Assessment of HILIC Anne Mack Mode and Stationary of HILIC Mode and Stationary Phase for UHPLC/MS Anne Mack Agilent Technologies Applications Chemist Pittcon 2012 March 12, 2012 Session

Assessment of HILIC

Mode and Stationary

Phase for UHPLC/MS

Anne Mack Agilent Technologies

Applications Chemist

Pittcon 2012 March 12, 2012

Session 420 - Liquid Chromatography

1

Agenda

What is HILIC

How it is different than RPLC

How it works

HILIC Considerations

Method Development

Advantages & Disadvantages

Why HILIC works well with MS

Review ESI-MS Source

Application Example

2

What is HILIC

Hydrophilic Interaction Liquid Chromatography

A liquid chromatographic technique that has been in limited

used for a couple of decades

Uses a polar stationary phase such as silica, amino, mixed

mode, zwitterionic, etc.

Uses a water-miscible, non-polar mobile phase containing a

small amount of water (at least 2-3% by weight)

May use volatile buffers for compatibility with MS detection

Complements reversed phase chromatography (RPLC); retains

hydrophilic compounds and often reverses elution order

3

HILIC versus RPLC

RPLC

Non-polar stationary phase (e.g., C18)

Polar mobile phase (i.e., H20/CH3OH, H20/CH3CN, etc.)

Decrease retention by decreasing polarity of mobile phase

(e.g., increase CH3CN in mobile phase to decrease retention)

HILIC

Polar stationary phase (e.g., silica)

Polar mobile phase (i.e., H20/CH3CN)

Decrease retention by increasing polarity of mobile phase

(i.e., increase H2O in mobile phase to decrease retention)

4

HILIC Retention Mechanism on Silica Based

Columns

O O O O

H2O H2O

H2O H2O H2O

H2O

H2O H2O

- - - -

H2O

3

CH 2 CHCH 3

NH +

ACN

ACN ACN

ACN ACN

ACN ACN

ACN ACN ACN

3

CH 2 CHCH 3

NH +

1. Partitioning in and out of adsorbed water layer

2. Ion exchange with silanols

1.

2.

5

HILIC Method Development: Mobile Phase

Considerations

Organic solvent concentration

Solvent strength in HILIC mode:

THF < Acetone < CH3CN < IPA < EtOH < MeOH < H2O

H2O is the strongest solvent—need >2-3% H2O for hydration of silica

Ionic strength of buffer

Concentration of (salt) buffer

Different anions and cations may can also affect analyte retention

Type of buffer

Acetates, formates good, soluble in CH3CN—also MS friendly

Phosphates are bad due to low CH3CN solubility

6

HILIC Method Development: Common LC

Parameters

Type of stationary phase

Vary retention mechanism and selectivity

Mobile phase pH

Controls ionization of silica and analytes

Temperature

Increasing temperature will decrease retention

Increasing temperature will increase column efficiency

Decreasing temperature can improve selectivity

7

Advantages of HILIC

Retention of polar compounds that would be unretained by RPLC

Complimentary selectivity to RPLC

Good peak shape for basic compounds where RPLC may give tailing or low efficiency

Higher flow rates, long columns can be used due to low viscosity mobile phases with high organic content; greater efficiency

Can simplify sample preparations

SPE, liquid/liquid extraction often end up in high organic mobile phases and you can directly inject, no need to dry down and reconstitute in high aqueous, like for RPLC

May eliminate some solubility issues

Enhanced detection sensitivity with MS

Efficient spraying and de-solvation in electrospray MS

8

Advantage of HILIC: Peak Shape, Retention of

Basic Compounds

LC/MS/MS Separation of Paroxetine and Ranitidine

Paroxetine

Antidepressant

MW 329.36

Ranitidine

Antiulcerative

MW 314.41

OO

NF

O

S

N NO

N

NO2

Basic portion of molecule, impacts HILIC retention

9

LC/MS/MS Separation of Paroxetine and Ranitidine

Advantage of HILIC: Peak Shape, Retention of

Basic Compounds

min 2 4 6 8 10 12

6 2 4 8 10 12 min

Agilent ZORBAX XDB-C18, 2.1 x 150 mm

Mobile phase: 5-90% B in 10 min

Agilent ZORBAX Rx-Sil, 2.1 x 150 mm

Mobile phase: 100-50% B in 10 min

Agilent 1100 Series LC System

Agilent 1100 Series LC/MSD Trap

A: 8 mM ammonium formate

B: 8 mM ammonium formate in acetonitrile /

water (95:5)

0.3 mL/min

Gradient elution

Injection Volume: 5 µL of 100 ppb sample

Column: 40 oC

MS: ESI+, MRM, 350 oC, 10 L/min, 45 psi

Sample:

Paroxetine, m/z 330192

Ranitidine, m/z 315176

Improved retention, peak

shape and sensitivity for

ranitidine in HILIC mode

10

Disadvantages of HILIC

Mechanism not entirely understood; mixed mechanisms

Column overloading can be a problem

Similar to silanol overloading in RPLC

Overloaded HILIC peaks show fronting

Equilibration times can be long for certain column types

Particularly true for bare silica columns – take longer to

equilibrate initially, will take longer to equilibrate when

mobile phase changes for gradients or method

development are required

Cannot inject strong solvent (H2O, CH3OH) without distorting

peaks

11

LC/UV Separation of Nucleobases

Disadvantage of HILIC: Column Equilibration and

Re-equilibration

min 2 4 6 8 10 12 14 16

mAU

0

50

100

150

200

250

min 2 4 6 8 10 12 14 16

mAU

0

50

100

150

200

250

Agilent 1200 Series Quat LC System

Agilent ZORBAX Rx-Sil, 2.1 x 150 mm, 5 µm

A: 25 mM ammonium acetate with 2.5 mM

ammonium formate

B: acetonitrile

0.1 mL/min

Isocratic elution, 90% B

Injection Volume: 0.5 µL of 0.4 mg/mL

sample

Column: 25 oC

DAD: Sig=254,4nm, Ref=360,100nm; 1 s

data collection rate; flow cell 3 mm, 2 µL

Sample:

Thymine, Uracil, Adenine, Guanine, Cytosine

Equilibration is quicker

when changing from

high to low aqueous

Equilibrating from 80 to 90% acetonitrile:

WORKS

Equilibrating from 95 to 90% acetonitrile:

DOES NOT WORK

12

LC/MS Separation of Vitamin B Related Compounds

Disadvantage of HILIC: Injection Solvent Effects

with H2O and CH3OH

x102

0

0.5

1 H2O

x102

0

0.5

1 H2O/CH3CN (1:1)

x102

0

0.5

1 CH3CN

Counts (%) vs. Acquisition Time (min)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9

x102

0

0.5

1 CH3OH

x102

0

0.5

1 CH3OH/CH3CN (1:1)

PW1/2 = 0.021

PW1/2 = 0.024 PW1/2 = 0.048

PW1/2 = 0.019

PW1/2 = 0.113

PW1/2 = 0.107 PW1/2 = 0.056

PW1/2 = 0.016

PW1/2 = 0.064

PW1/2 = 0.047 PW1/2 = 0.050

PW1/2 = 0.017

PW1/2 = 0.023

PW1/2 = 0.031 PW1/2 = 0.049

PW1/2 = 0.016

PW1/2 = 0.026

PW1/2 = 0.044 PW1/2 = 0.054

PW1/2 = 0.014

Agilent 1290 Infinity LC System

Agilent 6410A LC/MS

Agilent ZORBAX RRHD HILIC Plus 2.1 x 50

mm, 1.8 µm

Acetonitrile / 100 mM ammonium formate pH

3.2 (9:1)

0.4 mL/min, Pressure: 135 bar

Isocratic elution

Injection Volume: 1 µL of 5 µg/mL sample

Column: 25 oC

MS: ESI+, SIM, 200 oC, 10 L/min, 30 psi,

4000 V, 15 ms dwell time

Sample:

4-Aminobenzoic acid, m/z 138 (Frag 110 V)

Nicotinamide, m/z 123 (Frag 130 V)

Riboflavin, m/z 377 (Frag 160 V)

Nicotinic acid, m/z 124 (Frag 130 V)

Strong injection solvents

negatively affect peak

shape and retention

13

Electrospray Ionization Mass Spectrometry with

HILIC

Eluent is dispersed by electrospray into fine aerosol

Ion formation involves extensive evaporation

Volatile mobile phases needed

Acetonitrile or methanol

Compounds that increase conductivity are commonly added to

mobile phase

Also need to be volatile (formates, acetates, etc)

HILIC is a good match for ESI-MS

Typically uses highly organic, volatile mobile phases

14

HILIC versus RPLC Example

LC/MS Separation of Morphine and its Metabolites

Morphine

Opiate analgesic

MW 285.34

Morphine-3-β-D-glucuronide (M3G)

Non-active metabolite

MW 461.46

Normorphine

Intermediate

MW 271.312

Morphine-6-β-D-glucuronide (M6G)

Major active metabolite

MW 461.46

15

2 x10

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0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4

Improved LC/MS Performance with UHPLC

Columns


Agilent 6410A LC/MS

A: 10 mM ammonium formate pH 3.2

B: acetonitrile / 100 mM ammonium formate

pH 3.2 (9:1)

0.4 mL/min


Injection Volume: 2 µL

Column: 25 oC


4000 V, 200 V delta EMV, 20 ms dwell time

Sample:

Normorphine, m/z 272

Morphine, m/z 286

Morphine-6-β-D-glucuronide (M6G), m/z 462


Selectivity maintained

across particle sizes;

peak width is improved

with smaller particles

Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 5 µm

Pressure: 90 bar

Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 3.5 µm

Pressure: 160 bar

Agilent ZORBAX RRHD Eclipse Plus C18, 2.1 x 100 mm, 1.8 µm

Pressure: 480 bar

16

2 x10

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0.2

0.4

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1 1 1

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0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4

Improved LC/MS Performance with UHPLC

Columns


Agilent 6410A LC/MS



pH 3.2 (9:1)

0.4 mL/min



Column: 25 oC



Sample:


Morphine, m/z 286



5x increase in S/N with

1.8 µm compared to 5

µm

S/NM6G = 7.7

S/NM6G = 15.2

S/NM6G = 37.5

Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 5 µm

Pressure: 90 bar

Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 3.5 µm

Pressure: 160 bar


Pressure: 480 bar

17

2 x10

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HILIC versus RPLC: Column Selectivity


Mobile phase: 10% B

Pressure: 480 bar

Agilent ZORBAX RRHD HILIC Plus, 2.1 x 100 mm, 1.8 µm

Mobile phase: 70% B

Pressure: 350 bar


Agilent 6410A LC/MS



pH 3.2 (9:1)

0.4 mL/min

Isocratic elution


Column: 25 oC



Sample:


Morphine, m/z 286



Complimentary

selectivity between a

HILIC column and a C18

18

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HILIC versus RPLC: ESI-MS Sensitivity


Mobile phase: 10% B

Pressure: 480 bar


Mobile phase: 70% B

Pressure: 350 bar


Agilent 6410A LC/MS



pH 3.2 (9:1)

0.4 mL/min

Isocratic elution


Column: 25 oC



Sample:


Morphine, m/z 286



More efficient spraying

and de-solvation in ESI-

MS with HILIC mode

than with RPLC

19

HILIC versus RPLC: ESI-MS Sensitivity


Agilent 6410A LC/MS



pH 3.2 (9:1)

0.4 mL/min

Isocratic elution


Column: 25 oC



Sample:


Morphine, m/z 286



4x Improvement in S/N

for M6G in HILIC mode

Agilent ZORBAX RRHD Eclipse Plus C18

2.1 x 100 mm, 1.8 µm

Mobile phase: 10% B

Pressure: 480 bar

Agilent ZORBAX RRHD HILIC Plus

2.1 x 100 mm, 1.8 µm

Mobile phase: 70% B

Pressure: 350 bar

S/NM6G = 37.5

S/NM6G = 143.9

20

HILIC with UHPLC/MS versus RPLC with LC/MS:

ESI-MS Sensitivity

S/NM6G = 143.9

Agilent ZORBAX Eclipse Plus C18

2.1 x 100 mm, 5 µm

Mobile phase: 10% B

Pressure: 90 bar

Agilent ZORBAX RRHD HILIC Plus

2.1 x 100 mm, 1.8 µm

Mobile phase: 70% B

Pressure: 350 bar


Agilent 6410A LC/MS



pH 3.2 (9:1)

0.4 mL/min

Isocratic elution


Column: 25 oC



Sample:


Morphine, m/z 286



20x improvement in S/N

with HILIC as compared

to RPLC

S/NM6G = 7.7

21

2 x10

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1 Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 5 µm

Mobile phase: 10% B

0.4 mL/min

Pressure: 90 bar

2 x10

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HILIC with UHPLC/MS versus RPLC with LC/MS:

Analysis Time and ESI-MS Sensitivity

S/NM6G = 7.7

S/NM6G = 100.4


Mobile phase: 70% B

1 mL/min

Pressure: 810 bar


Agilent 6410A LC/MS



pH 3.2 (9:1)

0.4 mL/min

Isocratic elution


Column: 25 oC

MS (0.4/1 mL/min): ESI+, SIM, 250/325 oC,

11/12 L/min, 30/55 psi, 4000 V, 200 V delta

EMV, 20/10 ms dwell time

Sample:


Morphine, m/z 286



>10x more sensitivity in

half the time with HILIC

and UHPLC/MS

22

Conclusions

HILIC provides good peak shape and retention for polar compounds

HILIC uses highly organic mobile phases

Volatility allows for more efficient spraying in ESI-MS, can increase

MS sensitivity by 4x compared to a similar RPLC analysis

Low viscosity permits faster flow rates allowed with UHPLC columns

and LC systems, resulting in analyses accomplished in less time,

while sub-2-µm columns still improve MS S/N compared to RPLC

Sub-2-µm UHPLC columns can improve MS sensitivity by 5x compared to

traditional 5 µm columns, while maintaining the same selectivity

Transferring a method from RPLC mode with a traditional 5 µm column to

HILIC mode with a UHPLC column can improve MS sensitivity by 20x

23

Documents

Assessment of HILIC Anne Mack Mode and Stationary of HILIC Mode and Stationary Phase for UHPLC/MS Anne Mack Agilent Technologies Applications Chemist Pittcon 2012 March 12, 2012 Session