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[7, 20, 27], dialyses [5, 7, 20, 27, 30, 68–70], and membrane separation [71] (see Table 1.1).

      Ethanol precipitation appears to be a simple method for obtaining crude polysaccharides, but a further process must be performed to obtain a high purity product. The ion-exchange chromatography approach is time-consuming, and requires a high amount of organic solvent. Gel-permeation chromatography is an efficient purification method but requires costly and complex operations, limiting its large-scale application. Membrane separation is an effective method for purifying polysaccharides. However, it is essential to consider, gels from A. vera is very high viscous, and they can easily saturate the membrane without previous treatment [69].

Polysaccharide extraction is often carried out by homogenizing the pulp, filtration or centrifugation to remove insoluble fibers, and subsequent alcohol precipitation or direct chromatographic fractionation. When the alcohol precipitation method is applied, subsequent operations are performed such as centrifuging, dialysis [27] and drying. These fractions can also be obtained by immersing the samples in boiling ethanol [9, 27].

      The aqueous two-phase system (ATPS) extraction constitutes an efficient pretreatment method applied to separate and purify proteins, natural products, enzymes, aminoacids, many comprising a single-step procedure [69, 71]. In this regard, Xing & Li [71] developed an ATPS as a pre-treatment method using poly (acrylonitrile-acrylamide-styrene) membranes prepared using the phase inversion method. The polysaccharide was separated by a combination of aqueous two-phase extraction and membrane separation, yielding a polysaccharide of high purity. Tan et al. [69] developed a simple efficient, and emergent technique for the simultaneous extraction, and isolation of polysaccharides and proteins from A. vera. The polysaccharides migrated into the salt-rich phase, composed of (NH₄)₂SO₄ and NaH₂PO₄, whereas the major impurities of protein, minerals, and phenolic compounds were extracted into the ionic-liquid rich phase using 1-butyl-3-methylimidazolium tetrafluoroborate. Based on the investigation of the partitioning behavior of polysaccharides and proteins in the Ion Liquid Aqueous Two-Phase System, the extraction conditions obtained were optimal, confirming the efficiency of this method for obtaining high purity polysaccharides. The polysaccharides were submitted to dialysis and after this lyophilized.

1.4 Analytical Methods Applied

      1.4.1 Total Carbohydrates, Oligosaccharides, Acemannan and Free Sugars

      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]
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