III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography 2235

Conclusions: Summary of Main Trends tectors; Instrumentation; Mechanisms: Reversed Phases.
III / Polysaccharides: Liquid Chromatography.
A wide variety of HPLC methods are available to
analyse carbohydrates from various complex ma- Further Reading
trices. Aminopropylsiloxane-bonded silica of small
particle diameter (3 m), amine-bonded vinyl alcohol Alpert AJ (1990) Hydrophilic-interaction chromatography
copolymer packings, diol- and cyclodextrin-bonded for the separation of peptides, nucleic acids and other
silica, as well as macroporous cross-linked vinyl- polar compounds. Journal of Chromatography 499: 177.
Cheetham NHW and Teng G (1984) Some applications of
pyridinium polymers, are some of the columns of
reversed-phase high-performance liquid chromatogra-
choice for high selectivity in the HILIC of carbohy- phy to oligosaccharide separation. Journal of Chrom-
drates. Furthermore, a large number of columns may atography 336: 161.
be selected to separate carbohydrates on cation ex- Churms SC (1990) Recent developments in the chromato-
change resins carrying different counterions. Em- graphic analysis of carbohydrates. Journal of Chrom-
ploying these columns the chromatographic separ- atography 500: 555.
ation is governed by a combination of size exclusion Dreux M and Lafosse M (1995) Evaporative light scatter-
and ligand exchange mechanisms. In oligosaccharide ing detection of carbohydrates in HPLC. In: El Rassi
separations, size exclusion is the primary mechanism Z (ed.) Carbohydrate Analysis } High Performance
which involves the binding of hydroxyl groups of the Liquid Chromatography and Capillary Electrophoresis.
sugars with the Rxed counterion of the resin, thus Amsterdam: Elsevier.
El Rassi Z (1995) Carbohydrate Analysis } High Perfor-
they elute last.
mance Liquid Chromatography and Capillary Elec-
On the other hand, RP-HPLC with plain water as trophoresis. Amsterdam: Elsevier.
the eluent may be useful for the separation of various Giese R and Honda S (1996) Chromatographic and elec-
underivatized oligosaccharides. The mechanism gov- trophoretic analyses of carbohydrates (Special Issue).
erning this HPLC method produces separations that Journal of Chromatography 720.
complement those given by cation exchange columns Goulding RW (1975) Liquid chromatography of sugars and
and therefore the two techniques should be used in related polyhydric alcohols on cation exchangers } The
conjunction in isolation and analysis of carbohy- effect of cation variation. Journal of Chromatogra-
drates. phy 103: 229.
Column instability and low detection sensitivity are Herbreteau B (1992) Review and state of sugar analysis by
the major disadvantages using these HPLC methods. high performance liquid chromatography. Analusis 20:
355.
However, column and detection technologies to ana-
Honda S (1984) High-performance liquid chromatography
lyse carbohydrates are in continuous development. of mono- and oligosaccharides. Analytical Biochemistry
Mono- and oligosaccharides are detected without 140: 1.
any derivatization by PAD, which is sensitive and Hurst WJ (1997) Seminars in Food Analysis } HPAEC-
selective for detection of these analytes. The use of PAD in Food and Beverage Analysis. London: Chapman
HPAEC is now widely accepted as a powerful analyti- and Hall.
cal tool for carbohydrate analysis. However, HPAEC LaCourse WR (1993) Pulsed electrochemical detection at
methods require dedicated columns and instrumenta- noble metal electrodes in high performance liquid
tion. chromatography. Analusis 21: 181.
Lee YC (1990) High-performance anion-exchange chrom-
See also: II / Chromatography: Liquid: Detectors: Evap- atography for carbohydrate analysis. Analytical Bio-
orative Light Scattering; Detectors: Refractive Index De- chemistry 189: 151.

Thin-Layer (Planar) Chromatography

J. F. Robyt, Laboratory of Carbohydrate Chemistry differ in the number of carbon atoms, the conRgura-
and Enzymology, Iowa State University, Ames, IA, USA tion of the chiral centres and the size of the molecules,
Copyright ^ 2000 Academic Press that is to say whether they are di-, tri- and oligo-
saccharides. If two carbohydrates have any one of the
three characteristics in common, they can be difRcult
Introduction to separate. There are some carbohydrates that differ
Carbohydrates are some of the more difRcult com- by having a special functional group, for example
pounds to separate from each other. They primarily a carboxyl group (giving uronic, onic and aric acids),

and and convenient at that time. This led to a wide This solvent is frequently the Rrst choice when separ- variation in the quality of the plates and in the results ating unknown carbohydrates in a sample. A very sensitive detection system has recently been the column is irreversibly modiRed. dried not affected by other compounds that have previously and rapidly dipped into the reagent. a uronic acid. With solvent. Neverthe. once a sample has been added to a column. dis- graphic procedures developed. whether the carbohydrates are monosaccharides. Carbohy- Further. TLC plate. primarily by Whatman and Merck in the component. directly on the TLC samples must be separated and analysed in series on plate. The silica gel N-acetyl amino sugars). even within a single laboratory. or a phosphoric acid ester thin layer is usually uniformly 250 m thick over the group (giving carbohydrate phosphates). The separated in its own little column or lane. Figure 1 illustrates the separation of equipment. 2-N-acetylamino-2-deoxy sugars and sugar positions of the standards can be used for qualitative alcohols. HPLC. because carbohydrates play such an important achieved by the selection of a speciRc solvent system. TLC is also more reproducibile. neously separated on a single 20. commercially developed and produced at a reason.3% (w/v) of N-(1-naphthyl)ethylenediamine and feasible. whereas sample placed on to a TLC plate is 5% (v/v) concentrated sulfuric acid in methanol. Many was not until reproducible thin-layer plates were solvent systems contain three components: water. TLC is into the TLC tank for a second. the technique of TLC has now become the principal method of multiple ascents is frequently employed. A relatively simple solvent that has found valuable The use of TLC. dried and then heated at 1203C for The principal support material found most useful for 10 min. approximately 18 samples can be simulta. Further. chromatography (TLC) for separating carbohydrates. methods for veloped. the TLC plate. which is TLC plate is removed from the developing tank. TLC is much preferred evaporated from the plate. di. In this tech- choice for the separation of carbohydrates. and the detection relatively complex mixtures of carbohydrates using of the carbohydrates directly on the plate is more this technique with acetonitrile}water (85 : 15. valuable in the separation of carbohydrates is silica with detection in the 10}100 g range. or Early chromatographic methods in the 1950s for whether they contain a charged group such as separating carbohydrates used paper as the solid sup. as each laboratory had to trisaccharides is acetonitrile}water (85 : 15. uses much less sample. which can include standards. solution. an amino sugar or a carbohydrate port. in which a 25% (v/v) solution of sulfuric acid in methanol was sprayed on to the Detection Methods TLC plate. Solvents that were obtained. uses simple fourth ascent. for the separation of more complex mixtures can then The results were difRcult to reproduce from day to be developed using other organic compounds in com- day and particularly from laboratory to laboratory. trisaccharides or oligosaccharides. prepare its own thin-layer plates. many hundreds of ted in the 50}2000 ng range. forming furfurals from the carbohydrates by dehy- dration with the sulfuric acid. consisting the same column to make the procedure economically of 0. This is primarily due to the requirement of identiRcation. a stable and inert substance.20 cm TLC Most carbohydrates can be detected by using this plate. the solvent is allowed to ascend to the top of formance liquid chromatography (HPLC) has dis. v/v). It bination with the acetonitrile}water system. Solvents and sulfuric acid charring. whereupon the plate is removed from placed TLC in many laboratories. the . A simple com. nique. For maximum resolution. An older method used TLC Support Material. accharides. with the exception of 2-amino-2-deoxy parison of the migration positions with the migration sugars. The resolution of carbohydrates is less. This is a much less sensitive method. although for carbo. a water-soluble component and a water-insoluble able price.2236 III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography an amino or acetyl-amino group (giving amino and gel. The particular solvent used depends on separating them were some of the earliest chromato. The method uses a detecting reagent. In the early 1960s Thoma introduced thin-layer phosphate. was not particularly easy use in the separation of a number of mono-. Because of the developed in which most carbohydrates can be detec- high cost of the HPLC columns. role in living systems and comprise a large number of Many solvent systems have been empirically de- important naturally occurring products. reagent. High per. however. When compared with HPLC. the developing tank and the solvent is completely hydrates and other materials. The plate is dried been adsorbed and separated on the support material. that TLC became a viable and valuable various proportions so as to give a homogeneous procedure. v/v) sensitive. easier to perform. The plate is then put back over HPLC. third and/or much less expensive. The three components are combined in late 1970s. drates give blue-black spots on a white background. and placed in an oven at 1203C for 10 min.

depending on the desired number of saccharides to be separated. The carbohydrates were detected equivalent maltodextrin mixture. has been reported. primarily because by using the N-(1-naphthyl)ethylenediamine}sulfuric acid}meth. Figure 3 friendly than a dipping technique. Both can be used to detect ing these solvents. using Whatman K5 plates or Merck silica gel 60 plates and multiple ascents with acetonitrile}water systems. such as analysis of carbohydrates in foods. using silica-gel plates developed with butanol-1}ethanol}water. To obtain an equivalent separation for the isomaltodextrins. 35S. Many of these analyses involve the separation of a homolo- gous series of oligosaccharides containing one or pos- sibly two kinds of glycosidic linkages and as many as 20}30 monosaccharide residues of the same type in the oligosaccharides. containing 2}30 monosacchar- ides residues. Figure 2 illustrates the separation of maltodextrins using one to four Figure 1 Separation of various mono-. analysis of oligosaccharides from glycoproteins and glycolipids. -1P4. of differences caused by the -1P6 glycosidic link- anol dipping reagent. The amount of water (x) can be var- ied from 50 to 70 parts by volume. The largest saccharides can have 20 D-glucose residues. TLC plate. complex saccharides are separated from mixtures although the PhosphoImager is several orders of produced by the action of two different enzymes . -1P3. one to four ascents of acetonitrile}ethyl acetate}pro- mits several sensitive techniques to be used to detect panol-1}water in the volume proportions of and quantify the carbohydrates directly on the 85 : 20 : 50 : 90 at 20}223C. age vs the -1P4 glycosidic linkage. Likewise. or 32P radioactive isotopes. is illustrated in Figure 4 in which and quantitate 14C. The separation of a homologous series of saccharides. linked -1P2. Much faster systems. such as a TLC radioactive scanner The power of TLC separations of saccharides.and trisaccharides ascents of 85 : 20 : 50 : 60 volume proportions at on Whatman K5 TLC plates (18. III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography 2237 magnitude more sensitive and relatively easy to use quantitatively. the analysis of enzymatic synthesis of oligosaccharides and the analysis of the hydrolysis of polysaccharides. illustrates the separation of isomaltodextrins using The separation of radiolabelled carbohydrates per. the water content (x) of the solvent system has to be spraying technique is much less environmentally increased to 90 or 100 volume proportions.5 cm solvent path length for each 20}223C. di. using up to four ascents can increase the number of saccharides separated. and -1P6. several hours for one ascent. have been developed for the separation of maltodextrins (-1P4 linked glucosaccharides) and isomaltodextrins (-1P6 linked glucosaccharides). us- or a PhosphoImager. The separation of malto- dextrins was achieved using acetonitrile}ethyl acet- ate}propanol-1}water in volume proportions of 85 : 20 : 50 : x. The devel- opment time was however. TLC Separation of Homologous Series of Oligosaccharides The separation of oligosaccharides is important in a wide variety of applications. ascent). using four ascents of acetonitrile}water in volume pro. Isomaltodextrins migrate more slowly than an portions of 85 : 15 at 20}223C.

lane 2 shows an equivalent set of maltodextrin each ascent). B7 migrates behind G7 and faster than G8. Saccharides marked B6. containing D-glucose. These saccharides migrate in between the maltodextrins.5 cm solvent path length for dues. six and seven -1P4 glycosidic linkages. with each having two -1P6 glycosidic linkages and seven. Reproduced with permission from Robyt and Mukerjea tato amylopectin. The num- bers at the top of each chromatogram indicate the number of solvent ascents. maltose. Saccharides containing one D-galactose residue will usually migrate more slowly than an equivalent saccharide containing all D-glucose Figure 2 Separation of D-glucose and maltodextrins on What- man K5 TLC plates (18. The carbohydrates were detected using the N-(1-naphthyl)ethylenediamine}sulfuric acid}methanol dipping reagent.2238 III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography contain six. Saccharides marked BB9. maltotriose and so Figure 3 Separation of D-glucose and isomaltodextrins on forth down to maltodextrins with 14 D-glucose resi. Lane 1 on the plate is a set of maltodextrin standards. respectively. BB6 migrates behind G6 and faster than G7. agent. BB10 and BB11 contain nine. using 1}4 ascents of acetonitrile}ethyl acetate}propanol-1}water in volume proportions of 85 : 20 : 50 : 60 at 20}223C.5 cm solvent path length for each ascent). B7 and B8 (1994). . The saccharides were sep- arated by using four ascents of acetonitrile}ethyl acet- ate}propanol-1}water in volume proportions of 85 : 20 : 50 : 50 at 20}223C on a Whatman K-5 plate. for example. seven and eight D-glucose residues. Whatman K5 TLC plates (18. and so forth. using 1}4 ascents of acetonitrile}ethyl acetate}pro- saccharides that were obtained by the action of panol-1}water in volume proportions of 85 : 20 : 50 : 90. re- spectively having one -1P6 glycosidic linkage and Rve. The carbohydrates were detected using N-(1- the separation of products that resulted from the naphthyl)ethylenediamine}sulfuric acid}methanol dipping re- action of porcine pancreatic -amylase acting on po. lane 3 shows solvent ascents. eight and nine -1P4 glycosidic linkages. acting on polysaccharides. The numbers at the top of each chromatogram indicate the number of isoamylase acting on shellRsh glycogen. 10 and 11 D-glucose residues. Reproduced with permission from Robyt and Mukerjea (1994).

Alditols can be Figure 4 Separation of maltodextrins and maltodextrins.5 cm previously mentioned. water in volume proportions of 85 : 20 : 20 : 15. 1c had D-glucose transferred to C3 of the reducing-end glucose residue of maltotriose. Enzymes F and S only gave two initial products. alditols are not readily detec- solvent path length for each ascent) was irrigated four times with ted by the sulfuric acid-based reagents described acetonitrile}ethyl acetate}propanol-1}water in volume propor- above. 1b had D-glucose trans- ferred to C6 of the nonreducing-end glucose residue of maltotriose. To illustrate the tremendous power that TLC has in separating carbohydrate oligosaccharides with very similar structures. Whatman K5 TLC plate (18. 1a had D-glucose transferred to C6 of the reducing-end glu- cose residue of maltotriose. 3a. maltodextrins resulting from the ac. Lane 1. 3b and so forth are products resulting from the transfer to C6 of reducing-end glucose residue and to C6 of the nonreducing-end glucose residue. v/v/v) on a Whatman K5 TLC plate. D-Glucose was transferred from sucrose to maltotriose by the action of three enzymes (F. Enzyme I gave four initial products. containing the same number of D-glucose residues. ascents of acetonitrile}ethyl acetate}propanol-1} tion of isoamylase reaction with shellfish glycogen. but differing from each other by the location of the transferred D-glucose residue and the types of linkages. either -1P6 or -1P3. 1a}1d. The best detection system is an alkaline silver tions of 85 : 20 : 50 : 50 at 20}223C. Two different products were for- med by the transfer of D-glucose to the reducing end of the acceptor saccharide to form an -1P6 linkage (product-1a) and transfer of D-glucose to the non- reducing end of the acceptor saccharide to form - 1P6 linkage (product-1b). lane 2. and 1d had D-glucose transferred to C3 of the nonreducing- end glucose residue of maltotriose. glucose and mal- todextrin standards. lane 3. III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography 2239 residues. Reproduced with permission from detect alditols in the range of 500 ng to 1 g. Products 2a. and so forth. and so forth of the Rrst. 1a and 1b. Figure 5B shows the separation of an even more complex mixture of products. The carbohydrates were detected using the N-(1-naphthyl)ethylenediamine}sulfuric acid} nitrate}sodium thiosulfate dipping system that will methanol dipping reagent. I and S). second and third acceptor products. As amylase action on amylopectin. con- separated from their aldoses on TLC by using two taining one and two -1P6 linkages. These com- plex products were separated using four ascents of ethyl acetate}ethanol}water (2 : 2 : 1. All three enzymes gave higher products: 2a. Compare the migration of lactose with cel- lobiose in Figure 1. The Robyt and Mukerjea (1994). . 3a. dry TLC plate is dipped into a silver nitrate}acetone . 2b. Figure 5A shows the chromato- graphic separation of saccharides formed by the ac- tion of Leuconostoc mesenteroides B-512FM dex- transucrase transfer of D-glucose from sucrose to malto-oligosaccharide acceptors having two to eight D-glucose residues. Separation and Detection of Sugar Alcohols on TLC One of the more difRcult separations of carbohy- drates is that of separating aldoses from their corre- sponding alditols (sugar alcohols). 2b.

With G3 and higher. transferring D-glucose to the C6 position of the reducing-end glucose residue of the acceptor (product 1a) and the transfer of D-glucose to C6 position of the nonreducing-end glucose residue of the acceptor (product 1b). The reagent is prepared by adding a white background. solution for 5 min. sponding aldoses. and so forth acceptor products. followed by the dropwise addition of distil. The compounds were labelled with 14C and detected by autoradiography. and then the plate is washed saccharides are produced from the methylation of for 1 min in running water.4% NaOH (w/v) for 30 min. Figure 6 illustrates the separ- 1 mL of saturated aqueous silver nitrate to 200 mL of ation of seven different alditols from their corre- acetone. 3b and so forth represent transfer to C6 position of the reducing-end and the nonreducing-end glucose residues of the first.25 mol L NaHSO3. Reproduced with permission from Fu and Robyt (1991). The saccharides were separated on Whatman K5 TLC plates using four ascents of ethyl acetate}ethanol}water in volume proportions of 2 : 2 : 1 (18. O-Methylated mono- 0. maltose (G2) to maltooctaose (G8). led water until the silver nitrate is dissolved. the enzyme gave two products of the same size. giving black spots on oligosaccharides and polysaccharides followed by . in volume proportions of 2 : 2 : 1. Brown to black spots appear for the alditols. The Monosaccharides by TLC plate is then rapidly dipped into a 1. Products 2a. The plate is dried and then dipped into an alkaline methanol Separation of Methylated solution containing 0. Reproduced with permission from Fu and Robyt (1990). Streptococcus mutans mutansucrase (I). (B) Separation of the products from the transfer of D-glucose from sucrose to maltotriose by the action of three enzymes.08 mol L Na2SO3 and is especially important. second. and S.2240 III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography Figure 5 (A) TLC separation of a series of products produced by the action of Leuconostoc mesenteroides B-512FM dextransucrase catalysed transfer of D-glucose from sucrose to maltodextrin acceptors. The compounds were labelled with 14C and detected by autoradiography. The separation of O-methylated monosaccharides tion of Na2S2O3 containing 0. mutans dextransucrase (S).5 cm solvent path length for each ascent). 3a.5 mol L solu. Leuconostoc mesenteroides B-512FM dextransucrase (F). Whatman K5 TLC plate was irrigated with four ascents of ethyl acetate}ethanol}water. 2b.

because of the sensitivity of detecting the methylated monosacchar- ides (lower limits of 25}50 ng range) directly on the TLC plate. The easiest method of detecting and quantitating agent. if a gluco-polysaccharide contains 1P4 glycosidic linkages with 1P6 branch linkages. Further.3.5 cm solvent path length for each ascent) using two ascents of acetonitrile}ethyl acetate}propanol-1}water in volume proportions of 85 : 20 : 20 : 15 at 20}223C.6-tri-O-methyl-D-glucose (major). The methylated carbohydrates are detected by dipping the plate into the N-(1-naphthyl) ethylene- diamine}sulfuric acid}methanol reagent described Figure 7 Separation of O-methylated D-glucoses. Whatman K6 plates were irrigated (18. III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography 2241 Figure 6 Separation of alditols (sugar alcohols) from their corresponding aldoses on Whatman K5 TLC plates (18. The number of methyl groups and their location on the resulting monosaccharides indi- cate the positions of the linkages to the monosacchar- ides in the oligosaccharides or polysaccharides and whether or not the saccharides are branched. Figure 7 illustrates the chromatography of methylated D-glucoses on What- man K6 TLC plates. (1996). highly branched D-glucan. carbohydrates on a TLC plate is to use 14C-labelled . micro amounts of saccharide (10 g) can be methylated and analysed. The carbohydrates were detected by using the N-(1- naphthyl)ethylenediamine}sulfuric acid}methanol dipping re.4. The carbohydrates were detected by using the alkaline silver nitrate dipping reagents. there would be three kinds of methylated monosaccharides: 2. For example.5 cm solvent path length for each ascent) at 20}223C. followed by heating for 10 min at 1203C. 2.3.6-tetra-O- methyl-D-glucose (minor from the nonreducing-end D- glucose residues) and 2. The use of TLC to separate and analyse these methylated mono- saccharides has greatly simpliRed methylation analysis of oligo. using two ascents of acetonitrile} chloroform}methanol in volume proportions of 3 : 9 : 1 at 20}223C. using two as. acid hydrolysis. Quantitative Determination cents of acetonitrile}chloroform}methanol in volume proportions of Carbohydrates of 3 : 9 : 1. Lanes 1 and above. Reproduced with permission from Han and Robyt (1998).3-di-O-methyl-D-glucose (mi- nor from the branched D-glucose residues). lane 2 is the analysis of a complex. Reproduced with permission from Mukerjea et al. 3 are standards.and polysaccharides.

and so forth. such as N-(1-naphthyl)ethylenediamine re- agent described above. In this instance. a mixture are sufRcient. reagent. using a sulfuric acid-based chromatogram no. quantitated directly on the plate using a phos. is limited to carbohydrates that vidual saccharides that are separated in a mixture can can be obtained in radioactive form and therefore be scanned. or lytical TLC separations. using a Bio-Rad imaging densitometer. but substituting TLC plates . The relative weight per cents for each of the carbohydrates are shown at the top of each peak.2242 III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography Figure 8 Densitometric scan of TLC number 3 shown in Figure 2. the indi- hydrates. and the percentage of the individual compounds com- formed. The radioactivity can be detected and sucrose. Absolute. quantitative amounts can be determined using various amounts of Carbohydrates Using TLC the monosaccharide involved in the saccharides as By using the same solvent systems as is used in ana- standards. for many purposes. The more usual case is the chromatography puted by dividing the individual densities by the sum of nonlabelled carbohydrates. of the densities. 3 in Figure 2. down to a maltodextrin with 14 D-glucose residues. maltotriose. Figure 8 shows a density scan of hydrates on a TLC plate. followed by maltodextrins: maltose. carbohydrates. maltotetraose. however. The detection of carbo. D-glucose. can be quantiRed using Preparative Separation of a scanning densitometer. and so forth. D-mannose. However. The first peak is D-glucose. the relative amounts of the individual saccharides in phoimager. the densities of the compounds summed limited in the kinds of experiments that can be per. for example. The use of radioactively labelled carbo.

The plate is developed in the usual way. When many sam- ples are to be added to the TLC plate. The old idea that the TLC plate a hair-dryer is a fast and convenient method for should be removed from the solvent before the drying plates. keeping the size of the spot as small as pos- sible.5. A stand and a lid have to be fabricated from with the solvent over the entire surface of the plate. The multi-spotter is also convenient when a relatively large volume. Points to Consider to Obtain Good Separations 1. 15 mm above the bottom of a 20. In fact. 5. of sample is to be added. In developing the chromatogram. the TLC plate. The solvent and va. for example 10}25 L. A relatively small (1}25 L) measured amount of sample should be carefully added on to the TLC plate. 15 mm strips are cut off from each end of the chromatogram and dipped into the detecting reagent. Use no more than 15 mm margin from the bottom chamber (Figure 9). After develop- ment. III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography 2243 that have a much thicker silica gel layer (1000}2000 m). dry the TLC has become a powerful analytical tool for separ- plates thoroughly between each ascent.20 cm TLC plate. uniform spots with a minimum of effort. so as to give the maximum path length for the sol- vent. The aqueous extract can then be concentrated to dryness by evaporation. the development. The use of ating compounds with very similar chemical and . The use of a microlitre syringe pipette. In multiple ascending chromatography. The strips are lined up with the plate and the loca- tions of the desired compounds are marked. preparative separations of carbo- hydrates can be obtained. solvent has reached the top or just as the solvent 6. is desirable. it is best to allow reagent to obtain a uniform amount of reagent 5}10 min extra. The use of a narrow 1. before removing the plate. and the com- pound redissolved if necessary. polypropylene or similar material to hold the 4. be sure only to use pencil to mark the Conclusions and Future Prospects TLC plates. This 22. Approximately 25}50 L of a 20}30 mg sample of a mixture of carbohydrates is streaked along a sample application line. Allow the solvent to ascend to the top of the TLC plate to give the maximum path length for the solvent. after the solvent reaches the top of over the entire plate. of the plate to the point of sample application. 2. such as a Hamilton syringe. 3. a multi- spotting instrument can conveniently be used to make very small. The silica gel area containing the compound is scraped from the plate into approximately 2 mL of water. The suspen- sion is mixed and the silica gel removed by Rltration or centrifugation. Be sure that the developing tank is tightly sealed during development (vacuum grease can be used with glass-to-glass surfaces).22 cm glass chamber is convenient for dip- ensures that the plate has been completely saturated ping. rapidly and reaches the top of the plate is not valid. Also. if the carefully dip the irrigated plate into the detecting chamber is tightly sealed. Figure 9 Glass chamber and holder used to dip the TLC plates pour should be in equilibrium before beginning into the detecting reagent.

di-. and trisac- lend themselves to the usual physical detection and charides by thin-layer chromatography. irrigations. The use of 137}153. Tai H and Fisher EE (1968) chiral centres. Further. as in most areas of endeavour. FL: CRC Press. I. to analyse and compare compounds eas. The Han NS and Robyt JF (1998) Separation and detection of development of a wide variety of solvents has also sugars and alditols on thin-layer chromatograms. relatively inert materials. Archives of Biochemistry and Biophysics These reactions provide qualitative identiRcation of 321: 379}387. the compounds by speciRc colour production and the Fu D and Robyt JF (1991) Maltodextrin acceptor reactions quantitative determination of the compounds by the of Streptococcus mutans 6715 gluocosyltransferases. Fu D and Robyt JF (1990) Acceptor reactions of maltodex- allows chemical reactions to be conducted directly on trins with Leuconostoc mesenteroides B-512FM dex- the plate where the compounds have been separated. Journal of quantifying methods. The latter will then also provide for the a modiRed procedure and thin-layer chromatography. . There will probably be 145}157. Wise CS and Dimler RJ (1951) Improved tech- Future developments in TLC of carbohydrates may niques in paper chromatography of carbohydrates. The latter is parti- Gauch R. last 50 years. 2nd a wide range of laboratories. the use of rela- Block RJ. Gas Chromatography and Gas search 248: 339}348. New York: Academic Press. Carbohydrate Research 251: 187}202. But. trins and isomaltodextrins by thin-layer chromatogra- phy. Utamura T and Okada Y (1985) Analysis of is the necessity of invention. light scattering or refractive index. Durrum EL and Zweig G (1958) A Manual of tively simple and inexpensive materials permits Paper Chromatography and Paper Electrophoresis. reSectance and Suorescence ive determination of nanogram quantities of maltodex- in quantifying carbohydrates directly on the plate. need Koizumi K. Carbohydrate Re- ates: Electrophoresis. Carbohydrate Research 292: 11}20. Journal of Chromatography 321: matic modiRcation reactions. Liquid Chromato- opment of reliable and reproducible commercial TLC graphy. Leuenberger U and Baumgartner E (1979) cularly important for carbohydrates as they do not Quantitative determination of mono-. use of scanning densitometry. 170}214. permitted the separation of carbohydrates that differ Carbohydrate Research 313: 135}137. book of Chromatography: Carbohydrates. from each other in subtle aspects of conRguration of Huber CN. quickly and with high sensitivity. Preparative Thin-Layer (Planar) Chromatography. from the high-school edn. such as carbohydrates. Chromatography-Mass Spectrometry. III / Carbohydr. Ana- come slowly as much progress has been made in the lytical Chemistry 23: 415}418. development of new physical instrumentation using Robyt JF and Mukerjea R (1994) Separation and quantitat- lasers for scanning density. such as silica gel on glass. differences in the kinds of glycosidic Thin-layer chromatography of the malto-oligo and linkages present. Scobell HD. Spray Reagents. using the plate. teroides B-512FM dextransucrase.2244 III / CARBOHYDRATES / Thin-Layer (Planar) Chromatography physical properties. ily. Kim D and Robyt JF (1996) SimpliRed and sensitivity in the submicro range of carbohydrates on improved methylation analysis of saccharides. dextran and acceptor-products by Leuconostoc mesen- Radioactivity Detection. homogeneous D-gluco-oligosaccharides and -polysac- doubtedly be made in new solvents that will separate charides (degree of polymerization up to about 35) by new kinds of mixtures of carbohydrates obtained high performance liquid chromatography and thin-layer from natural products and from chemical and enzy. transucrase. teaching laboratory to quality control and research Churms SC (1982) In: Zweig G and Sherma J (eds). Jeanes A. The devel. See also: II / Chromatography: Thin-Layer (Planar): Su D and Robyt JF (1993) Control of the synthesis of Layers. vol. such as ultraviolet or visible Chromatography 174: 195}210. pp. plates has placed it at the forefront of separation methods that can be used for both qualitative and Further Reading quantitative determinations. absorbance. chromatography. Hand- laboratories. Developments will un. and differences in the size of the megalosaccharides with mixed support and multiple compounds. Boca Raton. detection methods developed that will give greater Mukerjea R. Analytical Chemistry 40: 207}209. pp. Carbohydrate Research 217: 201}211.