HPLC Method Development for Polar Compounds on Superficially Porous Particle Columns

Anne Mack, William Long, Jason Link, Stephen Luke 2850 Centerville Road, Wilmington, DE 19808

Abstract

Conclusions

Introduction

Results and Discussion Results and Discussion

2.5 μm

0.75 μm

0.75 μm

1.7 μm

0.5 μm

0.5 μm

Poroshell 120 2.7 µm Poroshell 120 4 µm

Best all around

Best for low

pH mobile

Phases

Best for high

pH mobile

phases

Best for

alternative

selectivity

Best for more

polar

compounds

Poroshell 120

EC-C182.7 µm, 4 µm

Poroshell 120

SB-C182.7 µm

Poroshell

HPH-C182.7 µm, 4 µm

Poroshell 120

Bonus-RP2.7 µm

Poroshell 120

SB-Aq2.7 µm

Poroshell 120

EC-C82.7 µm, 4 µm

Poroshell 120

SB-C82.7 µm

Poroshell

HPH-C82.7 µm, 4 µm

Poroshell 120

PFP2.7 µm, 4 µm

Poroshell 120

EC-CN2.7 µm

Poroshell 120

Phenyl-Hexyl2.7 µm, 4 µm

Poroshell 120

HILIC2.7 µm, 4 µm

min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

B1B2B3B5B6B7B9B10B12C

min0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

B1B2B3B5B6B7B9B10B12C

0

1

2

3

4

5

EC-C18, 90% pH 2.5 Buffer, 10% CH3CN HPH-C18, 90% pH 7.5 Buffer, 10% CH3CN

Retention Times (min) for Water Soluble Vitamins

A: 20 mM sodium phosphate pH 2.5 or 7.5,

B: acetonitrile, 0.5 mL/min, 30 C, 210 nm

B1 B2 B3 B5 B6 B7 B9 B10 B12 C

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

EC-C18, 90% pH 2.5

Buffer, 10% CH3CN

SB-Aq, 100% pH 2.5

Buffer

Phenyl-Hexyl, 100% pH

2.5 Buffer

Bonus-RP, 100% pH 2.5

Buffer

PFP, 100% pH 2.5

Buffer

EC-CN, 100% pH 2.5

Buffer

Retention Times (min) for Water Soluble Vitamins

A: 20 mM sodium phosphate pH 2.5, B: acetonitrile, 0.5 mL/min, 30 C, 210 nm

B1 B2 B3 B5 B6 B7 B9 B10 B12 C

Retention

Times

> 5 min

(k’ > 20)

k’ = 2

V0

min0.25 0.5 0.75 1 1.25 1.5 1.75 2

B1B2B3B5B6B7B9B10B12C

0

1

2

3

4

5

EC-C18, 90% pH 2.5 Buffer, 10% CH3CN EC-C18, 90% Ion Pair, 10% CH3CN

Retention Times (min) for Water Soluble Vitamins

A: 1.5 g sodium heptanesulfonate/0.2 mL triethylamine/

7.5 mL acetic acid/993 mL water,

B: acetonitrile, 0.5 mL/min, 30 C, 260 nm

B1 B2 B3 B5 B6 B7 B9 B10 B12 C

0

1

2

3

4

5

EC-C18, 90% pH 2.5 Buffer, 10%

CH3CN

HILIC, 97% CH3CN, 3% pH 3.0

Buffer

Glycan Mapping, 97% CH3CN, 3%

pH 3.0 Buffer

HILIC, 97% CH3CN, 3% pH 5.8

Buffer

Glycan Mapping, 97% CH3CN, 3%

pH 5.8 Buffer

Retention Times (min) for Water Soluble Vitamins

A: 100 mM ammonium formate pH 3.0 or 100 mM ammonium acetate pH 5.8,

B: acetonitrile, 0.5 mL/min, 30 C, 260/210 nm

B1 B2 B3 B5 B6 B7 B9 B10 B12 C

>

>

>

>

A: 20 mM NaH2PO4 pH 2.5

B: CH3CN, 10% B isocratic

0.5 mL/min, 30 C, 210 nm

2.1 x 50 mm, 2.7 μm

Poroshell 120 EC-C18

A: 20 mM Na2HPO4 pH 7.5

B: CH3CN, 10% B isocratic

0.5 mL/min, 30 C, 210 nm

2.1 x 50 mm, 2.7 μm

Poroshell 120 HPH-C18

A: Ion Pair Mobile Phase

B: CH3CN, 10% B for 1 min,

then 10-40% B in 1 min

0.5 mL/min, 30 C, 260 nm

2.1 x 50 mm, 2.7 μm

Poroshell 120 EC-C18

A: 20 mM NaH2PO4 pH 2.5

B: CH3CN, 0% B for 2 min,

then 0-30% B in 2.5 min

0.5 mL/min, 30 C, 210 nm

2.1 x 50 mm, 2.7 μm

Poroshell 120 Phenyl-Hexyl

Adequately retaining and separating small polar moleculeswith reversed-phase liquid chromatography (RPLC) is oftena challenging task. Alkyl phase LC columns, like C18, are acommon starting point for LC method development.However, highly polar analytes are poorly retained on non-polar C18 stationary phases. In order to increase retentionof these compounds, there are several techniques that canbe tried, such as: adjusting the mobile phase pH when theanalytes are ionizable, adding an ion pairing reagent to themobile phase, or selecting a more appropriate columnstationary phase for the analysis. The wide variety ofstationary phase chemistries currently available onsuperficially porous particle columns can facilitate methoddevelopment; several chemistries are well suited fortroublesome polar analytes and can be used under 100%aqueous conditions. Superficially porous particles areknown for their ability to generate high efficiency with lowback pressure. High efficiency can contribute to resolvingclosely eluting peaks, while low back pressure allows forflexibility with LC instrumentation. This work willdemonstrate a logical, stepwise methodology to enablechemists to retain and separate their polar analytes withsuperficially porous particle columns.

Agilent InfinityLab Poroshell 120 Columns:

Efficiency 90% of < 2 μm TPP Efficiency 2x 5 μm TPPPressure 50% of < 2 μm TPP Pressure often below 200 bar2 μm inlet frit 2 μm inlet frit

A variety of selectivities are available on InfinityLabPoroshell 120 Columns to meet almost any application need

These phases can be used with 100% aqueous mobile phasesto improve retention of highly polar analytes in RPLC mode

Compounds of Interest: Water-Soluble Vitamins

Thiamine, B1 Riboflavin, B2 Nicotinic Acid, B3

Pantothenic Acid, B5 Pyridoxine, B6 Biotin, B7

Folic Acid, B9 PABA, B10 Cyanocobalamin, B12

Ascorbic Acid, C

C18 columns are usually a good starting point for HPLC

method development. However, highly polar analytes can be

difficult to retain.

• Poor overall retention

• For 6 compounds, k’ < 2

• 4 unretained compounds

• Significant coelution

If analytes are ionizable, adjusting the mobile phase pH can

alter retention.

• At low pH, acids are neutral (unionized) and more retained.

• At high pH, bases are neutral and more retained.

• Non-ionizable compounds are unaffected by changes in pH.

• pH 2.5 mobile phase

allows slightly more

retention than pH 7.5

• Most compounds do not

change ionization state

between pH 2.5 and 7.5

• HPH-C18 is designed for

improved lifetime with

high pH applications

Decreasing the mobile phase strength improves retention.

C18 columns generally should not be used with too little

organic, due to the risk of dewetting. However, there are

several stationary phase options that can be successfully

used with 100% aqueous mobile phases.

• Dewetting occurs when the chromatographic pores dry out

and the non-polar surface expels the pure aqueous polar

mobile phase.

• When the pores dry out, the analyte cannot get in and will

not be retained by the column stationary phase.

• Dewetting can be observed by a reduction in V0 and a

sudden loss of retention.

• Low pH mobile phase was used for the above experiments

due to improved retention, compared to high pH

• Using a column compatible with 100% aqueous mobile

phase significantly improves overall analyte retention

• In this case, Phenyl-Hexyl has the best retention, with 7

compounds having k’> 2; this is improved from the original

C18 analysis where only 4 compounds had k’ > 2

Ion pairing agents can be added to mobile phases to helpimprove retention of polar analytes.• A traditional C18 or C8 column is typically used in ion

pairing to improve retention of charged polar molecules.• The ion pairing reagent has a charge at one end and a non-

polar tail.• The non-polar end sticks to the non-polar stationary phase,

while the charged end extends into the mobile phase.• For acids, a positively charged reagent should be used,

while a negatively charged reagent is optimal for retainingbases.

• The ion pairing reagent increased retention for most compounds

• 6 compounds have k’ > 2• B5 and B7 could not be

detected due to low signal and high background noise at 210 nm (not detectable at 260 nm)

If all efforts for reversed-phase liquid chromatography fail toretain troublesome polar analytes, then hydrophilicinteraction chromatography (HILIC) might be an option.• In HILIC mode, the stationary phase is a polar material.• Solvent elution strength is different compared to RPLC:

water> methanol>ethanol>IPA>acetonitrile>acetone• Use less polar solvent to increase retention of polar

analytes; HILIC mobile phases are typically highly organic(>80% CH3CN) with a smaller amount of aqueous mobilephase as the strong eluting solvent.

• Alternate selectivities are also available for HILIC mode• For ionizable analytes, mobile phase pH can also be

changed to manipulate retention in HILIC mode• At pH 5.8, all compounds have k’ > 2 in HILIC mode;

however selectivity is slightly better at pH 3.0 with lesscoelution of compounds

• C18 columns are usually a good column to try first for LCmethod development, however they may not provideenough retention for polar analytes

• For poorly retained ionizable compounds, mobile phase pHcan be used to adjust retention

• Several stationary phases are available that can be usedwith 100% aqueous mobile phase to improve retention ofpolar analytes without the risk of dewetting

• Ion pairing agents can be added to mobile phases toimprove retention of polar analytes

• If all else fails with RPLC, try HILIC for polar analytes• Similar method development techniques can be applied to

HILIC: adjust mobile phase pH & try alternate selectivities

A: 100 mM NH4HCO2 pH 3

B: CH3CN, 97% B for 2.5 min,

then 97-60% B in 5 min

0.5 mL/min, 30 C, 260/210 nm

2.1 x 50 mm, 2.7 μm

Poroshell 120 HILIC

min1 2 3 4 5 6 7

B1B2B3B5B6B7B9B10B12C

min0.5 1 1.5 2 2.5 3 3.5 4

B1B2B3B5B6B7B9B10B12C