nium chloride. The precipitate was collected and ignited a t 840’ C. Found: R203= 0.5270. Acid Soluble Calcium. The nickelfree filtrate from sample A containing gluconate was evaporated to 150 ml. and acidified. Ammonium oxalate and urea were added. The solution was diluted to 250 ml., heated, and treated with ammonia t o the first permanent turbidity. Heating was continued t o the methyl orange color change. The precipitate was collected, washed in the usual manner, and ignited at 510’ C. Found: Ca (as CaCOs) = 79.08%. EFFECT
OF
nium sulfate hexahydrate. Mohr’s salt (7.02 grams 1.00 grams Fe) was dissolved in 10 ml. of distilled water and 5 ml. of concentrated HN03. The solution was heated until evolution of NOz ceased and was diluted to 150 ml. Twenty-five milliliters of nickel stock solution was added. Further treatment of the solution and precipitation of the nickel were carried out using the procedure given by Willard and Diehl (9), but with the sodium salt of DMG and drying the precipitate at 170” C. Fe present, gram: 1.00; Ni taken, mg.: 25.0; Ni found, mg.: 24.9, 25.1 (tartrate); 25.1 (citrate); 25.2, 25.2 (gluconate).
LARGE AMOUNTS OF IRON
.4 nickel stock solution containing 1.00 mg. of Ni per milliliter waa prepared determinately using previously prepared and analyzed nickel ammo-
LITERATURE CITED
(1) Duval, C., “Inorganic Thermogravimetric Analysis,” pp. 230, 233, Elsevier,
New York, 1953.
(2) Ibid., p. 158.
(3) Hillebrand, W. F., Lundell, G. E. F.. “Applied Inorganic Analysis,” pp. 31517, Wiley, New York, 1929. (4) Kolthoff, I. M., Sandell, E. B., “Textbook of Quantitative Analysis,” pp. 689-90, 3rd ed., Macmillan, New York, 1952. (5) Smith, Rossman, Smith, and Underwood, Royal Oak, Mich., private communication. (6) Smith and Underwood, Royal Oak, Mich., Circular supplied t o purchasers of standardized samples. (7) Walton. H. F.. “Elementarv QuantitaQuantitative Analysis,” p. 883, D Nostrand, Princeton, N. J., 1943. (9) Ibid., p. 385. JAMESS. PROCTOR DAVIDC. FAR WELL^ Goessmann Laboratory University of Massachusetts Amherst, Mass. Present address, Box 46, Dover Road, Wilmington, Vt.
Microscopic Identification of Microgram Quantities of Mannoheptulose and Sedoheptulose SIB: Studies on the distribution of heptuloses in plants required sensitive tests to confirm chromatographic evidence for the occurrence of mannoheptulose (D-manno-heptulose) and sedoheptulose (D-altro-heptulose). This note describes an extension of the solvent diffusion technique for the identification of some pentoses (4, 6) and hexoses (6-7) to the identification of microgram quantities of these heptuloses. METHODS AND OBSERVATIONS
Prepare the plant extract and remove fermentable sugars (1, 8, 9). Chromatograph 10 pg. or more of mannoheptulose or 25 to 50 pg. or more of sedoheptulose on water-washed (7) Whatman No. 1 (chromatographic grade) sheets in ethyl acetate-pyridinewater (8 to 2 to 1) solvent until the maximum separation of the heptuloses from other sugars remaining in the plant extract has been effected. Wash the chromatogram with ethyl ether immediately after its removal from the tank. Locate the test area by means of adjacent guide strips using orcinol reagent (2) on some strips t o show the heptuloses and alkaline silver nitrate on others to show the nonheptulose components. Excise the test area eliminating as much nonheptulose material as possible. Using techniques described (6),elute the sugar with 3 drops of water and carry out the synthesis and observation of the sugar derivative using reagent A or B described below and 1 pl. of glacial acetic acid as the diffusing solvent. Compare the result-
ing products with those obtained with authentic heptuloses and with suitable blanks. To obtain the most sensitive, characteristic, and rapid tests, use reagents . - . A and B only for the sugar specified. Reanent A (for mannoheDtulose test). Grindvan equimolar mixt&e of recry& tallized 1-benzyl-1-phenylhydrazine hydrochloride and recrystallized phenylhydrazine hydrochloride and add, with gentle stirring, 1 mole of anhydrous spdium acetate for each mole of hydrazme. Reagent B (for sedoheptulose test). Grind recrystallized phenylhydrazine hydrochloride and stir in an equimolar amount of anhydrous sodium acetate. Prepare both reagent mixtures daily and keep dry.
-
-
1 Benzyl - 1 phenylhydrazinephenylhydrazine test for mannoheptulose. The compound synthesized in this test is mannoheptulose 1benzyl - 1 - phenylphenylosazone-a new derivative of this sugar. It appears in from 1 to 16 hours, depending on the amount of sugar present, as fine yellow needles growing from a common center in clusters. Growth is rapid after crystallization has started. With large amounts of sugar, the clusters are densely packed, regular in shape, and often have depressed centers. Small amounts of sugar produce larger, less densely packed clusters having needles of varying length. Dendritic formation or long runners may be present. As little as 1pg. of the pure mannoheptulose or 10 pg. (amount applied to the paper)
of the chromatographically separated sugar gives a characteristic product. Phenylhydrazine test for sedoheptulose. The reaction product is sedoheptulose phenylosazone. It appears in from 3 to 16 hours, depending on the amount of sugar present, as fragile, short, light yellow-orange needles in densely packed clumps with no apparent center of growth. The sensitivity of the test is 15 pg. for the pure sugar (5% aqueous sedoheptulosan monohydrate hydrolyzed 400 hours at room temperature with Dowex 50, hydrolysis assumed 10% complete) or from 25 to 50 pg. (amount applied to the paper) for the chromatographically separated sugar. The principal anhydrides of sedoheptulose, 2,7-anhydro-,%~-dtro-heptulopyranose and 2,7-anhydro-,%~-altroheptulofuranose, did not react under the conditions of either of the tests. DISCUSSION
Both mannoheptulose and sedoheptulose give a product in each of the tests described. However, they do not interfere in the tests for each other because they are readily separated chromatographically. Plant extracts contain other sugars that may react in one or both of the tests. Such interference can be eliminated or greatly minimized by fermentation of the plant extract, careful chromatographic separation, and judicious excision of the test area. A thorough study of interference by VOL 33, NO. 9, AUGUST 1961
0
1287
other heptuloses was not made because mannoheptulose and sedoheptulose are the only heptuloses that have been reported to occur in plants (8). Of the heptuloses that may occur in other sources, only very small amounts of D-allo-heptulose, D-gluco-heptulose, and Lgalacto-heptulose were available to us for study. Although each of these sugars reacted with one or both of the reagent combinations, only cgalactoheptulose gave sensitive rapid tests. It can be separated from both mannoheptulose and sedoheptulose by chromatography in ethyl acetate-pyridinewater (8 to 2 to l), and its reaction products differ sufficiently in appearance to preclude confusion. The tests described were used to confirm the occurrence of manno-
heptulose and sedoheptulose in the same fig leaf extract (1). ACKNOWLEDGMENT
We are indebted to Nelson K. Richtmyer for samples of the heptuloses used; and to Kenneth T. Williams and Arthur Bevenue for preparing the plant extracts and for suggesting the urgent need for these tests. LITERATURE CITED
(1) Bevenue, A., White, L. M., Secor,
G. E., Williams, K. T., J . Assoc. OB.
Aar. Chemists. in Dress. (2) Bevinie, A., williams, K. T., J. Chromatog. 4, 391 (1960). (3) Long, C., Sei. Prop. 41,282 (1950). (. 4.) Secor. G. E.. White. L. M., ANAL.
CHEM.27, 1998 (1955)‘
(5) White, L. M., Secor, G. E., Ibid., 27.1016 (1955). 28. 1052 lOk2 (1956). (1956). ( 6 ) ibid.. ibid., 28,
(7j zbg..; (7) Zbid., 31; 31, 1273 (i959j. (1959). ( 8 ) Wdliams, (8) Williams, K. K. T., Bevenue, -4 ., J. Assoc. Ofic. OJic. Agr. Chemists 34, 817 (1951): (9) Wdliams, K. T., Potter, E. F., Bevenue, A., Ibid., 35,483 (1952).
LAWRENCE M. WHITE GERALDINE E. SECOR Western Utilization Research and Development Division Agricultural Research Service U. S. Department of Agriculture Albany 10, Calif. REFERENCE to a company and/or product name by the department is only for purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be sllitable.
Improved Chromatographic Ana lysis of Petroleum Porphyrin Aggregates and Quantitative Measurement by Integral Absorption by W. Warren Howe SIR: I should like to comment upon the paper, “Improved Chromatographic Analysis of Petroleum Porphyrin Aggregates and Quantitative Measurement by Integral Absorption,” by W. Warren Howe [ANAL. CHEM.33, 255 (196l)l: The following remarks are listed in the order in which they appear in the text. 1. All currently known decomplexation methods fail to preserve certain sensitive porphyrin structures. The use of hydrogen bromide-acetic acid has often been recommended for splitting the complexes of very stable porphyrins, such as deoxophyllerythrin or its etioporphyrin. When applied to more sensitive compounds, this reagent is by no means inert and will produce artifacts. Thus, under the conditions used by Dr. Howe, the following conversions, picked a t random, will occur: chlorin es to oxy-meso-chlorin, pheoporphyrin e6 to phylloerythrin, mesopheophorbide to phylloerythrin, pyropheophorbide b to met hoxyet hyl-pheophorbide, rhodinporphyrin g7 to pheoporphyrin b,. In each of these instances the spectrum of the product is different from that of the original material. Aside from this, the hydrogen bromide-acetic acid reagent, if not absolutely free of bromine, will lead to brominated pigments, again of m e r e n t spectrum. 2. Dr. Howe apparently uses the terms “etiochlorin,” “rhodochlorin,” and 1288
ANALYTICAL CHEMISTRY
“phyllochlorin” to designate materials with (mostly hypothetical) spectra having etio-, rhodo-, or phyllotype aside from the chlorin-band. It should be pointed out that these three terms have commonly been used for certain chlorins, different from the ones isolated by Dr. Howe. Etiochlorin has an 8- or 10band spectrum, depending upon the solvent; phyllo- and rhodochlorin both have a spectrum of the intensity sequence (in Dr. Howe’s system) C, IV, I, 11, I11 and not C, IV, 111, 11, I and C, 111, IV, 11, I. I believe that Dr. Howe’s spectra were observed on mixtures or otherwise impure fractions where the relative band intensities were altered by the presence of impurities. 3. The statement that a pigment underwent reversion is a euphemism for its having been destroyed. The reverted pigments-eg., Table IV-are or contain artifacts which had not been present in the crude oil. 4 (Last paragraph, page 256). Etiochlorin I11 has an 8- or 10-band spectrum, depending upon the solvent. The extinction ratio of the 640- to the 500-mfi band is 4.5 in dioxane and 5.4 in hexane. The spectra of other etiochlorins are very similar. Dr. Howe’s pigment differs from etiochlorin in band positions, intensity sequence, and ratio of the chlorin to the 500-mp band (1.2). I believe that the identifica-
tion of his pigment &s an etiochlorin is premature or erroneous. 5. A chlorin “of imperfect spectrum” need not be called a pseudochlorin; a t best, it would be an impure chlorin, or it may be something completely different. 6. According to Dr. Howe, “the fractions are listed in the order in which they come off the column,” and “The position of the porphyrin fraction in the table indicates its polarity. The less polar fractions appear at the top.” In Table IV he lists this sequence as rhodo, etio, etiochlorin. In my own experience the pigments elute off alumina in the reverse order, etiochlorin, followed by etio, and this followed by rhodo. This is also the true order of polarities; etiochlorin has three aromatic pyrrole rings, etio has four pyrrole rings, and rhodo four pyrrole rings and a polar carbonyl group. How can these facts be brought into agreement? 7 (Last paragraph, page 257). I would assume, that the “brown phyllos,” “brown etios,” etc., which follow the elution of the “red pigments” are merely contaminated tail fractions, containing brown material in addition to the pigments. The use of this term does not seem to be justified. 8 (Page 258, bottom). The presence of bands a t 536, 568, and 638 (weak) are not sufficient to establish
