Merlin K. L. Bicking, Ph.D.

ACCTA, Inc.

Richard A. Henry, Ph.D.

Consultant Minnesota Chromatography Forum 2014 Session: 2D-LC and Novel LC Phases

1

Is A Second Retention Mechanisms in LC a

Curse or Cure?

Thank You! • Thank you for your interest in our research. This work was supported by

internal funding and the generous contributions of the organizations listed on the last page. We are providing this copy free of charge to help advance everyone’s understanding of HPLC columns, but also to demonstrate our ability to solve complex problems.

• We only have a few requests from you: • You are welcome to review this information and share it with your colleagues. Please retain all

references to ACCTA, Inc. in any information that you share. Our business is successful largely because of referrals and recommendations from customers.

• Please contact us if you would like to discuss these results in more detail.

• Please acknowledge the support of the sponsors by considering their products and services.

• Consider working with us to solve your specific problems. We are experts in providing the unique technical services that generate a positive ROI for laboratories. A small investment can mean a significant improvement in efficiency and a reduction of lab staff stress.

2

Merlin K. L. Bicking, Ph.D. ACCTA, Inc. St. Paul, MN USA 55125 Email: info@accta.com

Richard A. Henry, Ph.D. Consultant State College, PA USA 16801 Email: hyperlc@comcast.net

“Analytical Solutions to Analytical Problems”

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

20

40

60

80

100

120

140

160

Curse: The Effect of Silica Purity on Selectivity

Zorbax XDB C18 Moderate Purity Silica (fully endcapped)

ACE C18 High Purity Silica (fully endcapped)

Zorbax SB C18 Moderate Purity Silica (non-endcapped)

Zorbax ODS Low Purity Silica (fully endcapped)

9,700 pl/m

23,100 pl/m

63,400 pl/m

Decreasing silica purity can change selectivity, but poor chromatography and reproducibility may result from surface interactions.

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

50

100

150

200

250

300

350

400

0 2 4 6 8 10 12 14 16 18 20

50

100

150

200

250

300

350

400

450

500

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

50

100

150

200

250

300

2

3

4

5

1

2

3

4

5

1

2

3

4

5

1

3

A second retention mechanism from the silica affected the chromatography

Column: 250 x 4.6mm, 5m Mobile phase: 80:20 MeOH/25mM KH2PO4 (pH6.0) Flow: 1.00ml/min Components; 1: Norephedrine, 2: Nortriptyline, 3: Toluene, 4: Imipramine, 5: Amitriptyline

1, 2, 4 and 5 not eluted

Reproduced with kind

permission of Mac-Mod Analytical 3

min 0 0.5 1 1.5 2 2.5 3

mAU

0

20

40

60

80

100

120

min 0 0.5 1 1.5 2 2.5 3

mAU

0

20

40

60

80

100

120

Cure: Change Some Retention Times, Not All • Decreasing the acid content causes an increase in the retention of the

basic analytes (ionic effects). Since the acetonitrile content has not changed, the hydrophobic retention region does not change.

1 Creatinine 2 Procainamide 3 Protriptyline

Retention Window for Hydrophobic Components

1 2 3

1 2

3 0.10% Sulfuric Acid/ 80% Acetonitrile

0.01% Sulfuric Acid/ 80% Acetonitrile

Column: 4.6 X 50 mm PFPP

4

• When the presence of a second retention mechanism is part of the phase design, maybe we can use both mechanisms to improve the separation

• This is possible only if,

– We understand what the mechanisms are

– We understand how they behave

– We know how to control their behavior

So, is it a curse or cure?

5

Experimental Details • Equipment

– Agilent 1100 with autosampler, column compartment, and diode array

– OpenLab ChemStation software

• Mobile Phase Components – Acetonitrile (HPLC Grade) – Ammonium Formate

• 10, 20, and 30 mM

• Operating Conditions – Flow: 1.75 mL/min – Injection: 2 uL – Temperature: 35 C – Detection: 220 nm

Manufacturer Phase Dimensions Phase

Supelco Ascentis Express C18 4.6X50 mm, 5 um* Hydrophobic

Supelco Ascentis Express F5 4.6X50 mm, 5 um* Pentafluorophenylpropyl

Supelco Ascentis Express OH5 4.6X50 mm, 5 um* OH/Diol

Zirchrom Zirchrom-PBD 4.6X50 mm, 3 um Hydrophobic with adsorbed phosphate

Imtakt Scherzo SM-C18 4.6X50 mm, 3 um Hydrophobic + Cation Exchange + Anion Exchange

* Fused core design. Equivalent to 3 um particles. 6

• Test two mobile phase parameters that may affect retention

– Acetonitrile Content

– Buffer concentration

• Test high and low levels of each parameter

– Acetonitrile: 50% and 90%

– Buffer Concentration: 10 mM and 30 mM

22 Factorial Screening Design

7

• Why choose this design? – Factorials evaluate two

or more variables with a minimum number of experiments

• Why choose these conditions? – The choice is arbitrary

– Ideal for general LC and LC-MS applications

22 Factorial Screening Design

mM

Bu

ffer

% Acetonitrile

30mM

10mM

50% 90%

8

Procedure: Measure retention Calculate k Calculate Main Effects

• The Main Effect of a parameter is the difference between the results at high and low settings.

• Larger values mean a larger change in retention over the range studied.

• Small values mean that this variable does not produce a significant change in retention.

Calculating Main Effects

mM

Bu

ffer

% Acetonitrile

30mM

10mM

50% 90%

Ace

ton

itri

le H

igh

Ace

ton

itri

le L

ow

Buffer Low

Buffer High

mM

Bu

ffer

% Acetonitrile

30mM

10mM

50% 90%

High Average – Low Average

Hig

h A

vera

ge –

Lo

w A

vera

ge

9

0.407 (k=0.55)

0.452 (k = 0.72)

2.742 (k=9.34)

3.076 (k=10.6)

Example Calculations

mM

Bu

ffer

% Acetonitrile

30mM

10mM

50% 90%

Nicotinic Acid (OH5)

Main Effect for Acetonitrile

Main Effect for Buffer

(9.34+10.6) 2

(0.55+0.72) 2

- = +9.3

(0.55+9.34) 2

(0.72+10.6) 2

- = -0.7

• There is a significant increase in retention with an increase in acetonitrile.

• There is a small decrease in retention with an increase in buffer conc.

10

What Do Main Effects Mean?

Sign of the Main Effect

Negative Near Zero Positive

Acetonitrile Reversed Phase Trend

No effect Normal Phase Trend

Buffer Conc. Ion Exchange Trend

No effect Salting Out Trend

Important Note: the existence of a trend suggests, but does not prove, that the stated mechanism is responsible for the effect.

11

• How do we use this information? – We want to separate the polar compounds from the less polar 1-

ring aromatics. – Change buffer concentration to move the polar compounds, with

little change in retention for the aromatics. – Change the acetonitrile content to move the polar compounds in

the opposite direction from the aromatics.

Significant Main Effects for F5 (PFP) Compound Class Main Effects Trend

Acetonitrile Buffer

BTEX Aromatics* Non-Polar, neutral -2.9 -0.1 RP, No ionic

Benzyl Amine Polar, strong base +3.6 -2.6 NP, Ion Exchange

Phenethyl Amine Polar, strong base, more hydrophobic than Benzyl Amine

+4.2 -3.4 NP, Ion Exchange

BTMA** Polar, cation +6.0 -5.3 NP, Ion Exchange

12 *Benzene, Toluene, Ethyl Benzene, Xylenes **Benzyltrimethylammonium chloride

Improving the Separation on F5 (PFP)

min 0 0.5 1 1.5 2

mAU

-10

-5

0

5

10

15

20

25

1

2 3

90%, 30 mM

1 2

3

50%, 10 mM

min 0 0.5 1 1.5 2

mAU

-5

0

5

10

15

1 2

3

50%, 30 mM

min 0 0.5 1 1.5 2

mAU

-20

-10

0

10

20

Main Effects

Acn Buffer

BTEX -2.9 -0.1

Benzyl Amine (1) +3.6 -2.6

Phenethyl Amine (2) +4.2 -3.4

BTMA (3) +6.0 -5.3

13

90%, 10 mM

1

2

3

min 0 1 2 3

mAU

-2

0

2

4

6

8

10

12

14

30mM

10mM

50% 70%

Improving the Separation on PBD

1 2

3

50%, 10 mM

min 0 0.5 1 1.5 2 2.5 3

mAU

-15

-10

-5

0

5

10

15

20

1 2

3

50%, 30 mM

min 0 0.5 1 1.5 2 2.5 3

mAU

-20

-10

0

10

20

30

70%, 10 mM

1 2

3

min 0 0.5 1 1.5 2 2.5 3

mAU

0

5

10

15

20

25

1 2

3

70%, 30 mM

min 0.5 1 1.5 2 2.5 3 3.5

mAU

0

5

10

15

20

25

30

35

Main Effects

Acn Buffer

BTEX -1.6 -0.1

Benzyl Amine (1) +6.4 -8.0

Phenethyl Amine (2) +6.3 -8.8

BTMA (3) -0.1 -1.4

14

30mM

10mM

50% 70%

Probably not a linear

trend.

Improving the Separation on SM-C18 Main Effects

Acn Buffer

BTEX -10.0 -0.2

Creatinine (1) +0.30 0.0

Benzyl Amine (2) +2.1 -0.9

Phenethyl Amine (3) +0.5 -0.5

Nicotinic Acid (4) +2.4 -1.1

Benzene Sulfonate (5) 0.0 -1.1

1

2 3

50%, 30 mM

min 0 0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

-20

0

20

40

60

80

100

4

5

1 2 3

50%, 10 mM

min 0 0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

0

10

20

30

40

50

60

70

80

4

5

min 0 0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

0

5

10

15

20

25

30

35

40

90%, 10 mM

1

2 3

4

5

1

2 3

90%, 30 mM

min 0 0.25 0.5 0.75 1 1.25 1.5 1.75

mAU

0

10

20

30

40

50 4

5

15

30mM

10mM

50% 90%

Improving the Separation on OH5 Main Effects

Acn Buffer

BTEX 0.0 0.0

Creatinine (1) +2.1 0.0

Benzyl Amine (2) +2.3 -1.1

Phenethyl Amine (3) +2.3 -0.1

Nicotinic Acid (4) +9.3 -0.7

Phenylalanine (5) +9.6 +0.1

1,2,3

50%, 30 mM

4

5

min 0 0.5 1 1.5 2 2.5 3

mAU

0

20

40

60

80

100

1,2,3

50%, 10 mM

4

5

min 0 0.5 1 1.5 2 2.5 3

mAU

0

20

40

60

80

100

1

2

3

90%, 30 mM

4

5

min 0 0.5 1 1.5 2 2.5 3

mAU

0

5

10

15

20

90%, 10 mM

1

2,3

5,4

min 1 2 3 4

mAU

0

5

10

15

20

25

30

35

16

30mM

10mM

50% 90%

Main Effects Comparisons

17

BTEX Main Effect: ACN

SM-C18 -10.0

F5 -2.9

PBD -1.6

OH5 0.0

Benzyl Amine

Main Effect: ACN

F5 +6.4

PBD +6.4

OH5 +2.3

SM-C18 +2.1

Benzyl Amine

Main Effect: Buffer

PBD -8.0

F5 -2.6

OH5 -1.1

SM-C18 -0.9

• The 22 factorial method can be used to easily identify general retention trends.

• Each of the four columns studied demonstrated a unique combination of multiple retention mechanisms.

• When two mechanisms are present, it is possible to separately move classes of compounds in different directions by adjusting the mobile phase composition. – On a single mode column, this would only be possible by

changing the chemistry of the mobile phase components (e.g., change from acetonitrile to methanol).

– On a multi-mode column, the retention time selectivity can be changed by adjusting the composition of the mobile phase, without changing the chemistry.

Summary

18

Acknowledgements

Columns, reagents, and other resources were kindly provided by:

For more information, contact: Merlin K. L. Bicking, Ph.D. ACCTA, Inc. St. Paul, MN 55125 Email: mbicking2@accta.com Internet: www.accta.com

19

• ACCTA, Inc. provides “analytical solutions to analytical problems,” for those organizations who want to: – Better utilize their equipment and staff – Improve efficiency and productivity with better methods and processes, and – Have access to a range of support options.

About Us

20

Laboratory Consulting

Technical Training

Lab

Life-Line

Laboratory Best Practices

Recent Publications:

“Investing to Save - A Simple Tool for Calculating ROI can Determine Which Investments are Best for Your Lab” Lab Manager, 2012, 7 (11), 18-21. View the digital version.

http://www.accta.com/consulting.htmlhttp://www.accta.com/consulting.htmlhttp://www.accta.com/training.htmlhttp://www.accta.com/training.htmlhttp://www.accta.com/lablifeline.htmlhttp://www.accta.com/lablifeline.htmlhttp://www.accta.com/lablifeline.htmlhttp://www.accta.com/lablifeline.htmlhttp://www.accta.com/lablifeline.htmlhttp://www.labmanager.com/?articles.view/articleNo/33407/title/Investing-to-Save---A-Simple-Tool-for-Calculating-ROI-Can-Determine-Which-Investments-are-Best-for-Your-Lab/