9 North Harrison Road Bellefonte, PA -00 USA Telephone 00-- -9- Fax 00--0-9-0 email: supelco@sial.com sigma-aldrich.com/supelco HPLC Carbohydrate Column Selection Guide Because carbohydrates exhibit a significant degree of chemical and physical similarity, they are more difficult to analyze than most other classes of compounds. No single HPLC column or method is capable of separating all carbohydrates. Advantages, limitations, and applications for GEL and SIL columns specifically prepared for various carbohydrate analyses are summarized here. Key Words carbohydrates sugars saccharides monosaccharides disaccharides oligosaccharides Carbohydrate analyses are a challenge for the chromatographer. Diversity among compounds classified as carbohydrates (Table ) is far greater than among other classes of biochemicals. The potential for complexity in the structure of carbohydrate molecules is summarized by the fact that three amino acids can combine in a total of six different ways, while three monosaccharides can form more than 000 distinct trisaccharides. To further complicate the situation, chemical and physical properties among monosaccharides, disaccharides, and trisaccharides differ only slightly. Hence, HPLC separations of carbohydrates depend on differences in conformation, configuration, and bonding mode, and are more difficult than analyses of other classes of compounds. No single HPLC column or method is capable of separating all carbohydrates. For this reason, we offer a selection of columns specifically prepared for various carbohydrate analyses. Advantages and limitations of each column are summarized in this bulletin. Table summarizes applications for these columns; Table shows retention times for an extensive number of carbohydrates, under typical operating conditions. Table. Types of Carbohydrates Monosaccharides Pentoses (arabinose, ribose) Hexaoses (glucose, galactose, fructose) Disaccharides (sucrose, lactose, maltose) Trisaccharides (raffinose, melezitose) Tetrasaccharides (stachyose) Oligosaccharides (DP to DP0*) Polysaccharides (00 000 units) Polyhydric Alcohols/Sugar Alcohols (mannitol, sorbitol, polyols) *DP (degree of polymerization) = number of monosaccharide units in the molecule. The major advantage of RI detection is the universal response over a fairly wide linear range of analyte concentrations. Major disadvantages are poor sensitivity and baseline instability caused by temperature, solvent, or pressure changes (RI detection is impractical with gradient elution). Both positive and negative peaks can be present in a chromatogram. The abundance of carbohydrates in food and other samples, however, makes RI detection a suitable tool for these analyses. Figure A. Refractive Index Affords Superior Detection for Carbohydrates Column: SIL LC-NH, cm x.mm, µm particles Cat. No.: Mobile Phase: acetonitrile:water, : Flow Rate:.mL/min Temp.: ambient Inj.: 0µL, 0mg/mL each analyte in acetonitrile:water, : UV (90nm). Fructose. Glucose. Sucrose. Maltose. Lactose 0 0 9-00 Refractive Index Detection Because the UV wavelengths required to detect carbohydrates also will be strongly absorbed by impurities in the samples (Figure A), refractive index (RI) detection is most commonly used in analyses of carbohydrates. 0 0 9-00 sigma-aldrich.com/supelco
Table. Carbohydrate Column Applications and Operating Parameters GEL TM columns are resin-based; SIL TM LC-NH column is silica-based. Typical Maximum Sample Max. Mobile Flow ph Temp. Column Application Form Phase (ml/min) Range ( C) GEL K beet sugar, cane sugar, potassium 0mM K HPO. - 90 molasses, corn syrup GEL Pb monosaccharides, lead deionized water. - 90 xylose/galactose/mannose GEL Ca high fructose corn syrup, calcium deionized water. - 90 monosaccharides, sugar alcohols, oligosaccharides GEL C-0H organic acids, acid/carbohydrate/ hydrogen 0.% H PO alcohol mixes or H SO. - 0 GEL H organic acids, acid/carbohydrate/ hydrogen 0.% H PO alcohol mixes or H SO. - 90 GEL C- mono-, di-, and trisaccharides, divalent cations 0 - N NaOH. - galactose/mannose GEL Ag oligosaccharides, glycerol/ethanol, silver deionized water. - 90 beer, corn syrup, hydrolyzed starch SIL LC-NH mono-, di-, some trisaccharides aminopropyl silica % CH CN in water.0-0 Table. Typical Retention Times on Supelco Carbohydrate Columns GEL Columns SIL Ca C-0H H H Pb C- K Ag LC-NH Cat. No.: 90-U 90-U 90-U 9 9 90-U 9 9 Dimens. (mm): 00 x. 00 x. 00 x. 0 x. 00 x. 00 x. 00 x. 00 x. 0 x. Temp.: 0 C 0 C 0 C 0 C C 0 C C C ambient Mobile Phase: DH O 0.% H PO 0.% H PO 0.% H PO DH O 0 N NaOH mm K HPO DH O ACN: DH O (:) Flow Rate (ml/min): 0. 0. 0. 0. 0. 0. 0. 0..0 Det.: refractive index Compound Retention Times (min) Arabinose..9.. 9. 9.... Arabitol 9...9.....0. Betaine ND ND ND ND NR ND.0 ND ND Dulcitol.......9.9 9.0 Erythritol..0... 0..0..9 Ethanol 9.. ND ND ND.0 ND. NR Fructose.9...9 0. 0...0. Galactose..9.0..... 0. Glucose.0..9..9..0. 9. Glycerol..... 0.9.. NR Inositol.9.... 0... ND Isomaltose 9. 0. ND ND ND. ND. 9. Isomaltotriose. 9. ND ND ND. ND 9. NR Lactitol ND ND..0. ND 0. ND ND Lactose 0. 0. 0. 0... 0.9. 9. Maltitol..0 0. 0... 0..0. Maltoheptaose....9 9.... NR Maltohexaose..9.. 9..0.. NR Maltopentaose.9 9..9. 0.... NR Maltose 9. 0. 9.9 9.9.0. 0... Maltotetraose. 9....... NR Maltotriose. 9.. 9.0.0. 9. 9..0 Mannitol 9........ 9. Mannose...9. 9..9..9 9. Melezitose. 9.. 9.0 0... 9.. Psicose....9..9... Raffinose. 9...9... 9. 9. Ribitol..... 9... ND Ribose....0 0... 9..0 Sorbitol....9.9... 9.0 Stachyose. 9... 0..9.9.. Sucrose 9. 0. 9.9 9.9.. 0...0 Xylitol....0..0... Xylose......... NR - not recommended ND - no data available For optimal separations, allow at least minute between compounds.
SIL LC-NH Column This column, containing a silica-based, aminopropyl-modified phase packing, provides good separations of a variety of mono-, di-, and some trisaccharides found in foods. Separation is in order of increasing molecular weight, with monosaccharides eluting first (Figures B and C). Oligosaccharides are strongly retained and should not be analyzed on this column. Sample preparation is minimal. The sugars do not require derivatization and most samples require only dilution with deionized water, extraction of the sugars, and filtration (0.µm filter). Many juices and soft drinks can be analyzed undiluted, after degassing and filtration. Mobile phases typically contain acetonitrile and water in various proportions (: or 0:0 mixtures are recommended). Note that the disaccharides sucrose and lactose are best separated on the SIL LC-NH column. Figure B. Amino Column Test Mix Column: SIL LC-NH, cm x.mm, µm particles Cat. No.: Mobile Phase: acetonitrile:water, : Flow Rate:.0mL/min Temp.: ambient Inj.: 0µL, 0mg/mL each analyte in acetonitrile:water, : Figure C. 0 9-00 0 0. Fructose. Glucose. Sucrose. Maltose. Lactose Carbohydrate Standards on an Amino Column Column: SIL LC-NH, cm x.mm, µm particles Cat. No.: Mobile Phase: acetonitrile:water, : Flow Rate:.0mL/min Temp.: ambient Inj.: 0µL, 0mg/mL each analyte in water. Ribose. Arabinose. Galactose. Sucrose. Maltose. Isomaltose. Melezitose. Raffinose 0 9-00 Figure B shows an analysis of the test mix used to evaluate the performance of SIL LC-NH columns. The mix is available (Cat. No. ) for routine monitoring of the column in the laboratory. Figure C shows the variety of sugars that can be separated by using a SIL LC-NH column. Resolution between each pair of analytes is good. GEL Columns In contrast to their elution order on SIL LC-NH columns, carbohydrates elute in descending order of molecular size, monosaccharides last, from the resin-based GEL columns described below. The pores in the resins exclude polysaccharides and larger oligosaccharides, which elute first. Smaller di- and monosaccharides enter the pores, interact with the counterions, and are more strongly retained. Figures D and E show comparable separations on five columns. In addition to the differences in elution order and resolution, note the negative peak, a common characteristic of refractive index detection, in both figures. Also note that in Figure E the disaccharides sucrose and lactose are separated only on the SIL LC-NH column. Disaccharide separations are difficult to obtain with resin-based column packings. GEL Ca Column The GEL Ca column contains a polystyrene-divinylbenzene cross-linked resin in the calcium form. It separates oligo-, tri-, and disaccharides by class, using a mixed size exclusion/ion exchange mode, with the largest molecules eluting first. Its true separating power, however, is in its chromatography of monosaccharides and sugar alcohols a variety of monosaccharides can be separated, using only water as the mobile phase. The column can be operated at low temperatures, but separations are best at elevated temperatures. Figures F and G show a separation of carbohydrate standards and an analysis of high fructose corn syrup, respectively, on a GEL Ca column. Both figures were obtained using a temperature of 0 C and a mobile phase consisting solely of deionized water. GEL C- Column The unique polystyrene-divinylbenzene resin-based packing in this column contains two divalent cations, strontium and barium, rather than one. As with the other GEL columns, the separation mechanism is a combination of size exclusion and ion exchange modes. Disaccharides elute before monosaccharides, usually as a single peak. Sugar alcohols are strongly retained and elute with the monosaccharides. The column is compatible with many inorganic salts and can be used with dilute bases. Separations normally are carried out using a very weak basic solution, such as 0 - N sodium hydroxide, at 0 C. Analyses on a GEL C- column are temperature dependent. Figure I shows that resolution is improved as the temperature is increased. GEL Ag Columns These columns contain a silver-form styrene-divinylbenzene resin. Larger analytes elute before smaller analytes. The Ag column was developed specifically for separating the oligosaccharides in beer and corn syrup, and provides rapid separations of oligomers up to DP. It will separate ethanol and glycerol. The Ag column is better suited for larger oligosaccharides, resolving up to and including DP. Hydrolysis products and oligosaccharides can be analyzed on either an Ag or an Ag column. Figure J shows the carbohydrate profile of a domestic beer; Figures K and L show separations of corn syrup on GEL Ag and Ag columns, respectively.
Figure D. Carbohydrate Standards Figure E. Carbohydrates in Fruit Yogurt Flow Rate (SIL LC-NH column):.ml/min Inj.: 0µL,.mg/mL each analyte in water Other conditions listed in Table GEL Ca (Cat. No. 90-U). Maltotriose. Maltose. Glucose. Xylose. Arabinose. Ribitol. Arabitol. Xylitol Flow Rate (SIL LC-NH column):.ml/min Inj.: 0µL of 0g yogurt/00ml DI water, filtered (0.0µm filter) Other conditions listed in Table GEL Ca (Cat. No. 90-U). Oligosaccharides. Sucrose. Lactose. Glucose. Fructose GEL C- (Cat. No. 90-U) 9-00 GEL C- (Cat. No. 90-U) 9-00 GEL Ag (Cat. No. 9),, 9-00 GEL Ag (Cat. No. 9) 9-00 GEL C-0H (Cat. No. 90-U),, 9-00 GEL C-0H (Cat. No. 90-U), 9-00 9-009 9-00,,, SIL LC-NH (Cat. No. ) SIL LC-NH (Cat. No. ) 0 0 0 9-000 0 0 0 9-00
Figure F. Carbohydrate Standards on a GEL Ca Column Figure I. Temperature Affects Carbohydrate Analyses on GEL Columns Column: GEL Ca, 0cm x.mm Cat. No.: 90-U Flow Rate: 0.mL/min Temp.: 0 C Inj.: 0µL, mg/ml each analyte in water. Melezitose. Maltose. Glucose. Mannose. Fructose. Ribitol Column: GEL C-, 0cm x.mm Cat. No.: 90-U Mobile Phase: 0 - N sodium hydroxide Flow Rate: 0.mL/min Inj.: 0µL. Raffinose. Maltose. Glucose. Galactose. Mannose. Fructose C, 0 0 0 9-00 0 C Figure G. High Fructose Corn Syrup Column: GEL Ca, 0cm x.mm Cat. No.: 90-U Flow Rate: 0.mL/min Temp.: 0 C Inj.: 0µL of g corn syrup/ 0mL DI water, filtered (0.0µm filter) 0 C. DP. DP. Glucose. Fructose 0 0 0-09 Figure J. Domestic Beer Figure H. 0 0 0 Organic Acid Standards Column: GEL C-0H, 0cm x.mm Cat. No.: 90-U Mobile Phase: 0.% phosphoric acid Flow Rate: 0.mL/min Temp.: 0 C Det.: UV, 0nm Inj.: 0µL. Oxalic. Citric. Tartaric. Malic. Succinic. Formic. Acetic. Fumaric 9-00 Column: GEL Ag, 0cm x.mm Cat. No.: 9-U Flow Rate: 0.mL/min Temp.: 90 C Inj.: 0µL beer. DP. DP. DP. Maltotriose. Maltose. Glucose. Fructose. Glycerol 9. Ethanol 9 0 0 0 9-0 0 G00099
Figure K. Dark Corn Syrup Column: GEL Ag, 0cm x.mm Cat. No.: 9-U Flow Rate: 0.mL/min Temp.: 90 C Inj.: 0µL of g syrup/0ml DI water, filtered (0.0µm filter). DP. DP. DP. DP. DP. Glucose GEL C-0H Column This column contains a polystyrene resin in the hydrogen form. It is ideal for separating mixtures of organic acids (Figure H), fermentation products (e.g., alcohols), and carbohydrates. Such mixtures commonly occur in fruits, vegetables, and beverages. Larger and acidic analytes elute before smaller analytes. The column is stable between ph and, but results are best at low ph. A simple mobile phase containing 0.% H PO is suitable for a wide variety of analyses. GEL K Column A GEL K column separates raffinose, sucrose, glucose, fructose, and betaine, a trimethylammonium zwitterionic compound found in beet and cane sugars and widely distributed in other plants. 0 0 0 Figure L. Corn Syrup Column: GEL Ag, 0mm x.mm Cat. No.: 9 Flow Rate: 0.mL/min Temp.: 90 C Inj.: 0µL 9-009 GEL Pb Column The lead-form resin in GEL Pb columns provides the highest resolution and best selectivity for monosaccharides. GEL Pb columns provide excellent separation of xylose, galactose, and mannose, which are not completely resolved on calcium-form resin columns. GEL H Columns GEL H columns have the same particle composition, retention mechanism, performance, sensitivity, and applications as GEL C-0H columns. However, particle improvements have made it possible to pack the GEL H packing material efficiently into conventional.mm ID columns, to improve detection and reduce solvent consumption relative to.mm ID columns. -. DP-0. DP9. DP. DP. DP. DP 9. DP 0. Maltotriose. Maltose. Glucose - 9 0 G00099 Making a Decision To choose the best column for your particular carbohydrates analysis, we suggest you find the compounds of interest in Table and note their retention times on each column. Using this information, select the column that will separate the compounds of interest with at least minute between any pair. Guard Columns Although filtration removes particulate matter from a sample, food, beverages, and other samples often contain soluble components that can be strongly retained by the analytical columns described here. We strongly recommend using a guard column to protect the analytical column from these potentially damaging sample components. We offer cm x.mm Supelguard guard columns that are compatible with our GEL resin-based analytical columns and a cm x.mm Supelguard guard column for use with the SIL LC-NH silica-based column. Guard columns are listed under Ordering Information. Note that GEL Ca columns and GEL C- columns use the same guard column (Supelguard Ca). Suggested Reading Knight, P. Biotechnology, : (99). Parriot, D. A Practical Guide to HPLC Detection (Chapter ) Academic Press (99). Lehniger, A. Biochemistry (Chapter 0) Worth Publishers (9). Nollet, L. Food Analysis by HPLC (Chapter ) Marcel Dekker (99).
Ordering Information: GEL and SIL Carbohydrate Columns and Guard Columns Length ID Supelguard Column (cm) (mm) Cat. No. Guard Column Cat. No. GEL K 0. 9 K 9 GEL Pb 0. 9 Pb 9 GEL Ca 0. 90-U Ca 90-U GEL C-0H 0. 90-U H 99 GEL H 0. 90-U H 99 GEL H. 9 H 99 GEL C- 0. 90-U Ca 90-U GEL Ag 0. 9-U Ag 9-U GEL Ag 0. 9 Ag 9 SIL LC-NH. LC-NH (kit) 9 LC-NH (pk. ) 9 Carbohydrate/Organic Acid/Sugar Alcohol Reference Standards Prepared, tested, and packaged using rigorous manufacturing procedures. Description CAS No. Qty. Cat. No. Monosaccharides D-(-)Arabinose 9-- 00mg -U D-(-)Fructose -- 00mg D-(+)Galactose 9-- 00mg D-(+)Glucose (mixed anomers) 0-99- 00mg 9 D-(+)Mannose (mixed anomers) -- 00mg 0 D-Psicose (mixed anomers) -- 00mg D-(-)Ribose 0-9- 00mg D-(+)Xylose -- 00mg Disaccharides α-lactose 99-- 00mg -U Maltose -- 00mg Sucrose -0-00mg 9 Oligosaccharides Maltoheptaose (DP) 0-- 00mg Maltohexaose (DP) 0-- 00mg Maltopentaose (DP) 0-- 00mg Maltotetraose (DP) --9 00mg Stachyose (DP) 009-- 00mg 9 Maltotriose (DP) 09--0 00mg D-(+)Melezitose (DP) 000-- 00mg -U D-(+)Raffinose (DP) 9-0-0 00mg Isomaltotriose (DP) -0-00mg Organic Acids Acetic acid -9-00mg 9 Adipic acid -0-9 00mg 99 L-Ascorbic acid 0-- 00mg 90-U Benzoic acid --0 00mg 9 Butyric acid 0-9- 00mg 9 Citric acid -9-9 00mg 9 Formic acid -- 00mg 9-U Fumaric acid 0-- 00mg 9 Isobutyric acid 9-- 00mg 9 DL-Isocitric acid -- 00mg 9 L-(+)Lactic acid 9-- 00mg 9 Maleic acid 0-- 00mg 99 D-Malic acid -- 00mg 90-U Malonic acid -- 00mg 9 Oxalic acid -- 00mg 9-U Phytic acid 0-9-0 00mg 9-U Propionic acid 9-09- 00mg 9-U (-)Quinic acid -9-00mg 9-U Shikimic acid -9-0 00mg 9-U Succinic acid 0-- 00mg 9-U D-Tartaric acid -- 00mg 9-U Description CAS No. Qty. Cat. No.Sugar Alcohols D-(+)Arabitol -- 00mg 99-U Dulcitol (Galactitol) 0-- 00mg 90-U iso-erythritol 9-- 00mg 9 Glycerol -- 00mg 9 Maltitol -- 00mg 9-U D-Mannitol 9-- 00mg 9-U Ribitol (Adonitol) -- 00mg 9-U D-Sorbitol 0-0- 00mg 9-U Xylitol -99-0 00mg 9 Kits Description Cat. No. Monosaccharides Kit: each monosaccharide standard listed Disaccharides Kit: each disaccharide standard listed -U Oligosaccharides Kit: each oligosaccharide standard listed Organic Acids Kit: each organic acid standard listed Sugar Alcohols Kit: each sugar alcohol standard listed
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