HPLC Column Technology: Smaller
and Faster
Tuesday, May 13, 2008
HPLC 2008
Ronald E. Majors
Agilent Technologies
Wilmington, DE, USA
Outline of Talk*
• Drivers for HPLC Column Improvements
• Approaches to increase throughput in HPLC
• Small porous particles (10-  5-  3-  2-3- 
< 2-µm)
• Superficially porous** and non-porous particles
(40 1.5- 5-  2.7-µm)
• Monoliths
• Silica-based
• Polymer-based
• Brief Comparisons of above
• Parallel LC
* Focus on commercial products, not research products
** Also called pellicular, porous layer beads, fused core
1
Drivers for Improvements in HPLC Column
Technologies
1.
Productivity Gains (faster separations, rapid method development, automation,
time-to-market, QC department—quicker results)
2.
Improvement in Quality of Analysis [reproducible columns, improved recovery (biocompounds), lower activity (less tailing, especially for basic compounds}]
3.
Cost of Analysis (column lifetime & stability, guard columns, solvent reduction,
lower cost solvents, narrow bore and smaller, high throughput LC)
4.
Smaller, more complex samples, trace analysis (proteomics) (nanocolumns,
selectivity improvements-specialty columns, 2-D & comprehensive chrom./column
switching)
5.
Widespread use of LC-MS & LC/MS-MS (cap/nano columns, short columns,
smaller particles, packings with wider range of solvent compatibility, low bleed)
6.
Increasing Importance of Biologically -Derived Molecules (wide pore packings,
rugged packings, biocompatible surfaces, inert column materials)
7.
Environmental Reasons; solvent reduction (smaller diameter columns,
shorter columns, less toxic solvents, disposal costs)
Some Ways to Increase Sample Throughput
(and Resolution)

1) Shorter column lengths (to reduce analysis time) packed with small porous
particles (to maintain resolution). (Reasonable # of plates and reasonable
pressure, fast separation)
2) Longer column lengths (to increase efficiency) packed with even smaller porous
and non-porous particles (to maintain resolution), with the ultimate being the socalled “Ultra-High Pressure LC”. (Many plates, fast separation, high pressure)
3) Columns packed with various small superficially porous particles (pellicular) particle
sizes, pore sizes and phase thickness to allow the rapid resolution of
biomolecules such as proteins as well as small molecules. (Large and small
molecules, fast separations, lower pressure)
4) Columns designed with silica- and polymer-based monolith stationary phase formats
(fast separation, low pressure, in-series columns)
5) Parallel LC (multiple capillary columns/channels, increased samples/hour)
2
History of Commercial HPLC Particle Development
Year(s) of
Acceptance
Particle Size
Most Popular
Nominal Size
Irregularly
-Shaped
1950’s
Glass
Bead
1967
1972
1985
1992
Plates / 15cm
(Approximate)
100µm
200
50 µm (pellicular )
1,000
10 µm
6,000
5 µm
12,000
3-3.5 µm
22,000
1998*
1.5 µm* (non-porous) 30,000
1999
5.0 µm ( pellicular)
8,000**
2000
2003
2007/2008
2.5 µm
1.8 µm
2.7 µm (pellicular)
25,000
32,500
32,000 ***
* non-porous silica or resins
** 300 A pore for protein MW 5,700
*** 90-120 A pore
How to go Faster
With Minimal Loss of Resolution?
Shorter Columns with Smaller Particles
Plates
Rs =
N
N
4
Selectivity


k'
k'+1
L
Column Length =
N
Particle Size
=
N
Particle Size
=
P
dp
To Maintain Rs:
e.g.: L/2
-1

Retention
dp/2
000990P2.PPT
3
Van Deemter Curve: HETP vs. Volumetric
Flow Rate
0.0025
HETP (cm)
H = A + B/u + Cu
Column: ZORBAX Eclipse XDB-C18
Dimensions: 4.6 x 50/30mm
Eluent: 85:15 ACN:Water
Flow Rates: 0.05 – 5.0 mL/min
Temp: 20°C
Sample: 1.0L Octanophenone in Eluent
0.0030
0.0020
5.0m
3.5m
1.8m
0.0015
0.0010
0.0005
0.0000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Volumetric Flow Rate (mL/min)
Smaller particle sizes have flatter curves, minima shift out sli ghtly
Column Scalability: Change in Column Configuration
to Increase Speed While Maintaining Resolution
mAU
70
4.6 x 250-mm, 5 µm
1
60
50
40
R4,3 =9.30
R3,2 =3.71
R2,1 =4.31
2
3
30
20
8 min.
4
10
0
1
mAU
2
3
4
5
4.6 x 100-mm, 3.5 µm
6
7
8
R4,3 =8.53
R3,2 =3.37
R2,1 =3.69
60
50
40
30
min
3 min.
20
10
0
1
2
3
4
4.6 x 50-mm, 1.8 µm
6
7
8
R4,3 =9.30
R3,2 =3.61
R2,1 =3.87
mAU
60
50
5
1.5 min.
Column: Zorbax SB-C18
A= 0.2% FA, B=AcCN w 0.2%FA (98:2)
F=1.5 mL/min Inj. Vol: 2-,4-, 6-ul, respectively;
Detector: DAD, 254-nm; Flowcell: 3uL, 2 mm flow path
40
30
20
10
0
1
2
3
4
5
6
min
7
8
min
Solutes:
1-methylxanthine; 2) 1,3-dimethyluric acid; 3) 3,7-dimethylxanthine; 4) 1,7-dimethylxanthine
4
But What About Pressure? Pressure Increases
Dramatically with Decreasing Particle Size
Equation For Pressure Drop Across An HPLC Column
 L v
 dp2
P
=
P
= Pressure Drop

= Fluid Viscosity
L
= Column Length
v
= Flow Velocity
dp
= Particle Diameter

= Dimensionless Structural Constant of
Order 600 For Packed Beds in LC
 Many parameters influence column
pressure
 But the particle size and column length
are most critical
Long length and smaller particle size
mean more resolution and
pressure
 HPLC systems are now available that
can handle these higher pressures
Pressure and Efficiency Vary Inversely with dP
…but the effect of dP on pressure is greater
We need to try something more than continue to reduce particle
size; monoliths and nonporous particles may play a role.
25,000
N 
20,000
15,000
10,000
1
dp
bar
Efficiency
30,000
5,000
0
0
5
10
dp (μm)
15
20
As efficiency doubles,
pressure quadruples
400
350
300
250
200
150
100
50
0
1
P 2
dp
0
5
10
15
20
d p (μm)
Dr. Richard A. Henry, Supelco,
Minn. Chromatography Symp.
May, 2008
5
Pressures for Various Particle Sizes
Pressure,
1
N 
dp
Particle Size
1
P 2
dp
1
PNopt 
d3p
psi
Bar
N
1.8
5889
406
27,500
2.5
3089
213
20,000
3
2118
146
16,500
5
769
53
10,000
10
189
13
5,000
15
87
6
3,750
20
44
3
2,500
100x4.6mm column, 1.0 mL/min, 50/50
Water/ACN, 30
Dr. Richard A. Henry, Supelco,
Minn. Chromatography Symp.
May, 2008
Commercial Two and Sub-Two Micron Totally
Porous HPLC Columns (Pittcon ’08)*
Manufacturer
Product Name
Aver. d P, µm
Agilent
Zorbax Rapid Resolution HT
1.8
Alltech
VisionHT
1.5
Bischoff
ProntoPEARL TPP Ace-EPS
1.8
Macherey-Nagel
Nucleodur
1.8
MicroSolv Technology
Cogent Diamond & Silica-C
1.8
Orachem Technologies
Phenomenex
Emerald
Luna
1.7
2.0
Restek
Pinnacle DB
1.9
Sepax
Shant Laboratories
GP-8 and GP-18
Pathfinder
1.7
1.5
Thermo
Hypersil Gold
1.9
Tosoh Haas
TSKgel SuperODS
2.0
Waters
Acquity BEH
1.7
YMC
Ultra-Fast
2.0
* Non-porous & Superficially Porous Particles Not Included
6
Commercial Two to Three Micron Totally
Porous HPLC Columns (Pittcon ’08)*
Manufacturer
Product Name
Aver. dP , µm
Fortis Technologies
Fortis
2.1
Phenomenex
Luna HST and Synergi
2.5
Sepax
GP-8 and GP-18
2.2
Shimadzu
XR
2.2
Tosoh Bioscience
TSK-Gel ODS HTP
2.3
Varian
Pursuit UPS and XRS
2.4, 2.8
Waters
XBridge, SunFire
2.5
* Non-porous & Superficially Porous Particles Not Included
Some Ways to Increase Sample Throughput
(and Resolution)

1) Shorter column lengths (to reduce analysis time) packed with small porous
particles (to maintain resolution). (Reasonable # of plates and reasonable
pressure, fast separation)
2) Longer column lengths (to increase efficiency) packed with even smaller porous
and non-porous particles (to maintain resolution), with the ultimate being the socalled “Ultra-High Pressure LC”. (Many plates, fast separation, high pressure)
3) Columns packed with various small superficially porous particles (pellicular) particle
sizes, pore sizes and phase thickness to allow the rapid resolution of
biomolecules such as proteins as well as small molecules. (Large and small
molecules, fast separations, lower pressure)
4) Columns designed with silica- and polymer-based monolith stationary phase formats
(fast separation, low pressure, in-series columns)
5) Parallel LC (multiple capillary columns/channels, increased samples/hour)
7
Calculating Peak Capacity - Equations
Peak Capacity (Zp) - Isocratic Elution
Pc =
1
 N
k last :retention factor last peak determines:
N : efficiency
Rs : required resolution (base line separation: Rs 1.5)
P c : peak capacity
ln[1+klast ]
4RS
Peak Capacity (Zp) - Gradient Elution Equations
Equation 1 used in next examples
1. PC 1 
tg
1 
n
Pc =
tg =
w=
n
w
1
t
1  g
wav
peak capacity
gradient time (min)
peak width (min)
tR,n = last peak
tR,m = first possible peak (unretained)
tR,1 = first actual peak
2. Pc =
1+
3. Pc =
1+
tR,n – tR,m
W av
tR, n – tR,1
W av
W impacted by N, which includes column length
and particle size
Peptide Map of BSA for Three Different
Particle Size Columns
mAU
30
Zorbax SB-C18, 2.1x150mm, 5µm
Peak Capacity,
@5 sigma
25
20
231
Starting
Pressure
51
15
10
5
0
mAU
50
4
6
8
10
12
14
16
18
20
min
Zorbax SB-C18, 2.1x150mm, 3.5µm
40
103
348
30
20
10
0
4
mAU
6
8
10
12
14
16
18
20
min*
Zorbax SB-C18, 2.1x150mm, 1.8µm
60
50
442
40
340
30
20
10
0
4
6
8
10
12
14
16
18
20
min*
Gradient Time = 30 min
Temp. = 80°C
8
Gradient Reversed-Phase Separation of Licorice
Root Extract B on 15 cm, 1.8-μm Column
MWD1 B, Sig=254,16 Ref=450,100 (BUD\LIC000025.D)
mAU
450
Peak capacity (@ Rs= 1.0) = 426
400
G 14.6 min
350
300
250
200
GA 25.5 min
150
100
50
0
0
5
10
15
20
25
30
min
MWD1B,Sig=254,16Ref=450,100(BUD\LIC000025.D)
mAU
90
GA
80
Analysis of licorice root extract
B, using a Zorbax SB-C18
Rapid-Resolution HT , 1.8-um,
4.6 x 150 mm column.
70
60
50
40
30
20
16
18
20
22
24
26
28
30
32
34 min
Some Ways to Increase Sample Throughput
(and Resolution)

1) Shorter column lengths (to reduce analysis time) packed with small porous
particles (to maintain resolution). (Reasonable # of plates and reasonable
pressure, fast separation)
2) Longer column lengths (to increase efficiency) packed with even smaller porous
and non-porous particles (to maintain resolution), with the ultimate being the socalled “Ultra-High Pressure LC”. (Many plates, fast separation, high pressure)
3) Columns packed with various small superficially porous particles (pellicular) particle
sizes, pore sizes and phase thickness to allow the rapid resolution of
biomolecules such as proteins as well as small molecules. (Large and small
molecules, fast separations, lower pressure)
4) Columns designed with silica- and polymer-based monolith stationary phase formats
(fast separation, low pressure, in-series columns)
5) Parallel LC (multiple capillary columns/channels, increased samples/hour)
9
Optimum Chromatographic Efficiency
What is the optimum flow rate for a small vs. a large molecule?
Assumptions:
4.6mm i.d. Column
300 Angstrom Pore Diameter
5m Porous Packing, e 0.4
k 10, opt 13
Small Molecule
MW 100 g/mol
Dm 1x10-5cm2/sec
F opt=
opt Dm
r2
dp
ue = 0.26 cm/sec
Large Molecule
MW 10,000 g/mol
Dm 1x10-6cm2/sec

e
ue = 0.026 cm/sec
F opt = 1.04 mL/min
F opt = 0.104 mL/min
Large (e.g., proteins) should be chromatographed at low flow rates (slow
analyses) on conventional, totally porous packings for maximum resolution.
Effect of Increasing Flow Rate in Macromolecule
(Protein) Analysis with Use of Totally Porous Silica
mAU
1000
Totally Porous, 300 A
2.1x75mm, 5µm
3 mL/min
800
600
400
0-100%B in 0.67 min
200
0
mAU
1000
0
0.5
1
1.5
2
2.5
3
3.5
4
3
3.5
4
2 mL/min
800
600
400
0-100%B in 1 min
200
0
0
0.5
1
1.5
2
2.5
mAU
1000
1 mL/min
800
600
4.5
1.
2.
3.
4.
min
Neurotensin
Rnase A
Lysozyme
Myoglobin
4.5
min
4.5
min
4.5
min
0-100%B in 2 min
400
200
0
mAU
1000
0
0.5
1
1.5
2
1
800
600
2
2.5
3
3
4
3.5
4
0.5 mL/min
400
0-100%B in 4 min
200
0
0
0.5
1
1.5
2
2.5
3
3.5
4
Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Temp.: 70° C, Det.: 215 nm
Mobile Phase: A= 95% H2O, 5% AcN with 0.1%TFA; B= 5% H2O, 95% AcN, with 0.07%TFA
5 min.
10
Effect of Increasing Flow Rate in Macromolecule
(Protein) Analysis with Use of Totally Porous Silica
Resolution Loss
mAU
1000
800
600
400
200
0
0.25
Totally Porous, 300 A
2.1x75mm, 5µm
3 mL/min
0-100%B / 0.67 min
0.3
0.35
0.4
0.45
0.5
0.35
0.4
0.45
0.5
0.45
0.5
0.45
0.5
min
mAU
1000
800
600
400
200
0
0.25
mAU
1000
800
600
400
200
0
0.25
mAU
1000
800
600
400
200
0
0.25
2 mL/min
0-100%B in 1 min
0.3
1 mL/min
1.
2.
3.
4.
Neurotensin
Rnase A
Lysozyme
Myoglobin
min*
0-100%B in 2 min
0.3
0.5 mL/min
0.35
0.4
2
1
3
min*
4
0-100%B in 4 min
0.3
0.35
0.4
Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Temp.: 70°C, Det.: 215 nm
Mobile Phase: A= 95% H2O, 5% AcN with 0.1%TFA; B= 5% H2O, 95% AcN, with 0.07%TFA
min*
Aligned
Slower Diffusion of Large Molecules
Broadens Peaks at High Flow Rates
Decrease the diffusion time for macromolecules!
How?
• Increase the Diffusion Rate
• Elevate operating temperature
• Decrease solvent viscosity
• Decrease the Diffusion Distance
• Develop very small particles
• Limit diffusion distance into a particle
Poroshell Approach
11
Comparison of Diffusion Distances
Totally porous silica versus superficially porous silica
5 µm
Superficially Porous Particle
5 µm
Totally Porous Particle
Solid
Core
0.25 µm
2.5 µm
Maximum diffusion depth
for a macromolecule
Effect of Increasing Flow Rate in Macromolecule
(Protein) Analysis with Use of Superficially
Porous Silica
mAU
1000
3 mL/min
800
600
Poroshell 300SB-C18
2.1x75mm, 5µm
0-100%B
0.67 min
400
200
0
0
0.5
1
1.5
mAU
1000
2
2.5
3
3.5
4
600
2.5
3
3.5
4
3
3.5
4
4.5
min
4
4.5
min
0 -100%B
1 min
400
200
0
mAU
1000
4.5
min
2 mL/min
800
0
0.5
1
1.5
2
1 mL/min
800
600
0 -100%B
2 min
400
200
1.
2.
3.
4.
4.5
Neurotensin
Rnase A
Lysozyme
Myoglobin
min
0
0
0.5
1
1.5
2
mAU
1000
2
800
600
3
2.5
4
0.5 mL/min
0 -100%B
4 min
1
400
200
0
0
0.5
1
1.5
2
2.5
3
3.5
Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Temp.: 70°C, Det.: 215 nm
Mobile Phase: A= 95% H2O, 5% AcN with 0.1%TFA; B= 5% H2O, 95% AcN, with 0.07%TFA
5 min
12
Effect of Increasing Flow Rate in Macromolecule (Protein)
Analysis with Use of Superficially Porous Silica
VERY High
Linear
Velocity!
Flow = 3.0 mL/min
Sustained Efficiency and Resolution (5 - 100 %B / 0.67 min)
mAU
1000
800
600
400
200
0
0.25
mAU
1000
800
600
400
200
0
0.25
mAU
1000
800
600
400
200
0
0.25
mAU
1000
800
600
400
200
3 mL/min
Poroshell 300SB-C18
2.1x75mm, 5µm
0-100%B
0.67 min
0.3
0.35
0.4
0.45
0.5
0.35
0.4
0.45
0.5
0.35
0.4
0.45
min
2 mL/min
0-100%B
1 min
0.3
1 mL/min
0 -100%B
2 min
0.3
0.5 mL/min
0-100%B
4 min
0
0.25
1
0.3
0.35
Neurotensin
Rnase A
Lysozyme
Myoglobin
min*
0.5
min*
0.5
min*
4
3
2
1.
2.
3.
4.
0.4
0.45
Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Temp.: 70°C, Det.: 215 nm
Mobile Phase: A= 95% H2O, 5% AcN with 0.1%TFA; B= 5% H2O, 95% AcN, with 0.07%TFA
Aligned
High Flow Rates with 2.1 mm ID Poroshell 300
for High Resolution and Fast Separations
Columns:
Poroshell 300SB- C18
2.1 x 75 mm, 5 m
Mobile Phase:A: 0.1% TFA
B: 0.07% TFA in ACN
Gradient:
5 – 100% B in 1.0 min.
Flow Rate: 3.0 mL/min.
Temperature: 70°C
Pressure: 250 bar
Detection: UV 215 nm
0
4
Sample:
1. Angiotensin II
2. Neurotensin
3. Rnase
4. Insulin
5. Lysozyme
6. Myoglobin
7.Carbonic Anhydrase
8.Ovalbumin
5
3
1
2
7
6
8
0.5
1.0
Time (min)
• High efficiency at higher flow rates for extremely
rapid separations of proteins and peptides.
• This is due to more rapid mass transfer of the superficially porous particle
13
Comparison of Diffusion Distances
Totally porous silica versus superficially porous silicas
5 µm
Totally Porous Particle
5 µm
2.7 µm
Superficially Porous Particle Superficially Porous Particle
Poroshell 300 A
Poroshell 120 A
4.5 µm
0.25 µm
2.5 µm
1.7µm
0.50 µm
Required diffusion distance
for a molecule
What is Poroshell 120?
A new HPLC column with:
1. Sub-2 µm like high efficiency
2. At ~40-50% lower pressure
3. A 2.7 µm particle size with 1.0
µm porous shell
4. 120Å pores for small molecule
separations
5. A “standard” frit to reduce
potential clogging
Solid
Core
1.7um
14
Superficially Porous Column for Small Molecules
can Achieve Equal Performance to 1.8 µm
van Deemter
0.003
Eclipse Plus C18, 3.5 µm
Eclipse Plus C18, 1.8 µm
Poroshell 120 C18, 2.6um
Poroshell 120 C18, 2.6 µm
Poroshell 120 C18, 2.7 µm
0.0025
HETP (cm)
0.002
0.0015
0.001
0.0005
0
0
0.5
1
1.5
2
2.5
3
3.5
Flow rate (mL/min)
Comparing Efficiency and Pressure with Different
Types of Columns
Particle Size/Type
Pressure
Efficiency
LC Compatibility
3.5um – Totally Porous
123 bar
7,800
All 400 bar instruments
2.7um – Poroshell 120
180 bar
11,000
All LC’s
1.8um – Totally Porous
285 bar
11,000
All LC’s but pressure limits
may be reached early
Columns: 4.6 x 50mm,
Mobile Phase: 60% ACN:40% Water Flow Rate: 2 mL/min
15
Some Ways to Increase Sample Throughput
(and Resolution)

1) Shorter column lengths (to reduce analysis time) packed with small porous
particles (to maintain resolution). (Reasonable # of plates and reasonable
pressure, fast separation)
2) Longer column lengths (to increase efficiency) packed with even smaller porous
and non-porous particles (to maintain resolution), with the ultimate being the socalled “Ultra-High Pressure LC”. (Many plates, fast separation, high pressure)
3) Columns packed with various small superficially porous particles (pellicular) particle
sizes, pore sizes and phase thickness to allow the rapid resolution of
biomolecules such as proteins as well as small molecules. (Large and small
molecules, fast separations, lower pressure)
4) Columns designed with silica- and polymer-based monolith stationary phase formats
(fast separation, low pressure, in-series columns)
5) Parallel LC (multiple capillary columns/channels, increased samples/hour)
16
Monoliths as Separation Media
Monoliths: chromatography sorbents cast as homogeneous
phases into chromatographic columns
Typical Commercial Types:
1. Agglomerates of polyacrylamide particles (UNOBioRad Laboratories)
2. Polymethacrylate copolymerisates (CIM-BIA
Separations, Ljubljana, Slovenia; Dionex LC Packings)
3. PS-DVB Copolymerisates [Dionex LC Packings]
4 . Silica rod columns (Chromolith-Merck, Onyx-Phenomenex)
Characteristics of Silica-Based Monolithic Phases
Relative to Packed Microparticulate Columns
1. Flow-through pores with macroporosity (1-2-µm in
width)
2. Mesopore- diffusive pores, average pore diameter can
be controlled.
3. Higher porosity than packed microparticulate columns
(i.e. reduced pressure drop)
4. Efficiency about the same as a 3-5-µm silica gel
5. Flatter h vs. v curves than “efficiency-equivalent”
microparticulate packed column.
17
TM
Chromolith & Microparticulate HPLC Column
Comparison of Pressure vs. Flow Rate
Flow rate - Pressure
LiChrospher RP 18
SilicaROD RP 18
300
Pressure (b ar)
250
200
150
100
50
0
0
1
2
3
4
5
6
7
Flow rate (ml/min)
1. Chromolith TM RP 18 (100-4.6mm)
2. LiChroCART®(125-4mm) LiChrospher® RP 18, 5mm
Mobile Phase: Acetonitrile/ water (35/ 65; v/v)
Column
(courtesy of Merck,Darmstadt)
TM
Chromolith & Microparticulate HPLC Column
Comparison of Plate Height & Flow Rate
Flow rate - Plate Height
LiChrosphe r RP18
30
SilicaROD RP 18
Plate Heig ht ( µm)
25
20
15
10
5
0
0,0
1,0
2,0
3,0
4,0
5,0
6,0
Flow rat e (ml/min)
column:
eluent:
sample:
1. Chromolith TM RP 18 (100-4.6mm)
2. LiChroCART ® (125-4mm) LiChrospher® RP 18, 5um
Acetonitrile/ water (35/ 65; v/v)
Benzene
(courtesy of Merck,Darmstadt)
18
Separation Impedence: E vs. u as a Measure of
Monolith Column Performance
Chromolith® 100 x 3mm, i.d.
Separation
imp edance E
E* =
p · t0
N2 ·
*J.H. Knox
Chromatographia
1977
15000
10000
3m
5000
Chromolith, 3mm
0
0
2
4
6
8
10
Linear flow velocity (mm/ s)
Chromolith ® Performance RP-18e, 3mm i.d.
PurospherTM STAR RP 18e, 3m
(Courtesy of Merck KGaA)
Monolithic Silica Columns
Chromolith FastGradient – 2mm i.d.
25
10
4,6
3
2 mm
Silica-based monolithic columns of different diameters
[K. Cabrera, LCGC No. Amer. 26 (S4) 32 (2008)]
19
Interface of Silica Monolith and PEEK Encapsulation
SEM Picture
[K. Cabrera, LCGC No. Amer. 26 (S4) 32 (2008)]
With Chromolith PEEK Cladding-Minimized Wall
Effects
center region
wall region
Overlap of three chromatograms after manual injections of 0.1mL substance mixture
(toluene, nitrobenzene, 2-nitroanisole) on top of a silica-based monolith with a syringe
in the center or at the wall region. After replacement of the end fitting, connection to an
HPLC-system and operation with n-heptane/ dioxane (95/5; v/v) and a flow rate of
0,38mL/ min.
[K. Cabrera, LCGC No. Amer. 26 (S4) 32 (2008)]
20
Chromolith FastGradient
H/u and ΔP/u Curves
Chromolith FastGradient (50-2mm)
24
110,0
100,0
21
90,0
80,0
18
60,0
15
50,0
ΔP [bar]
H [µm]
70,0
40,0
12
30,0
20,0
9
10,0
6
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
0,0
14,00
lineare Fließgeschwindigkeit [mm/sec.]
[K. Cabrera, LCGC No. Amer. 26 (S4) 32 (2008)]
Van Deemter Plot A: HETP vs. Linear Velocity
Small Particle Columns including Monolith
0.0030
ZORBAX 5.0m
ZORBAX 3.5m
Vendor A 2.5m
Vendor B 2.0m
ZORBAX 1.8m
Monolith
HETP (cm/plate)
0.0025
0.0020
Dimensions: 4.6 x 50/30/20mm
Eluent : 85:15 ACN:Water
Flow Rates: 0.05 – 5.0 mL/min
Temp: 20°C
Sample: 1.0L Octanophenone in Eluent
0.0015
0.0010
0.0005
0.0000
0.0
0.2
0.4
0.6
0.8
1.0
Interstitial Linear Velocity (ue )
1.2
1.4
21
Chromolith® FastGradient (50-2mm)
Fast Analysis of Steroids (Doping Substances)
Mobile Phase:
A-ACN
B-H 2O
0
0,5
1
1,5
t [min]
2
Gradient:
t
[min]
A
[%]
B
[%]
0,0
0,4
2,0
2,5
2,6
3,0
15
15
100
100
15
15
85
85
0
0
85
85
Injection:
Detection:
Temperature:
0,5µl
240 nm UV
40°C
Sample:
1.) Fluoxymesterone
2.) Boldenone
3.) Methandrostenolone
4.) Testosterone
5.) Methyltestosterone
6.) Boldenone acetate
7.) Testosterone acetate
8.) Nandrolone propionate
9.) Testosterone propionate
10.) Nandrolone phenylpropionate
11.) Testosterone isocaproate
2,5
flow
[ml/min]
1,50
1,50
1,00
1,00
1,00
1,00
(Courtesy of Merck KGaA)
Example of Monolithic Polymer for
Biochromatography
CIM = Convective Interactive Media (BIA Separations)
- Crosslinked, porous monolithic polymer (GMA-EDMA)
- Available in disks and tubes
- IEX, HIC, RPC, affinity phases available
- Can be stacked and used for multi-modal separations
- Purification, process control, SPE, trace enrichment
22
Separation of Oligodeoxynucleotides on
CIM DEAE Disk
Separation Device: CIM DEAE Disk,
diameter: 16 mm, 3 mm thick,
active bed volumne: 0.34 mL
Instrument:: HPLC system with low dead
volume mixing chamber
Sample: 10-ug of each in 1 mL buffer A
Injection Volume: 20-uL
Buffer A: 20 mM Tris-HCl, pH 7.4
Buffer B: Buffer A + NaCl
Gradient: Shown on chromatogram
Flow Rate: 6 mL/min
Detection: UV at 260-nm
Sample:
No.of Bases
8
10
12
14
15
16
5'-3' Sequence
Short Name
CCA TGT CT
8mer
GTC CAT GTC T
10mer
AGG TCC ATG TCT
12mer
CGA GGT CCA TGT CT
14mer
CCGAGGTCC ATG TCT
15mer
GCCG AGG TCC ATG TCT 16mer
(Courtesy of BIA Separations)
Preparative Scaleup with CIM Column Modules
Latest Additions:
- 800-mL
- 8-L (200g protein,
2-L/min flow)
Tube, 80-mL
Tube, 8-mL
Disk, 0.34-mL
(Courtesy of BIA Separations)
23
(courtesy of Dionex)
24
Analysis of Alkylphenone Standard on a Set of 8
Columns of 25 cm x 2.1 mm i.d. Packed with 5 µm
Particles of Zorbax 300SB-C18 material.
DAD1A, Sig=245,8Ref=400,80(C:\CHEM32\1\DATA\8COL-TESTMIX\33PHENONES12006-07-18\8COL-2000007.D)
mAU
26.141
Plates: ~ 200,000
27.975
23.981
250
30.146
31.043
200
37.347
33.1 29
150
43.230
100
50
0
0
10
20
30
40
50
min
Isocratic (70% acetonitrile – 30% water), 0.2 mL/min, 60 °C
Compounds: acetanilide (23.98 min), acetophenone (26.14 min), propiophenone (27.97 min), butyrophenone (30.15
min), benzophenone (31.04 min), valerophenone (33.13 min), hexanophenone (37.35 min), heptanophenone (43.23
min).
(courtesy of Pat Sandra, RIC)
Analysis of Peptide Standard Mixture on a Set of
8 columns of 25 cm x 2.1 mm i.d. x 5 µm Zorbax
300SB-C18 material.
DAD1B, Sig=214,4Ref=off, TT(C:\CHEM32\1\DATA\BSA21MM\3BSA21MM_FINAL2006-06-12\FINAL-000003.D)
363.004
mAU
175
Peak Capacity: 900
150
125
100
319.255
361.883
271 .338
165.4 51
50
249.871
75
25.525
25
0
0
50
100
150
200
250
300
350
400
min
Standard mixture: Proteomix (containing 5 peptides and a-cyano-4-hydroxy-cinnamic acid,
LaserBio Labs, Sophia-Antipolis, France). Gradient: 2% to 70% acetonitrile (+0.1% TFA) in
water (+0.1 % TFA) in 500 min. Flow rate: 0.2 mL/min. Column temperature: 60°C.
(courtesy of Pat Sandra, RIC)
25
Some Ways to Increase Sample Throughput
(and Resolution)

1) Shorter column lengths (to reduce analysis time) packed with small porous
particles (to maintain resolution). (Reasonable # of plates and reasonable
pressure, fast separation)
2) Longer column lengths (to increase efficiency) packed with even smaller porous
and non-porous particles (to maintain resolution), with the ultimate being the socalled “Ultra-High Pressure LC”. (Many plates, fast separation, high pressure)
3) Columns packed with various small superficially porous particles (pellicular) particle
sizes, pore sizes and phase thickness to allow the rapid resolution of
biomolecules such as proteins as well as small molecules. (Large and small
molecules, fast separations, lower pressure)
4) Columns designed with silica- and polymer-based monolith stationary phase formats
(fast separation, low pressure, in-series columns)
5) Parallel LC (multiple capillary columns/channels, increased samples/hour)
What is Parallel LC?
Conventional Single-Channel
HPLC
8-Channel HPLC
Eksigent ®
Sepiatec®
One Lab
30” Benchtop
26
• Allows 8 independent methods to run
simultaneously
A
B
6
4
2
0
0
40
80
120
10
8
6
4
2
0
2
0
3
• High pressure binary mixing
10
8
6
4
2
0
0
40
80
120
10
8
6
4
2
0
0
40
80
4
2
0
4
0
120
(courtesy of Eksigent)
10
8
6
4
2
0
0
40
80
80
120
4
2
0
6
0
40
80
120
time, sec
120
flow ra te , µL /mi n
7
flo w ra te , µL/mi n
• 4 injections / minute
40
10
8
6
time, sec
• Dual Headed Autosampler
120
time, sec
flow ra te , µL /mi n
flow ra te , µL/mi n
5
• Independent UV detector arrays
80
10
8
6
time, sec
• Independent injector
40
time, sec
flow ra te , µL /mi n
time, sec
• Optimized for 300 m ID columns
• Each Channel Incorporates
10
8
fl ow ra te , µL /mi n
1
flow ra te , µL /mi n
• Eight Independent Channels
fl ow rate, µL /min
Eksigent ExpressLC-800 System Design
10
8
6
4
2
0
8
0
time, sec
40
80
120
time, sec
High Throughput Performance
of Parallel LC
Ch.
absorbance, a.u.
8
7
6
5
4
3
2
0
1
0.0
2.5
5.0
time, min
7.5
10.0
20
40
time, sec
0.3 x 50 mm; 5 µm C18
20 s equilibration
65  95 % ACN in 25 s
20 s hold;
12 µL/min
(courtesy of Eksigent)
27
Chiral Application
8 Channel Method Development – Isocratic
IPA
AD-H
Fenoprofen
EtOH
A: ETOH and IPA as marked
B: 0.1% TEA, 0.1% TFA, 5% IPA/hexanes
• isocratic 10% A; 4 µL/min
• detection at 220 nm
OD-H
AS-H
IPA
OJ-H
0
2
4
6
0
2
4
EtOH
AD-H
6
time, min
OD-H
AS-H
Atenolol
OJ-H
A: ETOH and IPA as marked
B: 0.1% TEA, 0.1% TFA, 5% IPA/hexanes
• isocratic 10% A; 7 µL/min
• detection at 230 nm
0
2
4
6
8
0
2
4
6
8
time, min
Parallel Screening Using 8 Different Conditions
Absorbance (mAU)
5000
H
4000
G
F
3000
E
D
2000
C
1000
B
A
1
8,3
7
4
9
2
5

LC-MS
0
0
5
10
15
20
25
min
Find one condition which gets most peaks resolved, then use LCMS to obtain the peak identity if needed
Data courtesy of Yining Zhao (Team leader)
Gang Xue, Nate Lacher of Pfizer, Groton,CT
28
Conclusions
• High-speed and/or high resolution HPLC separations can be performed
by a variety of approaches depending on time, sample complexity and
sensitivity requirements
•
Short columns, small particles (3.5- and sub-2-µm particles)
(reasonable # of plates, fast separations, reasonable pressure)
•
Long columns, smaller particles (down to 1.5-µm) (complex samples,
reasonable separation times, high- or ultra-high pressure)
•
Superficially porous particles for fast separations of both
biomolecules and small molecules
•
Polymeric or silica-based monoliths (low pressure drop, fast
separations, macromolecular or small molecule separations)
•
An alternative approach is to use parallel LC with capillary columns
• Instrumental configurations may have to be modified to make these
very fast separations possible and practical with available hardware.
These changes involve decreasing extra column effects, increasing
data rate, decreasing dwell volume, smaller flow cells and so on.
Acknowledgements
• Colleagues at Agilent-Cliff Woodward (deceased), Bill Long,
Maureen Joseph, John Henderson, Bud Permar and Bill Barber
• Manufacturers who supplied data on their products (Merck,
Eksigent, Supelco, BIA Separations)
• Yining Zhang at Pfizer for parallel LC data
AND LAST BUT NOT LEAST
• Thanks to you the audience for listening to my marathon
presentation!
29