low, mid and high ph recommendations...page 4 change in retention with ph for ionizable compounds is...

Simplified Method Development

Low, Mid and High pH Recommendations

pH – An Important Parameter for Method Development

Retention of ionizable compounds is strongly affected by pHIonizable compounds (acids and bases) may be analytes or matrix compoundsAccurate pH control improves method reproducibilityThe pH range from 1 – 12 provides maximum method development flexibility

Change in Retention with pH for Ionizable Compounds is Compound Dependent

0

2

4

6

8

10

12

pH 2.5 pH 6.5 pH 8 pH 11.5

pH

rete

ntio

n tim

e (m

in)

Acetylsalicylic acid

Pyridine

Codeine

Procainamide

Amphetamine

Caffeine

pKa 3.5pka 5.2pKa 8.0pKa 9.2pKa 9.9pKa 14

Mobile Phase: 45% MeOH: 55% 20 mM Phosphate Buffer

Change in Retention with pH for Ionizable Compounds is Key to Method Development

Non-charged analytes have better retention (i.e. acids at low pH and bases at high pH)

Silanols on silica ionize at mid-pH, increasing retention of basic analytes (i.e possible ion-exchange interactions)

Choose mobile phase pH and column type to optimize retention and selectivity during method development

Recommended Method Development Goals

Adequate resolution of all peaks, Rs ≥≥≥≥ 2.0

Retention of first peak preferred to be at least k=1

Analysis time below 30 minutes, 20 minutes preferred

Robust and rugged methods

Use buffered mobile phases and try low pH first

Why Develop Methods at Low pH?

Acids are protonated for maximum retentionSilica silanols are protonated thereby minimizing ion-exchange interactions with basic compounds

Good peak shape

Long term reproducibility

Excellent mobile phase choices

Method Development SchemeStart at Low pH

• Try ZORBAX Eclipse XDB-Phenyl, Bonus-RP• Try ZORBAX SB-Phenyl, SB-CN, SB-C3• Restart at STEP 1

Ion-Pairingof Basic

CompoundsChange Temperature

Bonus-RP orAdd TEA

STEP 1

STEP 4

Change % organic

• Change organic modifier (MeOH or THF)• Adjust % organic for 0.5 < k <20• Restart at STEP 2

STEP 2

STEP 3

Band spacing problems



STEP 1bSTEP 1a

STEP 2a Bandspacingproblems

Tailing peaks

Poorly retained compounds• pH 2.5 (2-3) 20 - 50 mM buffer,• T = 30°C (ambient – 60°C)• Adjust %ACN for 0.5 <k < 20

• ZORBAX Eclipse XDB-C18 or -C8 basesacids

• pH 1-3 20 - 50 mM buffer,• T = 30°C (ambient – 90°C)• Adjust %ACN for 0.5 <k < 20

• ZORBAX SB-C18 or SB-C8 MidpH


STEP 1c

Mid pHBand spacing problems

Separation Variable Preferred Initial ChoiceColumn

Primary Stationary Phase Eclipse XDB-C18 or Eclipse XDB-C8Secondary Stationary Phase SB-C18 or SB-C8Dimensions 4.6 x 75 mm or 4.6 x 150 mmParticle Size 3.5 µm 5 µmPore Size 80Å: M.W. ≤ 4000, 300Å: M.W. ≥ 4000

Mobile PhaseSolvents A-B Water-acetonitrile% B solvent VariableBuffer 25 mM NaH2PO4, pH ≤ 3

or 0.1% TFA or Formic acidAdditives i.e. amines and ion-pair reagents TEA, Hexane sulfonate as neededFlow Rate 1-2 mL/minTemperature 30 - 35°C

Recommended Starting Conditions for RP-HPLC Method Development Approach

Choose Eclipse XDB First at Low pH

Wide useable pH range – pH 2- 9 – can use for low and mid pH without changing columns

Fully-hydroxylated ultra-pure silica improves peak shape

Double endcapping for good peak shape of basic compounds at low and mid pH

Three different bonded-phases for selectivity options

Method Development – Eclipse XDB-C18 at Low pH - Separation of Flavones

min0 1 2 3 4 5 6

mAU

-10

0

10

20

30

40

50

60

70

min0 1 2 3 4 5 6

mAU

0

10

20

30

40

1

1

2

2

Eclipse XDB-C18

StableBond-C18 Tf = 1.11N = 9307

Tf = 1.03N = 9612

Tf = 1.21N = 8934

Tf = 1.09N = 9396

Conditions:Column: 4.6x75mm, 3.5µmTemp: AmbientEluent: 30% ACN

70% 25mM NaH2PO4pH 2.5

Flow: 0.75 mL/minInj. Vol.: 4 µL

Sample: 1) Luteolin2) Apigenin

Excellent Reproducibility of ZORBAX Eclipse XDB at Low pH

pH 3

pH 70 5 10 15 20

1Time (min)

0 5 10Time (min)

2 3

4

1

2 34

Conditions:Column: Eclipse XDB-C8

4.6x150mm, 5µµµµmTemp: 35ºCEluent: 15% ACN

85% 25mM PhosphateFlow: 1.0 mL/min

Sample: 1) Cefotaxime2) Cefoxitin3) Cefamandole4) Cephalothin

3 Lots % RSDk’ αααα

1) 3.1 -2) 2.6 0.563) 2.8 0.294) 2.5 0.34

3 Lots % RSDk’ αααα

1) 5.4 -2) 5.1 0.413) 4.6 0.594) 4.8 0.00

StableBond –Secondary Choice at Low pH

Superior column lifetime at very low pH – down to pH 1 –due to patented sterically protecting bonding technology

Fully-hydroxylated ultra-pure silica improves peak shape

Six different 80Å bonded-phases – SB-C18, SB-C8, SB-CN, SB-Phenyl, SB-C3, SB-Aq – provide optimum selectivity with exceptional lifetime

Four different 300Å bonded-phases for selectivity options with protein and peptide separations

Selectivity Options at Low pHStableBond vs. Eclipse

DAD1 A, Sig=254,40 Ref=360,100 (021000\LY000018.D)DAD1 A, Sig=254,40 Ref=360,100 (021000\LY000017.D)

Conditions:Column: 4.6x75mm, 3.5µµµµmTemp: AmbientEluent: 25% ACN

75% 25mM NaH2PO4pH 2.5

Flow: 0.75 mL/minInj. Vol.: 20 µµµµL

Sample: 1) Caffeic Acid2) Luteolin3) Naringenin4) Apigenin

min0 2 4 6 8 10 12

mAU

-10

0

10

20

30

min0 2 4 6 8 10 12

mAU

-10

-5

0

5

10

15

20

1 2

3,4

4

1 23

Eclipse XDB-C18

StableBond-C18

Method Development – SB-C18 at Low pH Separation of Plant Extract

Column: ZORBAX Rapid Resolution SB-C184.6 x 75 mm, 3.5 µµµµm

Mobile Phase: 22% ACN78% NaH2PO4, pH 2.5

Flow Rate: 1.0 mL/minTemperature: RTDetection: UV 254 nmSample: 1. Caffeic acid

2. Impurity3. Luteolin4. Naringenin5. Apigenin

• To obtain k=1 for caffeic acid requires 22 minute analysis time

Flavones, Flavanones, and Phenolic Esters

Time (min)0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

2

1

3

4

5

Method Development – Change Organic ModifierSeparation of Plant Extract

Column: ZORBAX Rapid Resolution SB-C184.6 x 75 mm, 3.5 µµµµm

Mobile Phase: 40% MeOH60% NaH2PO4, pH 2.5

Flow Rate: 1.0 mL/minTemperature: RTDetection: UV 254 nmSample: 1. Caffeic acid

2. Impurity3. Naringenin4. Luteolin5. Apigenin

• Methanol as the organic modifier changes selectivity and increases the analysis time.

Flavones, Flavanones, and Phenolic Esters

Time (min)350 5 10 15 20 25 30

1

2

3

4 5

Method Development – Change Bonded-Phase Separation of Plant Extract on SB-CN Flavones, Flavanones, Phenolic Esters

Column: ZORBAX Rapid Resolution SB-CN, 4.6 x 75 mm, 3.5 µµµµm Mobile Phase: ACN: NaH2PO4, pH 2.5 Flow Rate: 1.0 mL/minTemperature: RT Detection: UV 254 nm Sample: 1. Caffeic acid 2. Impurity 3. Luteolin 4. Naringenin 5. Apigenin

22% ACN: 78% Buffer 25% ACN: 75% Buffer

SB-CN with stronger mobile phase reduces analysis time by 50% and maintains retention of k=1 on 1st

peak.

Time (min) Time (min)0 5 10 15 20 25

3

4

5

1

2

0 2 4 6 8 10 12

2

314

5

StableBond 300SB Columns Ideal for Separations of High MW Analytes

300Å pore size necessary for good peak shape and high efficiency separation of proteins and polypeptides. Exceptional stability with low pH “TFA containing” mobile phases.Improve peak shape for lower molecular weight analytes with large hydrodynamic volume.Four different bonded-phases allow bonded-phase optimization of all high MW samples.

Comparison of Bonded-Phase Options – Affect on Selectivity and Retention of Polypeptides

300SB-C18

300SB-C8

300SB-C3

300SB-CN

Columns: ZORBAX 300SB, 4.6 x 150 mm, 5 µµµµmMobile Phase: Gradient, 0 - 26% B in 30 min.

A = 0.1% TFA in WaterB = 0.1% TFA in Acetonitrile

Temperature: 40°CSample: 2 µg of each peptideFlow Rate: 1.0 mL/minDetection: UV 210nm

Why Develop RP-HPLC Methods at Mid-pH?

Compounds of interest are unstable at low pH

Improved solubility of analytes at mid pH

Increase retention of basic compounds

May have better selectivity in the pH range 3 – 8

Method Development SchemeMid pH Range

• pH 7 (6-9) 20 - 50 mM buffer,• T = 30°C (ambient – 60°C)• Adjust %ACN for 0.5 <k < 20

• ZORBAX Eclipse XDB-C18 or C8

Change % organic

• Try ZORBAX Eclipse XDB-Phenyl or Bonus-RP • Restart at STEP 5


Ion-Pairing of Acidic Compounds

Change Temperature

Add 20 mM TEA Tailing peaks

Bandspacingproblems

STEP 5

STEP 6

STEP 7

STEP 8




STEP 5b

STEP 5a

STEP 6a

Poorly retained acidic compounds

Recommended Conditions for Mid-pH Method Development

Separation Variable Preferred Initial ChoiceColumn Stationary Phase Eclipse XDB-C18Dimensions 4.6 x 75 mm or 4.6 x 150 mmParticle Size 3.5 µm 5 µm

Mobile PhaseSolvents A-B Water-acetonitrile% B solvent VariableBuffer 25 mM Na2HPO4, pH =7

Acetate/acetic acidAdditives i.e. amines and ion-pair reagentsTEA, tetrabutylammonium as neededFlow Rate 1-2 mL/minTemperature 30 - 35°C

Choose Eclipse XDB for Mid-pH

Long lifetime at mid-pH with dense bonding and double endcappingStrong silica for long lifetimeDouble endcapping provides excellent peak shapeThree different bonded-phases (C18, C8, Phenyl) for selectivity optimization

Eclipse XDB-C18, 4.6 x 75 mm, 3.5 µµµµm

pH 3

0 1 2 3 4 5Time (min)

4

3

2

1

Low pH Provides Insufficient Selectivity for Some Samples

Mobile Phase: 20% Methanol: 80% 20 mM phosphate buffer Flow Rate: 1.0 mL/min Temperature: RTDetection: UV 254 nm Sample: 1. Nizatidine 2. Famotidine 3. Cimetidine 4. Pirenzipine

� Poor Selectivity with Eclipse at Low pH� Selectivity Improves Somewhat with StableBond

SB-C18, 4.6 x 75 mm, 3.5 µµµµm

pH 3

Time (min)0 2

2

1

3

4

4 6 8

Improved Selectivity and Resolution with Eclipse XDB-C18 at Mid-pH

Eclipse XDB-C18, 4.6 x 75 mm, 3.5 µµµµm

Mobile Phase: 20% Methanol: 80% 20 mM phosphate buffer Flow Rate: 1.0 mL/min Temperature: RTDetection: UV 254 nm Sample: 1. Nizatidine 2. Famotidine 3. Cimetidine 4. Pirenzipine

• Better selectivity and improved retention occur at pH 7• A little TEA also improves peak shape peak #4 (di-amine)

pH 7No TEA

pH 710 mM TEA

0 4 8 12Time (min)

2

1

3

4

0 5 10 15 20

2

1 3

4

Optimize Separations with Eclipse XDB Selectivity Options – Analysis of Sunscreens

min0 2 4 6 8 10 12 14 16

1

1

1

3

2

2

3

23

Eclipse XDB-Phenyl

Eclipse XDB-C8

Eclipse XDB-C18

Columns: 4.6 x 150 mm, 3.5 µµµµm Mobile Phase: 15% H2O: 85% MeOH Flow Rate: 1.0 mL/min Temperature: 30°CDetection: UV 310 nm Sample: 1. Oxybenzone 2. Padimate-O 3. Ethylhexylsalicylate

1. oxybenzone

2. padimate O

3. ethylhexyl salicylate

Bonus-RP Provides Alternate Selectivity at Mid-pH

Polar alkyl-amide bonded-phase for unique selectivity

Improves peak shape of basic compounds

Triple-endcapped for good lifetime at mid-pH

Enhanced low-pH stability (sterically protecting bonding) for alternate selectivity at low pH

Compatible with 100% aqueous mobile phases

Bonus-RP Provides Alternate Selectivity at Mid-pH

Mobile Phase: 75% mM NaCitrate, pH 625% MeOH

Flow Rate: 1.0 mL/min Temperature: AmbientDetection: UV 254 nmInjection Vol: 3 µLSample: Cephalosporins

1. Cephalexin2. Cephaclor3. Cephuroxime4. Cephoxitin

Bonus-RP

Eclipse XDB-C8

0 2 4 6 8 10

1

2

3

4

Time (min)

1

2

3

4

Why Develop RP-HPLC Methods at High pH?

Compounds of interest not soluble at lower pHCompounds of interest not stable at lower pH Increase retention of basic compounds by analyzing them in non-charged formImprove selectivity

Method Development at High pH

• ZORBAX Extend-C18• pH 10.5 (9-12) 5 mM ammonia, or TEA,

or 10 – 50 mM organic or borate buffers• T = 25°C (ambient – 40°C)• Adjust MeOH for 0.5 <k < 20

STEP 9

• Change organic modifier (ACN or THF)• Adjust for 0.5 < k<20

STEP 10Vary temperature within recommended

range for bonded phase

Band spacing problemsTry different HPLC mode

STEP 10a

Band spacing

problems


Separation Variable Preferred Initial ChoiceColumn Stationary Phase Extend-C18Dimensions 4.6 x 75 mm or 4.6 x 150 mmParticle Size 3.5 µm 5 µmPore Size 80Å: M.W. ≤ 4000, 300Å: M.W. ≥ 4000

Mobile PhaseSolvents A-B Water-methanol% B solvent VariableBuffer 20 mM TEA pH =11

ammonium hydroxide, pH 10Flow Rate 1-2 mL/minTemperature RT - 30°C

Recommended Conditions for High pH Method Development

Silica–Based HPLC Columns are Now a High pH Choice

New technologies to protect silica from dissolution provide good lifetimes at high pHSuperior efficiency of silica-based columns provides high resolutionRobust methods can be established using the same parameters as used at low pH

Choose Extend-C18 for High pH

Patented bidentate C18-C18 bonding for superior high pH stability – up to pH 11.5Improved performance over polymeric columnsExcellent peak shape with double endcappingLC/MS at high pH (ammonium hydroxide) with high efficiency

Silica support

Extend-C18Bidentate Structure O O

Si Si

C18 C18

0 5

1

2,3

4

5

Time (min)

7

6

0 5 10

1

2

3

4

Time (min)

6

5

7

High pH Increases Retention of Antihistamines

The retention of this sample of basic compounds increases at high pH.

Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 µµµµ m Mobile Phase: See Below Flow Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm

Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine

pH 730% 20 mM Na2HPO470% MeOH

pH 1130% 20 mM TEA70% MeOH

tR = 8.5 tR = 11.4

Extend-C18 Provides High Efficiency and Good Peak Shape

In comparison to polymeric columns, the Extend-C18 has superior efficiency and peak shape

Mobile Phase: 65% 20 mM TEA, pH 11: 35% MeOH Temperature: RT Detection: UV 254 nm Sample: 1. Pyridoxine 2. Pyridine 3. n-Methylbenzylamine 4. Procainamide 5. n-Acetylprocainamide

Polymeric-Based Column4.0 x 250 mm, 5 µm

Flow Rate: 0.5 mL/min

Extend-C184.6 x 250 mm, 5 µm


0 10 20 30Time (min)

1

2

34

5

0 5 10 15 20Time (min)

1

23

4

5

Match Method pH and Column ChoiceChoose the Best Bonded-Phase for Each pH Range

StableBond, pH 1-6Use at Low pH1. Uses bulky silanes2. Non-endcapped

Eclipse XDB, pH 2-9First choice at Low and Mid pH1. eXtra Densely Bonded dimethylalkylsilanes2. proprietary double-endcapping

001241P2.PPT

Bonus-RP, pH 2-8Use at Low and Mid pH1. polar alkyl phase2. triple endcapped3. uses bulky silanes

Extend-C18, pH 2-11.5Use at High pH1. unique bidentate structure2. double endcapped

**

O

R

R

Si

OH

Si

O

Si

R

R

R

O

OH

R

R1

R1

R1Si

OSi

O

Silica Support

C18C18

CH3

CH3

CH3

CH3

CH3

CH3

O

SiO

O

O

O

Si

Si

Si

CH3

CH3

CH3

CH3CH3

CH3

Si O R

Si

Si

O

Si

O R

R1

CH3

CH3

CH3

CH3

R

R1

CH3

R1

R1

R1

CH3

R1

Si

Si

PG

PG

PG

Summary

Low pH Eclipse XDB then StableBondMid pH Eclipse XDB or Bonus-RPHigh pH Extend-C18

This method development scheme follows an approach of trying different pH’s for ionizable compounds with an optimum bonded-phase for both small molecules and large biomolecules.

Appendix

Recommended Buffer Choices for High pH

Pyrrolidine 11.3 10.3 – 12.3Triethylamine (TEA) 10.7 9.7 – 11.71-methyl-piperidine 10.3 9.3 – 11.3glycine 9.8 8.8 – 10.8TRIS 8.1 7.1 – 9.1

Borate 9.2 8.2 – 10.2Ammonia 9.2 8.2 – 10.2Diethylamine 10.5 9.5 – 11.5

Buffer pKa Effective pH range

Amou

nt o

f Silic

a Diss

olve

d, m

g

001369P1.PPTVolume of Eluent, Liters0 2 4 6 8 10 12 14 16

0

20

40

60

80

100

120

140

160

180

Eclipse XDB-C8Eclipse XDB-C18Extend-C18

Good Lifetime of Extend-C18 at High pH

Columns: 4.6 x 150 mm, 5 µm

Purge: 50% ACN / 50% 0.02 M K2HPO4, pH 11


Temperature: 25°C

Detection: Silicate concentration bysilicomolybdate color reaction

Reduce Column “Secondary Interactions” at Low pHColumn: Eclipse XDB-C8, 4.6 x 150 mm, 5µm

Mobile Phase: 85% 25 mM Na2HPO4 : 15% ACN, Flow Rate: 1.0 mL/min, Temperature: 35°CSample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine

pH 3.0USP TF (5%)

4. 1.33

pH 7.0USP TF (5%)

4. 2.35

• Reducing the mobile phase pH reduces interactions with silanols that cause peak tailing.

Time (min)0.0 2.5 5.0

54

32

1

54

32

1

Time (min)0.0 2.5 5.0

Time (min)

1

2

34

5

0 5 10 15 20 25 0 2 4 6 8 10 12Time (min)

1

2

3

4

5

SB-CN Optimizes Retention and Resolution

SB-CN reduces analysis time by 50% and increases retention of early eluting peaks.Method development procedure followed to get to this point.

SB-C18 SB-CN

Columns: 4.6 x 75 mm, 3.5 µµµµm Mobile Phase: 30% ACN: 70% NaH2PO4, pH 2.5 Flow Rate: 1.0 mL/minTemperature: 35°C Sample: 1. Estriol 2. Daidzen 3. Quercetin 4. Genistein 5. Diethylstilbestrol

Phytoestrogens and Isoflavones

Why Choose 300Å Columns?

Molecules must enter pores to interact with bonded-phase.Molecules must freely enter and exit pores to maximize efficiency.

(C) Will be Excluded

(A,B) Enter Pores

B

C

A

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

300SB-C18 (300Å)SB-C18 (80Å)

PW 1/

2Leu

Enkep

halin

M.W. =

556

Angiote

nsin I

I

M.W. =

1046

Insuli

n BM.W

. = 3,4

96

Cyt CM.W

. = 12

,327

RNaseM.W

. = 13

,684

Lysozy

me

M.W. =

13,90

0

Why Choose 300Å Columns?

• Proper pore size selection results in sharper peaks for large molecules.

Effect of Pore Size and Molecular Size on Peak Width, Gradient Separations

Improved Peak Shape for Large Molecules in Solution

Columns: 4.6 x 150 mm, 5 µµµµm Mobile Phase: 60% MeOH: 40% 0.1% TFA Flow Rate: 0.75 mL/min Temperature: RT Detection: UV 282 nm Sample: Tylosin (MW 916)

SB-C3 (80Å) 300SB-C3 (300Å)

• The size of a molecule in solution determines which pore size column is best.• The narrower peak width indicates unrestricted access to the pores.

Pw1/2 = 0.442 Pw1/2 =0.125

O

O

NHOHO

CHO

HO

OHOH

O

OOO

O

O O

O

CH3

CH3CH3

OCH3OCH3

CH3

CH2

CH2

CH2

CH3

CH3

CH3

H3CH3C

H3C

0 5 10Time (min)

0 5 10Time (min)

Method Development SchemeStart at Low pH

• Try ZORBAX Eclipse XDB-Phenyl, Bonus-RP• Try ZORBAX SB-Phenyl, SB-CN, SB-C3• Restart at STEP 1

Ion-Pairingof Basic

CompoundsChange Temperature

Bonus-RP orAdd TEA

STEP 1

STEP 4

Change % organic


STEP 2

STEP 3




STEP 1b

STEP 1a

STEP 2aBand

spacingproblems

Tailing peaks

Poorly retained compounds

• pH 2.5 (2-3) 20 - 50 mM buffer,• T = 30°C (ambient – 60°C)• Adjust %ACN for 0.5 <k < 20

• ZORBAX Eclipse XDB-C18 or -C8 basesacids

• pH 1-3 20 - 50 mM buffer,• T = 30°C (ambient – 90°C)• Adjust %ACN for 0.5 <k < 20

• ZORBAX SB-C18 or SB-C8

MidpH

Band spacing

problems

STEP 1c

Mid pHBand spacing problems

low, mid and high ph recommendations...page 4 change in retention with ph for ionizable compounds is...

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