tn-1158 applications · 2016-12-16 · tn-1158 for additional technical notes, visit page 1 of 8...
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TN-1158
For additional technical notes, visit www.phenomenex.com Page 1 of 8
APPLICATIONS
Achieving Ultra-High Performance in Preparative Chromatography with the Combination of Axia™ Packing and Kinetex® Core-Shell Particle Technologies Marc Jacob, JT Presley III, Simon Lomas, and Jeff Layne
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
Abstract
Kinetex 5 μm is the first and only core-shell media available for lab-scale preparative purification. Chromatographers can now scale their ultra-high efficiency Kinetex 1.3, 1.7, 2.6, and 5 µm core-shell analytical columns directly to Kinetex Axia packed preparative col-umns. Other advantages resulting from the combination of those two award winning technologies are also presented.
Introduction
High performance HPLC/UHPLC core-shell materials are one of the latest technological advancements in chromatography. When used under analytical conditions, core-shell particles show improved efficiency and performance over fully porous particles of equivalent particle size.1,2 With the recent commercialization of a lower pressure 5 μm core-shell media, it is now possible to offer core-shell media in a preparative
Figure 1. Particle Morphology Comparison
format (>20 mm ID) that’s compatible with standard prep LC systems. In this technical study we will demonstrate that this new 5 μm core-shell particle size (Figure 1), available in four (4) bonded phases, can be packed efficiently in preparative formats with internal diameter greater than 2 centimeters. Additionally, we will highlight the advantage of such product and discuss applications that are pertinent for small scale drug discovery and peptide purifications.
Page 2 of 8
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Axia preparative columns (>20 mm ID) were introduced in 2005 and incorporate a patented Hydraulic Piston Compression technology that increases bed density and eliminates media bed collapse as a source of premature column failure. Unlike traditional pre-packed preparative column packing methods, the Axia packing method is completely automated and computer monitored which results in improved efficiencies, peak symmetries, column reproducibility and column overall performance (Figure 2).
To further study and explain the performance gains of core-shell particles, an investigation performed by F. Gritti and G. Guiochon1 analyzed the Van Deemter equation for fully porous and core-shell analytical columns to evaluate the contributions of the A, B, and C coefficients to plate height (H). From this study, they determined that core-shell media significantly reduces band broadening by affecting Eddy dispersion (A), and longitudinal diffusion (B), while mass transfer (C) was determined to be a minor component as de-picted in Figure 3A. The high column performance is explained essentially by a more homogeneous column packing.2 Combining 5 μm core-shell and Axia technology results in efficiency (N) that can reach up to 125,000 plates/meter for 21.2 mm ID prep columns (Figure 3B).
Material and Methods
All chemicals were obtained from Sigma Chemical Co. (St. Louis, Missouri). Solvents were purchased from EMD (San Diego, Cali-fornia). The Axia™ packed Kinetex® 5 μm C18 and XB-C18 as well as the analytical 2.6 μm C18, XB-C18, Phenyl-Hexyl, and C8 col-umns were obtained from Phenomenex (Torrance, California). The Waters® XBridge™ Prep C18 5 μm OBD™ were obtained from Waters (Milford, Ma.). The column dimensions and running condi-tions for the various assays are detailed in the figure captions. All analytical methods were performed using an Agilent® 1200 HPLC system. Preparative chromatography was performed using a Shimadzu® LC-20AP preparative chromatography system.
Results and Discussion
Core-shell particles are composed of an inner solid silica core sur-rounded by a layer of fully porous silica as shown in Figure 1. The initial core-shell chromatography particles were developed more than 50 years ago by C. Horvath1 and subsequently redeveloped and reintroduced in 2009 by Phenomenex due to the efficiency and performance advantages they offer over fully porous materials.
Packing piston locked. No media or pressure escape
Media maintains initial optimal packing density and shape
Silica media packed under piston pressure
Specialized computer control for ideal pressure
Col
umn
Figure 2. Axia Patented Packing Technology
For additional technical notes, visit www.phenomenex.com Page 3 of 8
TN-1158
Van Deemter Equation: H = A•dp + B/µ + C•de2•µ
Figure 3A. Particle Impact on Band Broadening
Figure 3B. Particle and Axia Technology Impact on Column Efficiency
Conditions for both columns:Columns: Kinetex 5 μm XB-C18 Axia Packed
Waters® XBridge™ 5 μm C18 Prep OBD™
Dimensions: 150 x 21.2 mm (Kinetex)150 x 19 mm (XBridge)
Mobile Phase: Water/ Acetonitrile (50:50)Injection Volume: 10 µL
Flow Rate: 25 mL/ minTemperature: Ambient
Detection: UV @ 254 nmSample: 1. Uracil
2. Acetophenone3. Toluene4. Naphthalene
2 4 6 8 10 12 14 min0
0
10
20
30
Res
pon
se -
Mill
ivol
ts
40
50
21456
150 x 19.0 mm N = 77,020 plates/meter
2 4 6 8 10 12 14 min0
0
10
20
30
Res
pon
se -
Mill
ivol
ts 40
50
21455
150 x 21.2 mm N = 121,620 plates/meter
Over 55% Increase in Efficiency
Fully Porous 5 μm C18 in Conventional Prep column Kinetex Core-Shell 5 μm XB-C18 in Axia Packed column
App
ID 2
1456
App
ID 2
1455
0 5 10 15 200
5
10
15
100 x 4.6 mm Kinetex® C18
Reduced velocity
Eddy dispersion Longitudinal diffusion Solid-liquid mass transfer
0 5 10 15 200
5
10
15
100 x 4.6 mm Luna® C18 (2)
Eddy dispersion
Longitudinal diffusionSolid-liquid mass transfer
Red
uced
pla
te h
eigh
t (h
)
Reduced velocity
Red
uced
pla
te h
eigh
t (h
)
( ) ( )
Comparative separations may not be representative of all applications.
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Figure 4. Peptide Purification
Preparative scale-up and fraction collection — Kinetex 5 μm XB-C18
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
mV
21454
FR
1
FR
2
FR
3
FR
4
Ap
p ID
214
54
Column: Kinetex 5 µm XB-C18 Axia PackedDimensions: 150 x 21.2 mm
Part No.: 00F-4605-P0-AXMobile Phase: A: 0.1% TFA in Water
B: 0.1% TFA in AcetonitrileGradient: Linear 85:15 (A/B) to 5:95 (A/B)
over 10 minutesInjection Volume: 1 mL
Flow Rate: 20 mL/ minTemperature: Ambient
Detection: UV @ 210 nmSample: Crude peptide mix
Analytical method — Kinetex 2.6 μm XB-C18
0 2 4 6 8 10 min
0
200
400
600
800
1000
mAU
21452
Ap
p ID
214
52
Column: Kinetex 2.6 µm XB-C18Dimensions: 150 x 4.6 mm
Part No.: 00F-4496-E0Mobile Phase: A: 0.1% TFA in Water
B: 0.1% TFA in AcetonitrileGradient: Linear 85:15 (A/B) to 5:95 (A/B)
over 10 minutesInjection Volume: 10 µL
Flow Rate: 1 mL/ minTemperature: Ambient
Detection: UV @ 210 nmSample: Crude peptide mix
Column: Kinetex 2.6 µm XB-C18Dimensions: 150 x 4.6 mm
Part No.: 00F-4496-E0Mobile Phase: A: 0.1% TFA in Water
B: 0.1% TFA in AcetonitrileGradient: Linear 85:15 (A/B) to 5:95 (A/B)
over 10 minutesInjection Volume: 10 µL
Flow Rate: 1 mL/ minTemperature: Ambient
Detection: UV @ 210 nmSample: Purified fractions
Analytical fraction analysis — Kinetex 2.6 μm XB-C18
3.5 4 4.5 5 5.5 min
21453
0
200
400
600
800
mAU
Ap
p ID
214
53
Fraction 2 = 97.3 % Purity
Fraction 1
Fraction 4
Fraction 3
Fraction 2
Results and Discussions (cont.)
The Kinetex® core-shell family of particles are designed such that regardless of particle size (1.3, 1.7, 2.6 and 5 µm), the retention and the selectivity remains consistent, allowing for excellent scalability of methods from particle and/or system platforms.
Figure 4 highlights the benefit of the 5 μm Kinetex core-shell particle to purify a crude peptide and achieve a purity greater than 97 % in one run. In this application, we take the ultra-high perfor-mance obtained by Kinetex Core-Shell Technology to develop a fast and effective analytical method (10 min gradient on 2.6 μm core-shell particle) and then directly scale up to the preparative level (5 μm core-shell particle packed in an Axia 150 x 21.2 mm column) to perform the purification. Finally, using that same ana-lytical method, the fraction purity can be analyzed without further development.
Comparative separations may not be representative of all applications.
For additional technical notes, visit www.phenomenex.com Page 5 of 8
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Figure 5A. Loadability of Fully Porous vs. Core-Shell Particle in Preparative Format
Conditions for both columns:Columns: Kinetex 5 μm C18 Axia Packed
Waters® XBridge™ 5 μm C18 Prep OBD™Dimensions: 50 x 21.2 mm (Kinetex)
50 x 19 mm (XBridge)Mobile Phase: A: Water with 0.5 % Formic acid
B: Acetonitrile with 0.5 % Formic acidGradient: Time (min)
0 8 11
% B20 50 50
Flow Rate: 30 mL/minTemperature: Ambient
Detection: UV @ 254 nmSample: 200 mg/mL in DMSO
1. Doxepin (From 1 - 500 mg on-column)2. Amitriptyline (From 1 - 500 mg on-column)
Fully Porous 5 μm C18 in Conventional Prep column
0 1 2 3 4 5 6 7 8 9 10 min
20775
50100150200250300350400450500550600650mV
0
App
ID 2
0775
0 1 2 3 4 5 6 7 8 9 10 min
50100150200250300350400450500550600650mV
0
20774
Increased Sensitivity and Excellent Loadability!
Kinetex® 5 μm C18 Core-Shell in Axia Packed column
App
ID 2
0774
Specifically looking at the peak shape for amitriptyline (Figure 5B), it is evident that the peak width is narrower on the Kinetex Axia packed column, and stays narrower across each sample load. This reflects the higher efficiency of the Kinetex 5 µm me-dia in combination with Axia technology, resulting in narrower peak widths. In addition, by observing the rate of peak distortion (shown by the slope of the increase in peak width as a function of sample load) of both columns, one can see that there is an increase of approximately the same rate for both medias. Peak dis-tortion can also be attributed to a function of sample overloading, thus we can infer that, for this sample and conditions, the Kine-tex 5 µm Axia packed core-shell column has a very similar loading capacity to the fully-porous XBridge Prep 5 µm C18 OBD 50 x 19 mm column. However, due to the narrower initial peak shape of the Kinetex 5 µm C18 Axia packed column, it provides better performance across the entire loading study.
Figure 5B. Plots of peak width as a function of sample load for amitriptyline using the 0.5 % FA modified mobile phase
1
0
0.2
0.4
0.6
0.8
1.2
1
10 100 1000mg on-column
Pea
k W
idth
X
XX
X
XX
XX
X
X
Fully Porous 5 μm C18 in Conventional Prep column
Kinetex 5 μm C18 Core-Shell in Axia Packed column X
Results and Discussions (cont.)
To further analyze core-shell media in a preparative setting, a small scale pharmaceutical purification loading study was performed with two common probes. From the results it’s apparent that with narrower peak widths than fully porous columns across every sam-ple load (1 to 500 mg on column), Axia™ packed core-shell 5 μm columns give the capability of increased sample load and higher throughput for improved purification performance (Figure 5A).
Comparative separations may not be representative of all applications.
Page 6 of 8
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Conclusion
With the release of Kinetex core-shell 5 µm media, it is now possible to take advantage of Core-Shell Technology for both analytical and preparative purposes allowing for easy scale up from analytical HPLC/UHPLC to preparative columns in 4 differ-ent bonded phases.
The combination of Kinetex core-shell 5 μm particle and Axia pat-ented packing technologies results in preparative columns with high efficiency in the range of 25-60 % greater than columns packed with fully porous media of equivalent particle size.
Axia preparative core-shell columns give equivalent or better loading capacities to columns packed with fully porous media.
References 1. F. Gritti and G. Guiochon LC-GC, 2012, 30, 586-5952. F. Gritti and G. Guiochon J. Chromatogr. A, 2013, 1230, 35–50.
Figure 6. Alternative Selectivities
Conditions for all columns:Column: Kinetex 2.6 μm C18
Kinetex 2.6 μm XB-C18Kinetex 2.6 μm Phenyl-HexylKinetex 2.6 μm C8
Dimensions: 50 x 2.1 mmMobile Phase: A: 0.1% Formic acid in Water
B: 0.1% Formic acid in AcetonitrileGradient: Time (min)
0.0 0.2 4.2 4.21 5.5
% B559555
Flow Rate: 0.8 mL/minTemperature: 30 ºC
Detection: UV @ 254 nm (ambient)Sample: 1. Pyridine
2. Acetaminophen3. Pindolol4. Quinidine5. Sulfathiazole6. Acebutolol7. Benzyl alcohol8. Chlorpheniramine9. Phenol
10. Triprolidine 11. Nortriptyline 12. Prednisolone 13. 3-Methyl- 4-nitrobenzoic acid 14. 2-Hydroxy- 5-methylbenzaldehyde 15. Diflunisal 16. Hexanophenone
Acids Bases Neutrals
Phenyl-Hexyl
XB-C18
C18
C8
App
ID 2
0856
App
ID 1
9532
App
ID 1
9533
App
ID 1
9539
Phenyl-Hexyl
TMSTMS
XB-C18
Si TMSTMS
C18
TMSTMS
C8
TMSTMS
As represented in Figure 6 Kinetex® core-shell 5 µm Axia™ packed columns are available in four different stationary phases that offer alternative selectivities and are fully scalable to all the available Kinetex particle sizes (1.3, 1.7, 2.6, 5 µm).
Comparative separations may not be representative of all applications.
0 1 2 min
1 2
3
4
5
6
7
8
9
10 11
12
13 14 15
0
12
7
8
9
10
11 12
14
13
15
16
6,4
3,5
1 2 min
20856
19532
12
4
3
5
8
69
710
11,13
12
14
15
16
0 1 2 min min
19533
1
2
3
4
5
6
7
8
9
10
11 12
13
1415
16
10 2 min min
For additional technical notes, visit www.phenomenex.com Page 7 of 8
TN-1158
5 µm Axia™ Packed Preparative Columns (mm)
SecurityGuard PREP Cartridges**
50 x 21.2 100 x 21.2 150 x 21.2 250 x 21.2 15 x 21.2
XB-C18 00B-4605-P0-AX 00D-4605-P0-AX 00F-4605-P0-AX 00G-4605-P0-AX AJ0-9145C18 00B-4601-P0-AX 00D-4601-P0-AX 00F-4601-P0-AX 00G-4601-P0-AX AJ0-9204C8 00B-4608-PO-AX 00D-4608-PO-AX 00F-4608-PO-AX 00G-4608-PO-AX AJ0-9205Phenyl-Hexyl 00B-4603-P0-AX 00D-4603-P0-AX 00F-4603-P0-AX 00G-4603-P0-AX AJ0-9147
for 18-29 mm ID
5 µm Columns (mm)
SecurityGuard™ ULTRA Cartridges*
SecurityGuard ULTRA Cartridges*
50 x 2.1 3/pk 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pk
XB-C18 00B-4605-AN AJ0-8782 00B-4605-E0 00D-4605-E0 00F-4605-E0 00G-4605-E0 AJ0-8768C18 00B-4601-AN AJ0-8782 00B-4601-E0 00D-4601-E0 00F-4601-E0 00G-4601-E0 AJ0-8768C8 00B-4608-AN AJ0-8784 00B-4608-E0 00D-4608-E0 00F-4608-E0 00G-4608-E0 AJ0-8770Phenyl-Hexyl 00B-4603-AN AJ0-8788 00B-4603-E0 00D-4603-E0 00F-4603-E0 00G-4603-E0 AJ0-8774 for 2.1 mm ID for 4.6 mm ID* SecurityGuard ULTRA cartridges require holder, Part No. AJ0-9000.
2.6 μm Analytical Columns (mm)SecurityGuard
ULTRA Cartridges*30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 3/pk
XB-C18 –– 00B-4496-E0 00D-4496-E0 00F-4496-E0 AJ0-8768C18 00A-4462-E0 00B-4462-E0 00D-4462-E0 00F-4462-E0 AJ0-8768C8 –– 00B-4497-E0 00D-4497-E0 00F-4497-E0 AJ0-8770Phenyl-Hexyl –– 00B-4495-E0 00D-4495-E0 00F-4495-E0 AJ0-8774
for 4.6 mm ID * SecurityGuard ULTRA cartridges require holder, Part No. AJ0-9000.** SecurityGuard PREP cartridges require holder, Part No. AJ0-8223.
Kinetex® Ordering Information
5 µm Axia Packed Preparative Columns (mm)
SecurityGuard PREP Cartridges**
50 x 30 100 x 30 150 x 30 250 x 30 15 x 30
XB-C18 00B-4605-U0-AX 00D-4605-U0-AX 00F-4605-U0-AX 00G-4605-U0-AX AJ0-9145C18 00B-4601-U0-AX 00D-4601-U0-AX 00F-4601-U0-AX 00G-4601-U0-AX AJ0-9204
for 18-29 mm ID
2.6 μm MidBore™ Columns (mm)SecurityGuard
ULTRA Cartridges*30 x 3.0 50 x 3.0 100 x 3.0 150 x 3.0 3/pk
XB-C18 00A-4496-Y0 00B-4496-Y0 00D-4496-Y0 00F-4496-Y0 AJ0-8775C18 00A-4462-Y0 00B-4462-Y0 00D-4462-Y0 00F-4462-Y0 AJ0-8775C8 00A-4497-Y0 00B-4497-Y0 00D-4497-Y0 00F-4497-Y0 AJ0-8777Phenyl-Hexyl –– –– 00D-4495-Y0 00F-4495-Y0 AJ0-8781
for 3.0 mm ID
2.6 μm Minibore Columns (mm)SecurityGuard
ULTRA Cartridges*30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
XB-C18 00A-4496-AN 00B-4496-AN 00D-4496-AN 00F-4496-AN AJ0-8782C18 00A-4462-AN 00B-4462-AN 00D-4462-AN 00F-4462-AN AJ0-8782C8 00A-4497-AN 00B-4497-AN 00D-4497-AN 00F-4497-AN AJ0-8784Phenyl-Hexyl 00A-4495-AN 00B-4495-AN 00D-4495-AN 00F-4495-AN AJ0-8788* SecurityGuard ULTRA cartridges require holder, Part No. AJ0-9000. for 2.1 mm ID
* SecurityGuard ULTRA cartridges require holder, Part No. AJ0-9000.
1.7 μm Minibore Columns (mm)SecurityGuard
ULTRA Cartridges*30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk
XB-C18 00A-4498-AN 00B-4498-AN 00D-4498-AN 00F-4498-AN AJ0-8782C18 00A-4475-AN 00B-4475-AN 00D-4475-AN 00F-4475-AN AJ0-8782C8 00A-4499-AN 00B-4499-AN 00D-4499-AN 00F-4499-AN AJ0-8784Phenyl-Hexyl –– 00B-4500-AN 00D-4500-AN 00F-4500-AN AJ0-8788
for 2.1 mm ID
1.7 μm MidBore Columns (mm)SecurityGuard
ULTRA Cartridges*30 x 3.0 50 x 3.0 100 x 3.0 3/pk
XB-C18 00A-4498-Y0 00B-4498-Y0 00D-4498-Y0 AJ0-8775C18 –– 00B-4475-Y0 00D-4475-Y0 AJ0-8775C8 00A-4499-Y0 00B-4499-Y0 00D-4499-Y0 AJ0-8777Phenyl-Hexyl –– –– –– AJ0-8781
for 3.0 mm ID
1.3 µm Columns (mm)
50 x 2.1
C18 00B-4515-AN
Page 8 of 8
TN-1158
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All other countries: Corporate Office USA (310) 212-0555 (310) 328-7768 info@phenomenex.comTerms and Conditions
Subject to Phenomenex Standard Terms and Conditions, which may be viewed at http://www.phenomenex.com/TermsAndConditions.
Trademarks Kinetex and Luna are registered trademarks, SecurityGuard and Axia are trademarks of Phenomenex. Waters is a registered trademark, XBridge and OBD are trademarks of Waters Corporation. Agilent is a registered trademark of Agilent Technologies. Shimadzu is a registered trademark of Shimadzu Corporation.
DisclaimersPhenomenex is not affiliated with Waters Corporation or Agilent Technologies. Comparative separations may not be representative of all applications.
Axia is patented by Phenomenex. U.S. Patent No. 7,674,383
© 2013 Phenomenex, Inc. All rights reserved.
TN14
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If Phenomenex analytical columns do not provide you with at least equivalent separations to a competing column of the same phase, particle size, and dimensions, return the column with comparative data within 45 days for a FULL REFUND.
SecurityGuard™ Column Protection
Protect your Kinetex and Axia Packed preparative columns from the damaging effects of mobile phase and sample chemical contaminants with SecurityGuard ULTRA and SecurityGuard PREP
• Dramatically extends column lifetime and performance
• Virtually no change in chromatography
• Simple to use
SecurityGuard ULTRAPart No. Description Unit Price
AJ0-9000 SecurityGuard ULTRA Holder for UHPLC/ HPLC Columns 2.1 to 4.6 mm ID
ea $ 102
SecurityGuard PREPPart No. Description Unit Price
AJ0-8223 SecurityGuard PREP HPLC Guard Cartridge Holder Kit, 21.2 mm ID, includes column coupler
ea $ 341
AJ0-8277 SecurityGuard PREP HPLC Guard Cartridge Holder Kit, 30 mm ID, includes column coupler
ea $ 341
** SecurityGuard PREP 15x21.2 mm cartridges require holder, Part No. AJ0-8223*** SecurityGuard PREP 15x30 mm cartridges require holder, Part No. AJ0-8277
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