Determination of Carbohydrates in Various Matrices by Capillary High-Performance Anion-Exchange...

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Terri Christison

Senior Marketing Specialist - IC/SP

Thermo Fisher Scientific

248th ACS 2014 National Meeting & Exposition

San Francisco, CA

August 14, 2014

Determination of Carbohydrates in Various Matrices by Capillary HPAE-PAD

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Outline

• What is High Performance Anion Exchange-Pulsed

Amperometric Detection (HPAE-PAD)?

• Capillary HPAE-PAD

• Available instrumentation

• Eluent generation

• Applications

• Beverage industry samples

• Milk products

• Nuclear medicine use

• Clinical research

• Conclusion

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High Performance Anion-Exchange with Pulsed Amperometric Detection

• HPAE-PAD is a sensitive, selective direct method without

costly and labor-intensive derivatization

• Carbohydrates are ionized by strong base

• Both neutral and charged carbohydrates are separated by

anion-exchange chromatography

• Charge, size, and composition

• Branching and linkage isomerism

• Detected by amperometry

• Au working electrode, Ag/AgCl reference electrode, counter electrode

• Four-potential waveform

• Pulsed twice in one second

• Specific for carbohydrates

• Stable, reproducible response

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Glucose Ionization at pH 12 - 13

Professor Patricia Shapley, University of Illinois, 2012

http://butane.chem.uiuc.edu/pshapley/GenChem2/B6/2.html

Sugar pKa

Fructose 12.03

Mannose 12.08

Xylose 12.15

Glucose 12.28

Galactose 12.39

Dulcitol 13.43

Sorbitol 13.60

-Methyl glucoside 13.71

Dionex (now part of Thermo Scientific) Technical Note 20

Analysis of Carbohydrates by High-Performance Anion-

Exchange Chromatography with Pulsed Amperometric

Detection (HPAE-PAD). Sunnyvale, CA, 2004.

http://butane.chem.uiuc.edu/pshapley/GenChem2/B6/2.html

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-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

0.00 0.10 0.20 0.30 0.40 0.50

Po

ten

tial (V

vs.

Ag

/Ag

Cl)

Time (Seconds)

Standard Quadruple Waveform for Carbohydrates

1. Detection potential (E1) 2. Duration time of E1 (t1)

3. Integration period (Integrate)

4. Reductive cleaning potential (E2) 5. Duration time of E2 (t2)

6. Oxidative cleaning potential (E3) 7. Duration time of E3 (t3)

8. Pre-detection (oxide reduction) potential (E4) 9. Duration time of E4 (t4)

E1 (t1)

E2 (t2)

E4 (t4)

E3 (t3)

Integrate

Time

(s)

Potential

(V)

Integration

0.00 -0.10

0.20 -0.10 Start

0.40 -0.10 End

0.41 -2.0

0.42 -2.0

0.43 0.60

0.44 -0.10

0.50 -0.10

Dionex (now part of Thermo Scientific) Technical Note 21 Optimal Settings for Pulsed Amperometric

Detection of Carbohydrates Using the Dionex ED40 Electochemical Detector. Sunnyvale, CA, 1998.

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Advantages of Disposable Electrodes

• Manufactured using electronics industry techniques

• Consistent grain structures with reproducible surface

• Excellent electrode-to-electrode reproducibility

• Easy installation – fast equilibration

• No conditioning required

• No hand polishing

• No messy polishing compounds

• No activation waveforms

• Cost effective

• Au on PET – Typically two week lifetime

• Au on PTFE – Typically three week lifetime

Cheng, J.; Jandik, P.; Avdalovic, N. Anal. Chem., 2003, 75, 572−579.

Cheng, J.; Jandik, P.; Avdalovic, N. Anal. Chim. Acta, 2005, 536, 267−274.

Working

Electrode

Au Contact Pad

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Advantages of Capillary IC

• “IC on demand”• System is available for your samples

• Higher laboratory productivity

• Less labor needed for calibration

• Eluent generation: Just Add Water

• Isocratic and gradient elution with eluent generation

• Lower cost of ownership

• Less eluent consumed and waste generated

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Dionex ICS-5000+ Reagent-Free HPIC System

The Thermo Scientific™ Dionex™

ICS-5000+ is a universal high-pressure

ion chromatography system

• Continuous operation up to 5000 psi

• High-pressure capable with both analytical

and capillary systems

• Increased productivity with fast run times

• Improved separations and higher

resolution with 4 µm particle columns

HPIC - high resolution, fast analyses

Thermo Scientific™

Dionex™ IC Cube™

Module

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Dedicated Capillary HPIC System

HPIC - high resolution, fast analyses

Thermo Scientific™ Dionex™

ICS-4000 Capillary HPIC™ System

• New level of resolution and speed

• Delivering best in class sensitivity

• Simplifies workflows

• Increases analytical efficiency

and productivity

• Small footprint

• Electrochemical, conductivity,

or charge detection

Thermo Scientific Dionex ED

Detector and Cell

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Data & System

Management

High-Pressure Non-Metallic Capillary Pump

EluentGenerator(OH– or H+)

Waste

Sample Inject(Autosampler)

H20

Separation Column

Degas

SuppressorBypass

CRDBypass

Regen flow in back

ED

CR-TC

Capillary HPAE-PAD System Flow Diagram

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Electrolytic Generation of KOH Eluents

Pt Cathode

(2H2O + 2e– 2OH– + H2)

Cation-ExchangeConnector

HydroxideGeneration ChamberPump

H2O

Pt Anode

(H2O 2H+ + ½O2 + 2e–)K+

ElectrolyteReservoir

K+

KOH + H2KOH

H2

DegasUnit

[KOH]Current

Flow Rate

Vent

CR-ATCAnion Trap

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Importance of HPAE-PAD to Beverage Industries

Beverages are complex sample matrices containing sugars or

sugar substitutes, organic acids, inorganic anions, inorganic

cations, preservatives, and flavor additives.

The determinations of these analytes are important for the

beverage industry to:

• Verify content needed for labeling and formulations

• Assure product quality

• Identify adulteration to illegally increase profits

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Which Sugar is Used by the Beverage Industry?

• Fruit juices

• Native sugars are primarily a mixture of glucose, sucrose, fructose

Glucose

Sucrose

Fructose

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Cane and Beet Sugars

• 100% sucrose

• Inverted sugar – partially converted to fructose and glucose

• Minimize crystallization and to increase sweetness

Glucose

Sucrose

Fructose

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Corn Sugars

• Corn syrup is 100% glucose which is not very sweet

• Convert 55% or 42% glucose to fructose HFC55 or HFC42

• Increase sweetness

Glucose Fructose

55%, 42%

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Six Carbohydrate Standard

Column: Thermo Scientific™ Dionex™

CarboPac™ PA20 with

guard, 0.4 mm

Eluent Source: Thermo Scientific Dionex EGC-KOH

(Capillary) cartridge

Eluent: 10 mM KOH

Flow Rate: 0.008 mL/min

Inj. Volume: 0.4 µL

Column Temp.:30 C

Detection: PAD, Au disposable, 4-Potential

Carbohydrate waveform

Gasket: 0.001” PTFE

Ref. Electrode:Ag/AgCl

Samples: 10 µM mixed standard

Peaks: 1. Fucose

2. Galactosamine

3. Glucosamine

4. Galactose

5. Glucose

6. Mannose

4

3

2

1

6

155 100Minutes

00

60

nC

5

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Native Sugars in Apple Cider

Column: Dionex CarboPac PA20 with

guard, 0.4 mm

Eluent Source: Dionex EGC-KOH (Capillary)

cartridge

Eluent: 10 mM KOH



Column Temp.:30 C





Sample Prep.: 5000-fold dilution

Peaks: 1. Void Volume -- µM

2. Galactose 0.1

3. Glucose 60

4. Mannose 2

5. Sucrose 20

6. Fructose 110

4

3

6

2

1

155 100

0

70

nC

5

Minutes

Thermo Scientific Technical Note 135 Determinations of Monosaccharides and Disaccharides in Beverages by

Capillary HPAE-PAD. Sunnyvale, CA 2013.

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HFC in a Carbonated Beverage


guard, 0.4 mm


cartridge

Eluent: 10 mM KOH



Column Temp.:30 C





Sample Prep.: 5000-fold dilution, degas

Peaks: 1. Glucose 98 µM

2. Fructose 95

2

1

155 100Minutes

0

90

nC



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HFC in a Carbonated Beverage -- 0.015” Gasket


guard, 0.4 mm


cartridge

Eluent: 10 mM KOH



Column Temp.:30 C

Detection: PAD, Au on PTFE disposable,

Four-Potential Carbohydrate

waveform



Sample Prep.: 500-fold dilution, degas

Peaks: mg/L % Ratio

1. Void Volume -- --

2. Glucose 102 58

3. Fructose 150 42

2

1

155 100Minutes

0

140

nC



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Cane Sugar in Coconut Water Flavored Beverage

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2

1

5


guard, 0.4 mm


cartridge

Eluent: 10 mM KOH



Column Temp.:30 C





Sample Prep.: 5000-fold dilution

Peaks: 1. Void Volume -- µM

2. Galactose 5

3. Glucose 23

4. Sucrose 7

5. Fructose 11

155 100Minutes

0

70

nC



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Lactose and Lactulose in Raw Unpasteurized Milk

Column: Dionex CarboPac SA10-4µm and

guard, 4 mm

Eluent Source: Dionex EGC 500 KOH cartridge

Eluent: 4 mM KOH


Inj. Volume: 10 µL

Column Temp.:35 C

Detection: PAD, Au on PTFE disposable,

Four-potential Carbohydrate waveform

Gasket: 0.002” thick PTFE

Ref. Electrode:pH-Ag/AgCl

Sample Prep.: Carrez digestion, centrifuge, filter,

Thermo Scientific™ Dionex™

OnGuard™ IIA cartridge

Sample: A: 100-fold diluted raw, unpasteurized

milk

B: Sample A + 0.5 mg/L lactulose

C: 0.5 mg/L carbohydrate standard

Peaks: A B

1. Sucrose -- -- mg/L

2. Galactose -- --

3. Glucose -- --

4. Lactose 3.75 3.77

5. Lactulose -- 0.48

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5

Minutes

1

A

B

C

82 640

30

50

nC

4

Thermo Scientific Technical Note 146 Fast Determinations of Lactose and Lactulose in Milk Products

Using HPAE-PAD. Sunnyvale, CA 2013.

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Clinical Research Applications

• Positron emission topography

• Abnormal glucose uptake

• Cancer, tumors, treatment course, response to treatment

• Schizophrenia, dementia, epilepsy

• Brain injuries

• Diabetes

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Column SuppressorED Detector

External

water

Waste

Injection

IC - Electrochemistry - Radiometry

High-Pressure Non-Metallic Pump

Radiometric

Detector

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Separation of Fluorodeoxyglucose (FDG) From Fluorodeoxymannose (FDM)

1

Minutes

205 15

25

32

nC

0

2

10

Cheng, J. Pharmaceutical Applications, Cap IC Library, Thermo Scientific, 2013


guard, 0.4 mm


cartridge

Eluent: 50 mM KOH



Column Temp.:30 C

Detection: PAD, Au disposable, Four-Potential




Peaks: 1. FDG 0.0025 mg/mL

2. FDM 0.0025

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Clinical Research Applications

Diagnosis of intestinal problems, leakage across membranes

• Carbohydrates as a probe

• Diagnose diseases and pharmaceutical therapies to small

intestine mucosa

• Celiac disease, rheumatoid arthritis, HIV, food allergies, drug

use, stress, NSAID

• Measuring D-mannitol/lactulose, L-rhamnose/lactulose

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Carbohydrates of Interest for Urine Analysis


guard, 0.4 mm


cartridge

Gradient: 10 mM KOH (-7 to 1 min),

10–30 mM KOH (1–9 min),

30–35 mM from (9–16 min)



Column Temp.:30 C





Peaks: 1. Mannitol

2. 3-O-Methylglucose

3. Rhamnose

4. Galactose

5. Glucose

6. Xylose

7. Sucrose

8. Ribose

9. Lactose

10. Lactulose

nC

4

3

2

5

1

6

0

Minutes

10

164 128

50

7

8 910

Thermo Scientific Application Note 1006 Determination of Carbohydrates in Urine by HPAE-PADD. Sunnyvale, CA

2012.

Thermo Scientific Technical Note 146 Fast Determinations of Lactose and Lactulose in Milk Products Using HPAE-

PAD. Sunnyvale, CA 2013.

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Carbohydrates in A) Water and B) Synthetic Urine


guard, 0.4 mm


cartridge

Gradient: 10 mM KOH (-7 to 1 min),

10–30 mM KOH (1–9 min),

30–35 mM from (9–16 min)



Column Temp.:30 C





Sample Prep.: 600-fold dilution in water

Samples: 1.67 µg/mL after dilution

A: in water, B: in synthetic urine

Peaks: 1. Void Volume

2. Mannitol

3. 3-O-Methylglucose

4. Rhamnose

5. Xylose

6. Lactulose

0

Minutes

5

40

nC4

3

2

5

1

6

164 128

A

B

Thermo Scientific Application Note 1006 Determination of Carbohydrates in Urine by HPAE-PADD. Sunnyvale, CA

2012.

Thermo Scientific Technical Note 146 Fast Determinations of Lactose and Lactulose in Milk Products Using HPAE-

PAD. Sunnyvale, CA 2013.

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Conclusion

• HPAE-PAD is a direct, selective and sensitive method

without costly and labor intensive derivitization

• Demonstrated carbohydrate determinations using capillary

format HPAE-PAD

• An RFIC™ system with electrolytically generated eluent

requires only adding water, thereby eliminating eluent

preparation, increasing reliability, stability, and ease-of-use

• Shown select interesting applications

• Beverage samples

• Milk products

• Urine samples

• Nuclear medicine samples

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Thank you!

OT71282-EN-0814S.

Determination of Carbohydrates in Various Matrices by Capillary High-Performance Anion-Exchange...

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