sugar & carbohydrate analysis - aafco.org · l. tucker aoac 22 pulsed amperometric detector...
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1 L. Tucker AOAC
Sugar & Carbohydrate Analysis
A Practical Perspective
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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
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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
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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
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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
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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
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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
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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
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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?