Ultrasound assisted extraction and determination of the
carbohydrate fraction in marine sediments
Mauro Mecozzi *, Patrizia Dragone, Marina Amici, Eva Pietrantonio
Istituto Centrale per la Ricerca Scienti®ca e Tecnologica Applicata al Mare, via di Casalotti n. 300, 00166 Rome, ItalyAbstract
In this paper, an ultrasound±acetic acid procedure for the simultaneous extraction and hydrolysis of carbohydrates in marine sediments prior to their colorimetric determination is described. The main advantage of the proposed pro-cedure is the improved analytical accuracy achieved. Extraction and hydrolysis are quantitative since the oxidative reactions which cause the underestimation of the total carbohydrate amount are minimized. Moreover the procedure is fast, requiring only 5 h for the whole analysis (extraction, hydrolysis and colorimetric determination by the phenol-sulphuric acid method). The proposed procedure has recoveries generally higher than 80% and gives comparable results with the conventional 24 h HCl extraction.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Carbohydrates; Ultrasound; Marine sediments; Colorimetric analysis
1. Introduction
Carbohydrates make up a signi®cant fraction of the organic matter present in soils and marine sediments and, due to their interaction with metals (Linnik and Vasil'chuk, 1995; Paciolla et al., 1999), they play a fun-damental role in the bioavailability of inorganic pollu-tants. For this reason, the quantitative determination of carbohydrates in environmental samples such as soils and marine sediments is extremely important for the characterisation of areas submitted to ecological studies. However, the determination of carbohydrates in solid samples is often a troublesome analytical procedure. Car-bohydrates are present in environmental samples as mono, oligo, and polysaccharides and all of these classes must be considered in order to perform an accurate analytical determination. The analytical accuracy of both colori-metric (Dubois et al., 1956; Miklestad et al., 1997) and chromatographic determinations (Sawardeker et al., 1965; Walters and Hedges, 1988; Han and Robyt, 1998; Jahnell et al., 1998) is strictly dependent on the quanti-tative conversion (i.e. hydrolysis) of polysaccharides
into monosaccharides and the avoidance of possible oxidative reactions of carbohydrates (Mecozzi et al., 1999).
The study of the carbohydrate fraction in soils and sediments is generally included in the characterization of humic substances which make up the main portion of naturally occurring organic matter in environmental samples (Klavins and Apsite, 1997).
The extraction of humic substances from marine sediments requires a 24 h acid pre-treatment and repe-ated 24 h alkaline extractions (Senesi et al., 1989; Cam-panella et al., 1995). Thus, the time consuming extraction is an additional disadvantage in the determi-nation of carbohydrates in solid samples. In a recent study, a microwave assisted procedure has been pro-posed to reduce the time needed for the analysis of car-bohydrates in soils (Gao et al., 1995). Because the solvent is in a sealed system, microwave heating allows a boiling point greater than that at atmospheric pressure (Barnabas et al., 1995). However the risk of partial oxidation of the carbohydrates due to heating in an aqueous acid medium cannot be excluded (Ledl and Schleicher, 1990).
Recently, ultrasound has been applied to the extrac-tion of polysaccharides in medicinal grass (HromaÂdovka et al., 1999), to improve the hydrolysis of poly-saccharides in environmental (seawater) and food (rice
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* Corresponding author. Tel.: 06-615-701-458; fax: +39-06-615-619-06.
and pasta) samples (Mecozzi et al., 1999), to hydrolyse lactose in milk fermentation products (Wang and Saka-kibara, 1997) and to hydrolyse native dextran synthe-sized by various microorganisms (Lorimer et al., 1995). The aim of this paper is to investigate the possible application of ultrasound to the simultaneous extraction and hydrolysis of carbohydrates in marine sediments. The proposed method is based on an ultrasound treat-ment coupled to an acid treattreat-ment, followed by the col-orimetric determination of carbohydrates according to the phenol±sulphuric acid method (Dubois et al., 1956). The procedure requires only 5 h for the whole analysis and gives results comparable to the conventional 24 h extraction by 1M HC1.
2. Experimental
2.1. Sampling
Terrigenous sediments were sampled oshore from the Continental Shelf of the West Italian coast (Tyr-rhenian Sea) by a Van Veen grab. Samples were super-®cial primarily clays with a concentration of organic carbon 0.680.5 w/w (Branca et al., 1996). The samples were stored frozen (ÿ20C) until analysis. The samples
were also lyophilized prior to analysis.
2.2. Reagents
All the reagents, i.e. soluble starch, standard carbo-hydrates, H2S04, HC1, CH3COOH, CF3COOH,
hydro-xylmethyl-2-furfural (HMF), phenol and KBr were of analytical reagent grade (Carlo Erba). Only MilliQ ultra-purity water was used for all the treatments.
2.3. Ultrasound treatment for extraction and hydrolysis
The ultrasound treatment was performed at room temperature in an ultrasonic cleaning bath (Elma model T890H 35 kHz) containing distilled water and ethylene glycol as antifouling additive. 1 M CH3COOH (50
ml0.05) was added to dried marine sediment (1.0 g0.01) in a 100 ml glass container. Each sample was sonicated at room temperature (4 h).
After sonication, the samples were centrifuged and the supernatant was used for the colorimetric analysis.
2.4. Instrumental
Colorimetric measurements were performed using a Varian DMS double beam UV±VIS spectrophotometer (0.1 nm resolution). Infrared measurements were per-formed using a Jasco 410 Fourier transform infrared spectrophotometer. Diuse re¯ectance spectra were collected at 4 cmÿ1resolution from 650 to 4000 cmÿ1
after 500 scans by using the cosine function as the apo-dization process.
2.5. Colorimetric analysis
Standard aqueous solutions of glucose (10±100 mg/l) were used for instrument calibration. Phenol (1.0 ml, 5% w/w) and concentrated H2SO4(5.0 ml) were added
to the supernatant (2.0 ml) or to a standard solution of glucose (2.0 ml) in glass tubes. The tubes were allowed to stand for 30 min in a shaker bath at 25±30C before
measuring the absorption.
Absorption was measured in a 10 mm quartz cell at 485 nm against a spectrophotometric blank consisting of deionized water (2.0 ml), phenol (1.0 ml, 5% w/w) and concentrated H2S04(5.0 ml). The limit of detection
of the procedure is 20 mg of carbohydrates expressed as glucose for 1 kg of sediment (20 mg/kg). Due to the average levels of carbohydrates in marine sediments, this value is satisfactory but the detection limit can be enhanced (down to 2 mg/kg) by using a 100 mm quartz cell and a calibration range for glucose within 0.1-1.0 mg/l.
2.6. FTIR measurements
FTIR spectra of soluble starch and sediment samples were collected before and after the sonication in the acid medium. The spectra of non-sonicated samples were collected on dried samples. The spectra of sonicated samples were collected after the evaporation of the acid medium. Pellets were prepared with a ratio of 100 mg of KBr and 20 mg of sample. The spectra were converted into Kubelka-Munch units (K/M).
3. Results and discussion
The extraction and hydrolysis of carbohydrates based on the application of ultrasound only, does not guaran-tee quantitative hydrolysis. Whilst ultrasound is able to accelerate the rate of hydrolysis, an acid medium is necessary to obtain the quantitative conversion of poly-saccharides to monopoly-saccharides. However, a procedure based on simultaneous extraction using ultrasound and sulphuric acid is not reliable because it produces strong oxidative conditions (Mecozzi et al., 1999). A single step extraction and hydrolysis of polysaccharides can be performed by using a non-oxidizing acid such as HCl, CH3COOH or CF3COOH and in preliminary
quantitative extraction and quantitative hydrolysis of carbohydrates without simultaneous oxidation. With respect to temperature, its enhancement can accelerate the extraction rate but it is also known to lower the reaction rate of hydrolysis of polysaccharides due to a reduction in phenomena related to the acoustic cavita-tion (Lorimer et al., 1995). Oxidative reaccavita-tions could also be enhanced by the high energy conditions of ultrasonic irradiation (Suslik, 1990; Price, 1992). Con-sequently, all of the experiments to identify the most suitable acid were carried out at room temperature only. In Fig. 1, the curves for the colour development of 50 mg/l solutions of soluble starch in dierent acid media are reported. They were compared to the colour devel-opment of a 50 mg/l soluble starch solution sonicated (without any acid) for three hours and then added to the phenol and sulphuric acid solutions. This curve was chosen as a reference because the treatment gives a quantitative conversion of starch to glucose (Mecozzi et al., 1999).
Although CF3COOH is used before the gas
chroma-tographic speciation analysis of carbohydrates (Walters and Hedges, 1988), it is not shown in Fig. 1 and it was excluded from further study owing to an interference with the reagents of the spectrophotometric blank in the Dubois method. HC1 and CH3COOH gave more
inter-esting results. Acoustic cavitation produces hydroxyl radicals and hydrogen peroxide (Suslik, 1990) but their amount can be inhibited by the presence in solution of
a radical scavenger (Henglein, 1987). Chloride ion is a good radical scavenger because it reduces the eciency of a persulfate in the determination of organic carbon in seawater (Peyton et al., 1993). The radical scavenging property of chloride ion is con®rmed by the results in Fig. 1. In fact, the higher the concentration of HC1, the slower is the colour development of the starch solutions and even the 0.5 M HCI solution, having the highest development rate, still has absorption values far from the reference solution. Hence, although HC1 is used for the extraction of humic substances in soils and sedi-ments (Campanella et al., 1995), its coupled use with ultrasound is not recommended because it does not guarantee a good recovery. The use of CH3COOH gives
better results. The maximum absorption of the starch solution hydrolysed with l M CH3COOH is reached
after 4±5 h. After 5 h a small reduction in the absorption is observed, followed by a rapid increase after about 6 h. The absorption for starch in 2 M CH3COOH shows a
reduction before reaching that of the reference solution and then surpassing it. Both the reduced and increased absorption depend on the oxidation of starch and the FTIR study of the products of the 2 M CH3COOH
ultrasonic treatment (Fig. 2) con®rms the presence of degradation products.
In Fig. 2 the IR spectra of sonicated starch in water without oxidative reactions (reference spectrum), soni-cated starch in 2 M CH3COOH (5 h) and
hydro-xylmethyl-2-furfural (HMF) are reported. HMF, one of
the most common compounds produced from the hexoses on heating in acid solutions (Ledl and Schlei-cher, 1990) has the typical absorption band due to the ± CO stretching at 1670 cmÿ1. The same absorption is
observed for the starch sample after the 5 h 2 M CH3COOH ultrasound treatment and the rest of the
spectra do not really correspond (essentially the 1100± 1200 cmÿ1 region). Consequently the results of Fig. 2,
con®rm the presence of products arising from oxidative reactions of carbohydrates. Moreover the presence of ±CO groups arising from oxidative reactions of carbo-hydrates could also explain the shape of the curve of colour development observed in Fig. 1 for the 2 M CH3COOH ultrasound treatment. In fact the ±CO
group of carbonyl compounds also reacts with the phe-nol±sulphuric mixture (Dubois et al., 1956) and the rapid enhancement of the visible absorption at 485 nm depends on the presence of degradation products having the ±CO functional group. This implies the colour yield of the oxidation products is higher than that of the original carbohydrates or that additional carbonyls are being formed.
On the basis of the results shown in Figs. 1 and 2, we studied the 1 M CH3COOH ultrasound treatment in
more detail. In Table 1, we report the comparison of the visible absorption for some standard solutions of car-bohydrates with and without the 4 h 1 M CH3COOH
ultrasound treatment. The comparable absorptions con®rm the absence of oxidative reactions of mono and disaccharides, so these conditions were applied to the extract and hydrolysis of carbohydrates in sediments. In Table 2, the concentrations of carbohydrates in some sediments, obtained by two sequential 4 h ultrasound treatments in 1 M CH3COOH are reported. Although
the results in Fig. 1 indicate that the treatment longer than 5 h causes the formation of degradation products, we submitted the samples to two sequential
treatments of 4 h each in order to evaluate the risk of carbohydrate oxidation in real samples and the percent recovery. The concentrations present in the second extraction were generally much lower than the con-centration in the ®rst extraction and sometimes were below the limit of detection. According to the results in Fig. 2, the absorption observed in the second extraction (i.e. for treatment longer than 5 h) must be produced by
Fig. 2. Evidence for the presence of oxidative reactions in a sample of starch sonicated for 5 h in 2 M CH3COOH. The band at 1670
cmÿ1in the HMF and sonicated CH
3COOH spectra is absent from the reference spectrum of starch sonicated in water.
Table 1
Comparison of absorption at 485 nm by Dubois method for some standard solutions of monosaccharides (fructose and glucose) and sucrose with and without 4 h treatment using the 1 M CH3COOH±ultrasound procedurea
Un-treated Treated
Glucose 0.5100.035 0.4800.026 Fructose 0.7950.050 0.8320.058 Sucrose 0.7580.040 0.7880.045
a The concentration is 40 mg/l each. The results are the
average of three repeated measurements.
Table 2
Concentrations of carbohydrates obtained by two sequential extractions with the ultrasound treatment in 1 M CH3COOH
for 4 h
Sample First extraction (% w/w)
Second extraction (% w/w)
1 0.52 N.D.a
2 0.20 N.D.
3 0.38 0.05
4 0.40 0.06
5 0.24 0.04
the degradation products of carbohydrates instead of by the actual carbohydrate content. In fact the degradation products such as HMF, including the reducing carbonyl group, react with the phenol±sulphuric acid reagent (Dubois et al., 1956). Hence it appears that the ®rst extraction gives recoveries higher than 80%.
It is also interesting to know the chemical origin of the carbohydrates determined with the proposed proce-dure. Carbohydrates can be present in all the fractions of humic substances. Humic substances are the main component of the organic matter in soils and sediments and cannot be extracted quantitatively because only the fulvic acids, soluble at any pH, and the humic acids, soluble at pH>2, can be extracted by an aqueous med-ium (Klavins and Apsite, 1997). The so-called ``bio-available fractions'' of organic matter consist of these two fractions while humin is the unavailable fraction because it is insoluble at any pH. Further studies are necessary to see if the carbohydrates extracted by means of the proposed procedure belong to all of the three fractions or whether they belong to the bio-available fractions alone. FTIR spectroscopy can give some help-ful indications. In Fig. 3, the IR spectra of a sediment before and after the proposed treatment are reported. The major evidence for the presence of carbohydrates is shown by the absorption due to the asymmetric stretching of the ±C±O±C group at 1156 cmÿ1(Smith,
1999). The absorption of this functional group is so sensitive (detection limit 0.02% v/v) that it is used for the quantitative estimation of non-ionic surfactants in waste water (Andrew, 1993) and for the estimation of icing inhibitor in aviation turbine fuels (Institute of Petroleum, London, 1992). The absence of this sensitive band in the treated sample suggests that the proposed procedure can hydrolyse carbohydrates in the
bio-available fractions and probably also carbohydrates in the humin fraction.
Finally, in Table 3 we report a comparison between the amount of carbohydrates determined by the pro-posed procedure and the 24 h 1 M HCl treatment at room temperature. The latter method is used to hydro-lyse chemical bonds between humic substances and metals present in clay and sand (Campanella et al., 1995) and to hydrolyse cellulose (Wayman, 1986). The two procedures give similar results but sometimes the HC1 treatment shows a spectral interference giving an absorption partially overlapping with the absorption at 485 nm for the carbohydrates (Fig. 4). This interference could depend on the presence of FeC13 having an
Fig. 3. Comparison between IR spectra of a sediment sample before and after the 1 M CH3COOH±ultrasound treatment. In the
spectrum of the untreated sample the absorption band of the C±O±C group (1156 cmÿ1) of polysaccharides is present but is absent
from the treated sample.
Table 3
Comparison between 24 h l M HCl extraction at room tem-perature and the proposed extractiona,b,c
Sample HCl extraction (% w/w)
Ultrasound extraction (% w/w)
Experimental
t-test
1 0.380.026 0.440.036 2.38 2 0.250.021 0.240.015 0.61 3 0.550.050 0.500.020 1.61 4 0.300.035 0.290.030 0.28 5 0.220.010 0.250.021 2.32
a The results are the average of three repeated
measure-ments.
b The concentrations of carbohydrates in the second
extrac-tions are not reported because the products were not detect-able.
c Because the critical t-test value at the 95% level of
intense absorption between 500 and 200 nm with a maximum at 315 nm.
The ultrasound±1 M CH3COOH procedure does not
have this spectral interference and so is more accurate and less time consuming than the HC1 extraction.
4. Conclusion
The proposed procedure based on the application of ultrasound±acid extraction prior to the colorimetric technique of Dubois is an accurate and rapid method for the analysis of extractable total carbohydrates in marine sediment samples. The analytical accuracy depends on several factors such as the absence of oxi-dative reactions, the quantitative hydrolysis resulting from the joint eect of ultrasound and the l M CH3COOH medium and the absence of spectral
inter-ference in the colorimetric analysis. Moreover, this pro-cedure can be also applied to the speciation of monosaccharides in polysaccharides by chromato-graphic methods (Walters and Hedges, 1988; Jahnel et al., 1998) where the derivatization procedure is per-formed in an alkaline medium and the neutralisation of a 1 M CH3COOH solution is less problematic than that
of samples hydrolysed by more concentrated acids (Wayman, 1986).
Finally, an additional advantage of the proposed procedure is that the use of the ultrasound cleaning bath allows the simultaneous treatment of several samples, which is essential in monitoring studies.
Acknowledgements
This study was supported by the scienti®c research program ``Mucillagini in Adratico e Tirreno'' ®nanced by the Italian Ministry of the Environment. The authors are grateful to Dr. S. Silenzi of ICRAM for the helpful information about the geological characteristics of marine sediments in the Italian West coast.
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