90
1
General Principles
with their own blood type, as they make antibodies against either the A- or B-types, and
AB-type donors, precipitating the blood [148,149,150].
There are additional blood group variations. A common variation is an isomerization in which
α-L-fucopyranose is moved from β-D-Galp to β-D-GlcNAcp and linked (1→4) to give the
Lewis-a blood type. A second and related variation is the addition of another α-L-fucopyranose
residue to β-D-GlcNAcp linked (1→4) to give two α-L-Fucp residues on the first chain, giving
Lewis-b blood type. These kinds of variations can occur for each of the ABO blood types,
giving O-type-Lewis-a, O-type-Lewis-b, A-type-Lewis-a, and so forth: O-Le a , O-Le b , A-Le a ,
A-Le b , B-Le a , B-Le b , AB-Le a , AB-Le b [150].
15 Separation and Purification of Carbohydrates
The source and the specific physical and chemical properties of carbohydrates determine the
methods that are used for their separation and purification. Mono-, di-, tri- and sometimes
higher-saccharides, for example maltodextrins, isomaltodextrins, and raffinose-sucrose dextrin series, are usually quite soluble in water. Carbohydrates, thus, are often obtained by the
extraction of natural materials with hot water. As many impurities as possible are removed in
an extraction mixture, such as salts, proteins, and lipids. Salts can be removed by precipitation and/or the use of ion exchangers. Lipids are removed with organic solvents, such as a 2:1
mixture of chloroform and methanol, and proteins are precipitated with acids and heat. High
amounts of alkali and acid, however, should be avoided. Frequently, some of the last impurities in the aqueous extract, especially colored yellow to brown materials, can be removed by
adding activated charcoal and filtering it out to give a clear solution before the extract is concentrated. The concentrated carbohydrate extract is obtained at an elevated temperature (50–
60 °C) and an organic solvent such as methanol or ethanol is slowly added to the point where
the clear solution just becomes cloudy. The solution is then cooled to ≈20 °C to give crystallization of the carbohydrate and then 4 °C to obtain additional crystals. Monosaccharides and
disaccharides will often crystallize, while higher oligosaccharides are frequently obtained as
amorphous precipitates that can be removed by centrifugation or filtration and dehydrated.
Many different chromatographic methods of separation (on charcoal, BioGel, silica gel,
hydroxyapatite, paper) can be used on a preparative scale to give pure materials that can be
studied and used even though they are not crystalline. Two typical examples are given for
the isolation, purification, and crystallization of a monosaccharide, α-D-xylopyranose, and
a disaccharide, lactose, from natural sources.
15.1 Isolation and Purification of α-D-Xylopyranose from Corn Cobs
Coarsely ground corn cobs or crude xylan can be used as starting materials. The xylan in either
source is hydrolyzed with 7% (v/v) sulfuric acid by refluxing for 2.5 h. The mixture is filtered
through cloth on a Büchner funnel with as much liquid as possible obtained by suction. The
residue is washed with an equal volume of water by suspension as thin slurry and then filtered.
A few drops of 1-octanol are added to the combined filtrates that are neutralized with barium
carbonate. The solids (primarily barium sulfate) in the mixture are filtered and the residue
washed by suspension in water and filtered. If corn cobs are used as the starting material,
1
General Principles
with their own blood type, as they make antibodies against either the A- or B-types, and
AB-type donors, precipitating the blood [148,149,150].
There are additional blood group variations. A common variation is an isomerization in which
α-L-fucopyranose is moved from β-D-Galp to β-D-GlcNAcp and linked (1→4) to give the
Lewis-a blood type. A second and related variation is the addition of another α-L-fucopyranose
residue to β-D-GlcNAcp linked (1→4) to give two α-L-Fucp residues on the first chain, giving
Lewis-b blood type. These kinds of variations can occur for each of the ABO blood types,
giving O-type-Lewis-a, O-type-Lewis-b, A-type-Lewis-a, and so forth: O-Le a , O-Le b , A-Le a ,
A-Le b , B-Le a , B-Le b , AB-Le a , AB-Le b [150].
15 Separation and Purification of Carbohydrates
The source and the specific physical and chemical properties of carbohydrates determine the
methods that are used for their separation and purification. Mono-, di-, tri- and sometimes
higher-saccharides, for example maltodextrins, isomaltodextrins, and raffinose-sucrose dextrin series, are usually quite soluble in water. Carbohydrates, thus, are often obtained by the
extraction of natural materials with hot water. As many impurities as possible are removed in
an extraction mixture, such as salts, proteins, and lipids. Salts can be removed by precipitation and/or the use of ion exchangers. Lipids are removed with organic solvents, such as a 2:1
mixture of chloroform and methanol, and proteins are precipitated with acids and heat. High
amounts of alkali and acid, however, should be avoided. Frequently, some of the last impurities in the aqueous extract, especially colored yellow to brown materials, can be removed by
adding activated charcoal and filtering it out to give a clear solution before the extract is concentrated. The concentrated carbohydrate extract is obtained at an elevated temperature (50–
60 °C) and an organic solvent such as methanol or ethanol is slowly added to the point where
the clear solution just becomes cloudy. The solution is then cooled to ≈20 °C to give crystallization of the carbohydrate and then 4 °C to obtain additional crystals. Monosaccharides and
disaccharides will often crystallize, while higher oligosaccharides are frequently obtained as
amorphous precipitates that can be removed by centrifugation or filtration and dehydrated.
Many different chromatographic methods of separation (on charcoal, BioGel, silica gel,
hydroxyapatite, paper) can be used on a preparative scale to give pure materials that can be
studied and used even though they are not crystalline. Two typical examples are given for
the isolation, purification, and crystallization of a monosaccharide, α-D-xylopyranose, and
a disaccharide, lactose, from natural sources.
15.1 Isolation and Purification of α-D-Xylopyranose from Corn Cobs
Coarsely ground corn cobs or crude xylan can be used as starting materials. The xylan in either
source is hydrolyzed with 7% (v/v) sulfuric acid by refluxing for 2.5 h. The mixture is filtered
through cloth on a Büchner funnel with as much liquid as possible obtained by suction. The
residue is washed with an equal volume of water by suspension as thin slurry and then filtered.
A few drops of 1-octanol are added to the combined filtrates that are neutralized with barium
carbonate. The solids (primarily barium sulfate) in the mixture are filtered and the residue
washed by suspension in water and filtered. If corn cobs are used as the starting material,