BEH Amide

Waters Amide Column Technology
For Food Analysis
Euan Ross
Business Development Manager
Chemistry Operations Europe
©2012 Waters Corporation
1
Overview
Overview of HILIC
Retention mechanisms and characteristics
Practical considerations
Implementing the Approach: Water Soluble Vitamins
What is the Amide Chemistry
BEH/Xbridge Amide – Carbohydrate Analysis
Further BEH/Xbridge Amide Application examples
©2012 Waters Corporation
2
What is a Polar Molecule?
 General chemistry definition:
– A molecule whose centers of positive and negative
charges do not coincide
– The degree of polarity is measured by the dipole
moment of the molecule
 Dipole moment is the product of the charge at
either end of the dipole times the distance
between the charges
– The unequal sharing of electrons within a bond
results in a separation of positive and negative
electric charge.
 Polarity is dependent on the electronegativity
difference between molecular atoms and
compound asymmetry
©2012 Waters Corporation
3
What is HILIC?
 HILIC - Hydrophilic Interaction Chromatography
– Term coined in 1990 to distinguish from normal-phase*
 HILIC is a variation of normal-phase chromatography
without the disadvantages of using solvents that are
not miscible in water
– “Reverse reversed-phase” or “aqueous normal-phase”
chromatography
 Stationary phase is a POLAR material
– Silica, hybrid, cyano, amino, diol
 The mobile phase is highly organic (> 80%) with a
smaller amount of aqueous mobile phase
– Water (or the polar solvent(s)) is the strong, eluting
solvent
*Alpert, A. J. J.Chromatogr. 499 (1990) 177-196.
©2012 Waters Corporation
4
Benefits of HILIC
 Retention of highly polar analytes not retained by reversed-phase
—Less interference from non-polar matrix components
 Complementary selectivity to reversed-phase
—Polar metabolites/impurities/degradants retain more than parent
compound
 Enhanced sensitivity in mass spectrometry
—High organic mobile phases (> 80% ACN) promotes enhanced ESIMS response
—Direct injection of PPT supernatant without dilution
—Facilitates use of lower volume samples
 Improved sample throughput
—Direct injection of high organic extracts from PPT, LLE or SPE
without the need for dilution or evaporation and reconstitution
©2012 Waters Corporation
5
Traditional SPE Methods for
Reversed-Phase Chromatography
 Traditional SPE methods often contain an elution step that
consists of high organic content
 To make this extracted sample compatible with your mobile
phase, you must first evaporate the high organic eluent then
reconstitute in some portion of aqueous
 Evaporation and reconstitution are often the most lengthy
steps in an SPE procedure*
 In HILIC, the high organic eluent can be directly injected on
the column, thus eliminating the need for evaporation and
increasing your throughput
*Jernal, M., Teitz, D., Ouyang, Z., J.Chromatogr. B, 732 (1999) 501.
©2012 Waters Corporation
6
When To Use HILIC
ESI-MS Response
excellent
When to Use HILIC:
HILIC
Reversed-phase
 Need improved MS response
for polar or ionizable
compounds
poor
Normal-phase
polar
 Need improved retention of
hydrophilic or ionizable
compounds
non-polar
 Need improved sample
throughput for assays using
organic extraction
Compound Index
©2012 Waters Corporation
7
Elution Strength in HILIC:
Solvent Selectivity
Water
Strongest
Methanol
Polar
Elution
Solvents
Use a less polar
solvent to
Increase
retention
of polar analytes
Ethanol
Isopropanol
Primary
Solvent
©2012 Waters Corporation
Acetonitrile
Weakest
8
Overview
Overview of HILIC
Retention mechanisms and characteristics
Practical considerations
Implementing the Approach: Water Soluble Vitamins
What is the Amide Chemistry
BEH/Xbridge Amide – Carbohydrate Analysis
Further BEH/Xbridge Amide Application examples
©2012 Waters Corporation
9
HILIC Retention Mechanisms are Complex
Combination of partitioning, ion-exchange and hydrogen bonding
•Polar analyte partitions between bulk mobile phase and partially
immobilized polar layer on material surface
• Secondary interactions between surface silanols and/or functional
groups with the charged analyte leading to ion-exchange
• Hydrogen bonding between positively charged analyte and negatively
charged surface silanols
©2012 Waters Corporation
10
Stationary Phases for HILIC separations
Hybrid HILIC Columns
(pH range 1 – 8 for BEH HILIC
pH range 2 – 11 for BEH Amide)
ACQUITY UPLC® BEH Amide
XBridgeTM Amide
ACQUITY UPLC® BEH HILIC
XBridgeTM HILIC
Silica HILIC Columns
(pH range 1 – 5)
Atlantis® HILIC Silica
©2012 Waters Corporation
11
2nd Generation Hybrid Particles:
Ethylene Bridged Hybrid (BEH) Particles
U.S. Patent No. 6,686,035 B2
Bridged Ethanes within a silica matrix
Anal.
Chem.
2003, 75, 6781-6788
©2012 Waters
Corporation
12
What is the BEH/Xbridge Amide
Chemistry?
 Ligand type:
Trifunctional Amide
 Particle size:
1.7 µm and 3.5 µm
 Ligand density:
6.3 µmol/m2
 Carbon load:
12%
 Endcap style:
None
 pH range:
2-11
©2012 Waters Corporation
13
Influence of Stationary Phase on Retention
1
4
2
5
ACQUITY UPLC BEH HILIC
2.1 x 50 mm, 1.7 µm
3
Unbonded hybrid with low silanol activity
1
2
4
5
ACQUITY UPLC BEH Amide
2.1 x 50 mm, 1.7 µm
3
Bonded hybrid
1
3
2
4
5
Atlantis HILIC Silica
2.1 x 50 mm, 3 µm
Unbonded silica with high silanol activity
0
1
Minutes
2
3
(1) acenaphthene (2) thymine (3) 5-fluoroorotic acid (4) adenine (5) cytosine; UV 254 nm
©2012 Waters Corporation
14
Overview
Overview of HILIC
Retention mechanisms and characteristics
Practical considerations
Implementing the Approach: Water Soluble Vitamins
What is the Amide Chemistry
Hilic Amide – Carbohydrate Analysis
Further Application examples
©2012 Waters Corporation
15
Before You Start:
Common HILIC mobile phases
 Common buffers/additives*
– Ammonium formate, ammonium acetate
– Formic acid, ammonium hydroxide, acetic acid
– Phosphate salt buffers ARE NOT recommended due to precipitation in the
highly organic mobile phase (phosphoric acid is OK)
 Recommended buffer concentration: 10 mM ON-COLUMN
 Recommended additive concentration: 0.2% ON-COLUMN
*The actual pH of the mobile phase may be 1 pH unit closer to neutral due to the highly organic mobile phase
Canals, I.; Oumada, F. Z.; Roses, M.; Bosch, E. J. Chromatogr. A. 911 (2001) 191-202.
Espinosa, S.; Bosch, E.; Roses, M. Anal. Chem. 72 (2000) 5193-5200.
©2012 Waters Corporation
16
Before You Start:
Column Equilibration and Wash Solvents
 Instrument Wash Solvents
– Strong needle wash: 9:1 acetonitrile: water
– Weak needle wash/purge solvent: initial mobile phase conditions [excluding salt,
additive or buffer]
 Brand new column
– Run 50 empty column volumes of 50:50 acetonitrile:water with 10 mM buffer or
0.2% additive solution
 Column equilibration
– Equilibrate with 20 empty column volumes of initial mobile phase conditions
 Gradient separations
– Re-equilibrate with 5 to 8 empty column volumes
As with any column, insufficient equilibration can cause drifting retention times
©2012 Waters Corporation
17
Before You Start:
Influence of Sample Diluent
 Sample diluent strongly influences solubility and peak shape
(just like reversed-phase)
 Sample diluent should be at least 75% acetonitrile or as close to
initial mobile phase conditions as possible
 However, polar analytes often have low solubilities in organic
solvents
 General purpose HILIC diluent
– 75:25 acetonitrile:methanol works for most polar analytes
– Offers a compromise between solubility and peak shape
– Adjust according to your analytes (add 0.2% formic acid to increase
solubility)
– In some cases, 25% methanol may be too polar to use as an injection
solvent
©2012 Waters Corporation
18
Overview
Overview of HILIC
Retention mechanisms and characteristics
Practical considerations
Implementing the Approach: Water Soluble Vitamins
What is the Amide Chemistry
Hilic Amide – Carbohydrate Analysis
Further Application examples
©2012 Waters Corporation
19
Implementing the Approach:
Example 1, Water Soluble Vitamins
N
N
CH3
OH
N
HO
OH
HO
NH2
OH
OH
N
H3C
N
O
N
O
NH
O
O
H3C
OH
O
Nicotinic acid
Riboflavin
Pyridoxal
Nicotinamide
O
O
H2N
CH3
O
H3C
N
NH2
HO
CH3
H2N
O
N
H3C
O
+
N
Cl
N
O
-
N
NH2
S
+
H3C
Co
H2N
O
HO
H3C
N
H2N
N
N
H
O
N
H3C
NH
CH3
O
CH3
OH
NH
CH3
O
O
H3C
NH
O
O
O
O
P
HO
OH
N
CH3
N
CH3
HO
-
O
Ascorbic acid
HO
NH2
OH
O
O
Thiamine
NH2
N
N
H
N
Folic Acid
OH
B12
©2012 Waters Corporation
20
Stationary Phase Selectivity at Low pH:
Water-soluble Vitamins
1
BEH Amide
pH 3
5
4
6
3
2
8
All 3 columns yield
different selectivity
7
1,6
BEH HILIC
5
4
7
3,2
8
1
5
4
6
0.50
1.00
1.50
Atlantis HILIC Silica
7
8
3,2
0.00
BEH Amide has greatest
resolution of all peaks
2.00
2.50
3.00
3.50
4.00
4.50
Compounds
1. Nicotinamide
2. Pyridoxine
3. Riboflavin
4. Nicotinic acid
5. Thiamine
6. Ascorbic Acid
7. B12
8. Folic Acid
50 µg/mL
50 µg/mL
30 µg/mL
50 µg/mL
50 µg/mL
50 µg/mL
50 µg/mL
25 µg/mL
5.00
Minutes
©2012 Waters Corporation
21
Stationary Phase Selectivity at High pH:
Water-soluble Vitamins
6
1
BEH Amide
4
2
Large selectivity
difference between the
two BEH chemistries
5
7
3
8
BEH Amide has greatest
resolution of all peaks
6
1
BEH HILIC
Compounds
1. Nicotinamide
2. Pyridoxine
3. Riboflavin
4. Nicotinic acid
5. Thiamine
6. Ascorbic Acid
7. B12
8. Folic Acid
4
2
5
3
7
8
0.00
pH 9
0.50
1.00
1.50
2.00
2.50
Minutes
©2012 Waters Corporation
3.00
3.50
4.00
4.50
50 µg/mL
50 µg/mL
30 µg/mL
50 µg/mL
50 µg/mL
50 µg/mL
50 µg/mL
25 µg/mL
5.00
22
Mobile Phase pH Selectivity:
Water-soluble Vitamins
BEH Amide
6
1
pH 9
Selectivity difference
between pH 3 and pH 9
5
4
2
3
0.00
0.50
7
1.00
1.50
2.00
2.50
Minutes
Adequate resolution for
both conditions
8
3.00
3.50
4.00
4.50
5.00
Greater sensitivity at pH 9
1
Compounds
1. Nicotinamide
2. Pyridoxine
3. Riboflavin
4. Nicotinic acid
5. Thiamine
6. Ascorbic Acid
7. B12
8. Folic Acid
pH 3
5
4
0.00
2
3
0.50
1.00
©2012 Waters Corporation
6
8
1.50
2.00
7
2.50
Minutes
3.00
3.50
4.00
4.50
50 µg/mL
50 µg/mL
30 µg/mL
50 µg/mL
50 µg/mL
50 µg/mL
50 µg/mL
25 µg/mL
5.00
23
Final Method:
Water-soluble Vitamins
Compounds
1. Nicotinamide
2. Pyridoxine
3. Riboflavin
4. Nicotinic acid
5. Thiamine
6. Ascorbic Acid
7. B12
8. Folic Acid
6
1
5
4
2
3
0.50
1.00
7
1.50
2.00
8
2.50
3.00
3.50
4.00
4.50
5.00
Minutes
©2012 Waters Corporation
24
Summary
 For HILIC retention and selectivity:
– ACN is used the primary [weak] solvent in HILIC
– Water, methanol, ethanol or isopropanol are strong [elution] solvents
– Stationary phase charge and bonded phase can impact retention and selectivity
– Analytes in their charged form exhibit greater retention [acids at high pH, bases at low
pH]
 Practical considerations:
– At least 10 mM buffer or 0.2% additive is recommended in mobile phase A and B
– Sample diluent should contain at least 75% acetonitrile for solubility and peak shape
– Weak needle wash must be in a high organic solution [90 – 95% ACN]
©2012 Waters Corporation
25
Overview
Overview of HILIC
Retention mechanisms and characteristics
Practical considerations
Implementing the Approach: Water Soluble Vitamins
What is the Amide Chemistry
Hilic Amide – Carbohydrate Analysis
Further Application examples
©2012 Waters Corporation
26
Typical Chromatographic Analysis
 Amino or polyamine columns
 RI detection is commonly used
– Carbohydrates have very weak UV absorbances
 Typical Problems Encountered
– Salt interferences
– Anomer mutarotation
– Schiff Base formation
– Loss of reducing sugars at elevated temperatures
– Shortened column lifetime
– Not compatible with gradient separations
©2012 Waters Corporation
27
Sample Preparation
 Very simple sample preparation
 Liquid Samples
– Dilute with 50:50 ACN/H2O
– Filter using 0.45µm PVDF syringe filter (if necessary)
 Solid Samples
– Weigh out sample (~3g) into 50mL centrifuge tube
– Add 25mL of 50:50 ACN/H2O and homogenize (mechanically)
– Centrifuge at 3200rpm for 30 minutes
– Collect supernatant and filter using 0.45µm PVDF syringe filter
 Depending on sample, additional sample dilutions may be
necessary
©2012 Waters Corporation
28
Carbohydrate Methods
Gradient Conditions
 Isocratic Conditions: 75% ACN (with 0.2% TEA) at 35°C
– For food sugars and other mono- and disaccharides
 Gradient Conditions: 80-50% ACN (with 0.2% TEA) at 35°C
– For larger polysaccharides
 Alternative Methods:
– Use Acetone instead of ACN (77% Acetone) at 85°C
o
Reduced mobile phase cost
– Use NH4OH instead of TEA as the mobile phase modifier
o
More compatible with Mass Spec
©2012 Waters Corporation
29
Stability Testing in ACN
BEH Amide 2.1 x 150mm Column
75% ACN with 0.2% TEA, 0.29mL/min at 35 C
900
3
1
800
2
5
700
4
Initial
LSU
600
After 100
Honey inj.
500
400
After 100
Molasses inj.
300
200
After 100
Cereal inj.
100
0.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Minutes
1)
Fructose,
2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (1mg/mL each)
©2012
Waters Corporation
5.0
5.5
6.0
6.5
7.0
7.5
8.0
30
Salt Interferences
ELSD using 75% ACN at 1.4mL/min, 35 C
4.6 x 150mm Columns, 10µL injection
5 Food Sugars
with *Salts on:
1
3
2
Aminopropyl Column
5
4
*
*
1
Polyamine Column
3
2
4,5
*
1
2
4
*
0.0
1)
Fructose,
2.0
2) Glucose,
* Salt Interferences
©2012 Waters Corporation
3
4.0
3) Sucrose, 4) Maltose,
6.0
Minutes
Polymeric
Amino Column
5
8.0
10.0
12.0
5) Lactose (each 1mg/mL),
31
Salt Interferences
ELSD using 75% ACN at 1.4mL/min, 35 C
4.6 x 150mm Columns, 10µL injection
BEH Amide
With 0.1% TEA Mobile Phase Modifier
Low Salt Retention = No Salt Interference
1
5 Food Sugars
3
with Salts
2
*
0.0
2.0
5
*
4
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
Minutes
1)
Fructose,
2) Glucose,
* Salt Interferences
©2012 Waters Corporation
3) Sucrose, 4) Maltose,
5) Lactose (each 1mg/mL),
32
Reducing Sugar Anomers
OH
H
HO
HO
H
H
H
OH
H
Mutarotation
O
H
OH
-D-Glucose
OH
O
HO
H
H
HO
OH
H
HO
HO
H
H
H
H
H
O
OH
OH
OH
H
-D-Glucose
H
O
H
O
HO
H
O
H
H
O
H
O
H
O
Sucrose
-Glucose
-Glucose
-Maltose
-Maltose
0.0
©2012 Waters Corporation
1.0
2.0
Minutes
3.0
Isocratic: 75% ACN
at 15 C
4.0
5.0
33
Anomer Collapse
0.2% TEA in 75% ACN
35 C
75% ACN
0.2% TEA in 75% ACN
25 C
75% ACN
Glucose
Sucrose
Maltose
0.2% TEA in 75% ACN
15 C
0.0
©2012 Waters Corporation
75% ACN
1.0
2.0
Minutes
3.0
4.0
5.0
34
Demonstration of Anomer Collapse
-D-glucopyranose
-D-glucopyranose
H
H OH
O
HO
H
HO H
H
H OHOH
OH
O
HO
H
H
HO
H
OH
OH
H
3
5
5
2
2
1
4
4
1
75% ACN
with no modifier
at 35 C
3
2
5
4
1
75% ACN
with 0.2% TEA
at 35 C
3
2
5
0.0
1.0
2.0
3.0
1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose,
2.1 x 150mm Column @ 0.29mL/min
©2012 Waters Corporation
4.0
Minutes
80% Acetone
with 0.05% TEA
at 90 C
4
5.0
6.0
7.0
8.0
5) Lactose (each 1mg/mL)
35
Commercial Food Samples on
BEH Amide Column
ELSD on ACQUITY UPLC® using 0.15mL/min at 85 C
Isocratic: 77% Acetone with 0.05% TEA
2.1 x 50mm Columns, 0.7µL injection
Strawberry
Smoothie
Hot Cross Buns
White Bread
Tomato Ketchup
Lamb Curry Meal
Raisin Bran Cereal
Energy Drink
1
0.00
3
4
2.00
Minutes
1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (1mg/mL each)
©2012 Waters Corporation
1.00
2
5
Food Sugars
3.00
4.00
36
Analysis of Beer
 Beer is made, in part, with malted barley. When the barley malt
is first cooked in the brewing process, the resulting liquid
contains maltose, other simple sugars and larger, more complex
carbohydrates. During fermentation, yeast consumes the
sugars, converting them to alcohol and natural carbonation.
 When finished, most beers contain little or no maltose or other
simple sugars, but retain many of the larger polysaccharides
©2012 Waters Corporation
37
Analysis of Beer
BEH Amide, 1.7µm (JTC-22-03), 2.1 x 100mm
10 minute gradient 75-45% ACN with 0.2% TEA at 35 C
0.13mL/min, 1.3µL injection volume
Samples: 50% in 50:50 ACN/H2O
Food Sugar Region
Wheat Beer
Pilsner
Dark Ale
Double Chocolate Stout
Malto-tri, tetra, penta, hexa, hepta-ose
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
Minutes
©2012 Waters Corporation
38
Analysis of Beer
Maltohexaose
Maltoheptaose
Maltotriose
Maltose
Lactose
Glucose
Xylose
Arabinose
Fucose
Gain Switching
Double Chocolate Stout
Maltopentaose
Maltotetraose
BEH Amide, 1.7µm (JTC-22-03), 2.1 x 100mm
10 minute gradient 75-45% ACN with 0.2% TEA at 35 C
0.13mL/min, 1.3µL injection volume
Samples: 100% Beer (No dilution or filtering)
Food Sugar Standard
Malto-tri, tetra, penta, hexa, hepta-ose
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
Minutes
©2012 Waters Corporation
39
Summary
 The BEH particle technology provides a stable and robust substrate
for the trifunctional amide bonding,
– provides extended column lifetime under a wide variety of conditions
(temperature & pH)
– Anomer peaks arising from reducing sugars can be collapsed into a single
peak by using either high temperature or high pH mobile phases
– Low salt retention eliminates salt interferences from complex sample
matrices
 Available in both HPLC (XBridge Amide, 3.5µm) and ACQUITY
UPLC® BEH Amide chemistries for easy method transfer
 Ideally suited for a wide variety of samples
– ELS detection allows gradient elution for analysis of more complex
polysaccharides
– Lower mobile phase consumption for sub-2µm particles results in reduced
cost and waste
©2012 Waters Corporation
40
The ACQUITY BEH Method Selector Tool:
ACQUITY UPLC® BEH Amide Column
©2012 Waters Corporation
42
Step 1
Select Column Dimensions
©2012 Waters Corporation
43
Step 1
Select Column Dimensions
©2012 Waters Corporation
44
Step 2
Determine the Sample Type
©2012 Waters Corporation
45
Step 3
Select Detector
©2012 Waters Corporation
46
Step 4
Select Temperature
©2012 Waters Corporation
47
©2012 Waters Corporation
48
Overview
Overview of HILIC
Retention mechanisms and characteristics
Practical considerations
Implementing the Approach: Water Soluble Vitamins
What is the Amide Chemistry
Hilic Amide – Carbohydrate Analysis
Further Application examples
©2012 Waters Corporation
49
Quality Control checks
Incoming raw materials
Stevia related compounds -
©2012 Waters Corporation
50
Ascorbic acid and Isoascorbic acid
©2012 Waters Corporation
51
Water Soluble Vitamins
©2012 Waters Corporation
52
Thank You?
©2012 Waters Corporation
53

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