Phase Separations Customer Education Program

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Phase Separations

_{Phase Separations}

Customer Education Program

Advanced HPLC Column Selection

Using the Hydrophobicity Chart

Time (min)

50 neutral

base

neutral

base

0

(2)

Course Library

* Strategies and Techniques for Faster HPLC

Methods Development

* Methods Development Optimization Software --HIPAC

* Advanced HPLC Column Selection Using The

Hydrophobicity Chart

* Solid Phase Extraction Technology

* Selecting Optimum HPLC Column Dimensions and

Stationary Phase Particle Size

* Troubleshooting Common HPLC Problems

* Purchasing High Performance Chromatographic Supplies

* Understanding RP-HPLC Separations -- Effect of Silica Type/Activity

* Reversed-Phase HPLC Methods Development

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Course List - Order Number & Duration

* Strategies and Techniques for Faster HPLC PSL908200 2.5 hours Methods Development

*Methods Development Optization Software -- HIPAC PSL908201 45min * Advanced HPLC Column Characterization & Selection PSL908203 1.5 hours

Using The Hydrophobicity Chart

* Solid Phase Extraction Technology PSL908204 4 hours * Selecting Optimum HPLC Column Dimensions and PSL908205 1 hour

Stationary Phase Particle Size

* Troubleshooting Common HPLC Problems PSL908206 2 hours * Purchasing High Performance Chromatographic PSL908207 7 hours Supplies

* Understanding RP-HPLC Separations PSL908208 1.5 hours -- Effect of Silica Type/Activity

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...because Chromatography is still a Science

SM

Reversed-Phase HPLC Methods Development

Examples are given, and a complete set of

handouts is provided for future reference.

A Certificate of Achievement will be given to all

attendees, which documents your participation in

this training program.

Order Course # PSL908202

This is a full day course is designed to provide

critical information important to those developing

new methods, as well as those redeveloping

existing methods to improve performance in the

areas of sensitivity, robustness and sample

throughput.

Key topics include:

• Impact of Particle Technology on

Chromatographic Performance

• Resolution

• Suggested Methods Development Strategy

• Benefits of Optimizing

Column Dimensions and Particle Size

• Optimization Software

• Troubleshooting Tips

Duration: 7 hours

Course Abstract:

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Advanced HPLC Column

Characterization & Selection

Using the Hydrophobicity Chart

. . . .. . . .

... .. .. . . ... .

.. . .. . . .

Time (min)

50 neutral

base

neutral

base

0

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Silica Pore Structure

* Porous Silica

> 99% of Surface Area in the Pores

* Pore Size

Surface Area

SA ~ 3

SA - Surface Area, m

2 _/g

SV - Specific Pore Volume, mL/g

PD - Pore Diameter, or nm

Typical Surface Area 100 -- 300 m

2 /g

SV

PD

100 10nm Å

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Silanol Types

H Si

O O

Si

O

Vicinal

Geminal

Lone

OH OH

Si

O

H H

O

Si

O

(8)

Bonded Phase: Trifunctional Synthesis

OH

+

_Cl

_Si

Cl

+ HCl

(Hydrolysis)

Si

_c

c

_c

c

_c

c

Si

o

Si

o

Si

o

OH

Si

c

_c

c

_c

c

_c

c

(9)

C

18

18 Bonded Pore

Bonded Pore

Si - OH = Silanol Group

Si-CCCCCCCCCCCCCCCCCC

= C18

25

Si - OH Si - O H Si - OH Si OH Si - OH

50

C H 3 Si O Si -C H3 CH 3 C H 3 Si O Si -C H3 C H 3 C H 3 Si O Si -C H3 CH 3 C_H3 Si O Si -CH3 CH3 CH3 Si - O - Si -CH3 CH3

Endcapped Silanol

Si-CCC CCCCCC CCCCCCC CC Si-CCC C C CCCC CC C CCCC CC Si-CCC CCCCCC CCCCCCC CC Si-CCCCCCC CCCCCCCCCCC Si-CCC C C CCCC CC C CCCC CC Si-CCC C C CCCC CC C CCCC C C Si-CCC CCCCCC CCCCCCC CC Si-CCC CCCCCC CCCCCCC CC

Å

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Ligand Density (Surface Coverage)

χ

₌

%C

100 SA

%C

100

]

1 -

[

MW - 1

nC 12

=

µ

moles/m

2 * Better measure of material's characteristics

SA

- Specific Surface Area

%C

- % Carbon Load

MW

- Molecular Weight of Ligand

nC

- # of Carbon Atoms in Ligand

Ligand Density

Primary Ligand Density

Silanols

Ligand Density

Hydrolytic Stability

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ION EXCHANGE

pH 14

pH 1

(-)

(+)

pKa 10.6

% Ionized

(-)

pKa 4.7

pH

12.6 Neutral

2.7 Neutral

8.6 ~100%

6.7 ~100%

pH 7

Cation

Anion

(12)

Terminology

Surface Silanol Groups

* Changes surface charge as pH 2 7

OH

O

+

H

+

Si

Behaves as a Cation Exchanger

(pH 2)

(13)

Tailing

vs

. Buffer pH

Buffer pH

Tailing

Factor

1

2

3

4

2

3

4

5

6

7

8 Conventional C

₁₈

Modern C

₁₈

Ideal

B. A. Alden, T. H. Walter

(14)

Effect of Mobile Phase pH on

Selectivity

Buffer pH

Ret. Time

0

4

8

12

3

4

5

6

7

8

9 base

neutral

acid

2 4 8

Acid

Neutral

Acid

Base

3 4 8

pH 3

pH 8

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Reversed-Phase Selectivity Chart

YMC-Basic 1 10 Platinum™ C18 Inertsil® C₈ Kromasil ® C₈ Zorbax® XDB C8 Symmetry ® C8 Hypersil ® BDS C₁₈ Inertsil® ODS-2 Prodigy™ C18 Symmetry ® C18 Kromasil ® C₁₈ YMC J'Sphere H80 Zorbax ® Rx C18 Nova-Pak® C18 Zorbax ® SB C18 Hypersil ® ODS Alltima™ C₁₈ YMC J'Sphere M 80

Waters Spherisorb ® ODS 2 µBondapak ® C18 Zorbax® SB C₈ YMC J'Sphere L 80 Lichrosorb ® Select B Waters Spherisorb ® C₈ Nova-Pak ® C₈ Hypersil ® BDS C8 Prodigy™ C₈ SymmetryShield ™ RP8 Zorbax ® Rx C8 Alltima™ C8 Lichrospher ® Select B Purospher® RP18 Nucleosil® C₁₈ Inertsil® ODS-3 1

Hydrophobicity

Silanol Act

ivity

Waters Spherisorb ® ODS 1 Resolve® C18

Zorbax® XDB C₁₈

Hypersil Hypurity Elite ®

2 3 4 5 6 7 8 9

(Log Scale)

2 3 4 5 6 7 8 9 20

(Log Sca

le)

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