alcohol in several additional determinations and gave nearly identical values. The Rosen ( 7 ) ninhydrin reagent is relatively stable. When it is used in the fraction collector method, it greatly reduces the need for checking reagent stability with an internal standard. Taurine used as an internal standard serves principally as a position marker in this method. B e t a - 2 - t h i e n y h ~ alanine (8),for instance, would not assist in determining buffer change since it (,lutes mitiwny in the second buffer and has less utility in the fract,ion collector method. 130th D-L-ortho phosphoscrine and taurine have some of the desired characteristics; however, the former elutes close to cysteic acid and is morc likrlly to be found in plants than taurine. Taurine elutes in an area where overlapping is not likely to occur (9). The compound is isolated from the muscle of certain shell fish and froin iiiaminalinn wastes. I t appcars to be relatively uncommon in plants. Red algae ( 2 ) and young peas ( I ) reportedly contain taurine. I n the latter case it was claimed t,o be an inter-
mediary in conversion of inorganic sulfur supplied as Xa2S3404. I n our laboratory column chromatography of free amino acid extracts of primary leaves of 10 bo 21-day old plants of Vigna sinensis var. Black and of %day old plants of Piswti sativum var. Alderman revealed no taurine (unpublished). Taurine may thus be eit,herinconspicuous or absent in legumes unless supplied with abundant inorganic sulfur. Contamination of plant extracts with tsurine present in animal origin fertilizers is unlikely with current plant growing practices. Synthesis of taurine is relatively easy and purity of a long term supply can be assured. I t has the desired properties of ninhydrin reaction and mobility stated for the natural product. As a reference point it will not replace aspartic acid or other internal standards for all column chromatography. I t can be helpful, however, to plant pathologists and plant physiologists interested in analysis of certain abnormal plants where a fraction collector system is used.
LITERATURE CITED
(1) Bisnas, B. B., Sen, S. P., Sei. Cult.
(Calcutla) 22, 697 (1957). (2) 'Lindberg, B., Acta Chem. Scand. 9, 1323 (1955). (3) Marvel, C. S., Bailey, C. F., "Organic Synthesis," Tol. 11, p. 558 Wiley, New York, 1943. (4) Marvel, C. S., Sparberg, M. S.,Ibid., p. 563. (5) Moore, S., S ackman, D. H., Stein, W.H., ASAL.$HEM. 30,1185 (1958). (6) Porter, C. A., Margolis, D., Sharp, P., Contrib. Boyce Thompson Insl. 18, 466 (1957). (7) Rosen, H., Arrh. Riochern. Biophys. 6 7 , 10 (1957). (8) Siegel, F. L., Roach, Mary K., ANAL. CHEY.33. 1628 i1961). (9) Zacharius, R. if.,Talley, E. A,, Ibid., 34, 1551 (1962). J. G . KAR.4s H. M. SELL C. L. HAMNER D. J. DEZEELT Departments of Botany and Plant Pathology, Horticulture, and Biochemistry Michigan State University East Lansing, Mich. RESEARCH supported in part by Xational Institutes of Health, Grants E-4581 and AI-04581-02. Published with the approval of the Director, Michigan Agricultural Experiment Station as Journal Article No. 3267.
Qualitative Analysis by Gas Chromatography Choice of a Retention Data System SIR: In a paper by Merritt and Walsh (18, 19), a system of retention data was suggested which they called Isothermal Retention Volume Ratio (IRVR) and Programmed Temperature Retention Volume Differences (PTRVD). In discussing their results and comparing them to existing systems they presented a very short and drastic rebuttal of the retention index (RI) system described by Kovats and Wehrli (14, 24) and completely ignored the Rx,g system described by Evans and Smith ( 7 ) . However, these two systems were the subject of a 1or.g discussion at' the Gas Chromatography Symposium held in 1962 in Hamburg (237, and the first one (RI) is currently used by a great number of workers (1, 6. 8, 10, 12, 13, 15, 16, 23> 26, 26). A lot of papers which appeared recently used ret'ention indices for identification purposes. Kaiser (13) used them to present a lot of retention data in his book. This shows t,hat RJ may be computed from relative retention voluirres if data on n-alkanes are given. Su-oboda (23) discussed the use of RI. Zulaica and Guiochon (25, 26) uscd them to identify diesters used as plasticizers in poly vinyl chloride and showcd that t,he indices of homologous comlioucds increase in a constant amount. of about 100, when one carbon is added to a normal chain. Kovats uses them to identify the numerous components of tangerine essential oil. Baron 1672
ANALYTICAL CHEMISTRY
and Maume ( I ) showed that the use of R I increment or A I-Le., the difference between the R I of a compound on polar and apolar liquid phases-may be used to distinguish radial and equatorial groups in cyclohexane derivatives. Loewenguth (16) used R I to identify C6-Cs olefins without needing the pure compounds: tentative identifications were ascertained by infrared spectromy 011 trapped compounds. I t is ieved that the choice by these authors of the R I system over any other existing system before the IRVR, was made because it is really a good system. I t may first be pointed out that the R I system is not limited to the use of -4piezon L and Emulphor 0. As first suggested by Kovats ( 1 4 ) this system was used with advantage with more than 20 different phases and a lot of retention data given in the literature as retention volumes (absolute or relative) may easily be recomputed as retention indices. I t would be erroneous to think that the R I system is so limited: in this case it would be far less useful. For identification purposes R I increments may be used as long as the R I themselves. These increments are the difference between the R I of one compound on two different, phases, whatever these phases. Of course the two phases used by Kovats and Wehrli are to be preferred because of the amount of data these authors collected and be-
cause the rules they gave to compute the A I a priori may be used easily (24). However these rules are still valid with a lot of other pairs of stationary phases and presumably with almost all possible pairs : the individual increments pertaining to the different adhering zones have only to be computed from previous measurements. The measurement of RI (as that of R,,g factor) may be made in several different ways depending on the complexity of the mixture. In easy cases, as the measurement of R I of pure compounds, it is possible to inject a mixture of the sample and the convenient nalkanes. Then the computation of the R I may be made directly from the logarithm of retention distances of the peaks, measured from the air peak. In more difficult cases it is possible either to inject in successive runs the sample and a mixture of the convenient nalkanes if the apparatus used is sufficiently stable or to allow for variations of retention distances by ,,introducing one n-alkane (or more) in the sample, the choice of the alkane(s) being made to have no interferences between it (them) and any compound of the sample. If no variation in the retention distance of this (or these) n-alkane is meawred, the computation of all the RI may be made from the logarithm of retention distances. In the other case retention volumes must be computed
or retention distances must be duly corrected. Even in the most difficult cases the introduction of n-alkanes in the sample may be made by using the method of Pitkethly ( 2 0 ) . By using a plot of logarithm of retention distances or retention volumes us. carbon number for the n-alkanes, the computation of R I may be made as rapidly as that of retention volumes. R I may be measured with great reproducibility on a given column, the absolute error being of some units. For dat,a obtained in different laboratories on the same phase, differences may be of 10 units or less if the analyses are made on columns using the same solid support and not too different liquid/solid rat'ios. The R I varies slowly and linearly with temperature which allows easy corrections. This slow variation and the fact that in programmed temperature gas chromatography the elution temperature of n-alkanes increases linearly with their carbon number, in a limited but sufficiently wide number range (9, 1 1 ) , allow one to define and use a programmed temperature retention index (6, 10) which may be shown to be equal to the isothermal R I a t the temperature 0.92 T R where T R is the absolute elution temperature (10).
The determination of the retention index may be made as easily from dat'a obtained with either capillary or packed columns provided they are corrected from gaseous volume of the column. The RI can give much useful information : (a) -4s was shown by Kovats (14) a correlation may be made between RI on an apolar liquid phase and boiling points. The correlation should probably be better between R I and vapor pressure at t'he column temperature. It is even possible to compute R I of hydrocarbons from their vapor pressure (26). The data obtained are in excellent agreement with those calculated from the relative retention volumes given by Desty ( 2 ) . This cannot be extended to other compounds because the variation of activity coefficient,s cannot be completely neglected (3). This is, however, a very convenient way to get an approximate boiling point for any compound. (b) One of the greatest difficulties in quantitative analysis is to match the peaks of two chromatograms obtained from two different columns. When matching the two series of R I is tried, a number of possible pairings may be eliminated because apolar R I are always less than polar ones, and because R I increments (difference between R I on two different liquid phases) cannot have any value. The numerous possibilities which remain, may be rapidly checked by using the rules that Iiovats (24) gave to compute increments for a given structure.
(c) Although Kovats (14, 24) emphasized the use of two st'ationary phases, .\piezon-L and Emulphor 0, R I are of course not limited to them but may be ext'ended to every phase and every compound, especially polyfunctional derivatives and compounds with isoalkyl chains. Structural contributions to the R I increments may also be used ( 1 ) . (d) I n homologous series R I may be computed by rules of additivity (14, 25). The logarithm of retention volume is proportional to the variation of free energy which occurs when a mole of solute vaporizes from the fixed to the mobile pha.;e (21). This A F is equal to the sum of different terms corresponding to each polar group and to the chain of the molecule, when the molecule is big enough so that these groups do not interfere too much ( 1 7 ) . So, with the exception of the first few terms, the R I as does the logarithm of absolute retent,ion volume, inrreases linearly with the carbon number. The increment by carbon number of the retention index is about 100 but the exact value depends on the polarity of t,he liquid phase. (e) The R I increment's b e h e e n two phases of very different polarity may vary slightly with the carbon number, and do not remain constant, as do RI increments between phases which are very alike: the difference in solubilit'y properties in this case comes mainly from the part of act'ivity coefficients which allows for the difference in molecular size of solvent ( 3 ) ,and have a very slight influence on the increment of AF (and R I ) with one carbon atom. However, if R I increments between polar and apolar columns vary widely with the carbon number for the first few terms, they often reach an approximative limit, which can be taken as a characteristic of the functional group studied and used in identificat,ion purposes. Some difficulties are encountered from hranched compounds, especially when there is some hindrance of the functional group by the lateral chain. At this point some comparison may be drawn between the RI system and the ot,her ones. Clearly this comparison must be made independently of the phases used by t'heir authors, a t least if t'he systems may be used with other phases. The R,,Qand the RI systems may be easily extended to any liquid phase. The IRVR can be used only with couples of phases which have more or less the same polarity; this could bring some limit,ation to its usefulness, but in fact a lot of such favorable couples might be found if necessary. Much more difficult is the application of this IRVR to isoalkyls and polyfunctional derivatives because homolog series of complex structures are very difficult to get, and this would be necessary to measure the IRVR inasmuch as the first few terms of a series always have retention dat,a which are very different from the values obtained by extrapolation of the retention data of the heavier hoinologs. and because t h e series of isomers bccome very numerous for eomplrx structures and may lead to spreading of TRVR and
to confusion between different functions. The IRVR looks very much like the R I increments for heavy normal homolog (n-C5or more) of the series and does not seem more useful in favorable cases. I n difficult ones, the R I remain very useful even when the IRVR is impossible t'o compute, and the R I increment may always be correlated with structure by using the rules given by Kovats, even if correlation with function is impossible. Last, the R I system is a very good one to present retention data and to use such data ga!ll;:c?d from different laboratories. Clearly in this field the IRVR system is not better than the retention volumes. For the above reasons we feel that it is erroneous to state that "RI increments. . . fail to provide either the required precision or the extent of numerical separation needed to distinguish important functional group types." The absolute error made in the determination of the R I increments is the sum of the errors made in the determination of each R I which in turn is bound to the efficiency of the column (22). The precision may be greatly enhanced by using better apparatus and a more efficient column. This has nothing to do with the retention data system used. The numerical separation also is not determined by t,his system: if the R I increments of two series of functional derivatives are not different enough a better result would certainly be obtained by usiiig another couple of stationary phases. The R I appears to be the most reproducible syst,em to report retention data (4). In conclusion, the R I system, which is the most useful for handling numerous retention data, is really worthwhile: those who publish data using t,he conventional system of relative retention volumes are urged to include the convenient n-alkanes in their tables to allow computation of R I or R x , Qby the users of these systems. LITERATURE CITED
( 1 ) Baron. C.. Maume. B.., 1j7111 ~ ~ - - S- n -- -c ('him. Fkanch 1962, 1113.( 2 ) Ilesty, 11. H., Goldup, .4., Swanton, W. T., "Gas Chromatography," p , 105, X. Brenner. J. E. Callen. >' 1). I. Weiss. eds. Academic Press. S e w York. 1062 \
,
)ool, H. van den, Kratz, P. D.,, J . Chromatog. 1 1 , 4 6 3 (1063). ( 6 ) Ilucros, P., l'hbse, Paris, 1962. ( 7 ) Evans, >I. R., Smith, J. F., J . Phromatog. 5 , 300 (1061): 6;. 293 (1961); 9 , 147 (1962). ( 8 ) Ferrand, R., Joumbes Intwn. de SPpnration Imm/dinte et (le ('hromatoyraphie, p. 132, G.A.lI.S., Paris. 1962, ( 9 ) Giddings, J . C., J . Chromcitoy. 4 , 11 (1960). (10) Guiochon, G., A 3 . i ~ CEiEv . 3 6 , (i6I (1964). VOL. 36, NO. 8, JULY 1964
1673
(11) Habgood, H. W., Harris, W. E., Ibid., 32,450 (1960); 34,882 (1962). (12) Huguet, M., JournBes Intern. de SBparation ImmBdiate et de Chromatographie, p. 69, G.A.M.S.,Paris, 1962. (13) Kaiser, R., “Chromatographie in
der Gas phase,” Bibliographisches Inatitut, A.G. Mannheim, 1962, Part 111, Tabellen. KID. 78-115: “Gas Phase Chromatography 111,” Butterwortha, London, 1963. (14) Kovats, E., Helv. Chim. Acta 41, 1915 (1958). (15) Landault, C., Guiochon, Chromatog. 9 , 133 (1962).
G., J.
(16) Loewenguth, J. C., Vth Petroleum
World Congress, Preprint, Frankfurt,
1963. (17) Martin, A. J. P., Biochem. SOC. Symposia 8 , 4 (1949). (18) Merritt, C., Walsh, J. T., ANAL. CHEM.34,903 (1962). (19) Ibid., 908. (20) Pitkettly, R. C., Ibid., 30, 1209 11958). ( 2 i ) Poher, P. E., Deal, C. H., Stross, F. H., J . Am. Chem. SOC. 78, 2999 (1956). (22) Strickler, H., Kovats, E., J . Chromatog. 8 , 289 (1962).
(23) Swoboda, P. A. T., “Gas Chromatography 1962,” p. 273, Van Sway, M . ed., Butterworths, London, 1963. (24) Wehrli, A,, Kovats, E., Helv. Chim. Acta 42, 2726 (1959). (25) Zulaica, J., Guiochon, G., Bull. SOC. Chim. France 1963, 1246. (26,) Zulaica, J., Guiochon, G., Unpublished data, Paris, France, 1963.
GEORGES GUIOCHON Laboratoire du Professeur L. Jacque Ecole Polytechnique Paris ( 5 e ) , France
Determination of Small Amounts of Manganese by Oxidation of 8-Aminoquinoline, Extraction, and Spectrophotometry SIR: Oxidations of the organic reagents o-tolidine (S), benzidine (9), tetramethyldiaminodiph e n y 1m e t h a n e (8),and tetramethyldiaminotriphenylmethane (4)with permanganate have been used as the basis for the determination of manganese. Although these are sensitive methods, the colored products are unstable. 8-Aminoquinoline is oxidized to a highly colored oxidation product with permanganate and the color formed is stable. 8-Aminoquinoline has been used for the gravimetric determination of copper ( I ) , and for the spectrophotometric determination of palladium (6) and iron (6). EXPERIMENTAL
Reagents and Apparatus. &AMINO-
QUINOLINE SOLUTION. 8-Aminoquino-
line, 50 mg., was dissolved in 1 ml. of concentrated sulfuric acid and diluted to 100 ml. with distilled water.
STANDARDPERMANGANATE SOLUA stock solution of Mn(VI1)
TIONS.
was prepared by dissolving 0.10 mole of reagent grade K M n 0 4 in distilled water and diluting to 1 liter. The stock solution was standardized against primary qtandard grade sodium oxalate (7‘). All other Mn(VI1) snlutions were prepared by appropriate dilution of the standardized stock solution. STANDARD M ~ ( 1 1 SOLUTIONS. ) Stock solutions of hln(I1) were prepared by
dissolving reagent grade M n S 0 4 in dilute sulfuric acid. The solutions were standardized spectrophotometrically after oxidation with bismuthate ( 2 ) . SOLVENTEXTRACTION SYSTEM.The benzyl alcohol-chloraform solution used for the extraction was prepared by mixing equal volumes of benzyl alcohol and chloroform. All solutions used in the interference studies were prepared by dissolving reagent grade salts in distilled water. Dilute HnSOa was added to prevent hydrolysis. The apparatus is the same as that described in a previous publication (6). Procedure. To a 50 ml. sample containing 1 to 30 pg. of h h ( I I ) , add 3 ml. of concentrated sulfuric acid and 0.2 gram of NaBiOs. Stir for 1 minute and filter through a 60-ml. sintered glass funnel into a 125-ml. filter flask. Wash the excess solid XaBiOs on the filter with two 10-ml, portions of 1M HzS04. Transfer the filtrate quantitatively to a 150-ml. beaker, add 3 ml. of the 8-aminoquinoline reagent solution, and then add solid NaOH until the p H is 11.0 or greater. Stir the solution for several minutes and adjust the p H to the interval 1.45 to 1.95 with 1 to 1 HzS04. Transfer the solution to a 125-ml. short stem separatory funnel, add 5.00 ml. of the benzyl alcoholchloroform solution and extract, shaking occasionally over a period of several minutes. Fill a 1-cm. absorption cell with the organic phase and measure the absorbance a t 550 mp against a blank. The hlank is prepared by treating 50 ml. of distilled water in the same manner as the manganese sample.
DISCUSSION A N D RESULTS
When Mn(VI1) reacts with 8-aminoquinoline, a red-colored oxidation product is formed which is soluble both in water and in a mixture of benzyl alcohol and chloroform. The absorption curve for the benzyl alcoholchloroform extract of the oxidation product is the same as that obtained when the reagent is oxidized by ferric iron (6), with maxima at 517 and 550 mp. The oxidation of 8-aminoquinoline with Mn(VI1) is sensitive to pH. The effect of the initial p H on the reaction was studied and the results are given in Figure 1. The solutions were prepared by adding a 50-ml. sample containing 14.7 pg. of Mn(VI1) to 3 ml. of the reagent solution. The pH of each of these solutions was adjusted to the desired value before they were mixed. After being mixed, the solutions were stirred for several minutes. The p H was then adjusted to 1.60, the solution was extracted with 5 ml. of the benzyl alcohol-chloroform solvent, and the absorbance was measured a t 550 mp. Oxidation is incomplete a t low p H values. If the reaction mixture is first made basic a t a p H of 10.8 or greater and then acidified to a p H of 1.60, the reaction proceeds quantitatively. Two 50-ml. samples that contained 44.1 pg. of Mn(VI1) were made basic to a p H of 11.O. They were added to 3 ml. of the reagent solution which was previously
Figure 2. Absorb-
;
0 4
Figure 1.
b
PH
A
Mn(VII) vs. pH
,b
_J I2
Effect of pH on oxidation
14.7 fig. of Mn(VII) used for oxidation
1674
ANALYTICAL CHEMISTRY
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