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Xanthan biopolymers: A review of methods for the determination of concentration and for the measurement of acetate and pyruvate content
Journal of Petroleum Science and Engineering, 9 ( 1993 ) 273-279
273
Elsevier Science Publishers B.V., Amsterdam
Xanthan biopolymers: A review of methods for the determination of concentration and for the measurement of acetate and pyruvate content Kevin C. Taylor and Hisham A. Nasr-E1-Din* Petroleum Recovery lnstitute, -#100, 3512-33rd Street NW,, Calgary, Alta. T2L 2A6, Canada
(Received February 25, 1993;revised version accepted April 8, 1993)
ABSTRACT Xanthan gum is used extensivelyfor enhanced oil recoveryas a mobilitycontrol agent, in drilling operations to increase the suspension capacity of the drilling mud, and in gels to improve the volumetric sweep efficiency.Flow properties, injectivity, and adsorption characteristics depend on acetate and pyruvate content of xanthan. This reviewdiscussesvarious methods and techniques available for measuring the concentration of xanthan and its pyruvate and acetate content in laboratory and field samples. It includes a description of the principles of each method, advantages, limitations, interferences, and other information necessaryto understand the strengths and weaknessesof each.
Introduction X a n t h a n gum has been used in m a n y oilfield operations including drilling, polymeraugmented water flooding, alkaline flooding, micellar flooding and profile modification (Chatterji and Borchardt, 1981; Nisbet et al., 1982; Sutherland and Kierulf, 1987). Its structure has been reported in detail (Jansson et al., 1975). Accurate m e a s u r e m e n t o f xanthan concentration is important both in field use and in laboratory studies. The pyruvate and acetate contents o f xanthan have a large effect on the rheological properties o f the polymer (Sandford et al., 1977; Smith et al., 1981, 1984; Callet et al., 1987a,b; Rochefort and Middleman, 1987; Kleinitz et al., 1989; Kulicke et al., 1990). X a n t h a n can be prepared with an absence o f either acetate or pyruvate groups, or with neither, by chemical modifi*Current address: Laboratories Department, P.O. Box 62, Saudi Aramco, Dhahran 31311, Saudi Arabia.
cation (Sloneker and Jeanes, 1962; Holzwarth and Ogletree, 1979; R i n a u d o et al. 1983; Cheetham and Punruckvong, 1985 ) or by genetic engineering (Hassler and Doherty, 1990). G r a h a m (1977), H o c k w i n (1974) and Jeanes et al. ( 1976 ) have published limited reviews of xanthan determination. These reviews are very limited in their scope, are no longer current, and do not include methods to measure acetate and pyruvate content. The present work reviews in detail methods for the determination o f xanthan concentration and the measurement o f acetate and pyruvate content. Principles and limitations of each method for use with oilfield samples are discussed.
Discussion A. Determination of xanthan Phenol-sulfuric a c i d m e t h o d
The phenol-sulfuric acid m e t h o d relies on the formation o f a colored complex from the
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274
reaction of sugars with a phenol compound or thioglycolic acid in concentrated sulfuric acid. The method measures the total carbohydrate content of a sample. Phenol, anthrone, orcinol, 3-phenyl phenol, and thioglycolic acid have been used in this method (Auerbach, pers. commun., 1991"*; Dubois et al., 1956; Jeanes et al., 1976; Philips et al., 1985; Quemener, 1986; Ranjbar-Hamghawandi, 1988, 1990, 1992). The method is accurate within _+2%, with a range of 2.5-35 mg/1 sugar (Dubois et al., 1956) and has been used for biopolymer determination in high salinity brine (Ranjbar-Hamghawandi, 1990). The concentration of glucose or other sugars must be known, because they will result in a positive interference (Jeanes et al., 1976). Color generation is affected by high salinity or the presence of Fe 3+ (Philips et al., 1985). Littmann et al. (1992) used the method to determine xanthan concentration in brine produced from a polymer flood field test. Samples were dialyzed prior to analysis to remove salts and potential interferences. The phenol-sulfuric acid method provides good sensitivity and reproducibility. However, the method is relatively slow, and the effect of interferences common in oilfield brines (especially formaldehyde) is largely unknown.
Size exclusion chromatography This method uses a column to separate the polymer from low-molecular-weight impurities. This allows the use of ultraviolet (UV) or refractive index detectors that would otherwise be unsuitable with unpurified samples. The UV detector is more sensitive, with detection limits as low as 0.6 mg/1 (Hunt et al., 1988). Beazley (1985) examined samples from a viscous emulsion produced by a partially hydrolysed polyacrylamide/sulfonate **M.H. Auerbach, pers. commun., 1991. Pfizer Procedure: "Total Carbohydrate Assay by Phenol-Sulfuric Acid Method". Pfizer Central Research, Easter Point Road, Groton, Conn., USA.
K.C. TAYLOR AND H.A. NASR-EL-DIN
surfactant flooding project and concluded that no interference with polymer determination occurred. Similar results are expected for emulsions containing xanthan. Size exclusion chromatography is ideally suited for highly contaminated samples. Sample throughput rates of three to five samples per hour are typical.
Radioactive labelling Carbon-14, a fl-ray emitter, was incorporated into xanthan by adding carbon-14-1abelled glucose to the fermentation broth late in the growth cycle (Sorbie et al., 1987). They produced a carbon-14-labelled xanthan with a specific activity of 1.8 MBq/cm 3 ( 48.6 X 10- 6 Ci/cm 3) at a xanthan concentration of 3200 mg/l. Quantitation is generally performed with a commercial liquid scintillation counter. Goodyear et al. (1992) used carbon-14-labelled xanthan in the measurement of polymer retention levels. Crushed rock with adsorbed polymer was heated to decompose the polymer into carbon dioxide and water. Radioactively labelled carbon dioxide was absorbed in a scintillation cocktail, and radioactivity was measured. The most significant drawback of radioactive labelling as a method for xanthan determination is that it can not be used for commercial samples. Labelled samples must be grown in culture, and their properties may not exactly match a commercial sample of interest.
Organic carbon content Measurement of total organic carbon in aqueous samples has been used by Lecourtier et al. ( 1987 ) and Muller et al. ( 1986 ) for xanthan determination. Solutions containing high amounts of salt may interfere with the complete combustion of carbon compounds, be-
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XANTHAN BIOPOLYMERS
cause sodium carbonate may be formed preferentially. Any carbon containing species will interfere with the method, making it unsuitable for the determination of polymer concentration in the presence of surfactants, biocides such as formaldehyde, or emulsions.
Metachromatic method The metachromatic acid method by Moraine and Rogovin ( 1971 ) uses the dye toluidine blue O which forms an insoluble complex with aqueous xanthan. When the mixture is extracted with a petroleum ether, the complex adsorbs at the interface. The method measures the decrease in absorbance of the aqueous phase as dye is lost due to the formation of the insoluble complex. It is reported to be of use at xanthan concentrations as low as 20 mg/l. The effect of interferences is not known.
Precipitation Xanthan can be purified and isolated by first dissolving the polymer in water and then precipitating it in a water-miscible nonsolvent such as isopropanol (Hassler and Doherty, 1990), methanol (Moraine and Rogovin, 1966) or ethanol (Jeanes et al., 1961; Milas and Rinaudo, 1984). For careful analytical work, several reprecipitations may be necessary. This method can be a useful technique for polymer concentrations greater than 10 g/l.
Viscosity This method requires that viscosity varies proportionally with polymer concentration, and assumes that the viscosity of xanthan solutions is independent of salinity. By measuring viscosity at a fixed shear rate for a range of polymer concentrations, Philips et al. (1985) obtained a different nonlinear calibration curve for each commercial polymer used. Any change in temperature, pH, shear rate, or mo-
lecular weight distribution will affect the solution viscosity, reducing the accuracy of measurement.
Refractive index This method requires polymer concentration to be proportional to the refractive index of the solution and has been used by Quemen e r ( 1 9 8 6 ) . The specific refractive index increment of xanthan was found to be 0.144 cm3/g at 633 nm (Hunt et al., 1988 ) and 0.155 cm3/g at 546 nm (Milas and Rinaudo, 1984). Lindstr~Sm and Srremark (1976) used a Knauer refractometer thermostatted to _+0.005°C, and claimed an accuracy of _+1 mg/1 for acrylamide copolymers in wood pulp suspensions in distilled water. The refractive index increment of this polymer is similar to that ofxanthan (Hunt et al., 1988). The use of refractive index for the determination of polymer concentration can produce acceptable results only when solutions of low, constant ionic strength are used. Temperature must be accurately controlled to reduce changes in refractive index. Ideally, only solutions of xanthan in distilled water or dialyzed samples should be considered for this method.
Hydrolysis with glucose determination Hassler and Doherty (1990) quantitated xanthan by first hydrolysing the polymer to its constituent sugars, then determining glucose concentration with an enzyme test kit (Sigma Procedure No. 16-UV) using hexokinase and glucose-6-phosphate dehydrogenase. This method is also useful for determining glucose impurities in xanthan. The efficiency and reproducibility of the hydrolysis step has not been established, nor has the method been evaluated for interferences common in oilfield brines.
276
Carbazole method Glucuronic acid ( 18.4 wt% ofxanthan) can be determined colorimetrically by the carbazole method (Wernau, 1981 ), which is similar to the phenol-sulfuric acid method. The original carbazole method of Dische (1947) was modified by Knutson and Jeanes (1968) for use with polysaccharides. The range is from 10 to 70 mg/l glucuronic acid. Bitter and Muir ( 1962 ) reported that glucose, formaldehyde, and salts interfere with the method.
Gas chromatography Gas chromatography has been used to determine xanthan concentration after hydrolysis and derivatization (Lawrence and Iyengar, 1985). Xanthan is hydrolysed and the resulting sugars converted into volatile aldonitrile acetates. Xanthan concentration is determined from the concentration of the constituent sugars. The presence of other carbohydrates will result in interference with the method. B. Measurement of acetate and pyruvate content 1. Methods for measurement of both acetate and pyruvate
K.C. TAYLOR AND H.A. NASR-EL-DIN
Nuclear magnetic resonance (NMR) spectroscopy Several groups (Morris et al., 1977; Smith et al., 1981 ) have used NMR to determine only the pyruvate to acetate ratio. They used an independent method to determine pyruvate content and then calculated the acetate content. Other groups have used an internal standard so that acetate and pyruvate can be measured directly (Paradossi and Brant, 1982; Lambert and Rinaudo, 1985; Milas and Rinaudo, 1986; Callet et al., 1987a; Milas et al., 1988). Rinaudo et al. (1983) and Kulicke et al. (1990) measured pyruvate and acetate content directly by relating the peak area of these groups to the only equatorial anomeric proton of the xanthan molecule. Kulicke et al. found good agreement between their NMR measurements and enzymatic methods. High measurement temperatures (90 °C) are required, and the polymer must be purified and dry. A measurement time of about 45 min is required after the molecular weight of the polymer has been reduced by ultrasonification. The method of Kulicke et al. (1990) is recommended. 2. Methods for measurement of acetate only
Hydrolys& and titration High performance liquid chromatography (HPLC) The HPLC method for the determination of acetyl and pyruvyl groups in xanthan uses a strong acid cation exchange resin as the column packing material. After hydrolysis of a small sample ( 10 to 20/tl of a 5 g/1 polymer solution), acetate and pyruvate groups are determined (Ash et al, 1983; Cheetham and Punruckvong, 1985; Hassler and Doherty, 1990).
Xanthan is hydrolyzed under basic conditions. Acetate content is determined by titrating the hydrolyzed product with acid (Sloneker and Jeanes, 1962; Rinaudo et al., 1983 ) or by acidification, distillation, and titration of resulting acetic acid (Schultz, 1965). Any acidic materials present which can react with base may interfere with the method. Pyruvate may decompose under acidic conditions to produce acetic acid, resulting in a positive interference.
277
XANTHAN BIOPOLYMERS
Hydroxamic acid method The acetate ester in xanthan reacts with hydroxamic acid to produce acetohydroxamic acid. This acid forms a water-soluble complex with iron that absorbs light at 520 nm. The method is applicable for the determination of 2 to 10 micromoles of acetate groups per mL (200 #1 sample ), with an accuracy within + 2% (Hestrin, 1949; McComb and McCready, 1957; Sutherland and Wilkinson, 1968). This method does not require hydrolysis of the acetate groups present in xanthan prior to the determination. Interferences, however, from species present in oilfield brines are unknown.
requires 2 ml of a sample containing approximately 2 g/1 of acid-hydrolyzed xanthan, and is reproducible to within 2%. The most serious disadvantage of the method is the relatively large number of steps involved and interference from formaldehyde (Hirase, 1957), which is usually added to xanthan as a biocide.
Oxidation and determination of carbon dioxide Hirase ( 1957 ) has determined the pyrnvate content of agar by oxidation with sodium metaperiodate and gravimetric determination of the liberated carbon dioxide. The same method can be used to determine pyruvate content in xanthan.
3. Methods for measurement of pyruvate only Conclusions
Lactate dehydrogenase method The lactate dehydrogenase method for the determination of pyruvate in polysaccharides is discussed in detail by Duckworth and Yaphe (1970). Application of the method to xanthan is discussed by Jeanes et al. (1976). Both methods are based on the work of Hadjivassiliou and Reider (1968 ). The pyruvate group in xanthan is first hydrolyzed using oxalic acid or dilute HC1. This is the method of choice for the determination ofpyruvate content in xanthan. It is specific, requires only 3 to 5 mg of polymer, and is readily carried out with a minimum of equipment.
2, 4-dinitrophenylhydrazine method (2, 4-DNP) Pyruvic acid reacts with 2,4-DNP to produce a 2,4-dinitrophenylhydrazone derivative which is measured colorimetrically. The method has been used extensively for the determination of pyruvate in xanthan (Koepsell and Sharpe, 1952; Sloneker and Jeanes, 1962; Sloneker and Orentas, 1962; Cheetham and Punruckvong 1985; Shatwell et al., 1990). It
The determination of concentration is important to the study of xanthan in enhanced oil recovery, in drilling operations and in gels. The measurement of acetate and pyruvate content is important because these two parameters have a large effect on the rheological behaviour, adsorption, and injectivity of xanthan solutions. Size exclusion chromatography with ultraviolet detection was found to be the most suitable method for xanthan determination in oilfield samples. The method is accurate and is not affected by impurities common in oilfield brines. The phenol-sulfuric acid method is acceptable if interferences from other carbohydrates, iron, and salinity are accounted for. Proton NMR is the method of choice for the simultaneous measurement of acetate and pyruvate content in xanthan. Alternately, pyruvate content can be measured using the lactate dehydrogenase method, or both acetate and pyruvate can be measured using HPLC. The effect of salts, divalent ions, and formaldehyde, common interferences in oilfield brines, is not generally known for methods that measure acetate and pyrnvate content in xanthan.
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Acknowledgements The authors would like to thank Janet Noy for conducting a literature search which identified many of the papers used in this report; Lynne Story and Wendy Faid, I.N. Mckinnon memorial library, for their help in obtaining many of the papers reviewed; and Prof. G.V. Chilingarian for useful discussions. References Ash, S.G., Clarke-Sturman, A.J., Clavert, R. and Nisbet, T.M., 1983. Chemical stability of biopolymersolutions. SPE 12085, 58th Annu. Tech. Conf. Exhib., San Francisco, Calif. Beazley, P.M., 1985. Quantitative determination of partially hydrolyzed polyacrylamide polymers in oilfield production water. Anal. Chem., 57( 11 ): 2098-2101. Bitter, T. and Muir, H.M., 1962. A modified uronic acid carbazole reaction. Anal. Biochem., 4: 330-334. Callet, F., Milas, M. and Rinaudo, M., 1987a. Influence of acetyl and pyruvate contents on rheological properties of xanthan in dilute solution. Int. J. Biol. Macromol., 9: 291-293. Callet, F., Milas, M. and Tinland, B., 1987b. Role of the xanthan structure on the rheological properties in aqueous solutions. 4th Int. Conf. Gums and Stabilisers for the Food Industry, Wrexham, Clwyd, Wales. Chatterji, J. and Borchardt, J.K., 1981. Applications of water-soluble polymers in the oil field. J. Pet. Technol., 33: 2042-2056. Cheetham, N.W.H. and Punruckvong, A., 1985. An HPLC method for the determination of acetyl and pyruvyl groups in polysaccharides. Carbohydr. Polym., 5: 399406. Dische, Z., 1947. A new specific color reaction of hexuronic acids. J. Biol. Chem., 167: 189-198. Dubois, M, Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Duckworth, M. and Yaphe, W., 1970. Definitive assay for pyruvic acid in agar and other algal polysaccharides. Chem. Ind. (June): 747-748. Goodyear, S.G., Johnston, J.D., Lawless, T.A. and Woods, C.L., 1992. Measurement of polymer retention levels representative of the formation. SPE/DOE 8th Symp. Enhanced Oil Recovery, Tulsa, Okla., SPE/DOE 24155. Graham, H.D., Editor, 1977. Analytical Methods for Major Plant Hydrocolloids. Avi Publ. Co. Inc., Westport, Conn., pp. 540-579. Hadjivassiliou, A.G. and Rieder, S.V., 1968. The enzy-
K.C. TAYLOR AND H.A. NASR-EL-DIN
matic assay of pyruvic and lactic acids: adefinitive procedure. Clin. Chim. Acta, 19:357-361. Hassler, R.A. and Doherty, D.H., 1990. Genetic engineering of polysaccharide structure: production ofvariants of xanthan gum in Xanthomonas campestris. Biotechnol. Progr., 6:182-187. Hestrin, S., 1949. The reaction ofacetylcholine and other carboxylic acid derivatives with hydro×ylamine, and its analytical application. J. Biol. Chem., 180:249-261. Hirase, S., 1957. Studies on the chemical constitution of agar-agar. XIX. pyruvic acid as a constituent of agaragar (Part 1 ). Identification and estimation of pyruvic acid in the hydrolysate of agar. Bull. Chem. Soc. Jpn, 30( 1 ): 68-79. Hockwin, O., 1974. Separation and determination ofpolysaccharides. Method. Chim., 1B: 1006-1013. Holzwarth, G. and Ogletree, J., 1979. Pyruvate-free xanthan. Carbohydr. Res., 76: 277-280. Hunt, J.A., Young, T.S., Green, D.W. and Willhite, G.P., 1988. Size-exclusion chromatography in the measurement of concentration and molecular weight of some EOR polymers. Soc. Pet. Eng. Res. Eng., 3(3): 835841. Jansson, P., Kenne, L. and Lindberg, B., 1975. Structure of the extracellular polysaccharide from Xanthomonas campestris. Carbohydr. Res., 45: 275-282. Jeanes, A., Pittsley, J.E. and Senti, F.R., 1961. Polysaccharide B-1459: a new hydrocolloid polyelectrolyte produced from glucose by bacterial fermentation. J. Appl. Polym. Sci., 5( 17): 519-526. Jeanes, A., Rogovin, P., Cadmus, M.C., Silman, R.W. and Knutson, C.A., 1976. Polysaccharide (xanthan) of Xanthomonas campestris NRRL B-1459: procedures for culture maintenance and polysaccharide production, purification, and analysis. ARS-NC51, USDA. Kleinitz, W., Littmann, W. and Herbst, H., 1989. Screening of xanthan-biopolymer for a high salinity oil reservoir. Proc. Symp. 5th Hung. Hydrocarbon Inst. Impr. Oil Recovery Europe. Budapest, Hungary, pp. 319328. Knutson, C.A. and Jeanes, A., 1968. A new modification of the carbazole analysis: application to heteropolysaccharides. Anal. Biochem., 24:470-481. Koepsell, H.J. and Sharpe, E.S., 1952. Microdetermination of pyruvic and a-ketoglutaric acids. Arch. Biochem. Biophys., 38: 443-449. Kulicke, W.M., Oertel, R., Otto, M., Kleinitz, W. and Littmann, W., 1990. Characterization of xanthan solutions for application in EOR. Erd61 Kohle Erdgas Petrochem. Brennst.-Chem., 43 ( 12 ): 471-476. Lambert, F. and Rinaudo, M., 1985. On the thermal stability of xanthan gum. Polymer, 26:1549-1553. Lawrence, J.F. and lyengar, J.R., 1985. Gas chromatographic determination of polysaccharide gums in foods after hydrolysis and derivatization. J. Chromatogr., 350: 237-244.
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Lecourtier, J., No'tic, C., Barbey, P. and Chauveteau, G., 1987. Semi-rigid polysaccharides for polymer flooding in high salinity reservoirs. Syrup. Proc. 4th BASF AG et al. Enhanced Oil Recovery Europe., pp. 105116. Lindstr~m, T. and SOremark, C., 1976. Adsorption of cationic Polyacrylamides on Cellulose. J. Colloid Interface Sci., 55(2): 305-312. Littmann, W., Kleinitz, W., Christensen, B.E. and Stokke, B.T., 1992. Late results of a polymer pilot test: performance, simulation, absorption, and xanthan stability in the reservoir. SPE/DOE 8th Symp. Enhanced Oil Recovery, Tulsa, Okla., SPE/DOE 24120. McComb, E.A. and McCready, R.M., 1957. Determination of acetyl in pectin and in acetylated carbohydrate polymers. Anal. Chem. 29(5): 819-821. Milas, M. and Rinaudo, M., 1984. On the existence of two different secondary structures for the xanthan in aqueous solutions. Polym. Bull., 12: 507-514. Milas, M. and Rinaudo, M., 1986. Properties ofxanthan gum in aqueous solutions: role of the conformational transition. Carbohydr. Res., 158:191-204. Milas, M., Linossier, J.L. and Contat, F., 1988. The effect of thermal aging on xanthan solutions. J.Appl. Polym. Sci., 35: 1115-1122. Moraine, R.A. and Rogovin, P., 1966. Kinetics of polysaccharide B- 1459 fermentation. Biotechnol. Bioeng., 8:511-524. Moraine, R.A. and Rogovin, P., 1971. Xanthan biopolymer production at increased concentration by pH control. Biotechnol. Bioeng., 13: 381-391. Morris, E.R., Rees, D.A., Young, G., Walkinshaw, M.D. and Darke, A., 1977. Order-disorder transition for a bacterial polysaccharide in solution. J. Mol. Biol., 110: 1-16. Muller, G., Anrhourrache, M., Lecourtier, J. and Chauveteau, G., 1986. Salt dependence of the conformation of a single-stranded xanthan. Int. J. Biol. Macromol., 8: 167-172. Nisbet, B.A., Bradshaw, l.J., Kerr, M. and Sutherland, I.W., 1982. Characteristics of microbial exopolysaccharides for E.O.R. use. Proc. 2nd Eur. Syrup. EOR, Paris, France, pp. 71-77. Paradossi, G. and Brant, D.A., 1982. Light scattering study of a series of xanthan fractions in aqueous solution. Macromolecules, 15: 874-879. Philips, J.C., Miller, J.W., Wernau, W.C., Tate, B.E. and Auerbach, M.H., 1985. A high-pyruvate xanthan for EOR. Soc. Pet. Eng. J., 25(4): 594-602. Quemener, B., 1986. Mrthodes d'identification et de dosage des polysaccharides. In: P. Colonna and J.F. Thibault (Editors), Propr. Fonct. Polysaccharides. Conf. Sess., pp. 333-364. Ranjbar-Hamghawandi, M., 1988. Quantitative photo-
279 metric microdetermination of polysaccharides. Fresenius Z. Anal. Chem., 331: 759-760. Ranjbar-Hamghawandi, M., 1990. Photometric determination of biopolysaccharides in brine. Fresenius. Z. Anal. Chem., 366: 41-42. Ranjbar, M., 1992. Colorimetric determination of xanthan with thioglycolic acid. Erdrl Erdgas Kohle, 108(2): 74-75. Rinaudo, M., Milas, M., Lambert, F. and Vincendon, M., 1983.1H and ~3C NMR investigation ofxanthan gum. Macromol., 16:816-819. Rochefort, W.E. and Middleman, S., 1987. Rheology of xanthan gum: salt, temperature, and strain effects in oscillatory and steady shear experiments. J. Rheol., 31: 337-369. Sandford, P.A., Pittsley, J.E., Knutson, C.A., Watson, P.R., Cadmus, M.C. and Jeanes, A., 1977. Variation in xanthomonas campestris NRRLB- 1459; characterization of xanthan products of differing pyruvic acid content. In: P.A. Sandford and A. Laskin (Editors), Extracellular Microbial Polysaccharides. Am. Chem. Soc., Washington, DC, Syrup. Ser., 45: 192-210. Schultz, T.H., 1965. Determination of acetyl in pectin. Methods Carbohydr. Chem., 5:187-189. Shatwell, K.P., Sutherland, I.W., Dea, I.C.M. and RossMurphy, S.B., 1990. The influence of acetyl and pyruvate substituents on the helix-Coil transition behaviour ofxanthan. Carbohydr. Res., 206: 87-103. Sloneker, J.H. and Jeanes, A., 1962. Exocellular bacterial polysaccharide from xanthomonas campestris NRRL B-1459. Can. J. Chem., 40: 2066-2071. Sloneker, J.H. and Orentas, D.G., 1962. Pyruvic acid, a unique component of an exocellular bacterial polysaccharide. Nature, 194: 478-479. Smith, 1.H., Symes, K.C., Lawson, C.J. and Morris, E.R., 1981. Influence of the pyruvate content of xanthan on macromolecular association in solution, Int. J. Biol. Macromol., 3: 129-134. Smith, I.H., Symes, K.C., Lawson, C.J. and Morris, E.R,, 1984. The effect of pyruvate on xanthansolution properties. Carbohydr. Polym., 4: 153-157. Sorbie, K.S., Parker, A. and Clifford, P.J., 1987. Experimental and theoretical study of polymer flowin porous media. Soc. Pet. Eng. Res. Eng., 2(3): 281-304. Sutherland, I.W. and Kierulf, C., 1987. Downhole use of biopolymers. Inst. Petrol. Microbiol. Comm. Microbial Problems in the Offshore Oil Industry. Int. Conf. Aberdeen, 1986, pp. 93-103. Sutherland, I.W. and Wilkinson, J.F., 1968. The exopolysaccharide of Klebsiella aerogenes A3 (SI)(Type 54). Biochem. J., 110: 749-754. Wernau, W.C., 1981. Xanthomonas biopolymer for use in displacement of oil from partially depleted reservoirs. US Patent No. 4,296,203.
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Elsevier Science Publishers B.V., Amsterdam
Xanthan biopolymers: A review of methods for the determination of concentration and for the measurement of acetate and pyruvate content Kevin C. Taylor and Hisham A. Nasr-E1-Din* Petroleum Recovery lnstitute, -#100, 3512-33rd Street NW,, Calgary, Alta. T2L 2A6, Canada
(Received February 25, 1993;revised version accepted April 8, 1993)
ABSTRACT Xanthan gum is used extensivelyfor enhanced oil recoveryas a mobilitycontrol agent, in drilling operations to increase the suspension capacity of the drilling mud, and in gels to improve the volumetric sweep efficiency.Flow properties, injectivity, and adsorption characteristics depend on acetate and pyruvate content of xanthan. This reviewdiscussesvarious methods and techniques available for measuring the concentration of xanthan and its pyruvate and acetate content in laboratory and field samples. It includes a description of the principles of each method, advantages, limitations, interferences, and other information necessaryto understand the strengths and weaknessesof each.
Introduction X a n t h a n gum has been used in m a n y oilfield operations including drilling, polymeraugmented water flooding, alkaline flooding, micellar flooding and profile modification (Chatterji and Borchardt, 1981; Nisbet et al., 1982; Sutherland and Kierulf, 1987). Its structure has been reported in detail (Jansson et al., 1975). Accurate m e a s u r e m e n t o f xanthan concentration is important both in field use and in laboratory studies. The pyruvate and acetate contents o f xanthan have a large effect on the rheological properties o f the polymer (Sandford et al., 1977; Smith et al., 1981, 1984; Callet et al., 1987a,b; Rochefort and Middleman, 1987; Kleinitz et al., 1989; Kulicke et al., 1990). X a n t h a n can be prepared with an absence o f either acetate or pyruvate groups, or with neither, by chemical modifi*Current address: Laboratories Department, P.O. Box 62, Saudi Aramco, Dhahran 31311, Saudi Arabia.
cation (Sloneker and Jeanes, 1962; Holzwarth and Ogletree, 1979; R i n a u d o et al. 1983; Cheetham and Punruckvong, 1985 ) or by genetic engineering (Hassler and Doherty, 1990). G r a h a m (1977), H o c k w i n (1974) and Jeanes et al. ( 1976 ) have published limited reviews of xanthan determination. These reviews are very limited in their scope, are no longer current, and do not include methods to measure acetate and pyruvate content. The present work reviews in detail methods for the determination o f xanthan concentration and the measurement o f acetate and pyruvate content. Principles and limitations of each method for use with oilfield samples are discussed.
Discussion A. Determination of xanthan Phenol-sulfuric a c i d m e t h o d
The phenol-sulfuric acid m e t h o d relies on the formation o f a colored complex from the
0920-4105/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved.
274
reaction of sugars with a phenol compound or thioglycolic acid in concentrated sulfuric acid. The method measures the total carbohydrate content of a sample. Phenol, anthrone, orcinol, 3-phenyl phenol, and thioglycolic acid have been used in this method (Auerbach, pers. commun., 1991"*; Dubois et al., 1956; Jeanes et al., 1976; Philips et al., 1985; Quemener, 1986; Ranjbar-Hamghawandi, 1988, 1990, 1992). The method is accurate within _+2%, with a range of 2.5-35 mg/1 sugar (Dubois et al., 1956) and has been used for biopolymer determination in high salinity brine (Ranjbar-Hamghawandi, 1990). The concentration of glucose or other sugars must be known, because they will result in a positive interference (Jeanes et al., 1976). Color generation is affected by high salinity or the presence of Fe 3+ (Philips et al., 1985). Littmann et al. (1992) used the method to determine xanthan concentration in brine produced from a polymer flood field test. Samples were dialyzed prior to analysis to remove salts and potential interferences. The phenol-sulfuric acid method provides good sensitivity and reproducibility. However, the method is relatively slow, and the effect of interferences common in oilfield brines (especially formaldehyde) is largely unknown.
Size exclusion chromatography This method uses a column to separate the polymer from low-molecular-weight impurities. This allows the use of ultraviolet (UV) or refractive index detectors that would otherwise be unsuitable with unpurified samples. The UV detector is more sensitive, with detection limits as low as 0.6 mg/1 (Hunt et al., 1988). Beazley (1985) examined samples from a viscous emulsion produced by a partially hydrolysed polyacrylamide/sulfonate **M.H. Auerbach, pers. commun., 1991. Pfizer Procedure: "Total Carbohydrate Assay by Phenol-Sulfuric Acid Method". Pfizer Central Research, Easter Point Road, Groton, Conn., USA.
K.C. TAYLOR AND H.A. NASR-EL-DIN
surfactant flooding project and concluded that no interference with polymer determination occurred. Similar results are expected for emulsions containing xanthan. Size exclusion chromatography is ideally suited for highly contaminated samples. Sample throughput rates of three to five samples per hour are typical.
Radioactive labelling Carbon-14, a fl-ray emitter, was incorporated into xanthan by adding carbon-14-1abelled glucose to the fermentation broth late in the growth cycle (Sorbie et al., 1987). They produced a carbon-14-labelled xanthan with a specific activity of 1.8 MBq/cm 3 ( 48.6 X 10- 6 Ci/cm 3) at a xanthan concentration of 3200 mg/l. Quantitation is generally performed with a commercial liquid scintillation counter. Goodyear et al. (1992) used carbon-14-labelled xanthan in the measurement of polymer retention levels. Crushed rock with adsorbed polymer was heated to decompose the polymer into carbon dioxide and water. Radioactively labelled carbon dioxide was absorbed in a scintillation cocktail, and radioactivity was measured. The most significant drawback of radioactive labelling as a method for xanthan determination is that it can not be used for commercial samples. Labelled samples must be grown in culture, and their properties may not exactly match a commercial sample of interest.
Organic carbon content Measurement of total organic carbon in aqueous samples has been used by Lecourtier et al. ( 1987 ) and Muller et al. ( 1986 ) for xanthan determination. Solutions containing high amounts of salt may interfere with the complete combustion of carbon compounds, be-
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XANTHAN BIOPOLYMERS
cause sodium carbonate may be formed preferentially. Any carbon containing species will interfere with the method, making it unsuitable for the determination of polymer concentration in the presence of surfactants, biocides such as formaldehyde, or emulsions.
Metachromatic method The metachromatic acid method by Moraine and Rogovin ( 1971 ) uses the dye toluidine blue O which forms an insoluble complex with aqueous xanthan. When the mixture is extracted with a petroleum ether, the complex adsorbs at the interface. The method measures the decrease in absorbance of the aqueous phase as dye is lost due to the formation of the insoluble complex. It is reported to be of use at xanthan concentrations as low as 20 mg/l. The effect of interferences is not known.
Precipitation Xanthan can be purified and isolated by first dissolving the polymer in water and then precipitating it in a water-miscible nonsolvent such as isopropanol (Hassler and Doherty, 1990), methanol (Moraine and Rogovin, 1966) or ethanol (Jeanes et al., 1961; Milas and Rinaudo, 1984). For careful analytical work, several reprecipitations may be necessary. This method can be a useful technique for polymer concentrations greater than 10 g/l.
Viscosity This method requires that viscosity varies proportionally with polymer concentration, and assumes that the viscosity of xanthan solutions is independent of salinity. By measuring viscosity at a fixed shear rate for a range of polymer concentrations, Philips et al. (1985) obtained a different nonlinear calibration curve for each commercial polymer used. Any change in temperature, pH, shear rate, or mo-
lecular weight distribution will affect the solution viscosity, reducing the accuracy of measurement.
Refractive index This method requires polymer concentration to be proportional to the refractive index of the solution and has been used by Quemen e r ( 1 9 8 6 ) . The specific refractive index increment of xanthan was found to be 0.144 cm3/g at 633 nm (Hunt et al., 1988 ) and 0.155 cm3/g at 546 nm (Milas and Rinaudo, 1984). Lindstr~Sm and Srremark (1976) used a Knauer refractometer thermostatted to _+0.005°C, and claimed an accuracy of _+1 mg/1 for acrylamide copolymers in wood pulp suspensions in distilled water. The refractive index increment of this polymer is similar to that ofxanthan (Hunt et al., 1988). The use of refractive index for the determination of polymer concentration can produce acceptable results only when solutions of low, constant ionic strength are used. Temperature must be accurately controlled to reduce changes in refractive index. Ideally, only solutions of xanthan in distilled water or dialyzed samples should be considered for this method.
Hydrolysis with glucose determination Hassler and Doherty (1990) quantitated xanthan by first hydrolysing the polymer to its constituent sugars, then determining glucose concentration with an enzyme test kit (Sigma Procedure No. 16-UV) using hexokinase and glucose-6-phosphate dehydrogenase. This method is also useful for determining glucose impurities in xanthan. The efficiency and reproducibility of the hydrolysis step has not been established, nor has the method been evaluated for interferences common in oilfield brines.
276
Carbazole method Glucuronic acid ( 18.4 wt% ofxanthan) can be determined colorimetrically by the carbazole method (Wernau, 1981 ), which is similar to the phenol-sulfuric acid method. The original carbazole method of Dische (1947) was modified by Knutson and Jeanes (1968) for use with polysaccharides. The range is from 10 to 70 mg/l glucuronic acid. Bitter and Muir ( 1962 ) reported that glucose, formaldehyde, and salts interfere with the method.
Gas chromatography Gas chromatography has been used to determine xanthan concentration after hydrolysis and derivatization (Lawrence and Iyengar, 1985). Xanthan is hydrolysed and the resulting sugars converted into volatile aldonitrile acetates. Xanthan concentration is determined from the concentration of the constituent sugars. The presence of other carbohydrates will result in interference with the method. B. Measurement of acetate and pyruvate content 1. Methods for measurement of both acetate and pyruvate
K.C. TAYLOR AND H.A. NASR-EL-DIN
Nuclear magnetic resonance (NMR) spectroscopy Several groups (Morris et al., 1977; Smith et al., 1981 ) have used NMR to determine only the pyruvate to acetate ratio. They used an independent method to determine pyruvate content and then calculated the acetate content. Other groups have used an internal standard so that acetate and pyruvate can be measured directly (Paradossi and Brant, 1982; Lambert and Rinaudo, 1985; Milas and Rinaudo, 1986; Callet et al., 1987a; Milas et al., 1988). Rinaudo et al. (1983) and Kulicke et al. (1990) measured pyruvate and acetate content directly by relating the peak area of these groups to the only equatorial anomeric proton of the xanthan molecule. Kulicke et al. found good agreement between their NMR measurements and enzymatic methods. High measurement temperatures (90 °C) are required, and the polymer must be purified and dry. A measurement time of about 45 min is required after the molecular weight of the polymer has been reduced by ultrasonification. The method of Kulicke et al. (1990) is recommended. 2. Methods for measurement of acetate only
Hydrolys& and titration High performance liquid chromatography (HPLC) The HPLC method for the determination of acetyl and pyruvyl groups in xanthan uses a strong acid cation exchange resin as the column packing material. After hydrolysis of a small sample ( 10 to 20/tl of a 5 g/1 polymer solution), acetate and pyruvate groups are determined (Ash et al, 1983; Cheetham and Punruckvong, 1985; Hassler and Doherty, 1990).
Xanthan is hydrolyzed under basic conditions. Acetate content is determined by titrating the hydrolyzed product with acid (Sloneker and Jeanes, 1962; Rinaudo et al., 1983 ) or by acidification, distillation, and titration of resulting acetic acid (Schultz, 1965). Any acidic materials present which can react with base may interfere with the method. Pyruvate may decompose under acidic conditions to produce acetic acid, resulting in a positive interference.
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XANTHAN BIOPOLYMERS
Hydroxamic acid method The acetate ester in xanthan reacts with hydroxamic acid to produce acetohydroxamic acid. This acid forms a water-soluble complex with iron that absorbs light at 520 nm. The method is applicable for the determination of 2 to 10 micromoles of acetate groups per mL (200 #1 sample ), with an accuracy within + 2% (Hestrin, 1949; McComb and McCready, 1957; Sutherland and Wilkinson, 1968). This method does not require hydrolysis of the acetate groups present in xanthan prior to the determination. Interferences, however, from species present in oilfield brines are unknown.
requires 2 ml of a sample containing approximately 2 g/1 of acid-hydrolyzed xanthan, and is reproducible to within 2%. The most serious disadvantage of the method is the relatively large number of steps involved and interference from formaldehyde (Hirase, 1957), which is usually added to xanthan as a biocide.
Oxidation and determination of carbon dioxide Hirase ( 1957 ) has determined the pyrnvate content of agar by oxidation with sodium metaperiodate and gravimetric determination of the liberated carbon dioxide. The same method can be used to determine pyruvate content in xanthan.
3. Methods for measurement of pyruvate only Conclusions
Lactate dehydrogenase method The lactate dehydrogenase method for the determination of pyruvate in polysaccharides is discussed in detail by Duckworth and Yaphe (1970). Application of the method to xanthan is discussed by Jeanes et al. (1976). Both methods are based on the work of Hadjivassiliou and Reider (1968 ). The pyruvate group in xanthan is first hydrolyzed using oxalic acid or dilute HC1. This is the method of choice for the determination ofpyruvate content in xanthan. It is specific, requires only 3 to 5 mg of polymer, and is readily carried out with a minimum of equipment.
2, 4-dinitrophenylhydrazine method (2, 4-DNP) Pyruvic acid reacts with 2,4-DNP to produce a 2,4-dinitrophenylhydrazone derivative which is measured colorimetrically. The method has been used extensively for the determination of pyruvate in xanthan (Koepsell and Sharpe, 1952; Sloneker and Jeanes, 1962; Sloneker and Orentas, 1962; Cheetham and Punruckvong 1985; Shatwell et al., 1990). It
The determination of concentration is important to the study of xanthan in enhanced oil recovery, in drilling operations and in gels. The measurement of acetate and pyruvate content is important because these two parameters have a large effect on the rheological behaviour, adsorption, and injectivity of xanthan solutions. Size exclusion chromatography with ultraviolet detection was found to be the most suitable method for xanthan determination in oilfield samples. The method is accurate and is not affected by impurities common in oilfield brines. The phenol-sulfuric acid method is acceptable if interferences from other carbohydrates, iron, and salinity are accounted for. Proton NMR is the method of choice for the simultaneous measurement of acetate and pyruvate content in xanthan. Alternately, pyruvate content can be measured using the lactate dehydrogenase method, or both acetate and pyruvate can be measured using HPLC. The effect of salts, divalent ions, and formaldehyde, common interferences in oilfield brines, is not generally known for methods that measure acetate and pyrnvate content in xanthan.
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Acknowledgements The authors would like to thank Janet Noy for conducting a literature search which identified many of the papers used in this report; Lynne Story and Wendy Faid, I.N. Mckinnon memorial library, for their help in obtaining many of the papers reviewed; and Prof. G.V. Chilingarian for useful discussions. References Ash, S.G., Clarke-Sturman, A.J., Clavert, R. and Nisbet, T.M., 1983. Chemical stability of biopolymersolutions. SPE 12085, 58th Annu. Tech. Conf. Exhib., San Francisco, Calif. Beazley, P.M., 1985. Quantitative determination of partially hydrolyzed polyacrylamide polymers in oilfield production water. Anal. Chem., 57( 11 ): 2098-2101. Bitter, T. and Muir, H.M., 1962. A modified uronic acid carbazole reaction. Anal. Biochem., 4: 330-334. Callet, F., Milas, M. and Rinaudo, M., 1987a. Influence of acetyl and pyruvate contents on rheological properties of xanthan in dilute solution. Int. J. Biol. Macromol., 9: 291-293. Callet, F., Milas, M. and Tinland, B., 1987b. Role of the xanthan structure on the rheological properties in aqueous solutions. 4th Int. Conf. Gums and Stabilisers for the Food Industry, Wrexham, Clwyd, Wales. Chatterji, J. and Borchardt, J.K., 1981. Applications of water-soluble polymers in the oil field. J. Pet. Technol., 33: 2042-2056. Cheetham, N.W.H. and Punruckvong, A., 1985. An HPLC method for the determination of acetyl and pyruvyl groups in polysaccharides. Carbohydr. Polym., 5: 399406. Dische, Z., 1947. A new specific color reaction of hexuronic acids. J. Biol. Chem., 167: 189-198. Dubois, M, Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Duckworth, M. and Yaphe, W., 1970. Definitive assay for pyruvic acid in agar and other algal polysaccharides. Chem. Ind. (June): 747-748. Goodyear, S.G., Johnston, J.D., Lawless, T.A. and Woods, C.L., 1992. Measurement of polymer retention levels representative of the formation. SPE/DOE 8th Symp. Enhanced Oil Recovery, Tulsa, Okla., SPE/DOE 24155. Graham, H.D., Editor, 1977. Analytical Methods for Major Plant Hydrocolloids. Avi Publ. Co. Inc., Westport, Conn., pp. 540-579. Hadjivassiliou, A.G. and Rieder, S.V., 1968. The enzy-
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