174
ANALYTICAL CHEMISTRY
these amino acids were present in mixtures, often they could not be located. As an example, tyrosine alone has an R/ value of 0.29 in m-cresol saturated with 9.0 p H buffer (paper buffered a t pH 9.0) and histidine an R / of 0.35. When these two amino acids were chromatogrammed together, histidine turned up a t Rt 0.14 and tyrosine a t R/ 0.26. This particular combination gave a clue, for when the tyrosine solution was prepared, it was acidified to get the tyrosine completely into solution. $pparently the acidity of the tyrosine solution was sufficient to override the buffered solutions and the buffered paper, so that when these amino acids were spotted together and chromatogrammed, a serious shift in R/ values resulted, particularly histidine. Subsequent experiments showed that when the solution or mixture of amino acids was first adjusted to a pH between 5.5 and 7.5 (1, S), the R, values of the amino acids in mixtures were reproducible. Unfortunately, this imposes another problem which has never been completely solved; in this pH range cystine is nearly quantitatively precipitated and tyrosine is rather insoluble. The only solution a t present seems to be to work with more dilute solutions and to build up a concentration on the paper by repeated spotting before chroniatograniming. SUMMARY
Each of twenty amino acids may be separated from all others by employing several one-dimensional buffered chromatograms. The paper chromatograms are buffered by dipping the paper into buffer of the desired pH and molarity and air-drying, and by equilibrating the solvent with the same buffer rather than with water. Different, but more reproducible, Rfvalues are obtained if all chambers are lined with a filter paper which dips into the solvent-saturated buffer in the bottom of the chamber. All solutions must be adjusted to a pH between 5.5 and 7.5 before spotting in order to prevent heading of some of the amino
acids in mixtures and to obtain reproducible R/ values for lysine, arginine, and histidine. The temperature in the room housing the chromatographic chambers must be thermostatically controlled to * 1O C.to prevent condensation on the paper and the resulting streaking and elongation of spots. Particularly with solvents such as collidine, the more the temperature varies, the more the R/ values of t,he amino acids vary. ACKNOWLEDGMEYT
The author is grateful to Gotfred Haugaard and R. A. Sullivan of the Sational Dairy Research Laboratory, Inc., for many helpful suggestions and criticisms, and to James A. Mills of this same laboratory, who did many of the routine experiments. LITER4TURE CITED
(1) Aronoff, S., Science, 110, 590 (1949). (2) Britton, H. T. S., “Hydrogen Ions,” New York, D. Van Nos-
trand Co., 1942. 13) . . Bull, H. B.. Hahn. J. W., and Baptist, V. R., J. Ant. Chenz. Soc.. 71,550 (1949). (4) Clark, TV. bl., “Topics in Physical Chemistry,” Baltimore, hld., Williams and Wilkins Co., 1948. (5) Consden, R., and Gordon, A. H., N a t u r e , 162, 180 (1948). (6) Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J . , 38, 224 (1944). (7) Haugaard, G., and Kroner, T. B., * J . Am. Chem. Soc., 70, 2135 (1948). ( 8 ) Karnovsky, M. L., and Johnson, M. J., ANAL.CHEW,21, 1125 (1949). (9) Miettinen, J. K., and Virtanen, A. I., Acta Chem. Scand., 3, 469 (1949). (10) Winsten, W.A , , Science, 107, 605 (1948). RECEIVEDApril 25, 1950. Presented before the Division of Biological Chemistry a t the 117th Meeting of the AMERICAN CHEMICALSOCIETY, Philadelphia, Pa.
Determination of Trivalent and Tetravalent Manganese W. S. FYFE C’niversity of Otago, D u n e d i n , N e w Zealand With acetylacetone, the manganic ion forms a stable complex which has no oxidizing action on acidified potassium iodide. In the presence of acetylacetone, manganese tetrachloride loses chlorine and forms the stabilized trihalide. The chlorine evolved in this reduction chlorinates the acetylacetone. This chlorination can be reversed in the presence of acidified potassium iodide in which case the iodine is liberated. Thus, by estimating the iodine liberated i t is possible to determine the tetrahalide in the presence of the trihalide.
A
C E T n A 4 C E T O x Eis selective in stabilizing the ferric and manganic ions (3). When manganese dioxide was dissolved in hydrochloric acid in the presence of acetylacetone, no chlorine was evolved; when treated mith potassium iodide the solution liberated iodine as follows:
+ 1/2Cl2 = KCI + ‘/Jz
MnC14 = iLInCl8 ‘/&I2
+ KI
The reactions involved in the solution of manganese dioxide in the presence of acetylacetone are: Mn02
+ 4HC1 = 1InCl4 + 2H20
+
21vInC14 z(CH,COCH&OCH,) 2MnC1,(CH&OCH~COCH3),-1
(3)
=
+ CH,COCHClCOCH, + HCl
(1)
(4) and with acidified potassium iodide:
(2)
The rate a t which this liberation of iodine occurs is identical with the rate a t which chlorinated acetylacetone reacts with potassium iodide, and is affected by both iodide and hydrogcn ion concentrations (6).
CH3COCHCICOCH3
+ 2HI
=
CHaCOCHzCOCH,
+ HCl + I1
(5)
Equation 5 involves a dechlorination; the chlorine evolved liberates iodine, which combines with potassium iodide to form polyiodides. These are not efficient iodinating agents. As the
V O L U M E 23, N O . 1, J A N U A R Y 1 9 5 1 trivalent complex formed in Equation 4 has no oxidizing action, the amount of iodine liberated is one half of that normally obtained from the reaction of manganese tetrachloride with potassium iodide. The acetj-lacetone is rapidly chlorinated by manganese tetrachloride, for i t normally exists to a large extent in the enolic form.
175 need be plotted. Acid and iodide concentrationp arc not critical, but kinetic experiments have demonstrated that, those chosen are suitable. Considerable latitude is also available n-ith regard to temperature-room temperature is suitable for most determinations. It is advisable to carry out a blank determination on the reagents. Analysis of Manganous Manganic Oxide. In a typical analysis 0.504 gram of manganous manganic oxide (hIn,Oa) was dissolved in hydrochloric acid with acetylacetone and made up to 100 ml. with potassium iodide. After 3 hours the iodine liberated by a 10ml. portion of the solution required 17.8 ml. of 0.01 N thiosulfatp, which corresponds to 0.0978 gram of tetravalent manganese. If hln30.ireacts like manganese dioxide and manganous oxide, the weight of tetravalent manganese would be 0.0975 gram. A comparison of the total oxidizing action of MnaOaon potassium iodide with ?vlnaOain the presence of acetylacetone indicated the absence of trivalent ions. Determination of Tetravalent Manganese in Presence of Iron. Ferric iron forms a deep red acetylacetone complex which is considerably less stable than the manganic complex. The acetylacetone complex oxidizes potassium iodide if the acidity is high. For compounds containing ferric iron, the solution obtained in hydrochloric acid in the presence of acetylacetone is reduced with stannous chloride until the red color of the ferricacetylacetone complex just disappears. Excess stannous chloride must not he added, as it will react with liberated iodine.
0
2
4 TIME
6 8 HOURS
IO Table T.
Results of Manganese Determinations
Figure 1. Analysis of Manganese Dioxide A.
B.
Reaction of manganese tetrachloride and acetylacetone on potassium iodide Reaction of chloroacetylacetone on potassium iodide
Using acetylacetone it is thus possible to estimate tetravalent manganese in the presence of trivalent manganese. The total amount of tetravalent manganese can be estimated from the oxidizing properties of the material on potassium iodide in the absence of acetylacetone. From these tvio results the amount of trivalent manganese can be estimated by difference. Several manganese oxides have been studied to determine the valence state of the manganese in these compounds; the results have been puhlished (4). EXPERIMENTAL
Dechlorination of Acetylacetone. Pure acetylacetone, 2 grams, was chlorinated with 50 ml. of 0.03 N chlorine solution in the preqence of hydrochloric acid. The mixture was shaken in a standard flask for about 10 minutes until no free chlorine remained. The solution was then made up to 100 ml. a t 25’ C., so that it was normal with respect to both potassium iodide and hydrochloric acid. The liberation of iodine was followed by titrating 10-ml. portions with 0.01 iV thiosulfate a t suitable intervals. Curve B, Figure 1, was obtained: the chloroacetylacetone was quantitatively dehalogenated.
Analysis 1 2
Substance Pyrolusite from thermal decomp. of RZn(Pu’03)2 Crude commercial pptd. ” 0.b y hydrolysis of MnOz -hl_.._. nlnCla (b-hInOd Pyrolusite MnO? hInz(SO4)a
+
30% hIn20a, 70% Fez03
11
(solid solution) 3In3Oa
0.1902
Determined, Gram 0 186
0,200
0 184
0.1:s
0 143
0.250 0.28 MnOz 0 , 3 9 8 sulfate 0,200 Mn09 0 . 3 0 0 FeCls 0.0098 0.433
0 245 0 272
0 0089 0 427
0.406
0.401
Weight of Sample, Gram
KO reaction, trivalent ions KO reaction
0 182
In actual practice it is advisable to reduce the ferric iron until the solution is faintly pink. At this stage only a trace of ferric iron will be present and it will have a negligible result on the analysis. After the iron has been reduced, potassium iodide is added and the analysis is carried out as in the previous examples. Table I gives some typical analyses. Various preparations of manganese dioxide differ widely in oxygen content; in MnO,, n may vary from 1.7 to 2.0 (1). For this reason it is difficult to record errors. Duplicate analyses on pure manganese compounds generally agree within a t least 0.2%. Analyses on compounds containing iron have larger errors.
ANALYSIS OF hIASGANESE DIOXIDE
I n a typical analysis, 0.192 gram of manganese dioxide was dissolved in 10 ml. of 10 N hydrochloric acid in the presence of 2 ml. of acetylacetone in a 100-ml. standard flask. After all the manganese dioxide had dissolved and the solution was a pale yellow color, the solution was made up with potassium iodide to 100 ml. 60 that i t was normal with respect to potassium iodide. The rpaction mixture was placed in a thermostat a t 25’ C . and 10-ml. portions were titrated a t suitable intervals, using 0.01 N thiosulfate and starch as an indicator. The results are plotted in curve A , Figure 1. The final titration value after 2 hours was 21.5 ml. Using acetylacetone, 1 gram-molecule of manganese dioxide yields 1 equivalent of iodine; hence 1 ml. of normal thiosulfate dorresponds to the formula weight of Mn02/1000. The amount of manganese dioxide determined was 0.190 gram, which was confirmed by standard methods. I n routine analysis the mixture can be made up and titrated after 2 hours and no curve
ACKNOW LEDGRl E S T
The author wishes to express his gratitude to F. G. Soper for his assistance and advice, and to Brian Mason of the University of Indiana for supplying some of the samples. LITERATURE ClTED
(1) Cole, W. F., Wadsley. A. D., and Walkley, Allan, Electrochemical SOC.,Preprint 92-2 (1947). (2) Dawson, H. hl., and Leslie, M. S., J . Chem. Soc., 1909, 1860. (3) Emeleus, H. J., and Anderson, J. S., “Modern Aspects of Inorganic Chemistry,” London, Routledge & Sons, 1938. (4) Fyfe, JT. S.,Xature, 164, 790 (1949). RECEIVEDAugust 29, 1949.
