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
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)
Top Related