ew Applications of Zirconia Based Stationary hases EAS 8 Bingwen Yan 1, Clayton V. Mceff 1, R.A. Henry, Dan owlan 1 1 ZirChrom Separations, Inc. 617 ierce St., Anoka, M 33 Independent Consultant, 983 Greenbriar Drive, State College, A 1681 Specialists in High Efficiency, Ultra-Stable hases for HLC
Surface Chemistry of Zirconia H H H H H H H HH Zr Zr Zr Zr Zr Zr Zr Zirconia chemistry is dominated by Lewis acid-base reactions Lewis Acid: Zr(IV): H + R 3 - Zr(IV): R 3 - + H ther Lewis base examples: 3-, RC -, Catechol
Interaction Strength of Lewis Bases with Zirconia 3 Interaction Strength Lewis Base (L) Strongest Weakest Hydroxide hosphate Fluoride Citrate Sulfate Acetate Formate itrate Chloride Water Small Lewis bases with high electron density and low polarizability interact more strongly with Zr atoms. 3 J.A. Blackwell and.w. Carr, "Development of an Elutropic Series for the Chromatography of Lewis Bases on Zirconium xide," Anal. Chem. 6, 863-73 (199). 3
Retention of Basic Analytes on ZirChrom -BD and ZirChrom -S Adsorbed Lewis base anion - - A + A + H A + A + olymer coating - H H H H H H Zr Zr Zr Zr Zr Zr Zr Zr BD, S Coating Reversed-hase (R) Moieties Lewis Base Anions Ion-Exchange (IEX) Sites Zr-L - :X + + A + = Zr-L - :A + + X + Zr- - :X + + A + = Zr- - :A + + X + A + : analyte cation, X + : counterion, L - : adsorbed Lewis base anion.
Effect of Ionic Strength on the Separation of Quaternary Amines Low Ionic Strength paraquat 3 nm diquat nm Column: Discovery Zr-S, 7.cm x.1mm ID, 3µ particles Mobile hase1: :, ( mm H 3, mm H HC, ph 7. w/ H H) : Acetonitrile Mobile hase: :, ( mm H 3, 1 mm H HC, ph 7. w/ H H) : Acetonitrile Flow:. ml/min Temp: as indicated Det: UV at nm & 3nm Inj: 1 µl Sample: diquat and paraquat in water; mg/l ea. 1 3 Time (min) High Ionic Strength k values for diquat are -3 at low ionic strength in % AC. paraquat 3 nm diquat nm k values for diquat decrease to about at high ionic strength without changing %AC. The classic method for reducing k in IE mode is to increase ionic strength, confirming IE mode. 6 8 1 Time (min) Data used by permission of Supelco
Effect of Reversed hase Character on the Separation of Quaternary Amines 3 % AC paraquat 3 nm diquat nm Column: Discovery Zr-S, 1cm x.1mm ID, 3µ particles Mobile hase 1:: : 3, ( mm H 3, 1 mm H HC 3, ph 7. w/ H H) : Water : Acetonitrile Mobile hase :: : 1, ( mm H 3, 1 mm H HC 3, ph 7. w/ H H) : Water : Acetonitrile Flow:.3 ml/min Temp: C Det: UV at nm & 3nm Inj: 1 µl Sample: diquat & paraquat in water; 1 mg/l ea. 6 8 1 1 1 Time (min) 1 % AC At 3% AC, the polymer coating adds very little to retention or selectivity for these ionic compounds. When nonionic compounds are present, changes in organic solvent strength will have a greater impact and can be used for optimizing resolution. 1 Time (min) Data used by permission of Supelco 6
Anticholinergics on Zr-BD 1 3 6 H H 1, ipenzolate ( mg/l), Scopolamine (1 mg/l) 6 8 1 1 1 16 18 Time (min) LC Conditions Discovery Zr-BD 1mm x.1mm i.d., 3 µm Mobile hase A: : [ mm H 3, ph 7. w/ H H]:water Mobile hase B: :3: [ mm H 3, ph 7. w/ H H]:water:AC Gradient 9:1 to :1 A:B over 18 minutes Temp = 8 o C, Flow =.3 ml/min, Inj vol = µl, UV nm, sample in ~6: Mobile phase A:MeH H H 3, Ipratropium (1 mg/l), Methscopolamine (1 mg/l) H, ropantheline ( mg/l) 6, xyphenonium (1 mg/l) Data used by permission of Supelco 7
MS Stationary hase Strategy 1. Chemisorb Ethylenediamine,,, - tetra(methylenephosphoni c)acid (EDTA) to the zirconia surface.. Quaternize amines on the zirconia surface with allyl iodide. zirconia - + H + - H BD BD n EDTA Chemisorptions BD/ dicumyl peroxide Crosslinking H H H H - + n Allyl iodide quaternization H + - H n 3. Coat polybutadiene (BD) on the chelator-modified zirconia surface and crosslink BD with allyl group and BD itself using dicumyl peroxide as initiator. 8
ZirChrom -MS Multi-Mode Character mau 17 1 /7 AC/mM H Ac ph=, T=3 o C, F=1mL/min, UV=1 H C C H C 1 1 7 1 3 1, Maleic acid, Benzonitrile H - 1 3 6 min 3, Benzyl amine mau 17 1 1 1 1 7 /7 AC/mM H Ac, mm H F ph=, T=3 o C, F=1mL/min, UV=1 3 Multi-mode retention on ZirChrom -MS (MS1-6) Addition of fluoride blocks Lewis acid sites, decreasing the ligand exchange between zirconia and maleic acid.. 1 1.. 3 3.. mi n 9
Retention Comparison: Si-C18 vs Zr-BD K' 18 16 1 1 1 8 6 yridine Lidocaine Basic harmaceutical retention in 8% AC yrilamine Tripeleneamine Methapyrilene Chlorpheniramine Thiothixene Doxepin Brompheniramine Solutes Atenolol Metoprolol xprenolol Amitryptyline Imipramine Alprenolol Desipramine ortryptyline Excess k due to IE mode Si-C18 Zr-BD LC Conditions: Machine-mixed 8/ AC/1 mm ammonium acetate ph=6.7 without ph adjustment; Flow rate, 1. ml/min.; Injection volume.1 ul; Temperature, 3 o C; Detection at nm; Columns, ZirChrom -BD, x.6 mm i.d. (3 um particles); Silica-C18 1 x.6 mm i.d., (3. um particles). 1
Beta Blockers on ZirChrom -MS mau 1 H 1 8 6 1 3. 7. 1 1. 1 17. min H H H H H H 1, Lidocaine, Atenolol 3, Metoprolol H H, xprenolol, Alprenolol LC Conditions: 6/3 AC/1 mm ammonium acetate ph=.; Flow rate, 1. ml/min.; Injection volume. µl; Temperature, 3 o C; Detection at nm; Columns, ZirChrom -MS, x.6 mm i.d. (3 um particles) H Multi-mode retention creates high k for bases even in high organic. Bases are charged at ph and can interact strongly with negative charges on phosphonate groups of EDTA. High organic mobile phase with ammonium acetate is ideal for LC- MS. 11
HLC Analysis of Cattle lasma Using ZirChrom -MS orm. 1 1 VWD1 A, Wavelength= nm (:\HCHEM\DATA\M31B\61116.D) VWD1 A, Wavelength= nm (:\HCHEM\DATA\M31B\61113.D) FMC-I Initial Separation Conditions A: AC B: mm H 3 ph=.8 /, 1 ml/min, T=3 o C. UV = nm C H 1 8 6 Di-FMC- Lys H H H C H ipecolic acid Lysine orm. 17. 1 1. 1 7.. VWD1 A, Wavelength= nm (:\HCHEM\DATA\M31B\6113.D) VWD1 A, Wavelength= nm (:\HCHEM\DATA\M31B\6113.D) 1 1 3 3 FMC-I Di-FMC- Lys Initial Separation Conditions A: AC B: 3 g mm ammonium acetate ph 3 adjusted with formic acid +.3 g H 3 o C min 3/6 A/B 611 min / A/B 1 min 6 8 1 Data used by permission of SarTec Corporation (Anoka, M) min min Analysis of pipecolic acid and lysine levels in cattle plasma ZirChrom -MS (MS1-16). IEX and R modes contribute to making a difficult separation. rganic solvent, ph and ionic strength are all important variables with ZirChrom-MS. 1
Surface Chemistry and Retention Mechanisms of QEI-Zirconia Anion-exchange Hydrophobic interactions Lewis acid-base interactions 13
Water-Soluble Vitamin Analysis on ZirChrom -SAX 1 3 1 - Thiamine (Vit. B 1 ) - yridoxine (Vit. B 6 ) 3 - icotinamide (form of Vit. B 3 ) - Riboflavin (Vit. B ) - icotinic acid (form of Vit. B 3 ) 6 - Ascorbic acid (Vit. C) 6 Vitamin C is strongly retained on ZirChrom -SAX 1 3 6 min. LC Conditions: Column: ZirChrom -SAX, 1 x.6 mm i.d. (part number: ZR6-16), Mobile hase: mm Ammonium dihydrogenphosphate, ph., Flow rate: 1. ml/min. Temperature: 3 o C, Injection Vol.:. µl, Detection: UV at nm 1
Summary and Conclusions Mixed-mode applications have become popular for difficult applications where compounds vary widely in chemical nature. Several ZirChrom phases, including Zr-BD, Zr- S, Zr-MS and Zr-SAX, are ideal for mixed-mode applications and show unique selectivity. ZirChrom phases are stable and reproducible over a wider range of ph and temperature than silicabased phases. 1
Acknowledgements The authors wish to thank Supelco for permitting the use of data on quaternary amine compounds and SarTec for use of data on cattle plasma compounds. Trademarks used include: ZirChrom, Discovery For more information contact ZirChrom support at www.zirchrom.com or stop by Booth. 16