Biofuels and Food Applications
Joachim Weiss, Ph.D.
Thermo Fisher Scientific GmbH, Dreieich, Germany
Proprietary & Confidential 2 Dionex Confidential
BIOFUELS ANALYSIS
Proprietary & Confidential 3 Dionex Confidential Dionex Confidential Dionex Confidential
Biomass to Biofuels Conversion Pathways
Solid Biomass
wood, straw
Resources Conversion Product Market
Wet Biomass
manure
organic waste
Sugar &
starch plants
sugar cane
Oil crops
rapeseed
sunflower
Steam
Liquid (BTL)
Hydrocarbons
Bio Oil
Bio Gas
Syngas
Bio-ethanol
Biodiesel
Digestion
Microbial
Fermentation
Biochemical
Distillation
Esterification
Fischer
Tropsch
Synthesis
Combustion
Gasification
Pyrolysis
Thermochemical
Fuel
Heat
Power HTU
Hydrolysis
Extraction
Proprietary & Confidential 4 Dionex Confidential Dionex Confidential Dionex Confidential
What is Biodiesel?
• Alternative fuel made from any fat or vegetable oil
• Used in diesel engine with no modifications
• Petroleum blends
• 20% blend is B20
• Pure form is B100
• Renewable fuel made from plant or animal oils
• Produced via transesterification reaction
Proprietary & Confidential 5 Dionex Confidential
ASTM Specifications for Biofuels
Parameter Bio-ethanol Biodiesel
ASTM D 4806-06c1
ASTM D 5798-062
DIN EN 14214 ASTM D 6751
Chloride (1 ppm) ASTM D 7328
ASTM D 4806 - -
Sulfate (4 ppm) ASTM D 7318
ASTM D 7319 - -
Group I Alkali Metals Na, K (5 ppm)
- EN 14108 UOP391
Group II Alkaline-Earth Metals
Ca, Mg (5 ppm) - EN 14108 TBD
Glycerol (2 ppm) - EN 14105/14106 D6584
Proprietary & Confidential 6 Dionex Confidential
Plant lb. oil/acre Gallons of
biodiesel/acre
Algae 6,757 700
Coconut 2,070 285
Jatropha 1,460 201
Rapeseed 915 126
Peanut 815 112
Sunflower 720 99
Soybean 450 62
Oil Seed Crop Production for Biofuels
Proprietary & Confidential 7 Dionex Confidential
ASTM Determination of Sulfate and Chloride in
Bio-ethanol
0 2.5 5 7.5 10 12.5 15
10
80
µS
Minutes
1
2
Column: IonPac AS22 + guard, 4 mm
Eluent: 4.5 mmol/L Na2CO3 +
1.4 mmol/L NaHCO3
Temperature: 30 °C
Flow rate: 1.2 mL/min
Inj. volume: 200 µL
Concentrator: TAC-ULP1
Detection: Suppressed conductivity,
AutoSuppression, recycle mode
Sample: Ethanol with 5% gasoline
spiked with aqueous standards
Peaks: 1. Chloride 4 mg/L
2. Sulfate 4
Proprietary & Confidential 8 Dionex Confidential
Determination of Glycerol
Column: IonPac ICE-AS1
Eluent: 5 mmol/L H2SO4/
10 mmol/L mannitol
Flow rate: 1 mL/min
Detection: D.C. amperometry, Pt, 0.6 V
Inj. volume: 50 µL
Peaks: 1. Glycerol
2. Sulfite
3. Ethanol
Minutes
0 2 4 6 8 10
0
35
nA
1
2
3
Proprietary & Confidential 9 Dionex Confidential
Determination of Cations in Biodiesel
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.2
2.0
µS
Minutes
1
2 3
4
Peaks: 1. Sodium
2. Potassium
3. Magnesium
4. Calcium
Proprietary & Confidential 10 Dionex Confidential
IC Determination of Cations in Biodiesel
Analyte #
Injections
Unspiked Sample
Concentration (ppb)
Measured Spiked Sample Concentrations
(ppb)
Unspiked Sample +
Spike (ppb)
% Recovery
Sodium 3 1021 1905 1880 103
Potassium 3 5.31 13.17 12.84 104
Magnesium 3 35.95 77.08 77.87 98
Calcium 3 173.6 340.3 337.0 102
Proprietary & Confidential 11 Dionex Confidential
Analysis of Organic Acids by IC-MS
Mucate
Quinate
2-methyllactate
Acetate
Glycolate
Propionate
Lactate Oxalate
2-hydroxyvalerate
Isovalerate Valerate
a-ketoglutarate
Tartrate
Oxalacetate
Glutarate
Malate
trans-Aconitate cis-Aconitate
Isocitrate
Citrate
SIM
Ch
rom
ato
gra
ms O
ve
rlay
Fumarate
Maleate
Succinate
Methylmalonate
Melonate
Adipate
Gluconate
Pyruvate
Butyrate
2-keto-D-gluconate
Galacturonate
5-keto-D-gluconate
Proprietary & Confidential 12 Dionex Confidential
1.0 40.0
1.0 39.0
60.0 38.0
60.0 36.0
30.0 28.0
15.0 18.0
1.0 8.0
1.0 –10.0
Conc. /mmol/L Time
Hydroxide Gradient
Column: IonPac AS11-HC
Temperature: 30 °C
Eluent: KOH gradient (EG)
Flow rate: 0.38 mL/min
Inj. volume: 25 µL
Sample: 50 ng of each analyte
1st Detection: Suppressed conductivity,
AutoSuppression,
External water mode
2nd Detection: MSQ Plus
Retention Time and SIM of 33 Small Organic Acids
1 2
3,4 5,6
7
8
10 12
13
14 15
16
17,18,19
20,21
23,24
25 26
27
28
30 31
32
33
0 5 10 15 20 25 30 35 40
µS
Minutes
8
29 22
9
11
1. Quinate
2. Gluconate
3. Lactate
4. 2-methyllactate
5. Acetate
6. Glycolate
7. Propionate
8. Formate
9. Butyrate
10. 2-keto-D-gluconate
11. 2-hydroxyvalerate
12. Pyruvate
13. Isovalerate
14. Valerate
15. Galacturonate
16. 5-keto-D-gluconate
17. Glutarate
18. Adipate
19. Mucate
20. Succinate
21. Malate
22. Methylmalonate
23. Melonate
24. Tartarate
25. Maleate
26. a-Ketoglutarate
27. Oxalate
28. Fumarate
29. Oxalacetate
30. Citrate
31. Isocitrate
32. cis-aconitate
33. trans-aconitate
Proprietary & Confidential 13 Dionex Confidential
IC-MS Determination of Organic Acids in Fermentation Broths
0
100
0
100
0
100 0
100 10.41
10.70
11.45
0
100 18.29
18.86
SIM ms [ [email protected]]
SIM ms [ [email protected]]
SIM ms [ [email protected]]
SIM ms [ [email protected]]
SIM ms [ [email protected]]
a-HBA
Acetate
Formate
Malate
Tartrate
Proprietary & Confidential 14 Dionex Confidential
Carbohydrate Analysis with HPAE-PAD
• Sensitive – 0.1 to 1 pmol detection limits
• High Carbohydrate Concentration Kit permits direct injection
of 10 to 80 g/L mixtures
• 0.2 μL injection loop
• 15 mil electrochemical detector gasket
• Direct detection – no derivatization necessary
• Accurate, reproducible analysis
• High-resolution separations, excellent peak efficiency for mono-,
oligo-, and poly-saccharides
Proprietary & Confidential 15 Dionex Confidential
Sugars: Key Feedstock of the Future
• Sugars are emerging as a key feedstock for the future – most
abundant resource on the planet
• Sugars are microbially fermented to yield basic chemical
building blocks:
• Alcohols
• Lactic acids
• Succinic acid
• Propylene glycol
• 3-Hydroxypropionic acid
• 1,3-Propanediol
• This enables low-cost production of bulk
organic chemicals
Proprietary & Confidential 16 Dionex Confidential
Shift from Hydrocarbon to Carbohydrate Economy
• Environmental, manufacturing, and sustainability costs are pulling a
variety of industrial processes toward carbohydrate feedstocks:
agro/wood
• Pulp/paper
• Fuel and energy
• Plastics – biochemicals/biopolymers
• Many of these processes are dependent on lignin, cellulose, and
hemicellulose carbohydrate chemistries and separation technologies
Proprietary & Confidential 17 Dionex Confidential
Bio-based Chemicals and Fuels from Sugars
Sugars Adipic acid
Polylactic acid
Glucaric acid
Succinic acid
Biochemicals
Food ingredients
Propylene glycol
1,3-Propanediol
Biofuels
Fuel alcohols
Biodiesel
Agricultural by-
products
Energy crops
Forestry waste
Food processing
waste
Industrial waste
Municipal waste
Chemo-Physical Treatment
and/or Enzymes
Biocatalysts
Microbial Organisms
Nutraceuticals
Drugs
Cosmetics
Pesticides
Antifreeze and deicing fluids
Paints and coatings
Nylon
Polyester
Textiles
Polymers and plasticizers
Surfactants/detergents
Ethanol
Propanol
Butanol
Dimethyl ether
Biodiesel
Proprietary & Confidential 18 Dionex Confidential
Analytical Needs for Biofuels Production
Challenge: to develop methods and standards for the measurement of the
hydrolysis products (sugars) of celluloses acting on lignocellulosic materials
• Published standards exist for carbohydrate analysis of biomass
• LC method E1758
• Poor peak resolution masks detection of minor sugars
• Long run times
• Anionic/cationic de-ashing columns required for desalting
• GC method E1821
• Complex, multistep derivatization
• Sugars have multiple peaks which introduces error
• HPAE-PAD methods are superior for profiling biomass carbohydrates
• Solves persistent problems of poor chromatographic fidelity and missed peaks
• CarboPac SA10 column increases throughput
• High Concentration Carbohydrate Kit solves sensitivity issue
Proprietary & Confidential 19 Dionex Confidential
Sources of Oligosaccharide Complexity
•Profiling of biomass carbohydrates
•Most abundant organic chemical on earth with annual biosphere production of ~90x109
metric tons
Essential Monosaccharides
• Chromatographic separations is key
• Similarity of carbohydrates (number of C, H, O) makes qualitative analysis by MS difficult
O
H
H
HO
CH3
OH H
H
OH
b-L-Fucose (Fuc)
CH2OH
HO
H
O OH
H
H OH
b-D-Galactose (Gal)
H
H
OH H
O
CH2OH
H
HO
H
H
OH
H
b-D-Mannose (Man)
OH HO
H
O
CH2OH
H
HO
H
H
OH
H
b-D-Glucose (Glc)
OH H
HO
Cellulose
Proprietary & Confidential 20 Dionex Confidential
Dissociation Constants of Some Common Carbohydrates
Sugar pKa
Fructose 12.03
Mannose 12.08
Xylose 12.15
Glucose 12.28
Galactose 12.39
Dulcitol 13.43
Sorbitol 13.60
α-Methyl glucoside 13.71
(in water at 25 °C)
At pH 13, most sugars deprotonate
to form anions
Even at pH 13, alditols are only weakly ionized
Proprietary & Confidential 21 Dionex Confidential
Separation of Biofuel Sugars on CarboPac SA10
Column: CarboPac SA10
Eluent: 1 mmol/L KOH (EG)
Flow rate: 1.5 mL/min
Inj. volume: 10 µL
Temperature: 45 °C
Detection: IPAD, Au working electrode
Sample: Biofuel sugar mix
Peaks: 1. Fucose 20 mg/L
2. Sucrose 20
3. Arabinose 20
4. Galactose 20
5. Glucose 20
6. Xylose 20
7. Mannose 20
8. Fructose 20
1
2
3
4 5
6
7
8
9 8 7 6 5 4 3 2 1 0
0
50
nC
Minutes
Proprietary & Confidential 22 Dionex Confidential
IC/MS Alditol Analysis of Microalgae Whole Cell Lysate
D+Arabitol
L-Arabitol
Mannitol
Algae Sample
0 10 20 30 40 50 60 70 80 90 100 110 120 130 141 –20
0
180
nC
7
6
5
4
3
2
1
Minutes
Xylitol Inositol
Dulcitol
Column: CarboPac MA1
Eluent: 500 mmol/L NaOH
Flow rate: 0.2 mL/min
Temperature: 30oC
Detection: PAD
Proprietary & Confidential 23 Dionex Confidential
High-Concentration Carbohydrate Analysis by HPAE-PAD
Column: CarboPac PA1, 4 x 250 mm
Eluent: 200 mmol/L NaOH
Flow rate: 1 mL/min
Inj. volume: 0.2 µL
Detection: IPAD, Au working electrode
Gasket: 15 mil UHMWPE cell gasket
0 1 2 3 4 5
0
5500
nC
Minutes
Glucose
Fructose
10-80 g/L Mixtures
• Reduces sensitivity
• Precludes dilution errors
Proprietary & Confidential 24 Dionex Confidential
FOOD & BEVERAGE ANALYSIS
Proprietary & Confidential 25 Dionex Confidential Dionex Confidential
Food & Beverage Components Analyzed by Ion Chromatography
• Inorganic anions
• Alkali- and alkaline-earth metals
• Organic acids
• Amines
• Carbohydrates
• Alcohols
• Amino acids
Proprietary & Confidential 26 Dionex Confidential
Food & Beverage Analytes Determined by IC
Inorganic Anions HPIC / Suppressed Conductivity, DC Amperometry, UV/Vis
IC Methodology Analyte
Alkali-/Alkaline-
Earth Metals HPIC / Suppressed Conductivity
Organic Acids HPIC / Suppressed Conductivity, HPICE / Suppressed
Conductivity
Aliphatic Amines HPIC / Suppressed Conductivity / Pulsed Amperometry
Carbohydrates HPIC / Pulsed Amperometry
Alcohols/Aldehydes HPICE / Pulsed Amperometry
Amino Acids HPIC / Pulsed Amperometry
Proprietary & Confidential 27 Dionex Confidential
Advantages of IC for Food & Beverage Analysis
• Sensitive Detection Samples can be diluted to decrease the concentration of
matrix components
• Specific Detection Low abundant analytes can be detected in presence of
large concentration of matrix components
• Analyte-Specific Separations The selectivity of the separator columns is tailored for the
specific requirements of the various analyte classes
Proprietary & Confidential 28 Dionex Confidential
• Meat and meat products
Simultaneous analysis of analytes in meat products:
Column: IonPac AS11 with AG11
Eluent: 5 mmol/L NaOH
Detection: UV, 225 nm
• Milk and milk products
Simultaneous analysis of analytes in milk products:
Column: IonPac AS7 with AG14
Eluent: 40 or 50 mmol/L KCl and 5 mmol/L Tris, pH 7.5
Detection: UV, 214 nm
IC Methods for Nitrite/Nitrate Analysis
Proprietary & Confidential 29 Dionex Confidential
• 10.0 g of sample in 100 mL of water
• Homogenize for 1 min
• Heat sample between 70°C and 80°C for 15 min
• Centrifuge at 5000 × g for 10 min
• Filter the supernatant
• Collect the filtrate for ion chromatography
Extraction of Nitrate and Nitrite from Meat Products
Proprietary & Confidential 30 Dionex Confidential
• Dissolve 1.0 g of milk powder into 20 mL of water
• Filter through a 1.2 µm filter
• Centrifuge the filtrate in a Centricon 3 filter at 5000 × g for 30 min
• Pass the Centricon 3 filtrate through OnGuard-RP cartridge
• Collect the filtrate for HPLC analysis
Extraction of Nitrate and Nitrite from Milk Products
Proprietary & Confidential 31 Dionex Confidential
Determination of Nitrate and Nitrite in a Meat Extract
Column: IonPac AS11 with AG11
Eluent: 5 mmol/L NaOH
Flow rate: 1 mL/min
Inj. volume: 25 µL
Detection: UV, 225 nm
Peaks: 1. Nitrite 1.16 mg/L
2. Nitrate 0.54
0 5 10
0.00
0.02
AU
Minutes
1
2
Proprietary & Confidential 32 Dionex Confidential
Sulfite in Food
• Sulfiting agents added to foods as antioxidants, preservatives,
and whiteners
• Added as sulfur dioxide, sodium sulfite, sodium and
potassium bisulfite, and sodium and potassium metabisulfite
• Many people suffer allergic reaction to ingestion of sulfites
• FDA requires warning labels on foods and beverages
containing 10 ppm of SO2
• AOAC contains several methods for determination of sulfites:
• Modified Monier-Williams method
• Ion exclusion-DC amperometric method (Kim & Kim)
Proprietary & Confidential 33 Dionex Confidential
Column: IonPac ICE-AS1
Eluent: 5 mmol/L sulfuric acid
Flow rate: 1.0 mL/min
Detection: DC amperometry,
Pt electrode, 0.6V
Inj.-volume: 50 µL
Peaks: 1. Glycerol
2. Sulfite 1.5 mg/L
3. Ethanol
Sample diluted 1:10. Sulfite concentration
was 15 mg/L in the undiluted sample
Separation of Sulfite, Glycerol, and Ethanol in White Wine
Minutes
0 2 4 6 8 10
0
35
nA
1
2
3
Proprietary & Confidential 34 Dionex Confidential
• Ion chromatography with suppressed conductivity or
UV detection
• Reversed-phase chromatography with DC amperometry
(silver electrode) (IDF/AOAC Method)
• Ion chromatography with DC amperometry
(platinum electrode)
• Ion chromatography with DC amperometry
(silver electrode)
Chromatographic Methods for Iodide Analysis
Proprietary & Confidential 35 Dionex Confidential
Iodide in Baby Formula
0 2 4 6 8 10
Minutes
Iodide
73 µg/L
Iodide
45µg/L
10
nA
-10
10
nA
-10
A.
B.
Column: IonPac AG11 with AS11
Eluent: 5 mmol/L HNO3
Flow rate: 1.5 mL/min
Inj. volume: 100 µL
Detection: DC amperometry, 0.05 V
Ag electrode
Samples: A. Milk-based baby formula
B. Soy-based baby formula
Proprietary & Confidential 36 Dionex Confidential
Colum: IonPac AS11 (2 mm)
Flow rate: 0.3 mL/min
Inj. volume: 20 µL
Detection: Suppressed conductivity
Peaks: 1. PO4
2. P2O7
3. P3O9
4. P3O10
5. P4O12
6. P4O13
Polyphosphate Analysis
Minutes
30
0
µS
0 10 5 15 25 45 40 35 20 30
1
3
2
4 6
5
Courtesy of E. S. Baluyot (Rhone-Poulenc Inc.)
^
Proprietary & Confidential 37 Dionex Confidential
Polyphosphates in Cheese
Column: IonPac AG11 / AS11 (2 mm),
Eluent: NaOH gradient
Flow rate: 0.3 mL/min
Inj. volume: 10 µL
Detection: Suppressed conductivity,
AutoSuppression, recycle mode
Peaks: 1. PO4
2. P2O7
3. P3O9
4. P3O10
5. P4O12
6. P4O13
A: Bad Quality
B: Good Quality
E. Baluyot and C.G. Hartford, J. Chromatogr. A 739 (1996) 217-222
Proprietary & Confidential 38 Dionex Confidential
Column: IonPac AS19 (0.4 × 250 mm)
and AG19 (0.4 × 50 mm)
Eluent: KOH (EG)
Gradient: 7 min 15 mmol/L isocratic,
in 18 min to 60 mmol/L
Temperature: 30 °C
Flow rate: 10 µL/min
Inj. volume: 0.4 µL
Detection: Suppressed conductivity
Sample: English breakfast tea
0 10 20 30
–10
90
µS
Minutes
Ch
lorid
e
Nitra
te
Su
lfa
te
Ma
late
Ort
ho
ph
osph
ate
Citra
te
Anions in Tea
Proprietary & Confidential 39 Dionex Confidential
Fast Analysis of Orthophosphate and Citrate in Cola Drinks
0 2 4 6 -5
35
1 2
3
Minutes
µS
4
Column: IonPac Fast Anion IIIA with guard
Eluent: 20 mmol/L KOH (EG)
Temperature: 30 °C
Flow rate: 1 mL/min
Inj. volume: 1.25 µL
Detection: Suppressed conductivity,
AutoSuppression, Recycle Mode
Peaks: 1. Unknown — mg/L
2. Unknown —
3. Orthophosphate 456
4. Citrate 52
Proprietary & Confidential 40 Dionex Confidential
Gradient Elution of Anions in Diet Coke
0 4 6 8
-5
40
Minutes
µS
2
1
2 3
4
5
6
Column: IonPac Fast Anion III with guard
Eluent: KOH (EG)
Gradient: 5 mmol/L from 0-2 min,
5-30 mmol/L from 2-5 min
Temperature: 30 °C
Flow rate: 1 mL/min
Inj. volume: 1.25 µL
Detection: Suppressed conductivity,
AutoSuppression, Recycle Mode
Peaks: 1. Unknown — mg/L
2. Chloride 20
3. Benzoate 78
4. Sulfate 102
5. Orthophosphate 227
6. Citrate 145
Proprietary & Confidential 41 Dionex Confidential
LC Methods for Organic Acid Analysis
• Ion-Exclusion Chromatography
• Anion Exchange Chromatography
• Reversed-Phase Chromatography
• Ion suppression techniques
• Mixed-Mode Chromatography
• Ion-exchange, reversed-phase
• HILIC
Proprietary & Confidential 42 Dionex Confidential
Column: Acclaim Mixed-Mode WAX-1, 5 µm
Dimensions: 150 mm × 4.6 mm i. d.
Temperature: 30°C
Eluent: 25 mol/L phosphate buffer, pH 6
Flow rate: 0.5 mL/min
Inj. volume: 10 µL
Detection: UV, 210 nm
Peaks:
1. Quinic acid
2. Shikimic acid
3. Glycolic acid
4. Lactic acid
5. Acetic acid
6. Formic acid
7. Ascorbic acid (Vitamin C)
8. Iso-ascorbic acid
9. Propionic acid
Separation of Monovalent Carboxylic Acids
AU
Minutes 0 5 10 15
1
2
3 6
7
9
8 4
5
Proprietary & Confidential 43 Dionex Confidential
Analysis of Organics in Soft Drink Products
Column: Acclaim Mixed-Mode WAX-1, 5 µm
Dimension: 150 mm × 4.6 mm i. d.
Temperature: 30°C
Eluent: 57/43 v/v MeCN / 120 mmol/L
phosphate buffer, pH 2.9
Flow rate: 2 mL/min
Inj. volume: 2.5 µL
Detection: UV, 210 nm
Sample: Direct injection of degassed sample
Peaks: 1. Caffeine
2. Aspartame
3. Sorbate
4. Benzoate
5. Citrate
6. Acesulfam K
0 1.5 3
Minutes
AU
Standard
Diet COKE
Diet PEPSI
2
3 6 4
1
5
2
4
1
5
2
4
1
5
Proprietary & Confidential 44 Dionex Confidential Dionex Confidential Dionex Confidential
HILIC – Hydrophilic Interaction Chromatography
• HILIC is a variation of normal-phase chromatography
without the disadvantages of using solvents that are not
miscible with water
• Also called “reverse reversed-phase” or “aqueous normal
phase” chromatography
• HILIC is used to retain very polar compounds
• Stationary phase is a polar material such as silica, cyano,
amino, diol, etc.
• Mobile phase is highly organic (>80 %) with a small
amount of polar solvent (e.g, H2O)
Proprietary & Confidential 45 Dionex Confidential Dionex Confidential Dionex Confidential
What are the Benefits of HILIC?
• Retain highly polar compounds that would not be retained by
reversed-phase chromatography
• Complementary selectivity to reversed-phase columns
• Enhanced sensitivity in mass spectrometry
• Utilizing high organic mobile phases (> 80 %) enhances ESI-MS
response
• Shortens sample preparation procedure
• Final SPE or liquid/liquid extraction step usually uses an organic
solvent (e.g., acetonitrile, isopropanol, etc.)
• Then organic solvent must be evaporated to dryness and
reconstituted in mobile phase before injecting onto a reversed-phase
HPLC column.
• Elimination of evaporation/reconstitution step by directly injecting the
sample
Proprietary & Confidential 46 Dionex Confidential Dionex Confidential Dionex Confidential
HILIC Types
• “Neutral” surface
• Diol group (simple retention mechanism, fast equilibration,
demonstrated applications)
• Cyano group
• “Ion-Exchange” surface
• Silanol group
• Amino group
• “Zwitterionic” surface
Silica
CN
Silica
NH
2
Silica
Silica
OH
OH
O
HO
HO
Silic
a
N SO3
Me
Me
+ -
Proprietary & Confidential 47
Acrylic Acid and Oligomers
Column: Acclaim HILIC-10, 3 µm
Dimensions: 150 mm × 4.6 mm i. d.
Eluent: 90/10 v/v MeCN/10 mmol/L (total) NH4OAc, pH5
Flow rate: 1 mL/min
Temperature: 30 °C
Inj. volume: 2 µL
Detection: UV, 230 nm
Peaks: (5 mg/mL in eluent)
1. Acrylic acid (monomer)
2. Dimer
3. Trimer
4. Tetramer
5. Pentamer
6. Hexamer
0 6 12 9 3 15
Minutes
mA
U
0
30
1
2
3
4
5 6
Proprietary & Confidential 48 Dionex Confidential Dionex Confidential Dionex Confidential
Surface Chemistry of Acclaim Mixed-Mode HILIC-1
Silica
OH
OH
OH
OH
OH
OH
OH
OH
Hydrophilic portion
Hydrophobic portion
Proprietary & Confidential 49 Dionex Confidential
Analysis of Fructooligosaccharides in Strawberry Joghurt
1 2
3
4
5
6 7
nC
40
-5
0 10 20 30
Minutes
Column: CarboPac PA1
Eluent: NaOH / NaOAc gradient
Flow rate: 1 mL/min
Inj. volume: 20 µL
Detection: Pulsed amperometry,
Au electrode
Sample
preparation: Carrez 1 & 2
Peaks: 1. 1-Kestose
2. Maltose
3. Nystose
4. Maltotriose
5. Inulotriose
6. Fructosylnystose
7. Maltotetraose
Proprietary & Confidential 50 Dionex Confidential
Column: CarboPac MA1
Eluent: 150 mmol/L NaOAc,
0.2 % (v/v) acetic acid, pH 5.5
Flow rate: 0.4 mL/min
Inj. volume: 25 µL
Detection: Integrated pulsed amperometry
Peaks: 1. 4-Cl-Galactose 7.8 mg/L
2. 1,6-di-Cl-Fructose 2.4
3. Sucralose
Analysis of Sucralose
nA
780
0
1
2
0
Minutes
30 40 50 60 10 20
3
Proprietary & Confidential 51 Dionex Confidential
Monosaccharides in Soluble Coffee
0 5 10 15 20 25 30 35 40 45 50
20
35
nC
1
Standard (1-9 µg/mL)
2 3
4 5
6
7
8 9 10
11 Column: CarboPac PA10 with guard
Eluent: 1 mmol/L KOH (EG)
Flow rate: 1 mL/min
Inj. volume: 5 µL
Detection: Pulsed amperometry,
Au electrode
Peaks :
1. Mannitol 7. Sucrose
2. Fucose 8. Xylose
3. Arabinose 9. Mannose
4. Rhamnose 10. Fructose
5. Galactose 11. Ribose
6. Glucose
0 5 10 15 20 25 30 35 40 45 50
Minutes
20
35
nC
Instant Coffee
(OnGuard RP Treatment)
1
2
4
3
5
6 7 8
9
10 11
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Conclusions
• IC is a powerful tool for the automated analysis of foods
and beverages
• Simple, direct methods with minimal sample
preparation can often be developed by using specific,
high sensitivity detection combined with column
selectivities tailored to the analytes
• Sample preparation for beverages can often be
reduced to only a dilution step
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Thank you for your kind attention!
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