Liquid Chromatography 2
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Liquid Chromatography 2. Lecture Date: April 14 th , 2008. Outline of Topics. UHPLC – ultra-high pressure liquid chromatography (also referred to as UPLC TM , as sold by Waters) Smaller particle packed columns Monolithic stationary phases: Dionex ProSwift TM Phenomenex Onyx TM 2D LC

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Liquid chromatography 2

Liquid Chromatography 2

Lecture Date: April 14th, 2008


Outline of topics

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


Particle size evolution

10 min

10 min

10 min

Particle Size Evolution

Late 1960’s

40µm pellicular non-porous coated

100-500 psi (7.1-35.5 bar)

1000 plates/meter

1m columns

Early 1970’s

10µm Irregular micro-porous

1000-2500 psi (71-177 bar)

25,000 plates/meter

3.9 x 300mm

Diagrams from Waters Inc.

1980’s to present day

3.5 - 5µm spherical micro-porous

1500-4000 psi (106.4-283.7 bar)

50,000 - 80,000 plates/meter

3.9 x 300mm

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

Smaller Particles

The evolution of the van Deemter plot

Diagram from Waters Inc.


Faster chromatography can reduce resolution

“Compressed Chromatography”

1

5um Reversed

Phase Column

* 50 mm column

* Higher Flow Rates

2

2.0 mL/min

1

2

3.0 mL/min.

Fails Rs Goal of 3

Limitation

0.0

3.0

Time in Minutes

Faster Chromatography Can Reduce Resolution

Run time is reduced, but resolution is lost!

Diagram from Waters Inc.


Uplc separations

UPLCSeparations

Diagram from Waters Inc.


Achieving speed without compression

2.1 x 50 mm, 5 µm

0.10

Peak Capacity = 153

AU

0.05

0.00

0.10

2.1 x 50 mm, 1.7 µm

Peak Capacity = 123

AU

0.05

0.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

Minutes

0.10

AU

0.05

0.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes

Minutes

Achieving Speed without Compression

6x Faster

3x Sensitivity

Diagram from Waters Inc.


Hplc and uplc tm

Rs = 2.30

0.30

Rs = 1.86

AUFS

0.30

Rs = 9.15

10.0

Rs = 4.71

AUFS

0.0

10.0

Time in Minutes

HPLC and UPLCTM

8 Diuretics + impurity

2.1x100mm 4.8µm

HPLC

2.1x100mm 1.7µm ACQUITY UPLC

More Resolution

ACQUITY UPLCTM

Diagram from Waters Inc.


Hplc and uplc tm1

0.30

AUFS

0.33

10.0

Rs = 9.15

Rs = 3.52

AUFS

Rs = 4.71

Rs = 1.82

0.0

3.5

Time in Minutes

HPLC and UPLCTM

2.1x100mm 1.7µm ACQUITY UPLC

ACQUITY UPLCTM

2.1x30mm 1.7µm ACQUITY UPLC

Scaled Gradient

Same Resolution as HPLC, Less Time

ACQUITY UPLCTM

Diagram from Waters Inc.


Technology requirements

Technology Requirements

  • 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


Creating a new particle technology

Creating a New Particle Technology

Advantages

Disadvantages

Inorganic

(Silicon)

  • Mechanically strong

  • High efficiency

  • Predictable retention

  • Limited pH range

  • Tailing peaks for bases

  • Chemically unstable

  • Wide pH range

  • No ionic interactions

  • Chemically stable

  • Mechanically ‘soft’

  • Low efficiency

  • Unpredictable retention

Polymer

(Carbon)

Hybrid (Silicon-Carbon) Particle Technology

Diagram from Waters Inc.


Bridged ethane silicon hybrid particles

C

Si

Si

O

Si

C

C

C

Si

C

O

O

C

Si

O

Si

Si

O

Bridged Ethane-Silicon Hybrid Particles

Bridged Ethanes in Hybrid Matrix

- Strength

- Good Peak Shape

- Wider pH Range

Diagram from Waters Inc.

Anal. Chem. 2003, 75, 6781-6788


Explaining uhplc with the resolution equation

Explaining UHPLC with the Resolution Equation

  • 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:

System Selectivity Retentivity

Efficiency

If N↑ 3x, then 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.


Improving resolution with smaller particles

Improving Resolution with Smaller Particles

  • For now, assume a constant column length

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

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.


Relationship between peak width and efficiency for constant column length

Relationship between Peak Width and Efficiency for Constant Column Length

  • 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

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 at constant column length

Back Pressure at Constant Column Length

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

  • Optimal flow rate is inversely proportional to particle size:

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.


Summary of effects at constant column length

Summary of Effects at Constant Column Length


Fixed column length flow rate proportional to particle size

0.050

0.040

0.030

AU

0.020

0.050

0.010

0.000

0.040

0.00

1.00

2.00

3.00

4.00

5.00

6.00

Minutes

0.030

AU

0.020

0.010

0.000

0.00

2.00

4.00

6.00

8.00

10.00

12.00

15.00

Minutes

Fixed Column Length: Flow Rate Proportional to Particle Size

1.5X Resolution

2.6X Faster

1.4X Sensitivity

22X Pressure

1.7 µm, 0.6 mL/min, 7656 psi

Theory:

1.7X Resolution

3X Faster

1.7X Sensitivity

25X Pressure

4.8 µm, 0.2 mL/min, 354 psi

2.1 x 50 mm columns

Diagram from Waters Inc.


Productivity improvements

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

Novel UHPLC Applications: High Resolution Peptide Mapping

0.08

HPLC

4.8 µm

Peaks = 70

Pc = 143

0.06

0.04

AU

0.02

0.00

0.08

UPLC™

1.7 µm

Peaks = 168

Pc = 360

2.5X increase

0.06

0.04

AU

0.02

0.00

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

Minutes

Diagram from Waters Inc.


Liquid chromatography 2

Drawbacks to UPLC

  • Cost

  • Solvent mixing problems

  • Lack of variety in commercial columns at 1.7 um

  • Baseline ripple – real data:

HPLC

UPLC


Liquid chromatography 2

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)


Liquid chromatography 2

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


Liquid chromatography 2

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

Two different flow rates on a monolithic columns (viper venom, a complex biological mixture)

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


Liquid chromatography 2

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

Fast RP LC dimension

Slower NP LC dimension

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


Liquid chromatography 2

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


Liquid chromatography 2

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)


Liquid chromatography 2

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


Liquid chromatography 2

Microfabrication

Detectors and Column


Liquid chromatography 2

Eskigent Express

  • 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.


Liquid chromatography 2

High Throughput HPLC: Eksigent Express 800

56 Chromatograms

10 Minutes

50 x .300 mm; 5 mm Luna C18(2)

Gradient: 65  95 % ACN in 25 s

Hold for 20 s; Equilibrate: 20 s

12 mL/min


Liquid chromatography 2

Another Example: The Nanostream PLC

Images courtesy of Nanostream Inc.


Liquid chromatography 2

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


Liquid chromatography 2

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


Liquid chromatography 2

Preparative Chromatography

Slide courtesy of Novasep


Liquid chromatography 2

The Langmuir Isotherm

Slide courtesy of Novasep


Liquid chromatography 2

Non-Linear Chromatography

Slide courtesy of Novasep


Liquid chromatography 2

Batch Preparative Chromatography

  • Inject and collect – delay between injections!

Inject

Inject again

Collect

Drawings courtesy Dr. G. Terfloth, GSK


Liquid chromatography 2

True Moving Bed Chromatography

  • What if we could move the SP backwards too?

Column 1

Column 2

Column 3

Column 4

Drawings courtesy Dr. G. Terfloth, GSK


Liquid chromatography 2

True Moving Bed Chromatography

  • What if we move the stationary phase backwards too?

inject

Column 1

Column 2

Column 3

Column 4

collect

collect

Drawings courtesy Dr. G. Terfloth, GSK


Liquid chromatography 2

SMB – Martin and Kuhn

  • Original Patent from 1940 (literally a moving SP):


Liquid chromatography 2

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

inject

Flow

Drawings courtesy Dr. G. Terfloth, GSK


Liquid chromatography 2

Simulated Moving Bed Chromatography

  • Step 2 – move injector, inject again

inject

Flow

Drawings courtesy Dr. G. Terfloth, GSK


Liquid chromatography 2

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

collect

Flow

inject

collect

Drawings courtesy Dr. G. Terfloth, GSK


Further reading

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)


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