An Acetate–Hydroxide Gradient for the Quantitation of the Neutral Sugar and Uronic Acid Profile of Pectins by HPAEC-PAD without Postcolumn pH Adjustment

Abstract

An HPAEC-PAD method was developed and validated to quantitate seven neutral sugars and two uronic acids of hydrolyzed pectic polysaccharides without postcolumn pH adjustment. Due to a short gradient phase minimizing the ion concentrations after equilibrating the CarboPac PA20 column with sodium acetate and hydroxide, subsequent isocratic separation of the neutral sugars was characterized by almost baseline resolution of rhamnose and arabinose (1.45 ± 0.15) and xylose and mannose (1.21 ± 0.02) at their maximal concentrations. Linearity was shown (R² = 0.9975-0.9998) for the relevant ranges (0.28-30.3 μmol L⁻¹); galacturonic acid, 1.7-128 μmol L⁻¹) above the limits of detection (30-81 nmol L⁻¹; galacturonic acid, 179 nmol L⁻¹) and ∼3.8 times higher limits of quantification. Conformity of the findings for four pectins after methanolysis plus hydrolysis in trifluoroacetic acid with those of reference procedures (total uronic acids, 95-102%; total neutral sugars, 97-105%) proved the accuracy.

Biopolymer production using fungus Mucor racemosus Fresenius and glycerol as substrate

“…For the identification of the uronic acids, hydrolysed and free acid samples were filtered through cellulose acetate membranes with pores of 0.22 μm, and for anion exchange chromatography using a Dionex ICS-5000 and CarboPac PA20 column, according to the methodology described by Nagel et al [15] .…”

Development of a new high-throughput method to determine the composition of ten monosaccharides including 4-<i>O</i>-methyl glucuronic acid from plant cell walls using ultra-performance liquid chromatography

“…The resolution (R P1, P2 ; Equation 1) between two adjacent peaks (P1 and P2) was deduced from the retention times of the two analytes (RT P1 , RT P2 ) and the baseline widths (W P1 and W P2 ) resulting from the tangents of the inflection points of each peak (Nagel et al 2014). Based on established methodologies for the ABEE labeling of monosaccharides (Kwon and Kim 1993;Yasuno et al 1997Yasuno et al , 1999, Kumagai et al (2012) determined the monosaccharide composition of slurry of Chamaecyparis obtusa (Hinoki cypress) wood after the method transfer from HPLC to UPLC.…”

“…On the other hand, high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) had been developed as a method to improve the sensitivity of monosaccharide detection without derivatization and is widely used for the plant cell wall analysis (De Ruiter et al 1992). A recent study improved the HPAEC-PAD method to quantify two acidic monosaccharides in addition to seven neutral monosaccharides taking only 60 min of analytic time with high resolution (Nagel et al 2014). However this advanced method has different linearity range for some monosaccharides, such as that for GalA (1.7-128 µmol l −1 ) is different from those of other sugars (0.28-30.3 µmol l −1 , Nagel et al 2014).…”

“…A recent study improved the HPAEC-PAD method to quantify two acidic monosaccharides in addition to seven neutral monosaccharides taking only 60 min of analytic time with high resolution (Nagel et al 2014). However this advanced method has different linearity range for some monosaccharides, such as that for GalA (1.7-128 µmol l −1 ) is different from those of other sugars (0.28-30.3 µmol l −1 , Nagel et al 2014). In addition, Chong et al (2013) has reported that the PAD response of 4-m-GlcA is one-third from that of GlcA.…”

“…However, the total run time of ABEE-labeled monosaccharide separation with HPLC is ca. 75 min for each sample analysis and therefore longer than the HPAEC-PAD method (Nagel et al 2014). Recently, Kumagai et al (2012) applied the ultra performance liquid chromatography (UPLC) system, which is a relatively new technique giving new possibilities in liquid chromatography by shortening run time, improving peak resolution, and sensitivity (Swartz 2005), to analyze the composition of trifluoroacetic acid (TFA)-hydrolyzable polysaccharides from Hinoki cypress (Chamaecyparis obtusa) slurry.…”