in 500 ml of distilled water and add 107 ml of glacial acetic acid. Dilute to 1 liter. FLUORIDE REAGENT.To 300 ml of reagent grade acetone, add 63 ml of the acetate buffer and shake. Follow this with 9.10 ml of A C solution and then 9.10 ml of cerium nitrate solution. Dilute to 500 ml with fluoride-free distilled water and shake well. The reagent is stable for at least five days at room temperature. STANDARD FLUORIDE SOLUTION (Primary). Dissolve 1.103 grams of anhydrous N a F in 1 liter of distilled water. The preparation of a secondary standard by further dilution will be necessary. The primary standard solution is stable for months. Procedure. Add 10 ml of seawater to 30 ml of fluoridefree distilled water (dilution is necessary to come within the concentration range where Beer’s law is followed). Follow this by accurately adding 20 ml of fluoride reagent. After at least 20 and not more than 60 minutes, read the absorbance of the samples and of a distilled water blank against distilled water at 625 nip, in a 2.5-cm cell (0.12-mm slit width). Determine the concentrations by reading from a distilled water (40-ml samples) curve plotted from sample values within the range of 600-1800 pg of F-/liter (these values are on the basis of 10 ml before dilution to 40 ml, as would be the case with 10-ml seawater samples before dilution). For the distilled water standard curve, add 20 ml of fluoride reagent to the 40-ml samples and allow to react for not less than 20 and not more than 60 minutes. The absorbancies of samples containing 600, 1200, and 1800 pg of F-/liter are approximately 0.13, 0.30, and 0.47, respectively (after subtraction of the large blank value of approximately 0.388). The distilled water standard curve is nonlinear below 80 pg of F-/ liter causing the intercept to be to the right of zero. Therefore, it is desirable to read the unknown values directly from the standard curve.
natural seawater. The artificial seawater contained 23.3 grams of NaCl, 10.63 grams of MgClz.6H20, 3.9 grams of Na2S04,1.11 grams of CaCL, 0.725 grams ofKC1,0.20 grams of NaHCO3,0.044 grams of H3B04, 0.0014 grams of LiC1, and 0.097 gram of KBr made up to 1 liter with distilled water. By effectively increasing the salinity to 58%, (salt content equal to that of a 10-ml seawater sample of salinity %go;,,after diluting to 40 ml as in the procedure), the slope remained identical to the natural seawater value, thus showing any salt effect to be quite small. pH and Time. The optimum pH for seawater analysis was found to be 3.85 to 4.40. A value of 4.35 is suggested to allow the use of the same reagent for seawater and distilled water (optimum pH range is 4.25-4.55) use. The optimum time for development of color in seawater samples, read against distilled water blanks, was 10-60 minutes, while distilled water blanks containing the reagent required 20-60 minutes. The time of 20-60 minutes is recommended in the procedure to allow for the slightly longer time required for stabilization of the distilled water reagent blank. DATA FROM THE COLUMBIA RIVER PLUME
The analysis of 196 samples of chlorinity range 17.99-18.99 from the Columbia River Plume region produced 27 samples 61 of in the fluoride/chlorinity range of 6.05-6.14 ( X the 5.95-6.04 (x lO-5), 31 of the 5.85-5.94 ( X lO-5), 46 of the 5.75-5.84 ( X lO-b), and 31 of the 5.65-5.74 ( X 10-5) range. ACKNOWLEDGMENT
DISCUSSION
The authors wish to gratefully acknowledge the technical assistance, helpful conversations, and critical evaluation of this work by Fred E. Palmer, Meredythe Meller, William W. Broenkow, Ralph W. Riley, Joel D. Cline, John C. Jorgenson, Robert V. Thurston, and Clifford A. Barnes.
Standard Curves and Salt Effect. The slope of the linear portion of the standard curve (80-2640 pg of F-/liter) is fairly constant from run to run (absorbance/F- in pg liter N 0.001066), being identical to that run in artificial seawater, and within 1.45% at the 95% confidence level to that run in
RECEIVED for review April 5, 1968, resubmitted December 5 , 1969. Accepted July 2, 1970. Work supported by the U S . Atomic Energy Commission, Grant AT(45-1)-1725 (ref. RLO1725-114). Contribution 453 from the Department of Oceanography, University of Washington, Seattle, Wash.
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Detection of Sugars and Sugar Derivatives by Perchloric Acid on Cellulose Thin-Layers Kinzo Nagasawa, Akira Ogamo, Harue Harada, and Kazuko Kumagai Faculty of Pharmaceutical Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo, Japan
DURINGTHE DETECTION of sugar phosphate esters on paper chromatogram by the Hanes and Isherwood reagent ( I ) , we found that 2-deoxyribose phosphate showed a specific and sensitive coloration different from other sugar phosphates. Later, it was found that aqueous perchloric acid alone, one of the components of the reagent, could cause this color reaction. In general, detection of sugars on chromatograms is based
on the reaction whereby furfural-type compounds resulting from sugars in a strong acidic condition reacted with aromatic amines, phenols, or aldehydes to form colored substances. A mixture of aqueous perchloric acid and vanillin was first reported as a specific spray reagent for sugar alcohols and ketose by Godin (2). A more detailed study revealed that deoxysugars besides sugar alcohols and ketoses were also
(1) C. S. Hanes and F. A. Isherwood, Nature, 164, 1107 (1949).
(2) P. Godin, Nature, 174, 134 (1954).
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ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
detected sensitively by this reagent (3, 4). The present authors confirmed that aqueous perchloric acid alone markedly reduced the sensitivity of sugar alcohols. Therefore, aqueous perchloric acid was expected to show more specificity for sugars, especially for deoxysugars. Weidemann and Fischer ( 5 ) reported the detection limit of three 2-deoxysugars on paper spot by acetone solution of perchloric acid. Comprehensive studies o n color reaction by aqueous perchloric acid were required for detection of sugars o n chromatogram. The present paper describes a systematic investigation of the color reaction of sugars, sugar derivatives, and related compounds by aqueous perchloric acid, the relationship between the chemical structure of sugars and sensitivity of the color reaction, and application of this reaction to detect sugars o n cellulose thin-layer. EXPERIMENTAL
Materials. All materials used were analytical reagent quality wherever possible, but otherwise of the best available grade. The following materials were prepared in this laboratory according to the references cited : Methyl 2-deoxyD-ribofuranoside 5-phosphate (6), methyl 2-deoxy-~-ribofuranoside 3,5-diphosphate (6), 2-deoxy-~-ribofuranoside 5-phosphate ( 6 ) , 2-deoxy-~-ribose diisopropylmercaptal (7), methyl 2-deoxy-~-ribofuranoside (8), methyl 2-deoxy-~ribopyranoside ( 8 ) , 3-deoxy-~-glucose (9, IO), 2-deoxy-~sorbitol (/I), 3-hydroxypropionaldehyde (12, 13), and 4hydroxybutyraldehyde (14). Procedure. THIN-LAYERCHROMATOGRAPHY OF SUGARS, SUGARDERIVATIVES, AND RELATED COMPOUNDS. Silica gel has been widely used in the thin-layer chromatography of sugars. Wolfrom et al. (15) reported that cellulose materials gave more effective separation of water-soluble sugars and sugar derivatives than silica gel, and the solvent systems reported for paper chromatography could be satisfactorily used for cellulose thin-layer chromatography. They also pointed out that “Avicel” (American Viscose Division of F M C Co., Marcus Hook, Pa.), a microcrystalline cellulose, was better than any other form of cellulose. In the present investigation, “Avicel SF” (Funakoshi Pharmaceutical Co. Ltd., and Asahi Kasei Co. Ltd., Tokyo, Japan), a finely powdered product of Avicel for thin-layer chromatography, was used as the adsorbent. PREPARATION OF AVICELCHROMATO-PLATE. A mixture of Avicel SF (15 g) in 60 ml of distilled water was homogenized in a glass homogenizer for about 30 sec. After deaeration with suction, the supension was spread evenly o n 20 glass plates (10 X 10 cm) with a suitable applicator, preset to give 0.25-mm thick layers. The coated plates were kept horizontal and allowed to dry overnight at room temperature and stored in a stocker containing silica gel. (3) M. G. Lambou, ANAL.CHEM., 29, 1449 (1957). (4) A. P. MacLennan, ibid., 31, 2020 (1959). (5) G. Weidemann and W. Fischer, 2. Phys. Ckem., 336, 189 (1964). (6) T. Ukita and K. Nagasawa, Chem. Pliarm. Bull., 7, 655 (1959). (7) H. Zinner, Chem. Ber., 90, 2696 (1957). (8) R. E. Deriaz, W. G . Overend, M. Stacey, and L. F. Wiggins, J . Cliem. Soc., 1949, 2836. (9) M . L. Wolfrom and A . B. Foster, J . Amer. Ckem. SOC.,78, 1399 (1956). (10) E. J . Hedgley. W. G. Overend, and R. A. C. Rennie, J . Cliem. Soc., 1963, 4701. (1 1) M. L. Wolfrom and A. Thommon. “Methods in Carbohydrate ‘ Chemistry,’’ Vol. 2, Academic Press, Inc., New York and London, 1963, p 60. (12) J. B. Lee, J . Clieni. SOC.,1960, 1474. (13) E. Pacsu, S. M. Trister, and J. W. Green, J. Amer. Chem. Soc., 61, 2444 (1939). (14) G. E. Arth, ibid., 75, 2413 (1953). (15) M. L. Wolfrom, D. L. Patin, and R. M. delederkremer, J. Chromatogr., 17, 488 (1965).
Table I. Color Reaction of Sugars by Perchloric Acid Classification Sugara Developed color Aldohexose D-Glucose ... D-Ribose Yellowish brown Aldopentose Ketohexose D-Fructose Dark green 2-Deoxy-~-ghcose Deep purple Deoxysugar 2-Deoxy-~-ribose Pale purple 3-Deoxy-~-glucose Yellow 2,6-Dideoxy-~-allose Dark blue ... D-Glucosamine Aminosugar N-Acetyl-D-glucosamine D-Glucuronolactone Uronic acid ... D-Gluconic acid Aldonic acid D-Mannitol Sugar alcohol a The amount of sugar is 30 pg. b The symbol . . . signifies that no spot is observed. SOLVENTS.The following solvents were used for the chromatography of each compound. Sugars, sugar derivatives, and related compounds: propanol-ethyl acetate-water ( 7 : l :2) (16). Nucleoside and Nucleotide: 0.005N HCl. DETERMINATION OF DETECTION LIMIT. The samples were dissolved in distilled water and 1 p1 of the test solution was spotted on the starting line 1.5 cm from the edge of the plate. The plate was developed ascendingly at 25 “C in a closed tank until the length of rim was 7 cm. The developed plate was moistened by spraying aqueous perchloric acid ( 5 %, w/v), dried at room temperature, heated for 10 min a t 80 “C, and observed for the appearance of spot. Rhamnose (50 pg) was developed in each case as a standard and the detectable limit of a sugar was determined by whether the appearance of the spot tested was faster than that of rhamnose (50 pg) or not. RESULTS AND DISCUSSION
Color Reaction of Sugars. The color reaction of typical simple sugars (30 pg) of various classes by aqueous perchloric acid was examined. Table I gives the positive sugars by tone of color. Deoxysugars and fructose underwent coloration. I n the aldose class, glucose was negative, but ribose was positive. Aminosugar, uronic acid, aldonic acid, and sugar alcohols were entirely negative t o coloration. Because the tone of color produced by aqueous perchloric acid was apt to fade with time, observation of coloration was made within 2 min after color appearance. The color resulting from deoxysugars and ketoses turned gradually dark brown. Therefore, characteristic colors of each class were recognized by comparing the initial tone. Relationship between the Chemical Structure of Sugars and Their Sensitivity of Color Reaction. 2-DEOXYSUGAR. 2Deoxyglucose and its anilide could be detected even at 0.5 pg, The derivatives of 2-deoxyribose, its anilide, diisopropylmercaptal, methyl glycoside, and phosphate esters, colored at the same sensitivity as the free type. The good sensitivity was maintained irrespective of ring form of the sugar. The detection limit of digitoxose, a 2,6-dideoxy sugar, was better than that of 2-deoxysugars. On the other hand, sensitivity of 3-deoxyglucose was markedly reduced to 10 pg, and the color (yellow) was rather different from those of 2-deoxysugars. Hence, the chromogen produced from 2-deoxysugar and that from 3-deoxysugar were considered t o be different. COMPOUNDS RELATEDTO ~-DEOXYSUGAR. Color reaction of compounds related t o 2-deoxysugar was investigated in order to assume the structural unit attributable t o the color reaction. Among the compounds containing a shorter carbon chain, 3-hydroxypropionaldehyde and 3-hydroxybutyralde(16) M. Tomoda, Yakrrgakir Zasshi, 37, 207 (1967).
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Table 11. Detection Limit of Sugars and Sugar Derivatives by Perchloric Acid Reaction Detection Compounds limit, pg Deoxysugar 2-Deoxy-~-glucose 0.5 anilide 0.5 2-Deoxy-D-ribose 1 anilide 1 diisopropylmercaptal 1 furanoside 5-phosphate 2 a$-methylfuranoside 1 a$-methylpyranoside 1 a$-methylfuranoside 5-phosphate 2 a$-methylfuranoside 3,5-diphosphate 2 3-Deoxy-~-glucose 10 2,6-Dideoxy-~-allose(Digitoxose) 0.25 Compounds related to 2-deoxysugar Acetaldehyde 30 Propionaldehyde 30 3-Hydroxypropionaldehyde 5 3-Hydroxybutyraldehyde (Aldol) 5 4-hydroxy butyraldehyde
Capronaldehyde Glutaraldehyde 1,3-Propanediol 1,3-Butanediol 2-Deoxy-~-ribitol 2-Deoxy-~-sorbitol P-Propionolactone y-Butyrolactone Malonic acid Malic acid L-Ascorbic acid Ketose D-Fructose L-Sorbose Dihydroxyacetone Aldose D-Glucose D-GalaCtOSe D-Ribose L- Arabinose D-XylOSe L-Erythrose D,L-Glyceraldehyde Glycolaldehyde L-Rhamnose L-Fucose Nucleoside Adenosine Guanosine Inosine Cytidine Uridine Deoxyadenosine Deoxyguanosine Thymidine Deoxycytidine Deoxyuridine Nucleotide Adenosine 5’-phosphate Guanosine 5’-phosphate Cytidine 5‘-phosphate Uridine 5’-phosphate Deoxyadenosine 5‘-phosphate Deoxyguanosine 5’-phosphate Thymidine 5’-phosphate Deoxycytidine 5‘-phosphate DNA (herring sperm) RNA (yeast)
10 30
20 Negativea Negative 10 20
Negative Negative 50 Negative 50 1 1 10 Negative Negative 25 25 25 1
15 10
50 50
Negativeb Negative Negative Negative Negative 1 1 3
10 5 Negative Negative Negative Negative 1.5 1.5 Negative Negative 5 Negative
Negative means that no spot was observed at 100 pg. * Negative means that no spot was observed at 80 pg. ~~
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hyde (aldol) were detected sensitively, while the sensitivity of 4-hydroxybutyraldehyde was low. The sensitivity of simple aldehydes (acetaldehyde, propionaldehyde, capronaldehyde) was lower than those of hydroxyaldehydes. The sensitivity was lowered when 2-deoxysugar was reduced to the corresponding sugar alcohol. The shorter-chain compounds, such as 1,3-propanediol and 1,3-butanediol, were negative. P-Propionolactone corresponding to 2-deoxyaldonic acid was also negative. I n general, sugars were degraded to furfural derivatives under acidic condition, followed by their polymerization t o form colored humin substances (17). 2-Deoxysugar was assumed to be degraded easily by perchloric acid. On the other hand, the shorter carbon chain compounds such as 3-hydroxypropionaldehyde and aldol which could not turn into furfural derivatives showed coloration at 5 pg. Therefore, the structure CHO-CH2-CHOH- was assumed to be related to the formation of colored substances. KETOSE. Fructose (dark green, 1 pg) and sorbose (dark blue, 1 pg) could be detected sensitively next to deoxysugar, and the dihydroxyacetone, the minimum unit of ketose, was also detected at 10 pg. The color obtained from dihydroxyacetone (yellow) was similar to that of aldose (see below). It is well known that ketoses are more easily degraded to furfural derivatives by acid than aldoses (18). ALDOSE. The sensitivity of aldoses to this color reaction was rather low. Carbon chain compounds shorter than pentose could turn into colored substances, and tetrose (1 pg) was detected more effectively than any other aldoses. The color obtained from aldoses (yellow t o yellowish brown) was different from that of 2-deoxysugar and ketose. Rhamnose (yellow) and fucose (grayish blue-green), which were terminal methyl derivatives of pentose were detected at 50 pg. Color Reaction of Nucleic Acid Constituents. While ribose colored at 25 pg, ribonucleoside and ribonucleotide could not be confirmed even at 100 pg. On the other hand, deoxyribonucleoside colored to perchloric acid, and the purine nucleoside was detected in several times the sensitivity of pyrimidine nucleoside. The difference in the reactivity of deoxyribonucleosides was much more marked in the case of nucleotides. It was noteworthy tht DNA from herring sperm colored sensitively ( 5 pg). Application of Color Reaction to Detection of Sugars. 2Deoxysugars and their derivatives could be detected most sensitively (0.5-2 pg) by aqueous perchloric acid as a purple spot o n Avicel SF layers. Ketoses were also detected sensitively (1-10 pg) next to 2-deoxysugars. The color (dark green to dark blue) resulting from ketoses was distinctly different from those of deoxysugars so that the discrimination of these two classes was possible. Avicel SF layers (10 x 10 cm, 0.25 mm thick) were suitable for the chromatography of sugars. Although the results of the experiments are not shown here, the Avicel SF layers brought about a good separation of sugars with shortening of the developing time and length of the run. The layers were also suitable for the color test by aqueous perchloric acid reagent because of the sensitivity to coloration and stability to the spray reagent. By means of the combination of Avicel SF layer and aqueous perchloric acid reagent, a minute amount of 2-deoxysugars and ketoses could be detected specifically and sensitively. RECEIVED for review March 13, 1970. Accepted July 7,1970. (17) F. H. Newth, Adcart. Carbohyd. Chem., 6 , 83 (1951). (18) W. N. Haworth and W. G. Jones, J. Chem. SOC.,1944, 667.
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