sign in sign up
<<
Home , Flumequine, Sulfamerazine, Sulfathiazole

APPLICATIONS Zeshan Aqeel Senior Application Scientist A Screen of 22 Common that Demonstrates Zeshan loves to collect watches and the Back to the the Unique Reversed Phase Selectivity and Improved Future Trilogy. He has twin boys who drive him crazy! He Chromatographic Performance for Bases using a is an Apple Fanboy for life and ® he likes being in the lab more Kinetex PS C18 HPLC/UHPLC Column than anywhere else. Zeshan Aqeel, Jeff Layne, and Ryan Splitstone Phenomenex, Inc., 411 Madrid Ave, Torrance CA 90501 USA

Overview

The Kinetex PS C18 is a USP classified L1 column, that provides Molecular Formula: C17H19FN3O3 both a unique polar/hydrophobic selectivity, and is 100 % aqueous Basic pKa: 8.77 stable. The column demonstrates enhanced selectivity and peak Acidic pKa: 5.56 shape for basic compounds under typical reversed phase condi- LogP: -0.86 tions. In addition, the solid support is a Kinetex core-shell (superfi- CH3 cially porous) particle morphology that provides ultra-high column + O S O 1 NH efficiency on any HPLC or UHPLC system. CH3 2 N N + O S O The mobile phase program chosen was a routine gradientNH of Acetonitrile with 0.1 % Formic Acid as the strong organic solvent2 and Water with 0.1 % Formic Acid as Nthe weak solvent.N The flow O F rate of 0.5 mL/min was used, and the column heater was set to ambient temperature (25 °C).O OH O F F OH Introduction OH O HO N In this application, 22 antibiotics were analyzed to demonstrate the F OH Kinetex PS C18 HPLC/UHPLC column’s unique multi-modal selec- F tivity and improved chromatographic performance for polar bases. HO N CI CI The unique selectivity and performance benefits were Fcompared to Florfenicol Molecular Formula: C H Cl FNO S another reversed phase C18 column of similar superficially porous H C 12 14 2 4 (core-shell) particle structure under the identical system and meth- Basic3 CI pKa: 13.6CI N F HO CI od conditions. The selected antibiotics were chosen due to their Acidic pKa: 8.49 H3C LogP: 1.49 N N associated challenges when analyzedN under reversed phase condi- tions. These challenges are primarily due to a wide variety of analyte F HO CI O N CI polarity, and are demonstrated by the compounds’N wideN range of O F H3C S O CI LogP values, from -0.52 to 2.32. In addition, several of these anti- N OH biotics were selected because they are polar basic compounds. O O OH O F H3C S CH OH 3 Following the analysis of the 22 mixture, threeO individualOH O antibiotic comparisons were run using ciprofloxacin, florfenicol, + O S O NH2 and to better demonstrate the relative peak shape per- formance and selectivity of the Kinetex PS C18. For comparison, a N N core-shell C18 column was also run under identical method condi- tions for the three individual antibiotics to validate stationary phase O selectivity differences. Difloxacin F Molecular Formula: C H F N O OH O 21 19 2 3 3 Experiment Basic pKa: 6.99 F OH Analytical reference standards for the 22 antibiotics and individual Acidic pKa: 5.52 HO N compounds (ciprofloxacin, florfenicol, and difloxacin) were ob- LogP: 1.75 tained through Sigma-Aldrich® (Saint Louis, MO). An Agilent® 1200 F Infinity HPLC system was used for this investigation and a SCIEX™ CI CI 4000 MS/MS was used for detection.

H3C N F HO CI N N O N CI O F H3C S OH O OH O

Having trouble reproducing this method? We would love to help! Visit www.phenomenex.com/LiveChat to get in touch with one of our Technical Specialists Page 1 of 4 Figure 1. A Screen of 22 Antibiotics.

16 2.2e6 Kinetex® 2.6 µm PS C18 2.0e6 Increased Selectivity 1.8e6 for Polar Compounds

1.6e6 2 1.4e6 1.2e6

1.0e6 3 ntensity, cps 8.0e5 1 15 6.0e5 7 6 17 5 10 4.0e5 4 12 8,9 18 19,20 13 22 2.0e5 11 14 21 App ID 25407 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min

1.8e6 Core-Shell 2.6 µm RP-MS C18 1.6e6

1.4e6

1.2e6

1.0e6

8.0e5 ntensity, cps 6.0e5

4.0e5

2.0e5 App ID 25408

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min

Conditions for all examples: Column: Kinetex 2.6 µm PS C18 Sample: 22 Antibiotics (Retention on Kinetex PS C18) Core-Shell 2.6 µm RP-MS C18 1. Sulfaguanidine (0.48 min) 12. Difloxacin (2.47 min) Dimensions: 50 x 2.1 mm 2. Lincomycin (1.86 min) 13. Valnemulin (2.49) Mobile Phase: A: Water with 0.1 % Formic Acid 3. (2.16 min) 14. Tylosin A (2.74 min) B: Acetonitrile with 0.1 % Formic Acid 4. (2.19 min) 15. (2.9 min) Gradient: Time (min) % B 5. (2.19 min) 16. Tiamulin (3.04 min) 0 3 6. (2.25 min) 17. Sulfaquinoxaline (3.16 min) 0.5 3 7. Ciprofloxacin (2.28 min) 18. (3.33 min) 5 100 8. Florfenicol (2.34 min) 19. (3.42 min) 5.1 3 9. (2.34 min) 20. (3.42 min) 8 3 10. (2.36 min) 21. (4.0 min) 11. Sulfamonomethoxine (2.42 min) 22. (4.03 min) Flow Rate: 0.5 mL/min Temperature: Ambient Detector: MS/MS SCIEX™ API 4000™

Discussion In Figure 1 the Kinetex PS C18 displayed improved selectivity and Under the routine and MS compatible mobile phase conditions several peak shape gains for the 22 polar basic antibiotics in com- used in this example, several of the antibiotics were either fully/ parison to a similar core-shell column with a C18 stationary phase. partially positively ionized chemically, which can contribute to peak shape problems. In these cases, the positive surface charge of the The positive surface charge and C18 ligand of the Kinetex PS C18 Kinetex PS C18 takes advantage of ionic repulsion to deliver im- demonstrates multi-mode interaction mechanisms which drive the proved polar basic peak shape. apparent chromatographic improvements and separation of the 22 polar antibiotics.

Page 2 of 4 Figure 2. Individual antibiotic comparisons. Kinetex® 2.6 µm PS C18 Core-Shell 2.6 µm RP-MS C18 5.4e3 Ciprofloxacin 5.0e3 Conditions for all examples: CH3 Column: Kinetex 2.6 µm PS C18 4.5e3 Core-Shell 2.6 µm RP-MS C18 + O S O Dimensions: 50 x 2.1 mm NH2 4.0e3 Mobile Phase: A: Water with 0.1 % Formic Acid CH 3.5e3 N 3 N B: Acetonitrile with 0.1 % Formic Acid Gradient: Time (min) % B + O S O 0 3 NH 3.0e3 2 O 0.5 3 2.5e3 F 5 100 N N 5.1 3 ntensity, cps 2.0e3 OH O 8 3 F OH Flow Rate: 0.5 mL/min O F 1.5e3 Temperature: Ambient HO N Detector: MS/MS SCIEX™ API 4000™ OH O 1.0e3 Sample: See Chromatograms F OH F

5.0e2 App ID 25409 HO N CI CI 0 F 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min H3C N CI CI F HO CI 2.3e5 Florfenicol N N H3C 2.1e5 O CI N N F HO OCI 1.9e5 H C S N N F 3 1.7e5 O OH O O N OH CI O 1.5e5 H C S F 1.3e5 3 OH O OH 1.1e5 O CH3 ntensity, cps 9.0e4 + O S O 7.0e4 NH2 5.0e4 N N

3.0e4 App ID 25410 O 1.0e4 F 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min OH O F OH

HO N 3.0e5 Difloxacin 2.8e5 F 2.6e5 CI CI 2.4e5 2.2e5 H3C 2.0e5 N 1.8e5 F HO CI 1.6e5 N N 1.4e5 O N CI ntensity, cps 1.2e5 O H C S 1.0e5 F 3 OH 8.0e4 O OH O 6.0e4

4.0e4 App ID 25411 2.0e4 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min

Conclusion Following the 22 antibiotic mixture, individual compound compar- with a similar particle morphology. Figure 2 depicts chromato- isons were evaluated with the antibiotics ciprofloxacin, florfenicol, graphic overlays, offset in order to aid in the visual comparison of and difloxacin to better demonstrate the relative peak shape the relative and corresponding chromatographic peak shape under performance and selectivity of the Kinetex PS C18. As before, the identical conditions. In all three examples, the Kinetex PS C18 dis- performance of the new stationary phase was compared to a C18 played demonstrates peak shape.

Having trouble reproducing this method? We would love to help! Visit www.phenomenex.com/LiveChat to get in touch with one of our Technical Specialists Page 3 of 4 ® Kinetex Core-Shell LC Column Ordering Information SecurityGuard™ 2.6 μm Minibore Columns (mm) ULTRA Cartridges‡ Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk PS C18 00A-4780-AN 00B-4780-AN 00D-4780-AN 00F-4780-AN AJ0-8951 for 2.1 mm ID

SecurityGuard 2.6 μm MidBore™ Columns (mm) ULTRA Cartridges‡ Phases 50 x 3.0 100 x 3.0 150 x 3.0 3/pk PS C18 00B-4780-Y0 00D-4780-Y0 00F-4780-Y0 AJ0-8950 for 3.0 mm ID

SecurityGuard 2.6 μm Analytical Columns (mm) ULTRA Cartridges‡ Phases 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pk PS C18 00B-4780-E0 00D-4780-E0 00F-4780-E0 00G-4780-E0 AJ0-8949 for 4.6 mm ID ‡ SecurityGuard ULTRA Cartridges require holder, Part No.: AJ0-9000.

Australia Mexico References t: +61 (0)2-9428-6444 t: 01-800-844-5226 1. Fabrice Gritti, Georges Guiochon. Facts and Legends About [email protected] [email protected] Columns Packed with sub-3-μm Core-Shell Particles. Austria The Netherlands LC-GC North America. 2012, 30(7), 586-595. t: +43 (0)1-319-1301 t: +31 (0)30-2418700 [email protected] [email protected]

Belgium New Zealand t: +32 (0)2 503 4015 (French) t: +64 (0)9-4780951 t: +32 (0)2 511 8666 (Dutch) [email protected] [email protected] Norway Canada t: +47 810 02 005 t: +1 (800) 543-3681 [email protected] [email protected] Portugal China t: +351 221 450 488 t: +86 400-606-8099 [email protected] [email protected] Singapore Denmark t: +65 800-852-3944 t: +45 4824 8048 [email protected] [email protected] ™ Spain Finland t: +34 91-413-8613 t: +358 (0)9 4789 0063 esp[email protected] BE-HAPPY [email protected] guarantee Sweden France t: +46 (0)8 611 6950 t: +33 (0)1 30 09 21 10 [email protected] Your happiness is our mission. Take [email protected] 45 days to try our products. If you Switzerland are not happy, we’ll make it right. Germany t: +41 (0)61 692 20 20 www.phenomenex.com/behappy t: +49 (0)6021-58830-0 [email protected] [email protected] Taiwan India t: +886 (0) 0801-49-1246 t: +91 (0)40-3012 2400 [email protected] [email protected] United Kingdom Ireland t: +44 (0)1625-501367 t: +353 (0)1 247 5405 [email protected] [email protected] USA Italy t: +1 (310) 212-0555 t: +39 051 6327511 [email protected] [email protected] All other countries Luxembourg Corporate Office USA t: +31 (0)30-2418700 t: +1 (310) 212-0555 [email protected] [email protected] Terms and Conditions Subject to Phenomenex Standard Terms and Conditions which may be viewed at www.phenomenex.com/TermsAndConditions. Trademarks Kinetex is a registered trademark and Be-Happy, SecurityGuard, and MidBore are trademarks of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc. Sigma-Aldrich is a registered trademark of Sigma-Aldrich, Inc. API 4000 is a trademark of AB SCIEX Ltd. AB SCIEX™ is being used under license. Disclaimer Comparative separations may not be representative of all applications. www.phenomenex.com Phenomenex is not affiliated with Agilent or Sigma Aldrich. Phenomenex products are available worldwide. For the distributor in your country, FOR RESEARCH USE ONLY. Not for use in clinical diagnostic procedures. contact Phenomenex USA, International Department at [email protected] © 2019 Phenomenex, Inc. All rights reserved. TN67910419_W Page 4 of 4