Combining High Temperature and Small Particles: The Advantages of Zirconia

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1 Combining High Temperature and Small Particles: The Advantages of Zirconia EAS 9 Dan owlan 1, Bingwen Yan 1, Clayton V. Mceff 1, R.A. Henry 1 ZirChrom Separations, Inc. 617 Pierce St., Anoka, M 5533 Independent Consultant, 983 Greenbriar Drive, State College, PA 1681 Specialists in High Efficiency, Ultra-Stable Phases for HPLC 1

2 Introduction Lately, efficiency has received most of the attention in HPLC. As we study and debate optimum particle geometry and instrument design, higher efficiency columns are being adopted by analysts to improve resolution, peak capacity, speed, sensitivity and solvent economy. Most of the progress with small particles has been made with silica RP columns so it is important to investigate whether the high efficiency observed with ultra-small silica RP particles can be translated to other substrates and phases, which may retain and separate by other selective modes. Zirconia phases often separate by a multi-modal mechanism so they are good candidates to see if the performance advantages of sub-µm particles can be observed (at ambient or elevated temperature) for other packings.

3 Addition of RP Behavior with Coated Zirconia Phases H Zr Zr R + H P R R H 3 + H H H H H H Zr Zr Zr Zr Zr R Polymer coating Ionic solute retention (and selectivity) is modulated by ph, buffer/salt type and concentrations, and temperature. RP solute retention is modulated by organic solvent. Five important mobile phase variables must be controlled. 3

4 Difficult Compounds for Silica ften Separate on Zirconia Quaternary amines paraquat and diquat are retained and resolved on Zr-PS (also Zr-PBD or bare Zr ) due to the cation exchange mechanism; 5% AC is useful to suppress or regulate retention by RP mode. Silica-C18: reversed-phase Zirconia-PS: primarily ion-exchange AU..1 Light retention and poor peak shape even in low organic (5% AC) AU Time (min) Time (min) column: Discovery C18, 15 cm x.6 mm I.D., 3µm mobile phase: 5% acetonitrile in 5 mm phosphate, ph 7 flow rate: 1 ml/min. temp.: 35 C det.: UV 9 nm column: Discovery Zr-PS, 7.5 cm x.6 mm, 3µm mobile phase: 5% acetonitrile in 5 mm phosphate, ph 7 flow rate: 3 ml/min. temp.: 65 C det.: UV 9 nm Data provided by Sigma-Supelco

5 Anticholinergics on Zr-PBD Quaternary amines and related compounds H H 1, Pipenzolate ( mg/l), Scopolamine (1 mg/l) Time (min) LC Conditions Discovery Zr-PBD, 1mm x.1mm i.d., 3 μm Mobile Phase A: 1 mm H P, ph 7. Mobile Phase B: 8/ mm H P, ph 7./AC Gradient: 1-1% B over 18 minutes Temp: 8 o C, Flow:.3 ml/min Inj vol: μl in 6% MeH Detector: UV@5 nm H H 3, Ipratropium (1 mg/l), Methscopolamine (1 mg/l) H 5, Propantheline ( mg/l) 6, xyphenonium (1 mg/l) Data provided by Sigma-Supelco 5

6 Plate Height, H (µm) 1 van Deemter Plots Reveal Column Performance H = A + B/ν + Cν (shown below for a single solute) Data plots move lower and become flatter for small particles due to combined effects of the equation terms. 1µm 5µm 3µm Idealized plot provided by Sigma-Supelco 6 Flow velocity, ν (mm/sec) Sub-µm goal: ca. 5, /m Instrument optimization very important! 6

7 Flow Studies on Sub-μm Zr-PBD: Instrument ptimization Plate Height Vs. Linear Velocity for a PBD Column Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene ml/min test conditions Plate Height Vs. Linear Velocity for a PBD Column Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene ml/min test conditions Plate Height, H (um) Plate Height, H (um) u (cm/s) u (cm/s) Factory Instrument Micro Cell nly Plate height based on van Deemter Equation vs linear velocity for retained solutes: Alkylbenzenes, Temperature 3 ºC, Mobile phase: 5/5 AC/water ZirChrom -PBD column: 5 x.6mm sub-μm (part #: ZR ), Agilent 11/UV 7

8 Flow Studies on Sub-μm Zr-PBD: Instrument ptimization Plate Height Vs. Linear Velocity for a PBD Column Plate Height Vs. Linear Velocity for a PBD Column Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene 1.6 ml/min test conditions Benzene Toluene Ethylbenzene Propylbenzene Butylbenzene 1.8 ml/min test conditions Plate Height, H (um) Plate Height, H (um) u (cm/s) u (cm/s) Micro cell + ptimized tubing (bypass instrument heat exchanger) Micro Cell + High pressure fitting + Heat exchanger Plate height based on van Deemter Equation vs linear velocity for retained solutes: Alkylbenzenes, Temperature 3 ºC, Mobile phase: 5/5 AC/water ZirChrom -PBD column: 5 x.6mm sub-μm (part #: ZR ), Agilent 11/UV 8

9 Alkylbenzenes on ZirChrom -PBD sub-μm and Silica-C8 orm , plates/m Silica-C8 5mm x.6mm, sub-3μm 65/35 AC/H F=1 ml/min UV=5nm T=3 o C min orm. 3 ZirChrom -PBD % less AC, plates/m 5mm x.6mm, sub-μm Part #: ZR /5 AC/H F=1 ml/min UV=5nm T=3 o C min 9

10 Drug Mix* Separation on Zr-PBD sub-μm Ambient mau bar 1 3 on-optimized Agilent 11 with micro cell * Mainly beta-blockers 8 153, plates/m 9 min Analytes 1=Labetalol =Atenolol 3=Acebutolol =Metoprolol 5=xprenolol 6=Lidocaine 7=Quinidine 8=Alprenolol 9=Propranolol Column: ZirChrom -PBD, 5 x.6 mm i.d., sub-μm; Part #: ZR Mobile phase: /76 AC/ mm K 3 P at ph=1; Flow rate: 1. ml/min; Temp.: 3 o C; Injection vol.:. µl; Detection: UV at 5 nm 1

11 Drug Mix on ZirChrom -PBD sub-μm, 75 o C mau bar 3% less AC Less than 1 min! 1 3 Agilent 11 ZirChrom -PBD 5mm x.6mm, sub-μm Part #: ZR ) 1/79 AC/mM K 3 P at ph=1 F=.5 ml/min UV=5nm (.1 sec response) T=75 o C Analytes 1=Labetalol =Atenolol 3=Acebutolol =Metoprolol 5=xprenolol 6=Lidocaine 7=Quinidine 8=Alprenolol 9=Propranolol 185, plates/m min 11

12 Toluidines Separation on sub-μm Zr-PBD: Temperature mau T=5 o C, 1 bar o m p USP.8 LC Conditions: 35/65 AC/ 5 mm HAc + 1 mm H H P ph=.67 F=1 ml/min, UV=5nm, T=5 o C 5x.6mm, 1.9 μm, μl inj Part #: ZR H H H 1 mau Minutes T=8 o C, 8 bar o m p USP 1.75 o m p LC Conditions: 1/9 AC/ 5 mm HAc + 1 mm H H P ph=.67 F= ml/min, UV=5nm, T=8 o C 5x.6mm, 1.9 μm, 7 μl inj Part #: ZR M i n u t e s 1

13 orm bar T=5 o C 1 Antihistamine Separation on sub-μm Zr-PBD 1 ml/min < 5.5 min α(,3) = S 1, Doxylamine, Methapyrilene Cl orm bar T=8 o C min.5 ml/min < 1. min α(,3) = 1. min 3, Chlorpheniramine, Triprolidine Column: ZirChrom -PBD, 5 x.6 mm i.d., sub-μm; Part #: ZR Mobile phase: 8/7 AC/5 mm TMA-H at ph=1.; Injection vol.:. µl; Detection: UV at 5 nm 13

14 orm. 16 bar 3 T=5 o C 5 15 Alkylbenzylamine Separation on sub-μm Zr-PBD: 5 and 5 o C 1 3 LC Conditions: 1/79 AC/ mm K 3 P ph=1 F=1. ml/min, UV=5nm, T=5 o C, 5x.6mm, 1.9 μm, 3 μl inj Part #: ZR min H 1 H orm bar 3 T=5 o C , plates/m < 1 min! min 3 LC Conditions: 1/79 AC/ mm K 3 P ph=1 F=1.5 ml/min, UV=5nm, T=5 o C, 5x.6mm, 1.9 μm, 3 μl inj Part #: ZR

15 Conclusions and Plans for Further HPLC Development with Zirconia Performance results with sub-µm Zr-PBD show comparable efficiency gains for the zirconia based sub-µm particles. The multi-modal separation mechanisms and high temperature stability of zirconia based sub-µm particles provide added resolution and lower back pressures. This improved performance enables the use of the particles on an optimized standard HPLC. The study of ultra-high speed applications using sub-µm Zr- PBD, especially at higher ph and temperature ( extreme conditions for silica ) will be continued; generic conditions for LC-MS will be investigated. ther sub-µm Zr phases (such as CARB) will be prepared and compared to Zr-PBD under ambient and extreme conditions. 15

16 Acknowledgements For more information contact ZirChrom support at or stop by Booth. The assistance of Supelco Division of Sigma-Aldrich is gratefully appreciated, including the use of a high-pressure column fitting. 16

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