Читать книгу Polysaccharides - Группа авторов - Страница 19

A number of different chemical methods have been used to determine the total carbohydrate content both in preparations of mucilaginous gels and isolated polysaccharides (Table 1.2). Analysis of the composition of total carbohydrates is extensively used and serves as a method of monitoring the quality of the products derived from the gel. The carbohydrates found in the composition of A. vera gel include polysaccharides, which are the major constituents from the dry matter, in addition to monosaccharides, free sugars and fibers.

Acid hydrolysis is the most commonly used method for determining the monomers present in the polysaccharide fractions or purified acemannan. Most acid hydrolysis is performed using hot sulfuric acid or the phenol-sulfuric acid assay method, anthrone-sulfuric acid method, besides hydrolysis with trichloroacetic acid (TFA) and also by enzymatic hydrolysis. Subsequent analysis is performed by High Performance Liquid Chromatography (HPLC) coupled to a Refraction Index detector [14, 26], Ultraviolet–Visible (UV-Vis) Spectroscopy [22, 25, 30, 37, 60, 61, 63, 69, 77], Gas Chromatography (CG) [7, 9, 20, 27, 34] and High Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAE-PAD) for analysis of free sugar after hydrolysis [19].

Using the acid hydrolysis method at high temperatures, Femenia et al. [20] employed electrophoresis to determine the carbohydrate composition of isolated and purified ace-mannan, where mannose was the most abundant component (82%), but was not the only monomeric component of acemannan, which also contained units of galactose (4.5%) and glucose (10%).

Table 1.2 Methods of chemical characterization of mucilaginous gel and its derivatives.

Analyte	Sample preparation	Technique	References
Total carbohydrates or monosaccharide	Acid hydrolysis	HPLC-RI	[26]
HPAE-PAD	[19]
composition		HPLC-UV	[69]
Thin Layer Chromatography (TLC)	[34]
CG	[5, 7, 9, 20, 71, 72]
CG-FID	[34, 40, 66, 70, 72]
CG/MS	[13, 38, 76]
UV	[22, 25, 30, 37, 60, 61, 63, 76]
Microplate reader – Color measurement	[4]
Fluorophore-assisted carbohydrate electrophoresis (FACE)/UV light	[4]
–	HPLC	[72]
HPSEC-UV	[38]
HPSEC-RI	[5]
HPSEC-RI	[21]
HPSEC-MALLS	[21]
HPSEC-MALLS-RI	[13, 75, 78]
FT-IR	[5, 7, 22, 28, 34, 37, 66]
ATR-FT-IR	[24]
1H NMR	[5, 13, 19, 38, 39, 76]
13C NMR	[5, 13, 76]
Comprehensive Microarray Polymer Profiling (CoMPP)	[85]
Analyte	Sample preparation	Technique	References
Oligosaccharides	Acid hydrolysis	CG/MS	[67, 76]
Enzymatic hydrolysis	CG/MS	[11]
ESI-MS	[40]
ESI-MS/MS	[40]
MALDI-MS	[40]
1H NMR	[11, 67]
13C NMR	[11, 67]
–	HPSEC-MALLS	[67]
Acemannan	Acid hydrolysis	UV	[69]
HPLC	[14]
CG	[7, 20]
CG-FID	[27]
–	UV	[24, 37, 68, 73, 84]
FT-IR	[7, 14, 30]
1HNMR	[14, 27]
HPSEC-RI	[14]
Free sugars	–	HPLC	[9]
HPLC-RI	[26]
HPAE-PAD	[19, 75]

(–) samples are not subjected to hydrolysis

Chokboribal et al. [14] also used the acid hydrolysis method to determine the composition of monosaccharides, structure and molecular mass of the alcohol precipitated polysaccharide performing these analysis using liquid chromatography with reflective index detector, Nuclear Magnetic Resonance Spectroscopy (¹H NMR), Fourier Transform Infrared Spectroscopy (FT-IR), and size-exclusion chromatography, respectively. The data obtained confirmed the polysaccharide was acemannan.

The presence of uronic acids together with trace levels of arabinose and ramnose is indicative of residual amounts of pectinic polysaccharides in the polysaccharide fraction, as previously observed in the hydrosoluble extracts from A. vera leaf pulp [4, 9].