Fast, Accurate and Direct Carbohydrate
Analysis Using HPAE-PAD
March 11, 2015
Michael G. Hvizd
Senior Manager, CoE Laboratories
1
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Agenda
• Outline
• What are Carbohydrates?
• High Performance Anion Exchange Chromatography (HPAE)
• Pulsed Amperometric Detection (PAD)
• Application Examples
• Total Solution from Thermo Fisher Scientific
2
Classification of Carbohydrates
• Monosaccharide
• Empirical formula (CH2O)n
• Each carbon except one contains a
hydroxyl group
• The remaining carbon atom has a
carbonyl oxygen
• If carbonyl is at the end of the chain,
monosaccharide is called an aldose
• If carbonyl is at any other position,
monosaccharide is called a ketose
• Based on number of carbons (3, 4, 5, 6),
a monosaccharide is a triose, tetrose,
pentose, or hexose.
3
H
O
CH2OH
C
C
O
HO
C
H
OH
H
C
OH
OH
H
C
OH
H
C
OH
HO
C
H
H
C
H
C
CH2OH
CH2OH
D-glucose
D-fructose
Aldoses
(e.g., glucose)
have an
aldehyde group
at one end.
Ketoses
(e.g., fructose)
have a keto
group, usually
at C2.
Classification of Carbohydrates
• Disaccharides
• Monosaccharides can be joined by means of a glycosidic bond
• Formed by the reaction of carbonyl carbon of one monosaccharide
with the hydroxyl group of the other monosaccharide
- H2O
glucose
4
glucose
maltose
Classification of Carbohydrates
• Oligo- and Polysaccharides
• Chains of monosaccharides joined by glycosidic bonds
• Since monosaccharides have multiple hydroxyl groups, oligo- and
polysaccharides can be highly branched
• Oligosaccharide
• Chain of 3–10 sugar molecules
• Polysaccharide
• Chain of 10+ sugar molecules
• Glycosidic bonds can be broken by:
• Acid digestion
• Enzyme digestion
5
CH2OH
CH2OH
O
O
OH
OH
HO
O
OH
OH
OH
n
Challenges with Carbohydrate Analysis
• Extremely Polar, Partly Ionic
• Many Similar and complex structures
• Non-Chromophoric
• Often present in complex matrices
• Often bonded to other molecules
(glycoproteins, glycolipids)
6
High-Performance Anion-Exchange
Chromatography with Pulsed
Amperometric Detection (HPAE-PAD)
77
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High Performance Anion Exchange Chromatography
• Separates neutral and charged saccharides
without derivatization
• Separates on the basis of:
• Charge, size, and composition
• Branching and linkage isomerism
8
Electrochemical Methods - PAD
• Electrochemical methods:
• Simple, direct detection method
• No sample derivitization
• High sensitivity
• Comparable to derivitization with fluorescence detection (fmol to low pmol).
• Minimal sample preparation
• Allows samples to be diluted 100 to 1000-fold, so simplifies sample prep
• Compatible with high pH anion exchange column technology
• Official methods (using Thermo Scientific™ Dionex™ CarboPac™ PA1
column)
• Detection is linear over at least 4 orders of magnitude
• PAD is a nondestructive method and can thus be used preparatively
or hyphenated to other detectors like MS
9
High Performance Anion Exchange Chromatography –Pulsed
Amperometry Detection (HPAE-PAD)
• Dissociation constants of
some common
carbohydrates
(in water at 25 °C)
10
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
• Carbohydrate are weak acids.
• At high pH, they are at least partially
ionized, and thus can be separated
by anion-exchange mechanisms
• HPAE uses NaOH/KOH as eluent,
sometimes with addition of Sodium
Acetate to increase ionic strength.
• Pulsed-amperometric detection is an
electrochemical detection method.
With new waveform it provides
direct, specific, highly-sensitive
detection for carbohydrates.
Bead Structure of Dionex CarboPac Columns
Latex Particle
( ≤ 0.1 µm)
Latex
CORE
5 - 10 µm
NR3+
SO3-
Sulfonated highly-crosslinked core
structure
Latex Microbeads with Anion Exchange
Functionalities
11
Dionex’s IC Columns for Carbohydrates Analysis
Column
FastDionex CarboPac
Time
(min)
Fast separation of N-acetyl- and N-glycolylneuraminic
Acids (Sialic acids)
<5
Dionex CarboPac
SA10 and Dionex
CarboPac
SA10-4μm columns
Fast and high capacity separation of mono and disaccharides in
biofuels, foods, and beverages.
<10
Dionex CarboPac
PA20 column
High-resolution separations of mono- and
disaccharides with optimized resolution of
glucosamine/galactose and glucose/mannose
peak pairs.
<20
Dionex CarboPac
PA200 column
High resolution separations of charged and
neutral oligosaccharides. Separation of neutral and sialylated Nlinked oligosaccharides from glycoproteins.
<50
Dionex CarboPac
PA1 column
For food nutrition labeling. Replaced by SA10 or PA20
Dionex CarboPac
MA1 column
Reduced mono- and disaccharide. alditol and galactosamine
PA20 column
Fast Sialic Acid
12
Applications
~
<50
Amperometry
• Measures current or charge resulting from
the oxidation or reduction of analyte on a
specific electrode surface
Amperometry
Electron Transfer
• Oxidation – electrons go from the analyte to
the electrode
e-
• Reduction – electrons go from the electrode
to the analyte
Analyte
eElectrode
Surface
13
Application Examples
14
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Rapid Monosaccharide Analysis with the Dionex
CarboPac PA10 Column
Column:
Eluent:
Flow Rate:
Detection:
2
100
1
3
4
nC
Peaks:
5
6
0
0
15
2
4
6
Minutes
8
10
Dionex CarboPac PA10 and guard
18 mM NaOH
1.5 mL/min
Pulsed amperometry,
gold electrode
1. Fucose
1 nmol
2. Galactosamine
1
3. Glucosamine
1
4. Galactose
1
5. Glucose
1
6. Mannose
1
Monosaccharide Analysis on Dionex CarboPac PA20
Column
18 mM
Column:
Dimension:
Eluent:
Flow rate:
Detection:
16 mM
Peaks:
20 mM
100
14 mM
nA
12 mM
0
10 mM
1
0
2
2
3
4
4
Minutes
16
5
6
6
8 mM
8
Dionex CarboPac PA20
150 mm x 3 mm ID
8-20 mmol/L NaOH
0.5 mL/min
Pulsed amperometry,
Au electrode
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
Separation of Reduced and Reducing Carbohydrates
Commonly Found in Foods and Beverages
0.2
Column:
Dionex CarboPac MA1
Eluent:
480 mM sodium
hydroxide
1
Flow Rate: 0.4 mL/min
Detector:
PAD (gold)
Peaks:
1. Inositol
2. Glycerol
3. Arabitol
4. Sorbitol
5. Dulcitol
6. Mannitol
7. Mannose
8. Glucose
9. Galactose
10. Fructose
11. Sucrose
2
µC
3
5
4
6
7
10
8
9
11
0
0
17
10
20
30
Minutes
40
50
18 mg/L
9
15
18
18
18
18
18
18
18
34
Maltodextrins—Used to Make Natural Soda
175
A: Maltrin M040
HPAE-PAD is the only way to develop
a quantitative analysis of the
polysaccharide chain link
nC
0
Column:
Dionex CarboPac PA10
Eluent:
Sodium hydroxide/
sodium acetate gradient
Flow Rate:
1 mL/min
Inj. Volume: 25 µL
175
B: Maltrin M700
Detection:
Pulsed amperometry,
Au electrode
nC
Maltrin is a trademark of Grain Processing Corp.
0
0
18
5
10
15 20
Minutes
25
30
35
Improved Chain-Length Resolution of Inulin Polymers
Columns:
180
PA200
Gradient:
Dionex CarboPac PA200 (3 × 250 mm)
CarboPac PA100 (4 × 250 mm)
120 to 320 mM NaOAc in
100 mM NaOH over 40 min
Flow Rate: Dionex CarboPac PA200: 0.5 mL/min
Dionex CarboPac PA100: 1.0 mL/min
Detection:
nC
0
PA100
–20
0 5 10 15 20 25 30 35 40 45 50 55 60
Minutes
19
Pulsed amperometry,
quadruple waveform,
gold electrode
Sialic Acid Analysis of Bovine Fetuin on Dionex
CarboPac PA20 Column
Column:
Gradient:
250
NANA
A
Flow rate:
Detection:
Dionex CarboPac PA20 (150 mm x 3 mm ID)
20-200 mmol/L NaOAc in 0.1 mol/L
NaOH over 10 min
0.5 mL/min
Integrated pulsed amperometry,
disposable Au electrode
nC
NGNA
0
A. 0.1 mol/L HCl fetuin hydrolysate
NGNA
B
NANA
B. NANA & NGNA standard
-150
20
0
2
4
6
8
10
12
Minutes
14
16
18
20
Fetuin N-Oligosaccharide Profiles: Dionex CarboPac
PA200 Column vs. Dionex CarboPac PA100 Colmun
Column:
160
Eluent:
3
4
Flow Rate:
Detection:
2
1
5
Peaks:
6
nC
3
2
1
20
0
10
20
1. Disialylated, triantennary
2. Disalylated, triantennary
3. Trisialyated, triantennary
4. Trisialyated, triantennary
5. Tetrasialylated, triantennary
6. Tetrasialylated, triantennary
4
2
5
30
Dionex CarboPac PA200 (3 x 250 mm)
Dionex CarboPac PA100 (4 x 250 mm)
Sodium acetate gradient in
100 mM sodium hydroxide
Dionex CarboPac PA200: 0.5 mL/min
Dionex CarboPac PA100: 1 mL/min
Pulsed amperometry,
Quadruple waveform,
Au electrode
Minutes
40
6
50
60
70
Fetuin N-Oligosaccharide Profiles: CarboPac PA200 vs. PA100
21
HPAE-PAD System Flow Diagram with RFIC
H20
High-Pressure
Non-Metallic Pump
Eluent (OH– )Generator
EGC
Data and System
Management
Recycle Mode
CR-TC
ED
Separation Column
Waste
Sample Inject
(Autosampler)
22
Electrochemical
Detector
Fast Separation of Food Sugars Using Dionex
CarboPac SA10 Column
100
Column:
Dionex CarboPac SA10-4µm
and guard, 4 mm
Eluent Source: Thermo Scientific Dionex EGC 500
KOH Cartridge
Eluent:
1 mM KOH
Flow Rate:
1.5 mL/min
Inj. Volume:
10 µL
Column Temp.: 40 °C
Detection:
PAD, Au on PTFE disposable,
Four-potential Carbohydrate waveform
Ref. Electrode: pH-Ag/AgCl
2
nC
1
3
Sample:
4
5
Peaks:
1.
2.
3.
4.
5.
6.
6
-10
0
2
4
6
Minutes
23
Food Sugars Standard, 5mg/L
8
10
Sucrose
Glucose
Fructose
Lactose
Cellobiose
Maltose
Separation of Coffee Sugars on the Dionex
CarboPac SA10 Column
2
80
3
4
1
5
Column:
Dionex CarboPac SA10 (4.0 × 250 mm)
Temp:
45 °C
Eluent:
1 mM KOH (EG/CR-TC)
Flow Rate:
1.5 mL/min
Inj. Volume:
10.0 µL
Det. Met.:
PAD (carbohydrate quadruple
waveform)
Electrode:
Au
Peaks:
1. Mannitol
6
nC
7
2. Arabinose
3. Galactose
20
4. Glucose
5. Xylose
0
24
1
2
3
4
5
Minutes
6
7
8
9
6. Mannose
7. Fructose
Carbohydrates in Instant Coffee
Column:
Eluent:
1000
Inj. Volume:
Detector:
2
1
Peaks:
nA
3
4
5 6
0
25
0
10
20
Minutes
30
7
40
Sample
Preparation:
Dionex CarboPac PA1
150 mM sodium hydroxide/
deionized water gradient
25 µL of 10 g/L solution
Pulsed amperometry,
Au electrode; postcolumn
addition of 0.3 M NaOH
1.
2.
3.
4.
5.
6.
7.
Mannitol
21 mg/L
Arabinose 140
Galactose 76
Glucose
44
Xylose
26
Mannose
51
Fructose
93
Phenolics removed with
Thermo Scientific™ Dionex™
OnGuard™ P cartridge
Six Carbohydrates in 10 min Using Capillary Dionex
CarboPac PA20 Column
60
Column:
Dionex CarboPac PA20, 0.4 mm
Eluent Source: Dionex EGC-KOH capillary with
capillary Thermo Scientific Dionex
CR-ATC Continuously
Regenerated Anion Trap Column
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
2
3
1
nC
4 5
6
Peaks:
0
0
26
5
10
Minutes
15
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
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
Flow Rate:
1.45 mL/min
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,
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
50
4
nC
2 3
5
1
C
B
A
Peaks:
30
0
2
4
Minutes
TN 146, Thermo Scientific, 2013
27
6
8
1. Sucrose
2. Galactose
3. Glucose
4. Lactose
5. Lactulose
A
---3.75
--
B
-mg/L
--3.77
0.48
Biofuel: Corn Stover Hydrolysate
180
Column:
Dionex CarboPac SA10,
Guard and Analytical , 2mm
Eluent Source: Dionex EGC 500 KOH cartridge
Eluent:
1 mM KOH
Flow Rate:
0.38 mL/min
Inj. Volume:
2.5 µL
Column Temp.: 45°C
Detection:
PAD, Au on PTFE disposable,
Four-potential Carbohydrate
waveform
Gasket:
15 mil thick PTFE
Ref. Electrode: pH-Ag/AgCl
4
nC
3
1
Sample:
2
5
Peaks:
0
0
2
4
Minutes
6
8
AN 282, Thermo Scientific, 2013
Dionex CarboPac SA10 Column Data sheet, Thermo Scientific, 2013
28
Corn Stover Hydrolysate
(150g/L, 1:200 Dilution)
1.
2.
3.
4.
5.
Arabinose
Galactose
Glucose
Xylose
Mannose
Native Sugars in Apple Cider
70
Column:
Dionex CarboPac PA20, 0.4 mm
Eluent Source: Dionex EGC-KOH capillary
cartridge with capillary Dionex
CR-ATC column
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
Sample Prep.: 5000-fold dilution
3
6
nC
1
5
Peaks:
4
2
0
0
5
10
Minutes
TN 135, Thermo Scientific, 2013
29
15
1. Void Volume
2. Galactose
3. Glucose
4. Mannose
5. Sucrose
6. Fructose
-- µM
0.1
60
2
20
110
Carbohydrates of Interest for Urine Analysis
50
8
9
6
45
10
7
nC
1
3
2
Column:
Dionex CarboPac PA20, 0.4 mm
Eluent Source: Dionex EGC-KOH capillary
cartridge with capillary Dionex
CR-ATC column
Gradient:
10 mM KOH (-7 to 1 min),
10–30 mM KOH (1–9 min),
30–35 mM from (9–16 min)
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
Peaks:
10
0
4
8
Minutes
TN146, Thermo Scientific, 2013
30
12
16
1. Mannitol
2. 3-O-Methylglucose
3. Rhamnose
4. Galactose
5. Glucose
6. Xylose
7. Sucrose
8. Ribose
9. Lactose
10. Lactulose
Sialic Acids (Neu5Ac and Neu5Gc) in Glycoproteins
Column:
Dionex CarboPac PA20
Fast Sialic Acid, 3 × 30 mm
Eluent:
70-300 mM acetate in
100 mM NaOH from 0−2.5 min,
300 mM acetate in 100 mM NaOH
from 2.5−2.9 min, 300−70 mM
acetate from 2.9−3.0 min; 1.5 min of
equilibration at 70 mM acetate
in 100 mM NaOH
Flow Rate:
0.5 mL/min
Inj. Volume:
4.514 µL (full loop)
Column Temp.: 30 °C
Detection:
PAD, Au on PTFE, 2 mil gasket
Ref. Electrode: pH-Ag/AgCl
N-acetylneuraminic acid (Neu5Ac)
N-glycolylneuraminic acid (Neu5Gc).
31
nC
1
A
B
Samples:
2
C
19
0
1
2
Minutes
AU 181, Thermo Scientific, 2011
31
3
A) h. α1-acid glycoprotein,
1:100 dilution (23 ng protein)
B) fetuin hydrolyzate,
1:100 dilution (18 ng protein)
C) s. α1-acid glycoprotein,
1:100 dilution (7.9 ng protein)
Peaks: (pmol)
1. Neu5Ac
2. Neu5Gc
A
13
—
B
C
5.6 6.1
0.20 1.2
Glycoprotein Monosaccharide Analysis
Column:
Dionex CarboPac PA20,
and Thermo Scientific™ Dionex™
AminoTrap™ column
Eluent Source: Dionex EGC-KOH cartridge with
Dionex CR-ATC column
Eluent:
10 mM KOH from 0−13 min
100 mM KOH from 13−16 min
10 mM KOH from 16−21 min
Flow Rate:
0.5 mL/min
Inj. Volume:
10 µL
Column Temp.: 30 °C
Detection:
PAD, Au disposable, 4-Potential
Carbohydrate waveform
Ref. Electrode: Ag/AgCl
70
2
Column
Regen.
3
nC
1
4
5
6
35
0
2
4
6
8
10
12
Minutes
TN 40, Thermo Scientific, 2012
32
14
16
18
20 21
Samples:
10 µM Mixed Standard
Peaks:
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
Monosaccharides in Digested Glycoprotein Samples
2
120
Column:
nC
Dionex CarboPac PA20,
and Dionex AminoTrap
Eluent Source: Dionex EGC (III)-KOH cargtridge
with Dionex CR-ATC column
Eluent:
10 mM KOH from 0−13 min
100 mM KOH from 13−16 min
10 mM KOH from 16−21 min
Flow Rate:
0.5 mL/min
Inj. Volume:
10 µL
Column Temp.: 30 °C
Detection:
PAD, Au disposable, 4-Potential
Carbohydrate waveform
Ref. Electrode: Ag/AgCl
3
1
4
B
30
130
2
nC
1
3
4
A
30
0
5
10
Minutes
TN 40, Thermo Scientific, 2012
33
15
21
Samples:
A: Fetuin HCl hydrolysate
B: Fetuin TFA hydrolysate
Peaks:
1. Galactosamine
2. Glucosamine
3. Galactose
4. Mannose
High-Pressure Ion Chromatography (HPIC) System
HPIC: A metal-free system which operates up to 5000 psi
Thermo Scientific™ Dionex™
ICS-4000 Capillary system
Thermo Scientific™ Dionex™ ICS-5000+ both
analytical and capillary systems
HPIC - High Resolution, Fast Analyses
34
Conclusions
• HPAE-PAD is a fast carbohydrate analysis method
• Directly quantify nonderivatized carbohydrates with high sensitivity and
selectivity
• HPAE-PAD is the one system for monosaccharides, sialic
and other sugar acids, sugar phosphates, sugar alcohols,
sulfate sugars, aminoglycoside antibiotics, oligosaccharides
(charged and neutral), and small polysaccharides
• With innovations, such as HPIC and CarboPac columns,
carbohydrates are analyzed in as little as 5 minutes
35