CHROMATOGRAPHIC SCIENCE SERIES A (e) MU] See): Thin Layer Chromatography TMH NER ai ie aa) oer Edited by ; Monika Waksmundzka-Hajnos Joseph Sherma Teresa KowalskaPrimary Metabolites 1 1 TLC of Carbohydrates Guilherme L. Sassaki, Lauro M. de Souza, Thales R. Cipriani, and Marcello lacomini CONTENTS: ILL Introduction... 256 H1A.L Sugar Chemistry 256 UL1.1.1 Mutarotatio oe 237 LL12 ‘The Reducing Sugary. 258 LL1.1.3 Bases for Sugar Detectio 258 111.2 Plant Carbohydrates .. 239 1241 Monosaccharides and Polyals 260 111.22 Oligosaccharides... 260 111.23 Polysaccharides . 265 266 267 267 268 268 ue Glycoconjugates (Glycolipids) . 11,2 Experimental TLC of Sugars . 11.2.1 Adsorbents and Solvents 11.2.2 Universal Reagents 11.2.3 Specific Reagents . 11.2.3. Preparation and Application of TLC Reeagemts..ecsennren en 268 11.2.4 Sample Preparati 270 11.241 Water Soluble Sugars (Mono. Oligo-, and Polysaccharides).. 270 Organie Soluble Compounds Containing Sugars wv.» 271 2s ative Analysis and Sugar Composition soneee BF 11.3 Conclusions. ml 7 ee 273 References ae e ee 255256 Thin Layer Chromatography in Phytochemistry 11.1 INTRODUCTION Carbohydrates are carbon compounds that contain farge quantities of hydroxyl groups. The simplest carbohydrates also contain either an aldehyde moiety (these are termed polyhydroxyaldehydes) or a ketone moiety (polyhydroxyketones). They are the most abundant biological molecules, and fill numerous roles in living organisms, such as the storage and transport of energy (starch, glycogen and sucrose) and structural companents (cellulose in plants, chitin in animals).'” All carbohyd- rates can be classified as monosaccharides, oligosaccharides, or polysaccharides. Anywhere from two to ten monosaccharide: units, linked by glycosidic linkage, make up an oligosaccharide. Polysaccharides are much larger, containing hundreds of monosaccharide units. The presence of the hydroxyl groups allows carbohydrates to interact with the aqueous environment and to participate in hydrogen bonding, both within and between chains. Additionally, carbohydrates and their derivatives play major roles in the functioning of the immune system, fertilization, pathogenesis, blood clotting, and development. Derivatives of the carbohydrates can contain nitrogens, phosphates, and sulfur groups. Some carbohydrates can attach by C-, O- lycosidic bonds, giving rise to complex structures namely glycoconjugates. When the sugar moiety is combined with lipid it forms a glycolipid and when it is combined with protein it forms another glycoconjugate molecule, a glycoprotein. The predominant carbohydrates encountered in the body are structurally related 10 the aldotriose glyceraldehyde and to the ketotriase dihydroxyacetone, All carbohyd- rates contain at least one asymmetrical (chiral) carbon and are, therefore, optically a In addition, carbohydrates can exist in cither of two conformations p ori, as determined by the orientation of the hydroxyl group about the farthest asymmetric carbon from the carbonyl group, With a few exceptions, thase carbohydrates that are of physiological significance exist in the p-conformation, ‘The mirror-image conformations, called enantiomers, arc in the (conformation. The carbohydrates found in plants are monosaccharides, disaccharides, higher oligosaccharides, poly- saccharides, and their derivatives: glycosides and glycoproteins, However, plants have many polysaccharides and glycoconjugates structures containing L-arabinose and t-fucose, which have been found as complex heteropolysaccharides, namely arabinogalactans andl gums, respectively,’ 11.1.1 Sucak CHewstey Monosaccharides are classified according to three different characteristics: the place ment and function of its carbonyl group, the number of carbon atoms, and its chiral center, Ifthe carbonyl group is an aldehyde, the monosaccharide is an aldose; if the carbonyl group is a kelone, the monosaccharide is a Kelose. The smallest possible monosaccharides, thase with three carhan atoms, are called trioses, just as those with four, five, and six are tetroses, pentoses, hexoses, and so en,'* Combining the function and number of carbons there is, for example, an aldahexose, aldapentose, and Ketohexose—glacose (six-carbon aldose), arabinose (five-carbon aldose) and fructose (six-carbon ketone), respectively (Figure 11.1), The carbon atoms of sugurs inked to a hydroxy! group (-OH), with the exception of the carbony ic groups andTLC of Carbohydrates 257 Aidohexose Ketohaxose Ho ay GH_OH H ‘OH 0 HO}—H = HO} Hon Hf on Hj—oH = HOH Fe=oy OH} «= ROH) = [HOH CH;OH (CH,OH CHs (CH;OH 0-Ghucose Fructose -Fucose L-Arabinose FIGURE 11.1 Examples of aldohexose, ketohexose, aldodeoxyhexase, and aklopentose Boxes indicate the last asymmetrical carbon, which define the absolute configuration, the last carbon, are asymmetric, making them stereocenters with two possible configurations. p-glucose is one isomer for an aldohexose (noncyclic form) with the formula (CH;0\, four of its six earbons atoms are chiral centers, making p-glucose one of the 2*= 16 possible stereoisomers in straight-chain form. Sugars belonging to the p-series are the most common isomers found in nature. The aldehyde or ketone group of a straight-chain monosaccharide will react reversibly with a hydroxyl group on a different carbon atom to form a hemiacetal or hemiketal, forming a heterocyclic ring with an oxygen bridge between two carbon atoms. Rings with five and six atoms are called furanose and pyranose forms, respectively, and exist in equilibrium with the straight-chain form. For exampl p-glucose can adopt two types of ring conformation, which result in the formation the b-glucopyranase (p-Glep) and p-glucofuranose (0-Glef); however, the pyranosyl conformation ix most commonly found in nature. For these reasons the ketone and aldehyde groups of carbohydrates make these structures highly reactive, so most of sugar reactions follow the classical reactions of these chemistry functions, being, catalyzed by acidic or basic compounds. 11.1.1.1 Mutarotation In water and polar solvents, the free reducing carbohydrates can convert the straight- chain form tseyelic form, The carbon atom containing the carbonyl axygen, called the anomeric carbon, becomes a chiral center with two possible configurations: the oxygen atom may take a position cither above or below the plane of the ring. The possible resulting stereoisomers are called anomers, In the « anomer, the -OH substituent on the anomeric carly rests on the opposite side of the ring from the CH;OH attached to the asymmetric carbon furthest from the anomeric carbon, The altemative form, in which the CHOH and the anomeric hydroxy! are on the sare side of the plane of the ring, is called the B anomer. Because the ring and straight-chain forms readily interconvert, both anomers exist in equilibrium. So the glucose mono- saccharide could be present in 10 possible structures, straight-chain forms of p- or Leglucose, a-D-Glep, B-o-Glep, a-1-Glep, Bt-Glep, e-0-Glef, B-D-Glef, a-L-Glef, and258 Thin Layer Chromatography in Phytochemistry -D-Glet Bo-Gker m-Gker Bu-Glor Glucose FIGURE 11.2 Possible isomers for glucose, B-L-Gilef (Figure 11.2), All of these characteristics made the carbohydrates the most complex molecules to study in nature, and the many possibilities af combinations, types of linkage and sequences of the sugar units give rise to many conformations and Uhese structures are used in many functions by the living organisms, 11.1.1.2 The Reducing Sugars ‘Carbohydrates that contain the free aldehydes or a-hydroxymethy! ketone groups can be oxidized by Cu(IT) ion and are classified as reducing sugars. They reduce the ‘Cu(l1) ion to Cu(D, the sugar is oxidized and its carbonyl group (i.c., aldehyde or ketone group) is converted to a carboxyl group. This reaction ean be performed by Benedicts reagent (CuSO,/citrate) and Fehlings reagent (CuSO, /tartrate) in aqueous alkaline solutions. So the oxidation will always occur when the hemi-acetal form (cyclic) became ring-open, giving the reactive aldehyde group (Figure 11.3), However, the glycosides (acetals) are nonreducing sugars (Figure 11.3B), unable to become ring-open, therefore, they cannot he oxidized. 1.1.1.3 Bases for Sugar Detection on TLC ‘The ketone and aldehyde groups of carbohydrates are the central point of reaction for detection in TLC or in paper chromatography (PC). However, the hydroxy! groups are very important in the formation of by-products in the dehydration of the sugars in the presence of strong inorganic acids and heat, as observed by the formation of furfuraldehyde (Figure 11.4). The classical work of Trevelyan, Procter & Harrison (1930) used silver nitrate-sodium hydroxide to detect sugars and polyalcohals in PC. The detection of sugar with silver nitrate would be performed on TLC using aqueous ammonia instead of sodium hydroxide, However, the most common tech- nique use the furfuraldehyde in conjunction with many phenolic reagents, which give rise to different colors, as observed by otcinol, «-naphtol, resorcinol, anisaldehyde,TLC of Carbohydrates 259 We on ou tion fron HOH 4 2004 6H ee fn #CHO HOO ss y ¥ H HoH Heri-acetal CH oon reducing sugar ‘Oxidized form co) @) FIGURE 11.3 (A) Example of oxidization reaction, (B) Nonreducing sugars. and aniline-diphenylamine, Another advamage of this type of detection is the peculiar color obtained for each manosaceharide, The most popular use of TLC af carbohydrates is performed in Silica Gel G-60 plates, followed by detection of the carbohydrates with Orcinol-H,SOy spray, since it is highly sensitive to sugars and each monosaccharide class gives rise to a specific color, However, depending on the sugar concentrations, the color intensities may vary, so some color variations may appear. 11.1.2 Peant Caenonvorares In plants, carbohydrates exist as monosaccharides, oligasaccharides, polysacchar- ides, and their derivatives, such as cyanogenic glycosides, phenolic and flavonoid glycosides, glycolipids, and glycoproteins. H o s0, [le Ho + shoe . Hydroxy-methyb furfural Phenolic reagent OH ‘Glucose Color products FIGURE 11.4 Furfuraldchyde formations during acidic treatment and reaction with phenolic reagents,260 Thin Layer Chromatography in Phytochemistry 1 Monosaccharides and Polyols There are several kinds of monosaccharides and they are the basic constituents of all carbohydrate structures, The main monosaccharides found in plants are glucose, fructose, galactose, mannose, rhamnose, arabinese, xylose, glucuronic acid, galac turonic acid, and 4-O-methylglucuronic acid, These monosaccharides are usually found as constituents of aligo- and polysaccharides, glycoproteins, glycolipids, etc The polyols are constituted by reduced forms of monosaccharides. Both acyclic {alditols) and cyclic <(cyclitols) polyols occur in plants, mostly in combined forms as glycosides, Glucitol, mannitol, dulcitol, and glycerol, for example, are alditols that may occur in free form. Inositols are the most widely occurring cyclitols, and anyo- inositol is the most important of them, playing various roles in plant metabolism.* An example of evaluation of polyos in plant extracts is shown in Figure 11.5, 11.4.2.2 Oligosaccharides As the name suggests, oligosaccharides (Greek oljgos—few) are carbohydrate struc- tures composed of few monosuecharides units, linked by O-glycosidic bonds, usu- ally consisting from two to ten monosaccharide residues, According to the number of monosaccharide contents per mole, the oligosaccharide may be classified as @ = Fhamnose *» 00 GOH eitesios oe = ee 123 FIGURE 11.5 Paper chromatography (WW! 40x. wilic fractions of the plant Maytenus iieiolia. Solvent buthanol:pyridine: HO (5:3:3 v/v), for 18h, and detection with silver nitrate-sodium hydroxide, Standards are listed from origin wo fromt: (1) Lac, Gal, Mam; (2) myo-inositol, Ara, Rha; (3) Gle, Xyl. Samples: (4) MeQH:H,0 (2:1 v/v extract; (5) McOH:H,0 (2:1 v/v) extract, after hydrolysis; (6) ethanolic supernatant of uqueous extraction; (7) ethanolic supematant of aqueous extraction, after hydrolysis,TLC of Carbohydrates 261 — on +0. 4 Abe. A. a ee wt ” R= c-oglcopyranosyl -> maltatriase ‘ay Sucrose 8) FIGURE 11.6 (A) Sucrose ~ [a-p-glucopyranosyl(1—2)-f-o-fructofuranoside). (B maltose -glucopyramosyl-(1—4}-a-o-glucopyranose] and maliofriose —[a-t-glucopyranasyl- +4 )-p-glucopyranosy-(1—-4)-c-cr-glicopyranose). Higher homologs comprise the chain elongation, disaccharide (two residues), trisaccharide (three residues), tetrasaccharide (four residues), and so on. Changing the number and the type of monosaccharide comtents, as well as, the glycosidic linkages may form several oligosaccharides. Two classes of oligosaccharides are found in plants: the primary oligosacchar- ides, which are always synthesized in vivo, and the secondary oligosaccharides, which are produced as result of polysaccharide, glycoprotein, or glycolipid deg- radation, in vive or in vitro. ‘The most common primary oligosaccharide, and widespread mainly by its sweet taste, is sucrose (Figure 11.64), a nonreducing disaccharide composed by glucopy- ranase and fructofuranose, However, some oligosaccharides may be found as primary or secondary products. Maltese and its higher homologs (Figure 11.6B) are examples, since they may be produced by enzymatic catalysis, through the action of glucosyltransferases on glucans degradation,” or in the laboratory by partial hydrolysis. On the other hand, maltose may arise from activated glucosyl units during the photosynthesis.* Sucrose is a very important plant disaccharide, since it is the main farm in which photosynthetic energy is transported throughout the plant. Moreaver, sucrose is the main precursor for synthesis of other oligosccharides. Two enzymes are known to be capable of sucrose synthesis in plants: sucrose synthase and sucrose-6-P synthase. Although sucrose is the most ubiquitous and abundant disaccharide, in pterido- phytas trehalose and selagiose (Figure 11.7) serve as the main soluble reserve of carbohydrates, replacing sucrose,” Many oligosaccharide families from plants, such as frictans or fructo- oligosaccharides and raffinase have sucrose as a common precursor. Fructans are fructose (Fru) polymers (oligo- or polysaccharides) synthesized from sucrose by a combined enzymes action, Sucrose:Sucrose 1-Fructosyl Trans- ferase (I-SST), and Fructam:Fructan 1-Fructosyl Transferase (1-FFT). In the first step, 1-SST synthesizes the |-kestose from two sucrose units and, in the second step, the |-FFT makes the chain elongation by transferring Fru units from ene fructan to another.!"-"? In some plants, such as grasses, or those from the families Asteraceae, Poaceae, and Liliaceae, fructans are the main energetic storage, *262 Thin Layer Chromatography in Phytachemistry R=H + hehalose D-glucopyranasyl ~> solagiose FIGURE 11.7 a.0-Trehalose [a-p-Glucopyranosy 1 I—|}-1-kostose (/sokestoso) Fi = urtructofuranoeyl - nystose FIGURE 11.8 Fructans series (Kestoses), (A) I-tesrase [a-o-glucopyranosyl-(1—+2)- B-o-fructofuranosyl-(1—»2)-B-o-fructofuranovide|, aysiose —x-b-glucopyranosyl-{1—-2-B- b-fructofuranosyl-(1—:2)-f6-b-fructofuranosyi-(1—»2).f-mructofuranaside]. (B) G-kestose [a-p-glacopyraniosy|-(1—+2)--0-fructofuranosyl-(6—2}-B-u-fructofuranoside). (C) meokestose fructofuranosyl4 2-—v6)a-0-g hicopyranosyl)-( 1-2}. ,b-p-fructofuranoside |TLC of Carbohydrates 263 uh Alt raffinose Fix 0-Galactopayranosy! > stachyose -b-Galactopayranosyl (1+) a-p-Galactopayranasy! => verhascose FIGURE 11.9 Raffinose oligosuccharides. If the fructosy! end is removed from the raffinose, it will give rise 10 a disac- charide, mefibiose, e-p-galaciopyanosyl4|—6)-o-glucopyranose, When the fructo~ syl end is removed from stachyose, it gives rise to the trisaccharide manninotriose, -a-p-galactopyanosy|-{--6)-a.-p-galactopyanosy]-{1—-6)-o-glucopyranose ‘There are many oligosaccharide series which also have:sucrose as a commen precursor, Some examples, including umbelliferase, planteose, dyehnose, and gentianose, are shown in Figure 11.10. Although not represented, these oligosaccharides may present higher homologs ** 1.1.2.4 Glycoconjugates (Glycolipids) Glycolipids are important membrane components in most living organisms, With other important lipids, such as phospholipids, sphingolipids, and sterols, they com- prise the main components of biological membranes, As their structural variability, their biological function is equally diversified, Plant glycolipids may be divided in two main classes, Glycoglycerolipids and Glycosphingolipids. Glycoglycerolipids may contain one or two fatty acid chains bound to hydroxyl groups of glycerol, as well as its sugar moiety, whose monosaccharide component may vary, but is usually compased by o-galactapyranosyl units or sulfoquinovasyl, a modified 6-deoxyglucopyranosyl containing a sulfonyl group linked to C-6, The carbohydrate moieties from glycolipids are usually refered as polar head group (PHG), and in most cases define the biological function of the lipid, The most common lipids of this class are: monogalactosyldiacylglyceral (MGDG), digalac- tosyldiacylglycerol (DGDG), trigalactosyldiacylglycerol (TGDG), and sulfoquiro- vosyldiacylglycerol (SQDG). These lipids may also occur in monoacylated form, in which only one fatty acid is linked to the primary hydroxyl group of glycerol. These lipids are called Iysotipids. Photosynthetic tissues from plants can contain high proportions of galactolipids such as MGDG and DGDG.*** The role of these lipids involves thylakoid aggre- gation and stacking."*-°* Glycosphingotipids consist of a sphingoide base (sphingosine), referred as long ‘chain base (LOB), characterized by an amide linkage with a faity acid, The hydroxyl group presents in C-1 links the glycosyl moiety to the sphingosine, As well as glycosylglycerides, glycosphingolipids may comain only one (cerebroside) or more sugar units and the glycosidic linkages vary according to the nature of the lipid, Glycosphingolipids di-, tri-, and tetrahexosides containing p-glucosyl and b-mannosyl units were isolated from wheat flour.”*" ‘There is a great variety of plant glycosphingolipids, some of them presenting a phosphate diester group, making the linkage between the sphingosine and the glycosyl moieties were isolated from leaves and seeds.“** These sphingolipids, called phytoglycolipids, isolated from Phaseolus vulgaris leaves have a complex glycosyl moiety. presenting a trisaccharide, inositol-hexuronic-hexosamine, attached to mannose, galactose, and arabinose. The main fatty acids found were 2-hydroxy Cay, Coa, and Cas and long chain bases dehydrophytosphingosine and phytosphin- gosine." The extraction and analytical methods for phytoglycolipids on chromato- graphic paper and TLC plates were previously described." Glycolipids structures are represented in Figure 11.12.TLC of Carbohydrates: 267 - 7 07 8 cH),—S Ae > MGDG YcH— cu, -A=a--galactopyranosyl > DGDG Ha nee 4 “k Ris a-0-galactopyranosyl-{16)-a-p- te — PH galactopyranosy! - TGDG ‘OH ‘= (A) i 0 om " fe ry of ;° BCH), om me Kyo “cis, on, we . ey uy Fiemannose, galactose, or arabinose . FN bc cr oO Wom, (2) ® Da FIGURE 11.12 Gilycolipids found in plants: (A) Galactolipids series, mona-, di-, and ti- galactosyldiacylglicerol, (B) Sulfonolipid ~ sulfoquinovasyldiacylglycerat, and (C) General structure of phytoglycolipids, a sphingolipid. Other glycoconjugates called heteroglycosides are very common in plant ‘extracts. In these compounds, the carbohydrate moiety presented glycosydic linkage to phenols, steroids, flavones, etc.** 11.2 EXPERIMENTAL TLC OF SUGARS 11.2.1 Apsorsents AND SOLVENTS Carbohydrates are very hydrophilic compounds and for this reason they a strongly to Silica Gel, Alumina, and Cellulose, so highly polar solvents we necessitry as mobile phase in TLC development, To date, the most common adsorbents are obtained from Merck, Sigma, and Carlo Erba. TLC aluminum or Glass plates containing Silica gel G 60 as adsorbent are widespread for rapid monosaccharide identification; the resolution and senstbili can be increased when HPTLC-Silica gel G 60 plates are used, Cellulose aluminum plates or PC (Whatman No. 1) have been used for monosaccharide and oligoxac- charide separation; however, the development is slower than Silica Gel G 60,268 Thin Layer Chromatography in Phytochemistry Since carbohydrates attach strongly to the adsorbents the mobile phase must be very polar. PC or Cellulose Aluminum plates have been developed with variations in the ratio of butanol:pyridine:H,O. Analytical chromatography for monosaccharide composition uses this solvent system in the ratio of 3 v/v in Whatman No, I, and Preparative isolation of oligosaccharides have been carricd out i dine: HO, 1:1:1, v/v, in Whatman No. 3 PC. The same solvent system has been used in Cellulose plates, sometimes small variations in the ratio are necessary, The development of Silica Gel G 60 plates are also made with polar solvents. ‘The literature shows the use of many organic mixtures of solvents systems; however, for plant monosaccharides the solvent system ethyl acctate:n-propanol:acetic acid: HO (4:2:2:1 v/v) has provided fast separation and high monosaccharides resolution (pentoses, hexoses, and deoxihexoses), as well as for sucrose and trealose aligosac- charides, Sometimes the plates of silica Gel G 60 were buffered with boric acid, sodium borate, and sodium acetate at ~0.02 M, this procedure enhances the separ- ation of the monosaccharide in TLC. The separation of oligosaccharides, such as aldobiuronic, kestoses, fructooligosaccharides, fructans series, and maltose series can be done with 4-propanol:H,0 (70:30 v/v). As the adsorbents and the mobile phase are important in the sugar separation and determination, the use of specific detection is applied, since differen reagents would be necessary to evaluate reducing or nonreducing sugars, cyclitols (inositol), and alditols, 11.2.2) Universat REAGents STAINING ‘The visualization of sugars by nonspecific reagents has been performed with sulfuric acid in silica gel and alumina, since they are inorganic compounds and are not carbonized by strong inorganic acids, Sugars, cyel bonized by solutions of 10%—-40% of H3SO, heating at 100°C for $40 min, The heating sulfuric acid concentration, General ing sugars are more sensitive than s and polyalcohols. [odine wapors have been applied ta detect carbohydrates; however, it is less sensitive than sulfuric acid, Although it is not destructive for 5-20 min of exposure, it can be used in quantitative and preparative chromatography. time is inversely proportional to the 11.2.3 Sreciric ReaGents The ketone and aldehyde groups of the reducing or nonreducing sugars are the preferential point to react specifically in the sugars, as described earlier. The detee- tion of sugars in TLC uses the furturaldehyde in conjunction with many phenolic reagents, which give rise to different colors, as observed by oreinol, a-naphtol, resorcinol, anisaldehyde, and aniline—diphenylamine (Table 11.1). 11.2.3. Preparation and Application of TLC-Staining Reagents Orcinol-H,S0, for Sugar: 250 mg dissolved in 95 mL EtOH:S mL Hz8O.. The TLC plates must be evenly sprayed or quickly dipped into the solution, dried, and heated at 100°C for 5-15 min. The solution is stable for a month if stored ar 4°C.TLC of Carbohydrates 269 TABLE 11.1 Calor Reagents of Carbohydrates on TLC Naphtol- Anisaldehyde-— Resorcinol Aniline Orcinol- Sulfuric Sugar Sulfuric Acid Sulfuric Acid =~ Diphenylamine Sulfuric Acid Acid 1thamnose Green Green Pale green Pale bewwn — Gray-brown p-ribose Blue Blue Gray-brown paylese Gry Ligh bie Bright blue Bue Gray-brown tearabinase ——Yellow-green Bright blue Blue Gray-brown mannose Green Vindet Gray brown beglucose Light blue Blue-violet == Gruy.green Viet Gray-trown p-pulatose —Gween-gmy —Mlueviolet © Gray-green Vaal Gray brown b-glucoronic — Blue = Blas Giray-trown acid galacturonic — Blue - Blue Gray trown acid Sucrose = = = Red-brown * Silver Nitrate-Sedium Hydroxide for Sugars and Polyols: Spray reagent !—1 mL. saturated aqueous silver nitrate solution is diluted to 200 mL with acetone and 5-10 mL. of water are then added until the precipitated has dissolved, * Spray reagent 2—0.5N aqueous-methanolic sodium hydroxide (20g sodium hydroxide are dissolved in a minimum of water and the solution diluted to 1 L of methanol). Spraying is carried out with reagent 1, the plate is dried, then sprayed with reagent 2, and finally heated 1-2 min at KC. To clarify the background the plate must be sprayed with a 5% aqueous sodium thiosulphate solution."° Naphtoresorcinel-HSO, for Sugar—Solution A: 200 mg of naphtoresorcinol dis- solved in 100 mL of ethanol, Solution B—20% H2SO, in E1OH (v/v), For staining, mixed solutions A and B (1:1 v/v) are sprayed on the plates, which are then heated at 100°C for 5-10 min.** Ninhydrin for Aminocompounds: 200 mg of ninhydrin dissolved in 100 ml. of acetone, The plates may be sprayed or quickly dipped into the solution, dried, and heated at 100°C, until the colors arise (5-10 min), Sulfirric Acit¢—Generat Surining: 10% of concentrate H;SO, dissolved in methanol (v/v). The users must take proper care during the mixture preparation to avoid bums. The plates are sprayed and heated at 120°C-140°C, for $15 min, Anisaldehyde-H;SO, for Sugars: a fresh solution of 0.5 mL of anisaldehyde is dissolved in 9 mL EtOH, 0.5 mL HySQ, and 0.1 mL of acetic acid, The TLC plates must be evenly sprayed ‘or quickly dipped into the solution, dried, and heated at 100°C for $-15 min,270 Thin Layer Chromatography in Phytochemistry Anisidine Hydrochloride far Reducing Sugars: 4 g of p-anisidine hydrochloride are dissolved in 4 ml. of HO and the volume completed with -butanol until 100 ml. TLC plates must he evenly sprayed or quickly dipped into the solution, dried, and heated at 100°C for 5-15 min, Aniline-Diphenylamine-Phospharic Acid for Reducing Sugars: 4 g of diphenyla- mine, 4 ml. of aniline, and 20 mL of 854% phosphoric acid (v/v) are dissolved in 20H) mL. of acetone. After the spray the plates must be heated at 85°C for 10 min."° 11.2.4 SAMPLE PREPARATION 11.2.4.1 Water Soluble Sugars (Mono-, Oligo-, and Polysaccharides) ‘The monosaccharide components of oligomers and polymers can be evaluated by their acidic hydrolysis with 2 M TFA at 100°C for 10 b. The product evaporated at room temperature and then dissolved in water and applied on TLC (Figure 11.13) or PC, and examined by silver nitrate or oreinol-H-SO, spray to detect carbohydrate spots or bands.*"7"* These reagents are more sensitive than a variety of methods that use aromatic amines combined with acids, including p-anisidine hydrochloride.” However, the latter and orvinol-H;SO, have great advantage of being more speci distinguishing by the color spots arising from hexose, pentose, and methylpentose. W124.1.1 Partial Acid Hydrolysis Partial hydrolysis can be effected by using conditions somewhat milder than those necessary for complete hydrolysis, for example 0.5 M TFA at 70°C-100°C for periods of 1-10 h, instead of 2M TFA at 100°C for 10 h, The optimum time for maximum production of oligosaccharide can be determined at intervals by PC or TLC (Figure 11.13B). Generally high quantities of polysaccharide are needed, since w aeons FIGURE 11.13 (A) TLC of total hydrolyzed arabinogalctan it LOO0°C, foe 8 h, with 2M TRA (determination of sugar composit ig the uronic acid), The TLC plates were devel- oped in ethyl acctate:a-propanolucetic acid:H0 (4:2:2:1 v/v) and stained with orcinol— H,SO,, (B) TLC of heworoxylan (partial acid hydrolysis using 0.5 M TRA at 100°C). AUT h of hydrolysis, oligoxiccharides are formed, in conjunction with more labile monosaccharides (generally nonreducing ends). After 5 h of hydrolysis only momosacchandes were observed.TLE of Carbohydrates 271 the predominant product is a monosaccharide, and in order to. isolate the minor oligosaccharide component a chromatography can be carried ont on a column of charcoal-Celite $45 (1:1 w/w). Monosaccharides are eluted with water and varying concentrations of aqueous EtOH up to 30%—40% have been used to elute oliga- saccharides.*” As the oligosaccharide produet is often mixed with cochited sificate, a further fractionation is generally carried out on a column of powdered Whatman CF-11 cellutose,“'** which has the advantage of being renewable after each run. Elution is recommended with relatively cheap acetone containing increasing propor- ions of water, which successively elutes monosaccharides (acetone-water, 10:1 v/v), disaccharide (7:1 to 4:1), trisaccharide (3:1), tetrasaccharide (5:2), etc.” The eluates from the charcoal or cellulose columns, when they contain mixtures af oligosaccharides, can be further fractionated on Whatman No. 3 filter paper Analysis of fractions can be carried out by TLC, PC, or HPLC, The latter has a great number of possibilities, Examples ‘of columns are Whatman Partisil-10 PAC for maltodextrins (DP 2-10), and cellodextrins (DP 2-6),"* Waters Bondapak for sucrose, raffinose, stachyose, and Bcyclodestrin, and Waters Altech Carbotydrate 10 for mannose-containing oligosaccharides.” Although preparative HPLC is pos- siti, the simpler and much more economical PC method is sometimes preferred, ¢ the resolution is often better.”” Also, direct cellulose column chromatography cam be employed hefore preparative PC, 11.2.4.2 Organic Soluble Compounds Containing Sugars 11.2.4.2.1 Glycolipids TLC for glycolipids analysis has been well developed using mixtures of organic solvents, These mixtures of solvents and HO, in appropriate amounts, may play an important role on TLC development, and it is possible to have a considerable gain in separation ratios on the plates. Moreover, it is possible to adapt the solvent according lo specific needs. ‘An important way of improving the results consists in the sample preparation, since many types of lipids may be present in plant extracts. Extraction methods consist in the dissolution of the material of interest in specific solvent or mixtures, uswally MeOH and CHC!), at different concentrations, which have shown good realis. in total lipid extraction. The extraction process may be performed at room temperature or under high temperature (60°C), and the process mast be repeated until the lipids are completely removed from the matrix. When the matrix for extraction consists of leaves or young stems, sevetal components may be removed together with the lipids, such as chlorophylls, and other pigmenis, low molecular mass organic compounds, 36 crude extracts should be pretreated in order to avoid interference, Fractionation on chromatographic column using high nonpolar solvents {ethers or hexanes) is employed to eliminate the pigments and other nonpolar components such as triglycerides. The increase of the polarity, by replacing the nonpolar solvent to CHCI, and gradually add MeOH (ic., $% to 50%), makes possible to gel lesser complex mixtures of lipids, which can be analyzed on TLC plates, Usually solvents such as CHCI,:MeOH (9:1, v/v) or CHCl.:MeOH:H,0 (65: v/v) are used as272 Thin Layer Chromatography in Phytochemistry ——* Front SQDG/TGDG + SOMG + —— : . + ——+ Origin (TLEN) (TLERI (NLE) (PFI) (FZ) (Oa00) FIGURE 11.14 Total lipid extract from Maytenus dicifolia (TLE); total lipid extract from Phytlanuhus nirurd (TLE2); neutral lipid extract (NLE}, and polar fractions (PF1 and PF2) after fractionation on DEAE-Sepharose; digalactosylglyceride standard (GDG). developers, To selectively visualize the glycolipids, the plate may be stained by corcinol/sulfuric acid method,”® specific for carbohydrate moieties. Free sugars and oligosaccharides may cause interference if present in the samples. Neutral, polar, and charged lipids may be separated on chromatographic columns as shown in Figure |,14, In this ease the lipids were fractionated on DEAI Sepharose column. The TLC was developed in CHCI)-MeOH:H50 (65:25:4, v/v) and stained by orcinol/H3SO,. A method for glycolipid analysis from invenebrates™* may be useful if applied in plant glycolipid analysis. The procedure was first carried out by suspension of the thawed sample in cold water followed by sonication, until the nematodes were broken, Into this solution CHCl, and MeOH were added, giving rise to final CHC1;:McOH:H30 ratio of 4:8:3, v/v, The solution was held for 4h at 37°C, and then centrifuged to remove the insoluble residues. The supernatant was partitioned in CHCly:MeOH:H,0 (4:8:5.6, v/v), and the phases were separated by centrifugation. ‘The lower phase was dried down and resuspended in 1:1 (CHCl):MeOH) for analysis, while the upper phase was appropriated desalted, dried down, and resus- pended in methanol, The two phases obtained were analyzed on HPTLC plates at different conditions. The lipid samples were developed in a TLC chamber containing (CHCl):MeOH:H,O (4:4:1, v/v) for upper phase lipids, or CHCly:MeOH:H,0 (45:18:3, v/v) for lower phase lipids, which were stained by the orcinal/H,SO,TLC of Carbohydrates 273 method. Glycolipids containing complex carbohydrate moicties were well resolved by this methodology. 11.2.5 Quantirarive Anatysis AND SuGar Composition Analysis on TLC (or HPTLC) provides a rapid and reproducible method for quan- rive andl qualitative analysis of carbohydrates and. glycoconjugates, since the methods are easier and cheaper than other common techniques, such as HPLC and GC. The qualitative method consists in the TLC development with the appropriate solvents and staining reagents (see section 11.2.1-1 1.2.3), containing authentic stand- ards. The A; values of standards are useful, since they are compared to the samples Ry values. Moreover, the color obtained by the staining reagents aid in the identification of sugars, To obtain a quantitative analysis on TLC it is necessary to create a calibration curve (i.¢., 0.1-1 g/aL. of known standards), After staining, the TLC is digitalized and the quantification is carried out by densitometry techniques. ‘To quantify sugars on TLC, it is necessary to examine the image using appro- priate software, Sassaki and coworkers" got accurate quantification for methyl- glycosides partially methylated using Scion Image Program (Scion Comporation, Maryland, USA), based on perception of the image of cach spot on TLC. TLC quantification methods have been employed in sucrose and fructo- oligosaccharides determination in order to provide a more rapid analysis than is possible by high-performance liquid chromatography (HPLC), since the time- consuming in samples pre-treatment is not needed,” $0, TLC scems to have some advantages over HPLC, such as in sample preparation, detection by specific reagents and the low consumption solvents and short time of analysis. 11.3, CONCLUSIONS ‘The great advantage in TLC analysis is its simplicity, In this review different solvents have been shown, since they are the simplest and easiest to obtain from chemical industries. These solvents present efficiency and high resolution for plant carbohydrates and glycoconjugates analysis. Specific detection of sugars on TLC uses the furfuraldehyde allied to orcinol, since it can resolve the most common monosaccharide classes present in plant by its peculiar colors and R, values. Since reduced sugars (alditols) are not stained by orcinal, the use of silver nitrate—sodium hydroxide can provide their detection, being useful to observe these components, ‘TLC of polysaccharides is not possible; however, it has been used te determine their monosaccharide composition after hydrolysis. The latter, also, has been monitored by TLC, provides the ideal time for oligosaccharides formation. Glycoconjugates have been isolated and characterized by TLC using standards. The presence of substitution in the sugar moiety, such as phosphate and amino groups, has been observed on TLC using specific s 1g methods, molybdate reagent™ and ninhy- i ely, in conjunction with oreinol visualization, However, TLC tech- nique does not provide information about O-glycasidic or N-glycosidie-linkages, position of substituents, and sugar configuration. 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