ANALYTICAL
BIOCHEMISTRY
189,178-181
(1990)
Further Studies on the Mechanism of Phenol-Sulfuric Acid Reaction with Furaldehyde Derivatives Pragna Rao and Thillaisthanam N. Pattabiraman Department of Biochemistry, Kasturba Medical College,Manipal-576 119, Karnataka, India
Received
February
20,199O
Even though the chromogens formed from mannose and galactose showed comparable absorbances at 480 nm in the conventional (developer present during heat of dilution) and modified (developer reacted at room temperature after cooling; t mannose = 13,700, galactose = 14,000) phenol-sulfuric acid reactions, shoulders in the region 420-430 nm were prominent in the former method. Fucose was 10 times less reactive in the modified method (t = 800) than in the conventional method. 2-Formyl-Sfuran sulfonic acid reacted equally efficiently in the two methods (t = 40,800). 5Methyl-2-furaldehyde, unlike the sulfonate derivative or 5-hydroxymethyl-2-furaldehyde, required heat for condensation with phenol. 2-Furaldehyde dimethylhydrazone reacted 26 times better to form a chromogen (c = 40,500) in the modified phenol-sulfuric acid method. The possible roles of intermediates between hexoses and furaldehydes in forming chromogens and the effect of substitution at the 2- and B-positions of furaldehyde on the rates of condensation with phenol for the observed differences between the conventional and the modified methods are discussed. 0 1990 Academic Press, Inc.
In an earlier communication, we reported a modified method for the estimation of hexoses and pentoses based on the original phenol-sulfuric acid procedure of Du Bois et al. (1,2). The modification involved dehydration of sugars in 75% H&SO, at an elevated temperature to form furaldehydes and their condensation with phenol at ambient temperature to form chromogens. The sensitivity for 2-furaldehyde and 5-hydroxymethyl-2-furaldehydeincreased6.0-foldand9.O-fold,respectively, in the new method. This was shown to be due to the abolition of p-sulfonation of phenol, which in the conventional method drastically reduced the availability of the developer. Further, the chromogens in the modified method had symmetrical spectra in the visible range with sharp absorption maxima around 475-485
nm. In the conventional method, a shoulder in the region 400-440 nm was discernible. However, the increase in color intensities in the modified method varied from 100% for glucose and arabinose to 270% for fructose, with other sugars tested occupying intermediate positions. The rates and amounts of furaldehydes formed from monosaccharides, the quantity of partially dehydrated derivatives and side products, and the possible sulfonation of these compounds in the conventional method could contribute to the observed differences. To evaluate these possibilities, we further investigated the phenol-sulphuric acid reaction using other hexoses and different furaldehyde derivatives. The results are presented here. MATERIALS
AND METHODS
5-Hydroxymethyl-2-furaldehyde (HMF);l 2-formylfuran-5-sulfonate, sodium salt (FFS); 2-furaldehyde dimethylhydrazone (FDMH); 5-methyl-2-furaldehyde (MF); 2-furaldehyde; L-fucose; D-mannose; and D-galactose were the products of Aldrich Chemical Co. (Milwaukee, WI). Sulfonated phenol was prepared in 75% H,SO, as described before (1). Other reagents were analytical grade chemicals. In the conventional phenol-sulfuric acid method, 0.05 ml of 80% phenol in water (W/W) was added to 1 ml of sugar solution followed by 3.0 ml of sulfuric acid (specific gravity 1.84) with mixing. The solution reached 115-118°C within 15 s and was allowed to cool at ambient temperature (28-30°C). After 30 min, the chromogens were analyzed. In the modified method the sugar solution was treated with sulfuric acid as described above. After 30 min standing at room temperature, the phenol solution was added. The chromogens were analyzed after 30 min. With furaldehydes the chromogens 1 Abbreviations used: HMF, 5-hydroxymethyl-2-furaldehyde; 2-formylfuran-5-sulfonate sodium salt; FDMH, 2-furaldehyde methylhydrazone; MF, 5-methyl-2-furaldehyde.
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SPECTROPHOTOMETRIC
400
I_~ __1_ !- -iL_1_ 520
440
480
400
440
480
FIG. 1. Spectra of chromogens formed sulfuric acid reaction. (0) Conventional method.
STUDIES
520 400
by hexoses method.
440
480
OF
520
in the phenol(0) Modified
were also formed without exposure to heat. The compounds were dissolved in ice-cold 75% sulfuric acid (VI V). The solution (4.0 ml) was brought to room temperature and 0.05 ml of 80% phenol was added. After 30 min, the chromogens were analyzed. Ultraviolet spectra of furaldehydes were studied in 75% sulfuric acid, after the solutions were prepared in cold 75% sulfuric acid. With hexoses, the aqueous solution (1.0 ml) was treated with 3.0 ml of sulfuric acid with the attendant heat of dilution. The solutions were cooled to 25°C in 30 min. The uv and visible spectral measurements were made at 25°C using a Uvikon 810P spectrophotometer. RESULTS
In Fig. 1, the spectra of the chromogens formed by mannose, galactose, and fucose in the phenol-sulfuric acid reaction are depicted. The absorbance values at absorption maxima (480 nm) for mannose and galactose in the conventional and modified methods were not significantly different. This is in sharp contrast to a two-fold increase for glucose in the modified method (1). Molar absorptivities for glucose, mannose, and galactose by the conventional method were 11.5 X 103, 14.4 X 103, and 12.7 X 103, respectively. To test whether complexation with phenol facilitates rapid dehydration of mannose selectively, the solutions of hexoses were incubated with 80% phenol for up to 30 min at 37°C before the addition of sulfuric acid. This however did not increase the absorbance or the special patterns. The shoulders in the region 400-440 nm were more prominent for mannose and galactose than for glucose in the conventional method as indicated by the ratios of absorbance at 430 and 480 nm (mannose 0.45, galactose 0.43, and glucose 0.31). It is probable, that the absorption in the lower wavelengths in the conventional method is due to the condensation products formed between phenol and partially dehydrated products of hexoses. In the modified
PHENOL-SULFURIC
ACID
179
REACTION
method, the spectra for mannose and galactose were sharper as it was with glucose, suggesting that the chromogen is mainly contributed by HMF. With fucose, the color intensity was very poor in the modified method. In the conventional method, the chromogen showed an absorption maxima at 480 nm with a prominent shoulder in the lower wavelength regions (Fig. 1). This suggests that MF arising from fucose reacts poorly with phenol at room temperature whereas MF and other dehydrated products derived from fucose may condense more efficiently with the developer at elevated temperature. In Fig. 2, the spectra of the chromogens formed by FFS, MF, and FDMH are shown. FFS showed almost identical patterns in the reactions at room temperature and in the conventional method (Fig. 2A). The spectral profile was very similar in the modified method (data not shown). It can be concluded that FFS reacts efficiently with phenol at ambient temperature like furaldehyde. Further, the rate of condensation of FFS with phenol at elevated temperature far exceeds the rate of sulfonation of phenol in situ, unlike with furaldehyde (1). When random sulfonated phenol (1) was used in place of phenol in reactions at room temperature, no chromogen was formed with FFS, confirming the above view. MF reacted feebly with phenol at room temperature (Fig. 2B). Nearly 40 times more (mole equivalent) MF was required to yield absorbance comparable to that of FFS at 480 nm. In the conventional method, the color yield (absorbance at 480 nm) increased by 74% for MF, suggesting that for optimal condensation of MF with phenol, heat is required. This is further supported by the observation that when MF (50 pg) is heated with phenol at 100°C in 75% sulfuric acid, absorbance at 480 nm increased from 0.11 at 0 time to 0.36 in 15 min and to 0.66 in 30 min without significant change in the spectral profile. FDMH, in which the aldehyde group is blocked, reacted poorly in the conventional phenol-sulfuric acid
~~~~A 400
440
480
520
400
440
480
520
400
440
‘lea
520
FIG. 2. Spectra of chromogens formed by furaldehyde derivatives in the phenol-sulfuric acid reaction. (0) Conventional method. (A) Modified method. (0) Reaction at room temperature. (A) FFS, 10 pg; (B) MF, 100 pg; (C) FDMH: conventional, 80 pg; modified, 6.0 gg; room temperature, 6.0 pg.
180
RAO
AND
PATTABIRAMAN
method (Fig. 2C). The fold increase in color in the modified method was nearly 24, the highest observed for any furaldehyde derivative. The data suggest that the rate of sulfonation of phenol in the conventional assay conditions is much faster than the hydrolysis of the hydrazone and the subsequent reaction of the resultant furaldehyde with phenol. FDMH however reacted efficiently with phenol at room temperature as shown in the figure, indicating that hydrolysis of FDMH in 75% H,SO, is almost complete in 30 min. In Fig. 3, the ultraviolet spectra of compounds derived from mannose, galactose, fucose, and FDMH in 75% sulfuric acid are compared with those of HMF, MF, and furaldehyde. The spectra of mannose and galactose products are not identical to that of HMF (Fig. 3A). The ratios of absorbance at 255 nm and 320 nm for mannose (0.57), galactose (0.53), and HMF (0.21) suggest that apart from HMF other products are formed in considerable amount from the hexoses. FFS does not appear to be formed from HMF in 75% sulfuric acid with heat of dilution since FFS in 75% sulfuric acid showed an absorption maxima at 285 nm. When HMF was subjected to heat (115-118°C) in 75% sulfuric acid, there was no alteration in the spectra. As a first approximation, based on optical density values at 320 nm, it can be calculated that mannose and galactose yielded 31.7% and 30.2% HMF, respectively. This agrees well with the color intensities formed in the modified phenol-sulfuric acid method (Table 1). The uv spectrum of products formed from fucose was also not identical to that of MF (Fig. 3B). On the basis of absorption at 330 nm, it can be computed that 59% of fucose is converted to MF in the presence of heat in 75% sulfuric acid. This is in fair agreement with the yield of chromogens formed from fucose in the modified method. The uv spectrum of FDMH is similar to that of furaldehyde (Fig. 3C). FFS showed an almost identical uv spectrum in 75% sulfuric acid, before and after exposure to heat of dilution. The absorbance at 285 nm was 4.0% higher after
FIG. 3. Ultraviolet spectra of compounds. A, (0) mannose, 50 pg. (0) Galactose, 40 pg. (A) HMF, 12.6 pg in 75% H,SO, after heat of dilution. B, (0) Fucose, 20 pg. (0) MF, 10 I.cg in 75% H,SO, after heat of dilution. C, (A) Furaldehyde, 9.5 pg. (0) FFS, 25 pg. (0) FDMH, 10 pg in 75% H,SO,.
TABLE
1
Molar Absorptivities of Chromogens Formed in PhenolSulfuric Acid Reactions and Furaldehyde Derivatives Phenol-sulfuric reaction absorptivity Compound HMF Furaldehyde MF FFS Mannose Galactose Fucose FDMH
acid molar X low3
A 47.6 42.4 1.01 40.8 13.7 14.0 0.8 40.5
B (485) (475) (480) (470) (480) (480) (480) (475)
14.4 12.7 7.67 1.66
(480) (480) (480) (475)
Ultraviolet absorption molar absorptivity x 10-s 20.4 19.2 21.6 10.5 6.05 7.56 12.1 19.9
(325) (315) (330) (285) (320) (320) (330) (315)
Note. A, Reaction at room temperature for furaldehyde derivatives and reaction at room temperature for hexoses and FDMH after exposure to heat of dilution in 75% H,SO,. B, Reaction by the conventional method. Values in parentheses indicate absorption maxima, and are expressed in nanometers.
heat treatment. Similar behavior was shown by MF, the absorbance at 330 nm increasing by about 5.0% after exposure to heat. FDMH also was found to be stable to heat treatment in 75% sulfuric acid. The absorbance at 315 nm increased by 1.6% after exposure to heat of dilution. The spectral profiles in cold 75% sulfuric acid and after exposure to heat were identical. On the other hand, with HMF and furaldehyde, even though the spectra were identical after heat treatment in 75% H,SO,, absorbance at 325 and 315 nm respectively, decreased by 33% and 25%, indicating partial destruction. In Table 1, the molar absorptivities of chromogens formed in the phenol-sulfuric acid reaction and of furaldehydes in the uv region are indicated. DISCUSSION The present studies provide additional evidence in support of the earlier conclusion (1) that the color intensities in the modified phenol-sulfuric acid method are an accurate reflection of HMF formed from hexoses in hot 75% sulfuric acid. On the other hand, the chromogens formed in the conventional method (2) represent, besides the condensation product of HMF with phenol, other partially dehydrated compounds of hexoses and probably other side products. Dische (3) and Love (4) indicated that in addition to HMF, sugars treated with strong sulfuric acid produce a large number of intermediate compounds related to or derived from HMF. Formaldehyde, propionaldehyde, 2-hydroxyacetyl furan, MF, and several other reducing compounds are shown to be formed from hexoses on acid treatment (5-8). 2-Formyl dihydrofuran derivatives are also envis-
SPECTROPHOTOMETRIC
STUDIES
OF
aged as intermediates during the degradation of hexoses (9). It is probable that some of these compounds form chromogens with phenol at elevated temperature. Fucase and its major dehydration product, MF, reacted poorly with phenol at room temperature. The color intensity of MF was nearly 50 times less than that of HMF. FDMH displayed a marked increase in color intensity in the modified method. This is of particular relevance for the determination of Amadori compounds since it has been proposed that during acid-mediated dehydration of 1-amino-1-deoxyfructose derivatives, imines are formed as intermediates (10). Substitution at the 5-position of furaldehyde was found to markedly affect the stability of the furaldehydes and their capacity to condense with phenol in 75% sulfuric acid. Substitution of a hydroxymethyl group marginally decreased the stability of the compound and the ability to condense with phenol in hot 75% sulfuric acid. The presence of a sulfonate group in the 5-position made the compound stable and FFS
PHENOL-SULFURIC
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REACTION
reacted efficiently with phenol. The presence of a 5methyl group also stabilized MF to acid dehydration but the compound reacted poorly with phenol. REFERENCES 1. Rao, P., and Pattabiraman, T. N. (1989) Anal. Biochem. 181, 18-22. 2. Du Bois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350-356.
3. Dische, Z. (1949) J. Biol. Chem. 181,379-392. 4. Love, R. M. (1953) Biochem. J. 55, 126-132. 5. Rice, F. A. H., and Fishbein, L. (1956) J. Amer. Chem. Sot. 78, 3731-3734. 6. Shaw, P. E., Tatum, J. H., and Berry, R. E. (1967) Carbohydr. Res. 5,266-273. 7. Feather, M. S., and Harris, J. F. (1973) Adu. Carbohydr. Chem. Biochem.
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O., and Nelson, 46, 273-325.
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9. Anet, E. F. L. J. (1964) Ado. Carbohydr. Chem. 19, 181-219. 10. Yaylayan, V., and Sporns, P. (1987) Food Chem. 26, 283-305.