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Soil Biology & Biochemistry 39 (2007) 26652669 www.elsevier.com/locate/soilbio

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Determination of neutral sugars in soil by capillary gas chromatography after derivatization to aldononitrile acetates
Wei Zhanga,b, Hongbo Hea, Xudong Zhanga,c,
a

Key Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China b Graduate University of Chinese Academy of Sciences, Beijing 100049, China c National Field Research Station of Shenyang Agroecosystems, Shenyang 110016, China Received 30 December 2006; received in revised form 13 March 2007; accepted 2 April 2007 Available online 11 May 2007

Abstract We have quantied ribose, rhamnose, arabinose, xylose, fucose, mannose, glucose, and galactose in soil by gas chromatography (GC) simultaneously after converting to aldononitrile acetate derivatives. A recommended single-hydrolytic step by 4 M triuoroacetic acid (TFA) at 105 1C for 4 h was more effective for releasing soil neutral sugars from non-cellulosic carbohydrates and better suited to our purication procedure compared with the sulphuric acid hydrolysis. Linearity of the GC detection for each neutral sugar was in the range of 10640 mg ml1 and the recovery of neutral sugars from the spiked soil samples ranged from 76% to 109.7%. The coefcients of variation of the neutral sugars in four soils were lower than 2.0% for the instrument and 4.67.6% for the whole determination procedures. Compared with the trimethylsilyl (TMS) derivatization, the recovery of our newly modied method was more satisfactory and the reproducibility of ribose was improved signicantly. Moreover, the aldononitrile acetate derivative was more stable than TMS derivative. Therefore, it is a promising approach suitable for a routine use in the quantitative analysis of soil neutral sugars, since it is fast, sensitive, and reproducible. r 2007 Elsevier Ltd. All rights reserved.
Keywords: Aldononitrile acetate; Gas chromatography; Soil neutral sugars; TFA hydrolysis; Determination

1. Introduction Soil carbohydrates, including neutral and amino sugar, represent 525% of soil organic matter (SOM) (Angers et al., 1988; Murata et al., 1999). In the carbon cycling in terrestrial system, neutral sugars constitute a signicant part of the labile pool of SOM and provide a major source of energy for microbial processes in soils (Martens and Loeffelmann, 2002). Moreover, they are the most crucial binding agents for soil aggregation (Cheshire, 1979; Oades and Waters, 1991; Puget et al., 1999). The origin and relative contribution of individual monosaccharides to the total carbohydrate pool may provide an indicator of organic matter origin (Hu et al., 1995). For instance,
Corresponding author. Key Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China. Tel.: +86 24 83970375; fax: +86 24 83970436. E-mail address: xdzhang@iae.ac.cn (X. Zhang).

arabinose (A) and xylose (X) are considered to be mostly originated from plant, whereas mannose (M) and galactose (G) are mainly of microbial origin in soil (Chantigny et al., 2000), and thus, the ratio of (G+M)/(A+X) is often used to characterize the origin of soil carbohydrates. When the ratio is lower than 0.5, the contribution is considered mainly from plant materials, whereas the ratio higher than 2 is a clue of microbial origin (Oades, 1984). Therefore, it is important to quantify accurately the neutral sugar compositions of soil carbohydrates. Capillary gas chromatography (GC) is a very universal tool to analyse carbohydrates in soil. In this case, the derivatization must be conducted before GC determination because saccharides are non-volatile. Alditol acetates derivatization have been used to derivatize soil neutral sugars (Oades, 1967; Dormaar, 1984; Cheshire et al., 1990); however, this method presents the disadvantage of a large number of manual preparation steps, which makes the procedure time consuming and tedious to be performed

0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.04.003

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2666 W. Zhang et al. / Soil Biology & Biochemistry 39 (2007) 26652669 Table 1 Some properties of soils (020 cm) Samples Organic C (g kg1) 34.00 12.51 9.55 7.68 Total N (g kg1) 2.49 1.52 0.86 1.07 pH DTPA-Fe (mg kg1) 86.65 48.38 67.57 166.91

(Zhang and Amelung, 1996). Rumpel and Dignac (2006) have tried to simplify the procedure by eliminating the removing step of borate salts during purication, but the recovery of ribose decreased signicantly (only 35% on average) and the coefcient of variation of fucose was too high (up to 45%). In order to overcome the inherent disadvantage of alditol acetates derivatization, trimethylsilyl (TMS) or O-methyl-oxime-TMS derivatization has been developed to quantify neutral sugars in soils by GC (Amelung et al., -Larrouy and Feller, 1997). Indeed, TMS 1996; Larre derivatization is much simpler, but it forms multiple peaks for each sugar, which made the GC separation more difcult and would increase the deviation of the determination (Andrews, 1989). In addition, TMS derivatives are -Larrouy and Feller, 1997); unstable during storage (Larre hence, the quantitative analysis is uncertain if the prepared samples are not determined within short time. In this context, still there a need to nd a derivatization procedure for more convenient and reliable determination of soil neutral sugars. Aldononitrile acetate derivatives have been tested to quantify neutral and amino sugars in biological samples (Guerrant and Moss, 1984) and amino sugars in soil samples by GC technique (Zhang and Amelung, 1996). Because the chemical structure of amino sugar and neutral sugar molecules involved in derivatization (i.e. CHO and OH) are similar, it is promising to extend the aldononitrile acetate derivatization to soil neutral sugars. According to Zhang and Amelung (1996), the aldononitrile acetate derivatization is sensitive and less time consuming than that of the alditol acetate derivatization. In addition, each neutral sugar produces single chromatographic peak. Therefore, it is worth evaluating this technique for the determination of soil neutral sugars. Traditionally, soil neutral sugars are released by H2SO4 (Cheshire, 1979), but it has been reported that TFA is more efcient than H2SO4 in releasing non-cellulosic saccharides from soils (Amelung et al., 1996). However, it is still not clear which hydrolytic procedure is better suited to the aldononitrile acetate derivatization and which purication procedure can be employed. Our objectives were therefore to evaluate the response and precision of aldononitrile acetate derivatization for GC determination of soil neutral sugars and then to nd out a suitable hydrolysis and purication procedure for the derivatization.

Endoaquepts Hapludolls Hapludalfs Hapludults

6.1 6.1 6.7 5.5

from the surface layer (020 cm), air-dried and ground to pass 250 mm sieve prior to hydrolysis. The weighted soil samples (containing about 4 mg organic C) were hydrolyzed in two ways: (1) 12 M H2SO4 (1 ml) in a closed hydrolysis ask at ambient temperature for 16 h followed by treatment with 1 M H2SO4 at 100 1C for 6 h (Hu et al., 1995). (2) 4 M TFA at 105 1C for 4 h (Guggenberger et al., 1994; Amelung et al., 1996). After hydrolysis, 100 ml of internal standard (adonitol of 1 mg ml1) was added to the hydrolysate, and then the mixture was ltered through a glass ber lters (GF 6, Schleicher and schuell, Germany). The soil neutral sugars liberated by H2SO4 were puried according to the procedure of Cheshire (1979) with slight modication. The key step was that the ltrate was neutralized with Ba(OH)2 to pH 6.66.8. For TFA hydrolysis, the slightly modied purication procedure of Zhang and Amelung (1996) was applied. Briey, the ltrate was dried completely by a rotary evaporator at 45 1C under vacuum. Thereafter, the residue was dissolved with 20 ml of distilled H2O and the pH was adjusted to 6.66.8 with 0.4 M KOH. The precipitates produced during purication either from H2SO4 or TFA hydrolysate were removed by centrifugation (3000 rpm for 10 min) and the clear supernatant was dried again by a rotary evaporator at 45 1C. The residue was dissolved with 4 ml distilled H2O, transferred to a Reacti-VialTM of 5 ml, and then freeze-dried completely under vacuum. Neutral sugars in the four soil samples after TFA hydrolysis were also determined as TMS derivatives by the method of Amelung et al. (1996), but the detail procedures are not described here because the TMS results were used for comparison only. Derivatization was conducted according to the procedure of Zhang and Amelung (1996). The second internal standard (myo-inositol) was added before derivatization and the area ratio of myo-inositol to adonitol represented the average recovery of the purication and derivatization. The derivation reagent (0.3 ml), which contained 32 mg hydroxylamine hydrochloride ml1 and 40 mg 4-(dimethylamino) pyridine (DMAP) ml1 in pyridinemethanol (4:1, v/v), was added to the Reacti-VialTM with a dry sample or a mixture of standards in it. The capped vial was shaken and heated for 30 min at 7580 1C (during heating, the vial was shaken once more). Then, the vial was cooled to room

2. Soils and methods Four soil samples with different organic carbon (OC) contents were used for neutral sugar analysis and the soils were classied according to US soil taxonomy (Soil Survey Staff, 2003) (Table 1). The Hapludults sample was collected from the Jiangxi Red Soil Research Station, the Hapludolls from Gongzhuling, the Endoaquepts from Hailun, and the Hapludalfs from Shenyang, China. All soils were sampled

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temperature and 1 ml of acetic anhydride was added. The vial was closed, shaken again, and heated for 20 min at 7580 1C. After cooling, 1.5 ml dichloromethane was added. The excessive derivatization reagents were removed by two washing steps. Firstly, 1 ml 1 M HCl was added, and the mixture was strongly shaken for 30 s, and then the upper aqueous phase was removed. Secondly, the organic phase was extracted three times with distilled water (1 ml each). After the last washing, the water was completely removed and the organic phase was dried with N2 at 45 1C, and then the residue was dissolved in 200 ml of ethyl acetate-hexane (1:1, v/v) for GC analysis. The derivatization procedure was less laborious and three times more rapid than that of alditol acetates derivatization (Guerrant and Moss, 1984). The GC analysis was performed on a HP 6890 GC equipped with ame ionization detector (FID) (Agilent, USA). A DM-1 fused silica capillary column (30 mm 0.25 mm 0.25 mm lm thickness) was used to separate the neutral sugar derivatives with a split ratio of 30:l. Nitrogen gas was used as a carrier gas at a column ow rate of 1.2 ml min1. The temperature programme was set as follows: the initial column temperature of 175 1C was held for 4 min and then the temperature was increased at 4 1C min1 to 225 1C, held for 2 min. Thereafter, the temperature was increased again at 50 1C min1 to 300 1C, held for 2 min. The inlet temperature was 250 1C and the temperature of detector was 300 1C. The results were processed using the Agilent ChemStation A.08.03. The retention times of the derivatives are listed in Table 2. Another rejected fused silica column HP-5 (30 m 0.32 mm 0.25 mm) was also tested, but baseline resolution was not obtained, in which ribose coeluted with rhamnose and fucose coeluted with xylose. The calibration (linearity) was carried out by using a standard mixture containing 10, 20, 40, 80, 160, 320, 640 and 800 mg of each sugar ml1 and 100 mg internal standard

(adonitol). We also estimated the recovery of each neutral sugar standards by spiking the standard to each soil hydrolysates in ve replicates (Table 2). Reproducibility of the method was evaluated by performing all analyses in triplicate. 3. Results and discussion Our purication procedure was faster and simpler compared with that of Zhang and Amelung (1996), because the methanol step was eliminated. Moreover, it is also better than the procedure of Amelung et al. (1996), who use two resin columns, which are not convenient for routine use. When using the TFA hydrolysis and aldononitrile acetate derivatization, baseline resolution was obtained for the derivatives of the eight neutral sugars and two internal standards and chromatograms were free of peaks of major contaminants (Fig. 1). The FID chromatogram of each compound has a single peak as conrmed by using gas chromatographymass spectrometer (GCMS) (Finnigan trace, Thermo Electron Finnigan Co. Ltd., USA). In addition, the ratios of individual neutral sugar to internal standard (adonitol) remained constant during the storage of the derivatives for 1 month, indicating that the aldononitrile acetate derivatives are more stable than O-methyl-oxime-TMS derivatives (Amelung et al., 1996) and our evaluation showed that the total amount of eight neutral sugars in the four soils increased 10% on average after 2-day storage due to signicant changes in glucose, fucose, and ribose. The response factors for the eight sugars was obtained within a short range (Table 2), regardless of sample concentrations and this was essential for quantitative determination of soil neutral sugars by the method. For the linearity evaluation, a concentration range of 10800 mg ml1 was satisfactory for each sugar (R240.990) but a better regression was obtained in the range 10640 mg ml1 (R240.999) since the linearity of fucose became worse slightly at the concentration4640 mg ml1. Hence, the range of 10640 mg ml1 is recommended for routine use. We did not evaluate lower concentration since 10 mg ml1 is low enough for determining soil neutral sugars according to Zhang and Amelung (1996). The coefcients of variation for the analysis of the eight neutral sugar standards were lower than 4.2%, on average (n 3), while those for the repeated instrumental analysis (n 3) were not bigger than 2.0%. The recoveries of each neutral sugar relative to adonitol after TFA hdrolysis from the spiked samples was 100.1% on average (Table 2), indicating that the adonitol behaved similarly to the sugars through the whole sample preparation, and thus any loss of the sugars during the sample preparation should be proportional to that of the adonitol. However, the recovery of ribose was only 76%. Our further experiment showed that when ribose and adonitol were added to the hydrolysate after ltration, the recovery relative to adonitol was improved to 90%. This proves that

Table 2 Retention times (RT), response factors (Rf) and recovery of eight neutral sugars in the spiked soila Compounds RT (min) 9.89 10.08 10.29 10.47 10.59 13.13 14.92 15.11 15.67 Rf (mean7SD, n 3) 1.6570.029 1.4370.010 1.5870.022 1.6970.027 1.7870.032 1 1.5670.027 1.4470.023 1.6970.026 Recovery (%) (mean7SD, n 5)b 76.076.9 99.473.4 100.074.1 104.579.6 109.3711.6 93.578.2 109.776.3 108.171.7

Ribose Rhamnose Arabinose Xylose Fucose Adonitol Mannose Glucose Galactose

a The sugars and internal standard were converted to aldononitrile acetate and then resolved by a DM-1 fused silica capillary column (30 mm 0.25 mm 0.25 mm lm thickness). b Recovery of neutral sugars relative to adonitol in TFA hydrolysates of spiked soils.

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Fig. 1. Gas chromatograms of aldononitrile acetates of neutral sugars. (a) Standards and (b) soil sample: (1) ribose, (2) rhamnose, (3) arabinose, (4) xylose, (5) fucose, (6) adonitol, (7) mannose, (8) glucose, (9) galactose, and (10) myo-inositol.

Table 3 Neutral sugar contents of four soils (020 cm) (means7SD, n 5) determined after aldononitrile acetate and trimethylsilyl derivatization (TMS) (mg kg1 soil) Samples Endoaquepts Aldononitrile acetate Ribose Rhamnose Arabinose Xylose Fucose Mannose Glucose Galactose Suma
a

Hapludolls TMS 79.7728.0 314.972.5 807.8719.3 611.3713.1 127.074.9 891.5787.7 1809.47109.1 939.5721.9 5581.1 Aldononitrile acetate 36.371.0 161.372.4 325.575.3 339.3712.5 86.771.6 360.876.3 625.5739.3 437.876.8 2373.2 TMS 32.074.4 149.8714.4 314.8728.6 376.3721.1 79.575.5 319.4760.3 588.4751.3 426.5742.0 2286.6

Hapludalfs Aldononitrile acetate 29.972.9 152.8715.3 310.0728.9 364.4735.9 107.9710.3 312.1732.2 656.8737.0 460.4732.8 2394.3 TMS 39.2714.2 181.5733.2 368.175.6 461.877.5 99.5725.7 351.2715.0 757.87148.7 486.5714.6 2745.5

Hapludults Aldononitrile acetate 28.172.0 114.878.4 191.2714.2 243.4720.3 81.376.5 250.8714.7 400.3719.3 312.5726.0 1622.4 TMS 17.272.2 127.876.1 222.873.7 273.1711.6 76.374.6 261.5722.2 426.774.5 355.5711.4 1760.9

122.5711.6 335.572.6 702.8745.3 510.4742.1 176.1719.8 898.1726.9 1599.3727.9 925.1722.4 5269.8

Sum of the eight neutral sugars.

low recovery of ribose is mainly due to the preferential adsorption to the soil mineral phase (Rumpel and Dignac, 2006). We also found that the recovery of adonitol relative to myo-inositol (the second internal standard) was higher than 85%, but that of TMS derivatization was as low as about 50%. In addition, the coefcients of variation of recovery in our method ranged between 1.6% and 10.6% (n 5), but as much as 13.8%, on average, has been reported by Amelung et al. (1996). All those ndings indicate that adonitol is a good choice of internal standard for the method. Unfortunately, when the H2SO4 hydrolysis was applied, the recovery of adonitol relative to myo-inositol was 51% probably due to co-precipitation of the sugars with BaSO4 during the purication procedure. Moreover, the coefcients of variation of the eight sugars ranged from 13% to 205%, which is really too large to be accepted. Thereby, TFA hydrolysis is

recommended if aldononitrile acetate derivatization is employed. When analysing the data in Table 3, we found that the total concentrations and compositions of the eight neutral sugars in the four soils determined by the aldononitrile acetate and TMS derivatization procedures were very close, ranging from 1622.4 to 5269.8 mg kg1 and 1760.9 to 5581.1 mg kg1 soil, respectively, and accounting for about 10% of soil organic matter, similar to the results of Amelung et al. (1996) and Murata et al. (1999). Arabinose, xylose, mannose, glucose and galactose were dominant and represented about 88% of the total amount of the eight neutral sugars, this composition was coincide with that of Angers et al. (1988). The coefcients of variation averaged from 4.6% to 7.6% for all neutral sugar quantied by the aldononitrile acetate derivatization, but higher variation, especially ribose was found by the TMS derivatization.

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In conclusion, we present a rapid method that can be used for routine analysis of neutral sugars in soil by GC after TFA hydrolysis and aldononitrile acetate derivatization. Our method is less laborious in the purication steps compared to both the alditol acetates method of Oades (1967) and the TMS method of Amelung et al (1996). The data obtained by our procedure are even more reliable than those by TMS derivatization if derivatized samples have to be stored before determination. Acknowledgements This work was jointly funded by the National Natural Science Foundation of China (project no.: 40535028) and the Knowledge Innovation Program of the Chinese Academy of Sciences (project no.: KZCX3-SW-433). References
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