Thin-Layer Densitometry of Certain Malto-Oligosaccharides C. T. Mansfield and H. G . McElroy, Jr. Research Department, R . J. Reynolds Industries, Inc., Winston-Salem, N . C. 27102
A MEANS OF ANALYZING large numbers of starch hydrolysates o r corn syrups for malto-oligosaccharides is often needed in product evaluation or in testing methods of starch degradation. Thin-layer methods have been described in the literature for the separation of malto-oligosaccharides which make use of silica gel (1-3))kieselguhr (3-6) or combinations of both (7). The separation system selected for use in our laboratory makes use of 3 parts of Silica Gel G t o 1 part of Kieselguhr G and a developing solvent system of butanol-ethanol-water (5 :3 :2) suggested by Weill and Hanke (6). After separation, the spots are made visible using a n aniline-diphenylamine spray reagent and evaluated using a double-beam, thin-layer densitometer. As described, the method is applicable to dextrose through maltopentaose in syrups or hydrolysates. I t is felt that the method described for the sugars in concern is a n improvement over previously described densitometry methods ( 2 , 3, 7 ) in that a double-beam densitometer and a different combination of developing solvents and visualization systems were used. EXPERIMENTAL Preparation of Thin-Layer Plates. Thirty grams of Silica Gel G and 10 g of Kieselguhr G (Merck) were weighed into a 125-ml Erlenmeyer flask and thoroughly mixed. Then, 85 ml of distilled water was added, and the slurry was shaken for 1 minute. The slurry was deposited o n 20 X 20 cm glass plates to a thickness of 0.5 m m using a Quickfit applicator. The plates were allowed to stand at room temperature for 20 minutes and then transferred to a forced air oven a t 110 O C for 20 minutes. O n cooling to room temperature, the plates were scored into 1-cm strips using a Schoeffel stripping device. Development. The plate was spotted with 1-pl portions of the standards and unknowns using a Hamilton Microliter Syringe. Each spot was dried as soon as possible after application with an air gun so as to keep the initial spot size as small as possible. Spots were placed o n alternate 1-cm strips and positioned 1 cm from the bottom of the plate. A Quickfit spotting template was used as a guide. The outermost strip position o n each edge was not used. After spotting, the plate was placed in a level developing chamber (Kontes Glass Co.) containing 150 ml of developing solvent which was composed of n-butanol-ethanol-water (5:3:2). The plate was allowed t o develop t o a line 15 cm above the spot origin and dried in a forced air oven for 5 minutes a t 110 "C. Visualization. After cooling to room temperature, the plate was sprayed with a color-developing reagent composed of 1 g of aniline, 1 g of diphenylamine, and 10 ml of phosphoric acid in 100 ml of ethanol, which is made fresh daily. Then, the plate was placed in an oven a t 110 "C for 60 minutes. O n removal from the oven, the plate was covered with (1) S. Chiba and T . Shimomura, Agr. Biol. Chem., 29,486 (1965). (2) V. A. de Stefanis and J. G. Ponte, Jr., J . Chromatogr., 34, 116
(1968). ( 3 ) P. Haytko, R. Burns, and C . E. Weill, Cereal Chem., 46, 177
(1969). N. 0. de Souza and A . Panek, J . Chromatogr., 15, 103 (1964). J. C. Shannon and R. G. Creech, ibid.,44, 307 (1969). C. E. Weill and P. Hanke, ANAL.CHEM., 34, 1736 (1962). C. N. Huber, H. Scobell, and Han Tai, Cereal Clzem., 43, 342 (1966).
(4) (5) (6) (7)
586
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
Table I. Recovery Study on 17 Known Samples Average Standard deviation, Sugar recovery Dextrose 105.1 6.37 Maltose 98.3 5.68 Maltotriose 100.4 7.60 Maltotetraose 103.0 9.18 Maltopentaose 98.5 9.4
z
z
a cover glass and allowed to cool to room temperature. Then, the cover glass was removed, and the plate was allowed to remain in contact with the air for 20 minutes. Densitometry. The plate was scanned at 520 nm using a Schoeffel SD-3000 densitometer which was connected to a Schoeffel SDC 300 density computer, a Varian 481 integrator, and a Varian 20 recorder. As a given strip was scanned, the integrator printed out a number directly related to the integrated absorbance of each spot. This, in turn, was proportional to concentration, and a standard curve was constructed for each sugar by plotting integrator reading cs. concentration for the standards. The concentrations of the sugars in the unknowns were calculated by referring the integrator readings of the sugars under consideration t o the respective standard curves. The standard curves were approximately linear. RESULTS AND DISCUSSION
The working range for this method was found to be from 0.1 to 10 mg per ml, with the optimum range being from 0.5 to 5 mg per ml for each of the sugars studied. The densitometer was operated on a range position so that one absorbance unit was full scale on the recorder. This working range could easily be extended by going to a more sensitive range position. Additional sensitivity was not needed on the samples studied in our laboratory. At least 3 standards were prepared covering the working range. Samples were diluted with distilled water so that those sugars of interest would be in the working range. A recovery study was run o n 17 samples, each containing known amounts of the 5 sugars shown. The results are given in Table I. The results in a one-time analysis of the 17 samples were used t o calculate average % recoveries and standard deviations. The average % recovery of dextrose was high due to some slight contamination of other sugars with dextrose. For most accurate work, three standards are spotted in duplicate on the strip positions nearest the sides of the plate, and three samples are placed on the center strip positions. For large numbers of samples, the duplication of spots on a plate can be eliminated, and a number of plates can be developed at the same time. The times required for oven drying of thin-layer plates during plate preparation and spot color development will depend o n the type of oven used. Less background is obtained if color development is carried out in a convection oven rather than a forced air oven. Rotation of plates during color
development is usually desired as most ovens d o not heat uniformly. After color development and removal of the cover glass, the plates are allowed t o remain in contact with the air for a few minutes prior t o scanning as some initial fading occurs. After this initial fading, the colors are stable for 60 t o 90 minutes. Spots should be kept as small as possible t o provide maxim u m separation. Tailing occurs if the spot is overloaded with a given sugar or if salts are present. Salts can be removed by pre-treatment with a mixed-bed ion exchange resin (8). I n some syrups containing large amounts of dextrose, the sample spot can be overloaded. Dextrose, in this case, can be analyzed using the glucose oxidase technique (9). Other sugars present can be analyzed by the method de(8) R. w. Scott, "Clinical Analysis by Thin-Layer Chromatography Techniques," Ann Arbor-Humphrey Science Publishers, Inc., Ann Arbor, Mich., 1969, p 55. (9) Method E-24, Standard Analytical Methods of the Corn Industries Research Foundation, Washington, D. C.
scribed after prior removal of dextrose by treatment with glucose oxidase. A recorder is not needed in the instrumental system, but is an asset in observing spot uniformity, base-line drift, and integrator triggering. After obtaining the SD-3000 densitometer, a slit height control was added. This device was used t o prevent scanning the rough edges of the 1-cm strips. A slit height of 7.5 m m was used. This was larger than the widest spot. This resulted in a smoother base line. This method has been extended to analysis of mono- and disaccharides by changing the thin-layer system. This work will be reported separately. This system has been found by us to be capable of handling a larger number of samples daily than other methods such as gas chromatography or column chromatography.
RECEIVED for review August 26, 1970. Accepted November 30,1970.
Quantitative Determination of DiaIkyI Phosphites and Dialkyl Alkylphosphonates by Gas-Liquid Chromatography Edwin J. Quinn and David H. Ahlstrom Research and Derelopment Center, Armstrong Cork Company, Lancaster, Pa. 17604
DURING A RECENT STUDY concerning the transesterification reaction between dialkyl alkylphosphonates, (RO)*P(O)CH2CHsC02R,and polyoxypropylene sorbitol and sucrose ( I ) , it was necessary to develop a rapid quantitative method t o determine unreacted phosphonate in the presence of volatile and nonvolatile reaction products. The dialkyl alkylphosphonates used in this study were prepared by addition of dialkyl phosphites t o unsaturated carboxylic esters. These dialkyl alkylphosphonates were subsequently added to sucrose and sorbitol polyols to form phosphorus-containing polyols for use in preparing polyurethanes. Previously, Sass and Cassidy ( 2 ) reported a colorimetric method for the detection and quantitative determination of dialkyl hydrogen phosphites in the presence of other phosphorus-containing compounds based on the reaction of the phosphites with trinitrobenzene. However, in our work the reaction of dialkyl alkylphosphonates with polyols gave yellow t o dark brown colored solutions preventing the use of colorimetric methods. Attempts to follow the transesterification reaction between the dialkyl alkylphosphonates and polyoxypropylene sorbitol and sucrose by determining the continuous decrease in hydroxyl value (acetic anhydridelpyridine method) with time were also unsatisfactory and time consuming (1 '/2 hours). Shipotofsky and Moser (3) demonstrated that mixtures of dimethyl and diethyl phosphites could be quantitatively analyzed using GLC. However, their method was not ex___ ~
~~~~
(1) E. J. Q u i n n , Abstracts, Fourth Middle Atlantic Regional Meeting of the American Chemical Society, Washington, D. C., February 1969, No. K17. (2) S. Sass and J. Cassidy, ANAL.CHEM., 28, 1968 (1956). (3) S. H. Shipotofsky and H. C. Moser, ibid., 33,521 (1961).
tended to other phosphites or phosphonates or reactions. Thus, a G L C technique was devised which gave excellent values for unreacted phosphonate within 15 minutes after sampling and was extended to include the quantitative determination of dialkyl phosphites. EXPERIMENTAL
Chromatographic Equipment and Conditions. A HewlettPackard Model 5750 dual column gas chromatograph, Hewlett-Packard Co., Avondale, Pa., equipped with thermal conductivity detectors and a Model 7128A strip chart recorder were used. A disc integrator, Disc Instruments, Inc., Santa Ana, Calif., attached to the recorder was used to measure chromatogram peak areas. The columns were 6 feet by 1/8-inch 0.d. stainless steel coils packed with 5% UCW-98 o n Chromosorb WAW, methyl vinyl silicone rubber on acidwashed white diatomite, Hewlett-Packard Co., Avondale, Pa. F o r the transesterification rate study, periodic reaction samples were taken, cooled in an ice-water bath to room temperature, and weighed into a sample vial. Dimethyl phthalate was accurately weighed into the vial and acetone was added. The acetone solution was then injected into the gas chromatograph for analysis. The column oven was maintained a t 200 " C while the injector and detector temperatures were 300 and 310 "C, respectively. Helium was used as the carrier gas at a flow rate of 20 ml per minute. Relative weight response factors and relative retention times were determined using dimethyl phthalate as the internal standard ( 4 ) . The column temperature was programmed a t a rate of 10" per minute from 100 to 350 "C. The injector (4) L. T. Sennello and C. J. Argondelis, ANAL.CHEM.,41, 171 (1969). ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
587
