Acetyl chloride → Hexuronic acid

Determination of the carbohydrate constituents of the mucoid antigens was performed by GLC analyses. Material to be analyzed was both the purified mucoid antigen and this same material reduced twice with water soluble carbodiimide and sodium borohydride by the method of Taylor and Conrad, Biochem., 11, 1383-1388 (1972). The use of EDTA in the extraction and purification steps of the preferred method of the invention markedly reduced the viscosity of the purified alginate, eliminating the need to perform a preliminary limited hydrolysis step previously described as necessary by others in order to reduce the exopolysaccharide with the carbodiimide reagent. Both intact and reduced exopolysaccharides were analyzed as the tri-methyl silyl (TMS) derivitized esters following hydrolyses for 24 hr in 12% methanolic HCl, prepared with acetyl chloride and methanol (2 ml acetyl chloride/10 ml methanol). The TMS procedure was chosen since both gulose and mannose in the carbodiimide reduced alginate could be measured, as well as gluluronic and mannuronic acid in the intact alginate. This procedure also allowed for measurement of the amount of reduction by the carbodiimide reagent. GLC was performed on a Packard model 421 using a 4 mm by 6 ft column packed with 3% SE-30 on Gaschrom Q. Nitrogen carrier gas flowed at 33 cc/min. The temperature program was 140° for 3 min followed by a rise of 5° C. per min to 245° C., which was held for 5 min. Injector temperature was 200° C., and the flame ionization detector was at 250° C. Data recording and analyses were performed with a Hewlett Packard model 3388A integrator. Quantitation was performed by comparisons of the areas obtained from analyzed samples with those obtained for authentic standards, except for guluronic acid, which was determined from authentic alginic acid (Sigma Chemical Co., St. Louis, Mo.) after subtraction of the mannuronic acid level.

1. extractionThe use of EDTA in the extraction and purification steps of the preferred method of the invention
2. customa preliminary limited hydrolysis step
3. customfor 24 hr
4. waitwas held for 5 min
5. customwas 200° C.
6. customwas at 250° C
7. customobtained

ORD record

ord-3742623ebbc94a1f96a80270fa5f6a33

Source dataset

uspto-grants-1986_03

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US04578458