sugar & carbohydrate analysis - aafco.org · l. tucker aoac 22 pulsed amperometric detector...

1 L. Tucker AOAC

Sugar & Carbohydrate Analysis

A Practical Perspective

2 L. Tucker AOAC

Liquid Chromatography System

Eluent Pump Injector Column DetectorAuto-Sampler

3 L. Tucker AOAC

Sample Preparation

4 L. Tucker AOAC

Sample Matrix

GrindExtractParticles – FiltrationMatrix elimination – extraction, dialysis, centrifuge, etc…

5 L. Tucker AOAC

Filtration

6 L. Tucker AOAC

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

13 L. Tucker AOAC

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

14 L. Tucker AOAC

Columns

Choice of vendorsSelectivity/separation neededIsocratic and gradient separations

15 L. Tucker AOAC

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

18 L. Tucker AOAC

Separation

19 L. Tucker AOAC

Separation

20 L. Tucker AOAC

Detectors

Refractive IndexPulsed AmperometricMass Spec

21 L. Tucker AOAC

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

24 L. Tucker AOAC

Mass Spec

25 L. Tucker AOAC

Mass Spec

26 L. Tucker AOAC

Summary

You have choices in hardware and separationChoose the right tools for your applicationWhat do you need?

27 L. Tucker AOAC

Thank You

Larry TuckerBrinkmann Instruments, Inc

ltucker@brinkmann.com281 330 9532