Physical and Chemical Properties of Oligosaccharides1
`
`J. A. JOHNSON and R. SRISUTHEPZ, Kansas State University, Manhattan 66506
`
`ABSTRACT
`
`Maltooligosaccharides (G1 to G12) from partially hydrolyzed amylose starch were partially
`separated on a Celite—carbon column and further separated and purified by macro-paper
`chromatography. The fractions were shown to be pure, straight-chain molecules of a
`homologous
`glucose
`series. Certain
`physical
`and chemical properties of
`these
`oligosaccharides were determined. Reducing power agreed with theoretical values and with
`values of some found in the literature. Specific gravity ofsolutions increased with chain length
`and concentration. Refractive indices did not
`increase with chain length but did with
`increasing concentrations. Solubility decreased with chain length. Oligosaccharides (G1 and
`Gm) did not completely dissolve at 8 to 10% concentrations. Relative viscosity and
`hygroscopicity increased with molecular weight of the oligosaccharides.
`
`Many procedures have been developed to separate maltooligosaccharides
`from starch hydrolysates and to measure certain physical and chemical
`properties of the fractions. Unfortunately, probably because of great difficulty in
`obtaining pure fractions, the values reported for physical properties vary widely.
`To establish physical and chemical properties of these substances would have
`obvious value to scientists and industrial users.
`Many investigators have used carbon columns and ethanol solutions to
`separate the maltooligosaccharides (1-1 l).They obtained pure fractions of the
`lower-molecular—weight sugars with ease, but experienced increasing difficulty as
`they attempted to separate the higher-polymer fractions. Carbon mixed with
`Celite (diatomaceous earth) with ethanol gradients has been used (3,6,10,13,14).
`Celite aids
`in maintaining a uniform flow-rate. Paper chromatography,
`including cellulose columns, with a wide range in solvents, has been used to
`separate maltooligosaccharides (15—22). These separation methods, while
`precise, usually are applied to small quantities of sugars and for identification.
`Other procedures for isolating oligosaccharides have included forming of the
`borate complex (23), carbon—aluminum oxide columns (24), polyacrylamide gels
`(25), cross-linked starch (6), and gas chromatography (26). Derivatives of the
`sugars have limited value when physical and chemical properties of pure sugar
`polymers are to be measured.
`Numerous investigators have measured the physical and chemical properties
`of the maltooligosaccharides but their data do not agree well. R1 values for
`maltooligosaccharides using paper chromatography with various solvents have
`been reported by Jeanes et a1. (17) and by French and Wild (18). Hoover et a1.
`(12) reported values for density, refractive index, viscosity, optical rotation,
`reducing power, and infrared spectra of the maltooligosaccharides from corn
`syrup. Some of their values for the higher—molecular-weight fractions were
`estimated by linear regression. Unusually high reducing values were reported.
`Generally, most measured values increased with chain length, but solubility
`decreased. Commerford and Scallet (27) found the dextrose equivalent to be
`higher than theoretical values for dextrose polymers, G1 to G6. They did not
`
`‘Contribution No. 840, Department of Grain Science and Industry, Kansas Agricultural Experiment Station,
`Manhattan. Condensed from thesis submitted by Rujira Srisuthep in partial fulfillment of requirementsfor Master
`01' Science degree. Kansas State University.
`chspcctively: Professor and Graduate Research Assistant.
`
`
`Copyright © 1975 American Association of Cereal Chemists, Inc., 3340 Pilot Knob Road, St. Paul,
`Minnesota 55121. All rights reserved.
`
`70
`
`

`

`January-February
`
`JOHNSON and SRISUTHEP
`
`71
`
`report on higher-molecular—weight polymers. Birch et a1. (28) suggested that
`copper oxidation was associated with oxidation beyond the terminal reducing
`group of the polymer. Donnelly et a1.
`(29) have shown that degree of
`polymerization has little relation to hygroscopicity.
`In View of the broad interest and wide variation in existing data on physical
`and chemical properties relating to the maltooligosaccharides, we attempted to
`obtain highly purified samples that could be used to measure the physical and
`chemical properties of the homologous glucose series.
`
`MATERIALS AND METHODS
`
`A partially hydrolyzed amylose starch (Morrex T. E.)3 was used as a crude
`source of maltooligosaccharides. Morrex is a dry powder consisting of glucose
`polymers from G1 to G14 but mainly G6 and G7.
`The polymers of Morrex were crudely separated on a large Celite—carbon
`column (6 in. X 12 ft.) filled with a mixture of Darco G—60 carbon, granulated
`carbon (20 mesh), and Celite 535 (4:422). The carbon-Celite was blended and
`water was added to form a thick slurry which was packed in the Pyrex-column.
`After being packed, the column was washed with one gallon of 40% hydrochloric
`acid, then with several gallons of distilled water until the eluant reached a pH of
`3.5. The Celite-carbon column was then loaded with 450 g. of Morrex dissolved
`in 1 liter of water. The column was further washed with 20 gal. of distilled water
`before beginning elution with 5% aqueous ethanol. An elution pressure was
`created by sealing the top of the column with an appropriate cap and elevating
`the eluant reservoir 6 ft. above the column cap. The flow rate was approximately
`0.3 gal. per hr. at first, but slowed to 0.08 gal. per hr. at the end of the elution with
`50% aqueous ethanol.
`Each gallon of eluant was analyzed for total carbohydrate by the method of
`Dubois et al.
`(30,31). Two milliliters of eluant of each gallon of the
`oligosaccharides was concentrated under reduced pressure and was spotted on
`No. 4 Whatman Chromatography paper, which was then irrigated with n-
`propanol-ethyl acetate—water (6:123) for 18 hr. After the paper was dried, the
`chromatogram was dipped successively in silver nitrate, sodium hydroxide in
`methanol and sodium thiosulfate solutions to detect and fix the sugar on the
`paper (32).
`After the eluant had been collected and analyzed for sugar content and type of
`oligosaccharides, the fractions of similar types were combined and concentrated
`in a vacuum evaporator (a dry milk evaporator operated at 26 in. of Hg vacuum
`and 1100 F.). The concentrated solution represented a mixture of neighboring
`polymers, cations, and anions originating from the Celite-carbon column.
`The concentrated solutions were deionized by passing through Amberlite IR-
`100 (H+ form) and Dowex IX-8
`(COs-form) columns. The deionized,
`concentrated solution was further concentrated in a rotary evaporator and
`finally dried to a white powder by lyophylization.
`The
`polymers were
`finally separated on washed 3MM Whatman
`Chromatography paper using the procedure of Commerford et a1. (21). The
`paper was irrigated with n-propanol, ethyl acetate, and water ( 14:3:7); it took 3 to
`
`3Corn Products International, Argo, Ill.
`
`

`

`72
`
`OLIGOSACCHARIDES
`
`Vol. 52
`
`5 days to separate G3 to G: and 8 to 14 days to separate G? to G12
`oligosaccharides. After separating and identifying the polymers, the remaining
`paper was cut into strips and the individual polymers eluted with 5 to [0 ml. of
`deionized water. The water solution ofthe individual polymers was freeze-d ried
`and collected in small vials, which were desiccated over phosphorus pentoxide.
`After collecting many samples ofeach oligosaccharide and combining them, they
`were dissolved in water, filtered and freeze-dried. The samples were further dried
`under vacuum and stored in a desiccator with phosphorus pcntoxide until
`physical and chemical properties could be determined.
`Reducing power was determined by the Somogyi method (33) with reagents
`prepared according to Hodge and Hofreiter (34). Calculations were made
`according to Commerford et a1. (21).
`
`REF.
`
`fill
`
`62
`
`G3
`
`64
`
`GS
`
`66
`
`REF.
`
`6?
`
`G!
`
`69
`
`510
`
`G“
`
`612
`
`REF.
`
`I?-.w
`
`-
`
`.r-
`
`
`
`—swine- 9S ‘a
`
`-..
`
`Fig. 1. Paper chromatograph of maltooligosaccharides separated from Morrax.
`
`

`

`January-February
`
`JOHNSON and SRISUTHEP
`
`-
`
`73
`
`Specific gravity was measured by dissolving samples in water and diluting to l,
`2, 4, 8, and 10% concentrations. Specific gravities were measured in a 2—ml.
`pycnometer at 200 C. The specific gravities were corrected for buoyancy
`according to Hann (35).
`The homogeneity of the fractions was established by paper chromatography
`(Fig. l). The curvilinear nature of the R{ suggested a homogeneous glucose series
`of relatively high purity. In addition, linearity of the individual oligosaccharides
`was established by B—amylolysis (29). The even—numbered oligosaccharides,
`after 24 hr. of B-amylolysis, produced only maltose when tested by TLC, whereas
`the odd-numbered oligosaccharides produced mainly maltose with traces of
`glucose and maltotriose.
`Hygroscopicity of the maltooligosaccharides was determined by placing a
`weighed quantity of each oligosaccharide in an aluminum dish that was placed in
`a desiccator containing sulfuric acid of known concentration to regulate the
`humidity at 60% r.h. (36). In addition, humidities were recorded with an Abbeon
`humidiscope and thermometer. Changes in weight of the dishes and polymers
`were determined periodically for 4.5 hr. Hygroscopicities were expressed as the
`percent increase in weight of the polymers by absorption of water.
`
`RESULTS AND DISCUSSION
`
`While glucose and maltose were present in Morrex in small quantities, they
`
`TABLE I. DEXTROSE EQUIVALENTS (D,E.) AND STOICHIOMETRY
`OF MALTOOLIGOSACCHARIDES
`
`Stoichiometry
`of Polymers
`Commerford
`Degree
`Hoover
`Present ——
`and
`of
`Polymerization
`et al,1
`Scallet2
`Theoretical
`Investigation
`Stoichiometry
`
`(12)
`(27)
`D. E. Value
`D. E. Values
`of Glucose3
`
`G‘
`
`Gz
`
`G3
`
`G4
`
`G5
`
`66
`
`G7
`
`GB
`
`69
`
`Gm
`
`10008
`
`100.00
`
`100.00
`
`100.39
`
`58.10
`
`39.50
`
`29.80
`
`24.20
`
`20.80
`
`71.95
`
`61.63
`
`52.27
`
`53.97
`
`41.14
`
`38.04
`
`3384
`
`31.83
`
`28.67
`
`52.63
`
`35.71
`
`27.03
`
`21 ,74
`
`181 8
`
`15.63
`
`13.70
`
`12.20
`
`10.99
`
`54.34
`
`3783
`
`29.58
`
`23.19
`
`20.36
`
`15.69
`
`13.65
`
`12.57
`
`10.91
`
`1.00
`
`0.98
`
`0.99
`
`1.03
`
`1.01
`
`106
`
`0.93
`
`0 93
`
`0.95
`
`0.93
`
`G“
`10.00
`9.63
`0.89
`G1Z 0.84 9.17 8.42
`
`1Ferricyonide procedure (12)
`2Lane and Eynon procedure (27).
`3Stoichiometry of the copper-sugar reaction (27).
`
`
`
`
`
`

`

`74
`
`OLIGOSACCHARIDES
`
`Vol. 52
`
`TABLE II. TRUE SPECIFIC GRAVITY‘ OF SOLUTIONS OF
`MALTOOLIGOSACCHARIDES AT 20°C.
`
`True Specific Gravity
`Degree of
`
`1%
`2%
`4%
`8%
`10%
`Polymerization
`
`G1
`
`G2
`
`G3
`
`G.
`
`G5
`
`Gs
`
`G7
`
`Ga
`
`1.0018
`
`1.0011
`
`1.0013
`
`1.0014
`
`1.0015
`
`1.0016
`
`1.0019
`
`10020
`
`1.0050
`
`1.0047
`
`1.0049
`
`1.0051
`
`1.0053
`
`1.0057
`
`1.0058
`
`1.0062
`
`1.0131
`
`1.0126
`
`1.0129
`
`1.0130
`
`1.0130
`
`1.0136
`
`1.0138
`
`1.0140
`
`1.0282
`
`1.0266
`
`1.0287
`
`1.0289
`
`1.0290
`
`1.0286
`
`1.0297
`
`1.0296
`
`1.0348
`
`1.0337
`
`1.0364
`
`1.0369
`
`1.0369
`
`1.0386
`
`1.0386
`
`(59
`1.0021
`1.0057
`1.0140
`G10 1.0139 1.0020 1.0056
`
`1Specific gravity in vacuo,
`
`
`
`
`
`were not collected when eluted by 10% aqueous ethanol. Both were obtained
`from commercial sources and purified with batch carbon treatment and
`crystallization. Maltotriose and maltotetraose were eluted together with 20%
`aqueous ethanol. Later
`fractions
`eluted with 20% ethanol contained
`maltotetraose, maltopentaose and maltohexaose. As
`the higher ethanol
`concentrations were used, higher members of the homologous glucose series
`were eluted but always as a mixture of the neighboring polymers.
`The proximities of the R1 values of the longer-chain polymers (Fig. 1) suggest
`why complete separation by Celite-carbon columns was impossible. The
`proximities also explain why good separation could be achieved with 3MM
`paper chromatographs irrigated 7 to 10 days to separate the G7 to G12 polymers.
`The dextrose equivalents of the 12 maltooligosaccharides calculated from the
`reducing power are compared with theoretical and literature values (12,27) in
`Table I. In general, the values agree well with those reported by Commerford and
`Scallet (27) for polymers up to G5 and with the theoretical values. High values
`reported by Hoover et a1. (12) suggest a low degree of purity in separation or
`perhaps, oxidation by ferricyanide beyond the terminal reducing group. The
`stoichiometry was in agreement with values reported by Commerford and Scallet
`(27). Samples G1
`through G5 gave approximately equal relative response
`(approximately 1.0) while G7 through G12 tended to be 10 to 15% less than an
`equivalent amount of glucose. Samples G7 through G12 may have needed longer
`time to react with copper than G1 through G6.
`The relationships between true specific gravity (corrected for buoyance) for 1
`to 10% sugar solutions and degree of polymerization are shown in Table II. There
`is a general
`linear
`relationship between specific gravity and degree of
`polymerization. With larger polymers (G111 through G12) the specific gravity
`
`

`

`January-February
`
`JOHNSON and SRISUTHEP
`
`75
`
`TABLE III. REFRACTIVE INDICES OF MALTOOLIGOSACCHARIDE SOLUTIONS AT 20°C.
`
`
`Refractive Index
`Degree of
`Polymerization
`Oligosaccharide concentration of solution
`1%
`2%
`4%
`8%
`10%
`
`G,
`
`(32
`
`(33
`
`G4
`
`G5
`
`G6
`
`(57
`
`G8
`
`G9
`
`1.3350
`
`1.3349
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3348
`
`1.3367
`
`1.3362
`
`1.3360
`
`1.3361
`
`1.3361
`
`1.3360
`
`1.3360
`
`1.3360
`
`1.3360
`
`1.3392
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3390
`
`1.3451
`
`1.3448
`
`1.3448
`
`1.3449
`
`1.3450
`
`1.3450
`
`1.3450
`
`1.3450
`
`1.3480
`
`1.3480
`
`1.3480
`
`1.3482
`
`1.3483
`
`1.3483
`
`1.3482
`
`(310
`1.3348
`1.3360
`1.3390
`
`
`a
`
`'0
`
`G9
`
`G7
`
`G6
`Gs
`
`G4
`
`63
`
`G2
`
`GI
`
`0
`\5
`k“
`
`.0500
`
`.0400
`
`.0300
`
`.0200
`
`2
`
`4
`
`6
`
`10
`
`CONCENTRATION
`
`(W/v)
`
`Fig. 2. The relationship of
`maltooligosaccharides.
`
`the ratio of specific viscosity to concentration of
`
`

`

`76
`
`OLIGOSACCHARIDES
`
`Vol. 52
`
`TABLE IV. HYGROSCOPICITIES OF MALTOOLIGOSACCHARIDES (G1 to G10)
`AT 60 i 5% RELATlVE HUMIDITY AT 26°C.
`
`
`Moisture, %
`Degree of
`
`Polymerization
`15 min.
`90 min.
`270 min.
`
`G1
`
`G2
`
`Ga
`
`G4
`
`6;,
`
`G6
`
`G7
`
`Ga
`
`G9
`
`1.72
`
`1.45
`
`3.82
`
`4.21
`
`6.21
`
`7.76
`
`4.01
`
`7.48
`
`9.62
`
`0.52
`
`0.33
`
`7.93
`
`9.22
`
`9.94
`
`9.14
`
`9.67
`
`11.53
`
`12.73
`
`0.43
`
`0.34
`
`11.27
`
`10.82
`
`10.10
`
`10.96
`
`11.44
`
`13.23
`
`14.32
`
`
`
`G10 13.92 8.90 12.30
`
`
`
`
`
`could not be measured because of the limited solubility of the polymers. As
`would be expected, the densities (Table II) of the solutions increased with
`concentration of solution.
`Refractive indices of maltooligosaccharides of 1 to 10% solution of G1 to G10
`are summarized in Table 111. These data indicate that refractive index did not
`increase as the size of the polymer increased but as expected increased with the
`concentration of the solution. Refractive indices of 8 and 10% concentrations of
`G9 and G10 could not be measured because of limited solubility.
`Viscosities of solutions of large polymers are defined as the ratio of sheer stress
`per square centimeter to the velocity gradient produced as the solution flows.
`Viscosities of various sugar solutions are listed in the International Critical
`Tables (37) and Viscosities of maltooligosaccharides have been reported by
`Hoover et al. (12). Specific viscosity (nsp.) frequently is used to express the
`relationship of polymer size to viscosity since specific viscosity depends on the
`volume occupied by the polymers (38). Specific viscosity can be used to express
`intrinsic viscosity of the polymers as the concentration approaches zero.
`The relationship of intrinsic viscosity to concentration of oligosaccharides is
`shown in Fig. 2. Maltooligosaccharides Gs to G10 and above had limited
`solubility. Therefore, few values were obtained for these maltooligosaccharides.
`The intrinsic viscosity increased linearly for
`the lower-molecular-weight
`oligosaccharides but for G7 to Go the intrinsic viscosity increased curvilinearly
`with increasing concentrations. The intrinsic viscosity values are generally higher
`than those reported by Hoover et al. (12) but lower than values reported for
`cellodextrins of equivalent chain length (39).
`Hygroscopicity is a characteristic of sugar polymers because of the many
`residual valence forces that attract water through hydrogen bonding. Table IV
`lists percentages of water absorbed by the maltooligosaccharides when exposed
`
`

`

`January—February
`
`JOHNSON and SRISUTHEP
`
`77
`
`to relative humidity of 60 i 5% at 26°C. These data indicate that hygroscopicity
`increased as the polymer became larger. Moisture increase was particularly large
`for polymers G3 to G10.
`
`SUMMARY
`
`Maltooligosaccharides G1 to G12 were separated by Celite-carbon column and
`further separated and purified by macro—paper chromatography. They were
`proved to be free of any branched fractions.
`Reducing power of the maltooligosaccharides agreed closely with theoretical
`values. Specific gravity tended to increase with increasing chain length and
`concentration but refractive indices were identical for all oligosaccharides and
`increased with concentrations. Intrinsic viscosity increased as polymerization
`and concentration increased. Hygroscopicity likewise increased with length of
`the glucose polymer.
`
`Literature Cited
`
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`of carbohydrates on charcoal. Chem. Ind. (London) 1956: 658.
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`J. H. Maltopentaose and crystalline octadea-O—
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`quantitative paper chromatography. Anal. Chem. 27: 1514 (1955).
`8. WHISTLER, R. L.,
`and HICKSON,
`J.
`L. Maltotetraose and
`pentadecacetylmaltotetraitol. J. Amer. Chem. Soc. 76: 1671 (1954).
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`

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`78
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`OLIGOSACCHARIDES
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`Vol. 52
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`
`[Received November 15, 1973. Accepted June 19, 1974]
`
`