Liquid Chromatography 2

Lecture Date: April 14th, 2008

Outline of Topics UHPLC – ultra-high pressure liquid chromatography

(also referred to as UPLCTM, as sold by Waters)– Smaller particle packed columns

Monolithic stationary phases:– Dionex ProSwiftTM

– Phenomenex OnyxTM

2D LC Micro-HPLC

– Eksigent Technologies 8-channel HPLC– NanoStream 24 column HPLC– Other examples

Preparative and Simulated Moving Bed (SMB) LC

10 min

1980’s to present day3.5 - 5µm spherical micro-porous1500-4000 psi (106.4-283.7 bar)50,000 - 80,000 plates/meter3.9 x 300mm

Early 1970’s10µm Irregular micro-porous1000-2500 psi (71-177 bar)25,000 plates/meter3.9 x 300mm

10 min

Particle Size EvolutionLate 1960’s40µm pellicular non-porous coated100-500 psi (7.1-35.5 bar)1000 plates/meter1m columns

10 min

Diagrams from Waters Inc.

J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A.

Smaller Particles

Smaller particles provide increased efficiency

With smaller particles this efficiency increase extends over a wider linear velocity

This provides the ability for both added resolution and increased speed of separation

Particles are central to the quality of the separation

The evolution of the van Deemter plot

Diagram from Waters Inc.

Faster Chromatography Can Reduce Resolution

* 50 mm column * Higher Flow Rates

2.0 mL/min

0.0

1

2

3.0 mL/min.

1

2

Time in Minutes 3.0

1 -- 0.4 0.12 3.3 0.3 0.3

Peak Rs RT %RSD

Area %RSD

1 -- 0.8 0.32 2.3 0.6 0.4

Peak Rs RT %RSD

Area %RSD

Fails Rs Goal of 3 Limitation

5um Reversed Phase Column

“Compressed Chromatography”

Run time is reduced, but resolution is lost!Diagram from Waters Inc.

UPLC Separations

Diagram from Waters Inc.

Achieving Speed without Compression

Peak Capacity = 153AU

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 SensitivityAU

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

Diagram from Waters Inc.

HPLC and UPLCTM

2.1x100mm 4.8µm

HPLC

0.30

AU

FS

Time in Minutes0.0 10.0

Rs = 4.71

Rs = 9.15

2.1x100mm 1.7µm ACQUITY UPLCMore Resolution

ACQUITY UPLCTM

0.30

AU

FS

10.0

Rs = 1.86

Rs = 2.30

8 Diuretics + impurity

Diagram from Waters Inc.

10.0

0.30

AU

FS

Rs = 4.71

Rs = 9.15

2.1x100mm 1.7µm ACQUITY UPLC

0.33

AU

FS

Time in Minutes0.0 3.5

Rs = 3.52

Rs = 1.82

2.1x30mm 1.7µm ACQUITY UPLCScaled Gradient

Same Resolution as HPLC, Less Time

ACQUITY UPLCTM

ACQUITY UPLCTM

HPLC and UPLCTM

Diagram from Waters Inc.

Requires improvements in the whole column:

– Sub 2 µm particles Porous for optimum mass transfer New bridged hybrid particle required for pressure

tolerance (up to 15000 psi) Sizing technology for narrow particle size

distribution

– Column hardware New frit technology to retain particles New end fittings for high pressure/low dispersion

operation

– Packing technology New column packing processes to optimize stability

Technology Requirements

Creating a New Particle Technology

Advantages Disadvantages

Inorganic (Silicon)

• Mechanically strong• High efficiency• Predictable retention

• Limited pH range• Tailing peaks for bases• Chemically unstable

Polymer (Carbon)

• Wide pH range• No ionic interactions• Chemically stable

• Mechanically ‘soft’ • Low efficiency• Unpredictable retention

Hybrid (Silicon-Carbon) Particle TechnologyDiagram from Waters Inc.

Bridged Ethane-Silicon Hybrid Particles

Anal. Chem. 2003, 75, 6781-6788

Si

Si

Si

Si

SiO

O

O

O

O

Si

Si

C

CC

CC

C

Bridged Ethanes in Hybrid Matrix - Strength - Good Peak Shape - Wider pH Range

Diagram from Waters Inc.

If N ↑ 3x, then Rs ↑ 1.7x

Explaining UHPLC with the Resolution Equation

System Selectivity RetentivityEfficiency

k

kNRs

11

4

NRs

J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A.

In UHPLC systems, N (efficiency) is the primary driver

Selectivity and retentivity are the same as in HPLC

Resolution, Rs, is proportional to the square root of N:

Improving Resolution with Smaller Particles

If dp ↓ 3X, then N ↑ 3X, and Rs ↑ 1.7X

J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A.

For now, assume a constant column length

From the van Deemter equation, we know that efficiency (N), is inversely proportional to particle size (dp):

ps d

R1

Relationship between Peak Width and Efficiency for Constant Column Length

J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A.

2

1

WN

WHeight

1

Efficiency (N) is inversely proportional to the square of Peak Width W:

Peak height is inversely proportional to peak width

Outcome – narrower peaks are taller, and easier to detect

If dp ↓ 3X, then N ↑ 3X, and Rs ↑ 1.7X

and sensitivity ↑ 1.7X

Back Pressure at Constant Column Length

If dp ↓ 3X, then P ↑ 27X

J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, “Advancing LC Performance with smaller particles and higher pressure”, Anal. Chem., 77 (2005) 460A-467A.

Back Pressure is proportional to Flow Rate (F) and inversely proportional to the square of particle size (dp):

pdFP

1

pdF

1

Optimal flow rate is inversely proportional to particle size:

Summary of Effects at Constant Column Length

  Resolution Improvement

Speed Improvement

Sensitivity Improvement

Back Pressure

1.7 vs. 5 µm particles 1.7x 3x 1.7x 25x

1.7 vs. 3 µm particles 1.3x 2x 1.3x 6x

Fixed Column Length: Flow Rate Proportional to Particle Size

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00

4.8 µm, 0.2 mL/min, 354 psi

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Theory:1.7X Resolution

3X Faster1.7X Sensitivity25X Pressure

1.5X Resolution2.6X Faster

1.4X Sensitivity22X Pressure

1.7 µm, 0.6 mL/min, 7656 psi

2.1 x 50 mm columnsDiagram from Waters Inc.

HPLC UPLC™

Cycle time (min) 27 3

# of Samples Run per Year 10,000 90,000

Productivity Improvements

UPLC™ gives 70% higher resolution in 1/3 the time

Target resolution is obtained 1.7x (+70%) faster

Method development up to 5x faster

Assume that an HPLC is running about 67% of the year, or 4,000 hr:

Diagram from Waters Inc.

Novel UHPLC Applications: High Resolution Peptide Mapping

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 = 168Pc = 360

2.5X increase

HPLC4.8 µm

Peaks = 70Pc = 143

Diagram from Waters Inc.

Drawbacks to UPLC

• Cost• Solvent mixing problems• Lack of variety in commercial columns at 1.7 um• Baseline ripple – real data:

min0 1 2 3 4 5 6 7 8

mAU

0

10

20

30

40

50

DAD1 A, Sig=220,4 Ref=off (OPEN_ACC\06080856.D)

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

Minutes

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

HPLC

UPLC

Monolithic Stationary Phases

Limitations of packed particle columns:

– Back pressure gets really high as the particle gets smaller – e.g. with UHPLC

What is a monolith?

– A continuous porous stationary phase or SP support

How are they made?

– Polymerization reactions that yield voids

Image from F. Svec, C. G. Huber, Anal. Chem. 78, 2100-2107 (2006)

Monolithic Stationary Phases

Typical monoliths (SEM images of the support for the stationary phase)

Both mesopores and micropores are apparent

http://www.iristechnologies.net/CIM/monolith_structure.gif

Advantages of Monolithic Stationary Phases Monolithic columns offer

several advantages over particulate columns

– The porous polymeric rod, which has no intra-particular void volumes, improves both mass transfer and separation efficiency

– Allow higher mobile phase flow rates with lower backpressure

– Stable over a wide pH range

Three Dionex monolithic columns

compared with a polymer bead

(particle) column

Figures from Dionex, Inc. application note, www.dionex.com

Two different flow rates on a monolithic

columns (viper venom, a complex biological mixture)

Two-Dimensional Liquid Chromatography (2D-LC)

● 2D LC: two LC experiments run back-to-back, with the effluent from the first LC column broken up and injected on a second LC column

P. Dugo et al., Anal. Chem. 78, 7743-7750 (2006).

Fast RP LC dimension

Slower NP LC dimension

Micro-LC

Micro (and nano) LC refers to precision microfluidic separation systems being developed for potential roles in drug discovery, miniaturized medical devices, enviromental and security applications, etc…

Micro-LC incorporates technologies such as:

– microfluidic flow control

– microscale pumping

– microfabrication

In other words, miniaturize the entire LC system

Eksigent Technologies: “Express”

Advantages of Miniaturization:

– Increase in the number of parallel analyses

– Decrease in analysis time

– Decrease in sample/reagent consumption, instrument footprint

– Increase in integrated system functionality

Barriers to Microscale HPLC

– Poor control of low flow rates

– Loss of separation efficiency from instrumental components

– Low sensitivity for absorbance detection (e.g. UV)

Microfluidic Flow Control

•Precise control of flow rate (1 nl/min to 100 µl/min) •Ability to pump against substantial back pressures (to 10,000 psi or more) •Active feedback for identification -and prediction- of leaks or blockages •Virtually instantaneous response to step changes in flow rate setpoint

Microfabrication

Detectors and Column

•microscale flow control increases in separation speed, system component optimized to minimize extra column variance.

•Advances allow typical gradient methods to be run at injection-to-injection cycles • 4-6 times faster than conventional analytical HPLC without a loss in resolution.

•This speed is a result of higher resolution in microscale formats, coupled with extremely rapid gradient mixing and column re-equilibration times.

column flow rates from 200 nl/min up to 20 ul/min.

Eskigent Express

High Throughput HPLC: Eksigent Express 800

56 Chromatograms

10 Minutes

0.0 2.5 5.0 7.5 10.0

8

7

6

5

3

2

4

Ab

sorb

an

ce

Time (min)

1

50 x .300 mm; 5 m Luna C18(2) Gradient: 65 95 % ACN in 25 sHold for 20 s; Equilibrate: 20 s12 L/min

0 10 20 30 40 50

0

500

1000

1500

Ab

sorb

an

ce (

mA

U)

Time (sec)

Another Example: The Nanostream PLC

Images courtesy of Nanostream Inc.

Nanostream PLC

Features of the Nanostream system include:

– 24 UV absorbance detectors

– A 8-head Autosampler

– Stationary phase – 10 m (Van deemter plot!)

– Column Length – 80 mm

– Equivalent i.d. – 0.5 mm

– Injection volumes 0.4-1.0 L

Preparative Chromatography

Preparative chromatography (and preparative separations sciences): the use of a separation method to isolate individual components of a material on a large scale

Can be used for both production and analysis

– Production: isolation of food, agricultural and pharmaceutical products, e.g. the recovery of sucrose is accomplished using prep SMB systems with capacilty of 500 tons/day feedstock (beet molasses)

– Analysis: the isolation and enrichment of impurities for chemical analysis

Preparative Chromatography

Slide courtesy of Novasep

The Langmuir Isotherm

Slide courtesy of Novasep

Non-Linear Chromatography

Slide courtesy of Novasep

Batch Preparative Chromatography

Inject and collect – delay between injections!

mobile phase

mobile phase

mobile phase

Inject

Inject again

CollectDrawings courtesy Dr. G. Terfloth, GSK

True Moving Bed Chromatography What if we could move the SP backwards too?

mobile phase

mobile phase

stationary phase

Drawings courtesy Dr. G. Terfloth, GSK

Column 1 Column 2 Column 3 Column 4

True Moving Bed Chromatography

What if we move the stationary phase backwards too?

Drawings courtesy Dr. G. Terfloth, GSK

mobile phase

stationary phase

Column 1 Column 2 Column 3 Column 4inject

collect collect

SMB – Martin and Kuhn

Original Patent from 1940 (literally a moving SP):

Simulated Moving Bed Chromatography Simulated moving bed (SMB) – a more practical way to

“move” the stationary phase, compatible with modern columns and pumps

Step 1 - inject

Drawings courtesy Dr. G. Terfloth, GSK

Flow

inject

Simulated Moving Bed Chromatography

Step 2 – move injector, inject again

Drawings courtesy Dr. G. Terfloth, GSK

Flow

inject

Simulated Moving Bed Chromatography Step 3 – collect, then move injector again, inject again

Continuous chromatography – keep moving, injecting, collecting as needed. Because it can go on for so long, it can separate closely-eluting compounds

Drawings courtesy Dr. G. Terfloth, GSK

Flow

inject

collect

collect

Further Reading

Please note that many other new LC technologies are being developed that are not discussed here!

For more about UHPLC, see:

– J. R. Mazzeo, U. W. Neue, M. Kele, and R. S. Plumb, Anal. Chem. 77 (2005) 460A-467A.

For more about monolithic materials in LC, see:

– F. Svec, C. G. Huber, Anal. Chem. 78, 2100-2107 (2006)

For more about SMB, see:

– F. Charton, R. M. Nicoud, J. Chrom. A 702, 97-112 (1995)