Anal. Chem. 1994, 66, 4514-4518
Colorimetric Determination of Carrageenans and Other Anionic Hydrocolloids with Methylene Blue Helena S. Soedjak
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Methylene blue interacts with carrageenans and other anionic hydrocolloids to form water-soluble metachromatic complexes at low concentrations of the reactants. The complexation results 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. The interaction between methylene blue and polyanions is reversible, electrostatic, and stoichiometric (1:1 ratio between the anionic sites and the bound dye molecules). The proportionality of 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 10~1 23% polyanion) may require a high dilution of samples. Consequently, potentially interfering compounds (e.g., acids, sugars, salts, milk, proteins, dyes, emulsifiers, 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 intercelnumerous species of red seaweeds, are extensively used as gelling, thickening, and stabilizing agents in
lular matrix materials in
the food, pharmaceutical, and cosmetics industries. Among the existing methods for determination of carrageenans and other polysaccharides are gas and liquid chromatography,12 which require hydrolysis of the samples prior to analysis. Other methods involve precipitation of the anionic polysaccharides using barium chloride,3 barium chloranilate,4 cetyl pyridinium chloride,5 alkyldimethylbenzylammonium chloride,6·7 and cationic dyes.8-10 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 Thier, H.-P. Z. Lebensm. Unters. Forsch. 1980, 170, 272-279. A G. J.; Schols, H. A; Pilnik, W. Prog. Food Nutr. Sci. 1982, 6,
(1) Glück, U.; (2) Voragen,
379-385.
. T.; Whitney, E. M./. Dairy Sci. 1960, 43, 175-186. (4) Graham, H. D.J. Dairy Sci. 1966, 49, 1102-1108. (5) Graham, H. D.J Food Sci. 1968, 33, 390-394. (6) Smith, F.; Montgomery, R. Chemistry of plant gums and mucilages; Rheinhold Publishing Corp.: New York, 1959.
(3) Hansen, P.
(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. /. Food Sci. 1961, 26, 365-372. (10) Yabe, Y.; Ninomiya, T.; Tatsuno, T.; Okada, T. J. Assoc. Off. Anal. Chem.
1991,
4514
74, 1019-1022.
Analytical Chemistry, Vol. 66, No. 24, December
15, 1994
Figure 1. Chemical structure
of methylene blue.
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.11 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-thiobarbituric acid 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). o-Tolidine reacts with carrageenans to produce a purple colored complex.13 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 10~3% hydrocolloid), fast, and reproducible quantification 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 Ewe15 as 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) (12) (13) (14) (15)
Edstrom, R D. Anal. Biochem. 1969, 29, 421-432. Anderson, W.; Bowtle, W. Analyst 1974, 99, 178—183. Graham, H. D.J. Dairy Sci. 1972, 55, 1675-1682. Cundall, R B.; Phillips, G. O.; Rowlands, D. P. Analyst 1973, 98, 857-862. Ewe, G. E. /. Am. Pharm. Assoc. 1930, 19, 568-570.
0003-2700/94/0366-4514$04.50/0
©
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 hydrocolloids and proteins were obtained from commercial suppliers: ¿-carrageenans (Lactarin MV406 and Viscarin GP109 from FMC), ¿-carrageenan (Viscarin SD389 from FMC), /¿-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 Rhóne-Poulenc), guar gum (Uniguar 150 from RhónePoulenc), 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 ¿-carrageenans, although they contain small amounts of /¿-carrageenans. 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 (i.e., 21 versus 129 cP at 14.4 s-1 shear rate and 26.7 °C). The viscosity was measured using a Vilastic capillary viscometer (tube radius, 0.0498 cm; tube length, 6.094 cm).
Methylene Blue Standard Assay Procedure. Hydrocolloid stock solutions16 were diluted to 0.02% with water. (a) Assay Procedure Using 100 mL of Reaction Solution. Pipet 0-10 mL17·18 of 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,19 and 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 10_3%), gum arabic (up to 4 x 10~3%), and agar (up to 18 x 10_3%) 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 vol %. (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 1 mL disposable polystyrene
absorbance at 559 nm (using cuvettes) against water blank.
(b) Assay Procedure Using 1 mL of Reaction Solution. Pipet 0-100 ¿¿L17'18 of 0.02% 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 (A55g) 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 10™3% Lactarin MV406) and 41 µ 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 1 mL of reaction solution. NaCl (2 M stock solution) was added incrementally to the complex solution to give final concentrations of 3.4-120 mM. Note that the total increase in volume was 0.993 (data not shown). This suggests that methylene blue assay is applicable for the polyanions, provided that samples with the same type of composition are used to prepare the standard curves. The binding reactivity of carrageenans toward methylene blue increases in the order of , t, and (see Table 1), consistent with the increasing sulfate content of these carrageenans (see Table 2). Carrageenan is structurally related to sulfated polysaccharides, such as furcellaran, fucoidan, iridophycan, funoran, hypnean, Eucheuma extract, and gums from lesser-known red algae.9'25™28 It is thus likely that these hydrocolloids also give the typical metachromatic complexes with methylene blue. Preliminary experiments confirm that the sulfated polysaccharide heparin can be quantified in microgram amounts (data not shown). The lightly sulfated agar exhibits very low reactivity, whereas the lightly carboxylated pectin shows no color reaction with the dye (data not shown). Neutral polysaccharides (e.g., locust bean cationic
dyes.21™24
gum, gellan gum, guar gum, propylene glycol alginate, and hydroxypropylmethylcellulose) do not form metachromatic complexes with methylene blue (data not shown). It is interesting to compare the reactivities of alginate versus propylene glycol alginate toward methylene blue. Alginate shows a high affinity toward the cationic dye. However, esterification of its carboxyl groups to produce propylene glycol alginate results in the loss of the anionic sites and consequently the affinity toward the dye. Similarly, a substitution of the anionic carboxyl group of car(21) Balasz, E. A.; Szirmai, J. A /. Histochem. Cytochem. 1958, 6, 278-289. (22) Klotz, I. M. In The Proteins. Chemistry, Biological Activity, and Methods', Neurath, H., Bailey, K., Eds.; Academic Press Inc.: New York, 1953; Vol. I, part B, pp 727-806. (23) Swift, H. In The Nucleic Acids, Chemistry and Biology, Chargaff, E., Davidson, J. N., Eds.; Academic Press Inc.: New York, 1955 Vol. II, pp 51-92. (24) Wiame, J. M./. Am. Chem. Soc. 1947, 69, 3146-3147. (25) Clingman, A. L; Nunn, J. R; Stephen, A M./. Chem. Soc. 1957,197—203. (26) Jones, J. . M; Smith, F. Adi Carbohydr. Chem. 1949, 4, 243-291. (27) Nunn, J. R; von Holdt, . M./. Chem. Soc. 1957, 1094-1097. (28) Painter, T. J. Can. ]. Chem. 1960, 38, 112-118.
boxymethylcellulose with the hydroxypropyl group, which is uncharged at the assay pH, also causes the loss of the binding affinity for methylene blue. These results substantiate the participation of the anionic groups of polysaccharides in the complex formation with methylene blue. Binding Ratio of Sulfate Residue of Carrageenans to Methylene Blue. The essentiality of the negative charge of hydrocolloids for binding to methylene blue suggests that complexation between the polyanions and cationic dye is due to a charge-charge interaction. In the case of carrageenans, it is likely that the sulfate residues are involved in the binding to methylene blue. It is therefore expected that the amount of carrageenans bound to methylene blue is governed by the sulfate content of the carrageenans. The binding ratio of carrageenans to methylene blue can be determined from titration curves (i.e., plots of A559 versus carrageenan concentrations). The carrageenan concentration at the saturation point reflects the amount of carrageenan required to occupy all binding sites of methylene blue. The data in Table 2 show that the calculated number of moles of sulfate at the saturation point corresponds well with the number of moles of methylene blue used. Nature of Interaction between Hydrocolloid and Methylene Blue. Methylene blue and carrageenan form a purple complex. The addition of NaCl results in a decrease of the absorbance of the complex at 559 nm and an increase in the absorbance of the free dye at 610 and 664 nm.20 In the presence of 120 mM NaCl, the purple color of the solution changes back to blue and the absorption spectrum of the free dye reappears.20 The reversal of the complex formation by electrolytes indicates an ionic interaction between carrageenan and methylene blue. This result supports the notion that the sulfate moities participate in the binding to the cationic dye. Effect of Phosphate. Phosphate buffers at pH 1.75-12.0 suppress the complex formation of alginate and carboxymethylcellulose with methylene blue.20 The inhibition of complex formation by phosphate might result from a competition between phosphate and carboxyl molecules for binding to the dye. In contrast, the dye-binding efficiency of A- (Lactarin MV406) and k(Gelcarin GP911) carrageenans is not affected by 40 mM phosphate for a pH range of 1.75-12.0. The absorbance at 559 nm is approximately identical to the value obtained when the assays are performed in water. This is readily explained by the fact that phosphate does not compete with the strongly acidic sulfate group for binding to methylene blue. The independence on the pH is expected, since the ionization of the sulfate group is not suppressed and the polysaccharide is still highly charged at low pH.29 The practical considerations arising from the difference in the responses of the two types of hydrocolloids to phosphate buffer are twofold. First, it can be used to identify the type of anionic residue of unknown hydrocolloid samples. For example, sulfated polysaccharides will deliver similar results when assayed in phosphate buffer and water, whereas carboxylated polymers are not reactive when assayed in phosphate buffer. Second, it allows a quantitative estimation of sulfated and carboxylated hydrocolloid mixtures, provided that the same polyanions are used to prepare the standard curves. The amount of the sulfated hydrocolloid can be derived from the A559 values from the assay in phosphate buffer, (29) Whistler, 1973; p 15.
R. L.
Industrial gums; Academic Press: New York and London,
Table 3. Compatible/lnterfering Compounds for Methylene Blue Assay of Lactarin MV406 sample contents
carrageenan detected (xl0_3%)
water acids/salts 1% citric acid 1% adipic acid 5% NaCl 5%
KC1
5% CaCl2 10% FeS04
10% ZnS04 carbohydrates 40% glucose 40% fructose
40% sucrose 40% maltodextrin 10% com starch
proteins 10% bovine serum albumin
1.00 1.00
0.98 0.97 0.97 0.96 1.00 0.99 0.99 1.01 1.00 1.00 1.01
10%
1.06 1.15 1.00 1.06
2%
0.96
10% 10%
casemate egg protein
whey protein gelatin emulsifiers 1% sucrose ester 1% lecithin 1% glyceryl monostearate
1.00 1.15 1.10
dyes 0.02% FD&C Red No. 40 0.002% FD&C Blue No. 1
neutral hydrocolloids 0.001% gellan gum 0.001% hydroxypropylmethylcellulose
1.02 1.00 1.10 0.98
whereas the amount of the carboxylated hydrocolloid can be calculated from the difference in the A559 values in water and phosphate buffer as described in the Materials and Methods section. When the method was applied for mixtures of sulfated (A-carrageenan Lactarin MV406) and carboxylated (alginate or carboxymethylcellulose) hydrocolloids with different ratios, the results show that the concentrations of both hydrocolloids can be individually estimated with ±85% accuracy.20 Effect of Common Substances. Under the assay conditions, several commonly encountered compounds (acids, salts, sugars, emulsifiers, dyes or proteins) do not interfere with methylene blue assay (see Table 3). According to a previous report,22 proteins interact with cationic dyes, but changes in the absorption spectrum of the dye are noticeable only at high protein concentrations. The presence of >5% citric acid or adipic acid in 0.4% carrageenan solution results in the formation of fine precipitate in the assay solution (data not shown); however, the interference can be overcome by adding NaOH when preparing the 20 times diluted carrageenan solution (e.g., 4 mL of 0.1 N NaOH in 200 mL of 0.02% carrageenan solution). Note that the compatibility of the assay is mostly due to the high sensitivity of the assay, which requires a high dilution of the carrageenan sample. The actual concentrations of the compounds in the assay solution are 400 times lower than those listed in Table 3. Some of the compounds are deliberately used at high concentrations to demonstrate the tolerance of the assay for samples which require lower dilution factors.
Methylene Blue Assay for Carrageenan in Milk. The determination of carrageenans in milk solutions using currently available methods requires special treatments of the samples prior Analytical Chemistry, Vol. 66, No. 24, December
15, 1994
4517
Table 4. Assay Sensitivity for Lactarin MV406 in Milk Solutions milk
(%)
0 15
30 50
slope ± SE (M559/10-3%) 0.47 0.42 0.31 0.19
± ± ± ±
0.01 0.01 0.01 0.01
to analysis, such as enzymatic hydrolysis of the proteins.513·30·31 The difficulties of determining carrageenans in milk solutions are reportedly due to the interaction between the hydrocolloids and milk proteins. To broaden the use of methylene blue assay, the sensitivity of the assay was tested for a -carrageenan (Lactarin MV406) in 1550% milk solutions. The results show that the sensitivity of the assay is lower for carrageenan in solutions with higher milk concentrations (see Table 4), suggesting that the milk solutions contain compounds, probably milk proteins, that either compete with carrageenan for binding to the dye or interact with the hydrocolloid. Despite the dependence of the standard curves on the milk concentrations, the linearity of the plot still exists in all cases, indicating that methylene blue assay is applicable, provided that the standard curves are prepared using samples with the same milk concentration. Note that for
