Improving the Analysis of Polar Analytes with a Novel ... 2017 - HILIC... · Improving the Analysis...
1
Improving the Analysis of Polar Analytes with a Novel HILIC Phase on Superficially Porous Particle LC Columns Anne 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 - H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O CH 2 CHCH 3 NH 3 + CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 3 CN CH 2 CHCH 3 NH 3 + H 2 O H 2 O min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 mV -100 0 100 200 300 400 min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 mV -50 -25 0 25 50 75 100 125 150 175 Na + Cl - Na + H 2 PO 4 - min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 mV -10 0 10 20 30 40 50 60 H 2 PO 4 - 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 o C/3.5 psi min 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 mAU 0 10 20 30 40 50 min 2.5 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 Acid Citric Acid Tartaric Acid Oxalic Acid Isocitric Acid Citric Acid min 2.5 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 min 0 5 10 15 20 25 30 35 mV 0 200 400 600 800 1000 min 0 5 10 15 20 25 30 35 mV 0 200 400 600 800 1000 160 column volumes 13680 column volumes Fructose Glucose Sucrose Maltose A: 1.0% ammonium hydroxide pH 12.0 in water, B: acetonitrile, 90% B for 40 min, 0.4 mL/min, 35 o C, ELSD, 60 o C, 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 o C, ESI-, MS2SIM, 200 o C, 5 L/min, 45 psi, 350 o C, 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 o C, 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 min 1 2 3 4 5 Sig=254,8 Ref=off Sig=308,8 Ref=off 2.7 μm Prototype P120 HILIC-Z (2.1 x 100 mm) Pmax = 196 bar min 1 2 3 4 5 Sig=254,8 Ref=off Sig=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 o C, ESI+, MS2SIM, 300 o C, 5 L/min, 45 psi, 400 o C, 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 major challenge in the separation and quantitation of the many highly polar analytes of interest in these areas. Adequately retaining and separating small polar molecules with reversed-phase liquid chromatography (RPLC) is often a challenging task. Alkyl phase LC columns, like C18, are a common starting point for LC method development, but highly polar analytes are poorly retained on these non-polar stationary phases. Several techniques have been developed to analyze polar compounds: pH adjustment, ion pairing, normal phase chromatography, derivatization, ion chromatography, and hydrophilic interaction chromatography (HILIC). Of these, HILIC has rapidly gained popularity among many chromatographers for its ability to retain and separate polar analytes. A main advantage of HILIC mode is that it uses the same solvents and system as RPLC. Unlike ion pairing, HILIC leaves no residue in the system and operates in both positive and negative modes on a mass spectrometer. Moreover, in HILIC mode, water is the strong eluting solvent and most analytes elute in >50% organic solvent. These high levels of organic have distinct advantages over traditional RPLC in both sample preparation and LCMS analytical sensitivity. Samples prepared in organic solvents can be injected directly without drying or solvent transfer, and the higher volatility of organic solvents improves the ionization efficiency and sample recovery on mass spectrometers. Superficially porous particles are known for their ability to generate high efficiency with low back pressure. This high efficiency is critical to resolving closely eluting peaks, while low back pressure allows for flexibility with LC instrumentation. These distinct advantages make superficially porous particles the ideal platform for developing a next generation HILIC chemistry. This work will demonstrate the direct analysis of highly polar analytes, without requiring the use of ion pairing or derivatization agents, by utilizing a novel HILIC phase on superficially porous particle columns. Possible retention mechanisms on a silica-based HILIC column: • A water layer is adsorbed onto the polar silica surface, creating a liquid/liquid extraction system • Polar analytes can partition into and out of the water layer, with more polar analytes having a stronger interaction (1) • Charged polar analytes can also undergo ion exchange with the silica 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-pH stable zwitterion stationary phase on 2.7 μm Agilent InfinityLab Poroshell 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 porous particle columns • Pressure is 50% of sub- 2-μm totally porous particle columns • 2 μm inlet frit to reduce clogging with dirty samples Metabolomics: Using an Agilent BioInert LC System with PEEK capillaries combined with a prototype PEEK-lined P120 HILIC-Z column can improve metabolite analysis, pH 6.8, 40 o C Additional improvements can be made to metabolite analytical sensitivity by choosing an appropriate mobile phase buffer pH, metabolites shown are at 5 ppm Amino Acids: Better resolution and 45% lower back pressure with InfinityLab Poroshell 120 compared to competitive amide column Pesticides: InfinityLab Poroshell 120 has better peak shape and better resolution for paraquat/diquat Glyphosate and AMPA are successfully analyzed with a prototype P120 HILIC-Z (2.1 x 100 mm) column Carbohydrates: 2.7 μm prototype P120 HILIC-Z (2.1 x 100 mm) column can be used to 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 organic acids compared to two competitive HILIC columns Inorganic Ions: 2.7 μm prototype P120 HILIC-Z (PEEK-lined 2.1 x 100 mm) column shows good separation of cations and anions • A novel high-pH stable zwitterion stationary phase was successfully bonded to 2.7 μm Agilent InfinityLab Poroshell 120 particles • The prototype P120 HILIC-Z phase has combined advantages of excellent chemical stability, batch-to-batch reproducibility, superior peak shape, and superficially porous particles • Polar molecules from various application areas are well retained and separated; these areas include but are not limited to metabolomics, pesticides, carbohydrates, organic acids, and inorganic ions • With PEEK-lined hardware, the column is also highly inert, enhancing analytical sensitivity for some analyses; analytical sensitivity can be further improved with an inert LC system and by using an optimal mobile phase For Research Use Only. Not for use in diagnostic procedures.
Transcript of 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.
![Liquid Chromatography: Injection Broadening in Ion ...€¦ · Liquid Chromatography (HILIC) is excellent in separating polar compounds which dissolve in water [5,9]. Ion-Exchange](https://static.fdocuments.in/doc/165x107/5f4978d8b32e7510da629122/liquid-chromatography-injection-broadening-in-ion-liquid-chromatography-hilic.jpg)
