Waters Amide Column Technology For Food Analysis · HILIC Retention Mechanisms are Complex Combination of partitioning, ion-exchange and hydrogen bonding •Polar analyte partitions

©2012 Waters Corporation 1

Euan Ross

Business Development Manager

Chemistry Operations Europe

Waters Amide Column Technology

For Food Analysis


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

Overview


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

What is a Polar Molecule?

http://upload.wikimedia.org/wikipedia/en/d/d9/Polarity_boron_trifluoride.png


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.


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 ESI-MS 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


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.


When To Use HILIC

When to Use HILIC:

Need improved retention of

hydrophilic or ionizable

compounds

Need improved MS response

for polar or ionizable

compounds

Need improved sample

throughput for assays using

organic extraction

Reversed-phase

polar non-polar

Compound Index

Normal-phase

ESI-

MS

Re

spo

nse

exce

llen

tp

oo

r

HILIC


Elution Strength in HILIC: Solvent Selectivity

Weakest

Strongest

Use a less polar solvent to Increase retention

of polar analytes

Water

Methanol

Ethanol

Isopropanol

Polar Elution Solvents

Acetonitrile Primary Solvent






BEH/Xbridge Amide – Carbohydrate Analysis

Further BEH/Xbridge Amide Application examples

Overview


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


Stationary Phases for HILIC separations

ACQUITY UPLC® BEH Amide XBridgeTM Amide

ACQUITY UPLC® BEH HILIC XBridgeTM HILIC

Atlantis® HILIC Silica

Silica HILIC Columns (pH range 1 – 5)

Hybrid HILIC Columns (pH range 1 – 8 for BEH HILIC pH range 2 – 11 for BEH Amide)


2nd Generation Hybrid Particles: Ethylene Bridged Hybrid (BEH) Particles

Anal. Chem. 2003, 75, 6781-6788

U.S. Patent No. 6,686,035 B2

Bridged Ethanes within a silica matrix


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


Influence of Stationary Phase on Retention

ACQUITY UPLC BEH HILIC 2.1 x 50 mm, 1.7 µm

Unbonded hybrid with low silanol activity

4 5

Minutes

1 2

3

3

1 2 4

5

1 2 3

4

5

0 1 2 3

ACQUITY UPLC BEH Amide 2.1 x 50 mm, 1.7 µm

Bonded hybrid

Atlantis HILIC Silica 2.1 x 50 mm, 3 µm

Unbonded silica with high silanol activity

(1) acenaphthene (2) thymine (3) 5-fluoroorotic acid (4) adenine (5) cytosine; UV 254 nm






Hilic Amide – Carbohydrate Analysis

Further Application examples

Overview


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.


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

Before You Start: Column Equilibration and Wash Solvents


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

Before You Start: Influence of Sample Diluent








Overview


Implementing the Approach: Example 1, Water Soluble Vitamins

O

OH OH

O

OH

OH

Ascorbic acid

O

N

NH2

Nicotinamide

N

O

OHOH

CH3

Pyridoxal

N N

N

NH

O

OCH3

CH3

OH

OH

OH

OH

Riboflavin

O

N

OH

Nicotinic acid

N+

CH3

SN NH2

N

OHCH3

Thiamine

Cl-

N N

N N

CH3

CH3

NH2

O

CH3

NH2

O

O NH2

O

NH2

O

NH2

CH3

CH3

O

NH2

NH

P

O

O

CH3

CH3

O-

O

O

O

OH

N

OH

Co+

N

N CH3

CH3

H

CH3

CH3

B12

N

NH

N

N

NH

O

O

O

NH2

NH

O

OHOH

Folic Acid


Stationary Phase Selectivity at Low pH: Water-soluble Vitamins

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

BEH Amide

BEH HILIC

Atlantis HILIC Silica

1

2

4

3 6

5

8 7

1,6

3,2

4

8

5

7

1

6

3,2

4

8

5

7

Compounds 1. Nicotinamide 50 µg/mL 2. Pyridoxine 50 µg/mL 3. Riboflavin 30 µg/mL 4. Nicotinic acid 50 µg/mL 5. Thiamine 50 µg/mL 6. Ascorbic Acid 50 µg/mL 7. B12 50 µg/mL 8. Folic Acid 25 µg/mL

pH 3 All 3 columns yield different selectivity

BEH Amide has greatest resolution of all peaks


Stationary Phase Selectivity at High pH: Water-soluble Vitamins

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

BEH Amide

BEH HILIC

1

2

3

4 5

6

7 8

6

4

5 2

8 7

3

1


pH 9 Large selectivity difference between the two BEH chemistries

BEH Amide has greatest resolution of all peaks


Mobile Phase pH Selectivity: Water-soluble Vitamins

Minutes 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

pH 3

Minutes 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

1

2

3

4 5

6

7 8

pH 9

1

2

4

3 6

5

8 7


BEH Amide Selectivity difference between pH 3 and pH 9

Adequate resolution for both conditions Greater sensitivity at pH 9


Compounds 1. Nicotinamide 2. Pyridoxine 3. Riboflavin 4. Nicotinic acid 5. Thiamine 6. Ascorbic Acid 7. B12 8. Folic Acid

1

2

3

4

5

6

7 8

Minutes

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Final Method: Water-soluble Vitamins


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]








Overview


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


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


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


Stability Testing in ACN

After 100 Molasses inj.

1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (1mg/mL each)

LSU

0.0

100

200

300

400

500

600

700

800

900

Minutes 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

1

2

3

4

5

BEH Amide 2.1 x 150mm Column 75% ACN with 0.2% TEA, 0.29mL/min at 35 C

After 100 Honey inj.

Initial

After 100 Cereal inj.


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: Aminopropyl Column

Polyamine Column

Polymeric Amino Column

1

2

3

4

5

1

2

3

4,5

1

2

3

4

5

Minutes

0.0

2.0

4.0

6.0

8.0

10.0

12.0

*

*

*

*

1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (each 1mg/mL), * Salt Interferences


*

*

Salt Interferences ELSD using 75% ACN at 1.4mL/min, 35 C 4.6 x 150mm Columns, 10µL injection

5 Food Sugars

1

2

3

0.0 Minutes

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

4

5

BEH Amide With 0.1% TEA Mobile Phase Modifier Low Salt Retention = No Salt Interference

1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (each 1mg/mL), * Salt Interferences

with Salts


Reducing Sugar Anomers

O

H

O

O H

H H

H

H

O H O H

O H H

O

H O H

O

H

O

H O H

O

H

O

H H O

O

H

O

O H O H

H H

H

H

H O H O H

O H

-D-Glucose

O

H O H

H H

H

H

O H O H

O H

O H

-D-Glucose

Minutes 0.0 1.0 2.0 3.0 4.0 5.0

-Glucose

-Glucose

-Maltose

-Maltose

Sucrose

Isocratic: 75% ACN at 15 C

Mutarotation


Anomer Collapse

Minutes 0.0 1.0 2.0 3.0 4.0 5.0

Glucose Sucrose

Maltose

15 C 75% ACN

0.2% TEA in 75% ACN

25 C 75% ACN

0.2% TEA in 75% ACN

35 C 75% ACN

0.2% TEA in 75% ACN


Demonstration of Anomer Collapse

1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (each 1mg/mL) 2.1 x 150mm Column @ 0.29mL/min

1

2

3

4

5

1

2

3

4

5

75% ACN with 0.2% TEA

at 35 C

80% Acetone with 0.05% TEA

at 90 C

1

3

2

4

2

4

5

Minutes

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

5

75% ACN with no modifier

at 35 C

-D-glucopyranose

-D-glucopyranose

O

H

O

H

H

H

H

H

O

H

O

H

O

H

O

H

O

O

H

O

H

H

H

H

H

H

O

H

O

H

O

H


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

1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (1mg/mL each)

Food Sugars

1 2 3 4 5

Energy Drink

White Bread

Lamb Curry Meal

Tomato Ketchup

Strawberry Smoothie

Hot Cross Buns

Minutes 0.00 1.00 2.00 3.00 4.00

Raisin Bran Cereal


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


Food Sugar Region

Analysis of Beer

Minutes

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

Malto-tri, tetra, penta, hexa, hepta-ose

Double Chocolate Stout

Dark Ale

Pilsner

Wheat 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


Gain Switching

Analysis of Beer

Minutes

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

Malto-tri, tetra, penta, hexa, hepta-ose

Double Chocolate Stout

Food Sugar Standard

Xylo

se

Arab

inose

Glu

cose

Malt

ose

Lacto

se

Fu

cose M

alt

otr

iose

Malt

ote

traose

Malt

op

en

taose

Malt

oh

exaose

Malt

oh

ep

taose

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)


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

The ACQUITY BEH Method Selector Tool:

ACQUITY UPLC® BEH Amide Column



Step 1 Select Column Dimensions


Step 1 Select Column Dimensions


Step 2 Determine the Sample Type


Step 3 Select Detector


Step 4 Select Temperature









Overview


Quality Control checks Incoming raw materials

Stevia related compounds -


Ascorbic acid and Isoascorbic acid


Water Soluble Vitamins


Thank You?

Documents

Waters Amide Column Technology For Food Analysis · HILIC Retention Mechanisms are Complex Combination of partitioning, ion-exchange and hydrogen bonding •Polar analyte partitions