oxygen, is 0.06%, half of which is lead. Even if all these impurities had a biased, cumulative effect proportionate to their individual concentrationh, which is not the case, the differential method should still be considered most satisfactory. Because of the possibility of applying the differential method to other than refined materials, such as crude tellurium and tellurium dioxide muds, the effect of significant amounts of foreign ions 011 tellurium analysis was considered (Table 111). Each impurity added represented 1.86% of the tellurium present. Antiniony and bismuth do not interfere, while the average interference for arsenic, silver, nickel, cadmium, lead, zinc, and palladium is less than 0.25aj, of the total tellurium present. In the case of lead, the main interfering impurity in rcfmed tellurium, the per cent error due to its presence is only one sixth its concentration. For an actual concentration of o.O3y0 lead in refined tellurium, the error in tellurium analysis n-ould be about 0.005%, Fhich is insignificant. For estimating tellurium in a number of tellurium refinery intermediate products containing relatively moderate amounts of tellurium and significant amounts of foreign ions, direct potentiometric titration of tellurium was considered (Table IV) . Each impurity added represented 4G.570 of the tellurium present; the
Table
V.
Typical Analyses of Refined Tellurium Products
%
Material Lot Tellurium 218
yo Te
99.93 99.91 219 99.84 99.75 220 99.93 99.85 221 99.95 100.00 222 99.61 99.64 223 99.61 99.64 224 99.59 99.29 225 99.r9 99.78 Tellurium R550-25 80.03 dioxide 79.99 R550-27 79.95 79.98 R550-29 80.11 80.11 Telluric R501-1654 68.24 acid 68.27 cast
deviation 0.01
0.045
tellurium present. Gold, copper, and iron interfere and some kind of complexing or masking agents mould have t o be introduced in their presence. However, for a higher telluriumforeign ion ratio than that considered, the effect of foreign ions would be less pronounced. RESULTS
0.04 0.025 0.015 0.015 0.00
0.005 0.02 0.015 0.00
0.015
>4v. 0.017 Std. dev. 0.023
tellurium-foreign ion ratio was approximately 2 to 1. Antimony, cadmium, and zinc had practically no effect on tellurium analysis. Bismuth, arsenic, lead, silver, and nickel had only a limited effect, causing an average error of 0.65% of the total
The differential potentiometric procedure for tellurium has been in daily use in our laboratory for almost a year and its reliability proved to our best satisfaction. The results from duplicate runs agree very closely, generally closer than in selenium analysis by the same technique. Some typical duplicate results for refined tellurium, tellurium dioxide, and telluric acid are shown in Table V. Eight of the 12 duplicate results reported agree within 0.015%, the mean deviation for all duplicate runs being 0.017% and the standard deviation 0.023%. The results shown were obtained by skilled technicians in regular routine analysis of current production lots. LITERATURE CITED
(1) Barabas, Silvio, Bennett, P. If7.,. I s a ~ . CHEM.35, 135 (1963). RECEIVEDfor review July 23, 1962. Accepted November 20, 1962. Thirteenth Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1962.
Spectrophotometric Determination of Iron with 2,6-Pyrid inedicarboxylic Acid and 2,4,6Pyrid inetrica r boxy1ic Acid ICHIRO MORIMOTO and SUSUMU TANAKA 1aboratory o f Chemistry, Gifu University, Gifu, Japan
b Color reactions between ferrous iron and 2,6-pyridinedicarboxylic acid, and between ferrous iron and 2,4,6pyridinetricarboxylic acid have been studied to determine the optimum conditions for the analytical use of these reagents. The iron complexes themselves are unstable in aqueous solutions, but could be stabilized for several days in the presence of hydroxylamine hydrochloride. Strongest color developed within the pH range from 5 to 6. Ferrous iron complexes have an absorption maximum at 4 8 5 mp in the case of 2,6-pyridinedicarboxylic acid, and at 520 mp in the case of 2,4,6-pyridine-tri-
carboxylic acid. The mole ratio of iron to the reagent in these complexes was 1 :2. The absorbance of these solutions at 5 17 mp obeyed Beer’s law in the ferrous iron concentration range of 1 to 2 0 p.p.m.
T
REDDISH-YELLOW COLOR produced by the interaction of a solution of ferrous salt with various pyridine-2-carboxylic acids was first observed by Skraup (8), and the color reaction was used for the determination of iron with picolinic acid (7) and quinolinic acid (4). We found that the HE
color reactions with 2,B-pyridinedicarboxylic acid and 2,4,6-pyridinetricarboxylic acid were more sensitive than those with picolinic acid and quinolinic acid in the paper chromatography of pyridinecarboxylic acids (6). 2’6-Pyridinedicarboxylic acid and 2,4,6pyridinetricarboxylic acid form soluble ferrous iron complexes of reddish-yellow or reddish-violet color. These complexes exhibit maximum absorption a t 485 and 520 mp between pH 5 and 6 . The reagents are specific for ferrous iron and hence can be used for the spectrophotometric determination of iron. The complexes obey Beer’s law a t 517 mp for ferrous iron concentraVOL. 35, NO. 2, FEBRUARY 1963
141
r
7 -
1.2
1
0.4
w
V z
40 500 > 100 > 100 > 100
>40 500 >100 > 100 > 100
100 25
Zn + 2
UO? + 2
c1-
F-
GO4 - 2
POI-
so,-2
15
500
500
Analysis of Synthetic Samples
(2,B-Pyridinedicarboxylic acid) Fe, p.p.m.
Found
2 0
1 9
4.0
3.9 8.0
8.0 12.0 20.0
25
50 500 25
Hg + l IIg +2
Added
100 25 25 15
25 25
C O +l
Table II.
REAGENT PER MOLE iROX
12 0
20.0
(2,4,6-Pyridinetricarboxylic acid) Fe, p.p.m. Added Found 2 0 4.0 5.0 5.0 5.0
1 9
3.9
4.9 5.0
4.9
-0Oc
--
-1.
- - -N$
0-c coo-
coo-
0
EFFECTOF DIVERSEIONS. The diverse ions were added, individually, to solutions containing 5.0 p.p.m. of iron and the color was developed. The results are shown in Table I. Tolerance limits were taken in such amount that the error was less than 3% for iron concentration. Interference of diverse ions could be reduced by the use of more than 5 nil. of each reagent. When 10 ml. of each reagent was used, interference of Znf2 (100 p.p.m.) and CuC2 (100 p.p.m.) was negligible. The results of the analysis of a series of synthetic samples containing iron by the above procedure are shown in Table 11. APPLICATIONS
This spectrophotometric method has been used to determine iron in limestone and manganese dioxide. Twenty grams of limestone was
Excess reagent (4 ml., 1 O-SM) Excessiron (1 ml., 10-aM) C. Excess reagent (4 rnl., 1O-2M) D. Excess iron (1 ml., 10-lM) ~ l r o n - 2 , 6 - p y r i d i n e d i c a r b o x y l i cacid o-lron-2,4,6~pyridinetricarboxylic acid
B.
dissolved in HC1 (1:1). After boiling, the solution was evaporated on a water bath and the residue was dissolved in water. The solution was filtered and the filtrate was made up t o 200 ml. Two grams of manganese dioxide was dissolved in hot 2N H2S04containing a few drops of 3% H202 and the solution was filtered. The filtrate was made up to 200 ml. and 25 ml. of this solution was made up t o 100 ml. Five milliliters of these solutions (contain250 pg. of iron) was used ing 125 for the spectrophotometric determination of iron. Iron hydroxide was precipitated by 6N N&OH from 5 ml. of these solutions, and was dissolved with several milliliters of HCl (1:3). Data for iron using comparative spectrophotometric determinations are shown in Table 111.
-
Table 111. Determination of Iron in Limestone and Manganese Dioxide Sample -
Reagent 2,6-Pyridinedicarboxylic acid 2,4,6-Pyridinetricarboxylic acid o-Phenanthroline Sulfosalicylic acid
Limestone, % 0,027 0,028
0,027 0,027 0.028 0.028 , ..
...
VOL. 35, NO. 2, FEBRUARY 1963
Manganese dioxide, % 1.85
1.83 1.78 1.82
1.90 1.87 1.86
1.80 143
CONCLUSIONS
These reagents are highly specific and can be used for the spectrophotometric determination of very small quantities of iron in the presence of many cations. C U +and ~ Hg+Zdo not interfere with the determination by the authors’ method, whereas they do with the more sensitive phenanthroline method. ACKNOWLEDGMENT
R e are indebted to Toshio Kitamura
at Industrial Research Institute of Gifu
Prefecture for the measurement of spectral absorbance of the iron complexes. LITERATURE CITED
(1) Black, G., Deep, E., Corson, B. B., J . Org. Chem. 14, 14 (1949). (2) Harvey, 8. E., Manning, D. L., J . Am. Chem. SOC.72, 4488 (1950); 74, 4744 (1952). (3) Jerchel, D., Bauer, E., Hippchen, H., Ber. 88, 156 (1955). ( 4 ) Majumder, A., Bag, S., Anal. Chim. Acta. 21, 324 (1959); 22, 549 (1960). (5) htorimoto. I.. Furuta.’ K.. ANAL. ’ CHEU.34, 1033 (1962).
(6) Sandell, E. B., “Colorimetric Determination of Trace of Metals,” 3rd ed., p. 83, Interscience, Sew York, 1959. (7)Shinra, K., Yoshikawa, K., J . Chem. SOC., Japan 75,44 (1954). ( 8 ) Skraup, Z. H., Monatsh. Chem. 7 , 212 (1886). (9) Soine, T. D., Buchdahl, RI. R., J . Am. Pharm. Assoc., Pract. Pharm. Ed. 39, 421 (1950). (10) T’osburg, W. C., Cooper, G. R., J . Am. Chem. SOC.63, 437 (1941). i. l l ). Yoe, J. H.. Jones, A. L.. ISD. ENQ. CHEM.,’ANAL.’ED. 16, 111 (1944). RECEIVEDfor review April 30, 1962. Accepted October 25: 1962.
A
Use of Ion Exchange Resin for Conversion, Separation, and Determination of Chlorophylls as Pheophytins JOHN R. WILSON’ AND MARVEL-DARE NUTTING Western Regional Research Laborafory, Western Utilization Research and Development Division, Agricultural Research Service,
U . S. Department of Agriculture, Albany, Calif.
b Ion exchange resin will effect a complete conversion of chlorophylls to pheophytins by removal of the magnesium and can b e used to measure total chlorophyll. The resin also provides a rapid method for separation of pheophytin a and b and removes cations and basic materials that could lead to the breakdown of pheophytins. The pheophytins obtained are more stable than those made by conversion with acid solutions.
S
and cellulose columns for the separation of components of chlorophyll have been used for a number of years (1, 6, and 10). The great amount of time involved and the difficulty of complete separation and quantitative recovery of the a and b components led to a search for a more rapid and accurate method. A technique is described for using ion exchange resin in the H+ form to remove magnesium from the chlorophyll components and convert them to pheophytins. Pheophytin a and b can then be determined in amounts of 1 to 10 pg. per ml. for the quantities of resin specified. UCROSE
EXPERIMENTAL
Apparatus. A Pyrex KO. 2700 West condenser of 1-cm. i.d. fitted with a fritted disk and a Teflon stopcock was used t o hold and t o heat the column of resin. A Cary 1 Present address: Wilson Analytical Laboratory, Albuquerque, N. M.
144
ANALYTICAL CHEMISTRY
Model 11 recording spectrophotometer with 1-cm., 5-m1. cells mas used to measure all absorption spectra. Reagents. Dowex 50K-X4 resin, analytical grade by J. T. Baker, 20- t o 50-mesh, in the H + form was used. Resins having more than 4Yc crosslinkage ( S ) , gave poor separation of the pheophytins. Acetone (lOOyc) was reagent grade. It contained 0.01 to 0 . 0 5 ~ c water as determined by the Karl Fischer (4) moisture methods. No special precautions mere necessary to maintain this water content other than keeping bottles glass-stoppered or tightly screwcapped. The SOTc acetone refers to 4 1acetone-water by volume. Tetracyanoethylene (TCSE), Eastman Kodak Co., No. 7883, melting point 200’ to 202” C. (sublimed under vacuum by one of us), was used. Preparation of Extract. Commercially frozen spinach (91 t o 92% water) of the Savoy variety, having 6.6% of the chlorophylls converted t o pheophytins ( 2 ) was used as starting material. One hundred grams was blended with 150 ml. of 100% acetone in a Waring Blendor for 3 minutes. iln additional 200 ml. of acetone was added to the mixture to bring the acetone concentration t o 80%. This slurry was stirred for 2 minutes more and then filtered through a coarse, sintered glass filter. The filter cake was washed with 50 ml. of 80% acetone. The filtrate and washings mere transferred to a 500-ml. volumetric flask and made t o volume with 80% acetone. An aliquot of 10 or 25 ml. of the original solution was evaporated to dryness on a water bath a t 20’ C. under reduced pressure (20 mm.). The chlorophyll and related pigments were then dissolved in 10 or 25 ml. of 100% acetone. Some nonchlorophyll components remained undissolved.
+
Procedure. Add Dowe.; 50K-X4 resin to a height of 20 to 24 em. in the water-jacketed column. To remove a n y metal ions present, wash the resin with 50 ml. of 10% XaOH. This causes a distinct reddening of the resin which should be washed clear with 150 t o 200 ml. of distilled water. Kext m s h the resin n-ith 50 ml. of 10% HC1 followed by distilled water until the eluate is neutral to p H paper. Then treat with 10- to 25-m1. portions of 85% acetone for a total of 150 ml. The column can be left 24 to 48 hr. in 85% acetone. To dehvdrate the resin for immediate use, wvasl; the column Kith 150 ml. of acetone containing 0.01 to 0.05% water. Put the acetone through in 10- to 25ml. portions. The resin contracts about 407, in volume in 100% acetone and should not be left over an hour because it will darken. After the resin has been dehydrated mith acetone, apply 1 to 3 ml. of the prepared extract to the column and allow it to move slonly downward until the liquid level just reaches the top of the resin bed. Add 5- to 10-ml.portions of lOO%l, acetone to elute the pheophytin b and contaminating carotenoids. Collect this brownish yellow solution in a 50-ml. volumetric flask a t a flow rate of 2 to 3 ml. per minute. A deviation of water content of the eluting acetone for pheophytin b beyond the range of 0.01 to 0.05~ccauses the release of some pheophytin a from the resin resulting in a mixture of pheophytin a and b. (Earlier it was noted that if the resin is wetted with 85% acetone before appIying the sample, conversion and elution of pheophytin a and b take place nithout separation.) Allow the interstitial 100% acetone to drain from the column just before elution of the material absorbed on the
