method development tools used in hydrophilic interaction

Method Development Tools Used in Hydrophilic Interaction

Chromatography (HILIC) for the analysis of Polar Basic Pharmaceuticals

Min Seok Changa, Kevin A. Schugb, and Ritu Aroraa

a: Varian, Inc., 25200 Commercentre Drive, Lake Forest, CA 92630, USA

b: Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX 76019, USAInspiring Excellence™

Ionic Strength - Salt Concentration Effect

Solvent Strength Effect

AbstractThe analysis of hydrophilic compounds is a major challenge to any researcher. Different modes of

chromatography are employed to gain retention of extremely polar compounds, e.g. ion exchange,

ion-pair reversed phase, or polar functionalized chemistries. Each mode suffers from different

limitations, be it high ionic strength, incompatibility with MS detection, insufficient retention, or time-

consuming sample preparation. HILIC (hydrophilic interaction chromatography) has been drawing a

great attention in various industries including pharmaceutical, environmental, and food industry for the

separation for hydrophilic compounds. Since HILIC-mode columns are inherently different from

conventionally used reversed phase or normal phase columns, a better understanding of the retention

mechanism involved and guidelines for method development are essential for their proper use and

maintenance.

The authors have screened several bonded phases for use in HILIC mode. Out of several options

available, we have selected a unique diol chemistry (Varian Prototype HILIC Diol) for investigating

different parameters used in method development for the analysis of popular polar basic

pharmaceuticals. Selectivity options have been explored using different tools such as organic:aqueous

modifier ratios, solvent strength, pH, ionic strength in terms of salt concentration of a single buffer

system and comparison of different buffer types. Besides these, selectivity differences between HILIC-

mode and C18 columns have also been looked into to demonstrate the benefit of HILIC-mode

columns over conventionally used reverse phase column for polar compound analysis. Data will be

presented illustrating the effect of different method development options available to an end-user

working with polar analytes and diol bonded phases.

54.84.64.44.243.83.63.43.232.82.62.42.221.81.61.41.210.80.60.40.20

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10mM Pursuit XRs Diol 3u_90B_0@4_beta blocker mix_3.DATAmAU

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54.84.64.44.243.83.63.43.232.82.62.42.221.81.61.41.210.80.60.40.20

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Mobile Phase A: 10, 50, 100, 250 mM ammonium formate (pH = 3.0) Column: Prototype HILIC Diol 3 , 100 X 2.0 mm

Mobile Phase B: Acetonitrile Flow rate: 0.4 mL/min

90% B Isocratic Detection: 272 nm

Flow rate: 0.4 mL/min

1. Acenaphthene

(t0 marker)

2. Pindolol 3. Practolol 4. Atenolol

log P 3.92 1.75 0.79 0.16

Conc. 0.2 mg/mL 0.1 mg/mL 0.3 mg/mL 1.1 mg/mL

1. Acenaphthene

(t0 marker)

2. Ascorbic acid 3. Cytosine

log P 3.92 -1.85 -1.73

Conc. 0.1 mg/mL 0.7 mg/mL 0.2 mg/mL

65.554.543.532.521.510.50

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250mM Pursuit XRs Diol 3u_90_5_5 H2O_0@4_beta blocker mix_4.DATAmAU

5:90:5 = Buffer:ACN:H2O1

2 34

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250mM Pursuit XRs Diol 3u_90_5_5 MeOH_0@4_beta blocker mix_4.DATAmAU

5:90:5 = Buffer:ACN:MeOH12 3

4

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250mM Pursuit XRs Diol 3u_90_5_5 IPA_0@4_beta blocker mix_6.DATAmAU

5:90:5 = Buffer:ACN:IPA12

3 4

Column: Prototype HILIC Diol 3 , 100 X 2.0 mm

Mobile Phase A: 50:50 premix of 250 mM ammonium formate

(pH = 3.0) and H2O

Mobile Phase B: ACN

C: organic modifier (MeOH, IPA, and H2O)

A:B:C = 5:90:5

Flow Rate: 0.4 mL/min

Temperature: Ambient

Detection: 272 nm

Samples: 1. Acenaphthene

2. Pindolol

3. Practolol

4. Atenolol

Fig 3. Compounds with positive log P values

Fig 4. Compounds with negative log P values

Solvent strength in HILIC mode

THF < Acetone < ACN < IPA < EtOH < MeOH < H2O (H2O is the strongest solvent in HILIC mode)

Higher ionic strength possibly suppresses electrostatic

interactions with the silica, as it removes some secondary

interaction modes with the analytes due to a shielding

effect. Retention times are lowered, but not too much,

especially for the ones that are more hydrophobic (pindolol

and practolol) which are less resistant to changes in buffer

strength. Interaction with the diol dominates at higher buffer

strength.

The hydrophilic molecules are induced to partition into a

water rich layer at the stationary phase surface that has a

higher proportion of salt associated with it. Interactions with

the higher concentration of salt in the “immobilized” water

layer may cause added retention.

Retention time of analytes changes

according to the solvent strength of mobile

phase. The use of a relatively weak solvent

like IPA increases retention of all analytes.

Complementary Selectivity to Reverse PhasePrototype HILIC Diol 3 100 X 2.0 mm

90:10 ACN:buffer Isocratic

1073 psi

Pursuit XRs C18 3 100 X 2.0 mm

60:40 ACN:buffer Isocratic

2075 psi

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100mM Pursuit XRs 3 C18_60B_0@4_atenolol_naphthalene_mix_4.DATAmAU

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100mM Pursuit XRs Diol 3u_90B_0@4_atenolol_naphthalene_mix_5.DATAmAU 1

1

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Columns: Prototype HILIC Diol 3 , 100 X 2.0 mm

Pursuit XRs C18 3 , 100 X 2.0 mm

Mobile Phase A: 100 mM ammonium formate (pH = 3.0)

B: Acetonitrile

90% B Isocratic (for HILIC, Prototype HILIC Diol 3 , 100 X 2.0 mm)

60% B Isocratic (for RP, Pursuit XRs C18 3 , 100 X 2.0 mm)


Detection: 272 nm

Temperature: ambient

1. Naphthalene 2. Atenolol

log P 3.30 0.16

Conc. 0.2 mg/mL 0.8 mg/mL

Complementary selectivity between reverse

phase column and HILIC column

Ionic Strength Effect – Different cation / anion effect in buffer

ACN vs. MeOH MS Application

a) Mobile Phase Effect

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100mM Pursuit XRs Diol 3u_90B_0@4_beta blocker mix_4.DATAmAUMobile Phase A: 100 mM ammonium formate (pH = 3.0)1

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formic acid buffer pH3_Pursuit XRs Diol 3u_90B_0@4_beta blocker mix_5.DATAmAU Mobile Phase A: Formic acid (pH = 3.0)1

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100mM KH2PO4 pH4_Pursuit XRs Diol 3u_90B_0@4_beta blocker mix_9.DATAmAU

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100mM ammonium acetate pH4_Pursuit XRs Diol 3u_90B_0@4_beta blocker mix_3.DATAmAU


Mobile Phase A: 100 mM ammonium acetate (pH = 4.0)

1

2 3

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Column: Prototype HILIC Diol 3 , 100 X 2.0 mm

Mobile Phase A: Various (Specified in the chromatograms.)

100 mM ammonium acetate (pH = 4.0)

100 mM ammonium formate (pH = 4.0)

100 mM ammonium formate (pH = 3.0)

Formic acid buffer (pH = 3.0)

Mobile Phase B: Acetonitrile

90% B Isocratic

Nicotinamide

Pindolol

Salbutamol

Atenolol

Mobile Phase A: 0.1% formic acid in H2O

Mobile Phase B: ACN

Sample solvent: 90:10 ACN:H2O (10% aqueous)

Data was irreproducible due to low ionic strength of mobile phase.

Mobile Phase A: 0.1% formic acid in H2O

Mobile Phase B: 0.1% formic acid in ACN


Data was irreproducible due to low ionic strength of mobile phase.

Typical MS mobile phase A and B for reverse phase separation

Mobile Phase A: 20 mM ammonium formate in H2O (pH = 3.0)

Mobile Phase B: ACN


Data was reproducible.

Ideal HILIC mobile phase condition on prototype HILIC Diol

Salbutamol Pindolol Atenolol Nicotinamide

Log P 0.64 1.75 0.16 -0.37

Structure

Conc.

(ng/mL)29.4 29.4 117.6 411.8

Sample

solvent

90:10 ACN:H2O (when mobile phase B was ACN-based) or

90:10 MeOH:H2O (when mobile phase B was MeOH-based)

Column: Prototype HILIC Diol 3 100 X 2.0 mm

Mobile phase A: Specified in the chromatograms.

Mobile phase B: Specified in the chromatograms.

A:B 10:90 Isocratic


Detection: Varian 320 LC/MS/MS


Inj. Vol.: 20 L

THF < Acetone < ACN < IPA < EtOH < MeOH < H2O (H2O is the strongest solvent in HILIC mode)

ACN Based Organic Mobile Phase

MeOH Based Organic Mobile PhaseColumn: Prototype HILIC Diol 3 100 X 2.0 mm


Mobile Phase B: MeOH


Data were reproducible.

Poor peak shape.

Significantly reduced retention due to the presence of a strong

organic solvent (MeOH) in mobile phase B.

Nicotinamide

Pindolol

Salbutamol

Atenolol

Formic acid alone cannot produce good peak

shapes for basic analytes due to a) low ionic

strength, and b) possible cationic exchange

effect with the sorbent (3 - 4). Both basic

analytes and silanol groups are ionized under

HILIC conditions with 0.1% formic acid. The

hydronium cation generated by formic acid is

not an effective competing cation for the

ionized silanol groups. Therefore, the ion

exchange component of the cationic basic

analyte is more influential on overall

separation performance. Peak shapes are

greatly improved if a cation with a higher

affinity for the ionized silanols, such as

ammonium, is added to the mobile phase.


Detection: 272 nm, Temperature: Ambient

Samples: 1. Acenaphthene, 2. Pindolol

3. Practolol, 4. Atenolol

Comparison with Competitor’s Column

a) HILIC-MS Application

Columns: Shown in the chromatograms


Mobile Phase B: ACN

A:B 10:90 Isocratic




Prototype HILIC Diol 3 100 X 2.0 mm 100Å

Competitor’s HILIC diol 3 100 X 2.0 mm 200Å

Nicotinamide

Pindolol

Salbutamol

Atenolol

b) Sample Solvent Effect

Columns: Prototype HILIC Diol 3 100 X 2.0 mm


Mobile Phase B: ACN

A:B 10:90 Isocratic


Temp: ambient

Detection: Varian 320 LC/MS/MS

Samples: Salbutamol, Pindolol, Atenolol, Nicotinamide

Sample Solvents: Seven different sample solvents were tested and they are specified in the chromatograms.

In reverse phase separation, it is desirable to have sample solvent weaker than initial mobile phase conditions.

In HILIC separation, it is a must to have sample solvent equal to or weaker than initial mobile phase conditions.

Evaporation / reconstitution or dilution with initial mobile phase may be necessary to produce good peak shapes.

The more aqueous

content in the sample

solvent, poorer the peak

shapes due to the strong

elution strength of water

in HILIC mode.

ACN Based Sample SolventSample solvent

100% ACN, 0% H2O

Sample solvent

90% ACN, 10% H2O

Sample solvent

75% ACN, 25% H2O

Sample solvent

10% ACN, 90% H2O

Sample solvent

100% MeOH, 0% H2O

Sample solvent

90% MeOH, 10%

H2O

Sample solvent

10% MeOH, 90%

H2O

Prototype HILIC Diol column showed

longer retention and better separation

compared to competitor’s HILIC column

with diol chemistry

MeOH Based Sample Solvent

MeOH is too strong to

be used as a sample

solvent. Poor peak

shapes were produced

as expected.

Conclusion• Varian prototype HILIC Diol showed complementary selectivity to reverse phase column.

• Solvent strength in HILIC separation is THF < Acetone < ACN < IPA < EtOH < MeOH < H2O and it was

confirmed by the data generated by using constant mobile phase composition of solvents differing in

solvent strengths.

• Ionic strength

• Buffer types with different cations / anions

• Bigger anion offers longer retention due to stronger ion-pairing effect and induction of greater

partitioning of analytes with the immobilized water layer on the stationary phase. Besides, its

greater hydrophobicity allows greater interaction of analytes with the stationary phase leading to

increased retention.

• Formic acid alone cannot provide good peak shapes for the basic compounds due to lack of

ionic strength leading to a cation-exchange effect.

• Salt concentration

• Retention time of analytes with positive log P values tends to decrease with increasing salt

concentrations.

• Retention time of analytes with negative log P values tends to increase with increasing salt

concentrations.

• MS compatiblility of prototype HILIC Diol:

• Mobile phase

• MeOH is a strong solvent in HILIC separation, hence, should be avoided due to drastic

reduction in retention times observed.

• For better peak shapes and reproducibility, addition of salt (~ 20mM) to the mobile phase is

needed.

• Sample solvent

• Sample solvent must be equal to or weaker than initial mobile phase conditions for good peak

shapes.

• MeOH is not recommended to be used as a sample solvent since it is a strong solvent in

HILIC separation and can generate poor peak shapes.

• Competitive data indicate prototype HILIC Diol yields a) longer retention & better separation and b)

better reproducibility possibly due to difference in pore size (prototype HILIC Diol is 100Å while

competitor’s HILIC with diol chemistry is 200Å.)

Acknowledgement• Dr. Huqun Liu, Varian, for synthesis of a very competitive Prototype HILIC Diol phase

• Samuel H. Yang, University of Texas at Arlington, in providing MS application of amino acids

• William Hudson, Varian, for help in generating MS sensitivity data

• Hema Chauhan, Varian, for help in screening of columns for HILIC applications

IntroductionHILIC columns can provide

Improved retention of polar analytes that would be hard to retain on RP columns (1)

Complementary selectivity to reversed-phase

Improved MS sensitivity due to high organic content of mobile phase (1)

Increased sample throughput, as direct injection of SPE eluates is possible without solventevaporation and reconstitution

Higher flow rates and fast separations due to low viscosity eluents

Unique benefits for preparative chromatography for purifying samples with poor water solubility

A good alternative for compounds with bad carryover in RP, the high organic eluents help withcarryover issues

To fully utilize the benefits of using HILIC columns, proper method development guidance and relatedmaintenance are necessary. Varian scientists have developed dedicated method development toolsfor HILIC applications with regards to the following items:

Organic / aqueous mobile phase ratios

Ionic strength in mobile phase in terms of

salt concentration

types of buffer counter ions

Solvent strength with different organic mobile phase modifications

ACN vs. MeOH

Mobile phase

Sample solventb) Reproducibility Test under Isocratic Condition

(48-hr Immersion Test)Columns: Prototype HILIC Diol 3 m 100 X 2.0 mm 100Å

Competitor’s HILC diol 3 m 100 X 2.0 mm 200Å

Mobile Phase A: 10mM ammonium acetate

Mobile Phase B: ACN

90% B Isocratic for 10 min



Detection: 220 nm

Sample: Mixture of neutral, basic, and acidic compounds

Columns were stored in mobile phase for 48 hrs after first analysis.

After being stored for 48 hrs in mobile phase, the same analysis was performed for inter-day immersion reproducibility.

1. Acenaphthene 2. Cytosine 3. Ibuprofen

log P 3.92 -1.73 3.97

Structure

Prototype

HILIC Diol

Competitor

HILIC Diol

Competitor’s HILIC (diol) 3 m 100 X 2.0 mm

Retention change: -4.6 %

Prototype HILIC Diol 3 m 100 X 2.0 mm

Retention change: 2.8 %

Day 1

1 2 3

Day 1

1 2 3

Day 3

1 23

Day 3

1 2 3

Fig 1. Complementary selectivity between HILIC and reverse phase columns

(Unlike reverse phase, non-polar compounds elute at t0 while polar

compounds are retained on prototype HILIC Diol.)

Column: Prototype HILIC Diol, 3 , 100 X 2.0 mm


Mobile Phase B: Acetonitrile


Detection: 270 nm


Samples 1. Pindolol

2. Practolol

3. Atenolol

As the aqueous content of the mobile phase is

increased, the observed retention of the analytes

decreases due to the strong elution strength of water

in HILIC mode.

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Pursuit XRs Diol 3u_85B_0@4_pindolol_practolol_atenolol_mix1_1.DATAmAU

85% ACN

15% Buffer

Isocratic

3

1

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Pursuit XRs Diol 3u_90B_0@4_pindolol_practolol_atenolol_mix2_3.DATAmAU

90% ACN

10% Buffer

Isocratic

1

2 3

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Pursuit XRs Diol 3u_95B_0@4_pindolol_practolol_atenolol_mix2.DATAmAU

95% ACN

5% Buffer

Isocratic

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Organic / Aqueous Modifier Ratios

Fig 2. Effect of organic / aqueous mobile phase ratio in retention time

Fig 5. Anion effect in buffer under same pH condition

Fig 6. Cation effect in buffer under same pH condition

Longer retention times were observed when

ammonium acetate was used compared to

ammonium formate, which can be explained by

the fact that a) acetate, being more

hydrophobic than formate, makes the analytes

interact with diol phase more, leading to longer

retention, and b) acetate can have a stronger

ion-pairing effect, facilitating increased

partitioning with the immobilized water layer,

resulting in increased retention.

Fig 7. Solvent strength effect with different organic phase modifiers

Fig 8. MS chromatograms with ACN as organic mobile phase (Sample

solvent conditions for a), b), and c) are given on the right.)

Fig 9. MS chromatograms with MeOH as organic mobile phase

(Compared to Fig 8. c), drastically reduced retention times and poor peak

shapes were observed with MeOH as organic mobile phase)

a)

b)

c)

Fig 10. MS chromatograms under ideal HILIC mobile phase conditions

with samples in ACN-based solvents

Fig 11. MS chromatograms under ideal HILIC mobile phase conditions

with samples in MeOH-based solvents

Fig 12. MS chromatogram comparison with Prototype HILIC Diol and

competitor’s HILIC column with diol chemistry

References1. Effect of stationary phase chemistry on selectivity of pharmaceuticals, pesticides, and oligosaccharides in HILIC

separations, Pittcon Poster #: 600 – 5P, Ritu Arora, Richard Robinson, Min Seok Chang, and Eugene Chang, Mar

2009

2. Nguyen, H.P., Schug, K.A. J. Sep. Sci. 2008, 31, 1465-1480.

3. Hydrophilic interaction chromatography, David McCalley, Chromatographyonline.com, April 2008

4. Deleterious Effects of Formic Acid without Salt Additives on the HILIC Analysis of Basic Compounds,

Phenomenex application note TN-1040

Fig 14. Histogram showing retention time shift from

reproducibility test for 48-hr immersion

Fig 13. Chromatograms of day 1 and 3 for reproducibility test

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Time (min.)

Inte

nsity / 1

06 Cys

[M + H]+

122.0 m/z

CSA

[M - H]-

152.0 m/z

Prototype HILIC Diol

R = 0.9908

k1 = 1.672

k2 = 2.259

HILIC-MS Application of Amino Acids

Fig 15. HILIC-MS application of cysteine and cysteine sulfinic acid on prototype HILIC Diol

Column: Prototype HILIC Diol 3 100 X 2.0 mm

Mobile phase A: 20 mM ammonium acetate

Mobile phase B: ACN + 0.5% acetic acid

A:B 25:75 Isocratic



Samples: 1. Cysteine (1 mM)

2. Cysteine Sulfinic Acid (200 M)

Injection volume: 10 l

10 mM

NH4COOH

(1 mM in flow)

1 23 4

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1 2 34

1 23

4

50 mM

NH4COOH

(5 mM in flow)

100 mM

NH4COOH

(10 mM in flow)

250 mM

NH4COOH

(25 mM in flow)

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10 mM

NH4COOH

(1 mM in flow)

50 mM

NH4COOH

(5 mM in flow)

100 mM

NH4COOH

(10 mM in flow)

250 mM

NH4COOH

(25 mM in flow)

Formic acid alone cannot provide sufficient ionic strength for

polar basic compounds, and addition of salt (~ 20mM) is needed

for good peak shapes and reproducible chromatography.

ros

Rectangle

ros

Temp Errata

method development tools used in hydrophilic interaction

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