Colorimetric Determination of Iron, Chromium, Thallium, and Aromatic and Alpha,BetaUnsaturated Aldehydes M. H. HASHMI, ABDUR RASHID, HAMID AHMAD, A. A. AYAZ and FAROOQ AZAM West Regional laboratories, Pakistan Council of Scientific and Industrial Research, Lahore, West Pakistan
b
Iron(lll), chromium(VI), and thallium
(111) give a pink color with an equimolar mixture of isonicotinic acid hydrazide and 2,3,5-triphenyltetrazolium chloride in dilute acid, and aromatic and cr,p-unsaturated aldehydes produce a yellow color with the same reagents. The reaction is developed into a colorimetric method for the determination of iron(ll1) separately, and in presence of iron(ll), as well as combined iron. Iron(ll) does not interfere up to 10-fold excess. Thallium can b e determined separately and in the presence of thallium(l), as well as in the combined form. Chromium has been determined quantitatively by this method. A spot test for a number of aldehydes is described. O n the basis of this spot reaction a colorimetric method is also described for the determination of a small amount of a number of aldehydes.
I
a method has been reported for the colorimetric determination of iodate and bromate (3). During a systematic study it was found that an equimolar mixture of isonicotinic acid hydrazide and 2,3,5triphenyltetrazolium chloride in dilute acid also gives a pink color with iron (111), chromium(VI), and thallium(II1). This color reaction has been developed for the quantitative determination of iron(II1) separately and in presence of iron(II), as well as combined iron. Similarly thallium(II1) and thallium(1) can be determined. Chromium(II1) can be determined after pre-oxidation with silver(I1) oxide. The method is very accurate and gives recoveries of better than 99%. The visual limit of identification of iron(II1) , chromium (VI), and thallium(II1) is 1, 5, and 1 pg./ml., respectively. Organic compounds and inorganic ions [except iodate and bromate ( S ) ] do not interfere. Isonicotinic acid hydrazide reacts with aldehydes to give corresponding hydrazones (7, 8). I t has been observed that isonicotinoyl hydrazones of aromatic and a$-unsaturated aldehydes give a characteristic yellow color in acid medium. Though this spot reaction N A PREVIOUS PAPER
can be used for the identification of a large number of aromatic aldehydes; the system does not obey Beer's law; however, conformance to Beer's law is achieved by addition of 2,3,5-triphenyltetrazolium chloride to the reaction mixture. The reaction is specific for aromatic and a,p-unsaturated aldehydes and a method for their determination is developed. The procedure can be used for the determination of aldehydes as small as 1 pg./ml. EXPERIMENTAL
Apparatus. All absorbance measurements were made with B. Lange's photoelectric colorimeter Model VI. To increase the sensitivity of the instrument, the colorimeter was connected with a lamp and scale type spot Cambridge galvanometer. A battery operated bench type Cambridge p H meter was used. Reagents. All reagents were. of analytical grade or comparable purity. The solutions of all aldehydes were made by weight in 25% alcohol by the procedure of Goodwin ( l ) , and the silver(I1) oxide was prepared according to the directions of Hammer and Kleinberg (2). Isonicotinic acid hvdrazide. 1% bv weight and 2,3,5-tri~henyltetrazdium chloride, 1% by weight were prepared (8).
Procedure. IRON.To a solution containing 16 to 160 pg. of iron(III), dilute nitric acid was added to adjust the pH to 2.03, and the volume of the solution was made to 5 ml. In the case of iron(II), bromine water was added and the excess was evaporated before adjusting the volume of the solution to 5 ml. A 2-ml. portion of the color producing reagent was added, and the solution was heated for 3 minutes a t 60-3" C. After cooling to room temperature, 5 ml. of n-amyl alcohol was added and the mixture was centrifuged. The lower aqueous layer was discarded while the upper layer was diluted to 100 ml. with ethyl alcohol, and the colorimetric measurements were made using 1-cm. cells and green filters. The experiment was repeated with different volumes of iron(II1) solution and a calibration curve was prepared. Iron(I1) does not interfere up to a 10-fold excess. Iron(I1) can be de-
termined by its oxidation with bromine water after the determination of iron (111); hence, the method can be used for the simultaneous colorimetric determination of iron(II1) and iron(I1). CHROMIUM.To a solution containing 5 to 15 mg. of chromium(III), 5 ml. of 6N nitric acid was added, followed by 0.5 gram of silver(I1) oxide ( 5 ) . After 10 minutes, excess silver(I1) oxide was destroyed by heating, and 3 ml. of 10% potassium chloride was added to precipitate silver chloride. The pH of the solution was adjusted to 5.5 with potassium hydroxide. Silver chloride was filtered off and the volume of the solution was made up to 100 ml. Colorimetric measurements were made a t room temperature by a procedure similar to that used for the determination of iron(II1). THALLIUM.To a solution containing 50 to 600 pg, of thallium(III), dilute nitric acid was added to adjust the pH of the final volume (20 ml.) to 2.67. A 2-ml. portion of the color producing reagent was added and the reaction mixture was shaken before setting aside for 3 minutes. The solution was diluted to 100 ml. with alcohol and colorimetric measurements were made a t room temperature by a method similar to that used for iron(II1). Thallium(1) was found by subtraction from total thallium(II1) after oxidation by bromine water. The effect of pH on color intensity was studied by varying the pH of the solution by means of nitric acid (0.25N). After adjusting the required pH, the color producing reagent was added. ALDEHYDES.A 1-ml. portion of 4N sulfuric acid was added to a solution (0.1 to 0.6 ml.) containing about 100 to 600 pg. of aldehyde. The total volume of the solution was made to 8 ml. with distilled water, and 2 ml. of an equimolar mixture of isonicotinic acid hydrazide and 2,3,5-triphenyltetrazolium chloride was added. The solution, which had a final pH of 1.1, immediately developed a yellow color. The solution was diluted to 100 ml. with distilled water, and absorbance was measured using a blue filter. Spot Test for Aldehydes. A drop of test solution containing aldehyde was mixed with 0.1 ml. of 2 N sulfuric acid and 0.5 ml. of 1% isonicotinic acid hydrazide on a spot plate. The characteristic yellow color appeared immediately. VOL. 37, NO. 8, JULY 1965
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CHROMIUM ( V I /RON( )
).*-e
CHROMlUM ( V I
......... 0 rti.u~/uH(m I.. ...A m
L
2
4
6
L
I
I
40
60
Effect of pH on color intensity
RESULTS A N D DISCUSSION
METALIONS. The effect of p H on color intensity is shown in Figure I . The effect of temperature on color intensity is given in Figure 2. The effect of time on color intensity before dilution is shown in Figure 3. Some typical results for the determination of iron, chromium, and thallium are given in Table I. Organic substances and inorganic ions (S),except iodate and bromate, do not interfere with these determinations. The mechanism of the reaction involves the formation of hydrazine from isonicotinic acid hydrazide which reacts with 2,3,5-triphenyltetrazolium chloride to give rise to pink colored formazan (3). Absorbance us. wavelength curve of the pink color shows maximum absorption at 480 mN and 308 mfi which closely resembles that at 485 mg reported by Johnson, King, and Vickers ( 4 ) . The color is stable for more than 24 hours.
Table I. Determination of Iron, Chromium, and Thallium pg./100 ml. Present Found Iron(111) 30 35 50 100 150
31 35 50 101 150
Thallium(II1) 50 60 80 100 300
50 60 81 100 305
kg./100 ml. Present Found Iron( 11) 120 100 40 90 10
1028
120 100 39 90 10
Thallium( I ) 40 40 50
41 40 49
70
70
250
252
Chromium 150 250 327 442 500
150 252 327 442 500
ANALYTICAL CHEMISTRY
m).,..A
1
I
84
100
TEMPERATURE
PH
Figure 1 .
)..a
..........0
THALLIUM(
L
I
I
/RO"l])
Figure 2.
ALDEHYDES. The visual limit of identification of various aldehydes is given in Table 11. The color is produced a t room temperature and remains stable on heating up to 90' C. The p H also has no effect on the intensity of color, except in the case of p-dimethylaminobenzaldehyde, where the color intensity decreases with decrease of pH. However, intensity of the color is reproducible at a particular pH and determinations of p-dimethylaminobenzaldehyde can be made a t any fixed pH. Some typical results for the determination of various aldehydes are given in Table 11. Alcohols, ketones, aliphatic aldehydes, amines, ethers, acids, carbohydrates, amino acids, and other organic compounds do not interfere. However, the pink color due to the formation of formazan (3) will be produced if any of the following compounds are present: IOa-, Tlf3, Cr04-*, Cr&-3, Be+* Isonicotinic acid hydrazide itself produces a yellow color in acidic medium even in the absence of aromatic aldehyde. The intensity of yellow color increases with the addition of aldehyde, but Beer's law is not obeyed. However, addition of 2,3,5triphenyltetrazolium chloride suppresses the yellow color produced by isonicotinic acid hydrazide alone and with the mixture of 2,3,5-triphenyltetrazolium chloride and isonicotinic acid hydrazide the yellow color is produced only in presence of aldehyde which obeys Beer's law. The minimum amount of 2,3,5-triphenyltetrazolium chloride which may be added to isonicotinic acid hydrazide should be in the ratio of 1 : 2, though up to &fold excess of 2,3,5-triphenyltetraeolium chloride does not interfere. However, in these investigations a n equimolar mixture of the two compounds was used. The mechanism of the reaction probably is as follows. Isonicotinic acid
Effect of temperature on color intensity
hydrazide condenses with aldehyde to form the corresponding isonicotinoyl hydrazone ( 7 ) .
N Isonicotinic hydrazide
Aldehyde
. acid
CONHN = CHR
N Isonicotinoyl hydrazone Isonicotinoyl hydrasones in acidic medium undergo reaction to yield a n extended conjugated system (6),the formation of which probably accounts for the characteristic yellow color:
0 II
C -NHN=CHR
N
\[+€I+
OH +C-NH I -N=CHR
OH I
Y=N-N=CHR
N Yellow colored compound
Figure 3. Effect of time on color intensity
t
0.2
a/
10
30
20
TIME, MINUTES
Table 11. Determination of Aldehydes Limit Limit of of identifiidentification,& pg./100 ml. cation,ll pg./lOO ml. Aldehyde pg./ml. Present Found Aldehyde pg./ml. Present Found Salicylaldehyde 20 500 495 Cinnamaldehyde 5 108 106
Anisaldehyde
7
Vanillin
5
T’eratraldehyde
6
Piperonal
5
300 150 595 425 170 200 500 700 107 268 535 150 300 500
297 150 600 422 170 200 500 700 108 268 532 152 300 500
Furfural p-Dimethylaminobenzaldehyde Citral
12 5
. 15
324 540 121 242 605 108 270 520 125 320 542
320 534 120 240 600 110 272 525 125 316 539
a Benzaldehyde, crotonaldehyde, and acrolein give a yellow color when these are present in 1 mg./ml. quantities ; o-, m-, p-nitrobenzaldehyde, and o-chlorobenzaldehyde do not respond to the color test.
The yellow-colored compounds, whether obtained directly by evaporating the yellow solution of isonicotinic acid hydrazide and aldehyde in acid medium, or indirectly from isonicotinoyl hydrazones by treatment with acid, have the same melting points (which were higher than the corresponding hydrazones). The compounds were purified by recrystallization from alcohol before determination of their melting points. The yellow color of the solution obtained by the action of isonicotinic acid hydrazide and aldehyde in acid medium disappears by neutralization with bicarbonate. However, the color reappears upon acidification. LITERATURE CITED
( 1 ) Goodwin, L. F., J . A m . Chem. SOC. 4 2 , 39 (1920). ( 2 ) Hammer, R. N., Kleinberg, J., “Inorganic Syntheses,” Vol. 4, p. 12, McGraw-Hill. New York. 1963. ( 3 ) Hashmi, M: H., Ahmad, H., Rashid, A., Azam, F., ANAL.C n m . 36, 2471 (1964). (4) Johnson, C. A., King, R., T’ickers, C., Analyst 85, 714 (1960). ( 5 ) Lloyd, C. P., Pickering, W. P., Talanta 11, 1409 (1964). ( 6 ) Packer, J., T’aughan, J., “Modern
Approach to Organic Chemistry,” p. 218, Clarendon Press, Oxford, 1958. ( 7 ) Sah, P. T., Peoples, S. A,, J. A m . Pharm. Assoc. 43, 513 (1954). (8) Yale, H. L., Losee, K., llartins, J., Holsing, M.,Perry, M., Bernstein, J., J . Am. Chem. SOC.75, 1935 (1953).
RECEIVED for review December 14, 1964. Accepted March 31, 1965.
Spectrophotometric Determination of Calcium and Magnesium in Blood Serum with Arsenazo and EGTA ERVIN G. LAMKIN’ and MAX B. WILLIAMS Department of Chemistry, Oregon State University, Corvallis, Ore.
A simple, rapid spectrophotometric procedure for the determination of magnesium and calcium in 1 ml. of blood serum using only three reagents is described. The total concentration of magnesium plus calcium is found by chelating with a glycine-buffered 0 - [ (1,8-disolution of Arsenazo, hydroxy 3,6 disulfo 2 naphthyl)azo] benzene arsonic acid, disodium salt. The magnesium concentration is found by sequestering the calcium in one half the above solution with EGTA, [ethylene bis(oxyethy1enenitrilo)] tetraacetic acid, which forms a colorless complex. The difference gives the calcium concentration. The reagents are all water soluble and stable. The complexes develop irn-
-
-
- -
does magnesium give a similar absorption spectrum to calcium with Arsenazo and obey Beer’s law at 580 mp and a p H of 9.6, but also various mixtures of calcium and magnesium follow Beer’s law for total concentration to about 0.1 N RECENT literature (6) the need for meq. per liter. If magnesium is to be improved methods for the deterdetermined, calcium may be sequestered mination of calcium and magnesium in with EGTA, [ethylene bis(oxyethy1eneblood serum has been indicated. A nitrilo)] tetraacetic acid ( 1 , 6, 7 , 9 ) , stable, sensitive reagent for calcium, leaving magnesium virtually unaffected. Arsenazo, o - [(1,8 - dihydroxy - 3,6The authors investigated methods of disulfo - 2 - naphthy1)azolbenzene armasking diverse ions and results showed sonic acid, disodium salt, has been rethat the following ions present in 20 ported by Jones ( S ) , Kirshen (4, p.p.m., Fe+3, C u f 2 , Ni+2, Mn+2, Hg+2, Polyak ( 8 ) , and others. Beer’s law is obeyed a t 580 mp although magnesium Present address, Department of and many other diverse ions interfere. Chemistry, Eastern Jlichigan University, It has been determined that not only Ypsilanti, Mich. mediately and are also soluble and stable. The analyses are very reproducible and do not require any extraction, precipitation, or separation steps.
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VOL. 37, NO. 8, JULY 1965
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