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Column Selection Reverse phase

Reversed Phase Chromatography

Introduction to chemically bonded phases.Fighting tailing Introduction to reversed phase selectivityIsocratic reversed phase separation development.

Some general guidelines for selecting RP-bonded phases are summarized below:• C18—Very hydrophobic, retentive, and stable phase. First choice for most separations.

• C8—Preferred for lower organic mobile phase applications for more polar solutes. Note that C8 has similar selectivity as C18 but is much less retentive.

• CN—Less retentive and different selectivity than C8. Note that many CN phases are much less stable than C18.

• Phenyl—For medium-polarity components. Unique selectivity for aromatics

Ligand (Bonded-Phase)

Reversed Phase Chromatography

Introduction to chemically bonded phases.Fighting tailingIntroduction to reversed phase selectivityIsocratic reversed phase separation development.

Minutes0 5 10 15 20 25

Peak Tailing

Integration errorsReduced resolution Reduced sensitivity

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

Minutes0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 5,50

Peak tailing

What Causes Tailing?

Mixed-Mode RetentionHydrophobic interaction with bonded phaseIon exchange interaction with charged sites

Na

RNO

N1

H

2 R3 Si O Si-

H ++

- +R R R1 2 3R

Cl-

+Na+Cl- +

pH 7Cation Exchanger

Why Do You See Poor Peak Shape? Answer: Ionization of Silanols

Surface silanol charge changes with mobile phase pH

OH O

H+Si Si

Behaves as a Cation Exchanger(pH 2) (pH 7)

Result: Strong interaction between ionized surface silanols and

basic analytes (positive charge)

Mixed-Mode Retention:Hydrophobic Interaction

with Bonded PhaseIon Exchange Interaction

with Charged Sites

O-SiO-SiO-O-SiO-SiO-O-SiO-O-SiO-SiO-SiO-

O--SiO-Si

O-SiO-SiOHO-SiO-SiOHO-SiOHO-SiO-SiO-SiOH

O-SiO-Si

(CH3)2HN+(CH3

)2

HN+

Mobile Phase pH < 3

Si - OH

-Mobile Phase pH > 3

Si – OBase

Base

(Strong Base -- Amitriptyline pKa 9.4)

2 3 4 5 6 7 8Buffer pH

1

2

3

4

Taili

ng F

acto

r

Ideal Behavior Pure Polymer – No Silanols

High ligand density and high purity silica gel: Modern C18

Low ligand density and/or high metal content silica gel: Conventional C18

Peak Shape Over Wide pH Range

Si CH3

H3CCH3

O

Endcap

C18 Bonded and “Fully Endcapped” Silica Gel Pore

SiCCCCCCCCCCCCCCCCCCH3C

CH3

O

25 Å“Steric Hindrance”

What do you still see? Silanols!

Fighting tailing

Ion suppressionIon Pairing

Ultra pure silica (Symmetry 1994,Sunfire 2004)Reduced tailing

Embedded Polar groups (Symmetry Shield 1997)Reduced tailingLower tendency to dewet/hydrophobic collapse

Hybrid materials: High pH Ion suppression (XTerra 2000)

Expanded pH rangeReduced tailingHigh efficiency and stability

Why Not Just Run at pH’s Less Than 3 –Ion suppression?

pH 7.0

pH 2.0

Selectivity !!

amitriptyline

nortriptyline

Retention as a Function of pHAcidic Compound

pH

Cap

acity

Fac

tor (

k)

Non-ionized Acid

Ionized Acid

Retention Map : Capacity Factor (k) versus pH

N

COOH

CH3

O

Non-ionized

N

COO

CH3

O

-

Ionized

pKa = 5.8

50%

50%

pKa = pH where 50% of the molecules are

non-ionized

Retention as a Function of pHBasic Compound

pH

Cap

acity

Fac

tor (

k)

Ionized Base

Non-ionized Base

NHCH3

CH3

NCH3

CH3

Non-ionized

Ionized

Reversed-Phase Retention Behavior of Basic Compounds Relative to

Changes in pH

pH

Cap

acity

Fac

tor (

k)

Ionized Base

Non-ionized Base

pKa

± 2± 1

> ± 2 pH units provides stable retention (better reproducibil-ity at flat portions of curve)

Reversed-Phase Retention Behavior of Acidic, Basic and Neutral Compounds Relative

to Changes in pH

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12

pH

Cap

acity

Fac

tor (

k) Acid

Base

Neutral

Note: Retention of neutral analytes not affected by pH

To increase retentionin these pH areas Ion-pair chromatographycan be used

Reversed Phase Chromatography

Introduction to chemically bonded phases.Fighting tailing Introduction to reversed phase selectivityIsocratic reversed phase separation development.

Column Selectivity

Hydrophobicity - carbon load.Silanol Group Interaction - selectivity chart.Alternative Column Chemistries

Different bonded phase functionality.Polymer based stationary phase.Hybrid stationary phases.

Different Selectivity and Great Peak Shape on XTerra™ Particles (pH 7 Evaluation Test)

5 10 15 20 250

XTerra™ RP18

Embedded Polar Ligand

TF USP = 1.1

Amitriptyline

Minutes10 20 30 400

XTerra™ MS C18

Straight Chain Ligand

TF USP = 1.3

Peaks1 Gallic Acid2 Epigallocatechin3 Catechin4 Caffeine5 Epicatechin6 Epigallocatechin Gallate7 Gallocatechin Gallate8 Epicatechin Gallate9 Catechin Gallate

Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

XTerra® RP865

4

32

1

7 89

XTerra® MS C8

6 54

32

17

89

XTerra® Phenyl65

432

1

Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

78

9

Reversed-PhaseChromatographyon Different Columns

What chromatographic parameter has primarily changed ?

Bonding Chemistries

SpacerO S iCH3

CH3

O CO

NH

CH2CH2CH2 CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3

O S iO

CH3

CH2 CHCH3

O S iCH3

CH3

O CO

NH

CH2CH2CH2 CH2CH2CH2CH2CH2CH2CH2CH3

C18

Spacer

C8

Ph

O S iO

CH2CH2CH2CH2CH2CH2CH2CH3

O

O S iO

CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3

O

Chromatographic Characterization

Mobile Phase: 65/35 Methanol/ 20 mM KH2PO4 Buffer pH 7.05 µm materials in 4.6 x 150 mm columns, 1.0 mL/min, 23°C

Naphthalene

Acenaphthene

N H+

Amitriptyline pKa = 9.4

O NH2+

OH

Propranolol pKa = 9.6

Uracil

HN

N H

O

O

O

O H

O

Butyl Paraben

Testing column characteristics

Tailing factor of two basesamitryptyline and propranolol

Hydrophobic retentionretention of acenapthene

Hydrophobic selectivityRelative retention acenapthene/naphalene

Silanol activityRelative retention acenapthene/amitryptyline and acenapthene/propranolol

Polar interactionRelative retention acenapthene/butyl paraben and acenapthene/dipropylpthalate

Reversed-Phase Selectivity Chart

Hydrophobicity (Log Scale)

YMC-Basic

1

10Platinum ™ C18

Inertsil ® C8

Kromasil ® C8Zorbax ® XDB C 8

Symmetry ® C8

Hypersil ® BDS C 18

Inertsil ® ODS-2Prodigy ™ C18

Symmetry ® C18

Kromasil ® C18

YMC J'Sphere H80

Zorbax ® Rx C 18

Nova-Pak ® C18

Zorbax ® SB C 18

Hypersil ® ODS

Alltima ™ C18YMC J'Sphere M 80

Waters Spherisorb ® ODS 2µBondapak ® C18

Zorbax ® SB C 8

YMC J'Sphere L 80Lichrosorb ® Select B

Waters Spherisorb ® C8

Nova-Pak ® C8

Hypersil ® BDS C 8

Prodigy ™ C8

SymmetryShield ™ RP8

Zorbax ® Rx C 8

Alltima ™ C8

Lichrospher ® Select B

Purospher ® RP18

Nucleosil ® C18

Inertsil ® ODS-3

1 10

Sila

nol A

ctiv

ityWaters Spherisorb ® ODS 1

Resolve® C 18

Zorbax ® XDB C18

Hypersi Hypurity Elitel®

2 3 4 5 6 7 8 9

2

3

4

5

6

7

8

920

(Log

Sca

le)

Reversed-Phase Column Selectivity Chart:The “C18 Zone”

-0.6

-0.3

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3

3.3

3.6

-1.5 -0.5 0.5 1.5 2.5 3.5

(ln [α

] am

itrip

tylin

e/ac

enap

hthe

ne)

(ln [k] acenaphthene)

The “C18 Zone”(~80% of all separations)

-0.6

-0.3

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3

3.3

3.6

-1.5 -0.5 0.5 1.5 2.5 3.5

(ln [α

] am

itrip

tylin

e/ac

enap

hthe

ne)

(ln [k] acenaphthene)

The Modern“C18 Zone”

Reversed-Phase Column Selectivity Chart:

The Modern “C18 Zone”

(ln [k] acenaphthene)

XTerra® MS C18

Hypersil® Elite C18

Inertsil® ODS-3Kromasil® C18

Inertsil® ODS-2Hypersil® HyPurity Elite C18

Luna™ C18YMC-Pack™ Pro C18™ Zorbax® Eclipse® XDB C18Zorbax® Rx C18

Prodigy™ C18

Symmetry® C18

SymmetryShield™ RP18

Supelcosil™ LC-ABZ+Plus

Supelcosil™ LC DB-C18

Zorbax® Extend C18

XTerra® RP18

Luna™ C18(2)

Polaris™ C18-A

-0.2

0

0.2

0.4

0.6

0.8

1 1.5 2 2.5 3 3.5

(ln [α

] am

itrip

tylin

e/ac

enap

hthe

ne)

1.0

1.2

Atlantis™ dC18YMC-Pack™ ODS–AQ™

EXPANDED VIEW

ACQUITY UPLC™ C18

ACQUITY UPLC™Resolution through

Efficiency

ChromolithTM RP-18

Nucleosil® C181.5The Modern “C18 Zone”

Resolution through Efficiency

(ln [k] acenaphthene)

XTerra® MS C18

Hypersil® Elite C18

Inertsil® ODS-3Kromasil® C18

Inertsil® ODS-2Hypersil® HyPurity Elite C18

Luna™ C18YMC-Pack™ Pro C18™ Zorbax® Eclipse® XDB C18Zorbax® Rx C18

Prodigy™ C18

Symmetry® C18

SymmetryShield™ RP18

Supelcosil™ LC-ABZ+Plus

Supelcosil™ LC DB-C18

Zorbax® Extend C18

XTerra® RP18

Luna™ C18(2)

Polaris™ C18-A

-0.2

0

0.2

0.4

0.6

0.8

1 1.5 2 2.5 3 3.5

(ln [α

] am

itrip

tylin

e/ac

enap

hthe

ne)

1.0

1.2

Atlantis™ dC18YMC-Pack™ ODS–AQ™

EXPANDED VIEW

ACQUITY UPLC™ C18

ACQUITY UPLC™Resolution through

Efficiency

ChromolithTM RP-18

Nucleosil® C181.5The Modern “C18 Zone”

Resolution through Efficiency

Column Differences within the Same Trademark

C18/C8little selectivity difference

Phenylsome selectivity difference

CNlarge selectivity differenceneeds redevelopment of mobile phase stability problems!!!

Embedded polar grouplarge selectivity difference

Reversed Phase Chromatography

Introduction to chemically bonded phases.Fighting tailing Introduction to reversed phase selectivityIsocratic reversed phase separation development.

Separation Development

Select a columnC18, Embedded polar group- ScoutingTest 3 pH rages – ScoutingTest MeOH, AcN - ScoutingOptimizeShort columns (50 to 100mm)are good for fast screening of separation conditions.

Developing a Separation

Our first goal is to adjust the mobile phase strength to elute the sample components at a reasonable k’

Use a high flow rate - k’ is independent of flow rate.Use a short column - k’ is independent of column length. This allows a relatively fast experiment.

Developing a Separation

Select an initial mobile phase.Sample solubilityCompatible with the detectortypically a blend of a “strong” eluent and a “weak” eluent

Always choose a mobile phase that is too strong for the first try. Why?

Developing a Separation

Experimental conditions:Symmetry C18 column, 100 x 4.6 mmMethanol and phosphate/acetate buffer, 1.5 ml / min. (about 1,5 column volumes / min)Alliance 2690 Separation Module UV detection at 250 nm.

5 minutes for new mobile phase to wash through.10 minute chromatogram = k’ of almost 15

Developing a Separation

With optimized k’s, we can look at the selectivity of the separation.

If necessary we can change the mobile phase or the column to change the selectivity as was noted in the theory chapter.

Developing a Separation

A reversed phase exampleIsocratic method development techniqueExcedrin brand analgesic and salicylamide

CaffeineAcetaminophenAspirinSalicylamide

Sample and Acetone Overlay

80% MeOH

Spiked Excedrin Sample

80% MeOH

60%MeOH

Spiked Excedrin Sample

60% MeOH

50% MeOH

Spiked Excedrin Sample

50% MeOH

40% MeOH

Spiked Excedrin Sample

40% MeOH

30% MeOH

Spiked Excedrin Sample

30% MeOH

20% MeOH

Void Volume Overlay Vs 30% MeOH

Vo

Optimizing pHpH 2.8 vs pH 3.3

pH 2.8

pH 3.3