Analysis of microbial extracellular polysaccharides in biofilms by HPLC. Part I: development of the analytical method using two complementary stationary phases

被引:20
作者
Meisen, Stefan [1 ]
Wingender, Jost [2 ]
Telgheder, Ursula [1 ]
机构
[1] Univ Duisburg Essen, D-47057 Duisburg, Germany
[2] Univ Duisburg Essen, Biofilm Ctr, D-47057 Duisburg, Germany
关键词
HPLC; uronic acids; monosaccharides; porous graphitic carbon; lead-form cation exchanger; alginates;
D O I
10.1007/s00216-008-2068-y
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The investigation of microbial extracellular polymeric substances (EPS) is helpful for the implementation of analytical methods which are suitable for biofilm analysis in order to understand the architecture and function of biofilms. A procedure for the qualitative and quantitative determination of various monosaccharides, oligosaccharides and uronic acids as important components of the carbohydrate fraction of microbial EPS by high-performance liquid chromatography (HPLC) and refractive index (RI)/UV detection is presented. Porous graphitic carbon and lead-form cation-exchanger have been examined as stationary phases. Therefore, two complementary HPLC methods are presented. To simulate the conditions of hydrolysis, the influences of various salts, acids and alkalis as matrix components have been investigated. Furthermore, the dependencies on the pH value and temperature of the mobile phase have been thoroughly studied. The results showed that the lead-form cation-exchanger is suitable for the separation of the neutral monosaccharides. However, for direct analysis after acidic hydrolysis with H2SO4, HCl or trifluoroacetic acid, an additional purification step, e.g., precipitation or lyophilization, is necessary when the cation-exchanger is used. With the exception of hydrolysis with HCl, the porous graphitic carbon stationary phase can be used without any further purification step and is appropriate for the separation of uronic acids and their gamma-lactones. Additionally, the separation of a single monosaccharide and its derivatives is possible. Analytical parameters including the sensitivities, repeatabilities, limits of detection and limits of quantification of both HPLC methods using the RI detector are presented. The optimized method has been applied for the characterization of alginates and is also suitable for other extracellular polysaccharides in biofilms.
引用
收藏
页码:993 / 1002
页数:10
相关论文
共 30 条
[1]  
ANZAI H, 1990, NIPPON SUISAN GAKK, V56, P73
[2]   Effects of current velocity on the nascent architecture of stream microbial biofilms [J].
Battin, TJ ;
Kaplan, LA ;
Newbold, JD ;
Cheng, XH ;
Hansen, C .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2003, 69 (09) :5443-5452
[3]  
Bellezza Simona, 2005, Archiv fuer Hydrobiologie Supplement, V159, P117
[4]   Exopolysaccharides of two cyanobacterial strains from Roman hypogea [J].
Bellezza, Simona ;
Albertano, Patrizia .
GEOMICROBIOLOGY JOURNAL, 2006, 23 (05) :301-310
[5]  
BeMiller J N, 1967, Adv Carbohydr Chem Biochem, V22, P25, DOI 10.1016/S0096-5332(08)60151-4
[6]   Study of sugar acids separation by high-performance anion-exchange chromatography-pulsed amperometric detection using alkaline eluents spiked with Ba2+, Sr2+, or Ca2+ as acetate or nitrate salts [J].
Cataldi, TRI ;
Campa, C ;
Casella, IG .
JOURNAL OF CHROMATOGRAPHY A, 1999, 848 (1-2) :71-81
[7]   Chemical and physical methods for characterisation of biofilms [J].
Denkhaus, Evelin ;
Meisen, Stefan ;
Telgheder, Ursula ;
Wingender, Jost .
MICROCHIMICA ACTA, 2007, 158 (1-2) :1-27
[8]  
Diepenmaat-Wolters MGE, 1997, J AM SOC BREW CHEM, V55, P147
[9]   COLORIMETRIC METHOD FOR DETERMINATION OF SUGARS AND RELATED SUBSTANCES [J].
DUBOIS, M ;
GILLES, KA ;
HAMILTON, JK ;
REBERS, PA ;
SMITH, F .
ANALYTICAL CHEMISTRY, 1956, 28 (03) :350-356
[10]  
Ferrier R. J., 1972, MONOSACCHARIDE CHEM