Exploring the Challenges of the Chromatographic Separation ... · Exploring the Challenges of the...

©2013 Waters Corporation 1

Exploring the Challenges of the

Chromatographic Separation

of Polar Compounds


Topics for “Meet the Experts” Today

Goal of “Meet the Experts” series

Overview of the key principles and practical aspects of

separation chemistry as a function of polarity

Comparison of chromatographic methods for retention of polar

compounds

Conclusions


Goal of Today’s Presentation

Obtain at least one idea that can make a positive change in

your workflow this week

Provide pertinent and timely scientific information to aide you in

finding possible solutions for challenges you are facing today or

might face in this year

Provide opportunity to see what other scientists are doing for

the analysis of compounds that are similar to yours


What is a Polar Molecule?

General chemistry definition:

– A molecule whose centers of positive and negative charges do not coincide

– The degree of polarity is measured by the dipole moment of the molecule

Dipole moment is the product of the charge at either end of the dipole times the distance between the charges

– The unequal sharing of electrons within a bond results in a separation of positive and negative electric charge.

Polarity is dependent on the electronegativity difference between molecular atoms and compound asymmetry

http://upload.wikimedia.org/wikipedia/en/d/d9/Polarity_boron_trifluoride.png


Challenges in Work Flow Processes

Number of analyses is often increasing due to increasing concern of

time to market, brand equity (effective and safe) and gov’t regulations

Costs per sample is increasing due to increasing costs of consumables,

resources necessary for lab (facilities, human, etc.)

Prepare standards and samples

Data acquisition and processing

Data compilation and management


Sample Preparation is a Major Source of Laboratory Costs

Sample preparation is the most often cited area of improvement to save time and operating costs

Most sample preparation involves being in an organic phase

– Liquid/Liquid, PPT, Soxhlet, Distillation, Evaporation and Reconstitution

Many matrices will respond best to organic phases (gels, blisters, ointments, synthesis solvents, etc.)

image from dyapharma.com image from sefetec.net image from tasnee.com


Typical Sample Preparation Choices

Soxhlet

Extraction

Distillation

Evaporation

Macro Micro

Liquid / Liquid

Drying Grinding SFE






Comparison of chromatographic methods for retention of polar

compounds

Conclusions


What Is a Good Chromatographic Separation Method? When Done?

Depends on the goals of the separation

– Resolution of all the analytes in a sample?

– Resolution of key analytes?

– Quantification of single analyte?

– Speed of separation?

– Other?

Use consistent, objective criteria for evaluating chromatographic

separation performance

– Individual peak attributes

– Relative peak attributes


Goals Today are a Combination of Business and Science

“Minimize the analysis time (or another overall cost function)

while meeting or exceeding the necessary effective resolution

around every peak of interest.

– Here, the target function is analysis time, and our optimization

goal is its minimization.”*

*Thomas L. Chester, Maximizing the Speed of Separations for Industrial Problems, J of Chrom A, 1261 (2012) 69– 77


Goals Today are a Combination of Business and Science

“Minimize the analysis time (or another overall cost function)

while meeting or exceeding the necessary effective resolution

around every peak of interest.

– Here, the target function is analysis time, and our optimization

goal is its minimization.”*

or

“Maximize the resolution of the least-resolved peak of interest,

relative to its specification, without exceeding an analysis time

limit.

– Here the optimization goal and target are the maximization of the

least-resolved peak of interest compared to its specification.”*

*Thomas L. Chester, Maximizing the Speed of Separations for Industrial Problems, J of Chrom A, 1261 (2012) 69– 77


Desirable Information is Critical in Separation Chemistry

Chemical properties [functional groups] – Ionizable species, polarity, pKa, molecular weight

Sample solubility – Log P

Number of compounds present – How many components are you trying to separate?

Sample matrix

Detection technique [UV, ELS, RI, FL, MS…etc.] – Based on available equipment or sensitivity requirements of assay

Criteria for success – Concentration range and quantitative requirements

– System suitability


Assessing Separation Attributes

Individual peak characteristics — shape, size, time

– Retention time

– Peak width

– Peak height

– Peak area

– Symmetry (USP tailing factor)

0


Assessing Separation Attributes

Relative peak characteristics

– Capacity factor

– Selectivity

– Resolution

– Number of peaks

– Column efficiency

0


Factors that Influence Chromatographic Resolution

Initial

Increase

Increase k

Increase N


Improving Resolution with Complementary Selectivity

1

1

k

k

4

NRs

Maximized in Separations by:

Range of column chemistries Multiple particle substrates Wide usable pH range High retentivity Wide range in selectivity

Maximized in Separations by:

Ultra-low dispersion system Smaller particles Higher pressure capability Well-designed columns


Factors that Influence Chromatographic Resolution

Initial

Increase

Increase k

Increase N

Efficiency Selectivity Retentivity


Chromatographic Resolution: Impacting Selectivity and Retentivity

Efficiency Selectivity Retentivity


Critical Components Chromatography

Define method objectives

Understand analyte properties and intended use of method

Devise initial LC conditions

Develop adequate separation by systematic screening or computer assisted development

Sample preparation procedure

Suitable sample clean-up procedure based on physical and chemical properties of matrix

Standardization [data processing]

Determine method linearity, accuracy and precision

Final method optimization/

robustness testing

Challenge method and identify weak spots

Method validation Prove assay meets

intended use






Comparison of chromatographic methods for retention of

polar compounds

Conclusions


Importance of Polarity

Related to the structure of the elements, and any electron charge distribution of that molecule

In order to create chromatographic separations, it helps to understand the polarities of the:

– Sample/analytes

– Mobile phase

– Stationary phase (packing material)


Polarity Defines the Modes of Chromatography

Competition between the stationary phase and the mobile phase creates a

separation of compounds in a sample

Typically, the polarity of the mobile phase is OPPOSITE that of stationary phase


Comparison of 4 Major Chromatographic Methods for Retention of Polar Compounds

Normal Phase Chromatography (NPLC)

Hydrophilic-Interaction Chromatography (HILIC)

Reversed-Phase Chromatography (RPLC)

Supercritical Fluid Chromatography (SFC)


Normal Phase Separation is Excellent for Polar Molecules

Normal Phase

Stationary Phase

Un-bonded

Silica (Polar) Surface

Packing Polarity Polar

Mobile Phase Polarity Non-Polar

Elution Order Most Non-Polar First

Effect of Increasing

Mobile Phase Polarity Reduces

Retention Time


Normal Phase LC

Normal phase conditions start with a

POLAR stationary phase and a NON-POLAR mobile phase


Normal Phase Benefits

There is a much larger choice of solvents

(compared to reversed-phase) to

manipulate selectivity

Widely applicable to a diverse range of

compounds in both polarity and functional group

Many organic compounds are more soluble in

normal-phase solvents

Ideal for separating positional isomers,

stereoisomers, diastereomers and chiral

compounds


Challenges Associated with Normal Phase Chromatography

Polar “contamination” in the stationary phase or mobile phase

will lead to:

– Difficulties in achieving reproducible retention times

– Difficulties in controlling and predicting solvent strength

– Lengthy equilibration times to achieve a stable baseline

– Solvent miscibility and proper mixing issues making gradients

impractical

Increased toxicity, flammability, solvent expense including

purchase and disposal

– Hexane, heptane

– Ethyl acetate, acids

– Chlorinated solvents


NPLC is Critical for Chiral Separations

Heightened awareness that enantiomers of racemic compounds

have different pharmacological activities, pharmacokinetics and

pharmacodynamics effects across all more products

Single enantiomers exhibit greater potency with fewer

side effects than most conventional molecules

Rigorous justification will be required for market approval of a

racemate of chiral products including drugs, pesticides, etc.


Normal Phase Use by Purpose, Industry, and Function

Purpose:

– Preferred method for sample that have limited water solubility

– Preferred method for separating isomers

Industry:

– Pharmaceutical: 22%

– Academia: 17%

– Chemicals: 15%

– Biotechnology: 11%

– CRO’s: 8%

– Other: 27%

Function:

– Applied R&D: 27%

– QC/QA: 22%

– Method Dev 14%

– Other 37%

SDi: Market Intelligence, Normal Phase HPLC Separations, July 29, 2010


If Not Traditional NPLC for Polar Compounds….Then What?


Reversed-Phase Chromatography



What is HILIC?

HILIC - Hydrophilic Interaction Chromatography

– Term coined in 1990 to distinguish from normal-phase*

HILIC is a variation of normal-phase chromatography without the

disadvantages of using solvents that are not miscible in water

– “Reverse reversed-phase” or “aqueous normal-phase” chromatography

Stationary phase is a POLAR material

– Silica, hybrid, cyano, amino, diol, amide

The mobile phase is highly organic (> 80% ACN) with a smaller

amount of aqueous mobile phase

– Water (or the polar solvent(s)) is the strong, eluting solvent

*Alpert, A. J. J.Chromatogr. 499 (1990) 177-196.


Benefits of HILIC

Retention of highly polar analytes not retained by reversed-phase – Less interference from non-polar matrix components

Complementary selectivity to reversed-phase – Polar metabolites/impurities/degradants retain more than parent

compound

Enhanced sensitivity in mass spectrometry – High organic mobile phases (> 80% ACN) promotes enhanced ESI-MS

response

– Direct injection of PPT supernatant without dilution

– Facilitates use of lower volume samples

Improved sample throughput – Direct injection of high organic extracts from PPT, LLE or SPE without the

need for dilution or evaporation and reconstitution


Expands the Selectivity Range with Polar Compounds

When to Use HILIC:

Need improved retention of

hydrophilic or ionizable

compounds

Need improved MS response

for polar or ionizable

compounds

Need improved sample

throughput for assays using

organic extraction

Reversed-phase

polar non-polar

Compound Index

Normal-phase

ESI-

MS

Re

spo

nse

exce

llen

tp

oo

r

HILIC


Influence of Polar Modifier on Retention and Selectivity

10 mM ammonium acetate with 0.02% acetic acid

Analytes:1: methacrylic acid 2: cytosine 3: nortriptyline 4: nicotinic acid

12 3

4

12

3

4

1 23

4

1

34

90:10 ACN:H2O

90:5:5 ACN:H2O:MeOH

90:5:5 ACN:H2O:EtOH

90:5:5 ACN:H2O:IPA

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Retention increases with decreasing solvent polarity


Compounds 1. Nicotinamide 2. Pyridoxine 3. Riboflavin 4. Nicotinic acid 5. Thiamine 6. Ascorbic Acid 7. B12 8. Folic Acid

1

2

3

4

5

6

7 8

Minutes

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Water-Soluble Vitamins Using HILIC Conditions


0.00 2.00 4.00 6.00 8.00 10.00 12.00

2

1

3

4

5

Compounds 1. PMPA 2. CMPA 3. MMPA 4. IMPA 5. EMPA 500 ng/mL each

Organophosphonic Acids Using HILIC Conditions







Reversed-Phase Chromatography for Polar Compound Retention

Non-polar stationary phase with >80% aqueous mobile phases

Strengths

– Familiar, well understood technique

– Many stationary phase choices

– Good reproducibility, stable equilibration

– High efficiency

Weaknesses of modern C18 phases designed for improved peak

shape for basic analytes: polar compound retention

– Poor retention of polar analytes on a high coverage, non-polar C18

stationary phase


Polar Retention: Why Does Atlantis® T3 Work?

Dominant retention mechanism is reversed-phase (van der

Waals forces – hydrophobic attraction)

– Retention maximized using 100% aqueous mobile phases

– Retention maximized by using reduced C18 coverage

o Polar analytes can “fit” between C18 ligands and interact with

pores of material

o Optimized particle morphology (i.e. pore diameter/volume)

Secondary interactions due to residual silanols that are more

accessible due to reduced C18 coverage

– Cation-exchange interactions

– Hydrogen bonding interactions


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22.00

Scalability to/from UPLC® Technology: T3 Bonding & Endcapping

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ACQUITY UPLC® HSS T3

HPLC Separation

UPLC® Separation

6

5

4

3

2 7

1

Compounds: 1. Norepinephrine 2. Epinephrine 3. Dopamine 4. 3,4-Dihydroxyphenylacetic acid 5. Serotonin (5-HT) 6. 5-Hydroxy-3-indoleacetic acid 7. 4-Hydroxy-3-methoxyphenylacetic acid

(HVA)

Atlantis® T3

T3 bonding and endcapping

present in both HPLC and UPLC

Technology







SFC as a Replacement for Normal Phase LC

Normal-Phase LC (NPLC) methods use solvents (aliphatic hydrocarbons and chlorinated solvents) that many laboratories would like to reduce for health, safety, environmental, and cost reasons

Since the principles of SFC are similar to those of NPLC, methods should be able to be converted to SFC

– Reduces solvent usage and disposal

– Lowers the cost per analysis while enhancing green initiatives

SFC offers significant performance advantages over NPLC

– Better reproducibility

– Ability to perform gradient separations

o most NPLC separations are isocratic

– Compatible with mass detection


Historical Issues with Analytical SFC

Large system volume

– Prevents the adoption of small particles packing materials

– Prevents high throughput analysis

Pumping stability at low co-solvent percentage

– Limits the range of applications possible

Injection accuracy and compatible format

– Limits quantitative applications

– Limits moving from Normal Phase LC to SFC

Poor detection sensitivity

– Limits are too high QA/QC in GMP environment


Normal Phase LC

Normal phase conditions start with a

POLAR stationary phase and a NON-POLAR mobile phase


Increasing Selectivity Space: Convergence Chromatography

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8



Solvent

Pentane, Hexane, Heptane

Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8


SFC Chromatography

Selectivity Space

Unlimited solvent

and stationary phase selectivity


Solvent


Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8

Weak

Str

ong

Supercritical CO2

Organic Modifier


Genesis of Innovation and Promise of Expanding the Selectivity Factors

Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland














UltraPerformance Convergence Chromatography

Convergence Chromatography is a category of separation science that provides

orthogonal and increased separation power, compared to liquid or gas

chromatography, to solve separation challenges.

UltraPerformance Convergence Chromatography [UPC2] is a holistically

designed chromatographic system that utilizes liquid CO2 as a mobile phase to

leverage the chromatographic principles and selectivity of normal phase

chromatography while providing the ease-of-use of reversed-phase LC.

The ACQUITY UPC2 System is built utilizing proven UPLC Technology to enable

scientists the ability to address routine and complex separation challenges while

delivering reliability, robustness, sensitivity and throughput never before possible

for this analytical technique.


Why the Name?

Giddings, J.C. (1965) A critical evaluation of the theory of gas chromatography. In Gas Chromatography. 1964, edited by A. Goldup, p. 3-24. Elsevier, Amsterdam

In this article Dr. Giddings stated “One of the most interesting features of ultra high pressure gas chromatography would be convergence with classical liquid chromatography.”

Prof. Calvin Giddings (1930-1996)


Harnessing the Promise of Smaller Particles

2.1 x 150, 1.7µm Flow = 1.4 mL/min

Caffeine, Carbamazepine, Uracil, Hydrocortisone, Prednisolone, and Sulfanilamide

2.1 x 150, 5µm Flow = 1.4 mL/min

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0.30

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NUSP(Sulfan) = 19,809

NUSP(Sulfan) = 6,561

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2 6

4

5

3

3X improvement in efficiency 1.7X increase in sensitivity 1.7X increase in resolution


ACQUITY UPC2 Binary Solvent Manager: Controlled Gradient Solvent Delivery

0.5% difference in programmed solvent composition (even below 5%) results in controlled retention shift and minimized baseline noise

n=10

RT %RSD: <0.15

Area %RSD: <0.67

n=10

RT %RSD: <0.34

Area %RSD: <0.69

1.0 - 20% B Gradient

1-C

oum

arin -

0.7

88

2-F

lavon

e -

1.0

72

3-C

aff

ein

e -

1.2

09

4-T

hym

ine -

1.5

59

5-P

apaveri

ne -

1.6

41

AU

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0.18

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0.24

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1-C

oum

arin -

0.8

40

2-F

lavon

e -

1.1

16

3-C

aff

ein

e -

1.2

47

4-T

hym

ine -

1.5

84

5-P

apaveri

ne -

1.6

64

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0.18

0.20

0.22

0.24

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1.5 - 20% B Gradient


UPC2 Provides Opportunity for Expanding the Selectivity Space

: CSH PFP

AU

0.000

0.012

0.024

0.036

0.048

: HSS C18 SB

AU

0.000

0.012

0.024

0.036

0.048

: BEH HILIC

AU

0.000

0.012

0.024

0.036

0.048

: 2-EP

AU

0.000

0.012

0.024

0.036

0.048

Minutes

0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00

ACQUITY UPC2 Hybrid 2-EP 1.7 µm

ACQUITY UPC2 Hybrid 1.7 µm

G A D(1,2)

C

H F

B

E

G A D

C

H F

B

E

G A

D C

H F

B E

G A

D C

H

F*

B

E

ACQUITY UPC2 CSH Fluoro-Phenyl 1.7 µm

ACQUITY UPC2 HSS C18 SB 1.7 µm

AP

I

AP

I

AP

I

AP

I


Orthogonal to RPLC Metoclopramide Related Substances

ACQUITY UPC2

Reversed-Phase

AU

0.000

0.013

0.026

0.039

0.052

Minutes

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1 2

3 4

5 6

8 9

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-0.003

0.000

0.003

0.006

0.009

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2

Metoclopramide

Metoclopramide

12 minutes

12 minutes


4.6 x 250 mm silica NPLC column (L3)

Hexane / Dichloromethane / glacial acetic acid

2.0 mL/min

Replacing NPLC with UPC2:

Anthralin USP Drug Substance Assay

Normal Phase HPLC

Cost approx: $0.92 per run

An

thra

lin

- 2

.212

IST

D -

5.0

55

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0.24

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10.0


4.6 x 250 mm silica NPLC column (L3)

Hexane / Dichloromethane / glacial acetic acid

2.0 mL/min

Viridis 2-EP 4.6 x 150 mm

CO2 / MeOH / glacial acetic acid

3.5 mL/min

Replacing NPLC with UPC2:

Anthralin USP Drug Substance Assay

SFC

Cost approx: $0.92 per run Cost approx: $0.04 per run

Suitability requirements met with NO change to

sample and/or standard

preparation

An

thra

lin

- 2

.212

IST

D -

5.0

55

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IST

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2.1

35

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0.000

0.010

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0.030

0.040

0.050

0.060

0.070

0.080

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10.0 6.0

Normal Phase HPLC


Replacing NPLC with UPC2: Low Level Impurity Analyses by UPC2

Compound RT %Area S/N

Unk. Impurity 6.24 0.006 2.9

Unk. Impurity Not Found --- ---

Unk. Impurity 10.86 0.01 2.7


Unk. Impurity 20.85 0.018 3

Unk. Impurity 26.63 0.021 3.2

Estradiol 30.86 99.87 ---

Main Impurity 36.81 0.077 9.2

6.23

7

10.8

55

20.8

50

26.6

32

30.8

56

35.8

19

AU

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

Minutes

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Normal Phase HPLC: Cost per run ~ $5.89

4.6 250 mm silica column

2,2,4-trimethylpentane / n-butyl chloride / MeOH, 2.0 mL/min

USP Method: Chromatographic Purity

of Estradiol

50.0


Replacing NPLC with UPC2: Low Level Impurity Analyses by UPC2


Unk. Impurity 6.24 0.006 2.9


Unk. Impurity 10.86 0.01 2.7


Unk. Impurity 20.85 0.018 3

Unk. Impurity 26.63 0.021 3.2

Estradiol 30.86 99.87 ---

Main Impurity 36.81 0.077 9.2


Unk. Impurity 2.26 0.012 3.4

Unk. Impurity 2.59 0.004 1.9

Unk. Impurity 3.34 0.01 3.1

Unk. Impurity 5.66 0.006 1.7

Unk. Impurity 6.15 0.016 5.5

Unk. Impurity 8.13 0.013 3.1

Estradiol 8.81 99.89 ---

Main Impurity 9.99 0.046 16

6.23

7

10.8

55

20.8

50

26.6

32

30.8

56

35.8

19

AU

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

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UPC² : Cost per run ~ $0.05

Normal Phase HPLC: Cost per run ~ $5.89

4.6 250 mm silica column

2,2,4-trimethylpentane / n-butyl chloride / MeOH, 2.0 mL/min

2.1 x 150 mm ACQUITY UPC² BEH, 1.7 µm CO2 / MeOH

USP Method: Chromatographic Purity

of Estradiol

Transferred UPC² Method

UPC2 Benefits: Additional Impurities Detected and Higher

Sensitivity

50.0

14.0


Chiral Separation in a Validated Method Provides Competitive Advantage and Brand Equity

Chiral screening

Chiral method development

– MS and UV detection

Chiral inversion studies

Enantiomeric excess

Pesticides

Drugs of Abuse

Beta-blockers

Binol

Warfarin

Benzyl Mandelate (enantiomeric excess)

Carprofen (chiral method development)

Pantoprazole and Oxfendazole (chiral

method development with MS)

Clenbuterol

Phenylalanine methyl esters

Flurbiprofen

Cyclometalated Iridium (III) Complexes

Fast Chiral Separations


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0.48

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Key advantages of moving to UPC2

– Results that are equal to or better

– 30X reduction in analysis time

– Nearly 75X reduction in solvent

• 135 µL of MeOH vs 10 mL of hexane/ethanol

Applicability of UPC2: Fast Chiral Screening

Providing meaningful impact to scientists from

discovery to QC based on the reducing of non-value

adding steps in analytical workflow process

Reduces the time consuming solvent mixing and

sample preparation so can reallocate resources to

other value adding analytical work

Increases the column lifetime so can reallocate

consumable budget

Reduces the cost of solvent investments of purchase

and removal

Reduces the complexity of instrument multi-method

use so can reduce the capital investments, or increase

value added human resources

Fast Chiral Screening UPC²

NPLC 15 min

0.5 min


Positional Isomers of DMBA

2.1

34

2.2

45

2.3

42

2.5

52

2.6

82

3.4

30

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

2,5

2,33,5

2,4

3,4

2,6

Mixture of 6 positional isomers of DMBA Sample: Each at 0.2 mg/mL in isopropanol (IPA) Column: 3.0 x 100 mm, 1.7 µm ACQUITY UPLC BEH125 Solvent: CO2 / MeOH with 0.2% formic acid


Increase in Resolution Power Provides Savings in Columns, Solvent and Time

AU

0.00

0.10

0.20

0.30

0.40

0.50

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

AU

0.0

2.5e-2

5.0e-2

7.5e-2

1.0e-1

1.25e-1

1.5e-1

1.75e-1

2.0e-1

2.25e-1

2.5e-1

2.75e-1

UPC2

IC + OJ-H

Traditional SFC

2 x IC + 2 x OJ-H

Baseline resolution of all isomers was achieved in less than 10 min using UPC² even with three chiral centers Compared to the chiral HPLC methods, the UPC2

method offers a better resolution and a much shorter run time The UPC2 method also eliminated the need for toxic hexane often used in NPLC methods


Simplicity in Workflow

Simplify the workflow with UPC2

– Any simplification in the whole workflow, from initial sample

collection and sample preparation to analysis, generally has the

greatest business impact in any market segment

Combining multiple techniques (LC and GC into CC)

Combining multiple methods (NPLC and RPLC into CC)

Reducing sample prep and analysis times

– Directly injecting organic solvent extracts (LLE, SPE, etc.)


Separation of Compounds from Matrix Interferences (Bioanalysis)

Clopidogrel (RPLC)

Clopidogrel (UPC2)

Interfering Phospholipids (RPLC)

Interfering Phospholipids (UPC2)


Combining Multiple Techniques for Lipid Analysis

Gas Chromatography Liquid Chromatography Convergence Chromatography

Free fatty acids are typically derivatized to form the methyl esters (FAMEs) Analysis time 30 min

Analyzed by both HILIC and RP HILIC separates lipid classes by polar head group RP separates based on acyl chain length and number of double bonds

Single methodology to separate complex lipids by class Faster baseline separation of lipids based on chain length and number of double bonds


UPC2 Analysis of a Mouse Heart Extract

PC

SM LPC

PE

TAG

TAG: Triacylglycerides PE: Phosphotidylethanolamine PC: Phosphotidylcholine SM: Sphynogomyelin LPC: Lysophosphotidylcholine

ACQUITY UPC2 BEH column 5-50% B


Time0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10

%

0

100

0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10

%

0

100

8:0

14:0

16:0

18:0

20:0

22:0

24:0

10:0

12:0

ESI negative mode Free Fatty Acids (FFA) containing 8-24 acyl chain

ESI positive mode Triacylglycerols (TG) and Cholesterol esters (CE)

1

7

2

8 3

4 5

6 11

10

13 12

9

Peak Lipid Species

1 15:0/15:0/15:0 TG

2 18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis) TG

3 16:0/16:0/16:0 TG

4 18:2(∆9,12Cis)/18:2(∆9,12Cis)/18:2(∆9,12Cis) TG

5 18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG

6 17:0/17:0/17:0 TG

7 18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG

8 18:3 CE

9 18:2 CE

10 17:0 CE 18:1 CE

11 18:0/18:0/18:0 TG

12 18:0 CE

13 19:0 CE

Neutral Lipids Based on Chain Length and Number of Double Bonds

ACQUITY UPC2 HSS C18 SB column 1-10% B


Official AOAC Method

Reducing Sample Prep and Analysis Time for -carotene Analysis

Dissolve/Hydrolyze

Extract

Dilute

Filter

LC Analysis

Modified AOAC Method

30 min

120 min

30 min

20 samples ~12.5 hrs

Dissolve/Extract

Filter

LC Analysis

30 min

20 samples ~10 hrs

UPC2 Method

Dissolve/Extract

Filter

UPC² Analysis

2 min

20 samples ~ 40 minutes

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50

AU

0.0

1.0e-1

2.0e-1

3.0e-1

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50

AU

0.0

1.0e-1

2.0e-1

3.0e-1

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50

AU

0.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

3.0e-1

0.91

0.91

0.91

0.77 1.31

-carotene standard

-carotene capsule

Carotenoids Mix

6 Replicates Peak Area Retention

Time (min)

Average 10326 0.91

RSD% 0.34 0

Label Claim: 15 mg/capsule

Assay

#1

Assay

#2

Assay

#3 Average RSD%

15.13 15.39 15.24 15.25 0.84%


Streamlining Sample Preparation of Creams and Ointments

Analysis of main API and impurities from creams and ointments involved a

tedious sample prep for QC assay

o Extraction from cream with organic solvent

o Back extraction into aqueous for RPLC analysis

Impact of UPC² in workflow

– Inject organic sample extraction directly

– Condense the sample preparation overhead

– Provide excellent reproducibility (peak area and retention time) and sensitivity

Imp 1 0.35%

Imp 2 0.11%

API 99.6%

*Standard sample of API & impurities

n = 5 injections Area %RSD < 1.0%


Monitoring Unstable Intermediates

Compound D sensitive to hydrolysis,

reverts to C

Could not use a single technique

– NPLC 90 min

– GC 18 min could not monitor B

Measure starting materials,

products and impurities

Analysis 8.5 min

Excellent sensitivity

Cpd A Cmpnd B

Cmpnd C Cmpnd D

Cmpnd E Cmpnd F

API

UPC2


EPA Method 8330B

C18, 4.6x250 mm

Cyano or phenyl hexyl column. 4.6x250 mm

• Typically analyzed by HPLC or GC • Long analysis times using HPLC or GC • Thermally labile compounds like tetryl

can not be done by GC • Inadequate baseline separation for

some of the compounds using official methods like EPA 8830B, requiring use of 2 orthogonal columns


UPC2 for 14 Explosives

Eliminates need for two analyses and two systems running one method each

Eliminates toxic solvents and concerns from waste disposal

Reduce time from 26 minutes (12 + 14) to 4 minutes with fast screening


Combining Multiple LC Methods Into One Fat Soluble Vitamins

Vitamin A Normal phase 12 minutes

Vitamin D3 Normal phase 20 minutes

Vitamin E Normal phase 30 minutes

Vitamin K1 Reversed-phase

12 minutes

β-carotene Normal phase 10 minutes

Lycopene Normal phase 10 minutes

Lutein Reversed-phase

10 minutes


Combining Multiple LC Methods Into One Fat Soluble Vitamins

Vitamin A Normal phase 12 minutes

Vitamin D3 Normal phase 20 minutes

Vitamin E Normal phase 30 minutes

Vitamin K1 Reversed-phase

12 minutes

β-carotene Normal phase 10 minutes

Lycopene Normal phase 10 minutes

Lutein Reversed-phase

10 minutes

Simultaneous Analysis of Fat-soluble Vitamins and Carotenoids in 10 minutes

Minutes

0.00 2.00 4.00 6.00 8.00 10.00

AU

Analyte Retention

Time (min) λ (nm)

(1) Vit A Acetate 3.14 345

(2) Vit E Acetate 3.76 263

(3) Vit K1 3.88 263

(4) Vit A Palmitate

4.34 345

(5) Vit E Tocopherol

4.82 263

(6) Lycopene 4.99 456

(7) Vit E Succinate

5.09 263

(8) β-carotene 5.12 456

(9) Vit D3 5.76 263

(10) Lutein 7.21 456

1

2 3

4

5

6

7,8

9 10


Improving Resolution Through Expanding Selectivity Space

1

1

k

k

4

NRs

Selectivity [α] and retentivity [k] impacted by: Stationary phase (column selectivity) Organic solvent (eluotropic series) Mobile phase additives (pH and ionic strength)

System efficiency [N] impacted by:

System dispersion Reduction in particle size

Impact on Rs % Improvement

Double N 20-40% Double k 15-20%

Double α > 400%

Convergence

Chromatography Selectivity Space

Unlimited solvent and stationary

phase selectivity

Solvent


Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8


Comparison of Chromatographic Methods for Retention of Polar Compounds

Technique Advantages Challenges

Traditional NPLC

There is a much larger choice of

solvents (compared to reversed-

phase) to manipulate selectivity

Widely applicable to a diverse range of

compounds in both polarity and

functional group

Many organic compounds are more

soluble in normal-phase solvents

Ideal for separating positional

isomers, stereoisomers, diastereomers

and chiral compounds

Long equilibration times Difficult to run gradients Not easily compatible with MS Difficult method development Purity of reagents Chemical modification of column

HILIC

High % organic mobile phases give

higher sensitivity in MS

Eliminate evaporation of SPE eluents

Sample and mobile phase solubility problems

Not well understood Not widely applicable

RPLC

Familiar technique

High efficiency

Rapid equilibration

Wide selection of columns

Dewetting under aqueous conditions Poor retention of polar compounds

Convergence

Chromatography and

UPC2

Familiar technique for purification

High efficiency

Rapid equilibration

Wide selection of columns that are

same as NPLC and RPLC

Uses same control, acquisition and

results sw

Initial learning of key method development factors as they are different from RPLC

Transfer of current methods need cross validation projects before in SOP


Supporting of Customers Seeking Theoretical and Practical Insight

Primary Theoretical Contributions

Developing the basis of the theoretical

support of the science

Provides basis of making next actions on

method development and key factors to

the separation

Practical & Business Contributions

Placing into workflow processes and

monitoring the metrics of impact based on

practitioners work

Best practices that are based on others

experiences including hyphenated

techniques (MS, ELSD, etc.)


Practitioners UPC2 Applications Expanding from the Customer’s Experiences

http://upc2.waters.com


Opportunity to Explore …

Frequent Concerns

Will my compound work with this

technology?

Is there any advantage to hyphenated

detection versus doing currently?

I do not have time to evaluate

technology as I have objectives to meet

in my job

Take the Challenge

Form to submit a sample for feasibility

analysis based on your criteria

Provides quick assessment before

considering more exploration how would

get to future state

Doesn’t interfere with taking care of your

current objectives, but doesn’t limit you

from future proofing your lab


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