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 cis 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 a n d t o heat t h e 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 t h e 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 t h e 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. P u t 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 i t 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
r
I
I
,
,
1
I
W A V E LENGTH, m p Figure 1 .
Spectra of pheophytins
-Pheophytin b carotenoids in 100% acetone Pheophytin b and carotenoids after treatment with TCNE (note absence of carotenoid shoulder at 4 7 5 mp) Curve omitted 5 7 0 - 7 0 0 mp; indistinguishable at this scale -.- Pheophytin a in 85% acetone Region from 4 6 0 - 6 4 0 mb was rerun with cell path length and concentration adjusted io show minor peaks more clearly
---
upper 2 to 3 em. of reiin. Immediately add 10 t o 1.5 ml. of 85Ye acetone at room temperature. The resin starts t o swell and the pheophytin a is eluted as a brownish gray solution. -4t this point any air entrapped in the resin due to reswelling should be removed by lowering and raising a glass rod gently up and down in the column. Add nearly boiling 8 S & acetone to the column and circulate water a t 50' =t 1.0' C. through the jacket. (All previous solutions were room temperature.) Continue to elute the pheophytin a with hot 85% acetone into a 100ml. volumetric flask. The complete elution usually requires about 1 hour. However, if the pheophytin a does not seem to be completely eluted, the resin may be allon-ed to soak a few minutes between each withdrawal of eluate until the resin becomes clear. At least three separations can be made with the same resin column before regeneration through a base and acid cycle becomes necessary. After each separation. wash the resin several times with distilled water t o reswell it to the original volume. Rewash with 85y0 acetone and subsequently dehydrate with 1 0 0 ~ acetone o just before the next separation. I t is not necessary to remove the resin from the column during the washing or regeneration cited under Procedure. Measurement of Absorption Spectra. The absorption spectra of both eluates were measured from 700 to 350 mp. Pheophytin b in the 100% acetone fraction was scanned. Then a few crystals of T C S E were added directly to the cuvette t o make the concentra-
tion 0.25 to 0.40 mg. per mi. (8). The mixture was alloived to react for 20 minutes and the absorption spectrum was rerun. Figure 1 shows the spectra of pheophytin b before and after removal of the 475-mp carotenoid peak. A typical spectrum of pheophytin a in 85% acetone is also shown. The absorbances of pheophytin a and b solutions were measured a t 667. 409, 654, and 436 mp, respectively: Absorptivities of 56.6, 130.9 liters per
Table 1.
Aliquota
Resin column
A
1 2
B
1 2
C
1 2
D
1 2
D
3 4
Av. Av.
Av. Av. Av.
gram-em. for a (7') and 35.3, 181.0 for b (8) were used in subsequent calculations of concentration. Concentration = A/ab, and A = absorbance, a = absorptivity, b = cuvette path in em. Table I gives results of duplicate determinations on four aliquots of a single acetone extract of spinach. The concentrations of pheophytin a and b from spectrophotometric maxima are given with the corresponding calculated chlorophyll a and b. A lOOyo acetone extract of spinach was used to test the accuracy of this method by comparison of spectral and magnesium measurements. Eight separations were made on resin columns over a period of several weeks. The chlorophyll was calculated using both the short and long wavelength maxima of the pheophytins. These gave averages of 0.432 mg. per ml. for the short and 0.438 mg. per ml. for the long wavelength maxima, with maximum variations of 3.5 and 6.3y0,.At the same time four duplicate magnesium determinations were made by the method of Young and Gill (9). The total chlorophyll calculated on the basis of 2.717, magnesium (2.6 to 1, a to 6 ) gave 0.432 mg. per ml. with 5,8yemaximum variation. Thus, conversion of chlorophyll with ion exchange resin and spectral measurement gives amounts of total chlorophyll comparable to magnesium determinations. To establish the identicalness of pheophytins prepared in two different ways. additional samples of pheophytin a and b were purified using sucrose columns. The spectra of pure pheophytin a and b were run before and after treatment of T C S E . As already reported (8),there was little change in absorptivities of either pheophytin a or b, but these must be taken into account in the estimation of pigment mixtures. Pure solutions ofchlorophyll a and b were also treated with TCNE. Only
Separated Pheophytins and Calculated Chlorophylls
Pheophytin a In In eluate spinach pg./ml. 4.13 4.20 4.16 3.97 4.20 4.08 4.28 4.28 4.28 3.97 3.97 3.97 3.74 4.28 4.01
Chloropbyll in spinach
mg./100 g. 69.3
71.2
68.0
69.9
71.3
73.3
66.6
67.9
66.8
68.7
-
Pheophytin b In In eluate spinach pg./ml. 5.41 5.33 5.37 6.18 5.86 6.02 5.50 6.35 5.92 6.02 5.86 5.94 6.57 5.39 5.95
Chlorophyll in
spinach
mg./100 g. 22.4
23.0
25.1
25.8
24.7
25.4
24.7
25.4
24.9
25.6
All aliquots made from the same acetone extract of spinach. The two runs of aliquots A , B , and C were put successively through the same pair of columns. For aliquot D, four freshly basic- and acid-cycled columns were used.
VOL. 35, NO. 2, FEBRUARY 1963
0
145
minor changes in absorptivities were observed in 20 minutes. This indicates little immediate deterioration or complex formation on the addition of TCNE (6). However, spectra of these solutions after 8 hours no longer resembled those of chlorophylls. DISCUSSION
Ion exchange resin dehydrated with acetone provides a rapid method of conversion, separation, and determination of chlorophylls as pheophytins. The pheophytins obtained by this method are more stable than those obtained by oxalic or mineral acid conversion. This was shown by the reproducibility of spectrophotometric curves over a period of 12 months on samples kept a t 0’ C. The chlorophyll a and b ratio can b e determined rapidly and with good precision by this method. This ratio includes both the
chlorophyll and pheophytin originally present in the blanched and frozen tissues. ACKNOWLEDGMENT
(32, Dow Chemical Co., Midland, Mich., Dowex: Ion Exchange, 1959. (4) . . Mitchell, J., Smith, E. M., “Aauametrv.” Interscience, New YorkLobdon, 1958. (5) Ozolins. Mara. Shenk. G. H.. ANAL. CHEM.33,1035 (1961). ‘ (6) Smith, J. H. C., Benitez, A,, ‘LChlorophylls: Analysis in Plant Materials.” Modern Methods of Plant Analysis Vol. IV, Paech, K., Tracey, M. V. (1955). (7) . . Vernon. L. P.. ANAL.CHEM.32. 1144 (1960). (8) Wilson, J. R., Nutting, M-D., Bailey, G. F., Ibid., 34, 1331 (1962). (9) Young, H. Y., Gill, R. F.: Ibid., 23, 751 (1951). (10) Zscheile, F. P., Botan. Rev. 7, 587 (1941). RECEIVEDfor review April 20, 1962. Resubmitted November 21, 1962. Accepted December 10, 1962. Reference t o a company or product by name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others which may also be suitable. .
The authors are indebted to Glen F. Bailey for helpful discussions of spectrophotometry, to Kenneth T. Williams and Allan D. Shepherd for their generous cooperation in obtaining materials and equipment necessary for this study, and t o Earl Potter and Fern Jantzeff for the magnesium determinations. LITERATURE CITED
(1) Anderson, J. M., “Research in Photc-
synthesis.” Ph.D. thesis, University of California (Berkeley) 1959. U. S. Atomic Energy Commission No. UCRL-
8870. (2) TDietrich, W. C., Boggs, M. M.,
hutting, M-D., Weinstein, W. E., Food TechnoI. 14,522 (1960).
I
Indirect Spectrophotometric Determination of Traces of Bromide in Water M. J. FISHMAN and M. W. SKOUGSTAD
U. S.
Geological Survey, Denver, Colo.
b A
rapid, accurate, and sensitive indirect spectrophotometric method for the determination of bromide in natural waters is based on the catalytic effect of bromide on the oxidation of iodine to iodate b y potassium permanganate in sulfuric acid solution. The method is applicable to concentrations ranging from 1 to 100 pg. of bromide per liter, but may b e modified to extend the concentration range. Most ions commonly occurring in water do not interfere. The standard deviation is 2.9 a t bromide concentrations of 100 pg. per liter and less at lower concentrations. The determination of bromide in samples containing known added amounts gave values ranging from 99 to 105% of the concentration calculated to b e present.
D
on the relative amounts of chloride, bromide, and iodide in natural water frequently are of great importance in hydrologic, geochemical, and other investigations. The sensitivity and the accuracy of ordinary chemical methods for determining traces of bromide in the presence of iodide and in the presence of much larger amounts of chloride generally are inadequate. Most available methods are based on the difference in oxidizing and reducing 146
ATA
ANALYTICAL CHEMISTRY
characteristics of the halogens and their behavior toward different oxidizing agents in carefully buffered solutions. A colorimetric method capable of detecting 0.1 p.p.m. of bromide ( I ) is based on the oxidation of bromide and subsequent bromination of phenol red in the presence of chloramine-T. The concentration of the reagents and the timing of the reaction are critical factors that limit the accuracy of the method, although modifications described by Goldman and Byles (8) are claimed to increase its precision and reliability. Iwasaki and others (3) developed a method for determining 0.1 to 50 p.p.m. of bromide by acid-permanganate oxidation of both bromide and iodide, the bromide being oxidized t o free bromine and the iodide to iodate. The resulting bromine was extracted with carbon tetrachloride and then determined indirectly by measuring the absorbance of the ferric thiocyanate complex formed in the aqueous layer upon shaking the extract with an alcoholic solution of ferric alum and mercuric thiocyanate. Shiota, Utsumi, and Iwasaki (4) noted that the oxidation of iodide or iodine t o iodate by acidpermanganate is catalytically promoted by traces of bromide and described a procedure for determining the concen-
tration of bromide in the range from 0.005 to 0.13 pap.m. They showed that under controlled conditions of pH, temperature, and concentration of reactants, and for any given reaction time, the concentration of unreacted iodine is inversely proportional to the concenTo determine tration of bromide. bromide, the oxidation reaction was stopped after a given time by extraction of the unreacted iodine with carbon tetrachloride. Then the amount of iodine in the extract was determined by adding an alcoholic solution of mercuric thiocyanate and ferric alum and measuring the absorbance of the resulting ferric thiocyanate complex. Bromide and iodide are readily oxidized by potassium permanganate in sulfuric acid solution, but chloride at ordinary temperatures is practically unaffected. The oxidation of bromide is complete with the formation of elemental bromine, but iodine is further oxidized to iodate. The oxidation of 1-1 to Io occurs rapidly, even a t 0” C.; the second step, oxidation of Io t o IO3-’ occurs only slowly at this temperature. The second step oxidation rate is dependent upon temperature, pH, and concentration of the oxidizing agent and is notably dependent upon traces of bromide ion, which acts as a catalyst. The greater the concen-
