Anal. Chem. 1994,66,4514-4518
Colorimetric Determination of Carrageenans and Other Anionic Hydrocolloids with Methylene Blue Helena S. Soedjak Krafi General Foods, Research, 555 South Broadway, Tarrytown, New York 10591
Methylene blue interacts with carrageenans and other anionic hydrocolloids to form water-soluble metachromatic complexes at low concentrations of the reactants. The complexationresults in a color change of the dye from blue (absorption maxima at 610 and 664 nm)to purple (absorption maximum at 559 nm). The anionic sites of the hydrocolloids appear to be primarily responsible for the dye-binding. ‘he interaction between methylene blue and polyanions is reversible, electrostatic, and stoichiometric (1:1 ratio between the anionic sites and the bound dye molecules). The proportionalityof the absorbance of the complex at 559 nm to polyanion concentrations allows a simple quantitative determination of the polymers. The high sensitivity of the assay (i.e., (0.2-2.0) x low3% polyanion) may require a high dilution of samples. Consequently, potentially interfering compounds (e.g., acids, sugars, salts, milk, proteins, dyes, emulsitlers, and neutral hydrocolloids) may be diluted out and become compatible with the assay. In addition, a selective dye-binding inhibition of carboxylated polymers by phosphate allows the analysis of mixed carboxylated and sulfated hydrocolloids. Because the colorimetric assay is simple, highly sensitive, fast, and reproducible, it can be useful for routine quantitative analysis. Carrageenans, sulfated polysaccharides that occur as intercellular matrix materials in numerous species of red seaweeds, are extensively used as gelling, thickening, and stabilizing agents in the food, pharmaceutical, and cosmetics industries. Among the existing methods for determination of carrageenans and other polysaccharides are gas and liquid chromatography,1s2 which require hydrolysis of the samples prior to analysis. Other methods involve precipitation of the anionic polysaccharides using barium chloride? barium chloranilate,4cetyl pyridinium ~hloride,~ akyldimethylbenzylammonium chloride,6j7and cationic dyes.8-1° In these methods, the polyanions are estimated from either the precipitate (e.g., analyzed gravimetrically or spectrophotometrically after solubilization in appropriate reagents) or the excess (1) Gliick, U.; Thier, H:P. Z.Lebensm. Unters. Forsch. 1980,170,272-279. (2) Voragen, A. G. J.; Schols, H. A; Pilnik, W. Prog. Food Nutr. Sci. 1982,6, 379-385. (3) Hansen, P. M. T.; Whitney, E. M.J. Dairy Sci. 1960,43, 175-186. (4) Graham, H. D. J. Dairy Sci. 1966,49,1102-1108. (5) Graham, H. D.]. Food Sci. 1968,33, 390-394. (6) Smith, F.; Montgomery, R Chemisty ofplant gums and muciluges; Rheinhold Publishing Corp.: New York, 1959. (7) Graham, H. D.; Thomas, L. B. J. Food Sci. 1962,27,98-105. (8) Graham, H. D. J. Food Sci. 1960,25, 720-730. (9) Graham, H. D.; Thomas, L. B. J. Food Sci. 1961,26, 365-372. (10) Yabe, Y.; Ninomiya, T.; Tatsuno, T.; Okada, T. J. Assoc. 08 Anal. Chem. 1991,74,1019-1022.
4514
Analytical Chemistry, Vol. 66,No. 24, December 15, 1994
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unreacted reagents. Unfortunately, these precipitation techniques are inconvenient for routine quantitative analysis, since they require centrifugation steps, a long incubation time (12-60 h), and large quantities of carrageenans (40 mg- 100 g). Complexing agents that form soluble complexes with anionic polysaccharides have been used to simplify the quantification methods. For example, the reaction of carbocyanine dye with carrageenans results in a shift of the absorption maximum of the cationic dye toward a longer wavelength.” This method allows rapid determination of acidic polysaccharides in low quantities but requires fresh preparation of the reagent for each assay. The method utilizing 2-thiobarbituricacid is based on its reaction with carrageenans to produce a yellow colored complex.12 Despite its high sensitivity, the method is inconvenient, since it requires tedious heating of the samples in barbituric acid reagent containing a high HC1 concentration (the heating and cooling cycle takes almost 1 h). @Tolidinereacts with carrageenans to produce a purple colored c0mp1ex.l~ This method is elaborate, since it requires three reagents which have to be added at specific time intervals and a pH adjustment of the mixture. Furthermore, the color development takes 30 min. Acridine orange binds to polyanions, resulting in a decrease of the fluorescence intensity of the free dye.14 This method is sensitive but requires a spectrofluorimeter. This paper describes a simple, highly sensitive (0.2 x hydrocolloid), fast, and reproducible quantiiication method for carrageenans and other anionic hydrocolloids that uses the cationic dye methylene blue (see Figure 1). The method is based on the shift of the absorption maxima of the dye from 610 and 664 nm to 559 nm due to the formation of a soluble complex. The absorbance of the complex at 559 nm is proportional to the hydrocolloid concentrations. The reaction between carrageenans and methylene blue to produce “blue insoluble clots”was initially recognized by Ewe15as early as 1930 and elaborated by Graham8 in 1960 to estimate carrageenans from the excess unreacted dye. The fundamental difference between the method previously developed by Graham and the one presented in this paper lies in the solubility of the dye-polyacid complex, which is achieved in (11) Edstrom, R D. Anal. Biochem. 1969,29, 421-432. (12) Anderson, W.; Bowtle, W. Analyst 1974,99,178-183. (13) Graham, H. D. ]. D a i y Sci. 1972,55, 1675-1682. (14) Cundall, R B.; Phillips, G. 0.; Rowlands, D. P. Analyst 1973,98,857-862. (15) Ewe, G. E. J. Am. Pharm. Assoc. 1930,19,568-570.
0003-2700/94/0366-4514$04.50/0 0 1994 American Chemical Society
the present study by working with low concentrations of dye and polyanions and a slight excess of the dye. The solubility of the complex thus eliminates the need for centrifugation steps and a long precipitation time. MATERIALS AND METHODS
Chemicals. Methylene blue (dye content, 86%) was purchased from Difco Laboratories. The following hydrocolloidsand proteins were obtained from commercial suppliers: Icarrageenans (Lactarin MV406 and Viscarin GP109 from FMC), Lcarrageenan Wiscarin SD389 from FMC), K-carrageenan (Gelcarin GP911 from FMC), alginate (Kelcosol from Kelco), propylene glycol alginate (Kelcoloid from Kelco) , sodium carboxymethylcellulose (Aqualon), hydropropylmethylcellulose (The Dow Chemical Co.),xanthan (Keltrol RD from Kelco) , gellan gum (Kelcogel from Kelco), locust bean gum (Sigma), gum arabic (SD No. 2 from RhBne-Poulenc), guar gum (Uniguar 150 from RhBnePoulenc), pectin (Mexpectin LC710 from Grindsted), agar (BBL Microbiology Systems), BSA fraction V (Fisher), whey protein (New Zealand Milk Product), and sodium caseinate (New Zealand Milk Product). In this paper, Lactarin MV406 and Viscarin GP109 are refered to as I-carrageenans, although they contain small amounts of Kmageenans. Homogenized milk was obtained from Crowley. Other chemicals were reagent grade. Water was of Millipore quality. Preparation of Hydrocolloid Solution Preparation. For accurate determinations, it is essential that the polymers are completely dissolved. Hydrocolloid powders (1 g) are best dissolved by stirring water (approximately 200 g) in a 300 mL beaker vigorously to form a vortex and gradually sprinkling the powders into this vortex so that the particles remain separate and are rapidly dispersed. The mixtures were boiled under continuous stirring for approximately 3 min or until the solutions became clear. After the solutions had cooled down, water was added to give a total weight of 250 g, and the beaker was covered to prevent evaporation.16 Hydrolyzed carrageenan (Lactarin MV406) solution was prepared according to the procedure mentioned above, except that water was replaced with 2.5 mM HC1 and the boiling time was extended to 25 min. Under these conditions, the carrageenan was degraded as confirmed by the much lower viscosity of the carrageenan in acid solution than in water (Le., 21 versus 129 CP at 14.4 s-1 shear rate and 26.7 OC). The viscosity was measured using a Vilastic capillary viscometer (tube radius, 0.0498 cm; tube length, 6.094 cm). Methylene Blue StandardAssay Procedure. Hydrocolloid stock solutions16were diluted to 0.02%with water. (a) Assay Procedure Using 100mL of Reaction Solution. Pipet 0-10 mLl7Jsof 0.02%hydrocolloid stock solution in 100 mL volumetric flasks. Add 75 mL of water, 10 mL of 0.41 mM (0.18 mg/mL) methylene blue stock solution,*gand more water to give ~
(16) All hydrocolloids were prepared at 0.4% (wt %), except locust bean gum and guar gum, which were prepared at 0.02% due to their lower solubility. (17)Xanthan (up to 3 x lo-%), gum arabic (up to 4 x and agar (up to 18 x lo-%) were used at higher concentrations because of their lower degree of color reaction with methylene blue. (18) The density of 0.02%hydrocolloid solutions is approximately equal to that of water. Thus the concentration of the hydrocolloid in the assay solution can be considered as wt % or ~ 0 1 % . (19) Methylene blue stock solution prepared in water at 0.41 mM is stable for at least 6 months based on its absorbance at 610 nm and response to hydrocolloids.
an exact volume of 100 mL. Mix the solutions and measure the absorbance at 559 nm (using 1 mL disposable polystyrene cuvettes) against water blank. (b) Assay Procedure Using 1 mL of Reaction Solution. Pipet 0-100 pL17J8of 0.0% hydrocolloid stock solution to 1 mL polystyrene cuvettes. Add water to give a total volume of exactly 0.9 mL, and then add 0.1 mL of 0.41 mM methylene blue. Mix the solutions and measure the absorbance at 559 nm against water blank. The standard curve, obtained by plotting the absorbances at 559 nm &59) versus the corresponding hydrocolloid concentrations, is used to determine the polymer concentration in the samples to be tested. The standard and test samples are prepared under the same assay conditions. Effect of NaCl. The effect of NaCl on the complexation of carrageenan (2.0 x Lactarin MV406) and 41 pM methylene blue was investigated by comparing the absorption spectrum of the complex before and after additions of NaCl. The complex was prepared as described for the standard assay procedure using 1mL of reaction solution. NaCl (2 M stock solution) was added incrementally to the complex solution to give hal concentrations of 3.4-120 mM. Note that the total increase in volume was
