Fused-CoreTM Particle Columns provide UHPLC performance
on any HPLC
Major Contributors and Co-Authors
D. Bell, W. Campbell, W. Way, K. Buckendahl
Supelco – a Division of Sigma Aldrich
Content
• Influence on Resolution & Van Deemter Curve
• HPLC Evolution of particle design
• Fused-CoreTM particle Technology
• Column performance
- System compatibility (pressure requirements)
- Comparison to Sub 2µm and 3µm materials
2
Fused-Core is a trademark of Advanced Materials Technology Inc.
Ascentis is a trademark of the Sigma-Aldrich Corp.
Resolution in HPLC
Key Influence Factors
3.0
Efficiency
Retention Selectivity
α
R=
N . k’ . α-1
k’+1
α
4
N
α
- Increase column length
- Decrease particle size (N ~ 1/dp)
Resolution (R)
2.5
- Change stationary phase
- Change mobile phase solvent
- Change buffer pH
k
- Increase (weaker solvent)
- Decrease (stronger solvent)
3
Zhao, J.H. and P.W. Carr. Analytical
Chemistry, 1999. 71(14): p. 2623-2632
2.0
N
1.5
1.0
k’
0.5
0.0
1.00
1.05
1.10
1.15
1.20
0
5000
10000
15000
20000
0
5
10
15
20
α
25000N
25 k’
1.25
van Deemter-Kurve: H = A + B/u + Cu
H
B = 2γDm
A = 2λdP
4
u
C = (f1dP2+f2df2)
Evolution of HPLC Column Particle Design
Irregular
Pellicular
Totally Porous
Fused-Coretm
5
Difficult to pack, easily clogged and not very rugged
Thin porous layer showed low capacity
Current HPLC workhorse
The future of HPLC columns?
Fused-Core is a trademark of Advanced Materials Technology, Inc.
Pressure & Efficiency are reciprocal to the particle size–
Influence of dP on P >> N
2x efficiency → 4x higher pressure
Effiiciency
30,000
1
N ∝
dp
25,000
20,000
15,000
10,000
dP (µm)
psi
Bar
N
5,000
1.8
5889
406
27,500
0
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
bar
0
5
10
400
350
300
250
200
150
100
50
0
15
20
1
P∝ 2
dp
10 cm column, 3 mm/s linear flow
0
6
Smaller particles are not the simplest
solution and have draw backs.
5
10
dp (μm)
15
20
How much separation power do you
have commonly on your HPLCSystem?
Fused Core-Technology – Higher Efficiency
1
4
2
Agilent 1200
Ascentis Express C18, 15cm x 4.6mm, 2.7 µm
1.0mL/min, 254nm, RT, 10µL inj
3
4. Toluol N = 30,738
3. Benzol N = 31,696
2. Acetophenon N = 33,786
1. Uracil (dead time marker)
Pressure = 183 bar (2690 psi)
0
7
1
2
3
4 min
Ascentis Express Fused-Core Particle Technology
• Fused-Core particle technology invented by Jack
Kirkland
• Porous silica layer fused to solid core
0.5 µm
• Ascentis Express HPLC columns use
this newly engineered particle
• 2.7 micron Silica particle
2.7 µm
1.7 µm
• 1.7 micron solid core
• 0.5 micron porous shell
• C18 and C8 phases available at launch
(RP-Amide later in 2007)
8
Fused Core Technology
• Reducing dispersion (increasing efficiency)
• A solid core at the center of a particle, the potential diffusion path length of an
analyte molecule is effectively shortened
• A larger particle then can theoretically generate similar efficiency of a smaller
totally porous particle without generating high backpressures
9
Fused-Core Vs. Totally Porous:
Improved Peak Shape
0.5 µm
2.7 µm
10
Migration Path
In Particle
1.7 µm
Shorter diffusion path reduces axial dispersion and
minimizes peak broadening
1.5 µm
System Compatibility
Comparison of Pressure for Different Particles
ΔP = 1000FηL
ΔP =
to πr2dp2
16,000
16,000
35.00
14,000
30.00
12,000
Pressure (psi)
HETP (μm)
H = A + B/u + Cu
25.00
20.00
15.00
10.00
10,000
8,000
2.7 µm FC
6,000
3 µm
4,000
2.7 µm Ascentis Express
5.00
1
2
3
4
5
Mobile Phase Velocity (mm/sec)
11
1.7 µm
2,000
2
4
6
8
10
Mobile Phase Velocity (mm/sec)
12
van Deemter – Ascentis Express 2.7 µm Vs. 3 µm
Small Molecule Study: Naphthalene: M.W. = 130
Naphthalene
8.00
7.00
Acentis C8 3 µm
Ascentis Express C18 2.7 µm
Ascentis Express C8 2.7 µm
5.00
h
h = H / dp
6.00
4.00
3.00
2.00
1.00
0.00
Reduced
plate hight
to compare
columns
with diff.
Particle
sizes
12
0.00
1.00
2.00
3.00
4.00
v (mm/s)
5.00
6.00
Columns:
50 x 4.6 mm
Mobile phase: 55/45 ACN/Water
Sample:
Naphthalene (20 µg/mL), dissolved in 55/45 ACN/water
Injection:
5 µL
Detection:
254 nm
Temperature: 30 °C
Instrument: Agilent 1100.
7.00
Smaller Particles Exponentially Increase Pressure
bar
Pressure Drop Vs. Particle Size
4 50
400
3 50
300
2 50
200
150
10 0
50
0
1.8 µm ~ 400 bar (6,000 psi)
3 µm ~ 150 bar (2,250 psi)
5 μm ~ 50 bar (750 psi)
0
5
10
dp (μm)
15
10 cm column, 3 mm/s linear velocity
13
20
25
Frit Requirements for HPLC columns
Column
Work
horse
Frit size
(μm)
5
2
Higher
Efficiency
3
0.5
UHPLC
1.6-2.0
0.2
Ascentis
Express
14
Particle
size (μm)
2.7
2
Ascentis Express
3µm
Porus material
• Ascentis Express’ narrow
particle distribution allows for
2 μm-Frits
• Like 5 μm-Columns
Ascentis Express-Columns
can efficiently be protected
by 0.5 μm-Inline-Filter.
High Definition (HD) Resolution
Compared to 5µm porus particle column
• Sub 2 µm columns can provide up to 1.55 x increase in
resolution for an 8 fold increase in pressure
• Ascentis Express Fused-Core (FC) columns can provide
1.55 x increase in resolution for only a 4 fold increase
in pressure
dp
5μm
3μm
2.7μm FC
1.8μm
15
N
Rs factor* Pressure (psi) Pressure (bar)
10.000
1
750
50
14.000
1,18
2.250
150
24.000
1,55
2.800
190
24.000
1,55
6.000
400
10cm columns
* RS normalised to 5µm column
Twice the Efficiency at the System Limit
2.7 µm Fused-Core Vs. 3 µm Totally Porous Particles
Ascentis Express C18 2.7 µm
65 % ACN
N = 237,740 p/m
or N = 35,661 p/col
Psi (approx 4,000)
Efficiency Comparison /
Columns: 150 x 4.6 mm
Mobile Phase: Acetonitrile / Water
Flow: 1.5 mL/min
Injection: 2.0 μL
Detection: 220 nm
Agilent 1100 HPLC System
2
1
4
1.
2.
3.
4.
3
0
2
Ascentis C18, 3 µm
72.5 % ACN
N = 140,631p/m
or N = 21,095 p/col
Psi (approx 4,000)
0
16
p-hydroxy ethylbenzene
Napthalene
p-Xylene
Biphenyl
4
2
4
1
3
2
4
NB: Ascentis C18 and Ascentis Express C18 have different Surface Chemistries
TO11/IP6A Carbonyl-DNPH Mix
120
140
Almost Twice the Sensitivity
20
40
mAU
60
80
100
Ascentis Express C18, 2.7 µm
Peak 8
N = 260,720 p/m
N = 39,108 p/col
8
Column: 150 x 4.6 mm I.D.
Mobile phase:
Ascentis Express C18 2.7 µm: 25:75, water: acetonitrile
Ascentis C18 3 µm: 30:70, water: acetonitrile
Flow rate: 1.0 mL/min.
Temp.: 30 °C
Det.: UV at 365 nm
Injection: 1 µL
Sample: 47285-U TO11/IP6A Carbonyl-DNPH Mix as indicated
below in 40:60, water: acetonitrile
0
Peak IDs
140
0
2
4
6
Time (min)
8
10
12
mAU
60
80
100
120
Sensitivity Gap
20
40
Ascentis C18, 3 µm
Peak 8
N = 146,587p/m
N = 21,988p/col
Formaldehyde-2,4-DNPH (105 µg/mL)
Acetaldehyde-2,4- DNPH (76.4 µg/mL)
Acrolein-2,4- DNPH (63.2 µg/mL)
Acetone-2,4- DNPH (61.5 µg/mL)
Propionaldehyde-2,4- DNPH (61.5 µg/mL)
Crotonaldehyde-2,4- DNPH (53.6 µg/mL)
Butyraldehyde-2,4- DNPH (52.5 µg/mL)
Benzaldehyde-2,4- DNPH (40.5 µg/mL)
Isovaleraldehyde-2,4- DNPH (46.4 µg/mL)
Valeraldehyde-2,4- DNPH (46.4 µg/mL)
o-Tolualdehyde-2,4- DNPH (37.5 µg/mL)
m-Tolualdehyde-2,4- DNPH (37.5 µg/mL)
p-Tolualdehyde-2,4- DNPH (37.5 µg/mL)
Hexaldehyde-2,4- DNPH (42 µg/mL)
2,5-Dimethylbenzaldehyde-2,4- DNPH (35 µg/mL)
8
0
17
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
0
2
4
6
8
Time (min)
10
12
NB: Ascentis C18 and Ascentis
Express C18 have different
Surface Chemistries
Higher Sample Throughput on Traditional
HPLC Systems*
0
2
4
6
Time (min)
0
0
2
4
6
Time (min)
0
2
Conventional C18
25 cm x 4.6 mm I.D., 5 μm
1.0 mL/min., N= 22147
Pressure: 128 bar (1880 psi)
4
6
Time (min)
10
0
2
4
6
Time (min)
20
0
2
4
6
Time (min)
30
2
4
6
8
0
2
4
Time (min)
6
4
6
Time (min)
Method requirement
was N >20.000
10
Ascentis Express C18
10 cm x 4.6 mm I.D., 2.7 μm
1.5 mL/min., N = 21297
Pressure: 248 bar (3645 psi)
18
2
40
Ascentis Express C18
10 cm x 4.6 mm I.D., 2.7 μm
1.0 mL/min., N = 22694
Pressure: 167 bar (2450 psi)
0
0
8
10
*Agilent 1100
HPLC System
High Speed: Increased Flow Rate
Ascentis Express Vs. sub-2 µm at System Pressure Limit
Ascentis Express C18
0.3 mL/min
45 % acetone.
2130 psi
N = 12,500
1
2
5
3
1.0
2.0
Time (min)
3.0
4.0
Ascentis Express C18
Sub-2 µm Column 2
0.3 mL/min
51 % acetone.
9500 psi
N = 12,170
2
4
5
3
19
0.6 mL/min
45 % acetone.
4450 psi
N = 8,500
1
1.0
Mobile Phase: water : acetonitrile;
isoelutropic for β-Estradiol
Columns: 100 x 2.1 mm
Flow: variable
Det: 200 nm
Inj: 1µL
Elution order:
1. Estriol
2. β-Estradiol
3. Contaminant
4. Estrone
5. Estrone degradant
4
2.0
Time (min)
3.0
4.0
1
2
4
3
1.0
Time (min)
5
2.0
All column efficiencies have been compromised by system dispersion, however
the pressure associated with the Ascentis Express column when run at twice
the flow rate. It is still at only half that of the sub 2 µm column.
Twice the Efficiency at the System Limit:
4,000 – 6,000 psi System
Ascentis Express C18
100 mm length
45 % acetonitrile
4500 psi
N = 8,500 p/col
1
2
4
5
3
1.0
Time (min)
2.0
Mobile Phase: water : acetonitrile;
isoelutropic for β-Estradiol
Columns: 2.1 mm ID
Flow: 0.6 mL/min
Det: 200 nm
Inj: 1µL
Elution order:
1. Estriol
2. β-Estradiol
3. Contaminant
4. Estrone
5. Estrone degradant
C18 Sub-2 µm Column
50 mm length
46 % acetonitrile
5200 psi
N = 3,790 p/col
1
2
4
3
20
0.2
0.4
0.6
0.8
Time (min)
Both column efficiencies have been compromised by system dispersion
however N is double for the longer column as would have been predicted
at the same pressure
5
1.0
1.2
21
Ascentis Express
Achieve 100,000 Plates under 7,000 PSI
Agilent 1200, 1.0mL/min, 254nm, ambient temp, 10uL
1 Column
15cm x 4.6mm
4. N Toluene = 30,738
3. N Benzene = 31,696
2. N Acetophenone = 33,786
Pressure = 183 bar (2690 psi)
N Toluene = 63,212
N Benzene = 64,785
2 columns
30cm
N Acetophenone = 67,390
Pressure = 350 bar (5145 psi)
N Toluene = 91,311
3 columns
45cm
0
22
2
N Benzene = 94,979
2
N Acetophenone = 101,586
4
Pressure = 473 bar (6953 psi)
3
4
6
8
10
12
14
Ascentis Express separates… deuterated Isomeres
Agilent 1200
55cm x 4.6mm (>100,000 N in 14 min)
60% Acetonitrile
1.1mL/min, 254nm, 50°C, 10uL
B
B
Toluene N = 104,235
4 columns
Benzene N = 106,096
DB
Acetophenone N = 117,475
Pressure = 473 bar (7,000 psi)
DB
Toluol N 1
=0
105,657
0
2
4
6
8
Time (min)
10
12
14
Benzol N = 106,260
D6 Benzol N = 105,235
Acetophenon N = 118,567
Pressure 1
=0
473 bar (7,000 psi)
0
23
2
4
6
8
Time (min)
10
12
14
Deuterated Analogues of Toluene and Benzene
H
H
H
H
H
D
D
H
H
D
D
D
D
H
D
H
CD3
CH3
CD3
CH3
H
H
H
H
D
D
D
D
H
H
H
H
D
D
D
D
H
24
D
D
H
D
D
High-Efficiency Separations: Deuterated Analogues of
Toluene and Benzene
Length: 45 cm
Pressure: 10500 psi
N (acetophenone) = 73400
0
1.8 µm, C18
10
20
Time (min)
2.7 µm, Fused Core
Length: 60 cm
Pressure: 10550 psi
N (acetophenone) = 113810
0
10
20
Time (min)
25
Further examples of high resolution
using Ascentis Express
26
Increased resolution over 3 µm particles
Ascentis Express C18 (2.7μm)
Efficiency (N)
Peak 9: 13160
2990 psi
6
0
2
4
Time (min)
C18 3 µm
6
0
27
2
4
Time (min)
6
Efficiency (N)
Peak 9: 8290
1750 psi
6
columns: 10 cm x 2.1 mm I.D.
mobile phase A: 100 mM ammonium acetate (pH 7.0;
titrated with ammonium hydroxide)
mobile phase B: water
mobile phase C: methanol
online mixing: Ascentis Express C18: A:B:C = 10:28:62
3 µm, C18: A:B:C = 10:25:65 (isoelutropic
for peaks 6 & 9)
flow rate: 0.3 mL/min.
temp.: 55 °C
det.: UV at 250 nm
injection: 1 µL
Peak ID
1. Uracil
2. Nordoxepin (50 mg/L)
3. Desipramine (50 mg/L)
4. Nortiptyline (50 mg/L)
5. Doxepin (50 mg/L)
6. Norclomipramine (50 mg/L)
7. Imipramine (50 mg/L)
8. Amitriptyline (50 mg/L)
9. Clomipramine (50 mg/L)
Carbonic Anhydrase Tryptic Digest
on Ascentis Express C18
40μL Injection 1.0mL/min flow @ 35ºC 215nm Detection
Response time >0.010min
Gradient: MP A → 5% ACN w/0.1% TFA MP B → 80% ACN w/0.095% TFA
2 min hold @ 0% B
58 min gradient from 0% B to 50% B
20 min gradient from 50% B to 85% B
Ascentis Express 15x4.6mm 2.7μm
0
10
28
20
30
40
50
60
C18 15x4.6mm 3μm
70
80 0
10
20
30
40
50
60
70
80
Greater peak resolution with Ascentis Express
Magnified regions:
each pair is at the same scale
12
14
12
16
14
18
16
18
20
20
22
24
22
26
24
26
28
28
C18 3μm
Ascentis Express C18
28
2829
30
30
32
32
34
36
38
40
34
36
38
40
HILIC on Ascentis Express
Erythromycin and Related Compounds by
O
3
erythromycins.esp
H3C
1
CH3
OH
CH3HO
OH
2
4
O
CH3
H3C
OH
H3C
H3C
N
O
O
O
O
CH3
CH3
O
O
0
30
5
10
Column Name
Length
Diameter
Particle Size
Mobile Phase A
pH Mobile Phase A
Mobile Phase B
Gradient
Temperature
Injection Volume
15
Retention Time (min)
Ascentis Express Si
15 cm
0.05 cm
2.7 um
H2O; 100mM ammonium formate
3
acetonitrile
90B
35 °C
5 uL
H3C
CH3 CH3
OH
CH3
Comparison of Ascentis Express HILIC and porous
particle based silica phases
Ascentis Express HILIC
1
Ascentis Silica
1. Norephedrine
4
4
2. Ephedrine
1
3. Pseudoephedrine
4. Synephrine
2
2
3
0
2
4
6
Time (min)
Column
31
8
10
0
2
4
Plates/Column Synephrine
Ascentis
Express HILIC
18500
Ascentis Silica
12878
Other HILIC
10013
3
6
8
10
Time (min)
4
1
12
14
Other porous HILIC
2
0
2
4
3
6
8
10
Time (min)
12
14
Ascentis Express Stability
A Customers Experience
32
Summary
• Ascentis Express columns with 2.7 µm Fused-Core Particles
offer the chromatographer a real ‘Speed Advantage’
- Greater resolving power per unit pressure on any system
• Almost double the efficiency of 3 µm columns with totally porous particles
• Close to half the back pressure of sub 2 µm particles
→ possible use of 2x longer columns
- Increase the speed of analysis while maintaining resolution
• Use a column of half the length or smaller while maintaining the same or
better resolving power than 5 µm & 3 µm columns
• Get the same speed as sub 2 µm columns but at half the pressure
• Or increase flow and double the speed (or better) at the same pressure
- Benefit from the ruggedness and durability of traditional HPLC
columns
33
Acknowledgements
• Dr. Hillel Brandes
• Dr David Bell
• Hugh Cramer
• Dr. Richard A. Henry
• Dr. Wayne Way
• Dr. Russel Gant
• Dr. Paul Ross
• Dr. William Campbell
• Dr. A. Daniel Jones, MSU
34
Thank you!
• C18
• C8
• HILIC
• RP-Amide (coming soon)
35
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