- Email: [email protected]
Polymer formation from aqueous solutions of α-D-glucose by ultrasound and λ-rays
Polymer formation from aqueous solutions of -D-glucose by ultrasound and -rays* G. Portenl~inger and H. Heusinger Institut for Radiochemie, Technische Universit~it MLinchen, Walther-Meissner-Str. 3, D-85747 Garching, Germany The production of polymeric material from aqueous glucose solutions treated by ultrasound or 7-rays was investigated by size-exclusion chromatography (SEC). The polymers show strong electrostatic interactions with the SEC-columns and the non-ideal elution behaviour indicates that charged molecules are produced during irradiation. By optimization of the size-exclusion separation applying salt solutions in the mobile phase it was possible to determine the molecular weight (MW) as a function of irradiation dose. For both types of irradiation in the presence of oxygen, products in the molecular weight range up to 4000 dalton were formed. For sonolysis in the absence of oxygen, the same effect was observed due to the fact that small amounts of oxygen are formed by ultrasound irradiation of water. In the case of 7-irradiation in the absence of oxygen there is no inhibition of the polymerization resulting in higher molecular weights of many thousand dalton. The resulting molecular weights depend on dose and dose rate.
Keywords: ?-rays; glucose; polymerization; size-exclusion chromatography
Interest in the radiation chemistry of carbohydrates resulted from considerations of food preservation using ultrasound and ionizing radiation. Research was performed on the chemical changes caused by the irradiation of ct-D-glucose. The low molecular weight products formed by ultrasound irradiation of aqueous glucose solutions are similar to those formed by exposure to 7-irradiation~ -3, because ultrasound initiated reactions are very similar to those induced by 7-rays 4- 6. OH- and H-radicals produced by ultrasound and 7-rays in aqueous solutions react with carbohydrates by abstracting an H-atom from carbon. In D-glucose, attack at the various positions is almost random even though there appears to be a slight preference for attack at C1-H (being the most loosely bonded) and C6-H which carries two H-atoms. Starting from all these radicals the products formed are mainly governed by reactions of the primary radicals by disproportionation and by elimination of water. Solvated electrons, produced only in the case of ~-irradiation, do not react with carbohydrates at an appreciable rate. For sonolysis 6 and radiolysis 1 in the presence and absence of oxygen, the mechanisms leading to products with six carbon atoms or less are well established. However, the yield of these low molecular weight products from ~;-irradiation in the absence of oxygen was found to be 50% lower than for 7-irradiation in the presence of oxygen 7. Polymeric material was found to be produced by passing aqueous glucose solutions under a beam of high * Paper presented at the "Free radicals and Ultrasound in Chemistry and Medicine" conference held at the Scientific Societies Lecture Theatre, London, 17-18 December 1992
1350-4177/94/020125-05 © 1994 Butterworth-HeinemannLtd
energy electrons in the absence of oxygen8'9. For these polymers produced by y-irradiation in the absence of oxygen, very different molecular weights are given in the literature 1°'~1. For sonolysis, the formation of higher molecular weight products was also observed 12, However, for all irradiation conditions no exact information on the molecular weights of these polymers exists. Due to the fact that glucose is unable to undergo a polymerization reaction producing higher molecular weight products directly, neither the structure of the polymer nor the structure of the monomer unit is known. Using techniques for particle size analysis such as light scattering or, for molecular weight determination, osmometry, no information on the molecular weight distribution is obtained. The molecular weight (MW) average can be the same for different MW-distributions. Using size-exclusion chromatography (SEC) it is possible to evaluate all the MW-averages and the molecular distribution from one measurement. SECcolumns are normally calibrated by the elution times at the peak maximum for a series of narrow standards. Small molecules can penetrate the pore volume of the chromatographic support completely and they elute later than larger molecules which cannot penetrate the total volume of the pore structure. Very large molecules elute at an early stage due to exclusion from the pore structure. For a homologous series of dextrane standards assuming constant flow rate, the elution time is proportional to the logarithm of the molecular weight, as shown in Figure 1. By plotting molecular weight versus elution time it is possible to determine the MW-averages and the MWdistribution of an unknown sample in the linear range
Ultrasonics Sonochemistry 1994 Vol 1 No 2
S125
Polymer formation from glucose by ultrasound and 7-rays: G. Portenlanger and H. Heusinger l0
7
. . . .
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D e t e c ~ f r a c t i v e Index @ 2x 10 2
....
7
, ....
8
~
. . . . . . . . . . . . . . . .
9 10 11 12 elution time in minutes
"~'i i
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13
F i g u r e 1 Multilinear calibration of the Ultrahydrogel 250 SECcolumn using dextrane standards
of the calibration curve. The purpose of the present work was to determine the molecular weights of the polymers formed by irradiation of glucose with ultrasound or 7-rays and to obtain information on the reaction mechanisms of polymer formation.
Experimental details Sonolysis was carried out using a 800 kHz ultrasound generator (Physikalische Werkst/itten, PHYWE GmbH, G&tingen). The transducer had a diameter of 2.5 cm and an average spatial intensity of 2 W cm-a (as calibrated by the manufacturer). A schematic diagram for the ultrasound irradiation experiment has been given in an earlier publicationla. Gamma-irradiations were conducted in a 6°Co source using a 50 ml Schlenk reaction vessel. All irradiations were performed at 21°C in doubly distilled water and at a dose rate of 0.6kGyh -~, determined by the Fricke iron(II)-sulphate dosimeter. 500 #1 samples were evaporated in a vacuum centrifuge and resolved with the HPLC-solvent for chromatography. Columns of the Ultrahydrogel-series (4.8 × 300mm, Millipore) were used for SEC-measurements coupled with a differential refractometer RI 401 (Waters Associates Inc) and a diode array detector 440 (Kontron Instruments). The flow rates were optimized with respect to separation efficiency and peak resolution. The columns were heated to 65 °C because the adsorption of sample molecules is known to be reduced at higher temperatures. The MW-averages and MW-distributions were determined using the computer program GPC Station 4.0 (Polymer Laboratories).
Results and discussion After irradiation of aerated and argon-saturated aqueous solutions of a-D-glucose (5 mM) size-exclusion chromatography (SEC) was performed with an Ultrahydrogel 250 A column, which is designed for the size determination of water-soluble polymers. Dextrane standards, which are
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Ultrasonics Sonochemistry 1994 Vol 1 No 2
polysaccharides containing predominantly straight chains of glucose with a linkage of the alpha- 1,6-type, were used for a multilinear calibration of the SEC-columns. By SEC-analysis of the sonolytically and the radiolyticaUy produced higher molecular weight products from c~-Dglucose with bidistilled water as a mobile phase all higher molecular weight compounds elute near the exclusion limit and gave results greater than the real molecular weight. It is known 13 that this type of non-ideal size-exclusion chromatography is observed if the polarities of the chromatographic support and the mobile phase are very different or if strong electrostatic interactions of the chromatographic support with the sample molecules take place. Commercially available high-performance SEC-columns have anionic character 14. In the case of the crosslinked methacrylate gel packing of our columns, this charge is the result of residual carboxyl groups. By addition of salt to the mobile phase, it is possible to suppress the non-ideal effects15 and to separate the broad peak near the exclusion limit into low molecular weight acidic products and different polymeric fractions. Good high performance SEC-columns will only resolve a mixture into separated peaks if the molecular weights of the succeeding peaks differ by at least a factor of two 13 In order to optimize the size-exclusion separation different salts were investigated as the mobile phase
(Figure 2). Due to the fact that a linear change in elution time results in a logarithmic change in molecular weight (Figure 1) the dependence of elution time on concentration of salt in the mobile phase should be as small as possible. In this respect ammonium formate gave the best results. In addition, ammonium formate can be removed after size-exclusion separation by sublimation at room temperature under high vacuum.
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;
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:
- ~" Na2 03
100 1000 concentration of salt in the mobile phase in mmol/l
Figure 2 Effect of salt concentration in the mobile phase on the elution time of a radiolytically formed polymer from glucose (argon dose rate 2.0 kGy h -1 - dose 380 kGy) with Ultrahydroge1500 (flow rate 0.780 ml min-~; temperature 65 °C; inject 120/~1; detection UV at 254 nm): (1) sodium acetate; (2) sodium dihydrogenphosphate; (3) ammonium acetate; (4) sodium carbonate; (5) ammomum formate
Polymer formation from glucose by ultrasound and 7-rays: G. Portenlanger and H. Heusinger For a polymer from glucose formed by y-irradiation in the absence of oxygen on a logarithmic scheme the elution time shows an almost linear dependence on concentration of ammonium formate in contrast to neutral molecules like dextranes (Figure 3). Due to the constant elution time of the dextrane standards, an interaction between the mobile phase and the chromatographic support cannot be responsible for the elution behaviour of the higher molecular weight products produced by irradiation. Therefore, the chemical structure of the sample molecules must be the origin of the non-ideal elution behaviour on the SEC-columns. This observation is in conformity with the known acidity of the polymers produced by the ?-irradiation of glucose in the absence of oxygen 1~,~6,w However, even for ammonium formate the molecular weight determination for this polymer depends on the salt concentration in the mobile phase. Therefore, other methods for molecular weight determination were applied
(Figure 4). Laser light scattering experiments were used for the molecular weight determination of a polymer formed by y-irradiation of a glucose solution in the absence of oxygen. Intensities of the scattered light were found which result in a molecular weight (Mw) of 97000 dalton compared with light intensities of the dextrane standards. Because a suitable organic solvent could not be found, only water was used for the experiments. At the applied laser wavelength of 495 mm, the polymer shows a slight UV-absorbance, resulting in a decrease of scattered light. Therefore, the real molecular weight should be a little higher. By ultra-filtration with different membrane types and subsequent SEC-measurements we obtained a molecular weight in the range 100000 to 300000 dalton compared with dextrane standards. By ion moderated partition chromatography on a column for carbohydrate analysis (Aminex HPX-87C, Fa. BioRad) we found the elution of the polymer in the region
11
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1000
[ NH4OOCH ] in mMol/l Figure 3 Elution behaviour of a radiolytically produced polymer ( ~ ) from glucose as a function of ammonium formate concentration in the mobile phase compared with dextrane standards ( 0 and [ ] )
Laser Light Scattering (495nm)
Ultrafiltrations !
~
Ion Moderated Partition Chromatography
! i
I
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I
I
I
200000
I 300000
I
I 400000
I
I
I
500000
I 600000
I
I 700000
Nominal molecular weight in dalton
F i g u r e 4 Comparison of molecular weight determination by different methods: (a) size-exclusion chromatography; (b) laser light scattering; (c) ultrafiltration; (d) ion moderated partition chromatography
near the exclusion limit. This column separates molecules by multiple mechanisms depending on the chemical characteristics of each compound and its hydrodynamic volume. The elution time for the unknown polymer compared with dextrane standards was found to be in the range from 80 000 to 270 000 dalton. From these data (Figure 4) based on the data of light scattering, ultra-filtrations and ion moderated partition chromatography, we suggest the real molecular weight is in the range around 130000 dalton. Conducting measurements with ammonium formate concentrations in a range between 15 and 40ram the molecular weight of the unknown polymer compared with dextrane standards was found to be in the range between 80 000 and 150 000 dalton (marked with a circle in Figure 3). We have adjusted the molecular weight average Mw of the unknown polymer by the ammonium formate concentration to the nominal Mw of 135000 dalton. The corresponding concentration of 25ram ammonium formate was used for all investigations on the molecular weights of the polymers produced by irradiation. For ?-irradiations in the presence of oxygen we observed with increasing dose the known decay of glucose and the formation of three different polymer fractions. A fraction in the range of 2000 dalton was already found at low doses. This fi'action decreased at higher doses when two other fractions with molecular weights of 4000 and 500 dalton are formed from the 2000 dalton fraction (Figure 5). The formation of these higher molecular weight products could be detected on the SEC-columns, in contrast to polymer separation by dialysis in the earlier work of Barker et al. 8- lo, which was not appropriate to remove such polymeric material of relatively low molecular weight (Mw < 4000). For 7-irradiations in the absence of oxygen the formation of two fractions in the same molecular weight range as for radiolysis in the presence of oxygen at low
Ultrasonics Sonochemistry 1994 Vol 1 No 2
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Polymer formation from glucose by ultrasound and 7-rays: G. Portenlanger and H. Heusinger doses was observed, but only traces of the 500 dalton fraction appeared (Figure 6). In contrast to irradiation in the presence of oxygen, polymeric species with very different molecular weights were formed at higher doses. The molecular weight increased with dose as expected, but stops at around 65 000 dalton and remains constant up to many hundred kGy. The polydispersity tended towards a single fraction with Mw/Mn = 1.0 (i.e. all molecules becoming a similar hydrodynamic shape). Applying ultrasound in the presence of oxygen in analogy to 7-irradiation, the formation of the three fractions with 500, 2000 and 4000 dalton is also observed
CoIL Tem Ftov
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(Figure 7). 6
JII;t Col, Terr Flo~ Elm Irra, Dos
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7
elution time in minutes
Figure 5 Polymer formation from D-glucose in aqueous oxygen saturated solutions treated by 7-rays as a function of dose
Column: Ultrahvdro~,el® 250~ Tem I Flow Eluel lrrad Dose
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6
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time in minutes
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S128
Ultrasonics Sonochemistry 1994 Vol 1 No 2
7
e[U[IL)II IlIIIC 111 [IIIIIIILC5
Figure 8 Polymer formation from D-glucose in aqueous oxygen free solutions treated by ultrasound as a function of dose
Figure 8 shows the results of ultrasound irradiation in the absence of oxygen. We observed at low doses only two higher molecular weight fractions with 2000 and 4000 dalton, arising from glucose in an analogous way to sonolysis in the presence of oxygen. At higher doses the fraction with 500 dalton appeared, probably by decay of the two polymer fractions with 2000 and 4000 dalton. Small amounts of oxygen produced by ultrasound ~8 in deaerated aqueous solutions are supposed to be responsible for the formation of this product. Owing to the larger oxygen content at higher doses, this effect increases with dose. The lower molecular weights produced in the case of sonolysis cannot be generated by shear forces from traces of higher molecular weight products x9 because the same effect is observed by radiolysis in the presence of oxygen. Many years after the formation of polymers by 7-irradiation in the absence of oxygen was detected 8, we found the same effects for sonolysis. In addition, we were able to show that even in the presence of oxygen, higher molecular weight compounds are produced. By chromatographic methods, the molecular weight averages and the molecular weight distribution for these compounds, produced by irradiation of a-D-glucose in aqueous solutions, could be determined. From all these results we discuss the following mechanisms for the formation of polymeric species from glucose by y-rays and ultrasound in the absence and presence of oxygen based on chromatographic data and on line UV-spectra of different molecular weight fractions. For the structure of the polymer only some preliminary results can be presented at the moment. Applying HPLC and GC/MS-techniques, compounds with molecular weights corresponding to dimeric or trimeric species were not detected, in contrast to earlier suggestions of Barker and Snell ~°'~. Therefore, an intermediate (glucose is transformed by radical reactions of OH- and H-radicals) must be assumed, which produces the polymer not by radical recombination but by a polyaddition reaction. The on-line UV-detection of the polymer fractions demonstrates the existence of e,flunsaturated carbonyls like -C--C-CO- (205-215 nm) and enolic structures like -C--C-OH (250-270nm). This suggests a polyreaction of a conjugated system. By irradiation in the presence of oxygen, very similar UV-spectra were found for 2000 and 4000 dalton fractions, indicating a nearly identical chemical structure. Because the 4000 dalton fraction appeared at a higher
Polymer formation from glucose by ultrasound and ~-rays: G. Portenl~nger and H. Heusinger irradiation dose (after glucose consumption) and under no-irradiation conditions before the 2000 dalton fraction, the 4000 dalton fraction may just be a dimer of the 2000 dalton fraction. The formation of the 2000 dalton fraction is observed for 7- and ultrasound irradiation in the presence and absence of oxygen. Long-term storage of glucose solutions leads exactly to the same 2000 dalton fraction. Thus, the sonolytically and radiolytically induced formation could originate in an acceleration of natural processes, The present work underlines the sugestion that '...the polymer may have originated from the products of oxidation or degradation of glucose by polyaddition reactions'12. In the case of 7-irradiation in the absence of oxygen a polymerization to very high molecular weights takes place. Due to the narrow molecular weight distribution of the polymers (Mw/M, tends towards 1.0) we suggest not a radical polymerization but a polyreaction to the low molecular weight polymer species (Mw < 4000). This proposed mechanism involves radicals only in the step of linkage formation to very high molecular weights. By chromatographic separation and the subsequent removal of ammonium formate we intend to isolate greater amounts of the different polymer fractions (500, 2000 and 4000 dalton) for investigations using NMR, FTIR and mass spectrometry techniques.
2 3 4 5 6 7 8 9 10 11 12 13 14 15
Acknowledgements Financial support from the Deutsche Forschungsgemeinschaft (DFG) is gratefully acknowledged. Mr R. Knopp is thanked for his assistance in performing the laser light scattering experiments.
16 17 18
References yon Sonntag, C. Free-radical reactions of carbohydrates as studied by radiation techniques Adv Carbohydr Chem Biochem (1980) 37 7-77
19
Heusinger, H. A comparison of the degradation products formed in deaerated aqueous or-D-glucose solutions Z Lebensm Unters Forsch (1987) 185 447-456 Heusinger, H. Action of ultrasound on deoxygenated aqueous solutions of D-glucose Carbohydr Res (1988) 181 67-75 El'Piner, I.E. Ultrasound: Physical, Chemical and Biological Effects Consultant Bureau, New York, USA (1964) Henglein, A. Sonochemistry: historical development and modern aspects Ultrasonics (1987) 25 6-16 Heusinger, H. Comparison of the reactions induced by ultrasound and gamma rays in aqueous lactose solutions Ultrasonics (1990) 28 30-36 yon Sonntag, C. The chemical basis of radiation biology Carbohydrates (Chapter 12) Taylor & Francis, London (1986) Barker, S.A., Grant, P.M., Stacey, M. and Ward, R.B. Effects of )'-irradiation. Part I. Polymer formation from sugars, hydroxyacids and amino-acids J Chem Soc (1959) 2648-2658 Barker, S.A., Lloyd, I.R. and Stacey, M. Polymerisation of glucose induced by gamma irradiation Radiat Res (1962) 16 224-231 Barker, S.A,, Lloyd, I.R. and Stacey, M. Structure of a radiationinduced polymer from glucose Radiat Res (1962) 17 619-624 Snell, J.B. Polymer production from aqueous solutions of D-glucose by high-energy radiation J Polym Sci (1965) 3A 2591-2607 Heusinger, H. Gel-permeation chromatography of solutions of D-glucose after irradiation by ultrasound and 'g-rays Carbohydr Res (1991) 209 109-118 gopaeiewiez, W. and Regnier, F.E. Nonideal size-exclusion chromatography of proteins: effects of pH at low ionic strength Anal Biochem (1982) 126 8-16 Pfannkoch, E., Lu, K.C., Regnier, F.E. and Barth, H.G. Characterization of some high performance size-exclusion columns for water-soluble polymers J Chromatogr Sci (1980) 18 43(L441 Size exclusion chromatography of proteins Influence of salt concentration in the mobile phase, Hewlett-Packard HP Peak (1992) 2 6-7 Phillips, G.O. and Criddle, W.J. Radiation chemistry of carbohydrates. Part VIII. The action of "g-radiation on deaerated solutions of D-sorbitol J Chem Soc (1961) 3763-3770 Phillips, G.O. and Criddle, W.J. Radiation chemistry of carbohydrates. Part IX. The action of )'-radiation on deaerated D-mannose solutions J Chem Soc (1962) 2733-2739 Gutierrez, M., Henglein, A. and Fischer, Ch.-H. Hot spot kinetics of the sonolysis of aqueous acetate solutions Int J Radiat Biol (1986) 50313 321 Lorimer, J.P., Mason, T.J. and Cuthbert, T. The effect of ultrasound on the degradation of aqueous native dextran Ultrasonics International Conference Proceedings (1991) 649-654
Ultrasonics Sonochemistry 1994 Vol 1 No 2
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Keywords: ?-rays; glucose; polymerization; size-exclusion chromatography
Interest in the radiation chemistry of carbohydrates resulted from considerations of food preservation using ultrasound and ionizing radiation. Research was performed on the chemical changes caused by the irradiation of ct-D-glucose. The low molecular weight products formed by ultrasound irradiation of aqueous glucose solutions are similar to those formed by exposure to 7-irradiation~ -3, because ultrasound initiated reactions are very similar to those induced by 7-rays 4- 6. OH- and H-radicals produced by ultrasound and 7-rays in aqueous solutions react with carbohydrates by abstracting an H-atom from carbon. In D-glucose, attack at the various positions is almost random even though there appears to be a slight preference for attack at C1-H (being the most loosely bonded) and C6-H which carries two H-atoms. Starting from all these radicals the products formed are mainly governed by reactions of the primary radicals by disproportionation and by elimination of water. Solvated electrons, produced only in the case of ~-irradiation, do not react with carbohydrates at an appreciable rate. For sonolysis 6 and radiolysis 1 in the presence and absence of oxygen, the mechanisms leading to products with six carbon atoms or less are well established. However, the yield of these low molecular weight products from ~;-irradiation in the absence of oxygen was found to be 50% lower than for 7-irradiation in the presence of oxygen 7. Polymeric material was found to be produced by passing aqueous glucose solutions under a beam of high * Paper presented at the "Free radicals and Ultrasound in Chemistry and Medicine" conference held at the Scientific Societies Lecture Theatre, London, 17-18 December 1992
1350-4177/94/020125-05 © 1994 Butterworth-HeinemannLtd
energy electrons in the absence of oxygen8'9. For these polymers produced by y-irradiation in the absence of oxygen, very different molecular weights are given in the literature 1°'~1. For sonolysis, the formation of higher molecular weight products was also observed 12, However, for all irradiation conditions no exact information on the molecular weights of these polymers exists. Due to the fact that glucose is unable to undergo a polymerization reaction producing higher molecular weight products directly, neither the structure of the polymer nor the structure of the monomer unit is known. Using techniques for particle size analysis such as light scattering or, for molecular weight determination, osmometry, no information on the molecular weight distribution is obtained. The molecular weight (MW) average can be the same for different MW-distributions. Using size-exclusion chromatography (SEC) it is possible to evaluate all the MW-averages and the molecular distribution from one measurement. SECcolumns are normally calibrated by the elution times at the peak maximum for a series of narrow standards. Small molecules can penetrate the pore volume of the chromatographic support completely and they elute later than larger molecules which cannot penetrate the total volume of the pore structure. Very large molecules elute at an early stage due to exclusion from the pore structure. For a homologous series of dextrane standards assuming constant flow rate, the elution time is proportional to the logarithm of the molecular weight, as shown in Figure 1. By plotting molecular weight versus elution time it is possible to determine the MW-averages and the MWdistribution of an unknown sample in the linear range
Ultrasonics Sonochemistry 1994 Vol 1 No 2
S125
Polymer formation from glucose by ultrasound and 7-rays: G. Portenlanger and H. Heusinger l0
7
. . . .
~
i
. . . . .
~
1
. . . .
h '
~
i
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~
=
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L
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)
...........................
i~
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D e t e c ~ f r a c t i v e Index @ 2x 10 2
....
7
, ....
8
~
. . . . . . . . . . . . . . . .
9 10 11 12 elution time in minutes
"~'i i
~
13
F i g u r e 1 Multilinear calibration of the Ultrahydrogel 250 SECcolumn using dextrane standards
of the calibration curve. The purpose of the present work was to determine the molecular weights of the polymers formed by irradiation of glucose with ultrasound or 7-rays and to obtain information on the reaction mechanisms of polymer formation.
Experimental details Sonolysis was carried out using a 800 kHz ultrasound generator (Physikalische Werkst/itten, PHYWE GmbH, G&tingen). The transducer had a diameter of 2.5 cm and an average spatial intensity of 2 W cm-a (as calibrated by the manufacturer). A schematic diagram for the ultrasound irradiation experiment has been given in an earlier publicationla. Gamma-irradiations were conducted in a 6°Co source using a 50 ml Schlenk reaction vessel. All irradiations were performed at 21°C in doubly distilled water and at a dose rate of 0.6kGyh -~, determined by the Fricke iron(II)-sulphate dosimeter. 500 #1 samples were evaporated in a vacuum centrifuge and resolved with the HPLC-solvent for chromatography. Columns of the Ultrahydrogel-series (4.8 × 300mm, Millipore) were used for SEC-measurements coupled with a differential refractometer RI 401 (Waters Associates Inc) and a diode array detector 440 (Kontron Instruments). The flow rates were optimized with respect to separation efficiency and peak resolution. The columns were heated to 65 °C because the adsorption of sample molecules is known to be reduced at higher temperatures. The MW-averages and MW-distributions were determined using the computer program GPC Station 4.0 (Polymer Laboratories).
Results and discussion After irradiation of aerated and argon-saturated aqueous solutions of a-D-glucose (5 mM) size-exclusion chromatography (SEC) was performed with an Ultrahydrogel 250 A column, which is designed for the size determination of water-soluble polymers. Dextrane standards, which are
$126
Ultrasonics Sonochemistry 1994 Vol 1 No 2
polysaccharides containing predominantly straight chains of glucose with a linkage of the alpha- 1,6-type, were used for a multilinear calibration of the SEC-columns. By SEC-analysis of the sonolytically and the radiolyticaUy produced higher molecular weight products from c~-Dglucose with bidistilled water as a mobile phase all higher molecular weight compounds elute near the exclusion limit and gave results greater than the real molecular weight. It is known 13 that this type of non-ideal size-exclusion chromatography is observed if the polarities of the chromatographic support and the mobile phase are very different or if strong electrostatic interactions of the chromatographic support with the sample molecules take place. Commercially available high-performance SEC-columns have anionic character 14. In the case of the crosslinked methacrylate gel packing of our columns, this charge is the result of residual carboxyl groups. By addition of salt to the mobile phase, it is possible to suppress the non-ideal effects15 and to separate the broad peak near the exclusion limit into low molecular weight acidic products and different polymeric fractions. Good high performance SEC-columns will only resolve a mixture into separated peaks if the molecular weights of the succeeding peaks differ by at least a factor of two 13 In order to optimize the size-exclusion separation different salts were investigated as the mobile phase
(Figure 2). Due to the fact that a linear change in elution time results in a logarithmic change in molecular weight (Figure 1) the dependence of elution time on concentration of salt in the mobile phase should be as small as possible. In this respect ammonium formate gave the best results. In addition, ammonium formate can be removed after size-exclusion separation by sublimation at room temperature under high vacuum.
11
10
'
!
!
0)
;
ati: ~""
i/i
"~= 9
E
, "4
..... ~ ........ 3 i
7
;
~
10
/
...~.. NH4OAc C
:
- ~" Na2 03
100 1000 concentration of salt in the mobile phase in mmol/l
Figure 2 Effect of salt concentration in the mobile phase on the elution time of a radiolytically formed polymer from glucose (argon dose rate 2.0 kGy h -1 - dose 380 kGy) with Ultrahydroge1500 (flow rate 0.780 ml min-~; temperature 65 °C; inject 120/~1; detection UV at 254 nm): (1) sodium acetate; (2) sodium dihydrogenphosphate; (3) ammonium acetate; (4) sodium carbonate; (5) ammomum formate
Polymer formation from glucose by ultrasound and 7-rays: G. Portenlanger and H. Heusinger For a polymer from glucose formed by y-irradiation in the absence of oxygen on a logarithmic scheme the elution time shows an almost linear dependence on concentration of ammonium formate in contrast to neutral molecules like dextranes (Figure 3). Due to the constant elution time of the dextrane standards, an interaction between the mobile phase and the chromatographic support cannot be responsible for the elution behaviour of the higher molecular weight products produced by irradiation. Therefore, the chemical structure of the sample molecules must be the origin of the non-ideal elution behaviour on the SEC-columns. This observation is in conformity with the known acidity of the polymers produced by the ?-irradiation of glucose in the absence of oxygen 1~,~6,w However, even for ammonium formate the molecular weight determination for this polymer depends on the salt concentration in the mobile phase. Therefore, other methods for molecular weight determination were applied
(Figure 4). Laser light scattering experiments were used for the molecular weight determination of a polymer formed by y-irradiation of a glucose solution in the absence of oxygen. Intensities of the scattered light were found which result in a molecular weight (Mw) of 97000 dalton compared with light intensities of the dextrane standards. Because a suitable organic solvent could not be found, only water was used for the experiments. At the applied laser wavelength of 495 mm, the polymer shows a slight UV-absorbance, resulting in a decrease of scattered light. Therefore, the real molecular weight should be a little higher. By ultra-filtration with different membrane types and subsequent SEC-measurements we obtained a molecular weight in the range 100000 to 300000 dalton compared with dextrane standards. By ion moderated partition chromatography on a column for carbohydrate analysis (Aminex HPX-87C, Fa. BioRad) we found the elution of the polymer in the region
11
t
10
,...,
e*t o ,*a00ar Mw= 809OO
f~..,#~
~-----;--i--~-~-~+~. . . . . . .
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[ NH4OOCH ] in mMol/l Figure 3 Elution behaviour of a radiolytically produced polymer ( ~ ) from glucose as a function of ammonium formate concentration in the mobile phase compared with dextrane standards ( 0 and [ ] )
Laser Light Scattering (495nm)
Ultrafiltrations !
~
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! i
I
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I
200000
I 300000
I
I 400000
I
I
I
500000
I 600000
I
I 700000
Nominal molecular weight in dalton
F i g u r e 4 Comparison of molecular weight determination by different methods: (a) size-exclusion chromatography; (b) laser light scattering; (c) ultrafiltration; (d) ion moderated partition chromatography
near the exclusion limit. This column separates molecules by multiple mechanisms depending on the chemical characteristics of each compound and its hydrodynamic volume. The elution time for the unknown polymer compared with dextrane standards was found to be in the range from 80 000 to 270 000 dalton. From these data (Figure 4) based on the data of light scattering, ultra-filtrations and ion moderated partition chromatography, we suggest the real molecular weight is in the range around 130000 dalton. Conducting measurements with ammonium formate concentrations in a range between 15 and 40ram the molecular weight of the unknown polymer compared with dextrane standards was found to be in the range between 80 000 and 150 000 dalton (marked with a circle in Figure 3). We have adjusted the molecular weight average Mw of the unknown polymer by the ammonium formate concentration to the nominal Mw of 135000 dalton. The corresponding concentration of 25ram ammonium formate was used for all investigations on the molecular weights of the polymers produced by irradiation. For ?-irradiations in the presence of oxygen we observed with increasing dose the known decay of glucose and the formation of three different polymer fractions. A fraction in the range of 2000 dalton was already found at low doses. This fi'action decreased at higher doses when two other fractions with molecular weights of 4000 and 500 dalton are formed from the 2000 dalton fraction (Figure 5). The formation of these higher molecular weight products could be detected on the SEC-columns, in contrast to polymer separation by dialysis in the earlier work of Barker et al. 8- lo, which was not appropriate to remove such polymeric material of relatively low molecular weight (Mw < 4000). For 7-irradiations in the absence of oxygen the formation of two fractions in the same molecular weight range as for radiolysis in the presence of oxygen at low
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Polymer formation from glucose by ultrasound and 7-rays: G. Portenlanger and H. Heusinger doses was observed, but only traces of the 500 dalton fraction appeared (Figure 6). In contrast to irradiation in the presence of oxygen, polymeric species with very different molecular weights were formed at higher doses. The molecular weight increased with dose as expected, but stops at around 65 000 dalton and remains constant up to many hundred kGy. The polydispersity tended towards a single fraction with Mw/Mn = 1.0 (i.e. all molecules becoming a similar hydrodynamic shape). Applying ultrasound in the presence of oxygen in analogy to 7-irradiation, the formation of the three fractions with 500, 2000 and 4000 dalton is also observed
CoIL Tem Ftov
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7
elution time in minutes
Figure 5 Polymer formation from D-glucose in aqueous oxygen saturated solutions treated by 7-rays as a function of dose
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7
6
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time in minutes
Figure 6 Polymer formation from D-glucose in aqueous oxygen free solutions treated by y-rays as a function of dose
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s a t u r a t e d s o l u t i o n s t r e a t e d by u l t r a s o u n d a s a f u n c t i o n of d o s e
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Ultrasonics Sonochemistry 1994 Vol 1 No 2
7
e[U[IL)II IlIIIC 111 [IIIIIIILC5
Figure 8 Polymer formation from D-glucose in aqueous oxygen free solutions treated by ultrasound as a function of dose
Figure 8 shows the results of ultrasound irradiation in the absence of oxygen. We observed at low doses only two higher molecular weight fractions with 2000 and 4000 dalton, arising from glucose in an analogous way to sonolysis in the presence of oxygen. At higher doses the fraction with 500 dalton appeared, probably by decay of the two polymer fractions with 2000 and 4000 dalton. Small amounts of oxygen produced by ultrasound ~8 in deaerated aqueous solutions are supposed to be responsible for the formation of this product. Owing to the larger oxygen content at higher doses, this effect increases with dose. The lower molecular weights produced in the case of sonolysis cannot be generated by shear forces from traces of higher molecular weight products x9 because the same effect is observed by radiolysis in the presence of oxygen. Many years after the formation of polymers by 7-irradiation in the absence of oxygen was detected 8, we found the same effects for sonolysis. In addition, we were able to show that even in the presence of oxygen, higher molecular weight compounds are produced. By chromatographic methods, the molecular weight averages and the molecular weight distribution for these compounds, produced by irradiation of a-D-glucose in aqueous solutions, could be determined. From all these results we discuss the following mechanisms for the formation of polymeric species from glucose by y-rays and ultrasound in the absence and presence of oxygen based on chromatographic data and on line UV-spectra of different molecular weight fractions. For the structure of the polymer only some preliminary results can be presented at the moment. Applying HPLC and GC/MS-techniques, compounds with molecular weights corresponding to dimeric or trimeric species were not detected, in contrast to earlier suggestions of Barker and Snell ~°'~. Therefore, an intermediate (glucose is transformed by radical reactions of OH- and H-radicals) must be assumed, which produces the polymer not by radical recombination but by a polyaddition reaction. The on-line UV-detection of the polymer fractions demonstrates the existence of e,flunsaturated carbonyls like -C--C-CO- (205-215 nm) and enolic structures like -C--C-OH (250-270nm). This suggests a polyreaction of a conjugated system. By irradiation in the presence of oxygen, very similar UV-spectra were found for 2000 and 4000 dalton fractions, indicating a nearly identical chemical structure. Because the 4000 dalton fraction appeared at a higher
Polymer formation from glucose by ultrasound and ~-rays: G. Portenl~nger and H. Heusinger irradiation dose (after glucose consumption) and under no-irradiation conditions before the 2000 dalton fraction, the 4000 dalton fraction may just be a dimer of the 2000 dalton fraction. The formation of the 2000 dalton fraction is observed for 7- and ultrasound irradiation in the presence and absence of oxygen. Long-term storage of glucose solutions leads exactly to the same 2000 dalton fraction. Thus, the sonolytically and radiolytically induced formation could originate in an acceleration of natural processes, The present work underlines the sugestion that '...the polymer may have originated from the products of oxidation or degradation of glucose by polyaddition reactions'12. In the case of 7-irradiation in the absence of oxygen a polymerization to very high molecular weights takes place. Due to the narrow molecular weight distribution of the polymers (Mw/M, tends towards 1.0) we suggest not a radical polymerization but a polyreaction to the low molecular weight polymer species (Mw < 4000). This proposed mechanism involves radicals only in the step of linkage formation to very high molecular weights. By chromatographic separation and the subsequent removal of ammonium formate we intend to isolate greater amounts of the different polymer fractions (500, 2000 and 4000 dalton) for investigations using NMR, FTIR and mass spectrometry techniques.
2 3 4 5 6 7 8 9 10 11 12 13 14 15
Acknowledgements Financial support from the Deutsche Forschungsgemeinschaft (DFG) is gratefully acknowledged. Mr R. Knopp is thanked for his assistance in performing the laser light scattering experiments.
16 17 18
References yon Sonntag, C. Free-radical reactions of carbohydrates as studied by radiation techniques Adv Carbohydr Chem Biochem (1980) 37 7-77
19
Heusinger, H. A comparison of the degradation products formed in deaerated aqueous or-D-glucose solutions Z Lebensm Unters Forsch (1987) 185 447-456 Heusinger, H. Action of ultrasound on deoxygenated aqueous solutions of D-glucose Carbohydr Res (1988) 181 67-75 El'Piner, I.E. Ultrasound: Physical, Chemical and Biological Effects Consultant Bureau, New York, USA (1964) Henglein, A. Sonochemistry: historical development and modern aspects Ultrasonics (1987) 25 6-16 Heusinger, H. Comparison of the reactions induced by ultrasound and gamma rays in aqueous lactose solutions Ultrasonics (1990) 28 30-36 yon Sonntag, C. The chemical basis of radiation biology Carbohydrates (Chapter 12) Taylor & Francis, London (1986) Barker, S.A., Grant, P.M., Stacey, M. and Ward, R.B. Effects of )'-irradiation. Part I. Polymer formation from sugars, hydroxyacids and amino-acids J Chem Soc (1959) 2648-2658 Barker, S.A., Lloyd, I.R. and Stacey, M. Polymerisation of glucose induced by gamma irradiation Radiat Res (1962) 16 224-231 Barker, S.A,, Lloyd, I.R. and Stacey, M. Structure of a radiationinduced polymer from glucose Radiat Res (1962) 17 619-624 Snell, J.B. Polymer production from aqueous solutions of D-glucose by high-energy radiation J Polym Sci (1965) 3A 2591-2607 Heusinger, H. Gel-permeation chromatography of solutions of D-glucose after irradiation by ultrasound and 'g-rays Carbohydr Res (1991) 209 109-118 gopaeiewiez, W. and Regnier, F.E. Nonideal size-exclusion chromatography of proteins: effects of pH at low ionic strength Anal Biochem (1982) 126 8-16 Pfannkoch, E., Lu, K.C., Regnier, F.E. and Barth, H.G. Characterization of some high performance size-exclusion columns for water-soluble polymers J Chromatogr Sci (1980) 18 43(L441 Size exclusion chromatography of proteins Influence of salt concentration in the mobile phase, Hewlett-Packard HP Peak (1992) 2 6-7 Phillips, G.O. and Criddle, W.J. Radiation chemistry of carbohydrates. Part VIII. The action of "g-radiation on deaerated solutions of D-sorbitol J Chem Soc (1961) 3763-3770 Phillips, G.O. and Criddle, W.J. Radiation chemistry of carbohydrates. Part IX. The action of )'-radiation on deaerated D-mannose solutions J Chem Soc (1962) 2733-2739 Gutierrez, M., Henglein, A. and Fischer, Ch.-H. Hot spot kinetics of the sonolysis of aqueous acetate solutions Int J Radiat Biol (1986) 50313 321 Lorimer, J.P., Mason, T.J. and Cuthbert, T. The effect of ultrasound on the degradation of aqueous native dextran Ultrasonics International Conference Proceedings (1991) 649-654
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