1

Part 1. General Chromatographic Theory

Part 2. Overview of HPLC Media

Part 3. The Role of the Mobile Phase in Selectivity

Part 4. Column Care and Use

2

HPLC Particle Technology

2

Core-Shell Particle

Fully porous Particle

3

Tetraethoxysilane Silica Sol-Gel

Polymerization

Alkaline

3

99.9% of reactive surface area is internal

Fully Porous Silica

44

There is a high amount of variability

in the final physicochemical qualities of silica made by different manufacturers. These differences in the base silica itself help account for the wide variance in the behavior of different brands of HPLC columns. The principle variables are:

• Particle Shape (Irregular versus spherical)

• Purity

(metal content)

• Surface area

• Pore size

• Carbon load

Fully Porous Silica

5

Advantages:• Ability to derivatize with numerous bonded phases• High mechanical strength• Excellent efficiency• Highly amenable to modulation of material characteristics (pore size, surface area, etc.)

Disadvantages:• Dissolution of silica at pH > ~7.5 (may extend with bonded phase)• Hydrolysis of bonded phase at pH <1.5

5

Fully Porous Silica

66

Conventional Silica Particle Organosilica Hybrid Particle

Dissolution at pH > 7.5 Stable to pH ~12

SiloxaneBridge

Ethane linkage

Organosilica

Hybrid Particle

7

Advantages:• Extended pH range from 1-12• Performance and strength of conventional silica particle• Unique selectivity

Disadvantages:• Fewer stationary phases available compared to conventional silica (e.g. cyano, amino)

7

Organosilica

Hybrid Particle

8

LC/MS Analysis of Nicotine & Metabolites

Gemini-NX 3 µm

C18 100 x 2.0mm

MP: A = 10mM NH4 HCO3B = Acetonitrile

Gradient: 10-75 %B in 3 minFlow rate: 500 µL/min

1. Nornicotine2. 3-OH-Cotinine3. Anabasine4. Cotinine5. Nicotine

8

Organosilica

Hybrid Particle

9

Advantages:• Extremely low pressure• Macroporous structure allows direct injection of dirty samples • No bed shifting/instability of packed columns

Disadvantages:• Fewer stationary phases• Efficiency less than conventional particles (< 3m performance)

9

Monolithic Silica Rod

10

Onyx C18 100 x 4.6mm 15 µL

injections of human plasma; filtered; no PPT

1. H = Hypoxanthine2. U = Uric acid3. X = Xanthine4. A = Adenosine5. I = Inosine

10 Journal of Chromatography B, Volume 854, Issues 1-2, 1 July 2007, Pages 158-164

Monolithic Silica Rod

11

1.9 µm Solid Core

0.35 µm Porous Shell

2.6 µm Core-Shell Particle

11

Core‐Shell Particle

12

“Conventional”

Core-Shell 2.6 m:• 2.6 m total particle diameter• Pressure ~50% less than sub-2 m fully-porous• Efficiency = 260 – 300,000 P/m• UPLC efficiency using conventional HPLC systems*

“UHPLC”

Core-Shell 1.7 m:• 1.7 m total particle diameter• Efficiency = 280 – 320,000+ P/m• Highest efficiency media currently available

12

Core‐Shell Particle

13

Agilent®

1100 Agilent®

1100

Core-Shell 2.6 µm C18 150 x 4.6 mm

N = 295,343 p/m

Traditional 3 µm C18 150 x 4.6 mm

N = 166,502 p/m

13

Increased peak height

Core‐Shell Particle

Agilent is a registered trademark of Agilent Technologies, Inc. Phenomenex is in no way affiliated with Agilent Technologies, Inc..

14

Fully porous 1.7 µm C18 50 x 2.1 mm 0.6 mL/minEfficiency = 272,080 p/m

Core-Shell 2.6 µm C18 50 x 2.1 mm 0.6mL/minEfficiency = 267,720 p/m

Core-Shell 1.7 µm C18 50 x 2.1 mm 0.6mL/minEfficiency = 318,680 p/m

14

Core‐Shell Particle

15

Advantages:• 3x the efficiency of 5 m fully- porous media & 2x the efficiency of 3 m media• Pressures compatible with conventional HPLC systems*

Disadvantages:• Pressure is still higher than 3 m media• More sensitive to system extra- column volumes• More sensitive to overload in some cases

15

Core‐Shell Particle

1616

Drug Impurity Profiling:MP: A = 40mM KH2 PO4 pH 7.2,

B = AcetonitrileGradient: 5-35%B over 30minFlow rate: 0.2 mL/min

AU0.00

0.02

0.04

0.06

Minutes10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00

Fully porous 1.7 µm

C18 150x2.1mm

AU

0.00

0.02

0.04

0.06

Minutes10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00

Core-Shell 2.6 µm

C18 150x2.1mm

634 Bar

372 Bar

Core‐Shell Particle

17

Fully-porous Silica Particle:• General workhorse• Many particle sizes and stationary phases• Low sensitivity to system dead volume

Organosilica

Hybrid Particle:• Expanded pH stability range• Same performance characteristics as fully-porous• Optimal for high pH applications

Monolithic Rod:• Low back-pressure; reduced susceptibility to physical clogging• Ideal for analysis of “dirty” samples (e.g. plasma)• Efficiency ~3um fully-porous spherical silica

Core-Shell Particle:• UPLC performance using conventional HPLC systems• Should be the new “go-to” choice for method development in place of fully-porous media

17

Review

18

Reversed‐Phase Bonded Phases

18Granick

Research Group

University of Illinois at Urbana-Champaign

Once you have decided upon the optimal HPLC particle, the next decision will be to choose an appropriate stationary phase. This choice will greatly influence the final selectivity of your separation.

Choice of particle technology:• Fully-porous• Core-shell• Monolithic rod• UPLC

Choice of bonded phase:• Alkyl-bonded phases• Phenyl phases• Polar-embedded phases

19

Bonded Phases

20

1. Bonding main stationary phase ligand

2. Endcapping residual silanols

HCl

The Bonding Reaction

21

Alkyl bonded phases (C18, C8, C4):

Phenyl phases (Phenyl, PFP):

F F

F

FF

Polar-embedded phases:

Fusion

Polar-endcapped

phases:

Hydro

RP Stationary Phase Classes

22

Primary Mode of Interaction

Alkyl Bonded Phases

23

The primary mechanism of retention in reversed-phase chromatography is based upon hydrophobic interactions

between the analytes and the bonded phase. Therefore, bonded phases that exhibit strongly hydrophobic nature tend to perform well.

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO HCH 3

OH O H OH O H O H O H OH O H O H O H

O O O- O H O O OO

S iS i S i

S iS iCH 3

C H 3

CH 3C H 3

CH 3C H 3

CH 3 C H 3

C H 3C H 3 C H 3 CH 3

C H 3

C H 3

C H 3O H

OH

N

CH3

CH3

CH3CH3

Hydrophobic Interactions

24

We use the methylene selectivity test to determine the ability of stationary phase to separate molecules based upon differences in their hydrophobic character. In general, very hydrophobic bonded phases (e.g. C18) will display higher levels of methylene selectivity than less hydrophobic phases.

m in2 4 6 8 1 0

m A U

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

m i n2 4 6 8 1 0

m A U

0

1 0 0

2 0 0

3 0 0

4 0 0

C18

Phen

ylMethylene Selectivity

25

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.160

0.180

0.200

Luna C18(2)

Synergi Hydro-RP

Jupiter C18

Synergi Max-RP

Luna C8(2)

Luna C5 Luna Phe-Hex

Jupiter C4

Synergi Polar-RP

Prodigy Phenyl

Luna Cyano

Luna Amino

Slop

e of l

og k

vs. #

-CH2

-uni

ts

C18 > C8 > C5 ≥

Phenyl > CN > Amino

Methylene Selectivity

26

Columns: 5m C18

150x4.6mm 5m C8

150x4.6mm5m Phenyl

150x4.6mm

Mobile phase: 65:35 Acetonitrile:WaterFlow rate: 1 mL/min

Components: Two steroids:1. Testosterone2. Methyltestosterone

O

H H

CH3 H

CH3

OHCH3

Methyltestosterone

O

H H

CH3 H

CH3

OH

Testosterone

Methylene Selectivity

• C18• Rs

= 3.39• High Selectivity

• C8• Rs

= 1.78• Medium Selectivity

• Phenyl• Rs

= 1.06• Low Selectivity

Testosterone

Met-Testosterone

27

Methylene Selectivity

Phenyl phases are significantly less hydrophobic than C18 phases, but offer a potential selectivity that can be quite distinct due to the capacity of the phase to engage in pi-pi interactions with analyte molecules. It should be considered a complementary selectivity to conventional alkyl-bonded (e.g. C18) phases.

F F

F

FF

Modes of Interaction

28

Phenyl Phases

While C18 phases excel at separating molecules that differ primarily in the hydrophobic character, phenyl phase often display an enhanced ability to separate molecules that differ in polar functional groups. This enhanced polar selectivity is probably attributable to interactions with the pi electron cloud of the phenyl ring.

29

Phenyl Selectivity

Columns: C18Phenyl

Dimensions: 150 x 4.6 mmMobile phase: 75:25 Methanol:waterFlow rate: 1 mL/min

Components: 1. Estrone2. Estradiol

OH

H H

OHCH3

H

Estradiol

OH

H H

OCH3

H

Estrone

30

Phenyl Selectivity

31

m in1 2 3 4 5

m A U

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

1 7 5

m in1 2 3 4 5

m A U

0

2 0

4 0

6 0

8 0

1 0 0

C18

Phenyl

1+2

1

2

OH

H H

OHCH3

H

Estradiol

OH

H H

OCH3

H

EstroneC18

Phenyl

Phenyl Selectivity

32

The residual silanol groups of any silica-based RP sorbent give some degree of hydrophilic interaction. The presence of additional polar-functional groups (endcapping or embedded within the bonded phase) further increases interactions with polar compounds. These phases are typified by:

a. Polar selectivity for some polar analytes b. Stability in 100% aqueous mobile phasesc. Improved peak shape for basic drugs (minimal silanols)

Polar‐Embedded Phases

33

Nucleic Acid Bases:

Day 2

min2 4 6 8 10 12 14

0

Day 1

2 4 6 8 10 12

0

Phase Collapse!

Luna®

C18(2)

Day 1

0 2 4 6 8 10 12

0

Day 6

2 4 6 8 10 12 14

0

Polar-Endcapped

C18

Aqueous Stability of Embedded Phases

34

LC/MS/MS Analysis of ETG & ETS in Urine:

XIC of -MRM (6 pairs): 221.200/75.000 Da ID: ETG-1 from Sample 6 (P-2_Hyro-RP_100x4.6_4u_FR600... Max. 9020.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.549 96 144 191 239 287 334 382 429 477 525 572 620

Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Intensity, cps

ETGETS

XIC of -MRM (6 pairs): 221.200/75.000 Da ID: ETG-1 from Sample 6 (P-2_Hyro-RP_100x4.6_4u_FR600... Max. 9020.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.549 96 144 191 239 287 334 382 429 477 525 572 620

Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

Intensity, cps

ETG

ETS

Polar-Endcapped

2.5 µm

C18 100x3.0mm

10mM Ammonium formate

1. Ethyl glucuronide2. Ethyl sulfate

ETG

ETS

Aqueous Stability of Embedded Phases

35

min0 0.5 1 1.5 2 2.5

mAU

-20

0

20

40

60

80

100

120

140

DAD1 A, Sig=210,4 Ref=360,100 (DJ031111\CATECHOL000065.D)

Polar-Embedded Phenyl 150x4.6mm

Epinephrine

Norepinephrine Dopamine

min0 0.5 1 1.5 2 2.5

mAU

0

25

50

75

100

125

150

175

DAD1 A, Sig=210,4 Ref=360,100 (DJ031111\CATECHOL000066.D)

C18 150x4.6mm

Enhanced Retention of Polar Analytes

36

Oxymetazoline and oxidation product:

min2 2.5 3 3.5 4 4.5

mAU

-3

-2

-1

0

1

2

3

4

3.2

33 3

.336

C18

Rs

1.45

min2 2.5 3 3.5 4 4.5

mAU

-3

-2

-1

0

1

2

3

4

3.6

84

3.9

02

Phenyl

Rs

2.1

min2 2.5 3 3.5 4 4.5

mAU

-1

0

1

2

3

4

5

6

3.0

49

3.5

63

Polar-Embedded Phenyl

Rs

3.2

Oxymetazoline

• Improved Rs with Phenyl • Optimal Rs with polar- embedded and endcapped Phenyl Phase

Enhanced Polar Selectivity

37

1. Choice of Particle Technology

will have a dramatic effect on of critical components.

• Particle selection will usually be either fully-porous silica or core-shell media

• Monolithic rod for extremely dirty samples• Organosilica hybrid for high pH

• Differences in pore size and surface area may give columns from different manufacturers very different performance characteristics

2. The most common stationary phase for RP methods is C18, and it is logical to begin screening with a C18 phase

• Phenyl phases offer an alternative selectivity, especially for polar aromatics• Polar-endcapped or polar-embedded phases offer another selectivity option, and stability in 100% aqueous mobile phases

Stationary Phases Summary

38

Does sample require 97+%aqueous mobile phase?

Does sample require basic pH mobile phase?

No Is your matrix extremely dirty?

No

Organosilica hybrid particle

Yes

Monolithic HPLC Column

Yes

Particle size & Length

Bonded phase screening

No

Conventional or Core-Shell

HPLC Column

Polar-embedded or Polar-endcapped phase

Yes

Insufficient Resolution? Alternate SP

Column Selection Model

39

Method Development Exercise 2:              Media Selection and Phase Screening

40

HH

CH3

O

CH3OH

Testosterone, C19 H28 O2

Monoisotopic mass: 288.21 Da

Epitestosterone, C19 H28 O2

Monoisotopic mass: 288.21 Da

• Epimers = stereoismers that differ in configuration of one stereogenic center (Carbon 17)• Identical mass, so much be separated by HPLC for accurate quantitation using MS

Testosterone Epimer

Analysis

41

Does sample require 97+%aqueous mobile phase?

Does sample require basic pH mobile phase?

No Is your matrix extremely dirty?

No No

Conventional or Core-Shell

HPLC Column

Particle size & Length

Bonded phase screening

Core-shellParticle

100x2.1mm; C18, XB-C18, C8

Particle Selection

42

Core-Shell C18

Core-Shell XB-C18

Core-Shell C8

Testo Epi-

Alkyl Phase Screening

43

Does sample require 97+%aqueous mobile phase?

Does sample require basic pH mobile phase?

No Is your matrix extremely dirty?

No

Organosilica hybrid particle

Particle size & LengthFlow rate

Bonded phase screeningMobile phase optimization

No

Conventional or Core-Shell

HPLC Column

Polar-embedded, or polar-endcapped phase

Insufficient Resolution? Alternate SP

Alternative Column Screening

44

Core-Shell XB-C18

Core-Shell PFP

Evaluation of Phenyl Phase

45

Core-Shell XB-C18

Organosilica

Hybrid C18

Polar-endcapped

C18

Evaluation of Embedded and Hybrid

46

Core-Shell XB-C18 (Primary)

Organosilica

Hybrid C18 (Back-up)

Columns:

Core-Shell 2.6 µm

XB-C18 100x2.1mmOrganosilica

Hybrid 3 µm

C18 100x2.1mm

MP A:

0.1%FA, 1 mM Ammonium Formate in WaterMP B:

0.1% FA, 1 mM Ammonium Formate in ACNFlow rate

0.4 mL/minStep

Time, min

%B0 0 301 1 752 3 753 3.1 30

Final Method

47

End of Part II