Table I.
Composition of Chromatographic Solvent
Dioxane, 65 ml. Distilled water, 27 5 ml. Trichloroacetic acid, 5 grams , Animonium hydroxide, 0 25 ml.
oxide G (13rinkmann) did not yield separations of these compounds using the Kolloff (6) solvent s j stem. Glass plates, 200 mm by 50 mm., were coated nith a 250-micron thick cellulose adsorbent Is?er S o attempt n a s made to determine the phosphorusc a r q ing capacitj of the cellulose laker because phosphorus-32-tagged compounds nere being used and the only requirement ma> that a spot should be registered on the x-ray film \hen exposed to the chromatogram sample volume of 1 to 5 pl. n a s used in applying the radioactir e solution of lou phosphorus concentration.
The time for development of a chromatogram was approximately one hour. These chromatograms were developed at room temperature. To minimize tailing of the spots, the Kolloff solvent system was altered t o that given in Table I. Figure 1 shows a typical autoradiogram for this system. Also during the course of this investigation, it was observed that the phosphate-specific chromatographic sprays of Hanes and Isherwood ( 2 ) and Kolloff ( b ) ,which are successfully used to locate the phosphate spots in paper chromatography, did not yield a suitable reaction in the thin layer chromatographic method. The reasons for this behavior were not ascertained because it was the authors' intention to use labeled phosphate compounds for these experiments, More work should be done to determine if this thin layer technique is applicable to the separation of condensed phosphates.
LITERATURE CITED
(1) Campbell, D. O., Kilpatrick, M. L., J . Am. Chem. SOC.7 6 , 893 (1954). ( 2 ) Hanes, C. S., Isherwood, F. A , , Sature 164, 1107 (1949). (3) Hettler, H., J . Chrornatog. 1, 389
(1958). (4) Karl-Kroupa, E., h . 4 ~ .Cmnx. 28, 1091 (1956). (5) Kolloff, R. H., Ibid., 33, 373 (1961). ( 6 ) Lowenstein, J. hf.> Bwchem. J . 65, 197 (1957). ( 7 ) Ohashi, S.,Van Waaer, J. R., ANAL. CHEM.35, 1984 (1963). (8) Westman, A. E. R., Scott, A. E., S a t u r e 168, 740 (1951). KICHOLASL. CLESCERI' G. FREDLEE Hydraulic and Sanitary Laboratory University of Wisconsin Madison, Wis. 53706 ISVESTIGATIOS supported in part by Public Health Service Fellowship, k4'PPM-10 713, and Public Health Service Training Grant, ITI-JVP-22-01, Division of Rater Supply and Pollution Control, Public Health Service, JVashington 25, D . C. 1 Present address, Swiss Federal Institute of Technology, Zurich, Switzerland.
Separation of Planktonic Algal Pigments by Thin Layer Chromatography S I R : I n the course of an investigation on planktonic pigments, a rapid method for separation of small quantities of these pigments was needed. search of the literature revealed several papers on the application of column and paper chromatography ( 2 , 4-8) for the separation of plastid pigments. This paper describes the separation of the chloroplast pigrnenh of algae in thin layers of powdered cellulose. The separations are better than those obtained in thin layers of other adsorbents ( 1 , 3 ) . Moreover, alteration, as indicated by the formation of pheophytins, is at, a minimum in the cellulose. I t was therefore decided to investigate the separation of these pigments by thin layer chromatography. During the course of this investigation two papers (I, 3 ) were published that use thin layer procedures for separating plastid pigments. The method reported below was superior to either of the reported methods for thin layer separation of plastid pigments.
sodium chloride solution was added, the solution was mixed by gentle swirling, and the aqueous layer was removed. The deep green petroleum ether solut,ion was washed a number of times with the above salt, solution. The extract was protected from bright light to reduce photodecomposition of the pigments. Preparation of Thin Layer Plates. 4 slurry of the adsorbent was prepared by mixing the following: cellulose powder, 8 grams (AIS cellulose powder 300, Urinkmann Instruments, Inc.); sugar, 2 grams (C Bi H sugar, confectioners, powdered) ; 3Yc starch (potat,o, I3aker ;lnalyzed); and 50
N-
G
CHLOROPHYLL
a
ml. distilled water. The mixture was blended in a Karing Blendor for a few minutes, and well mixed to prevent 1uml)iness before application to the plate. The 200- X 200-mm. glass plates were washed with petroleum ether and air dried. The absorbent was spread on the glass plates nith a Erinkniann Instrument thin layer apparatus llodel 250015. After applying the coating, the plates were dried at, room t,emperature for 15 minutes and finally dried in an oven a t 100" C. for 15 minutes. Separation Procedure. One-tent'h milliliter of the petroleum ether extract, solution was spotted on the adsorbent coated plate. The plate was dried under a st,ream of nitrogen gas and placed in a tightly covered, wide-mouth, rectangular (230 X 230 x 50 mm.) jar containing approximately 300 ml. of developing solvent0.5yc n-propanol in petroleum ether. Kitrogen gas was passed through the jar to displace the air in the jar. The chromatogram was developpd a t 5" C. in the dark. The deidopment was stopped when the solvent front was about 18 em. from the bottom of the plate.
EXPERIMENTAL
Separation of Plastid Pigments. A pure culture of Scenedesmus quadricauda u-as centrifuged, water decanted, and suspended in a 150-ml. mixture of methanol and petroleum ether (3 parts methanol to 1 part P-ether). Extraction was complete in 15 minutes. The extract was filtered through glaqswool into a separatory funnel, 500 ml. of lOyo 2208
e
ANALYTICAL CHEMISTRY
RESULTS
Y A't?O.%'Ah'TMN
ond
Y/OLA YANTC//N
Figure 1 . pigments
Chromatogram of plastid
Figure 1 shows the separation of the pigments in the folloii ing sequence from top to bottom, carotenes, chlorophyll a, lutein k zeaxanthin. chlorophyll b, violaxanthin, and neoxanthin. Time to achieve separation was about 45 to .50 minuteb. For the identification of each pigment
the colored zone was scraped off the illate, washed in acetone, and filtered through a cotton plug. The absorption sl)ectruni for each colored zone was obtained on a recording spectrophotometer. The absorption spectrum obtained for each spot matched the spectra rellorted in the literature. ACKNOWLEDGMENT
The assistance given to this project by G . P. Fitzgerald is greatly appreciated.
LITERATURE CITED
(1) Anwar, M. H., J . Chern. Educ. 40, 29 (1963). ( 2 ) Lind, E. F., Lane, H. C., Gleason, L. S., Plant Physiol. 28, 325 (1953). 13) Rollins. C.. J . Chert. Educ. 40. 32 ( 4 ) S,porer, A . H., Freed, S., Sancier, M.,
Sczence 119, 68-9 (1954). (5) Strain, H. H., J . Phys. Chem. 57, 638 (1953). 1~, 6 ) Strain. H. H.. Thirtv-Second Priestlev Lecture,' University Park, Pa., 1958. " ( 7 ) Strain, H. H., Murphy, G. W., ANAL. CHEM.24, 50 (1952).
18) . . Strain. H. H.. Sato. T. R.. b'. S . i l t . Energy Comrn. R e p t . No. TID7512, 1956.
HAKUMAT RAI G. FREDLEE
Water Chemistry Program Hydraulic and Sanitary Laboratory University of Wisconsin Madison, Wis. 53706
INVESTIGATION supported in art by a grant from the Wisconsin Afumni Research Foundation and P. H. S. Training Grant No. ITI-WP-22-01, Division of Water Supply and Pollution Control, Public Health Service.
Determination of Serum Creatinine by Ion Exchange Chromatography and Ultraviolet Spectrophotometry SIR: Serum creatinine methods now in use are based on the colorimetric measurement of a red chromogen formed by reaction of the compound with alkaline picrate [the Jaffe reaction ( 5 )J in a protein-free filtrate. Sumerous investigators (Z-i+] 6! 7 ) have demonstrated the Jaffe reaction to be nonspecific and unreliable without prior removal of interfering substances. Many biologically occurring compounds other than creatinine contribute to the Jaffe reaction, and attempts to remove these substances, as well as the serum proteins, by precipitation techniques are often accompanied by serious manipulative loss. h method recently reported by the authors for the determination of urinary creatinine involved preliminary separation of the compound by an anion exchange resin and subsequent ultraviolet spectrophotometric measurement of its concentration ( 1 ) . That procedure was not directly applicable to serum because of the high concentration of protein arid the relatively low creatinine concentration (about one twentieth that of urine). The quantitative separation of creatinine on a strong cation exchange resin from substances which interfere with the Jaffe reaction was recently descritjed by Teger-Kilsson ( 7 ) . His proredure, however, proved unsatisfactory for direct ultraviolet spectroIihotoinetric studies because of high intcrfering absorbance at 220 to 260 nip. In the present investigation, a two-column resin system was successfully employed for elimination of t,he substances responsible for the interfering absorbance, thus making feasible a ~iiectrophotometricdetermination of serum creatinine. Weakly acidified serum was ai)plietI to a cation exchange resin column which retained the creatinine, while wrum Iiroteins, uric acid, and some of the other interfering sub-
AIR
PRESSURE
4.
APPARATUS A Figure 1. Apparatus for Dowex 50 cation exchange resin column
stances were removed by washing. Creatinine was then eluted directly from that resin column through an anion exchange resin and the eluate quantitatively collected for ultraviolet spectrophotometric measurement. EXPERIMENTAL
Reagents. WATER. Deionized water was used throughout for all reagents, washing the resins, and final rinse of glassware.
CREATININE.A stock standard solution was made up in O.1N HC1 at a concentration of 1 mg. per ml. ALKALINE AMMONIUM CHLORIDEREAGENT. Ammonium chloride, 1.33 gram, was dissolved in about 800 ml. of water; 15 ml. of concentrated ammonium hydroxide were added and the volume was adjusted to 1 liter. Apparatus. U.V. SPECTROPHOTOMETER. Beckman Model DU or DK-2 with semimicro-absorption cells, 1-em. light path. T h e recording model was used for this investigation. MANIFOLD AND AIR PRESSURE REGULATOR. A glass manifold equipped with a stopcock a t each port opening was employed for independent control of the air pressure to each resin column. The regulator used for control of air pressure to the manifold was the Kullmatic .\ir Pressure Model 4015, Moore Products Co., Philadelphia. APPARATUSFOR RESIN COLUMNS. The design of the equipment for chromatography with Dowex 50 resin is shown in Figure 1. The narrow-bore section a t the bottom for sulqiort of the resin was provided by the glass tubing attached to the socket portion of a ball and socket joint. The reservoir a t the top for accommodation of the larger amounts of wash solutions could be attached by glass joint connections to the resin column below and to the manifold above. The column for the Dowex 2 resin is shown in Figure 2 . Procedure. P R E P l R A T I O N A K D REG E N E R A T I O N O F DOWEX 50 R E S I N COLUMN.The resin (Dowex 50 W-Xl2, H + form, 200 to 400 mesh, small porosity) was introduced as a slurry into the narrowbore column of ailparatus * I (Figure I ) with a filling pipet such as the 11-introbe. The ball and socket joint was then sealed with a Teflon-base sealant (Hoke Slic-Seal), secured with a pinch clamp, and the column w,s allowed to stand 24 hours to ensure drying of the sealant before the column was subjected to air pressure. .'\fter a column had been used for a serum creatinine analysis, the resin was regenerated by 1)assing VOL. 36, N O . 1 1 , OCTOBER 1964
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