HPAE-PAD analysis of Carbohydrates
Michael G. HvizdManager, Laboratory OperationsCenters of ExcellenceDionex Corporation
Laboratory
Presentation Outline
Principles of High Performance Anion Exchange
Column Technologies
Principles of Pulsed Amperometric Detection
Applications using HPAE-PAD
22060
Carbohydrates -Features of HPAE-PAD
Simple, Direct Method– No derivatization is necessary
High Sensitivity– comparable to derivatization with fluorescence detection (fmol to low pmol). – Allows samples to be diluted 100 to 1000-fold, so simplifies sample prep
Minimal Sample Preparation is necessaryUnique column technologyOfficial Methods (using CarboPac PA1)
Resolving Power of HPAE-PAD
Comparison of chromatographic techniques used for the analysis of a hydrolyzed glucose syrup illustrates the remarkable resolving power of the HPAE-PAD technique. Elution order is reversed with the CarboPac® PA1 column as compared with conventional metal loaded cation-exchange columns.
Rapid Monosaccharide Analysis Rapid Monosaccharide Analysis with the CarboPacwith the CarboPac™™ PA10PA10
Column: CarboPac PA10 and guardEluent: 18 mM NaOHFlow Rate: 1.5 mL/minDetection: Pulsed amperometry,
gold electrodePeaks: 1. Fucose 1 nmol
2. Galactosamine 13. Glucosamine 14. Galactose 15. Glucose 16. Mannose 1
22
00 88 101000
100100
nCnC
11
3344 55
66
MinutesMinutes664422
1179011790--0101
Column: CarboPac™ PA10, PA10 guard,Borate Trap®
Eluent: 52 mM sodium hydroxideFlow Rate: 1.5 mL/minInj. Volume: 5 µLDetection: Pulsed amperometry, gold electrode
Detection of Food Sugars and Sugar AlcoholsPeaks: 1. Glycerol 4.0 mg/mL
2. Xylitol 2.03. Sorbitol 1.54. Mannitol 3.05. Glucose 5.06. Fructose 8.07. Sucrose 2.08. Lactose 8.0
15150-01
50
nC
4
5
6 78
12
3
0
Minutes5 10 150
–2
–1
0
1
0 0.2 0.4
Poten
tial (V
)
Time (s)
Column: CarboPac™ MA1
Eluent: 480 mM sodium hydroxide
Flow Rate: 0.4 mL/min
Detector: PED (gold)Peaks: 1. Inositol 18 mg/L
2. Glycerol 93. Arabitol 154. Sorbitol 185. Dulcitol 186. Mannitol 187. Mannose 188. Glucose 189. Galactose 18
10. Fructose 1811. Sucrose 34
Minutes0 10 20
0
µC
0.2
30 40 50
1
34
5
68 9
10
11
7
2
Separation of Reduced and Reducing CarbohydratesCommonly Found in Foods and Beverages
8460-03
Column: CarboPac™ PA-100 and guardEluent: NaOH/sodium acetate gradientFlow Rate: 1.0 mL/minInj. Volume: 10 µLDetection: Pulsed amperometry, Au electrode
Peaks: 1. Glucose2. Maltose3. Maltotriose4. Maltotetraose5. Maltopentaose6. Maltohexaose7. Maltoheptaose8. Maltooctaose9. Maltononaose
10. Maltodecaose
Separation of Maltose Oligomers
10927-02Minutes
2
3
4
0 5 10 15 20 25
0
70
nC
1
5
67
89
10
Column: CarboPac™ PA10Eluent: Sodium hydroxide/
sodium acetate gradientFlow Rate: 1 mL/minInj. Volume: 25 µLDetection: Pulsed amperometry,
Au electrode
Maltrin is a trademark of Grain Processing Corp.
0
175
nC
A: Maltrin M040
Minutes0 5 10 15 20 25 30
B: Maltrin M700
0
175
nC
35
Maltodextrins—Maltrin® M040 and M700
13026-01
Retention Time (min)
From: Koizumi, K., Fukuda, M., Hizukuri, S.. J. Chromatography. 1991, 585, 233–238
Distribution of Chain Length of Amylopectins by HPAE-PAD
Rice12
6
20
30 40 50
12
6
20
30 40 50 60
Corn
0 10 20 30 40
60504030
20
12
6
EdibleCanna
0 10 20 30 40
12
6 20
30 40 50 60
Sweet Potato
9661-01
Improved Chain-Length Resolution of Inulin Polymers
Columns: CarboPac™ PA200 (3 x 250 mm) CarboPac PA100 (4 x 250 mm)
Gradient: 120- to 320-mM NaOAc in 100 mM NaOH over 40 min
Flow Rate: PA200: 0.5 mL/minPA100: 1.0 mL/min
Detection: Pulsed amperometry,quadruple waveform, gold electrode
180
0 5 10 15 20 25 30 35 40 45 50 55 60–20
nC
Minutes
PA200
PA1000
20608-01
Column: CarboPac™ PA-100Eluent: 0 to 250 mM NaOAc over
110 min in 100 mM NaOHFlow Rate: 1 mL/minDetector: PAD (Gold)Peaks: 1. Fucosylated Man3GlcNAc2
2. Man3GlcNAc23. Asialo, agalacto bi, core fuc4. Asialo, agalacto bi5. Asialo bi, core fuc6. Asialo bi7. Man9GlcNAc28, 9. Disialylated tri (reduced)10, 11. Trisialylated tri (reduced)12, 13. Tetrasialylated tri (reduced)
00 1010 2020 3030 4040 5050 6060 7070
200 200
nAnA
MinutesMinutes
1122
33
4455
667
8899
10101111
12121313
NANA NANA —— Gal Gal —— GlcNAc GlcNAc —— ManMan……α2−6α2−6
Peak 10Peak 10
NANA NANA —— Gal Gal —— GlcNAc GlcNAc —— ManMan……α2−3α2−3
Peak 11Peak 11
Separation of N-Linked Oligosaccharides
Official Food Methods Using IC/PAD
AOAC Method 2000.17CarboPac PA1Low-Level Glucose and Fructose in Raw and Refined Sugar
AOAC Method 2001.02CarboPac PA1Transgalacto-oligosaccharides
AOAC Method 2000.11CarboPac PA1Polydextrose
AOAC Method 997.08CarboPac PA1Fructans in Food and Food Products
AOAC Method 995.13ISO 11292 ApprovedCarboPac PA1Carbohydrates in
Soluble Coffee
AOAC Method 996.04ICUMSA Method Approval 1994
CarboPac PA1Sugars in Molasses
Official MethodCarboPac™ ColumnAnalysis
20611-01
High Performance Anion Exchange Chromatography with High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEPulsed Amperometric Detection (HPAE--PAD) PAD)
1032810328
Separates neutral and charged saccharideswithout derivatizationSeparates on the basis of:– Charge, size and composition– Branching, linkage isomerism
Dissociation Constants of Some Common Carbohydrates
13.71 α-Methyl glucoside13.60Sorbitol13.43Dulcitol12.39Galactose12.28Glucose12.15Xylose12.08Mannose12.03FructosepKaSugar
(in Water at 25 °C)
8709-03
86748674--BB
Polymeric Nonporous (“Polymeric Nonporous (“PellicularPellicular”) Packing”) Packing
Highly Highly CrosslinkedCrosslinked CoreCore
Latex Latex MicrobeadsMicrobeadswith Anion Exchange with Anion Exchange
FunctionalitiesFunctionalities
Pulsed Amperomerty Detection
Advantages of PAD for Carbohydrate Detection– No derivatization is necessary– Low detection limits: low picomole to high femtomole– Detection is linear over at least 4 orders of magnitude– PAD is a non-destructive method and can thus be used preparatively– Highly specific– Using optimized pulse settings, PAD is highly specific for carbohydrates– Versatile: isocratic or gradient elution, high pH or neutral pH (with post column base
addition)– Specific for electroactive species– Both amino acids and carbohydrates can be detected in a single run
Disadvantages of PAD for Carbohydrate Detection ***– Each sugar gives a different signal, thus calibration curves must be run for quantitative
results
– *** True for every quantitative analysis
13450
Principal of Pulsed Amperometric Detection (PAD)Quadruple Potential Waveform for Carbohydrates
–2
0
0.8
0.0 0.1 0.2 0.3 0.4 0.5Time (seconds)
Poten
tial (V
vs. A
g/AgC
l)
Electrode Cleaning
Au Oxide Reduction,Electrode Activation
Au Oxide Formation Time Pot. Integ.0.00 +0.10.20 +0.1 Begin0.40 +0.1 End0.41 -2.00.42 -2.00.43 +0.60.44 -0.10.50 -0.1
Integrate
Pulsed Amperometric Detection (PAD)Waveform and Integration Period
Carbohydrate Waveform
– 3
– 2
– 1
0
1
0 0.2 0.4 0.6 0.8 1.0Time (s)
Poten
tial (
V )
Amino Acid Waveform
0 0.2 0.4 0.6 0.8 1.0Time (s)
Poten
tial (
V )
– 3
– 2
– 1
0
1
Integration Period
22136
Column: CarboPac™ PA1 and guardEluent: 150 mM NaOHFlow Rate: 1.0 mL/minInj. Volume: 50 µLDetection: Pulsed amperometry
Peaks: 1. Glucose 4.39%2. Fructose 6.673. Lactose (int. std.) –4. Sucrose 30.8
Sugars in Sugar Cane Molasses
2 4 6 8Minutes
2
1
3
4
µA
3
8
010930-03
Carbohydrates in Instant CoffeeColumn: CarboPac™ PA1 Eluent: 150 mM sodium hydroxide/
deionized water gradientInj. Volume: 25 µL of 10 g/L solutionDetector: Pulsed amperometry,
Au electrode; postcolumn addition of 0.3 M NaOH
Peaks: 1. Mannitol 21 mg/L 2. Arabinose 1403. Galactose 764. Glucose 445. Xylose 266. Mannose 517. Fructose 93
Sample Preparation: Phenolics removed with
OnGuard® P cartridge0 10 20 30 40
12
34
5 6 7
1000
0
nA
Minutes
6007-03
Sucralose in Diet Drink and Artificial Sweetener Product
Column: CarboPac™ PA20 with guard Eluents: 40 mM sodium hydroxide/
75 mM sodium acetateTemperature: 30 °CFlow Rate: 0.5 mL/min, 10-g/L solutionInj. Volume: 25 µLDetection: Pulsed amperometric
detection, carbohydrate waveform, disposable gold working electrode
0 1 2 3 4 5 6 7 8 930
65
Sucralose
Red Raspberry Diet SodaDiluted 50-Fold
0Minutes
2 4 6 8 10 12
Sucralose
20
nC
16
Splenda® Sweetener100 µg/mL
Splenda is a registered trademark of McNeil-PP
20607-01
Column: CarboPac PA-100Eluent: Sodium hydroxide/
sodium acetate gradientFlow Rate: 1 mL/minConc.: 2 mg/mLInj. Volume: 25 µLDetection: Pulsed amperometry,
Au electrode
5
0
µA
A. Pure Orange Juice
5
0
µA
0 10 20 30Minutes
B. Orange Juice Adulterated with Beet Medium Invert Sugar
Oligosaccharide Profiles of Pure and Adulterated Orange Juice
11399-01
Apple Juice
Column: CarboPac™ MA1Eluent: 500 mM sodium hydroxideFlow Rate: 0.4 mL/minDetection: Pulsed amperometryPeaks: 1. Sorbitol 14 µg
2. Glucose 1303. Fructose 364. Sucrose 0.2
0.2
µC
0
0 30 60Minutes
1
2
3
4
2010 50408588-03
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