improving the analysis of polar analytes with a novel ... 2017 - hilic... · improving the analysis...

Improving the Analysis of Polar Analytes with a Novel HILIC Phase on

Superficially Porous Particle LC ColumnsAnne Mack, William Long, Adam Bivens, Jason Link

HPLC 2017

Prague, Czech Republic

Introduction

Conclusions

Experimental

Results and Discussion Results and Discussion

1.7 μm

0.5 μm

0.5 μm

(1)

(2) O-O-O-

O-

H2O

H2O

H2O H2O H2O

H2O

H2O

H2O

H2O

CH2CHCH3

NH3+

CH3CN

CH3CN

CH3CN

CH3CNCH3CN

CH3CN

CH3CN

CH3CN CH3CN CH3CN

CH3CN

CH3CNCH3CN

CH2CHCH3

NH3+

H2O H2O

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mV

-100

0100

200

300

400

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mV

-50-250

255075

100125150175

Na+Cl-

Na+ H2PO4-

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mV

-100

102030405060

H2PO4-

K+

A: 20 mM ammonium formate pH 3.0 in water, B: acetonitrile, 80-20% B in 10 minutes, 0.4 mL/min, ELSD, 40 oC/3.5 psi

min2.5 3 3.5 4 4.5 5 5.5 6 6.5 7

mAU

01020304050

min2.5 3 3.5 4 4.5 5 5.5 6 6.5 7

mAU

0

10

20

30

40

50

1.7 μm Totally Porous Particle Competitive Amide (2.1 x 100 mm)

Press = 267 bar

2.7 μm Prototype P120 HILIC-Z (2.1 x 100 mm)

Press = 148 bar

Tartaric

Acid

Oxalic Acid

Isocitric AcidCitric Acid

Tartaric

Acid

Oxalic Acid

Isocitric Acid Citric Acid

min2.5 3 3.5 4 4.5 5 5.5 6 6.5 7

mAU

0

10

20

30

40

50

3.5 μm Totally Porous Particle Competitive Zwitterion (2.1 x 100 mm)

Press = 90 bar

Tartaric

Acid

Oxalic Acid

Isocitric Acid Citric Acid

A: 200 mM ammonium acetate pH 6.8 in water, B: acetonitrile, 70% B isocratic, 0.3 mL/min, 220 nm

min0 5 10 15 20 25 30 35

mV

0

200

400

600

800

1000

min0 5 10 15 20 25 30 35

mV

0

200

400

600

800

1000 160 column volumes

13680 column volumes

Fructose

GlucoseSucrose

Maltose

A: 1.0% ammonium hydroxide pH 12.0 in water, B: acetonitrile,

90% B for 40 min, 0.4 mL/min, 35 oC, ELSD, 60 oC, 3.5 psi

Counts (%) vs. Acquisition Time (min)0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2 4.6 5 5.4 5.8 6.2 6.6 7 7.4 7.8 8.2 8.6 9 9.4 9.8

A: 100 mM ammonium acetate pH ~10 in water, B: acetonitrile, 80-70% B in 10 minutes, 0.4 mL/min, 30 oC, ESI-,

MS2SIM, 200 oC, 5 L/min, 45 psi, 350 oC, 11 L/min, -4000 V, 1 μL injection of 0.4 mg/mL glyphosate + 0.5 mg/mL

AMPA in acetonitrile/water (3:2)

Compound MW M-H Frag Dwell

Glyphosate 169 168 70 200

AMPA 111 110 70 200

Paraquat

(254 nm)

Diquat

(308 nm)

Tail Factor 1.55 1.93

Peak Width 0.037 0.045

Resolution 5.10 5.08

Paraquat

(254 nm)

Diquat

(308 nm)

Tail Factor 2.74 2.86

Peak Width 0.046 0.067

Resolution 1.36 1.46

A: 10 mM ammonium formate pH 3.0 in water, B: 10 mM ammonium formate pH 3.0 in acetonitrile/water

(9:1), 90-40% B in 6 minutes, 0.4 mL/min, 40 oC, 254 and 308 nm, 80 Hz, 5 μL injection of Sigma Aldrich

Standard paraquat/diquat/glyphosate in water (QC1435-2ML), diluted 1:10 in acetonitrile

1.7 μm Totally Porous Particle Competitive Amide (2.1 x 100 mm)

Pmax = 372 bar

min1 2 3 4 5

Sig=254,8 Ref=offSig=308,8 Ref=off

2.7 μm Prototype P120 HILIC-Z (2.1 x 100 mm)

Pmax = 196 bar

min1 2 3 4 5

Sig=254,8 Ref=offSig=308,8 Ref=off

1.7 μm Totally Porous Particle Competitive Amide

(2.1 x 100 mm)

Pmax = 410 bar

2.7 μm Prototype P120 HILIC-Z

(2.1 x 100 mm)

Pmax = 220 bar

Counts (%) vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14

A: 10 mM ammonium formate pH 3.0 in water, B: 10 mM ammonium formate pH 3.0 in acetonitrile/water (9:1),

100-70% B in 15 minutes, 0.4 mL/min, 15 oC, ESI+, MS2SIM, 300 oC, 5 L/min, 45 psi, 400 oC, 11 L/min, 3500 V, 5

μL injection of Agilent Amino Acid standard, 1 nmol/μL (5061-3330), diluted 1:10 in acetonitrile

Amino Acid M+H

Glycine 76

L-histidine 156

L-tyrosine 182

L-methionine 150

L-serine 106

L-alanine 90

L-phenylalanine 166

L-glutamic acid 148

L-proline 116

L-iso/leucine 132

L-arginine 175

L-threonine 120

L-valine 118

L-lysine 147

L-aspartic acid 134

The rapidly advancing fields of metabolomics, pharmaceuticals,pesticide analysis, and food testing have encountered a majorchallenge in the separation and quantitation of the many highlypolar analytes of interest in these areas. Adequately retaining andseparating small polar molecules with reversed-phase liquidchromatography (RPLC) is often a challenging task. Alkyl phase LCcolumns, like C18, are a common starting point for LC methoddevelopment, but highly polar analytes are poorly retained on thesenon-polar stationary phases.

Several techniques have been developed to analyze polarcompounds: pH adjustment, ion pairing, normal phasechromatography, derivatization, ion chromatography, and hydrophilicinteraction chromatography (HILIC). Of these, HILIC has rapidlygained popularity among many chromatographers for its ability toretain and separate polar analytes.

A main advantage of HILIC mode is that it uses the same solventsand system as RPLC. Unlike ion pairing, HILIC leaves no residue inthe system and operates in both positive and negative modes on amass spectrometer. Moreover, in HILIC mode, water is the strongeluting solvent and most analytes elute in >50% organic solvent.These high levels of organic have distinct advantages overtraditional RPLC in both sample preparation and LCMS analyticalsensitivity. Samples prepared in organic solvents can be injecteddirectly without drying or solvent transfer, and the higher volatilityof organic solvents improves the ionization efficiency and samplerecovery on mass spectrometers.

Superficially porous particles are known for their ability to generatehigh efficiency with low back pressure. This high efficiency iscritical to resolving closely eluting peaks, while low back pressureallows for flexibility with LC instrumentation. These distinctadvantages make superficially porous particles the ideal platform fordeveloping a next generation HILIC chemistry.

This work will demonstrate the direct analysis of highly polaranalytes, without requiring the use of ion pairing or derivatizationagents, by utilizing a novel HILIC phase on superficially porousparticle columns.

Possible retention mechanisms on a silica-based HILIC column:

• A water layer is adsorbed onto the polar silica surface, creatinga liquid/liquid extraction system

• Polar analytes can partition into and out of the water layer, withmore polar analytes having a stronger interaction (1)

• Charged polar analytes can also undergo ion exchange with thesilica surface (2)

• Elution is typically from least to most polar, opposite of RPLC

• Solvent strengths in HILIC mode are:

• THF < acetone < acetonitrile < isopropanol < ethanol <methanol < water

This work will use a prototype column with a novel high-pHstable zwitterion stationary phase on 2.7 μm Agilent InfinityLabPoroshell 120 particles (prototype P120 HILIC-Z).

Structure of a 2.7 μm Agilent InfinityLab Poroshell 120 particle:

• Efficiency is 90% of sub-2-μm totally porousparticle columns

• Pressure is 50% of sub-2-μm totally porousparticle columns

• 2 μm inlet frit to reduceclogging with dirtysamples

Metabolomics:

Using an Agilent BioInert LC System with PEEK capillariescombined with a prototype PEEK-lined P120 HILIC-Z column canimprove metabolite analysis, pH 6.8, 40 oC

Additional improvements can be made to metabolite analyticalsensitivity by choosing an appropriate mobile phase buffer pH,metabolites shown are at 5 ppm

Amino Acids:

Better resolution and 45% lower back pressure with InfinityLabPoroshell 120 compared to competitive amide column

Pesticides:

InfinityLab Poroshell 120 has better peak shape and betterresolution for paraquat/diquat

Glyphosate and AMPA are successfully analyzed with a prototypeP120 HILIC-Z (2.1 x 100 mm) column

Carbohydrates:

2.7 μm prototype P120 HILIC-Z (2.1 x 100 mm) column can be usedto separate sugars, and is robust and chemically stable at pH 12,35°C for at least 14000 column volumes

Organic Acids:

InfinityLab Poroshell 120 column shows faster separation of organicacids compared to two competitive HILIC columns

Inorganic Ions:

2.7 μm prototype P120 HILIC-Z (PEEK-lined 2.1 x 100 mm) columnshows good separation of cations and anions

• A novel high-pH stable zwitterion stationary phase wassuccessfully bonded to 2.7 μm Agilent InfinityLab Poroshell120 particles

• The prototype P120 HILIC-Z phase has combined advantagesof excellent chemical stability, batch-to-batch reproducibility,superior peak shape, and superficially porous particles

• Polar molecules from various application areas are wellretained and separated; these areas include but are notlimited to metabolomics, pesticides, carbohydrates, organicacids, and inorganic ions

• With PEEK-lined hardware, the column is also highly inert,enhancing analytical sensitivity for some analyses; analyticalsensitivity can be further improved with an inert LC systemand by using an optimal mobile phase

For Research Use Only. Not for use in diagnostic procedures.