec. - 1 1 2 ; h = 80 em. hmperometric titrations with rotated platinum wire as indicator electrode were carried out using an alternating current motor of 1800 r.p.m. to provide constant rotation for the platinum wire electrode. Reagent was added from 5-, 2-, and 1-ml, semimicroburets divided into 0.01 ml. The pH was measured with a Beckman p H meter, laboratory niodel G. ARIPEROMETRIC MERCURIMETRIC TITRATION OF OXIDIZED GLUT4THIONE IN PRESENCE OF CYSTINE
Figure 2.
Current-Voltage Curves
Of 5 X 10-5 M GSSG a n d of 1.25 X 10-6 M . HgClz in alkaline chloride solution (2 M KC1 0.026 M NaOH) a t rotating platinum elec: trode A . Supporting electrolyte B . 5 x 10-5 .VI GSSG C. 1.5 ml. 10-3 M HgClz added t o B D . 0.5 ml. 10-3 M H g C h added t o C E . 1.25 X 10-5 M Hg(I1)
another method for the determination of oxidized glutathione and cystine in a mixture of both is described in this paper. Both oxidized glutathione and cystine give reduction waves a t the dropping mercury electrode (5,f2), but no sharp separation of the two waves is observed when both substances are present together in solution. Capillary active substances like thymol displace both the oxidized glutathione and cystine waves to more negative potentials ( 5 , 1 2 ) . The authors found t h a t the displacement of the cystine waves occurs a t thymol concentrations a t which the oxidized glutathione wave is hardly affected. Thus by working at the proper thymol concentration it is possible to get separate oxidized glutathione and cystine waves in a mixture of both. The analytical methods developed were successfully applied to the quantitative determination of disulfide-containing peptides and of cystine in solutions of partially hydrolyzed proteins. It is planned to make practical use of these methods in studies on the rate of cystine formation upon denaturation and hydrolysis of proteins. MATERIALS USED
Cystine was a reagent grade Alerck product. Cysteine, which was used in the form of its hydrochloride, was a C.P. product from Pfanstiehl. Stock solutions of cysteine and cystine were prepared as described reviously ( 7 ) . Glutathione in the reduced state was a Pfanstieffl product. The purity of this product was 99% as determined by titration with cupric copper (6). A 2 X 10-2 . I Istock solution of oxidized glutathione was prepared as described (12). A Kahlbaum product of thionalide was used in the form of a 2% (0.092M) solution in glacial acetic acid. 9 1 1 other chemicals were commercial C.P. products. EXPERIhl ENTA L
Current voltage curves were measured a t 25" C. with the manual apparatus and circuit described by Lingane and Kolthoff (f 0) and automatically mith a Heyrovsky self-recording polarograph. A11 reported values of i d were corrected for the residual
In mercurimetric titrations the platinum electrode after prolonged use is coated with mercury and then virtually behaves like a mercury electrode. S e w indicator electrodes which have been cleaned nith nitric acid and rinsed with water proved to be insensitive in the first two or three titrations, but responded normally thereafter. Apparently a uniform coating of mercury is necessary for the proper functioning of a platinum electrode in mercurimetric titrations. I n order to obtain a firmly adhering niercury deposit, it was found to be expedient to use platinum electrodes which had been coated n-ith a thin layer of silver which forms an amalgam nith mercury. The silver coating was obtained by electrolysis of a X ammoniacal silver solution (0.1 ~ l fammonia, 0.1 Jf ammonium nitrate) a t -0.3 volt for half an hour with the platinum electrode as cathode. After the first two or three titrations, perfectly reproducible results were obtained with such an electrode, which has been used for more than 100 titrations. Current-voltage curves obtained with this electrode in ammoiiiacal medium are given in Figure 1. It is seen that oxidized glutathione in weakly alkaline medium (0.013 M sodium hydroxide, 0.1 Jf ammonia) does not give an anodic diffusion current. At the relatively low alkalinity of the solution the equilihrium of Equation 3 is a t the left side and the concentration of thiol compound is too small to be measured. This has also been found with the dropping mercury electrode. A solution containing oxidized glutathione and enough mercury to react completely with reduced glutathione formed according to Equation 3 [ 1GSSG : O.iBHg] gives a current voltage curve (Figure 1, C) a t the rotated platinum electrode which is similar to that of the supporting electrolyte, and does not show a reduction wave of mercury glutathionate. The addition of an excess of mercuric chloride to the solution of mercury glutathionate which also contains glutathione sulfinic acid (GSOOH) results in the appearance of a cathodic diffusion current (Figure 1, D). Under proper conditions and within a limited range of excess of mercury this current is found to be proportional to the concentration of the mercury added a t potentials between -0.3 and -0.5 volt. From Figure 1, E, it is seen that mercuric chloride (1.26 X 10-6 M )added to the supporting electrolyte in the absence of oxidized glutathione gives a diffusion current which is greater than that of the excess mercury (Figure 1, D )of the same molar concentration. Excess mercury reacts with mercury glutathionate to form loose complex compounds in which additional mercury is bound to the carboxyl groups of glutathione. Evidence for the formation of these compounds was obtained from unfinished polarographic studies of the mercury-glutathione system. Current voltage curves of oxidized glutathione in nonammoniacal medium before and after addition of mercuric chloride with a large concentration of alkali chloride [2 M potassium chloride, 0.026 Af sodium hydroxide] are illustrated in Figure 2. The curves are similar to those given in Figure 1, but the diffusion current of mercury is better defined in the presence of ammonia. The information obtained from the current-voltage curves can be made use of in the titration of oxidized glutathione. The best results are obtained in ammoniacal medium a t an applied poten-
ANALYTICAL CHEMISTRY
1052 tial of -0.3 volt. a t which potential moqt of the titrations weie performed. -4great of titrations ac: out under varJ.ing conditions. The effects of the conrentration of alkali, chloride ammonia. and cystine were studied.
Figure 3.
Remove air with purified nitrogen, which must be passed through two bottles with 0.1 Jf ammonia to maintain a fairly constant ammonia concentration in the titration mixture Pass nitrogen through the mixture during the entire titration. To the air-free solution add so much of the that the oxidized concentration in the mixture is between 2 X 10-5 and IO-' 31. Immerse the salt bridge and tio of the buret in the solution and titrate with a TO-3 -11mercuric chloride solution a t an applied potential of -0.3 to -0.4 volt 2's. S.C.E. .Add mercuric chloride solution slowly to the titration mixture. After addition of an increment of titrating agent, wait 1 or 2 minutes until the current remains constant. After the end point, mercuric chloride can be added rapidly to the titration mixture. One milliliter of 10-3 -11mercuric chloride solution corre$ponds to 0.816 mg. of oxidized glutathione. The sum of bot,h oxidized glutathione and cystine can tie determined in a separate sample by an amperometric titration in ammoniacal medium a t pH 9 with cupric copper as reagent ( 2 ) . I n contrast t o cystine, oxidized glutathione cannot be titrat,ed successfully with cupric copper a t p H markedly higher than 9.
Amperometric Titration of Oxidized Glutathione with .M Mercuric Chloride
A t rotating platinum electrode as indicator electrode a t applied potential of -0.3 volt. Volume of mixture. 40 ml. A . 0.61 nig. GSSG (initial GSSG concn. 2 . 5 X 10-5 .li) in 0.026 .M K a O H , 0.05
M KCI
B . 0.61 mg. GSSG in 0.026 M S a O H . 2 M KCI C . 0.306 ing. GSSG, 0.48 nig. RSSR (1.28 x 10-5 .cI GSSG, 5 X 10-5 M RSSR) in 0.1 M NHs, 0.006 M S a O H , 0.1 IM KaSOg CI. Blank. same electrolyte as in C , no RSSR C?. Blank, saine electrolyte as in C plus 5 X 10-5 .If RSSR
The effect of concentration of chloride ion in a nonanimoniacal solution is illustrated in Figure 3 (compare -4 and B ) in which titration$ of oxidized glutathione in 0.05 Jf and 2 .I1 potassium chlori,le are reproduced. The alkali concentration was 0.026 .If in both titrations. The end point corresponds to the formation of mercury glutathionate [Hg(GS),]. It is seen that a sufficient exc'ess of chloride is important in order to obtain a well defined end point ( B ) . Under these conditions t,he reagent line corresponds to the diffusion current of the complex HgCI,-- and also to complex mercury glutathione compounds which are in equilibrium with HgCla--. .IZt a low chloride ion concentration the formation of Hg2(GS)?is favored after the first end point is reached. This results in a curved excess reagent line (curve d j which a t least in its loxer part corresponds to the electroreduction of a mercury glutathione complex in which the mer(-ury is bound partly to the carboxyl groups of the peptide. Curve C in Figure 3 illustrates the titration of a mixture of oxidized glutathione in the presence of cystine in ammoniacal medium, containing a small concentration of free alkali hut no chloride. A sharp end point [Hg(GS)r] is obtained in this medium. Titrations carried out in phosphate buffers a t pH IO and 11 and in an ammonia solution of p H 11.3 indicated that the hydrolytic fission of glutathione is so slow in this p H region that it is of no practical analytical use. I n dilute sodium h>,tlroxide solutions (0.02 to 0.03 M ) as well as in ammonia solut,ions containing a small amount of sodium hydroxide (0.02 to 0.006 W)the titration can be cirried out wit'hin 15 to 30 minutes. Generally it has been found that the reaction is somewhat, faster in sodium hydroxide-chloride than in ammoniacal medium, but t'he titration curves are better defined and t.he excess reagent line is steeper in the presence of ammonia. The procedure is therefore given for ammoniacal medium.
Procedure. Introduce 40 ml. of a solution which is 0.1 M in sodium nitrate, 0.1 M in ammonia, and 0.006 t o 0.02 Jf in sodium hydroxide into a 150-ml. beaker which is provided with a rubber stopper with holes for electrode, salt, bridge, buret, and inlet tube for nitrogen. Immerse a platinum wire electrode in the solution.
Table I gives the results of amperometric titrations of oxidized glutathione in the absence and presence of cystine. The accuracy and precision of the oxidized glutat'hione titration are about =k3% in the concentration range between 1.25 X and 1.25 X IO-' JI, and are not affected by cystine even if the concentration of the latter is twenty t.imes greater than that of oxidized glutathione. It is seen from Table I that the accuracy is better if t,he oxidized glutathione concentration is 5 X IO-j .If or larger. The accuracy is also better in the presence of ammonia than in its absence. The proper adjustment of the alkali concectration is important. Small alkali concentrations reduce the rate of the reaction but improve the reproducitiility of the results. POLAROGRAPHIC DETERMINATION OF CYSTINE AFTER R E M O Y I L OF OXIDIZED GLUTATHIONE
In a reaption mixture of a coxposition given in the procedure above, the mercury waves given by an excess of mercuric chloride and by the mercury glutathionate would make it impossible to determine polarographically with reasonable accuracy small concentrations of cystine when the initial concentration of oxidized glutathione is large. Therefore, it was decided to remove the soluble mercury, so that cystine would be the oniy compound left reducible at the dropping electrode. Hydrogen sulfide in acetic acid medium was found unsuitable for this purpose, as it slowly reduces cystine. The mercury in mercuric glutathionate and the excess mercuric chloride can be removed by precipitating uith thionalide (TSH) in acetic acid medium, whereupon the cathodic cJ.stine wave can be determined a t t,he dropping mercury elertrode. Thionalide is slightly soluble in weakly acid aqueous medium. Apparently, it is strongly capillary-active and it displaces the cystine wave to more negative potent'ials, but to a m u c h larger extent than substances like thymol, caffeine ( j j , etc. Consequently, t'he diffusion current region of the displaced cystine wave often is poorly defined in the presence of thionalide, which afferts the accuraq- of the polarographic cystine determination. The disturbing effect of thionalide can be eliminated by treatment of the solution with +dichlorobenzene, which readily dissolves the excess thionalide. o-Dichlorobenzene, which is slightly soluble in water, displaces the cystine wave to a much smaller extent than thionalide and the diffusion current region of t'he cystine wave is well defined in the presence of t,his solvent. The aqueous solution, which is milky after addition of thionalide, becomes perfectly clear after treatment with dichlorobenzene and the precipitate of mercurj- thionalide adheres firmly to the surface of the solvent, which after stirring collects a t the bottom of the cell. The polarogrsms in Figure 4, which were obtained with a mix-
V O L U M E 2 5 , NO. 7, J U L Y 1 9 5 3
1053
Jf) and cystine (2 X ture of oxidized glutathione ( 5 X .lf) after decomposition of oxidized glutathione with mercury and precipitation of the latter with thionalide, illustrate the effect of thionalide on the cystine wave. Polarogram A was obtained before and polarogram B after removal of thionalide with o-dichlorobenzene. The only cathodic Tvave in these polarogranis is that of cystine. This wave is much better defined in I3 than in A . Polarogram A exhibits two anodic waves. From a blank with thionalide alone it is found that the small anodic wave with a hnlfwave potential of about -0.375 volt corresponds to that of the sulfhydryl group of thionalide in its saturated solution. o-Dicahlorobenaene removes the thionalide almost, completely from the aqueous solution and therefore no anodic thionalide wave is seen in polarogram B. In both A and B an anodic wave with a half-wave potential of about -0.17 volt is found. This wave is clue to the reduced glutathione (GSH), formed on the addition of thionalide to mercury glutathionate according to: Hg(GS)*+ 2TSH
=
Hg (TS)Q 4
+ 2GSH
measured at 0 volt and found to be 2.00 pa., corresponding to 7.50 X 10+ Jf reduced glutathione or an i d / c of 2.67, a value that is in fair agreement \vit,h that obtained previously ( 1 8 ) (2.53) with reduced glutathione in an acetate buffer of pH 5.1 using the same capillary. Experiments have been carried out with various mixture. of oxidized glutathione ( 5 X M) and cystine ( I O - ' to 2 X to 4 X .If). The alkali concentration used in these experiments was 0.02 to 0.03 31 sodium hydroxide. At this alkalinity and in the presence of a slight excess (6 to 8%) of mercuric. c~liloride, oxidized glutathione decomposes completely according to Reaction 3 Fvithin about 5 minutes, while cystine is unattackc-tl.
(4)
The diffusion current of the reduced glutathione wave was E de,
Figure 5 .
2
KILT
Polarogram
Of 19.96 iiil. 5 X l o - < .If G S S G , 2 X 10-4 .If RYSR in 0.026 .lf NaOH, 0.5 .Vf KC1, 0.0025% gelation after reaction with 0.16 nil. 0.05 .I!HgC19 I
W
The complete decomposition of oxidized glutathione can be testeJ I)>- measuring the anodic reduced glutathione wave after precipi0 tation of the mercury with thionalide as illustrated in Figure 4 u or by measuring the rathodic wave of mercury glutathionate heI Core addition of thionalide. A polarogram obtained with tliv same mixture as in Figure I before precipitation of the rnei~c~qy is shown in Figure 5 . The first small wave is given by the slight 2 excess of mercuric chloride (6.7%) which had been added. The mercury glutathionate wave starts a t a potential of about -0.4 volt and has a well defined difiusion current region. The curi,ent 3 +2 * I 0 ~l .2 -3 - 4 - 5 .6 -7 -8 - 9 -10 - 1 1 4 2 -13 -14 a t -0.8 volt was found to be 1.72 pa. after correction for the E d e , VOLT residual current and volume change. This current should correspond to 3.75 X lo-' -limercury glutathionate if all the oxidizefl Figure 4. Polarograms ( 5 X 10-4A1f)had been decomposed. The idle (curOf 101m1.5 x ~ O - + . T ~ G Y S C x ; . ~1 0 - ~ . 1 f ~ ~ ~ ~ , 0 . 0 2 6 ~. ~ MiE~ ;0~~~, o0 . glutathione 1 after reaction with 0.1 nil. 0.05 .I!HgC12 and 0.18 nil. 2% TSH rent corresponding to a 10-3 11f solution) of mercury glutaA . Before thionate should therefore be 4.57, which compares favorably with B . After treatment with 5 nil. o-dichlorobenzene the value of 4.2 obtained with a -~ -~ ~solut,ion of mercury glutathionate in boras a t p H 6.9 ( 2 2 ) . Table I. Amperometric Titration of 40 311. of Oxidized Glutathione Solution in The polarogram in Figure 5 -4bsence and Presence of Cptine with M Mercuric Chloride also shows the cystine \~-ave' Initial (:SSG Concn. of Soln. Concn. of GSSG GSSG nhich starts a t about -1.2 ~ ~ ~ k: ~ , ~ ~-i\.erage d ~ , , Coniposition of Electrolyte, If S o . of Titrated, .If, RSSR, ~- volts and is poorly defined i n Detns Approx. .If, -4pprox. 31g. 1 1 ~ . Error, "c NaOH KC1 S a S O z S I I a 4 . 0 0.026 2 . 0 . . . . . alkaline medium. 2 1 25 x 10-5 Sone 0.298 0 286 6 2 50 x 10-5 Sone 0.596 O596to -3.4 0.026 2.0 ... I n order to work under the 0.563 2 3 . 7 5 x 10-5 Sone 0 891 0.893 -0 1 0.026 2.0 ... most uniform conditions, the 1 Sone 1.19 1.17 -1.7 0.026 2.0 ... a x 10-4 same volume (0.6 ml. per 10 ml. 1 1 25 X 10-5 4 X 10-5 0.298 0 291 -2.3 0.026 2.0 ,. 1 2 5 X 10-5 1 . 2 5 X 10-5 0.595 0.628 +5,5 0 026 2 0 . . . of test solution) of thionalide 1 2 . 5 x 10-5 0.026 2.0 ... 2 . 5 x 10-6 o 571 -4.0 0,595 0 026 2.0 . .. reagent (2% t h i o n a l i d e i n 7 2 . 5 x 10-5 0.595 0.5~0 -0.8 5 x 10-6 1 2 5 X 10-5 7 . 5 X 10-5 0.595 0.612 +2.8 0.026 2.0 , . glacial acetic acid) was added 1 2 . 0 x 10-5 1 x 10-4 0.595 0.571 -4.0 0.026 2.0 .. 1 1 . 2 5 x 10-5 Xone 0 298 0.310 +4.0 0.026 0.1 b' 5 to various mixtures. Since 1 1 25 x 10-5 Sone 0.298 0.286 -4 0 0.026 0.1 . . 0.1 1 2 5 x 10-5 Sone 0.595 0 592 -0.5 0.026 0.1 .. 0 5 the oxidized glutathione con1 3 75 x 10-5 Sone 0,893 0 894 +O 1 0.026 0 1 0.1 centration was varied from 5 1 1 . 2 5 x 10-5 5 x 10-6 0.298 o 290 -2.7 0.0065 ... o:i o 1 I 1 . 2 5 X 10-5 2 . 5 X 10-5 0.298 0 294 -1.3 0.52 ... 0 1 0 1 X 10-4 to 2 X 10-3 31, the > 3 . 7 5 X 10-5 5 X 10-6 0.893 0 890 -0.3 0.026 ... 0 1 0 1 1 3 . 7 5 X 10-5 5 X 10-6 0.893 0 850 -4.8 0.026 0.1 . . 0 1 amount of thionalide added 1" 3 . 7 5 x 10-5 1 x 10-4 0.893 0.939 +Ll 0.026 0 1 0.1 corresponded to an excess of 1 3 X 10-5 5.34 X 10-4 0.893 0.882 -1 2 0.006 ... 0.1 0 1 1 -0.1 0.006 ... 0.1 0 1 400 to loo%, respectively, 1 . 0 7 X 10-8 0.893 0 892 1 5 X 10-5 1 19 1.184 -0.5 0.006 ... 0 1 0 1 1" 7 5 x 10-5 I x 10-4 1.78 I.&? +2.8 0.026 ... 0.1 0 1 over the mercury to be precipi1 1 . 2 5 X 10-4 5 X 10-6 2 97 2 96 -0.3 0 006 .. 0.1 0 1 tated f r o m t h e s o l u t , i o n . 10 1 25 x 10-4 1 x 10-4 2 97 3.04 +2.4 0.026 , . . 0 1 0.1 a Titration was carried o u t at -0.4 volt 1,s. S.C.E. Flagg ( 2 ) r e c o m m e n d s a threefold excess of thionalide W
% o
; 2
~~~~
~~~
~
~
~~~~~~
1054
ANALYTICAL CHEMISTRY
for the precipitation of mercury. The authors did not find a mercury wave after addition of a 100% excess of the reagent and thus assumed the precipitation to be complete. The volume of o-dichlorobenzene used for the extraction of thionalide from the aqueous solution was kept constant. Five milliliters of solvent was always added per 10 ml. of aqueous solution, while a vigorous stream of nitrogen was bubbled through the mixture for 5 minutes, after which time no more thionalide could be detected polarographicallp in the solution.
€de.,
Figure
"OLT
6. Current-Voltage Curves h~wroximatelv10 ml.
I n 0.026 M S a O H , 0.1 M NaNOs after complete reaction of GSSG with appropriate amount (according t o Equation 3) of 0.05 .M HgClz, precipitation of mercury with 0.6 ml. 2% TSH. and removal of the latter with 5 rnl. o-CsH4Ch
The accurate estimation of cystine from its cathodic: diffusion current requires the value of the residual current. Blank experiments were run with cystine-free alkaline solutions of oxidized glutathione a t various concentrations (5 X 10-4 to 2 X l O - 3 M ) after addition of the appropriate amounts of mercuric chloride, thionalide, and o-dichlorobenzene. From Figure 6, which illustrates cathodic current-voltage curves obtained with these mixtures, it can be seen that the residual current is small and fairly const.ant up to an applied potent'ial of -0.7 volt but increases appreciably a t more negative potentials as the concentration of osidized glutathione is increased. Thus a t -1.05 volts the residual current is found to be 0.16, 0.23, and 0.31 pa. a t oxidized glutathione conc,entrat'ions of 5 X and 2 X lO-3MI respectively. This variation of the residual current involves limitations of t,he polarographic cystine determination. For an accurate determination of small concentrations of cystine it would be necessary to run blanks a t various oxidized glut'athione concentrations and to obtain calibration curves. However, for practical analytical purposes and within a limited concentration range of oxidized glutathione the determination of the residual current can be avoided by making use of an extrapolation methocl, as indicated i n Figure 6. From polarograms BI and Bz. which were obtained with mixtures of the same oxidized glutathione (10-3 M) but of different cystine concentrations ( and 3 x lO-'M), it is seen t,hat the diffusion current lines of the cystine waves are practically parallel to the line presenting the residual current, B, at the same oxidized glutathione concentration. By extending the diffusion current plateau of curves B1 and Bz to a potential of -0.8 volt as indicated by the dotted lines in Figure 6 current values (0.55 and 1.55 pa.) were obtained, which are almost equal to the values of the corrected diffusion current measured a t -1.05 volts (0.51 and 1.55 pa.). Experiments with various osidized glutathione concentrations revealed that the extrapolation method gives good results only a t osidized glut,athione Concentrations not markedly larger than 10-3 iM. At higher oxidized glutathione concentrations the extrapolation values are high-illustrated in polarogram CI of Figure 6, M . in oxidized glutathione obtained with a mixture 2 x M in cystine. The extrapolated value of id is 0.68 pa., and while the corrected id a t a potential of -1.05 volts is 0.56 pa.
Procedure. Introduce 0.2 mi. of 5 .M sodium nitrate and 0.1 to 0.15 ml. of 2 X sodium hydroxide into a polarographic cell, provided with gas inlet and outlet tubes. Add 9 ml. of distilled water, and cover the cell with a rubber stopper with holes for the salt bridge, dropping mercury electrode, and a pipet. Introduce the salt bridge, stopper the holes for pipet and electrode, place the cell in a thermostat a t 25' C., and make the solution air-free. While bubbling nitrogen through the air-free solution add a given volume of the sample, so as to make the solution not more than 10-3 M in oxidized glutathione. If the oxidized glutathione con-11,blanks must be centration is considerably greater than run with oxidized glutathione solutions alone of various oxidized glutathione concentrations in order to determine the residual current. Add a measured volume of a 0.05 J1 mercuric chloride solution, corresponding to 6 to 8% excess of the quantity required to react completely with the reduced glutathione in the solution. The oxidized glutathione in the mixtuie can be determined by an amperometric titration a-ith the rotating platinum electrode a3 described above. After addition of the mercuric chloride, nait 5 minutes while passing nitrogen through the solution and precipitate the mercury by adding 0.6 ml. of a freshly prepared 2% thionalide solution in glacial acetic acid. Add 5 ml. of o-dichlorobenzene while bubbling nitrogen through the solution. The bubbling of nitrogen not only serves to prevent air ovidation but also brings about an intimate mixing of the two liquidp. Five minutes of thorough stirring is sufficient to obtain a clear aqueous layer. Khile passing nitrogen over the surface of the solution, introduce the dropping mercury electrode and run a polarogram between -0.7 and - 1.2 volts. The practically straight diffuqion current line of the current voltage curve is extended to -0.8 volt and the current read a t this point. Refer the current to a volume of 10 mi., accounting for change in volume after the addition of sample, mercuric chloride, and thionalide solution. From the characteristics of the capillary and from the diffusion coefficient of cystine the i d / C value of cystine can be calculated. The diffusion coefficient of cyytine under the conditions of the present experiments has been found to be i . 0 X 10-6 cm.2 sec-1. I f t h e characteristics of the capillary are not known, measure the diffufiion current of a 5 X 10-4 M cystine solution and of the residual current, using the same wpporting electrolyte as for the sample in the presence of o-dirhlorobenzene. From the Z d / C value of cystine and from the current measured in the sample, the concentration of cystine is calculated Note. The amount of mercuric chloride required to react completely with reduced glutathione can be determined with the dropping mercury electrode. For this purpose a separate sample muqt be t i t r a t d in the Fame supporting electrolyte as given in the
Figure 7. Effect of Thymol at Varying Concentrations on Reduction Waves Of 5 X 10-4 A4 RSSR and of 5 X 10-4 ~k'GSSG in 0.1 .\I CHaCOOH, 0 1 M KC1.
RSSR A . Nothymol B. 2.8 x 10-8 AM thymol C. 8.4 X 10-6 M thymol D . 1.7 x 10-4 M thymol E 2.8 x 10-4 M thymol
.411 curve6 start a t zero volt
GSSG 1. Xothymol 3. 2 . 1.7 8.4 X 10-4 10-6 M thytl1ol thymol
4.
J.
2.8 x 10-4 M thymol 5.1 X 10-4 41 thymol
V O L U M E 25, NO. 7, J U L Y 1 9 5 3
1055
Table 11. Diffusion Current of Cystine in Mixtures of Cystine and Oxidized Glutathione According to Procedure
GSSG Concn., A! 2 x 10-3 I x 10-2 1 1
5 5
x lo-' x 10-8 x 10-4 x
lo-'
(All ad values corrected for change in volume) RSSR Ware Values Correcred Extrapolated RSSR for Residual Values Concn., Jl Id Id C td Zd/C 1 x 10-4 2 7 0,693 6.93 0 .ii 1 3
x
x
0 52 1.64 2 28 0.57 1.18
10-4 10-4
4 X IO-'
1 X 10-4 2 x 10-4
a ?
5 5
5 7 5 7 5.9
0,56 1.64 2.36
E5 9a
054
5.4 5.6
1 12
waves. Thus at pH marke,lly higher than 3 the cystine wave becomes drawn out and exhibits a prewave ( 5 ) which makes inipossible an accurate measureinent of the oxidized glutathione wave in the presence of cystine. The best separation of the peptide oncl amino arid is ohtairied in dilute acetic acid.
I
procedure, using the dropping mercury electrode as indicator electrode at a potential of -0.3 volt. The bottom of the titration vessel must be covered with chloroform to prevent the mercury metal from reacting with the mercury in the solution. A 0.05,1_1 mercuric chloride solution is added to the mixture from a 1-ml, buret. .4fter the end point a cathodic current is observed which is proportional to the excess mercury added.
Ed., VOLT
Figure 9.
l'olarograms
Of mixtures of same RSSR concentration ( 5 X 10-6 -If) and varying GSSG concentrations
A . 10-4.W B . 2 x 10-4 c. 3 x 10-4
D. 5 x E.
i
x
10- . lo-48M
Supporting electrolyte: 0.1 ,M CHICOOH, 0.1 .M KCI, 1.7 x i n - 4 .M tilylnoi
Figure 8.
Polarograms
Of mixture of 5 X 10-4 4 !. GSSG and 5 X 10-4 iM RSSR In 0.1 M CHKOOH. 0.1 M KC1 in presence of varying concentrations of thymol. A11 curves s t a r t a t zero volt A . Nothyniol B . 5.6 X 1 0 - 6 -11 thymol C.
D. E.
1.' 1.r
2.2
X 10-4 X 10-4 10-4
x
.M thymol .M tliymoi
,If thymol
The results of the polarographic determination of cystine :ire 3ummarized in Table 11, which gives the diffusion current of the cystine wave as measured a t -1.05 volts and corrected for the residual current and also the extrapolated values of id as illustrated in Figure 6. For a better comparison of solutions of different cystine concentration the values of i d l c (diffusion current cystine solution) are also given in Table calculated for a 10-3 11. It is seen that good results are obtained with mixtures of oxidized glutathione and cystine a t concentration ratios of 20 to 2.5. If the concentration of oxidized glutathione is markedly larger than ?.I, the extrapolated values of id are high. Under such circumstances the residual current must be determined in separate experiments. POLAROGRAPHIC DETERMINATION OF CYSTINE AND OXIDIZED GLUTATHIONE IN PRESENCE OF EACH OTHER
Experiments have been carried out with mixtures containing varying ratios of oxidized glutathione and cystine in the presence of thymol, c d e i n e , gelatin, ethyl alcohol, and amyl alcohol as papillary active substances a t varying pH. All these substances have similar effects on the oxidized glutathione and cystine waves and displace the cystine wave to a larger extent than the oxidized glutathione wave. Under favorable conditions two v-ell-defined waves are obtained, the first one corresponding to the reduction of oxidized glutathione, the second to that of cystine. For analytical purposes thymol was found to be the most suitable substance, since most of the other compounds give rise to drawn out waves and to irregularities in the current-voltage-curves. I n the presence of surface active compounds the p H of the solution also affects the appearance of the oxidized glutathione and cystine
Figure i illustrates the cliff erence in the behavior of osidized glutathione and of cystine, both at' the same concentration ( 5 X 10-4 31) in separate solutions in the presence of varying concentrations of thymol in 0.1 II/ acetic acid. The addition of increasing amounts of thymol to a cystine solution results in the supprepsion of the maximum and in a displacement of the wave t.0 more negative potentials. The sniall premave noticeable a t a thymol concentration of 8.4 X IO-5 Af is completely eliminated upon furt8heraddition of thymol. Considering glutathione, it is found that the suppression of the oxidized glutathione maximum red f ) than quires a much larger thymol concentration (1.4 X that of cystine (2.8 X 10-6 JI thymol). On the addition of more thymol to the oxidized glutat,hione solution, the cathodic wave is split into two waves. The first wave is not displaced to more negat.ive potentials, but decreases i n height on the addition of increasing amounts of thymol and a t thymol concentrations M reseinbles the prewave of cystine. On greater than 5 X furt.her increase of the t>hyniolroncentration this wave is eliminated and only one oxidized glutathione wave appears in the polarogram. The mechanism of the electroreduction of oxidized glutathione in the presence of thymol is very complicated, since free radical reactions (13) are involved in this reduction. The total height of both the cystine ant1 oxidized glutat'hione waves is hardly affected by the presence of t.hymo1. A4n inspection of Figure 7 indicates that a thymol concentration which is just sufficient to eliminate t,he osidizcd glutathione maximum (Figure 7-33 displaces the cystine wave (Figure 7 , D ) to a potential negative enough to give riPe to t'wo separate waves in mixtures of the peptide and amino acid. This is illustrated in Figure 8, which presents polarograms of a mixture of cy.stine and oxidized glutathione of t,he same molar concent,rat,ion( 5 X 10-4 .If)a t varying thymol concentrations. At a thymol concentration of 1.7 X 1 0 - ~ df the diffusion current of ositlizetl glut.at,hione is well defined ant1 corresponds to an i d / c of 4.7 at a potential of -0.5 volt. This value is in fair agreement with the idle of 4.56 of oxidized glutathione in an acetate buffer, os reported previously (fa). Coruparing the oxidized glutathione wave in the absence and presencr of cystine under otherwise identical conditions, it is seen from Figure 7-3 that the osidized glutathione wave in the absence of cystine is poorly defined a t a thymol concentration of 1.7 X M but is perfect,ly normal in the presence of cystine (Figure 8, D 1. Apparently cystine diminishes the effect of th-mol on the oxidized glutathione wave.
1056
ANALYTICAL CHEMISTRY
Polarograms obtained with mixtures of the same cystine (5 x -11) and thymol (1.7 X 1 0 - 4 M ) content but of varying oxidized glutathione concentrations to T X 21) are reproduced in Figure 9. Il'ith the exception of curve E , the oxidized glutathione waves are found t o be well defined at various t-oncentrations of the peptide. The rounded niariniuni in E. which does not affect the diffusion current a t -0.5 volt, rould be eliminated by a slight increase of the thymol concentration (from 1 .i to about 1.9 X M). The diffusion current a t -0.5 volt has been found to be proportional to t.he oxidized glutathione (soncentration within the entire concentration range investigated. The same was also found at higher (10-3 Jf) and lower (2.5 X 1 0 - ~21) cystine concentrations. Thus two n-ell defined iyaves can be obtained with various mixtures of cystine and oxidized glutathione in dilute acetic. acid and with the proper thymol concentration. Procedure. Intioduce 2 nil. of 1 dl acetic acid and 2 nil. of 1 Jf potassium chloride into a polarographic cell nhich is p ~ o vided with gas inlet and outlet tubes. Add enough sample t o make the total disulfide concentration not lower than about 5 X ;If and not larger than 1.5 X M . Fill up n ith a measured volume of distilled water to a total of about 20 ml. Cover the cell with a rubber stopper provided with holes for the diopping mercury electrode, the salt bridge, and a pipet. Introduce the electrode and salt bridge, stopper the hole for the piprt, place in a thermostat at 25' C., and make the solution air-free bv _ passing _ purified nitrogen. From a graduated 1-ml. pipet add 0.6 to 0.7 ml. of a saturated thvmol solution (5.66 X 10-3M a t 20" CJ. This amount is sufficient if the oxidized glutathione Concentration is not la1ger than about 7 X M . At larger oxidized glutathione concentrations more thymol must be added to suppress the maximum of the peptide wave. Continue the bubbling of nitrogen not longer than 1 minute after addition of thymol. While passing nitrogen over the surface of the solution, measure the diffusion currents a t -0.5 and -0 9 volt, respectively. Correct for the residual current and refer the currents to a volume of 20 rnl , accounting for change in volume after addition of thymol. I n separate experiments measure the diffusion currents of a 5 X 10-4 -11 ovidized glutathione and a 5 X Jf cystine solution and of the residual current, using the same supporting electrolyte (with thymol) as for the sample. From the % d / C values of oxidized glutathione and cystine, both measured at -0.9 volt, and from the currents measured with the mixture at -0.5 and -0.9 volt the concentrations of the peptide and of cystine are calculated. The correction for m2/3tils (3)is very small in this procedure and ran be neglected in the calculation of the diffusion rurrent of cystine. Results of the polarographic determination of oxidized glutathione and cystine are listed in Table 111. The accuracy of the oxidized glutathione determination is within 5% and is neither affected by the concentration ratio of oxidized glutathione and cystine which has been varied from 0.1 to 2.4 nor by the concentration of cystine (2.5 X 10-4 to 10-3A14'). The results of the cystine determination are high (6%) at smaller cystine concentrations (2.5 X lo-* Jl), while the accuracy is better than 3% a t larger cystine concentrations.
C
I
2
;-3 W 4 P
-4
I!
6
-1
Cone RSH IM)
Figure 10.
Current-Voltage Curves
9. GSH. R S H , and their mixtures in acetate buffer (0.4 M CHrCOOH. 0.05 .If CHsCOOSa. 0.1 M S a N 0 3 ) a t pH 3.67 1. 2 . 6 x 1 0 - 4 M R S H 2. 10-5 M R S H 3. 10-3 -VI GSH .ill others 10-3 M G S H plus
R SH
+l--iSjx
10-4
M RSH
2.3 X 10-4 Jf R S H 3.75 X 10-4 .M R S H 2 10-4 M RSH , . a X 10-4 iM R S H lo-'JMRSH 1.25 x 10-3 M RSH 11. 1.75 X 10-3 , I !RSH Diffusion current (mea\iired a t +0.1 volt) of mixtiires of 4 cio. runcentration of R S H J.
6. 7. 8. 9. IO.
B.
Table 111. Diffusion Current of Oxidized Glutathione and Cystine in llixtures of Both (Supporting electrolyte 0.1 .U CHzCOOH, 0.1 .M KCL 1.7 X IO-' .If
All current sallies corrected for residual and for change in volume) Diffusion Currents of GSSG and RSSR .It - 0.5 Volt A t -0.9 Volt GSSG RSSR (id)C, iTb. (IdJCy'. Concn., .\f Cuncn., .II Ira. (id)G/Ca Ira. Ira. (id)Cy'Ca None 5 x 10-4 ... ... 2.97 5 94 2.97 5 x 10-4 None 2.18 4.37 2.38 1.55 6.20 0.906 4.53 2.46 2 x 10-4 2 5 x 10-4
thymol.
4 x 10-4 6 X 10-4
2 5 5
1
?
x 2 x 3 x 5 x 7 x 1x 3 x 5 x
10-4 10-4
10-4 10-4
!
10-4
pJ
lo-'
1
10-4 10-4
1 1
x x x x
x
x x x x x
10-4 10-4 10-4
10-4 10-4 10-4 10-4
10-3 10-3
1.84 2.79 0.468 0,955 1.398 2.36 3.32 0,485 1 37 2.34
4.59 4.66 4.58 4.78 4.66 4.73 4.74 4.85 4.57 4 68
3.42 4.38 3.44 3.92 4.37 5.37 6.29 6.36 7.11 8.09
1 58 1 58 2.98 2 96 2.97 3.00 2.97 5 88 .5 74 5.75
6.32 6.32 5 96 5 93 5.94 6 00 8 95 5.88 p 74 5.75
10-3 a n d (,id)cu/care values of diffusion current of GSSG and RSSR respectively, calculated f o r concentration of 10-3 .If. i~ is tbtal diffusion current a t - 0.9 volt.
a (id)G/c b
ANODlC WAVES OF REDUCED GLUTATHlOYE AND CY STElYE IN MIXTURES OF BOTH
I n connection with the polarographic analysis of mixtures of oxidized glutathione and cystine i t was of interest to study polarographically the reduced forms of the peptide (GSH) and the amino acid (RSH), as these compounds also differ in their polarographic behavior within a certain p H range. While reduced glutathione gives a normal anodic diffusion current throughout the entire p H range ( 1 2 ) cysteine behaves normally only at p H lower than 2 and higher than 8 ( 4 ) . I n the p H range between 2 and 8 cysteine gives a small anodic limiting current which is constant and independent of the concentration a t cysteine conrentrations larger than 2.5 X 10-4X. The behavior of the two compounds was studied in mixtures
of 130th in an acetate buffer of p H 3.67. Polarograms of mivtures of constant reduced glutathione ( 10-3M) and varying cysteine (1.25 X 10-4 to 1.75 X l O - 3 M ) concentrations are shown in Figure 10. The behavior of cysteine is entirely changed in the presence of reduced glutathione, the diffusion current being well defined (Figure 10, -4) and proportional to the cysteine concentration (Figure 10, B ) . As the cysteine concentration becomes markedly larger than that of glutathione, the cysteine wave is ill defined again and finally decreases in height. Further study is required to account for the remarkable effert of reduced glutathione on the anodic wave of cysteine.
V O L U M E 25, N O . 7, J U L Y 1 9 5 3
1057
ACKNOW LEDGXIEST
This invest,igation was supported by a research grant from the Sationnl Cancer Institute, U. S. Public Health Service. LITERATURE CITED
(1) Cecil, R., Biochem. J . , 47,572 (1950). ( 2 ) Flagg, J. F., “Organic Reagents,” p. 275, Sew York, Interscience
Publishers, 1948. (31 Kolthoff, I. LI,,IXD.EXG.CHEX.,AKAL.ED.,14, 195 (1942). (4) Kolthoff, I. LI., and Barnurn, C..J . A m . Chem. Soc., 6 2 , 306
(1940).
fhid., 63, 520 (1941). Kolthoff, I. 31.,and Stricks, W., . h - . i L . CHEW,2 3 , 763 (1951). Kolthoff, I. X , , and Stricks, R., J . Am. Chein. Soc., 72, 1952 (1950). Lee, T. S., Ph. D. thesis, University of Alinnesota, 1949. Lee, T. S., and Kolthoff, I. JI., Ann. S. 1.. A c n d . S c i . , 5 3 , 1093 (1951). Lingane, J. J., and Kolthoff, I. AI., J . A m . Cheni. Soc., 61, 825 (1939). Schoherl, h.,and Hornung, T., Liebigs Ann., 534, 210 (1934). Stricks. if*., and Kolthoff, I. 31., J . Am. Chem. Soc., 74, 4040 (1952). R E C E I V Efor D review January 26, 1953.
.Xcrei)ted .\larch 27. 19.53
Properties of High Boiling Petroleum Products High Aromatics Characterization by Chromatography L. T. ERY Oil Deuelopnien t Co., Linden,
Esso Laboratories, Standard
.Y.J .
Little information is available on compositions of petroleum products boiling above 600” F. .A rapid method has been developed for characterizing the aromatic and other fractions with the object of predicting carcinogenic potencies. The refractibe index of the aromatic fraction was found useful, even when that of the whole sample was valueless. The aromatic fraction was obtained by a chromatographic separation. The procedure employs an elution method with silica gel adsorbent and three selecti\e sol>ents-n-heptane, benzene, and pyridine. Nonaromatic, aromatic, and o x ) fractions were obtained. Typical results are described for a wide variety of refinery products. Data obtained by this procedure have been found useful in various processing studies involFing high boiling petroleum products.
A
SULIBER of chromatographic procedures have been employed for the analysis of petroleum products (1-21). -4 more satisfactory general procedure for inspection purposes was drsired for high boiling petroleum products, especially those boiling above 600” F. It was required that.the method be as simple and short as possible, b u t that it should provide isolated fractions of the nonaromatic, aromatic. and “oxy” (fraction elut>ed with pyridine) fractions, as \vel1 as quantitatively estimate the amount present. Because of high melting points and high viscosities of the samples and their fractions, displacement techniques were unsatisfactor!.. even with hot columns or with various added solvents. hfter more elaborate procedures involving combinations of elution and displacement techniques had been tried. the folloiving method was developed as most satisfactory for a general procedure. It has been used on a large number of high boiling petrolruni products for purposes of characterization. The fractions f i ~ ~ this m method have been used for spectral investigation as well as for the refractive indices that are presented here. Little emhas heretofore been given to the osy fraction. For the latter, pyridine has been found to be the most, satisfactory eluent twc*auseof its high solvent power, absence of oxygen, and strong odor for detection. This method has been named the “high aromatics characterization.” because it was of interest to characterize the high boiling ni,oniatirs. The information obtained from this method is: 1. The per cent of nonaromatics, which includes paraffin$, olefins, naphthenes, and some polyalkylated aromatics which ran be removed from silica gel with n-heptane. 2.. The per cent of aroniaticp, which includes the compounds that are not removed with n-heptane but nre easily eluted with benzene.
3 . T h per cent of oscy material, which ini*lutlrq all the compounds not eluted with n-heptane or benzene but whirh are removed with pyridine. I n petroleum fractions, these are mainly compounds containing oxygen, as well as some sulfur and nitrogen. 4. The per rent loss. This represents volatility, or lower hydrocarbons, although it may represent nonvolatile material left on the column after washing with pyridine. There is very little organic matter that is not removed by elution with pyridine, but the nonvolatile loss may include insoluble fibers, inorganic material, et?. 5 . The refractive indes a t 50” C. of the nonaromatic fraction. This temperature was cho9en because of the waxy character of many of these fractions a t room temperature. 6. The refractive indes a t 20” C. of the aromatic fraction. This may be taken directly, or in a benzene or toluene solution. -is many of the refractive indices encountered were beyond the range of the instrument, a st’andard procedure was employed for this measurement in a benzene solution. A refractometer designed to use reflected light rather than transmitted light should be employed for correct refractive indices of the highly colored fractions ( 1 7 ) .
.is there is usually a fair-sized fraction after evaporation, it is possitilr to carry out other analyses for special characterization purposes-e.g., the spectral investigations mentioned above. The isolation of any one or two of the fractions may be omitted where only partial information is required. PROCEDURE
Chemicals. n-Heptane, pure grade from Phillips Petroleum co. Benzenr, thiophene-free grade. Pyridine, 2’ grade. Silica gel, 28 to 200-mesh commercial desiccant from Davison Chemical Corp., heated overnight and stored in tightly stoppered, narrow-mouthed bottles. Hyflo, diatomaceous earth filter aid. Adsorption Tube. Four-foot, glass column. 35 nim. in outside
