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Fast determination of the degree of methyl esterification of pectins by head-space GC
Food Hydrocolloids 18 (2004) 665–668 www.elsevier.com/locate/foodhyd
Fast determination of the degree of methyl esterification of pectins by head-space GC M.M.H. Huisman, A. Oosterveld, H.A. Schols* Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Received 18 August 2003; accepted 4 November 2003
Abstract A new, fast method for the quantitative analysis of methoxyl groups in pectin using head-space gas chromatography (HS-GC) has been developed. With this method, results were obtained which were in reasonable agreement with the conventional HPLC method, and the reproducibility of the measurements is high. The advantages of the HS-GC method are that only a small amount of sample (2 mg) per analysis is needed, the chromatogram shows a nice symmetrically shaped methanol peak which is very easy to integrate, the sample preparation for HS-GC is short and easy, and for soluble pectins the sample in the head space vial can also directly be used for analysis of the galacturonic acid content and the degree of acetylation. q 2003 Elsevier Ltd. All rights reserved. Keywords: Pectin; Methyl esterification; Head-space GC
1. Introduction Homogalacturonan is the most well known part of pectic substances. It consists of (1,4)-linked a-galacturonic acid residues. Methyl esterification of the carboxyl groups is the most common substitution of homogalacturonan, while acetyl esterification is also reported for pectin from various sources. The degree of methyl esterification (DM) is expressed as the percentage of the total number of galacturonic acid residues esterified with a methoxyl group. Several methods exist to determine the degree of methylation (DM) of pectins. One commonly used method within the food industry is the titration method proposed by Food Chemical Codex (1981). This procedure involves titration of a pectin suspension with sodium hydroxide before and after saponification of the suspended pectin. The first endpoint indicates the unesterified carboxyl groups and the second endpoint (following the removal of all esters) shows the total number of carboxyl groups. In the case where acetyl groups are present in the pectins a too high saponification equivalent is obtained. Acetyl groups must * Corresponding author. Tel.: þ 31-317-482239; fax: þ 31-317-484893. E-mail address: [email protected] (H.A. Schols). 0268-005X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2003.11.006
then be determined separately and corrected for. Relatively large quantities of pectin are needed for this method, although this usually is not a problem for commercial pectins. Another method to measure the DM is to de-esterify the pectins and determine methanol for the quantification of pectin methyl ester content. Wood and Siddiqui (1971) used a colorimetric procedure for the analysis of the methanol content by oxidising methanol to formaldehyde with potassium permanganate, followed by condensation with 2,4-pentanedione and ammonia to yield the coloured product 3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine. More recently, an improved method was reported employing an enzymatic procedure for oxidising methanol to formaldehyde (Klavons & Bennet, 1986). Another method to directly quantify the amount of methanol after pectin de-esterification uses gas chromatography (Walter, Sherman & Lee, 1983). The availability of ion-exchange resin columns made it possible to determine simultaneously acetyl and methoxyl content of pectin fractions by HPLC (Voragen, Schols & Pilnik, 1986). The pectin is saponified and precipitated with isopropanol, the supernatant was injected on an Aminex column and the amount of methanol and acetic acid calculated. Drawbacks of this
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M.M.H. Huisman et al. / Food Hydrocolloids 18 (2004) 665–668
HPLC method are that it needs a large amount of material (20 –40 mg per analysis), the elution pattern shows a negative carbonate peak near the methanol peak in the chromatogram hampering the integration of the methanol peak, the method is rather time consuming and not very reproducible, pectins sometimes form a gel in the tube from which no sample for HPLC analysis can be drawn. The amount of material needed for the HPLC method was recently reduced to 5 mg by Levigne, Thomas, Ralet, Quemener and Thibault (2002), the use of CuSO4 as a precipitant for the saponified pectin and isopropanol as an internal standard enabled them to shorten the analysis time on a reversed-phase HPLC column using RI-detection. The latest method to measure the degree of esterification of commercial pectin samples is diffuse Fourier transform infrared spectroscopy (DRIFTS). The ester carbonyl band area (CyO) occurring at a mean frequency of 1756 cm21 correlates well with the mean DE ( ¼ degree of esterification) of the bands observed. DRIFTS is a rapid method, but spectral variations due to sample source have to be considered in developing prediction equations using FTIR (Gnanasambandam & Proctor, 2000). Drawbacks of this method when working with pectins are that the pH of the sample is very important in these measurements, the apparatus has to be calibrated with known well-characterised pectins, as well as with other esters (like feruloyl esters). Other acids (like glucuronic acid) interfere with the measurements. This paper describes a head-space GC method for the determination of methanol released from pectic material by saponification and presents the results obtained for a number of well-characterised commercial pectin preparations and pectins extracted from various sources. Quantification was carried out by using HS-GC equipped with a flame ionisation detector. This technique involves thermostatisation and precise injection of the sample, and it is a very fast analysis method.
2. Materials and methods 2.1. Materials Eight different pectins having known characteristics were used in this study: polygalacturonic acid (PGA; DM ¼ 0), soluble pectins with a wide range of DM: C30, C70, C74, M93 (Daas, Boxma, Hopman, Voragen & Schols, 2001), soy ChSS (Huisman, Schols & Voragen, 1998), acid extracted beet pectin (Rombouts & Thibault, 1986), and completely insoluble pectin-rich material soy WUS (Huisman et al., 1998). The beet pectin was a gift from Dr J.-F. Thibault from INRA, Nantes (France), and is composed of 66.0% (w/w) GalA, 2.7% Rha, 1.6% Ara, 0.3% Xyl, 0,2% Man, 11.9% Gal, 0.3% Glc, 0.79% ferulic acid, DM is 41.6%, and DA is 15.5%.
2.2. Uronic acid content The uronic acid content of the soluble pectin samples was determined by analysing the sample solution after determination of the DM with the automated colorimetric mhydroxydiphenyl assay (Blumenkrantz & Asboe-Hansen, 1973; Thibault, 1979) using an auto-analyser (Skalar Analytical BV, Breda, The Netherlands). The uronic acid content of the insoluble pectin samples was determined after H2SO4 hydrolysis with the same method. 2.3. Acetyl content The acetyl contents of the samples were determined in triplicate after saponification (0.1N NaOH, 4 8C, 16 h) using an acetic acid kit (Bergmeyer & Mo¨llering, 1974a,b) (Boehringer Mannheim, Mannheim, Germany) according to the instructions of the manufacturer. 2.4. Determination of DM with head-space gas chromatography 2.4.1. Sample preparation Two milligrams of each sample was weighed into a headspace vial (in quadruplicate). To the samples (duplicate) 1 ml of 0.1N NaOH (4 8C) and to the blanks (duplicate) 1 ml of water was added. The head-space vials were sealed with a Teflon lined rubber septum. Samples and blanks were kept at 4 8C for 1 h, kept overnight at room temperature, and subsequently analysed using head-space GC. 2.4.2. Quantitative head-space gas-chromatography Gas chromatography was run on a HS-GC equipped with a flame ionisation detector and an automatic injection system. Column: DB-WAX 30 £ 0.53 £ 1.0. The following conditions were used: helium as carrier gas with a flow rate of 20 ml/min. Column temperature was set at 40 8C for 5 min and then programmed to 150 8C at a rate of 10 8C/min. The injector was set at 200 8C and the detector performed at 225 8C. The integration was achieved using ChromCard software. Samples were heated at 50 8C for 10 min in the head-space sampler. Two ml of the head-space volatiles was automatically injected in 10 s on the column.
3. Results and discussion The determination of the methoxyl content of pectin fractions by HPLC is very laborious and time-consuming. New ways to measure the methanol content of samples may provide an alternative to this HPLC method. It appeared that HS-GC offers a nice possibility of measuring the methanol content of large numbers of samples. The head-space GC elution patterns of one standard (6.18 mmol/ml) and one actual sample (soy ChSS) are shown in Fig. 1. Methanol elutes as a nice symmetrical
M.M.H. Huisman et al. / Food Hydrocolloids 18 (2004) 665–668
Fig. 1. HS-GC elution pattern of a methanol standard ( – ) and soy ChSS (—).
peak, at all concentrations used in the standards (0.25 – 12.36 mmol/ml), which enable easy integration of the peak and accurate determination of the peak area. In the tested samples from soy bean and beet, no disturbing peaks are detected. Three calibration curves were measured, each time with approximately 50 samples (one day) in between, to determine the reproducibility of the method. The calibration curve of the three measurements—including the error—is shown in Fig. 2. A linear relationship between the methanol concentration and the peak area was found, it has a high R-squared value ðR2 ¼ 0:9954Þ: The reproducibility of the methanol analysis is high; the error (expressed as percentage of the average) is typically below 2%. The determination of the DM was performed with different sample concentrations to determine the lowest sample concentration still giving an accurate value for the DM. C30 was checked at concentrations of 5, 2, 0.5, and 0.2 mg/ml. From the results it was concluded that 2 mg/ml was the lowest concentration still giving an accurate value with low standard error: DM ¼ 30.4% ^ 0.7. Decreasing the sample concentration gave a huge rise in the standard error, partly explained by the inaccuracy of weighing such small amounts of sample.
When lower concentrations of pectin have to be analysed, samples can be prepared from a stock solution to avoid inaccuracy of weighing, but the final amount and NaOH concentration of the samples should be as described in Section 2. From a set of samples, which vary in galacturonic acid content (from 16 – 85%w/w) and differ in origin, the characteristics of the pectins were determined. The DM was determined in triplicate with HS-GC, and the DA was determined in triplicate using an acetic acid test kit. The results are shown in Table 1. The values for the DM found by the HS-GC method are in reasonable agreement with the values determined by HPLC as described in literature for the same samples. An important advantage of the HS-GC method is that the standard error (0.3 – 6.6%) is much lower that the error of the HPLC method (typically 3– 20% (absolute value)). An additional advantage of the new method is that it takes only 2 mg per analysis, whereas HPLC analysis takes 5 –40 mg per analysis. In addition, after analysis on HS-GC the sample in the head space vial can be used for analysis of the galacturonic acid content. However, this is only true for soluble pectins. Another advantage of the HS-GC method was already mentioned above, and is the nice symmetrically shaped peak, which is very easy to integrate. The integration of the obtained HPLC chromatograms is rather difficult, and it is our experience that it is often necessary to reintegrate the chromatograms manually using specific software. The sample preparation for HS-GC is much easier and less time consuming than sample preparation for HPLC. The HS-GC method only needs weighing and pipeting the NaOH solution or water, and HS-GC analysis. The HPLC method, on the other hand, needs weighing, adding water – isopropanol or NaOH – isopropanol solution, regularly Table 1 Characteristics of various pectin samples Sample
GalA DM DM content (HPLC) (HS-GC) (% w/w) (%) (%)
Soy ChSS (Huisman et al., 1998) PGA Beet pectin (Rombouts & Thibault, 1986)# C30 (Daas et al., 2001) C70 (Daas et al., 2001) C74 (Daas et al., 2001) M93 (Daas et al., 2001) Soy WUS (Huisman et al., 1998)
16
35a
36.4 (1.8)b 36a
30
75 66
n.d. 42c
0 n.d. 35.1 (0.3)b 16c
1 12
79 85 85 79 17
30a 70a 74a 93a 36a
30.4 62.8 65.1 99.1 32.1
(0.7)b (1.0)b (1.4)b (6.6)b (2.4)b
DA DA (HPLC) (enz) (%) (%)
n.d. n.d. n.d. n.d. 49a
0 0 2 0 n.d.
n.d. ¼ not determined. As published before by the authors in the first column. b The standard error is presented in brackets. c Extraction according to Rombouts and Thibault (1986), values for this batch). a
Fig. 2. Calibration curve of the relationship between methanol concentration and peak area.
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mixing, centrifuging, transferring samples to HPLC vials, prior to HPLC analysis. A drawback of the method is that the degree of acetylation (DA) cannot be determined in the same analysis, because that would need a much longer analysis time, acetic acid gives a very asymmetrical, tailing peak, and has a very low response factor since it is not very volatile under alkaline conditions. However, determination of the DA with the acetic acid test kit is an accurate and convenient method, while few pectins will contain relevant levels of acetylation.
Acknowledgements The authors wish to thank A. Legger and J. Cozijnsen for their assistance with the analyses.
References Bergmeyer, H. U., & Mo¨llering, H. (1974a). In H. U. Bergmeyer (Ed.), Grundlagen der enzymatischen Analyse: Messungen mit Hilfe gekoppelter Reaktionen (2nd ed.) (Vol. 1) (pp. 119 –125). Methoden der enzymatischen Analyse, New York: Academic Press. Bergmeyer, H. U., & Mo¨llering, H. (1974b). In H. U. Bergmeyer (Ed.), Substanzen im Kohlenhydrat-Stoffwechsel: Acetat (2nd ed.) (Vol. 2) (pp. 1566–1574). Methoden der enzymatischen Analyse, New York: Academic Press. Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484 –489.
Daas, P. J. H., Boxma, B., Hopman, A. M. C. P., Voragen, A. G. J., & Schols, H. A. (2001). Nonesterified galacturonic acid sequence homology of pectins. Biopolymers, 58, 1–8. Food Chemical Codex (1981) 3rd ed., National Academy of Sciences, Washington, DC. Gnanasambandam, R., & Proctor, A. (2000). Determination of pectin degree of esterification by diffuse reflectance Fourier transform infrared spectroscopy. Food Chemistry, 68, 327–332. Huisman, M. M. H., Schols, H. A., & Voragen, A. G. J. (1998). Cell wall polysaccharides from soybean (Glycine max.) meal. Isolation and characterisation. Carbohydrate Polymers, 37, 87–95. Klavons, J. A., & Bennet, R. D. (1986). Determination of methanol using alcohol oxidase and its application to methyl ester content of pectins. Journal of Agricultural and Food Chemistry, 34, 597–599. Levigne, S., Thomas, M., Ralet, M.-C., Quemener, B., & Thibault, J.-F. (2002). Determination of the degrees of methylation and acetylation of pectins using a C18 column and internal standards. Food Hydrocolloids, 16, 547 –550. Rombouts, F. M., & Thibault, J.-F. (1986). Sugar beet pectins: chemical structure and gelation through oxidative coupling. In M. L. Fishman, & J. J. Jen (Eds.), Chemistry and function of pectins (pp. 49 –60). ACS Symposium Series 310, Washington: JAI Press Ltd. Thibault, J.-F. (1979). Automatisation du dosage des substances pectiques par la me´thode au me´ta-hydroxydiphenyl. Lebensmittel Wissenschaft und Technologie, 12, 247 –251. Voragen, A. G. J., Schols, H. A., & Pilnik, W. (1986). Determination of the degree of methylation and acetylation of pectins by h.p.l.c. Food Hydrolcolloids, 1, 65 –70. Walter, R. H., Sherman, R. M., & Lee, C. Y. (1983). A comparison of methods for polyuronide methoxyl determination. Journal of Food Science, 48, 1006–1007. Wood, P. J., & Siddiqui, I. R. (1971). Determination of methanol and its application to measurement of pectin ester content and pectin methyl esterase activity. Analytical Biochemistry, 93, 418–428.
Fast determination of the degree of methyl esterification of pectins by head-space GC M.M.H. Huisman, A. Oosterveld, H.A. Schols* Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Received 18 August 2003; accepted 4 November 2003
Abstract A new, fast method for the quantitative analysis of methoxyl groups in pectin using head-space gas chromatography (HS-GC) has been developed. With this method, results were obtained which were in reasonable agreement with the conventional HPLC method, and the reproducibility of the measurements is high. The advantages of the HS-GC method are that only a small amount of sample (2 mg) per analysis is needed, the chromatogram shows a nice symmetrically shaped methanol peak which is very easy to integrate, the sample preparation for HS-GC is short and easy, and for soluble pectins the sample in the head space vial can also directly be used for analysis of the galacturonic acid content and the degree of acetylation. q 2003 Elsevier Ltd. All rights reserved. Keywords: Pectin; Methyl esterification; Head-space GC
1. Introduction Homogalacturonan is the most well known part of pectic substances. It consists of (1,4)-linked a-galacturonic acid residues. Methyl esterification of the carboxyl groups is the most common substitution of homogalacturonan, while acetyl esterification is also reported for pectin from various sources. The degree of methyl esterification (DM) is expressed as the percentage of the total number of galacturonic acid residues esterified with a methoxyl group. Several methods exist to determine the degree of methylation (DM) of pectins. One commonly used method within the food industry is the titration method proposed by Food Chemical Codex (1981). This procedure involves titration of a pectin suspension with sodium hydroxide before and after saponification of the suspended pectin. The first endpoint indicates the unesterified carboxyl groups and the second endpoint (following the removal of all esters) shows the total number of carboxyl groups. In the case where acetyl groups are present in the pectins a too high saponification equivalent is obtained. Acetyl groups must * Corresponding author. Tel.: þ 31-317-482239; fax: þ 31-317-484893. E-mail address: [email protected] (H.A. Schols). 0268-005X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2003.11.006
then be determined separately and corrected for. Relatively large quantities of pectin are needed for this method, although this usually is not a problem for commercial pectins. Another method to measure the DM is to de-esterify the pectins and determine methanol for the quantification of pectin methyl ester content. Wood and Siddiqui (1971) used a colorimetric procedure for the analysis of the methanol content by oxidising methanol to formaldehyde with potassium permanganate, followed by condensation with 2,4-pentanedione and ammonia to yield the coloured product 3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine. More recently, an improved method was reported employing an enzymatic procedure for oxidising methanol to formaldehyde (Klavons & Bennet, 1986). Another method to directly quantify the amount of methanol after pectin de-esterification uses gas chromatography (Walter, Sherman & Lee, 1983). The availability of ion-exchange resin columns made it possible to determine simultaneously acetyl and methoxyl content of pectin fractions by HPLC (Voragen, Schols & Pilnik, 1986). The pectin is saponified and precipitated with isopropanol, the supernatant was injected on an Aminex column and the amount of methanol and acetic acid calculated. Drawbacks of this
666
M.M.H. Huisman et al. / Food Hydrocolloids 18 (2004) 665–668
HPLC method are that it needs a large amount of material (20 –40 mg per analysis), the elution pattern shows a negative carbonate peak near the methanol peak in the chromatogram hampering the integration of the methanol peak, the method is rather time consuming and not very reproducible, pectins sometimes form a gel in the tube from which no sample for HPLC analysis can be drawn. The amount of material needed for the HPLC method was recently reduced to 5 mg by Levigne, Thomas, Ralet, Quemener and Thibault (2002), the use of CuSO4 as a precipitant for the saponified pectin and isopropanol as an internal standard enabled them to shorten the analysis time on a reversed-phase HPLC column using RI-detection. The latest method to measure the degree of esterification of commercial pectin samples is diffuse Fourier transform infrared spectroscopy (DRIFTS). The ester carbonyl band area (CyO) occurring at a mean frequency of 1756 cm21 correlates well with the mean DE ( ¼ degree of esterification) of the bands observed. DRIFTS is a rapid method, but spectral variations due to sample source have to be considered in developing prediction equations using FTIR (Gnanasambandam & Proctor, 2000). Drawbacks of this method when working with pectins are that the pH of the sample is very important in these measurements, the apparatus has to be calibrated with known well-characterised pectins, as well as with other esters (like feruloyl esters). Other acids (like glucuronic acid) interfere with the measurements. This paper describes a head-space GC method for the determination of methanol released from pectic material by saponification and presents the results obtained for a number of well-characterised commercial pectin preparations and pectins extracted from various sources. Quantification was carried out by using HS-GC equipped with a flame ionisation detector. This technique involves thermostatisation and precise injection of the sample, and it is a very fast analysis method.
2. Materials and methods 2.1. Materials Eight different pectins having known characteristics were used in this study: polygalacturonic acid (PGA; DM ¼ 0), soluble pectins with a wide range of DM: C30, C70, C74, M93 (Daas, Boxma, Hopman, Voragen & Schols, 2001), soy ChSS (Huisman, Schols & Voragen, 1998), acid extracted beet pectin (Rombouts & Thibault, 1986), and completely insoluble pectin-rich material soy WUS (Huisman et al., 1998). The beet pectin was a gift from Dr J.-F. Thibault from INRA, Nantes (France), and is composed of 66.0% (w/w) GalA, 2.7% Rha, 1.6% Ara, 0.3% Xyl, 0,2% Man, 11.9% Gal, 0.3% Glc, 0.79% ferulic acid, DM is 41.6%, and DA is 15.5%.
2.2. Uronic acid content The uronic acid content of the soluble pectin samples was determined by analysing the sample solution after determination of the DM with the automated colorimetric mhydroxydiphenyl assay (Blumenkrantz & Asboe-Hansen, 1973; Thibault, 1979) using an auto-analyser (Skalar Analytical BV, Breda, The Netherlands). The uronic acid content of the insoluble pectin samples was determined after H2SO4 hydrolysis with the same method. 2.3. Acetyl content The acetyl contents of the samples were determined in triplicate after saponification (0.1N NaOH, 4 8C, 16 h) using an acetic acid kit (Bergmeyer & Mo¨llering, 1974a,b) (Boehringer Mannheim, Mannheim, Germany) according to the instructions of the manufacturer. 2.4. Determination of DM with head-space gas chromatography 2.4.1. Sample preparation Two milligrams of each sample was weighed into a headspace vial (in quadruplicate). To the samples (duplicate) 1 ml of 0.1N NaOH (4 8C) and to the blanks (duplicate) 1 ml of water was added. The head-space vials were sealed with a Teflon lined rubber septum. Samples and blanks were kept at 4 8C for 1 h, kept overnight at room temperature, and subsequently analysed using head-space GC. 2.4.2. Quantitative head-space gas-chromatography Gas chromatography was run on a HS-GC equipped with a flame ionisation detector and an automatic injection system. Column: DB-WAX 30 £ 0.53 £ 1.0. The following conditions were used: helium as carrier gas with a flow rate of 20 ml/min. Column temperature was set at 40 8C for 5 min and then programmed to 150 8C at a rate of 10 8C/min. The injector was set at 200 8C and the detector performed at 225 8C. The integration was achieved using ChromCard software. Samples were heated at 50 8C for 10 min in the head-space sampler. Two ml of the head-space volatiles was automatically injected in 10 s on the column.
3. Results and discussion The determination of the methoxyl content of pectin fractions by HPLC is very laborious and time-consuming. New ways to measure the methanol content of samples may provide an alternative to this HPLC method. It appeared that HS-GC offers a nice possibility of measuring the methanol content of large numbers of samples. The head-space GC elution patterns of one standard (6.18 mmol/ml) and one actual sample (soy ChSS) are shown in Fig. 1. Methanol elutes as a nice symmetrical
M.M.H. Huisman et al. / Food Hydrocolloids 18 (2004) 665–668
Fig. 1. HS-GC elution pattern of a methanol standard ( – ) and soy ChSS (—).
peak, at all concentrations used in the standards (0.25 – 12.36 mmol/ml), which enable easy integration of the peak and accurate determination of the peak area. In the tested samples from soy bean and beet, no disturbing peaks are detected. Three calibration curves were measured, each time with approximately 50 samples (one day) in between, to determine the reproducibility of the method. The calibration curve of the three measurements—including the error—is shown in Fig. 2. A linear relationship between the methanol concentration and the peak area was found, it has a high R-squared value ðR2 ¼ 0:9954Þ: The reproducibility of the methanol analysis is high; the error (expressed as percentage of the average) is typically below 2%. The determination of the DM was performed with different sample concentrations to determine the lowest sample concentration still giving an accurate value for the DM. C30 was checked at concentrations of 5, 2, 0.5, and 0.2 mg/ml. From the results it was concluded that 2 mg/ml was the lowest concentration still giving an accurate value with low standard error: DM ¼ 30.4% ^ 0.7. Decreasing the sample concentration gave a huge rise in the standard error, partly explained by the inaccuracy of weighing such small amounts of sample.
When lower concentrations of pectin have to be analysed, samples can be prepared from a stock solution to avoid inaccuracy of weighing, but the final amount and NaOH concentration of the samples should be as described in Section 2. From a set of samples, which vary in galacturonic acid content (from 16 – 85%w/w) and differ in origin, the characteristics of the pectins were determined. The DM was determined in triplicate with HS-GC, and the DA was determined in triplicate using an acetic acid test kit. The results are shown in Table 1. The values for the DM found by the HS-GC method are in reasonable agreement with the values determined by HPLC as described in literature for the same samples. An important advantage of the HS-GC method is that the standard error (0.3 – 6.6%) is much lower that the error of the HPLC method (typically 3– 20% (absolute value)). An additional advantage of the new method is that it takes only 2 mg per analysis, whereas HPLC analysis takes 5 –40 mg per analysis. In addition, after analysis on HS-GC the sample in the head space vial can be used for analysis of the galacturonic acid content. However, this is only true for soluble pectins. Another advantage of the HS-GC method was already mentioned above, and is the nice symmetrically shaped peak, which is very easy to integrate. The integration of the obtained HPLC chromatograms is rather difficult, and it is our experience that it is often necessary to reintegrate the chromatograms manually using specific software. The sample preparation for HS-GC is much easier and less time consuming than sample preparation for HPLC. The HS-GC method only needs weighing and pipeting the NaOH solution or water, and HS-GC analysis. The HPLC method, on the other hand, needs weighing, adding water – isopropanol or NaOH – isopropanol solution, regularly Table 1 Characteristics of various pectin samples Sample
GalA DM DM content (HPLC) (HS-GC) (% w/w) (%) (%)
Soy ChSS (Huisman et al., 1998) PGA Beet pectin (Rombouts & Thibault, 1986)# C30 (Daas et al., 2001) C70 (Daas et al., 2001) C74 (Daas et al., 2001) M93 (Daas et al., 2001) Soy WUS (Huisman et al., 1998)
16
35a
36.4 (1.8)b 36a
30
75 66
n.d. 42c
0 n.d. 35.1 (0.3)b 16c
1 12
79 85 85 79 17
30a 70a 74a 93a 36a
30.4 62.8 65.1 99.1 32.1
(0.7)b (1.0)b (1.4)b (6.6)b (2.4)b
DA DA (HPLC) (enz) (%) (%)
n.d. n.d. n.d. n.d. 49a
0 0 2 0 n.d.
n.d. ¼ not determined. As published before by the authors in the first column. b The standard error is presented in brackets. c Extraction according to Rombouts and Thibault (1986), values for this batch). a
Fig. 2. Calibration curve of the relationship between methanol concentration and peak area.
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mixing, centrifuging, transferring samples to HPLC vials, prior to HPLC analysis. A drawback of the method is that the degree of acetylation (DA) cannot be determined in the same analysis, because that would need a much longer analysis time, acetic acid gives a very asymmetrical, tailing peak, and has a very low response factor since it is not very volatile under alkaline conditions. However, determination of the DA with the acetic acid test kit is an accurate and convenient method, while few pectins will contain relevant levels of acetylation.
Acknowledgements The authors wish to thank A. Legger and J. Cozijnsen for their assistance with the analyses.
References Bergmeyer, H. U., & Mo¨llering, H. (1974a). In H. U. Bergmeyer (Ed.), Grundlagen der enzymatischen Analyse: Messungen mit Hilfe gekoppelter Reaktionen (2nd ed.) (Vol. 1) (pp. 119 –125). Methoden der enzymatischen Analyse, New York: Academic Press. Bergmeyer, H. U., & Mo¨llering, H. (1974b). In H. U. Bergmeyer (Ed.), Substanzen im Kohlenhydrat-Stoffwechsel: Acetat (2nd ed.) (Vol. 2) (pp. 1566–1574). Methoden der enzymatischen Analyse, New York: Academic Press. Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484 –489.
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