APPLICATIONS TN A Comparison of Various Kinetex C18 Phases USP: L1

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1 APPLICATIONS A Comparison of Various Kinetex C18 Phases Zeshan Aqeel and Jeff Layne Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 91 USA Introduction When performing reversed phase LC separations, there is a natural tendency of method developers to first test a C18 phase, the most commonly used phase in LC applications. Today, there are many different C18 phases available to method developers so choosing a simple C18 column may prove to be more challenging than one had originally thought. This technical note explores the different C18 phases within the Kinetex core-shell family of HPLC/UHPLC columns to determine the differences as well as to provide a general guideline of how to select the most appropriate C18 phase for a variety of applications. Materials and Methods To provide a streamlined method development approach, the following cases were studied to determine the most appropriate C18 phase for each application: Acid, Base, Neutral Panel Highly Polar Analytes Highly Polar Bases (Catecholamines) Highly Polar Bases (Nucleotides) Hydrophobic Neutrals and Acids (Cannabinoids) Three different Kinetex core-shell C18 phases were screened under the same conditions as well as optimized conditions in some cases: Polar Kinetex Polar C18 Combined C18 and polar modified surface that provide polar and non-polar retention alongside 1 % aqueous stability Polar Kinetex C18 Balanced C18 phase that provides the highest degree of hydrophobic selectivity relative to the other Kinetex phases Si Kinetex XB-C18 Unique C18 phase that yields increased hydrogen bonding with hydrophobic selectivity, resulting in improved peak shape for basic compounds and increased retention of acidic compounds Page 1 of 8

2 Results and Discussion I. Acid, Base, Neutral Panel using a Formic Acid Gradient In general, gradients will reduce selectivity differences between phases as the gradient rate itself will play a large role in analyte elution But, particularly when using formic acid and other weak buffers, differences in ion-exchange and polar interactions will still be apparent, giving subtle (or not so subtle) differences in selectivity These differences will be less apparent when using more strong buffer salts like phosphate, or when using ion-pairing agents (e.g. hexane sulfonic acid) or ion-masking agents (TEA; TFA) Kinetex 2.6 µm Polar C min 2 17 Dimensions: x 4.6 mm Mobile Phase: A:.1 % Formic acid in Water B:.1 % Formic acid in Acetonitrile Gradient: Time (min) B (%) Flow Rate: 1.8 ml/min Temperature: 3 C App ID Sample: 1. Pyridine 2. Acetaminophen 3. Sulfathiazole 4. Quinidine. Quinidine Impurity 6. Acebutolol 7. Chlorpheniramine 8. Phenol 9. Triprolidine 1. Prednisolone 11. Nortriptyline 12. -Methylsalicaldehyde 13. Hexanophenone II. Highly Polar Analytes Water-Soluble Vitamins Take advantage of the aqueous stability of Kinetex Polar C18 to perform your analyses in 1 % aqueous conditions to maximize retention of polar analytes Traditional C18 phases like Kinetex C18 or XB-C18 should not be used below 2-3 % organic to maintain phase stability (mobile phase = 1% 2 mm Potassium phosphate, ph 3.) ' 6 min Dimensions: x 4.6 mm Mobile Phase: 2 mm Potassium phosphate, ph 3. / Methanol (97:3) (except where noted) Temperature: 3 C Detection: 21 nm Sample: : 1. Thiamine 2. Nicotinamide 3. Pyridoxal 4. Pyridoxine min App ID App ID min App ID min App ID min App ID min App ID Page 2 of 8 Page 3 of 8

3 III. Highly Polar Bases Catecholamines Take advantage of the aqueous stability on Kinetex Polar C18 to perform your analyses in 1 % aqueous conditions to maximize retention of polar analytes Traditional C18 phases like Kinetex C18 or XB-C18 should not be used below 2-3 % organic to maintain phase stability (mobile phase = 1 % 2 mm Ammonium acetate, ph 6.9) Dimensions: x 4.6 mm Mobile Phase: 2 mm Ammonium acetate, ph 6.9 / Methanol (98:2) (except where noted) Temperature: Ambient Detection: 28 nm Sample: 1. Norepinephrine 2. Epinephrine 3. Dopamine 4. Serotonin. -Hydroxy-3-Indoleacetic Acid 6. 4-Hydroxy-3-Methoxyphenylacetic Acid IV. Highly Polar Bases Nucleotides Take advantage of the aqueous stability on Kinetex Polar C18 to perform your analyses in 1 % aqueous conditions to maximize retention of polar analytes. The trade-off can be an increase in tailing, as is apparent below, but we include the data as a logical extreme; one would expect to optimize their method to find the right balance of retention and peak shape The key is that operating in highly aqueous conditions is only possible with the Kinetex Polar C18 phase Dimensions: x 4.6 mm Mobile Phase: 2 mm Ammonium acetate, ph 6.9 / Methanol (97:3) (except where noted) Temperature: Ambient Detection: 28 nm Sample: 1. Thiourea 2. -Fluorocytosine 3. Adenine 4. Thymine. Guanosine--Monophosphate min App ID (mobile phase = 1 % 2 mm Ammonium acetate, ph 6.9) min App ID min App ID min App ID min App ID min App ID min App ID min App ID 2439 Page 4 of 8 Page of 8

4 V. Hydrophobic Neutrals and Acids Cannabinoids Even very hydrophobic molecules will display selectivity differences on different C18 phases due to structural (e.g. positional isomers) or polar functional groups In this case, although selectivity was similar overall, the traditional Kinetex C18 provided better selectivity for one critical pair (CBN and CBGA), which is interesting because they are totally different structurally one would think could easily separate min min Sample: 1. CBDV Dimensions: x 4.6 mm Mobile Phase: A:.1 % Formic acid in Water Gradient: B:.1 % Formic acid in Acetonitrile 4. CBD Time (min) B (%). CBG CBDA Flow Rate:. ml/min Temperature: C Detection: 23 nm min 7. CBN 2. CBDVA 8. CBGA 3. THC-V 9. D9-THC App ID App ID App ID D8-THC 11. CBC 12. THCA-A Conclusions You will find differences in selectivity between the Kinetex C18, Kinetex XB-C18, and Kinetex Polar C18 stationary phases based upon the differences in surface chemistry Under identical running conditions, the differences may be subtle these are after all still all C18 phases but subtle differences can make the difference between resolution and co-elution, or lead to reversals in elution order that can help to resolve a target analyte from matrix interferences With the newest Kinetex Polar C18 phase, however, you can operate in 1 % aqueous conditions, whereas the traditional C18 phases should not go below 2-3 % organic. This opens the Kinetex Ordering Information 1.3 μm Minibore Columns (mm) Phases 3 x 2.1 x 2.1 C18 A-4-AN B-4-AN 1.7 μm MidBore Columns (mm) Phases 3 x 3. x 3. 1 x 3. 3/pk XB-C18 A-4498-Y B-4498-Y D-4498-Y AJ-877 C18 B-447-Y D-447-Y AJ-877 for 3. mm ID 1.7 μm Minibore Columns (mm) Phases 3 x 2.1 x x 2.1 x 2.1 3/pk EVO C18 B-4726-AN D-4726-AN F-4726-AN AJ-9298 XB-C18 A-4498-AN B-4498-AN D-4498-AN F-4498-AN AJ-8782 C18 A-447-AN B-447-AN D-447-AN F-447-AN AJ μm Analytical Columns (mm) Phases 3 x 4.6 x x x 4.6 x 4.6 3/pk EVO C18 B-47-E D-47-E F-47-E AJ-9296 Polar C18 B-479-E D-479-E F-479-E AJ-93 XB-C18 B-4496-E C-4496-E D-4496-E F-4496-E AJ-8768 C18 A-4462-E B-4462-E C-4462-E D-4462-E F-4462-E AJ μm MidBore Columns (mm) Phases 3 x 3. x 3. 7 x 3. 1 x 3. x 3. 3/pk EVO C18 B-47-Y D-47-Y F-47-Y AJ-9297 Polar C18 B-479-Y D-479-Y F-479-Y AJ-931 XB-C18 A-4496-Y B-4496-Y C-4496-Y D-4496-Y F-4496-Y AJ-877 C18 A-4462-Y B-4462-Y C-4462-Y D-4462-Y F-4462-Y AJ-877 for 3. mm ID 2.6 μm Minibore Columns (mm) Phases 3 x 2.1 x x x 2.1 x 2.1 3/pk EVO C18 A-47-AN B-47-AN D-47-AN F-47-AN AJ-9298 Polar C18 A-479-AN B-479-AN D-479-AN F-479-AN AJ-932 XB-C18 A-4496-AN B-4496-AN C-4496-AN D-4496-AN F-4496-AN AJ-8782 C18 A-4462-AN B-4462-AN C-4462-AN D-4462-AN F-4462-AN AJ-8782 potential to generate significant changes in retention for polar molecules. A thorough method development project should involve screening each of these phases, but if only one column could be screened, it is recommended that it be the Kinetex Polar C18, with its ability to operate in 1 % aqueous conditions, followed by either the Kinetex C18 or Kinetex XB-C18 to complement it The Kinetex Polar C18, with its aqueous stability and special surface modification, may provide a benefit for analyzing polar analytes, whereas the standard Kinetex C18 or XB-C18 phases, may be advantageous for separations based upon primarily hydrophobic differences Although it was not included in this study, our Kinetex EVO C18 is the obvious choice for operating in alkaline mobile phases, or when you need improved peak shape for tailing basic analytes, especially when using weakly-buffered mobile phases like.1 % formic acid 1.7 μm Microbore Columns (mm) Phases x 1. 1 x 1. x 1. EVO C18 B-4726-A D-4726-Y F-4726-A More Ordering Information ULTRA Cartridges require holder, Part No.: AJ-9 Page 6 of 8 Page 7 of 8

5 APPLICATIONS Kinetex Ordering Information 2.6 Microbore Columns (mm) Phases x 1. 1 x 1. x 1. XB-C18 B-4496-A F-4496-E F-4496-A 3. μm Analytical Columns (mm) Phases 1 x 4.6 x 4.6 3/pk XB-C18 B-4496-A D-4796-E AJ-8768 Australia t: +61 () f: +61 () au Austria t: +43 () f: +43 () anfrage@phenomenex.com Belgium t: +32 ()2 3 4 (French) t: +32 () (Dutch) f: +31 () be Canada t: +1 (8) f: +1 (31) China t: f: +86 () phen@agela.com Denmark t: f: nordic Finland t: +38 () f: nordic France t: +33 () f: +33 () france Germany t: +49 () f: +49 () anfrage@phenomenex.com India t: +91 () f: +91 () india Ireland t: +33 () f: eire Italy t: f: italia Phenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at international@phenomenex.com Page 8 of 8 Luxembourg t: +31 () f: +31 () nl Mexico t: f: tecnicomx@phenomenex.com The Netherlands t: +31 () f: +31 () nl New Zealand t: +64 () f: +64 () nz Norway t: f: nordic Puerto Rico t: +1 (8) 41-HPLC f: +1 (31) Spain t: f: esp Sweden t: +46 () f: nordic United Kingdom t: +44 () f: +44 () uk USA t: +1 (31) 212- f: +1 (31) All other countries Corporate Office USA t: +1 (31) 212- f: +1 (31) μm Minibore Columns (mm) Phases 3 x 2.1 x x 2.1 x 2.1 3/pk EVO C18 A-4633-AN B-4633-AN D-4633-AN F-4633-AN AJ-9298 XB-C18 A-46-AN B-46-AN D-46-AN AJ-8782 C18 A-461-AN B-461-AN D-461-AN F-461-AN AJ-8782 μm MidBore Columns (mm) Phases x 3. 1 x 3. x 3. 3/pk EVO C18 B-4633-Y D-4633-Y F-4633-Y AJ-9297 XB-C18 B-46-Y D-46-Y F-46-Y AJ-877 C18 B-461-Y D-461-Y F-461-Y AJ-877 for 3. mm ID μm Analytical Columns (mm) Phases x x 4.6 x 4.6 x 4.6 3/pk EVO C18 B-4633-E D-4633-E F-4633-E G-4633-E AJ-9296 XB-C18 B-46-E D-46-E F-46-E G-46-E AJ-8768 C18 B-461-E D-461-E F-461-E G-461-E AJ-8768 μm Semi-Preparative Columns (mm) SemiPrep Cartridges*** Phases x 1 x 1 1 x 1 EVO C18 F-4633-N G-4633-N AJ-936 C18 F-461-N G-461-N AJ-9278 for 1 mm ID μm Axia Packed Preparative Columns (mm) PREP Cartridges* Phases x x 21.2 x 21.2 x 21.2 x 21.2 EVO C18 B-4633-P-AX D-4633-P-AX F-4633-P-AX G-4633-P-AX AJ-934 XB-C18 B-46-P-AX D-46-P-AX F-46-P-AX G-46-P-AX AJ-914 C18 B-461-P-AX D-461-P-AX F-461-P-AX G-461-P-AX AJ-914 for 21.2 mm ID μm Axia Packed Preparative Columns (mm) PREP Cartridges** Phases x 3 1 x 3 x 3 x 3 x 3 EVO C18 B-4633-U-AX D-4633-U-AX F-4633-U-AX G-4633-U-AX AJ-93 XB-C18 B-46-U-AX D-46-U-AX F-46-U-AX G-46-U-AX AJ-924 C18 B-461-U-AX D-461-U-AX F-461-U-AX G-461-U-AX AJ-924 ULTRA Cartridges require holder, Part No.: AJ-9 * PREP Cartridges require holder, Part No.: AJ-8223 ** PREP Cartridges require holder, Part No.: AJ-8277 *** SemiPrep Cartridges require holder, Part No.: AJ-9281 for 3 mm ID If Kinetex core-shell analytical columns do not provide at least an equivalent separation as compared to a competing column of the same particle size, similar phase and dimensions, return the column with comparative data within 4 days for a FULL REFUND. Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at Trademarks Kinetex is a registered trademark and, Axia, and MidBore are trademarks of Phenomenex. Axia column and packing technology is patented by Phenomenex. U.S. Patent No. 7,674,383. Kinetex EVO is patented by Phenomenex. U.S. Patent Nos. 7,63,367 and 8,68,38 and foreign counterparts. 217 Phenomenex, Inc. All rights reserved. TN17_W