Saccharides, Sugar Alcohols, Aminosugars and Amino Acids

When Asahipak NH2P-50 columns are used, the retention time of sugar alcohols as well as saccharides becomes longer as the acetonitrile concentration in the eluent becomes higher.

Sample :
Arabitol
Fructose
Galactose
Glucose
Glycerol
Lactose
Maltose
Mannitol
Mannose
Raffinose
Rhamnose
Ribitol
Sorbitol
Sucrose
Xylitol
Xylose

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : CH3CN/H2O
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 30deg-C

SUGAR SC1211 which allows easy adjustment of separation by adding some quantities of organic solvent is suitable for the analysis of sugar alcohols. Monosaccharides and disaccharides are eluted first in a group.

Sample :
Maltotriol
Glycerol
meso-Erythritol
Adonitol
Maltitol
myo-Inositol
Arabitol
Mannitol
Xylitol
Dulcitol(Galactito)
Sorbitol

Columns      : Shodex SUGAR SC1211 (6.0mmID*250mm)
Eluent       : H2O/CH3CN
Detector     : Shodex RI
Column temp. : 40deg-C

SUGAR SP0810 allows the analysis of saccharides and sugar alcohols in a single run with simple aqueous eluent though the column can be used only for limited combinations of saccharides and sugar alcohols because no organic solvent can be added to the eluent.

Sample :
1. Pullulan P-10, 2. Stachyose
3. Raffinose, 4. Sucrose
5. Lactose, 6. Glucose
7. Lactulose, 8. Galactose
9. Arabinose, 10. Fructose
11. Glycerol, 12. Mannitol
13. Xylitol, 14. Sorbitol

Columns      : Shodex SUGAR SP-G (6.0mmID*50mm) + SP0810 (8.0mmID*300mm)
Eluent       : H2O
Flow rate    : 0.5mL/min
Detector     : Shodex RI
Column temp. : 80deg-C

Three saccharides can be separated using one column of SUGAR KS-801 with water as the eluent.

Sample : 1% each, 8micro-L
(A)
1. Mannitol
2. Sorbitol
3. Xylitol
(B)
1. Mannitol
2. Pentaerythritol
3. myo-Inositol

Column       : Shodex SUGAR KS-801 (8.0mmID*300mm)
Eluent       : H2O
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 80deg-C

Sugar alcohols were analyzed using SUGAR SC1211 (a column for saccharides analysis). When the concentration of acetonitrile in the eluent is increased, the retention of sample becomes stronger and separation is improved though the neccessary time for the analysis becomes longer.

Sample :
1. Maltitol
2. Glycerol
3. Mannitol
4. Xylitol
5. Sorbitol

Column       : Shodex SUGAR SC1211 (6.0mmID*250mm)
Eluent       : (A); H2O, (B); H2O/CH3CN=80/20, (C); H2O/CH3CN=60/40 
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 70deg-C

SUGAR SZ5532 is suitable for analyzing saccharides and sugar alcohols in a single run. Separation can be adjusted by changing the quantity of acetonitrile in the eluent.

Sample :
Sugar alcohols
a. Glycerol
b. Xylitol
c. Mannitol
d. Sorbitol
e. Maltitol
Saccharides
1. Xylose
2. Arabinose
3. Fructose
4. Glucose, Mannose
5. Galactose
6. Sucrose
7. Maltose
8. Lactose

Column       : Shodex SUGAR SZ5532 (6.0mmID*150mm)
Eluent       : H2O/CH3CN=20/80
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 60deg-C

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In the Guidelines on Nutrition Labelling in Japan, ligand-exchange columns of 7.8-8.0mmID x 300mm Length are adopted for the analysis of sugar alcohols. SUGAR SP0810 is a polymer-based (sulfonated polystyrene gel) ligand-exchange column having counter ions of Pb2+. This chromatogram shows the analysis result of typical saccharide and sugar alcohol standards using SUGAR SP0810.

Sample : 1% each, 5micro-L
1. Sucrose, 2. Glucose
3. Xylose, 4. Galactose
5. Fructose, 6. meso-Erythritol
7. Lactitol, 8. Mannitol
9. Xylitol, 10. Sorbitol

Column       : Shodex SUGAR SP0810 (8.0mmID*300mm)
Eluent       : H2O
Flow rate    : 0.6mL/min
Detector     : Shodex RI
Column temp. : 85deg-C

In the Guidelines on Nutrition Labelling in Japan, ligand-exchange columns of 7.8-8.0mmID x 300mm Length are adopted for the analysis of sugar alcohols. SUGAR SC1011 is a polymer-based (sulfonated polystyrene gel) ligand-exchange column having counter ions of Ca2+. This chromatogram shows the analysis result of typical saccharide and sugar alcohol standards using SUGAR SC1011.

Sample : 1% each, 5micro-L
1. Sucrose
2. Glucose
3. Lactitol
4. Fructose
5. meso-Erythritol
6. Mannitol
7. Sorbitol

Column       : Shodex SUGAR SC1011 (8.0mmID*300mm)
Eluent       : H2O
Flow rate    : 0.6mL/min
Detector     : Shodex RI
Column temp. : 85deg-C

Ten components of saccharides and sugar alcohols were well separated by SUGAR SZ5532. The counter ion of SUGAR SZ5532 is Zn2+.

Sample : 0.5% each, 20micro-L
1. Rhamnose
2. Xylose
3. Arabinose
4. Fructose
5. Glucose
6. Galactose
7. Arabitol
8. Xylitol
9. Mannitol
10. Sorbitol

Column       : Shodex SUGAR SZ5532 (6.0mmID*150mm)
Eluent       : H2O/CH3CN=20/80
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 65deg-C

Asahipak NH2P-50 4E can separate saccharides and sugar alcohols such as glucose and sorbitol or lactose and lactitol which cannot be separated by silica based amino columns.

Sample :
1. Sorbitol
2. Glucose

Sample :
1. Lactitol
2. Lactose

Columns      : Shodex Asahipak NH2P-50 4E (4.6mmID*25-mm) , Silica based amino column (other manufacturer)
Eluent       : CH3CN/H2O=75/25 
Flow rate    : 0.6mL/min (NH2P-50), 1.0mL/min (Silica based amino column)
Detector     : Shodex RI
Column temp. : 25deg-C

Saccharides and sugar alcohols were analyzed using a polymer-based amino column, Asahipak NH2P-50 and LC/MS.

Sample :
1. Sorbitol (m/z=218)
2. Glucose (m/z=216)

Sample :
1. Xylitol (m/z=188)
2. Fructose (m/z=216)
3. Glucose (m/z=216)
4. Sucrose (m/z=378)

(HPLC)
Instrument   : Agilent 1100
Column       : Shodex Asahipak NH2P-50 2D (2.0mmID*150mm)
Eluent       : H2O/CH3CN=25/75
Flow rate    : 0.2mL/min
Column temp. : 40deg-C
(Post column)
Reagent      : CH3CN/CHCl3=50/50
Flow rate    : 0.2mL/min
(MS)
Instrument      : Agilent 1100MSD
Mass range      : 100-500(m/z)
Ionization      : APCl
Mode            : SIM(M + Cl)
Polarity        : Negative
Fragmentor      : 20V
Nebulizer       : N2(40psi)
Drying gas      : N2(10L/min, 350deg-C)
Corona current  : 30micro-A
Vaporizer temp. : 400deg-C

Amino sugars were analyzed using OHpak SB-802.5HQ (a column for GFC separation).

Sample :
1. D-Glucosamine
2. N-Acetyl-D-glucosamine

Columns      : Shodex OHpak SB-G (6.0mmID*50mm) + SB-802.5 HQ (8.0mmID*300mm)
Eluent       : 0.2M CH3COOH aq.
Flow rate    : 0.8mL/min
Detector     : Shodex RI
Column temp. : 50deg-C

Generally, ligand exchange mode columns are used with aqueous eluent, but the addition of the same cation as the counter ion enables the columns to separate samples under ion exchange mode additionally to the ligand exchange mode. Pb(NO3)2 aqueous solution is used as an eluent for SUGAR SP0810 because the its counter ion is Pb2+. This means that ligand exchange occurs between the counter ions of the packing material and the hydroxyl groups of saccharides and ion exchange occurs between the sulfo groups and the amino groups of the saccharides. Meanwhile, as Pb(NO3)2 contains lead, the waste liquid should be disposed of in accordance with legal procedures.

Sample :
1. Glucose
2. Galactose
3. Mannose
4. Chloride anion
5. Glucosamine
6. Galactosamine

Column       : Shodex SUGAR SP0810 (8.0mmID*300mm)
Eluent       : 20mM Pb(NO3)2(pH3.8)
Flow rate    : 0.7mL/min
Detector     : Shodex RI
Column temp. : 60deg-C

Asahipak NH2P-50 4E with evaporative light scattering detector (ELSD) is suited to the separation of saccharides, amino sugars and N-acetyl amino sugars.

Sample :
1. N-Acetyl-D-galactosamine
2. N-Acetyl-D-glucosamine
3. Galactosamine
4. Glucosamine
5. Galactose
6. Glucose

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : CH3CN/H2O=80/20
Flow rate    : 1.0mL/min
Detector     : ELSD

Generally, ligand exchange mode columns are used with aqueous eluent, but the addition of the same cation as the counter ion enables the columns to separate samples under ion exchange mode additionally to the ligand exchange mode. CaSO4 aqueous solution is used for SUGAR SC1011 as its counter ion is Ca2+. This means that ligand exchange occurs between the counter ions of the packing material and the hydroxyl groups of saccharides and ion exchange occurs between the sulfonate groups and the amino groups of the saccharides. For sensitive measurement, attention should be paid to the positions of minus peak due to the cations in the eluent.

Sample :
1. Maltose
2. Glucose
3. Fructose
4. Glutamic acid
5. Aspartic acid
6. Threonine
7. Serine
8. Glutamine
9. Alanine

Column       : Shodex SUGAR SC1011 (8.0mmID*300mm)
Eluent       : 0.01M CaSO4(pH6.0)
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 80deg-C

The chromatogram shows an example of separating taurine and saccharides by SUGAR SC1211 with ligand exchange and normal phase modes.

Sample :
1. Sucrose
2. Glucose
3. Fructose
4. Taurine
5. myo-Inositol

Columns      : Shodex SUGAR SC1211 (6.0mmID*250mm)
Eluent       : H2O/CH3CN=60/40
Flow rate    : 0.6mL/min
Detector     : Shodex RI
Column temp. : 70deg-C

According to USP method (United States Pharmacopeia 23), mannitol should be analyzed using a column which can separate mannitol and and sorbitol with R (resolution) of equal to or greater than 2.0. USPpak MN-431 is a column specially designed for mannitol analysis, whcih satisfies this criterion.

Sample :
1. Mannitol
2. Sorbitol

Column       : Shodex USPpak MN-431 (4.0mmID*250mm)
Eluent       : H2O
Flow rate    : 0.5mL/min
Detector     : Shodex RI
Column temp. : 60deg-C

The measurement of mannitol by USP method should be done at 30 to 85 deg-C within 2 deg-C.

Palatinit is a sugar alcohol which can be obtained by reducing palatinose, and it is used as a low-calorie and anti-decay food additive. It is composed of two isomers, 6-O-alpha-D-Glucopyranosyl-D-glucitol and 1-O-alpha-D-glucopyranosyl-D-mannitol. SUGAR SP0810, SUGAR SC1011 and SUGAR SZ5532 can separate the isomers.

Sample : Palatinit

(above); 5mg, 5micro-L/mL
        Column       : Shodex SUGAR SP0810 (8.0mmID*300mm)		
        Eluent       : H2O		
        Flow rate    : 1.0mL/min		
        Detector     : Shodex RI		
        Column temp. : 80deg-C
(center); 5mg, 5micro-L/mL
        Column       : Shodex SUGAR SC1011 (8.0mmID*300mm)
        Eluent       : H2O
        Flow rate    : 1.0mL/min
        Detector     : Shodex RI
        Column temp. : 80deg-C
(below); 2.5mg, 10micro-L/mL
        Column       : Shodex SUGAR SZ5532 (6.0mmID*150mm)
        Eluent       : CH3CN/H2O=75/25
        Flow rate    : 1.0mL/min
        Detector     : Shodex RI
        Column temp. : 60deg-C

Xylitol is officially permitted as a food additive by the Ministry of Welfare in Japan on April 17, 1997. The chromatogram is an analysis of xylitol in tablet candy. Asahipak NH2P-50 4E column is quite useful for the analysis of sugar alcohols including xylitol. NH2P-50 is a polymer-based amino column, and packed with the polyvinylalcohol gel bonded with polyamine. It is good for quantitative analysis and can be washed with alkaline solvents.

(Sample pretreatment)
2% aqueous solution of the sample was passed through a 0.45 micron filter,
and the same volume of acetonitrile was added.

Sample : Tablet candy, 20micro-L
1. Xylitol
2. Sorbitol

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : H2O/CH3CN=25/75
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 30deg-C

Soft drink containing erythritol was analyzed using Asahipak NH2P-50 4E (a column for saccharides analysis).

Sample : Soft drink, containing erythritol

20micro-L
injection		 Concentration
(mg/mL)		 Contents
in 100g (g)
1.15 2.3
Indicated Value by Producer
Component (in 100mL)
Calorie
0kcal
 Saccharides 1
0g
Proteins
0g
 Sucrose
0g
Lipids
0g
 Vitamin B6
1.0mg
Saccharides 2
2.9g
 Vitamin C
1.0mg
Sodium
7.0mg
 Vitamin E
0.3mg

Saccharides 1 : Mono- and di- saccharides
Saccharides 2 : Total saccharides

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : CH3CN/H2O=75/25
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 25deg-C

When analyzing saccharides in food, it is necessary to remove the substances besides saccharides before the analysis. This procedure is called as sample pre-treatment. Here, the analysis result of saccharides in food using Asahipak NH2P-50 4E is shown together with the sample pre-treatment procedure.

(Sample pre-treatment for apple juice)
1) Put 5g of apple juice in a 50mL a messflask.
2) Dilute with ultra pure water to make up a 30mL solution.
3) Neutralize with 10%(W/V) NaOH solution.
4) Apply ultrasonic vibration for 30 minutes using an ultrasonic vibrator.
5) Add ultra pure water to make up a 50mL solution.
6) Filtrate the solution with a paper filter (No.5B).
7) Take a part of filtrated solution and add the same volume of acetonitrile.
8) Pass through a 0.45microm membrane filter before injecting into HPLC.

Sample : Apple juice, 20micro-L
1. Fructose
2. Sorbitol
3. Glucose
4. Sucrose

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : CH3CN/H2O=75/25
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : Room temp.(25deg-C)

Saccharides and a sugar alcohol contained in apple juice were analyzed using SUGAR SC1011 (a column for saccharides analysis).

(Sample pretreatment)
1) Take 5g of apple juice in a beaker, add 30mL of pure water and neutralize with 10%(W/V) NaOH aq. cooled with ice.
2) Apply ultrasonic vivration for 30 minutes and make 50mL solution with pure water.
3) Filtrate with 0.45 micron membrane filter.

Sample : Apple juice 5micro-L
1. Sucrose
2. Glucose
3. Fructose
4. Sorbitol

Column       : Shodex SUGAR SC1011 (8.0mmID*300mm)
Eluent       : H2O
Flow rate    : 0.6mL/min
Detector     : Shodex RI
Column temp. : 85deg-C

Three kinds of Japanese sake (wine), sweet, medium and dry, were analyzed using SUGAR SC1211 (a column for saccharides analysis). It was confirmed that the sweet sake had the largest sugar content.

Sample : Sake
1. Maltotriose
2. Maltose
3. Glucose
4. Glycerol
5. Ethanol

Column       : Shodex SUGAR SC1211 (6.0mmID*250mm)
Eluent       : H2O 
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 80deg-C

According to EP method (Reference No. 0559, Version Date 04/2009), mannitol should be analyzed using a column which can separate mannitol and sorbitol with resolution of equal to or greater than 2.0. EP SC1011-7F is a column specially designed for mannitol analysis, whcih satisfies this criterion.

Sample : 5% each, 5micro-L
1. Mannitol
2. Sorbitol

Sample : 0.1% each, 20micro-L
Mannitol
Sorbitol
Maltitol
Isomalt (=Palatinit)

Column       : Shodex EP SC1011-7F (7.8mmID*300mm)
Eluent       : H2O
Flow rate    : 0.5mL/min
Detector     : Shodex RI
Column temp. : 85deg-C

According to EP method (Reference No. 1805, Version Date 01/2008), mannitol should be analyzed using a column which can separate myo-Inositol and mannitol (impurity A) with resolution of equal to or greater than 4.0. EP SC1011-7F is a column specially designed for myo-Inositol analysis, whcih satisfies this criterion.

Sample :
1. myo-Inositol
2. Mannitol
3. Glycerol

Column       : Shodex EP SC1011-7F (7.8mmID*300mm)
Eluent       : H2O
Flow rate    : 0.5mL/min
Detector     : Shodex RI
Column temp. : 85deg-C

Pinitol, a compound present in pine and legumes, is a methylester of chiro-inositol (an optical isomer of myo-inositol) effective in lowering blood glucose levels. For this reason, it is sold in combination with myo-inositol (a compound known for inhibiting fat storage) as a diet supplement for treating metabolic syndrome. Here, RSpak DC-613 columns for sugar analysis were used here to analyze pinitol and myo-inositol, chiro-inositol simultaneously with mono- and disaccharides.

Sample : 0.1％ each, 10micro-L
1. Pinitol
2. Fructose
3. Glucose
4. Sucrose
5. chiro-Inositol
6. myo-Inositol

Column       : Shodex RSpak DC-613 (6.0mmID*150mm)
Eluent       : H2O/CH3CN=25/75
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 70deg-C