advantages of using monodisperse particles in u/hplc columns · 2016. 2. 28. · the kirkland 2.7...
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sigma-aldrich.com/analytical
© 2014 Sigma-Aldrich Co. All rights reserved.
Advantages of Using Monodisperse Particles in U/HPLC Columns
rhenry@psualum.com
Richard A. Henry, Technical AdvisorSupelco, Div. of Sigma-AldrichBellefonte, PA 16823 USA
T414098
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
The Trend to Small Particle Columns, High Separation Speed and High Pressure
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Small Particle Columns are Not New in HPLC
• 3 µm particles yield lower HETP and more efficiency per unit column length.
• Optimum flow rate is higher for 3 µm columns.
• 3 µm columns maintain high efficiency better as flow increases.
• PE was first in offering high speed systems, but Supelco was a close second with 3 x 3 and 5 x 5 HPLC columns (now 2 x 2).
In 1984, 3 µm particle columns were exploited for Fast LC2, which is a precursor to UHPLC; 2 µm particles were also developed by then.
2. M. W. Dong and J. R. Gant, LC, 2(4), 294-303 (1984).
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HPLC Column Performance Has Steadily Improved
ColumnParticles
Plate HeightH (or HETP)
Efficiency*N/m
Efficiency of a10cm column
Plates/cm estimate*
5 µm 10 µm 80-100,000 8-10,000 1000
3 µm 6 µm 120-160,000 12-16,000 1500
2 µm 4 µm 200-250,000 20-25,000 >2000*
Sub-2 µm 3 µm 300-350,000 30-35,000 >3000*
* Calculations are based upon the assumption of H = 2dp for a uniform, tight particle bed and a small volume of ideal solute introduced to the column under optimum mobile phase conditions. Efficiencies observed in normal practice may be significantly lower due to many variables that contribute to peak broadening and departure from ideal conditions, especially at higher performance levels.
4* Instrument factors are important
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
New Interest in Monodisperse HPLC Particles
• In 1999 and 2002, Knox (3-4) suggested that contributions from the A term in the van Deemter equation had been underestimated and that more uniform particle beds should have advantages.
• In 2006, Kirkland (5) commercialized a spherical Fused-Core® silica with very narrow size distribution (PSD) that showed surprising performance and became widely accepted.
• In 2010, Desmet, et. al. (6-7) observed a trend between narrow silica particle size distribution (PSD) and better column performance and suggested that porous particles should benefit.
• In 2012, Titan particle launched (9) to explore performance advantages for columns with porous particles having a very narrow PSD equal to Fused-Core.
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What Can We Learn from Van Deemter Plots?
• Chromatographic band spreading happens in mobile zones (around the particle exterior) and static zones (inside particle pores or other non-convective transport regions). Monodisperse Fused-Core particles showed improved (lower) values for all three terms.
• Band spreading processes outside the particle (A term) have been the subject of recent research (3-6) that has resulted in preparation of more uniform beds with higher column efficiency.
• With monodisperse (highly regular) particles now becoming available in commercial quantities, further advances in HPLC column performance are anticipated as we gain better understanding of A, B and C terms in the van Deemter equation (Knox variation shown).
H = Au1/3 + B/u + Cu
Flow Uniformity Axial Diffusion Radial Diffusion
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The Kirkland 2.7 µm Fused-Core Particle (2007)
Reproduced by permission from ref (5). 7
2.7 µm Ascentis® Express C8
Kirkland and AMT blew through the previous barrier of h = 2, suggesting that a limit of h = 1 might be attainable.
B term region A term region C term region
Regions shown where different terms in the van Deemter equation dominate.
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Comparison: Fused-Core Particle vs Totally Porous Particles
Columns: 4.6 x 150 mm; Instrument: Agilent® 1100, autosamplerVerapamil - Mobile phase: 35% acetonitrile/65% 0.1% aqueous trifluoroacetic acid;
Temperature: 40 C; Fused-Core k = 2.8, totally porous k = 6.31-Cl-4-Nitrobenzene - Mobile phase: 50% acetonitrile/50% water;
Temperature: 30 C: Fused-Core k = 2.7, totally porous k = 4.3
Mobile Phase Velocity, mm/sec
0 1 2 3 4 5 6 7 8 9 10
Red
uced
Pla
te H
eigh
t, h
1
2
3
4
5
6verapamil: 5 m Halo fused-coreverapamil: 5 m totally porousData fitted to Knox equation1-Cl-4-nitrobenzene: 5 m Halo fused-core1-Cl-4-nitrobenzene: 5 m totally porous
verapamil: 5 µm Fused-Coreverapamil: 5 µm totally porousData fitted to Knox equation1-Cl-4-nitrobenzene: 5 µm Fused-Core1-Cl-4-nitrobenzene: 5 µm totally porous
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0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90Time, seconds
1
3
4
5 6
2
Abs
orba
nce
/ %B
Column: 2.1 x 20 mm, Ascentis Express C18, 5 µmFlow Rate: 2 mL/min/Pressure: 172 barMobile Phase: 11/89: A/BA = Water/0.1% TFAB = Acetonitrile/0.1% TFAGradient: 5-50% B in 60 seconds
Temperature: 40 CDetection: UV 254 nm, PDAInjection Volume: 0.5 μLFlow Cell: 1 μL microLC System: Shimadzu Nexera
Peak Identities:1. Atenolol2. Pindolol3. Propranolol4. Indoprofen5. Naproxen6. Coumatetralyl
Fast Gradient with 5 µm Fused-Core Column*
Time (s) % ACN0 5
60 5063 9566 9569 587 5
Gradient Program
* Unpublished data supplied by Advanced Materials Technology. 9
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Evolution of HPLC Particles to Smaller Size and Narrower Distribution
2.7 µm Fused Core
5 µm Fused Core
5.0 µm 3.0 µm ≤2 µm 2.5 µm
Higher Backpressure
≤2 µm Fused Core
• Core-type particles generate higher efficiency and speedat lower pressure.
• Porous particles also have high efficiency plus greater retention and sample capacity.
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Monodisperse (Narrow PSD) C18 Silica Particles
1.9 µm Titan Porous, ca. 5% RSD (D90/10 < 1.15).
2.7 µm Fused-Core, ca. 5% RSD (D90/10 < 1.15).
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Polydisperse 1.7 and 1.8 µm C18 Silica Particles*
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* Particles removed from commercial columns.
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0
2
4
6
8
10
12
14
16
0 1 2 3 4 5 6
number
count
(
%)
Particle Size (m)
Particle Size Distribution Data for Silicas
Titan 1.9
Fused-Core 2.7Fused-Core 5
Comparing Size Distribution for Different C18 Silicas
13
“Narrow PSD columns will prove to be more efficient, reproducible and stable. They should replace broad PSD in analytical columns.”Conventional
silicas have broad PSD
1.9 µm porous (Titan)1.7 µm porous2.7 µm Fused-Core3 µm porous5 µm Fused-Core5 µm porous
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Titan Porous Silica Characteristics
Particle Size1µm
Pore DiameterÅ
Surface aream2/g
Pore Volumecc/g
1.9 80 410 0.76
1. EcoporousTM synthesis results in very narrow distribution (D90/10 < 1.15) without additional sizing.
Monodisperse particles can be defined as having a narrow PSD of 5-10% RSD. Modern core-type silicas are monodisperse. Until recent developments at Supelco, porous silica has been polydisperse with much larger PSD of 20-40% RSD.
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TitanTM Performance Results
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Evidence for a Monodispersity Advantage
0.000
2.000
4.000
6.000
8.000
10.000
12.000
0.000 2.000 4.000 6.000 8.000 10.000 12.000
h
mm/s
Toluene (reduced plate height)
Titan C18 1.9um
Competitor B C18 1.7um
Ascentis Express C18 2.7um
Porous Polydisperse 1.7
Porous and Core-type Monodisperse 1.9 and 2.7
Lower h values means more plates for a given particle size; Titan and Ascentis Express advantages can be attributed to monodispersity that creates very uniform beds (low A term).
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1.0 2.0
NN= 13,573N/m = 271,460h= 1.90= 1.03Pressure: 3,100 psi
50 x 2.1 mm0.4 mL/min
= 3.29 mm/s
1.0 2.0
NN= 14,710N/m = 294,200h= 1.75= 1.05Pressure: 4,100 psi
50 x 3.0 mm0.9 mL/min
= 4.07 mm/s
2
1.0 2.0Time (min)
NN= 15,220N/m = 304,400h= 1.69= 1.02Pressure: 6,030 psi
50 x 4.6 mm2.4 mL/min
= 4.50 mm/s
1
3
45
Dionex 3000 UHPLCMobile Phase: 60% AcetonitrileTemp: 35 CFlow Rate: As IndicatedDetection: 254 nmLow D Connecting TubingInlet: 35 cm x 75 mOutlet: 35 cm x 75 m
QC Test1.Uracil2.Diazepam3.Toluene4.Naphthalene5.Biphenyl
Titan C18 Performance in a Modern UHPLC Instrument
Titan C18 1.9 µm: 13-15,000 plates expected for 5 cm columns; system pressure includes instrument blank pressure.
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Independent Evaluation of Titan Kinetic PerformanceKinetic plot limit for Pmax = 600 bar (Ascentis, Poroshell, Waters XP)and Pmax = 1000 bar (Titan and Waters BEH)
Titan offers best kinetic performance over entire t0 or N range for Pmax = 1000 bar
Acknowledgement to U. Brussels (Desmet Group)
18
0.1
1.0
10.0
100.0
10000 100000 1000000N (/)
t 0(m
in) Ascentis 2.7µm
Waters XP 2.5µmPoroshell - Agilent 2.7µmTitan - 1.9µmWaters BEH 1.7µm
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Independent Evaluation of Titan Pressure Drop
Lower P for Titan compared to similar sized particles: Kv0is 25% higher than Waters BEH and only 7 % lower than2.5µm XP column
Lower P of superficially porous particles due to lower T (higher u0)
Acknowledgement to U. Brussels (Desmet Group)
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0
150
300
450
600
750
0 1 2 3 4 5 6
u0 (mm/s)
H (µ
m)
Ascentis 2.7 µmPoroshell - Agilent 2.7 µmWaters XP 2.5µmTitan - 1.9µmWaters BEH 1.7µm
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Dionex 3000 (Low D tubing)Column: 50 x 2.1 mmMobile Phase:60% Methanol40% 0.1% Ammonium Acetate (pH = 7.1)Temp: 35 CFlow Rate: 0.25 mL/minDetection: 220 nm
1.Uracil2.Quinidine3.Diphenhydramine4.Nordiazepam5.Diazepam
Pressure= 5,370 psi
Pressure= 5,220 psi
Pressure= 4,210 psi
1.0 2.0 3.0
1.0 2.0 3.0
1.0 2.0 3.0
1
23
4
5
Titan C18 Performance Comparison in MeOH
20
Efficiency and asymmetry reported on peaks 3 and 5
N= 9,644= 1.15
N= 11,485= 1.00
N= 6,603= 1.02 N= 8,094
= 1.02
N= 7,420= 1.72
N= 9,470= 1.13
Titan C18 1.9 m
Competitor B C18 1.7 m
Competitor A 18 1.8 m
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Titan C18 1.9 mDionex 3000 (Low D tubing)Column: 50 x 3.0 mmMobile Phase: 60% AcetonitrileTemp: 35 CFlow Rate: 0.9 mL/min (4 mm/s)Detection: 254 nm
1.Uracil2.Diazepam3.Toluene4.Naphthalene5.Biphenyl
NNap= 9,783Nnap/m= 195,700 h= 2.84= 1.16Pressure= 4,900 psi
NNap= 9,260NNap/m= 185,200h= 3.18= 1.02Pressure= 4,650 psi
Titan C18 Performance Comparison in ACN
21
1.0 2.0
NNap= 14,710N/m = 294,200h= 1.75= 1.05Pressure= 4,100 psi
1.0 2.0
1.0 2.0
1
23
4 5
Data used in subsequent van Deemter plots Competitor B C18 1.7 m
Competitor A 18 1.8 m
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Columns: 50 x 3.0 mm60% Acetonitrile
Performance (h) of Porous Titan and Fused-Core Ascentis Express for Different Molecules
22
See poster 0905
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
9.000
10.000
0.000 2.000 4.000 6.000 8.000 10.000 12.000
Titan C18 1.9 µm Toluene
Ascentis Express C18 2.7 µm Toluene
Titan C18 1.9 µm Diazepam
Ascentis Express C18 2.7 µm Diazepam
Toluene / Diazepam ComparisonReduced Plate Height
h
(mm/s)
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Columns: 50 x 3.0 mm60% Acetonitrile
Performance (H) of Porous Titan and Fused-Core Ascentis Express for Different Molecules
23
DiazepamN/m = 295,000plates/m
TolueneN/m = 293,000plates/m
0.000
2.000
4.000
6.000
8.000
10.000
12.000
14.000
16.000
18.000
20.000
0.000 2.000 4.000 6.000 8.000 10.000 12.000
Titan C18 1.9 µm Toluene
Ascentis Express C18 2.7 µm Toluene
Titan C18 1.9 µm Diazepam
Ascentis Express C18 2.7 µm Diazepam
Toluene / Diazepam ComparisonPlate Height
(mm/s)
H (m)
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
24
HminHmin
Optimum Linear Velocities are Solute-Dependent
TolueneN/m = 293,000 plates/mat Hmin = 4.873 mm/s
Flow Rate at Hmin = 1.1 mL/minP = 5050 psi
DiazepamN/m = 295,000 plates/m at Hmin = 1.796 mm/s
Flow Rate at Hmin = 0.4 mL/min P = 1810 psi
Columns: 50 x 3.0mm60% Acetonitrile
Every separation has a different velocity tolerance (poster 0905)
0.000
2.000
4.000
6.000
8.000
10.000
12.000
14.000
16.000
18.000
20.000
0.000 2.000 4.000 6.000 8.000 10.000 12.000
Titan C18 1.9 µm Toluene
Titan C18 1.9 µm Diazepam
Toluene / Diazepam ComparisonPlate Height
H (m)
(mm/s)
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Further Evidence for a Monodisperse Advantage*
25*Unpublished data supplied by Supelco Division of Sigma-Aldrich.
-1.000
1.000
3.000
5.000
7.000
9.000
11.000
0.00 2.00 4.00 6.00 8.00 10.00 12.00
h
mm/s
Column G TolueneColumn G NaphthaleneColumn G BiphenylColumn A TolueneColumn A NaphthaleneColumn A BiphenylTitan TolueneTitan NaphthaleneTitan Biphenyl
Columns: porous C18, 50 x 3.0 mmTest conditions 60% acetonitrile
Column G 1.9 µm, D (90/10) ca.1.4Column A 1.9 µm, D (90/10) ca.1.4Titan 1.9 µm, D (90/10) ca. 1.1
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TitanTM Selectivity and Applications
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0 2 4 6 8Time (min)
0.46
3
2.52
3
2.93
1
3.34
93.
473
4.64
14.
847
5.30
6
5.56
5
7.78
8
TolueneD8
H8
Naphthalene
D8
H8
P-Xylene
D10
H10
Dionex 3000 UHPLC Column: Titan C18, 1.9 mDimension: 100 x 2.1 mmMobile Phase: 50% ACNTemperature: 35 CFlow Rate: 0.4 mL/minDetection: UV / 254 nmPressure: 5650 psi/390 bar
Dia
zepa
m
N,N
-Dim
etyl
anili
ne
Bip
heny
l
N = 25,530N/m = 255,000
Titan C18 1.9 µm: High Speed and High Resolution
Separation of deuterated isomers
27
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Comparison of Porous and Core-Shell UHPLC Columns
0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)
12
3
4
5
6
Titan 1.9 µm C185800 psi (400 bar)
0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)
12
3
4
5
6
Core-Type 1.7 µm C187455 psi (514 bar)
LC-MS Analysis of Benzodiazepine Drugs and Metabolites
instrument: Agilent 1290 TOF 6210column(s): 5 cm x 3.0 mm, as indicated
mobile phase: (A) 0.1% formic acid in 95:5, water:acetonitrile;(B) 0.1% formic acid in 5:95, water:acetonitrile;
gradient: 35% B to 60% B in 1 min; 60% B held for 0.5 min
flow rate: 0.6 mL/mincolumn temp.: 35 C
detector: MS-TOF, ESI+, XIC, 100 -1000 m/z injection: 2 µLsample: 300 ng/mL in 97:3, water:methanolelution: 1. Oxazepam glucuronide (463 m/z)
2. Lorazepam glucuronide (497 m/z)3. Temazepam glucuronide (477 m/z)4. Oxazepam (287 m/z)5. Lorazepam (321 m/z)6. Temazepam (301 m/z)
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column: 5 cm x 3.0 mm, 1.9 µmmobile phase: water:acetonitrile, pH=3.0 (formic acid)
flow rate: 1.0 mL/mindet.: 225 nm
Selectivity Comparisons of Phenols: Titan C18 vs Titan C18-Amide (in the pipeline- see poster 0915)
29
Titan C18-Amide30% Acetonitrile
p-H p-NO2p-Me
p-OMe
Titan C1825% Acetonitrile
Iso-eluotropicp-CN
p-Hp-NO2
p-Me
p-OMe
p-CN
1.0 2.0
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
p-Me
p-NO2
p-OMe
p-H
p-CN
Selectivity Comparisons para Substituted Benzoic Acids
30
column: 5 cm x 3.0 mm, 1.9 µmmobile phase: water:acetonitrile, pH=3.0 (formic acid)
flow rate: 1.0 mL/mindet.: 240 nm
Titan C18-Amide25% Acetonitrile
Titan C1822% Acetonitrile
Iso-eluotropic
p-Mep-
NO
2
p-OMe
p-H
p-CN
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
p-Mep-NO2
p-CN
p-Mep-NO2
p-OMe
p-CN
Selectivity Comparisons for para Substituted Anilines
31
p-OMe
column: 5 cm x 3.0 mm, 1.9 µmmobile phase: water:acetonitrile, pH=6.5 (ammonium formate)
flow rate: 1.0 mL/mindet.: 230 nm
Titan C18-Amide25% Acetonitrile
Titan C1825% Acetonitrile
Iso-eluotropic
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C
Z
OCH3O
Selectivity Comparison of Methylbenzoates
32
p-Me
p-N
O2
p-OMe
p-H
p-CN
p-CN
p-Me
p-OMe
p-H
p-N
O2
column: 5 cm x 3.0 mm, 1.9 µmmobile phase: water:acetonitrile, pH=3.0 (formic acid)
flow rate: 1.0 mL/mindet.: 235 nm
Titan C18-Amide38.5% Acetonitrile
Titan C1840% Acetonitrile
Iso-eluotropic
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
1.0 2.0
1.0 2.0
column: 5 cm x 3.0 mm, 1.9 µmmobile phase: 65:35 water:acetonitrile, 0.1% formic acid
flow rate: 1.0 mL/mindet.: 240 nm
Titan C18-Amide
Titan C18
OH
O
H3CO
OCH3
O
O2N
Selectivity Comparisons Polar Mix
33
Hydrophobic selectivity (logP or logD)
Dual mode selectivity
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
1. Piroxicam2. Ketorolac 3. Tolmetin4. Ketoprofen5. Naproxen6. Oxaprozin7. Nabumetone8. Fenoprofen9. Etodolac
10. Flubiprofen11. Indomethacin12. Ibuprofen13. Diclofenac
0 2 4 6 8 10
1
34
2
5
6
7
8
9
10
11
12
13
Imp
0 2 4 6 8 10
2
6
8,10,9
13
12
13,4
5
7
11
column: 5 cm x 3.0 mm, 1.9 µmmobile phase: 55:45, 0.1% H3PO4:acetonitrile
flow rate: 1.0 mL/mindet.: 270 nm
Titan C18-Amide
Titan C18
Applications: NSAIDS (Non Steroidal Anti-Inflammatory Drugs- Carboxylic Acids)
34
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35
Application: Tetrahydrocannabinol and Metabolites
1,2
3
12
3
Titan C18
Titan C18-Amide
column: 5 cm x 3.0 mm I.D., 1.9 µm particles mobile phase: (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile
gradient: 60% B to 100% B in 6 min; held at 100% B 1.5 minflow rate: 400 μL/min
temp.: 35 Cdet.: UV at 220 nm
injection: 2 µLsample: 50 µg/mL in 95:5, 0.1% formic acid in water:0.1% formic acid in acetonitrile
1. 11-HYDROXY-DELTA-9-THC2. 9-CARBOXY-11-NOR-DELTA-9-THC3. (-)-DELTA-9-THC
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Instrument Designs and New Challenges
36
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Instrument Designs Must Keep Pace with Columns
• It has been known for over forty years, since the beginning of modern HPLC1, that instruments would have to be steadily improved as smaller column particles became available.
• New instrument requirements include higher pressure operation and streamlined small-volume flow paths from injector to detector. As a result, instrument cost has increased dramatically to more that $100,000.
• Modern columns cannot be used effectively with just any instrument. Development of UHPLC methods requires that higher performance instruments be available to all departments and be validated often. Chemists may have to develop methods for both HPLC and UHPLC instruments.
• The best modern instruments still do not perform perfectly (zero dispersion cannot be achieved) with small ID columns!
37
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1.0 2.0
NN= 13,573N/m = 271,460h= 1.90= 1.03Pressure: 3,100 psi
50 x 2.1 mm0.4 mL/min
= 3.29 mm/s
1.0 2.0
NN= 14,710N/m = 294,200h= 1.75= 1.05Pressure: 4,100 psi
50 x 3.0 mm0.9 mL/min
= 4.07 mm/s
2
1.0 2.0Time (min)
NN= 15,220N/m = 304,400h= 1.69= 1.02Pressure: 6,030 psi
50 x 4.6 mm2.4 mL/min
= 4.50 mm/s
1
3
45
Dionex 3000 UHPLCMobile Phase: 60% AcetonitrileTemp: 35 CFlow Rate: As IndicatedDetection: 254 nmLow D Connecting TubingInlet: 35 cm x 75 mOutlet: 35 cm x 75 m
QC Test1.Uracil2.Diazepam3.Toluene4.Naphthalene5.Biphenyl
Titan C18 Performance in a Modern UHPLC Instrument
Titan C18 1.9 µm: 13-15,000 plates expected for 5 cm columns; system pressure includes instrument blank pressure (increases with flow rate) .
38
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Flow - Pressure Comparison for Same 1.9 µm Titan C18 4.6 mm Column with HPLC vs UHPLC InstrumentSee QC Test on previous slideMobile Phase: 60% ACNTemperature: 35 CFlow Rate: As IndicatedDetection: 254 nm
Agilent 1290 Connecting Tubing:Injector to Column:
35 cm x 75 µmColumn to Detector:
25 cm x 75 µm(blank P about 600 bar at 5 mL/min)
Agilent 1200SL Connecting Tubing:
Injector to Heat Exchange:400 x 0.17 mm
Heat Exchange to Column:150 x 0.127 mm
Column to Detector:280 x 0.17 mm
(blank P about 100 bar at 5 mL/min)
0
2000
4000
6000
8000
10000
12000
14000
0
100
200
300
400
500
600
700
800
900
1000
0.0 1.0 2.0 3.0 4.0 5.0
Pres
sure
(PSI
)
Pres
sure
(bar
)
Flow Rate (mL/min)
Pressure vs Flow on the Agilent 1200SL and Agilent 1290
Linear (1200 Instrument)
Linear (1200 Instrument + Titan C18 50 x4.6mm Column)
Linear (1290 Instrument)
Linear (1290 Instrument + Titan C18 50 x4.6mm Column)
UHPLC
HPLC
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Efficiency Performance for HPLC vs UHPLC Instrument
Agilent 1200SL Agilent 1290
k’ N k’ N
Diazepam 1.90 8187 2.25 10107
Toluene 3.14 11982 3.75 15407
Naphthalene 4.11 12524 5.01 14603
Biphenyl 6.14 12648 7.56 14227
40
Same Titan C18, 50 x 4.6 mm; flow = 2.4 mL/min
1200SL is rated at 600 bar; 1290 is rated at 1200 bar; see previous slide to estimate system operating pressure.
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Performance Plot for HPLC vs UHPLC Instrument
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0 2 4 6 8
N
k'
Agilent 1200SL: N vs k' at 2.4 mL/min
Diazepam
TolueneNaphthalene Biphenyl
Uracil
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0 2 4 6 8N
k'
Agilent 1290: N vs k' at 2.4 mL/min
Diazepam
TolueneNaphthalene Biphenyl
Uracil
More loss at low k’ due to larger volume flow path.
41
Titan C18, 50 x 4.6 mm
Plates are wasted here
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
How Particles and Plates Impact Resolution for a Difficult Separation with Selectivity (α) of 1.1
42
More plates in shorter columns allow faster separation of difficult pairs.
Example for α = 1.1 at different k values from 2-7, where α = k2/k1.
The perfect experiment has zero instrument dispersion uniform column performance. Working at low k values is further hampered by wasting plates due to extra-column effects.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 5000 10000 15000 20000 25000 30000
Rs
N
Rs vs N for selected k and α
α=1.1 k=7
α=1.1 k=6
α=1.1 k=5
α=1.1 k=4
α=1.1 k=3
α=1.1 k=2
Rs = √N/4 + k/(1+k) + (α-1)/α
Target resolution
k = 7
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Variables That Limit Modern Column Performance
1. Peak bandwidth increases with sample injection volume; focusing techniques (weak solvent injection and gradients) become important.
2. With particles from sub-2 µm to 3 µm, peaks are so narrow that any excess sample flow path outside of the column creates bandspreading (called extra-column effect); very low internal volume instruments are required; instrument designs still limit performance.
3. With sub-2 µm particles, high pressure creates frictional heat within the column, especially at high mobile phase velocities; uneven temperature and flow across the column increases bandwidth.
4. For certain (non-ideal) solutes, mass transfer can be slow due to adsorption or restricted diffusion within particle pores; slow mass transfer can increase peak bandwidth and discount advantages of small particle columns operating at high flow rates and pressures.
5. Problems 3 and 4 will be difficult to solve, but most problems do not require UHPLC designs. Don’t sell your HPLC instruments yet!
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Instrument Designs Must Keep Pace with Columns
• It has been known for over forty years, since the beginning of modern HPLC1, that instruments would require steady improvement as smaller column particles became available.
• New instrument requirements include higher pressure operation and streamlined small-volume flow paths from injector to detector. As a result, instrument cost has increased dramatically to more that $100,000.
• Modern columns cannot be used effectively with just any instrument. Development of UHPLC methods requires that higher performance instruments be available to all departments and be validated often. Chemists may have to develop methods for both HPLC and UHPLC instruments.
• The best modern instruments still do not perform well enough with 2.1 mm I.D. columns!
44
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Conclusions on Monodisperse Particles in HPLC
• A new process for making porous silica has been developed that matches the narrow size distribution of Fused-Core particles; no extra sizing step is required; no silica is wasted.
• Particles with 80 Å pores and 410 m2/g have been prepared in 1.9 µm with a 6% standard deviation in PSD.
• Performance results for Titan 1.9 µm particles:• Efficiency matches or exceeds porous particles of 1.7 and 1.8 µm size
while pressure drop for the larger Titan particle is lower.• Uniform Titan particles pack easily into rugged column beds that are
stable over a range of UHPLC flow and pressure conditions. • Excellent reproducibility observed for silica bonded phases.• High stability noted for phases within the pH range 2-8.
• HPLC research will continue about equally on both Fused-Core and Titan porous particle platforms.
45
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
References1. J. H. Knox and M. Saleem , J. Chromatogr. Sci., 7, 614-622 (1969).2. M. W. Dong and J. R. Gant, LCGC, 2(4), 294-303 (1984).3. J. H. Knox, Band Dispersion in Chromatography- A New View of the A Term,
Journal of Chromatography A, 831 (1999) 3–15. 4. J. H. Knox, A Universal Expression for Bandspreading in the Mobile Zone,
Journal of Chromatography A, 960 (2002) 7–18.5. J. J. Kirkland, T. J. Langlois and J. J. DeStefano, Fused-Core Particles for
HPLC Columns, American Laboratory, 39 (February 2007), 18-21.6. D. Cabooter, A. Fanigliulo, G. Bellazzi, B. Allieri, A. Rottigni, G. Desmet,
Relationship between Particle Size Distribution and Chromatographic Performance, J. of Chromatography A, 1217 (2010) 7074–7081.
7. K. Broeckhoven, D. Cabooter and G. Desmet, Kinetic Performance Comparison of Fully and Superficially Porous Particles, Journal of Pharmaceutical Analysis 2013;3(5):313–323.
8. Dorina Kotoni, HPLC 2013 Meeting, Amsterdam Download files at: www.sigmaaldrich.com/hplc2013.
9. R. A. Henry, et. al., Monodisperse Particles, poster, HPLC 2012 Anaheim. 46
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Acknowledgements
Assistance from the following groups for evaluating particles and columns is acknowledged and greatly appreciated:• Ken Broeckhoven and Gert Desmet, Free University of Brussels, Department
of Chemical Engineering, Brussels, Belgium. • Francesco Gasparrini, Sapienza University, Rome, IT.• Luigi Mondello and Paulo Dugo, U. Of Messina. Messina, IT.• Fabrice Gritti and Georges Guiochon, U. of Tennessee, Knoxville, TN.
The assistance of many dedicated analytical chemists at Supelco and Fluka Divisions of Sigma-Aldrich is also appreciated.
Ascentis is a registered trademark of Sigma-Aldrich Co. LLC; Titan is a trademark of Sigma-Aldrich Co. LLC; Fused-Core is a registered trademarks of Advanced Materials Technology, Inc.
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© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.
Acknowledgements and Trademarks
The assistance of Ken Broeckhoven and Gert Desmet, Vrije Universiteit Brussel, Department of Chemical Engineering, Belgium in evaluating columns is greatly appreciated.
The assistance of William Campbell and many other Supelco scientists is also greatly appreciated.
Ascentis is a registered trademark of Sigma-Aldrich Co. LLC; Titan, Ecoporous and BIOshell are trademarks of Sigma-Aldrich Co. LLC. Fused-Core is a registered trademark of Advanced Materials Technology, Inc., Wilmington, DE. The assistance of Advanced Materials Technology for supplying data on Fused-Core particles is greatly appreciated.
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Thank You!
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