titrated. The titration is essentially quantitative but precision was poor; values ranged from 98 to 107% of theory for the total titration. The results approached a 1 : l ratio for the two species. Methyl sulfate and bisulfate salts gave similar H S P and E P but the results were not quantitative with values ranging from 45 to 85y0of theory. The titration of the high molecular weight nitrogen compounds except for the sulfates is quantitative and the standard deviation calculated from replicate analyses of the ditallowdimethylammonium chloride is 1.47% a t the l00Y0 level. The half-neutralization potential ranges of the nitrogen compounds examined are shown in Figure 1. The data graphically show those compounds whose H S P are sufficiently separated to allow differential titration in mixtures, such as tertiary amines and quaternary ammonium chlorides or tertiary and secondary amines. The information in Figure 1 together with the AEeP given in Table I1 shows that the workable range of the HC104 in dioxane titrant with the acetic anhydride solvent is -50 to -800 mv. As would he espected, the A K e p value for each compound is dependent upon the H S P and becomes smaller as the N T P apl~roaches -800 mv. The reproducibility of the H S P was determined from replicate analysis of the
Table 111.
Titration of Mixtures
Mg.
Compounds Compounds Ditallow methyl amine and Ditallow amine Uitallow dimethyl amm. chloride and Ilitallow dimethyl arnni. bromide Ditallow methyl amine and Ditallow dimethyl amm. chloride
EP
HSP
A E e p Expected Found
-313
-114 -656 -295 -450
325 150 50 325 221 290
- 725
-375 - 710 -232 -595
ditallowdimethylamnionium chloride. The standard deviation is 15.4 my. Three samples of mixed nitrogen compounds were titrated and the results are given in Table 111. Good agreement was obtained between the expected and experimental reault,s. The halfneutralization potential of each component is approximately the same as when titrated alone but the end point potential of the coxiiiiouiici titrated first changed api)ro?iimately 100 to 200 mv. as would be expected. The diffwences in H S P and AE,,, for the two compounds titrated in the first arid third mixture are ideal for analyzing such samples. Since the sampler titrated were comniercial samples, the observed differences were expected. In the second mixture, the small millivolt differences between H S P and
ca.-100
-385
56 1 46 4 82 61 97
82
57.8 45 2 83 8 64 5 99 5 83 5
the lower A E c p make the end points less sharp but the result\ show that the method is quantitative and it is possible to perform differential titrations of mixtures of quaternaries. LITERATURE CITED
(1) Fritz, J. S., Fulda, 11. O., ANAL. CHEX 25, 1837 (1953). ( 2 ) Harlow, G. A , , Ibid., 34, 148 (1962). (3) Murray, R. W.,Iteilley, C. S.,lbicl., D. 313R. ( 4 ) Pifer, C. W., Wollish, E. IT., Ibi-d., 24, 300~ fl9.52).~ ~ , ( 5 ) Iiiddirk, J. A , . Ibid., 32, 178R (1960). ( 6 ) Ibid., p. 1771t. ( 7 ) Streuli, C. A.,Ibid., 30, 997 (1958). (8) \\:?mer, D. C., Ibid., p. 77. ( 9 ) Vi imer, I>. C., Ibid., 34,873 (1962).
MARYE L L E N P Z X H O F F J . H. BENEDICT Ivorydale Technical Center Procter & Gamble Co. Cincinnati, Ohio
Thin Layer Chromatographic Separation of Orthophosphate and Pyrophosphate SIR: Recently the application of chromatographic techniques to difof phosphate mixtures has been made. 1 %use ~ of paper chromatography, \Yestman and Scott (8) have determined members of the family of chain phosphates as high as the dodeca1)hosphate and a number of ring phos1)hates. Two-dimensional chromatography as described by KarlKroupa ( i t ) , can be wed to distinguish clearly betlveen the family of ring and the family of chain phosphates. Paper chroniatogralihy has also been successful in the se1)aration of the various phosphate esters of both orthophosphate and condensed phosphates. Hanes and Isherwood ( 2 ) develolied this method for seliaration of the ~ ~ h o s p h a esters. te Recently. Ohaehi and Iran Wazer (?) have reported the separation of long chain phosphates by paper chromatogral)hy. Hettler (3) compiled a refercnce list including all the work until I958 on the chromatographic separation of phosl)hates. S o method for the separation of
phosphate mixtures by thin layer chromatography was found in the literature. Therefore, a method was developed for the thin layer chromatographic separation of phosphates. This method was evaluated by using phosl)horus-32tagged c o m p u n d s and autoradiograms of the chromatogram. Examination of the autoradiogram was made to decide when adequate separation of the compounds had been achieved. The film used for the autoradiograms was Kodak Royal Medical x-ray film. Time of exposure is dependent on the level of activity on the chromatogram, but in general, the film was exiiosed for 24 to 48 hours. Ph o s 1) h or u s - 32 -1 a be 1e d p y r o p h o sphate was prepared using an adaptation of the procedures given by Lowenstein (6) and Campbell and Kilpatrick ( 1 ) . Cellulose powder (13rinkmann Instruments;, Inc.) was a satisfactory adsorbent for thin layer separation of orthophosphate and pyrophosphate using the solvent system suggested by Kolloff ( S ) . Silica gel G and aluminum
Solvent tront
OO
0 +
+
Orthophosphate
Pyrophosphate
oriqin
Figure 1 . Autoradiogram of orthophosphate and pyrophosphate Chromatogram development time, 1 hour; exposure time for x-ray film, 2 4 hours; temperature, 2 3 ' C.; autoradiogram, 2 0 0 mm. by 50 mm.; solvent front, 1 3 5 mm. from origin; origin, 1 0 mm. from bottom of chromatogram; R j values, 0 . 8 3 (ortho) and 0 . 6 1 (pyro).
VOL. 3 6 ,
NO. 1 1 ,
OCTOBER 1964
2207,
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
