Table VI. Mg
+
MgO 3.18 3.06 3.18 3.01
3.07 2.96 3.05 3.05
Precision Data
0.76 0.77 0.78 0.76 0.74
0.82 0.76 0.78
The method described has been satisfactory for the analysis of slags from experimental reductions in our laboratories. An extensive study into the
effect of diverse ions on the determination has not been undertaken, since the slags analyzed contained only the compounds listed. Precision Data. Precision studies of t h e described methods were made by analyzing one sample of slag eight times (Table VI). T h e standard deviations for t h e magnesium plus magnesium oxide and magnesium metal determinations were 0.076 a t a level of 3.O7ye and 0.027 a t a level of 0.77%, respectively. The standard deviation of the difference [1/(0.076)2+ (0.027)2] was 0.081. Correcting for molecular weights, the standard deviation for the determination of mag-
nesium oxide in magnesium fluoride slag was 0.13 at a level of 3.83%. LITERATURE CITED
(1) Allsopp, H. J., Analyst 81, 469 (1956). (2) Bricker, C. E., Parker, G. H., ANAL. CHEM.29,1470 (1957). (3) Oda, N., Sawabe, S., J. Chem. SOC. Japan, Ind. Chem. Sect. 59, 1445 (* 1- -956). --,
(4) Scribner, W. G., ANAL. CHEM.31, 273 (1959). (5) Tartakovsky, V. J., Trans. Inst. Econ. Mineral. (U.S.S.R.) 1934, KO.64. 16) Welcher. F. J.. “Ethvlenediaminetetraacetic Acid,’’ pp. “103-42, Van Yostrand, Princeton, N. J., 1957. ~
R~~~~~~~for review October 19, 1959. hccepted Lfay 2, 1960.
Spectrophotometric Determination of iron and Copper with Methyl-2-pyridyl Ketoxime and Their Simultaneous Determination in Mixtures D.
K. BANERJEA and K. K. TRIPATHI
Department o f Inorganic and Analytical Chemistry, Indian Association for the Cultivation o f Science, Calcutta 32, India
b Methyl-2-pyridyl ketoxime gives a red-violet color with iron(l1) and a yellowish green color with copper(1) in alkaline media. This affords a convenient method for the determination of these metals by measurement of the absorbances at 525 and 410 mp, respectively. Copper(1l) produces the same color because of its reduction to copper(1) by an excess of the reagent. The optimum pH range for development of the iron color i s from 10.5 up (to 6 N caustic alkali), and for copper from 10.0 to 11.7. Color development i s instantaneous in both cases, and at room temperature the colors are stable for over 24 hours. Beer’s law i s obeyed by both systems (0.05 to 10 p.p.m. for iron and 0.5 to 37 p.p.m. for copper) with an optimum range of 1 to 4 p.p.m. for iron and 6 to 12 p.p.m. for copper. In both cases the colored species are anionic with a composition (metal to reagent) 1 to 3 for the iron(l1) and 1 to 2 for the copper(1) complex; the former has a high degree of thermodynamic stability ( K , = 5.32 X 10l2). Most of the common anions are without effect on both the colors and many of the cationic interferences can be effectively masked with tartrate. The method is also applicable for the simultaneous determination of iron and copper in mixtures of varying proportions (1 to 13 to 6 to 1 ). 1 196
ANALYTICAL CHEMISTRY
S
U B S T A N C E ~ such
as 1,2-dipyridine, 1,lO-phenanthroline, and related compounds, il-hich belong to the class of substances known as a-diimines, and also the l,2-dioximes such as dimethylglyoxime, all of which contain the characteristic grouping
-C(:N-).C(:X-)give highly sensitive color reactions with iron and copper in solution. However, slight alterations in the structures of molecules containing any particular reactive grouping often produce marked changes in their behavior towards metal ions, particularly with respect to their selectivity and sensitivity. Since methyl-2-pyridyl ketoyime
Q C(:NOH)
C H ~
is intermediate in its structural character to 1,Zdipyridine and dimethylglyoxinie, its reactions with metal ions were investigated (Table I), The color reactions given by iron, copper, and rhenium are highly sensitive and are suitable for the spectrophotometric determinations of these metals. This communication records the results of investigations of the iron and copper color systems and procedures for their determination. APPARATUS AND REAGENTS
All absorbance measurements were made with a Unicam SP 600 spectro-
photometer using 0.5-, 1-, or 2-cm. glass cells. A Cambridge pH meter was used for pH measurements. Methyl-2-pyridyl ketoxime was prepared from methyl-2-pyridyl ketone (Light PE Co., England), hydroxylamine hydrochloride, and sodium carbonate in aqueous solution ( 1 ) . The crude material \vas recrystallized several times fiom hot water to obtain it in pure form (white solid, m.p. 120’ C,). A freshly prepared 0.5% aqueous solution was used as the reagent. Standard iron(I1) solution was prepared by dissolving a R eighed quantity of pure iron wire (G. R., E. llerck, Germany) in water containing sufficient sulfuric acid to give a final acid concentration of about 0.1I14, and making up the volume with water. Standard iron(I1I) solution was made by oxidizing an aliquot of the iron(I1) solution by boiling with nitric and sulfuric acids, and then diluting to requisite volume with water. Standard copper solution was prepared by dissolving a weighed quantity of electrolytic copper (G. R., E. llerck, Germany) in 1 to 1 nitric acid and diluting to a definite volume with water after expelling the nitrous fumes by boiling. Solutions of foreign ions were made from suitable reagent grade chemicals and standardized by usual methods. All other chemicals were of reagent quality. Ordinary distilled water redistilled with potassium hydroxide and potas-
I
I
051
TO 6 M K O H )
1 4
W A V E LENGTH, m/
Figure 1 .
O f t
1
Absorption spectra
Cell, 1 -cm. A. Fe(ll), 4.1 X 1 O-5M; NHpOH, 0.1 M ; AH, 3 X 1 O-3M; pH, 4.0 B. Same as in A at pH 10.7 C. Sample A after 4 8 hours D. Fe(lll), 4.1 X 1 O-jM; AH, 3 X 1 O-3M; pH, 10.8 E. Cu, 1.1 X 1 O-4M; AH, 3 X 1 O-3M; pH, 1 1 .O F. Cu, 5 X 10-5M; NHzOH, 0.2M; AH, 3 X 10-3M; pH, 10.8 G. AH, 6 X 1 O-3M; pH, 1 1 .O; (AH = methyl-2-pyridyl ketoxime)
siuin permanganate in a n all-glass apparatus was used for making the solutions. IRON-(METHYL-2-PYRIDYL KETOXIME) SYSTEM
Recommended Procedure. An acid solution (5 nil.) of t h e sample, containing 0.01 t o 0.2 mg. of iron, free from interfering ions, is treated ITith a n excess of hydroxylamine hydrochloride and warmed t o reduce a n y iron(II1) present t o iron(I1). T o this solution is then added about 1 giani of Rochelle salt (potassium sodium tartrate) as a masking agent, if necessary, followed by 10 ml. of reagent and the pH is adjusted to over 10.5 by adding either sodium or potassium hydroxide solution. This is then diluted with water to 25 ml. and the absorbance of the final solution is measured a t 525 mp against distilled water. The amount of iron is then evaluated either directly from a calibration curve piepared with known amounts of the metal. or else calculated using Beer's Ian and t h e kn0ir.n absorbance index. Results and Discussion. ABSORPSPECTRA. M e t hyl-2-pyridyl ketoxime (AH) reacts with iron(I1) to give a n orange-red color in acid medium (pH < 5.0) and a red-violet color in alkaline niedium (pH > 7.0). I n Figure 1 the charactcristic spectra of these colors are shown by A and B having absorbance maxima a t 475 and 525 m p , respectively. (Henceforth, these are referred to as acid and alkali colors. respectively.) Honever. the acid color is stable for only about 15 to 20 minutes and then gradually changes, resulting in a shift of the , , ,A from 475 to 525 nip (Figure 1,C). Iron(II1) nhen trmted with the reagent in acid or alkaline mcdia gives only a faint yellow color (Figure 1,D). This gradually changes to the iron(I1) color by reduction in acid medium and TIOX
, 2
4
Table I.
Figure 2.
Color in
Light green ppt. (pH 3-5)
cu Rln +2
... ...
c o '2
X I 0 +6
w u +e f6
Re +7
comparable conditions, has no absorbance a t any wave length over 400 mp in the visible region (Figure 1,G). EFFECT OF PH. Solutions containing iron(I1) and excess reagent were prepared a t different pH's and their absorbances were measured at 475 and 525 m p . Results (Figure 2) show that the acid color reaches its maximum in-
Reactions of Methyl-2-pyridyl Ketoxime with Metal Ions
c u +2
Pd + 2 y +5
L
I4
Cell, 1 cm. Fe(ll), 4.1 X IO-SM; Cu, 1.1 X 10-4M
Fe +2 Fe + 3
Xi + 2
12
Effect of pH
Acid medium Orange-red Faint orange
+
10
P"
extremely slowly in alkaline medium. Because of its stability and sensitivity only the iron(I1) color obtained in alkaline medium is suitable for the spectrophotometric determination of the metal. The reagent itself, under
Metal Ion
8
6
...
Alkaline medium Red-violet Faint orangeyellow Yellon-ish green Same Dirty brown ipH > 6)
Remarks Slowly reduced to Fe (11)color Dark brown ppt. on adding iodine
Light brownish yellow (pH
Color intensifies on exposure to air or on adding an oxidizing agent Buff colored ppt. on adding iodine
Light orangeyellow (pH
Dark red crystalline ppt. on adding iodine
> 6)
> 6)
Light yellow iPH > 6) ... ...
Same as blank Orange-yellow ... Pink when tin(I1) chloride is added to a solution containing excess reagent and HC1 KO reaction in acid or alkaline medium ... Light yellow (PH > 6 ) Pink when tin(I1) chloride is added to a solution containing excess reagent and HC1. Color develops slowly in cold and rapidly on warming. At higher acid concentration, green color is formed immediately. At intermediate acid concentrations, green color first formed gradually changes to pink. Under identical conditions Re(VI1) alone, in absence of reagent, is very faintly yellow
VOL. 32, N O . 9, AUGUST 1 9 6 0
1197
tensity in the p H range from 3.2 to 4.6, while the alkali color reaches its maximum intensity a t p H 10.5 and remains unaltered even in 6M potassium hydroxide solution. EFFECTOF REAGENT.An excess of the reagent over ten times the molar amount of iron present has no effect on the intensity of the alkali color. The same is also true for the acid color. RATE O F REACTION AXD STaBILITY OF COLOR. Under optimum conditions both the acid and alkali colors develop instantaneously. At room temperature the alkali color is stable for over 24 hours, while the acid color is stable only for 15 to 20 minutes. The intensity of the alkali color remains unaltered in the temperature range from 10’ to 40’ C. BEER’SLAW. The alkali color system strictly adheres to Beer’s lam in the range of 0.05 to 10 p.p.m. of iron with an optimum range of 1 to 4 p.p.m. of the metal, evaluated b y Ringbom’s graphical method ( 3 ) . SENSITIVITY. The color system is highly sensitive; spectrophotometric 41being 0.004 scnsitivity [Sandell ( pg. of Fe per sq. em., the visual identification limit is 0.05 pg. of Fe per nil. (1 to z x 107). NATUREOF COLORED SPECIES. Compositions of the colored species formed in acid and alkaline media were evaluated by Job’s method following Vosburgh and Cooper ( 6 ) . For both species a 1 to 3 ratio of metal to reagent (AH) was indicated (Figure 3). Since both the colored species are highly soluble in water and nonextractable into organic solvents, it was inferred that they are probably ionic in nature ( 2 ) . The anionic nature of the species formed in alkaline medium I\ as, however, proved conclusively by the fact that i t was taken up from solution by an anion exchange resin (Amberlite IRA-400, O H form). It seems very likely that in alkaline medium the complex formed is I (abbreviated as Fe=ia-\ nhile in acid medium we get I1 [abbreviated as
Table II. Formation Constant of Iron(ll)-(Methyl-2-pyridyl Ketoxime) Fe] [AHl, 1.83 X 10-iJl. Molar absorbance index (525 mp), 11370. pk,, 9.07. pH, 10.82 i 0.02. u, 0.5.21 (KC1). Temp., 28” C. [AH]/ Absorbance ([Fel (1 Cm., I2 . ann2. C h c n ~ 115, . 332
(1939). (4) Pandell, I:.
R., ',Colorimetric Determination of Traces of lletals," 3rd ed., p. 83, Interscience, Sew York, 1959, ( 5 ) Vosbiirgh, IT. C., Cooper, R. G., J . :1m. Cheni. Sor. 6 3 , 437 11941).
LITERATURE CITED
(1) Engler, C., Rosumoff, P., Rer. 24, 2528 (1891); BeilPtein, Bd. 21, p. 279.
RECEIIED for revien February 24, 1OtiO. Accepted l l a y 24, 1960.
Microchemical Detection of Characteristic Functional Groups on the Steroid Nucleus LEONARD R. AXELROD and JEAN E. PULLIAM Department of Physiology and Biochemistry, Southwest Foundation far Research and Education, Sun Antonio, Tex.
b Procedures for the qualitative determination of specific functional groups on the steroid nucleus are useful in the fields of steroid metabolism. Methods have been devised for the selective microchemical identification of specific functional groups found in steroid compounds. These groups include 1,2diketones, o-unsubstituted phenols, ohydroxyphenols, homoannular 1,3dienes, ditertiary double bonds between bridgeheads, a carboxyl group, 3a- and 36-hydroxyl groups, and 17a-and 176-hydroxyl groups. Most group identifications can b e made with 10 pg. or less of the compound per sq. cm. of filter paper test strip.
A
COMhIUXICATION described the detection of characteristic steroid side chains ( I ) . Other investigations have since presented a number of spot tests for steroid detection ( 2 , 8, IO). This paper describes methods of utilizing known chemical reaction for micro quantities of steroids applied to filter paper or developed on chromatograms. Each series of reactions leads to the specific and selective detection of a characteristic functional group on the steroid nucleus and uses less than 15 pg. of the compound. The tests can be used in the field of paper chromatography as an aid in the identification of biochemical intermediates and in steroid organic chemistry. PREVIOUS
EXPERIMENTAL
K h a t m a n KO. 1 filter paper is cut into strips and the steroid applied as described (1) mithin a 1-cm. circle. A stream of nitrogen is used intermittently to evaporate the solvent. The various chemical reactions are carried out by 1200
ANALYTICAL CHEMISTRY
PROCEDURE. h few milligrams of reagent A are dissolved in 10 ml. of reagent B, which gives a light violet color to the solution. The strip is dipped into the solution and held in the air until maximum color develops; Test I, 1,2-Diketones. REAGEXTS. this takes about 3 minutes. The reagent imparts a deep purple color with A . A solution of 1 gram of hydroxyla tan cast to the test spot and requires amine hydrochloride and 1 gram of less than 8 pg. of steroid per sq. cm. of sodium acetate in 2 ml. of water paper. prepared just before use. B. A 5y0 Test IV, Homoannular 1,a-Dienes. solution of nickel acetate in distilled R E r l G E N T S . Ai. A 40% solution O f water. Solution B is stable. PROCEDURE. The paper strip is trichloroacetic acid in absolute methpassed through the hydroxylamine reaanol kept in a tightly stoppered brown gent, A, placed on a glass plate, and bottle. PROCEDURE. The test strip is dipped heated to dryness on a hot plate (surface into the reagent X and placed on a temperature about 80" to 85" (2.). preheated glass plate for 1 minute The dried strip is then passed through (surface temperature of hot plate is the nickel acetate solution. An immediabout 90" C,). A blue-green color ate yellow color ensues which deepens appears which turns purple. A second to maximum intensity in 1 minute, dipping and heating intensifies the against a very light green background. colors. The test is sensitive to 8 to 10 The background color can be rinsed pg. of compound per sq. cm. of paper. out m-ith water. Similar results can be Test V, Ditertiary Double Bonds obtained b y substituting a 1% solution between Bridgeheads. of cobalt acetate in water for the nickel RErlGEXTS. A. A 4% solution of bromine in acetate solution. This test is sensitive chloroform (V./V.). B. Glacial aceto less than 10 pg. of compound per sq. tic acid. cm. of paper. The test strip is dipped PROCEDURE. Test 11, Phenolic Groups with Unsubstituted Ortho Positions. REA- into a freshly prepared mixture of 2 volumes of reagent X and 1 volume of GENTS. A . A 5% solution of sodium reagent B. As the hroniine evaporates cobaltinitrite in distilled water. B. from the paper a yellon--green color Glacial acetic acid. develops a t the site of the compound. PROCEDURE. The test strip is dipped The color then fades and returns with into a mixture of equal volumes of both redipping. The sensitivity is 7 to 9 reagents, A and R.prepared just before pg. per eq. em. use. It is then placed on a glass d a t e Test VI, Compounds with Acidic and heated (surface temperature of hot GrouDs-Phenols and Carboxylic d a t e 90" to 100" C.1. I n 30 to 60 ieconds a yellow-brown color appears Acids. REBGEXT A. Lk buffered on a light purple background. The bromocresol purple solution prepared by adding 100 mg. of C . P . boric acid spot is soluble in chloroform but not in and 7 , s ml. of a 1% aqueous borax \Tater. Sensitivity of this reaction is 8 solution t o 100 ml. of a 0.047, solution to 10 pg. per sq. cm. of paper. of bromocresol purple in absolute Test 111, o-Hydroxyphenols. REAmethanol. This buffer solution is GENTS. A . Phloroglucinol ( N a t h e stable for over a year in a tightly son, Coleman and Bell). B. 1 S stoppered bottle. aqueous sodium hydroxide. immersion of the filter paper bearing the steroid, into the reagent contained in a porcelain evaporating dish. The tests described below correspond numerically to the structures in Figure 1.
