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1 L. Tucker AOAC
Sugar & Carbohydrate Analysis
A Practical Perspective
2 L. Tucker AOAC
Liquid Chromatography System
Eluent Pump Injector Column DetectorAuto-Sampler
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Sample Preparation
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Sample Matrix
GrindExtractParticles – FiltrationMatrix elimination – extraction, dialysis, centrifuge, etc…
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Filtration
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Dialysis
Sample in (csmpl)
Sample out
Ion
Ion
Ion
Ion
Ion
Ion
Ion
Ion
Ion
XIon
Ion
Acceptor outcacc < csmpl
Acceptor in
7 L. Tucker AOAC
Dialysis
Sample in (csmpl)
Sample out
Ion
Ion
Ion
Ion
Ion
Ion
Ion
Ion
Ion
XIon
Ion
Acceptorcacc. ~ csmpl
(after 10 min.)
X
X
8 L. Tucker AOAC
Dialysis
•particles
•high molecular weights
•high organic load
•oils
•fats
•proteins
•dyestuffs
9 L. Tucker AOAC
C
Dialysis, dilution 1:100
26155Sucrose8
18820Fructose7
n.d.1
1107Inositol2
n.d.3
n.d.4
n.d.5
19214Glucose6
100 mmol/L NaOH
Metrosep Carb1 – 250 ppm
Carbohydrates: orange juiceCarbohydrates: orange juice
10 L. Tucker AOAC
Dilution 1:1000, dialysis
53Glucose10
35625Lactose11
47Mannose9
52Glycerin1-3
35Inositol4
28Glycerin5
21Xylitol6
6Arabitol7
51Arabinose8
100 mmol/L NaOH
Metrosep Carb1 – 250 ppm
Carbohydrates: milkCarbohydrates: milk
11 L. Tucker AOAC
C
Dilution 1:10, dialysis
1.60Fructose13838.96Lactose14
2.09Xylose12
1.50Inositol30.26Arabitol60.40Sorbitol70.77Mannitol81.29Arabinose101.37Glucose11
100 mmol/L NaOH
Metrosep Carb1 – 250ppm
Anions, pulsed Anions, pulsed amperometric amperometric detectiondetectionCarbohydrates: milk chocolateCarbohydrates: milk chocolate
12 L. Tucker AOAC
Auto-Sampler
Sugars and Carbohydrates fuel biological processesReduce temperature in autosampler to slow / prevent change in samplePeltier cooling or external cooling
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Eluent
Specific for column and separationIsocratic for simple sugarsGradient for complex mixturesFollow manufacturer recommendations
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 min
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
nA
Cell
1
2
3 4
man
nito
l
sucr
ose
arab
inos
ega
lact
ose
gluc
ose
xylo
se man
nose
fruc
tose
Column:METROSEP Carb 1 4X250mm
Eluent: 2.5 mmol/L sodium hydroxide + 0.65 mmol/L sodium acetate in ultrapure water
Post column reagent: 300 mmol/L sodium hydroxide in ultra pure water
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Columns
Choice of vendorsSelectivity/separation neededIsocratic and gradient separations
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Column Selection
Sugar Class Separation Mode
Ligand Exchange +GFCMonosacharides
Disacharides Ligand Exchange +Reversed Phase
Normal Phase
Sacharides & Sugar Alcohols Ligand Exchange +GFC
Ligand Exchange + Normal Phase
Oligosacharides Ligand Exchange +GFC
Amino Sugars Ligand Exchange +GFCNormal Phase
Acidic Sugars Ion ExclusionIon Exchange
Sacharides & Organic Acids Ion Exclusion+GFC
Sugar Alcohols & Organic Acids Ion Exclusion+GFC
16 L. Tucker AOAC
Isocratic Separation
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 min
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
nA
Cell
gluc
ose
fruc
tose
sucr
ose
Sample: Vodka
Column: Metrosep Carb 1 4X150mm
Mobile Phase: 100mM NaOH
Loop: 20uL
Detector: Metrohm PAD
17 L. Tucker AOAC
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 min
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800nA
Cell
rham
nose
man
nose gl
ucos
ega
lact
ose
fruc
tose
lact
ose
sucr
ose
mal
tose
Separation
Sample: Sugar Standards
Column: Metrosep Carb 1
4x250mm
Mobile Phase: 200mM NaOH
Loop: 20uL
Detector: Metrohm PAD
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Separation
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Separation
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Detectors
Refractive IndexPulsed AmperometricMass Spec
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Detectors
Selectivity Sensitivity Cost Separation Refractive Index Little 0.1 to 0.05% Moderate Isocratic
Pulsed Amperometric Some ng/g, ppb Moderate Isocratic, GradientMass Spec Most ng/g, ppb Expensive Isocratic, Gradient
22 L. Tucker AOAC
Pulsed Amperometric Detector
Measuring principleMeasuring principleA defined decent potential is applied to a three electrode system consisting of reference-, working- and auxiliary electrodes. If any oxidizable substance pass these electrodes an oxidation process takes place. A current corresponding to the concentration of therespective substance is measured.
Simple connection to any IC- or HPLC-system
Large variety of electrode materials (glassy carbon, carbon paste, silver, gold, etc.)
Highly sensitive
Very selective
23 L. Tucker AOAC
Mass Spec
Electrospray ionization– Operating temp 250 to 300°C– Sugars oxidize– Form adduct with HCl
HCl Adduct formation– HCl is volatile– Source of Cl¯ for adduction– Post column addition
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Mass Spec
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Mass Spec
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Summary
You have choices in hardware and separationChoose the right tools for your applicationWhat do you need?
