Modern Approaches to Chromatography - Waters Corporation · 2014-12-12 · Modern Approaches to Chromatography Guy Wilson UPC2 & PurificationBusiness Development, Northern Europe,

©2013 Waters Corporation 1

Modern Approaches to Chromatography

Guy Wilson UPC2 & Purification Business Development,

Northern Europe, Waters Ltd


Chromatography Today


What is UPLC?


Understanding How a Chromatographic

Column Works -- “BANDS”

We create a separation by changing the relative speed of each analyte band (competition between the mobile phase and stationary phase)

Yellow is the earliest eluting analyte “band” (it will be broader in the column), but moving fastest – it “likes” the mobile phase

Blue is well retained, it will be in a more focused, narrower band, near the inlet and move the slowest in the column – it “likes” the particles

X


Narrow Band – Narrow Peak More Concentrated

– Increased Peak

Height/Sensitivity More Resolution Capability

Broad Band – Broad Peak Less Concentration – Less Sensitivity Less Resolution Capability

Broader “Band” More “Band Spreading” Broader Peak

Narrower “Band” Less “Band Spreading” Narrower, Taller Peak

Mobile

Phase

Mobile

Phase

Mobile

Phase

Mobile

Phase

Peak Widths are Reduced with Less Band Spreading

Less Concentrated

0 0

More Concentrated

You must reduce and control Band Spreading in order to improve LC performance.


Fundamental Resolution Equation


Increased Efficiency with Small Particles UPLC Technology Maintains Resolution

dpN

1

Efficiency (N), is inversely proportional to particle size, dp

If dp 3x Rs 1.7x N 3x

and

1

1

4

k

kNRs


Particle Size

Images are on

same scale

(Bar = 10 μm)

5 μm

Analytical Particles

(can fit 12 across a hair)

1.7 μm

ACQUITY UPLC™ Particles

(can fit 35 across a hair)

Optimal Particle Size

Distribution For Maximum Efficiency

at a given Pressure

60 μm Human Hair (very fine hair)


“Extra Column”

(Instrument)

- Injector

- Tubing (ID x L) - Connectors - Detector Cell

- Data Capture Rate

Contributors to Band Spreadin

Column

- End Fitting Design - Packed Bed Uniformity - Column Volume - Particle Size

- Linear Velocity - Mass Transfer

Reduced Band Spreading = Increased Plate Count = Increased Resolution

For optimal performance, BOTH CONTRIBUTORS to Band Spreading MUST BE DECREASED


Potential for Band Spreading

Band Spreading will occur along the flow path from the Injector (“Sample Band), into, through and out of the column (“Analyte Bands”), and then into the Detector


AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

Minutes

0.00 1.00 2.00 3.00 4.00

UPLC® Columns on HPLC Systems

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

Minutes

0.00 1.00 2.00 3.00 4.00

ACQUITY UPLC® BEH C18

2.1 x 50 mm, 1.7 µm F = 0.3 mL/min PSIMAX = 4,200

T (4) = 1.63 N (4) = 5,400

Rs (2,3) = 0.97

XTerra® MS C18

2.1 x 50 mm, 2.5 µm F = 0.5 mL/min PSIMAX = 3,950

T(4) = 1.30 N (4) = 4,100

Rs (2,3) = 1.10

1

2

3

4

1

2

3

4

Fully optimized HPLC system:

Minimal benefits realized with 1.7 μm particles


The promise of the van Deemter plot

Smaller Particles The enabler of productivity

Optimal velocity range

Typical back pressure : 700-1000 bars


Loss in Performance running an

UPLC® Column on a HPLC - van Deemter Curves H

eig

ht E

quiv

ale

nt

to T

heore

tical P

late

Linear Velocity

HE

TP

u {mm/sec}

H

1.7 µm ACQUITY UPLC ® Column on

an ACQUITY UPLC® Instrument

1.7 µm ACQUITY UPLC® Column on a

80 µl Bandspreading (5 Peak Width) HPLC

2.5 µm HPLC Column on a 80 µl Bandspreading (5 Peak Width) HPLC

Different Instrument and Column Types

Pressure Limitation

Column ID: 2.1mm


AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00

The Benefits of the Holistically Designed ACQUITY UPLC® System


2.1 x 50 mm, 1.7 µm HPLC

F = 0.3 mL/min

PSIMAX = 4,200 T (4) = 1.63

N (4) = 5,400 Rs (2,3) = 0.97


2.1 x 50 mm, 1.7 µm ACQUITY UPLC®

F = 0.6 mL/min

PSIMAX = 8,400 T (4) = 1.02

N (4) = 10,100 Rs (2,3) = 2.25

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00

1

2 3

4

HPLC Non - Optimal Linear Velocity

UPLC® Separation Optimal Linear Velocity

1

2 3 4

Less Band Spreading


High Resolution Peptide Mapping: Influence of Particle Size

AU

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.02

0.04

0.06

0.08

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

UPLC®

1.7 µm Peaks = 168 Pc = 360 2.5X Increase

HPLC 5 µm Peaks = 70 Pc = 143

More information in the same amount of time


Similar Peak Capacity – Less Time

Peak Capacity = 153 AU

0.00

0.05

0.10

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00

2.1 x 50 mm, 5 µm

Peak Capacity = 123

AU

0.00

0.05

0.10

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00

2.1 x 50 mm, 1.7 µm

6x Faster

3x Sensitivity

AU

0.00

0.05

0.10

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

The same information in less time

Expanded View


ACQUITY UPLC


ACQUITY UPLC


ACQUITY UPLC H-Class


ACQUITY UPLC I-Class

The ACQUITY UPLC I-Class System represents: • Evolution of UPLC

• Pinnacle of performance

• Based on seven years

of engineering innovation

• Fueled by customer input

The ACQUITY UPLC I-Class System accomplishes: • Maximum peak capacity

• Enhances the

performance of any Mass Spectrometer

• Better Data Quality





Efficiency vs. Backpressure Comparisons

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

0 10000 20000 30000 40000 50000 60000 70000 80000

Backpressure (psi)

Eff

icie

nc

y (

N/m

) Influence of Particle Size on

Backpressure & Efficiency

1.4 μm

1.7 µm

3.5 μm

5.0 μm

10.0 μm

2.5 μm

1.8 µm

1.0 μm

21% more Efficiency 79% more Backpressure

70% more Efficiency 390% more Backpressure

Assume: 1) 100 mm length columns 2) Pmax at uopt 3) ACN/H2O Gradient, 40oC

UPLC 15,000 psi


Fundamental Resolution Equation




SFC - An introduction

Supercritical Fluid Chromatography (SFC)

– Supercritical Fluid: a substance above its critical temperature and

pressure


SFC - An introduction

Why do Supercritical fluids make good mobile phases for chromatography?

Diffusivity describes the rate at which one substance can move through another Viscosity is resistance to flow High diffusivity, and low viscosity combine in SFC to give fast, efficient chromatography


Understanding the Terminology

Conventional SFC terms such as solvent, co-solvent and

modifier ALL refer to the primary liquid component(s) of mobile

phase B

– This co-solvent (mobile phase B) is the strong eluting solvent in UPC2

– It is typically methanol but can also be other organic solvents such

ethanol, 2-propanol, acetontrile, etc. (or combinations)

An additive is a salt or liquid added to the co-solvent at a low

concentration in order to improve peak shape(s) or analyte

solubility and may influence selectivity

– Examples of typical additives include diethyl amine, ammonium

hydroxide, formic acid, trifluoroacetic acid, water, etc.

– Typical additive concentrations are ≤ 2% or 10 mM


How an ACQUITY UPC2 System Works

Inject valve

Auxiliary Inject valve

Column Manager

PDA detector

Back Pressure Regulator (Automated and Static)

Waste

Make-up Pump

Mass Spec

Splitter

Modifier CO2 Supply CO2

Pump Modifier

Pump

mixer Thermo-electric heat exchanger


Ultra Performance Chromatography


Expand Selectivity… …Uncover Orthogonality

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

Purnell Equation

Provide selectivity and

resolution

– Compare Intra- and Inter-

separation techniques

Tune retention

– Reduce or increase retention

based on the retention

mechanisms

Identify hidden components

Benefits:

– Better understand your process

– Eliminate reaction scheme

inhibitors

– Provide flexibility for scale-up

process

Rs1

1

k

k

4

N


Convergence Chromatography

Selectivity Space

Unlimited solvent

and stationary phase selectivity

Addressing Selectivity: Convergence Chromatography

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

Weak

Str

ong

Supercritical CO2

Organic Modifier


CC compared to RPLC Rosuvastatin Synthesis

JM141-75_06252013_11:00am

Time-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 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

AU

0.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-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 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

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

-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 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

AU

0.0

1.0e-2

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

CRT_RXN8_Path2_T0-1-2 3: Diode Array 270

Range: 8.268e-2

0.53

0.01

1.77

CRT_RXN8_path2_UPLC_T0-2 Diode Array 270

Range: 3.052e-1

1.04

0.92

0.19 1.26


Range: 3.273e-1

1.05

1.17

SM1 = m/z 352 Da

SM2 = m/z 535 Da SM2

SM1

SM2

SM2

Low pH UPLC

High pH UPLC

UPC2

2.5 min

1.5 min

1.5 min


CC compared to RPLC Rosuvastatin Synthesis

JM141-75_06252013_5:00pm

Time-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 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

AU

0.0

5.0e-3

1.0e-2

1.5e-2

2.0e-2

2.5e-2

3.0e-2

3.5e-2

4.0e-2

4.5e-2

-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 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

AU

-5.0e-3

0.0

5.0e-3

1.0e-2

1.5e-2

2.0e-2

2.5e-2

3.0e-2

3.5e-2

4.0e-2

-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 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

AU

-5.0e-3

0.0

5.0e-3

1.0e-2

1.5e-2

2.0e-2

2.5e-2

3.0e-2

3.5e-2

4.0e-2

4.5e-2

5.0e-2

CRT_RXN8_Path2_T5-1-2 3: Diode Array 298

Range: 5.798e-2

0.69

0.53

0.01

1.76


Range: 5.239e-2

1.04

0.190.21

1.08


Range: 4.845e-2

1.05

0.950.20

1.07

1.25

SM1 SM2

PDT

PDT + SM1 Co-elution

2.5 min

1.5 min

1.5 min

Low pH UPLC

High pH UPLC

UPC2


Hydrophilic Compounds


UPC2 Publications






Figure 1D shows that the average

Ce in SFC was equal to 22 and 30%

with the 2-EP and hybrid phase,

respectively.

As shown in Fig. 1D, the analyzed

compounds were well distributed

over the gradient, from 5 to 34%

MeOH on the 2-EP phase and from

11 to 40% MeOH on the bare

hybridphase.

Sensitivity

Between SFC (...) and RPLC at pH 9, the

average gain in sensitivity was ∼7.

However, the peak area enhancement

was found to be much more compound

dependent.



Lipophilic Compounds


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

5%

A comparison of GC-MS and UPC2-MS

42

RT: 0.00 - 49.99 SM: 15G

0 5 10 15 20 25 30 35 40 45

Time (min)

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

NL:2.40E4

m/z= 73.50-74.50+504.24-505.24 MS 2012July13 CME 100 ppm_N27

C14:0

C16:0

C18:0 C18:1

%

100

100

Time

C8:0 C10:0

C12:0

C14:0

C16:0

C18:0

C18:1

C18:2

GC-MS IP 585/10 method

UPC2-MS method

C17:0 (IS)


Natural Compounds


Neutral and Acidic Compound Mix: (Food Analysis)

Vanillin C

om

pounds

Vanillin

piperonal

ferulic acid

coumarin p-coumaric acid

vanillic acid 4-hydroxybenzoic acid

3,4-dihydroxybenzaldehyde 4-hydroxybenzalcohol

1 3 2

4 5

7

6

8

9 10

ethyl vanillin


Screening: Acidic Additives

Methanol

Acidic additive improves peak shape of phenol compounds

Column: 2-EP 3.0 x 100 mm, 1.7 µm; Wavelength: 260 nm-Compensated

20mM Citric Acid in Methanol

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

AU

0.00

0.20

0.40

0.60

0.80

Minutes

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

1 5

8 10

4

2,9

7 6

3

1 5

8 10

4,3

2,9

7 6

1-vanillin

2-4-hydroxybenzalcohol

3- 3,4-

dihydroxybenzaldehyde

4-vanillic acid

5-ethyl vanillin

6-4-hydroxybenzoic acid

7-coumaric acid

8-coumarin

9-ferulic acid

10-piperonal


AU

0.00

0.20

0.40

0.60

0.80

AU

0.00

0.20

0.40

0.60

0.80

1.00

AU

0.00

0.50

1.00

Minutes

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20

Effect of Temperature: Optimization

Temperature changes selectivity of critical pair

Solvent effect for piperonal observed at lower temperatures

Column: 2-EP 3.0 x 100 mm, 1.7 µm; Wavelength: 260 nm-Compensated

40 ° C

50 °C

30 ° C 1 5

8

10

4

2

7

6

3

9

1 5

8

10

1 5

8

10

7

6

7

6

4

3

9,2

4

3

9,2

1-vanillin

2-4-hydroxybenzalcohol

3- 3,4-

dihydroxybenzaldehyde

4-vanillic acid

5-ethyl vanillin

6-4-hydroxybenzoic acid

7-coumaric acid

8-coumarin

9-ferulic acid

10-piperonal


Chiral Compounds


Chiral Separations

Key advantages of moving to UPC2

– Results that are equal to or better than NPLC

– Drastic reduction in analysis time (up to 30X)

– Nearly 75X reduction in solvent

– Drastic reduction in cost of analysis (up to 100X)

o Waste generation and disposal

AU

0.00

0.12

0.24

0.36

0.48

Minutes

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

AU

0.00

0.30

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

UPC²

NPLC

11 min

0.3 min

Chiral screening

Chiral method development

– MS and UV detection

Chiral inversion studies

Enantiomeric excess

Fast Chiral Separations


Preparative Chromatography


A Typical Workflow to Support Synthesis

Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

AU

0.0

1.0e-2

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

8.0e-2

9.0e-2

1.0e-1

1.1e-1

1.2e-1

1.3e-1

1.4e-1

1.5e-1

1.6e-1

1.7e-1

1.8e-1

1.9e-1

2.0e-1

2.1e-1

2.2e-1

2.3e-1

2.4e-1

2.5e-1

2.6e-1

2.7e-1

2.8e-1

2.9e-1

3.0e-1

3.1e-1

3.2e-1

3.3e-1

3.4e-1

3.5e-1

3.6e-1

Timed

1#1,1:47

Timed

1#1,1:48

Timed

1#1,1:49

Timed

1#1,1:50

Timed

1#1,1:51

Timed

1#1,1:52

Timed

1#1,1:53

Timed

1#1,1:54

Timed

1#1,1:55

Timed

1#1,1:56

6.022.83

4.08

12.439.23

7.26 10.47

15.63

13.6816.89

AU

-0.0035

0.0000

0.0035

0.0070

0.0105

AU

0.0000

0.0076

0.0152

0.0228

0.0304

AU

-0.004

0.000

0.004

0.008

0.012

Minutes0.00 0.30 0.60 0.90 1.20 1.50 1.80 2.10 2.40 2.70 3.00 3.30 3.60 3.90 4.20 4.50 4.80 5.10 5.40 5.70 6.00

Timothy M. Shoup, David F. Lee Jr., Rui Chen, Marc D. Normandin, Ali A. Bonab, Georges El Fakhri, John P. McCauley and Neil Vasdev, “Synthesis of the dopamine D2/D3 receptor agonist (+)-PHNO via supercritical fluid chromatography: Preliminary PET imaging study with [3-11C]-(+)PHNO, Tetrahedron Letters, 2013.

Synthesis

AU

0.000

0.010

0.020

0.030

0.040

AU

0.000

0.010

0.020

0.030

AU

0.000

0.010

0.020

0.030

0.040

AU

0.00

0.02

0.04

0.06

Minutes0.000.200.400.600.801.001.201.401.601.802.002.202.402.602.803.003.203.403.603.804.004.204.404.604.805.00

(A) AD-H

(B) OD-H

(C) OJ-H

(D) AS-H

Analysis

Purification

Fraction Analysis

A workflow across many industries

o Pharmaceutical

o Chemical material

o University/Research Institute

Targeting the main product

Productivity/ROI is the key


Preparative LC


Preparative SFC


Enhanced Purification Workflow for Medicinal Chemistry

Dry Down Fraction

Purify by Preparative LC-MS Purify by Preparative SFC-MS

Use tR to DetermineLC Segmented Gradient

Use tR to DetermineSFC Segmented Gradient

Best Results Obtained by

Analysis by UPLC and UPC2

Sample Preparation

UPLC UPC230% 70%

In collaboration with Gerard Rosse and Maria Ramirez, Dart Neuroscience, LLC


Column: Chiralpak AD-H Eluent: CO2/ EtOH 90/10 Flow rate: 50ml/min T°c: 35°C Outlet pressure: 100b

Chiral Separation : SFC vs HPLC

Courtesy of D. Speybrouck, Janssen Cilag

HPLC SFC

Column ID 50 mm 20 mm

Mobile Phase Heptane 95% Ethanol 5%

CO2 90% Ethanol 10%

Flow rate 180 ml/min 50 ml/min

Injected amount 500 mg 88 mg

Cycle time 27 min 4.5 min

Productivity 1000 mg/hour 2030 mg/hour

Solvent consumption

9.72 L CO2 : 1.33 L Ethanol : 0.15L

22 g racemic mixture

Solvent consumption

214 L CO2 : 29.2 L Ethanol : 3.3 L

Fraction A 47.5 L 1 L

Fraction B 71 L 1.5 L


Thank You

Questions?

Modern Approaches to Chromatography - Waters Corporation · 2014-12-12 · Modern Approaches to Chromatography Guy Wilson UPC2 & PurificationBusiness Development, Northern Europe,

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