Zirconia-Based Phases as a Powerful Complement to Silica-Based Phases for LC and LC-MS under Non-extreme Mobile Phase Conditions
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1 Zirconia-Based Phases as a Powerful Complement to Silica-Based Phases for LC and LC-MS under on-extreme Mobile Phase Conditions Richard A. Henry, Shawn R. Wyatt, Carmen T. Santasania and David S. Bell Supelco 595. Harrison Rd. Bellefonte, PA 16823
2 Abstract Highly stable zirconia-based HPLC stationary phases have important advantages over other HPLC phases when column operation at elevated temperature or extreme ph is desired. The unique selectivity of polymer-coated zirconia phases may also be exploited in certain cases at nearambient temperature with similar mobile phase ph and composition to that employed for popular silica phases. This paper will briefly describe the origins of unique selectivity for Discovery Zr phases and compare performance to Discovery silica phases for a selection of compounds under non-extreme operating conditions.
3 Importance of LC/MS LC-MS with C8 and C18 silica-based columns has become extremely popular; however, there is still a need for stable, efficient columns that show different selectivity than silica packings with pure alkyl stationary phases. While silica packings with polar phases fulfill some of the need for different selectivity, they can show excessive bleed and high background ionization in LC-ESI-MS even under non-extreme mobile phase conditions. A study of the utility of several zirconia phases for LC-ESI- MS under normal (non-extreme) operating conditions will be included.
4 Background Stationary phases based on silica supports remain the workhorse for liquid chromatography (LC) and LC/MS. Zirconia phases with phosphate buffers are becoming a popular alternative to silica for LC analyses which employ UV detection. Important advantages include: - ability to withstand extreme ph and temperature conditions - unique selectivity and retention for various classes of compounds (focus of this paper) There is still a need for stable, selective alternatives to the current silica phases for LC/MS, where volatile mobile phases are required.
5 Hydrophobicity of Discovery RP Columns Discovery HS C18 Discovery C18 Discovery Zr-CarbonC18 Discovery RP Amide C16 Discovery BI Wide Pore C18 Discovery HS F5 Discovery C8 Discovery BI Wide Pore C8 Discovery Zr-Carbon Discovery BI Wide Pore C5 Discovery Zr-PBD Discovery Cyano Discovery Zr Zr phases are are different from from each other and and from from other Discovery RP RP columns. Discovery HS PEG Discovery Zr-PS k Butylbenzene (40:60 H2:CH3C, 30 C)
6 Silica C18 Structure CH 2 CH 2 CH 2 CH 2 10 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 10 CH 2 CH 2 Strong efforts have been made by the silica-based column manufacturers to mask silanol effects, causing columns having the same phase functional groups to become increasingly similar to each other. CH 2 CH 2 CH 2 CH 2 CH 2 CH Si CHCH 2 CH 2 CH Si CHCH 2 H H H There is still a strong need for stable columns which have different selectivity than silicabased bonded phases. Silica Substrate
7 rigins of Unique Selectivity on Zirconia Bronsted base and acid - P Lewis acid - A + H H nly ions can interact with phosphate layer H A + B H 2 H H H 2 H 2 Both ions and neutrals can interact with hydrophobic polymer layer (PBD, PS, Carbon, CarbonC18) Zr Zr Zr Zr Zr Zr Zr Zr Zirconia, a transition metal oxide, has very rich surface chemistry. Coated zirconia (Carbon and PBD) exhibits mixed-mode surface properties (RPC and IEC) which allow simultaneous nonpolar and polar interaction with solutes. Retention of various basic and acidic analytes can be fine-tuned by changing ph, buffer and buffer concentration.
8 eutral Analyte Selectivity: Zr-PBD Similar to Silica-C18 (o Phosphate Required) Butylbenzene Pentylbenzene Column: 150 x 4.6mm, 5 µm Mobile Phase: 20:80 water:methanol Flow Rate: 1.0mL/min Temperature: 40ºC Injection Volume: 5µL Detection: UV, 254nm o-terphenyl Triphenylene Discovery C18 Discovery Zr-PBD AU ) Butylbenzene 2) o-terphenyl 3) Pentylbenzene 4) Triphenylene Time (min) AU ) Butylbenzene 2) Pentylbenzene 3) o-terphenyl 4) Triphenylene Time (min) 3 4
9 eutral Diastereomers on Discovery Zr- CARB (o Phosphate High ph ) F C18 does not resolve mau Time (min.) LC Conditions: Column, 150 mm x 4.6 mm i.d. ZirChrom-CARB; Mobile phase, 35/35/30 AC/Butanol/10mM Diethylamine, ph 11.2; Flow rate, 2.00 ml/min.; Temperature, 80 o C; Injection volume, 5 µl; Detection at 265 nm.
10 Ionic Solute Selectivity- Very Different Absorbance (mau) , 2 Antihistamines 5 6 3, 2, Silica-C18 30 o C Discovery Zr-PBD 30 o C 9 log k C18 Silica R 2 =0.147 S= log k Discovery Zr-PBD Mobile Phase: 40% C in 25 mm phosphate (ph 7) The selectivity of zirconia-based RP columns towards basic compounds becomes very different from that of traditional silica-based RP columns when phosphate is employed in the mobile phase under acidic and neutral conditions, and the Zr-PBD column becomes comparable in retention to a typical Silica-C18.
11 Impact of Sample Types on Selectivity Discovery Zr-PBD vs. Silica-C18 R 2 = 0.98 R 2 = logk (DS) log k' (DS at 40 o C) 1.0 Antihistamines logk (PBD-Zr 2 ) onionic solutes: Columns are very similar due to retention by RP mode only, but Zr-PBD is less retentive log k'(pbd-zr2 at 40 o C) Ionic solutes: Columns are very different due to retention by combined RP and IEC modes
12 Advantage of Ion-Exchange on Zirconia Quaternary amines paraquat and diquat are retained and resolved on Zr-PS because of the mixed-mode RP and ion-exchange. Silica-C18: reversed-phase only Zirconia-PS: reversed-phase and ion-exchange AU Light retention and poor peak shape AU Time (min) Time (min) C18-silica conditions: Discovery C18, 15cm x 4.6mm, 3µm particles; 5% C in 25mM H 3 P 4 (to ph 7 with H 4 H); 35 C, 1mL/min, UV 290nm Zr-PS conditions: Discovery Zr-PS, 7.5cm x 4.6mm, 3µm particles; 50% C in 25mM H 3 P 4, 25mM H 4 F, (to ph 8 with H 4 H); 65 C, 3mL/min, UV 290nm
13 Discovery Zr for LC/MS Many applications for neutral compounds on Discovery R Zr phases do not require phosphate and are LC/MS compatible; however, guards and regular washes/regenerations are recommended. Most applications for ionic compounds are not LC/MS compatible due to the requirement of high ionic strength phosphate buffers, especially under desired acidic conditions for optimum sample detection by ESI+. This study was aimed at: - Assessing the need for phosphate in various systems - Determining if Discovery R Zr phases can be made LC/MS compatible for ionic samples - Investigating Lewis acid endcapping as a viable solution
14 Bases on Discovery Zr-PBD with Phosphate Discovery Zr-PBD 15cm x 4.6mm ID, 5µm particles (70:30) 25mM potassium phosphate, ph 3.0: acetonitrile 1.0 ml/min UV, 220nm 35 o C 25µg/mL diltiazem, metoprolol in (50:50) 25mM potassium phosphate, ph 3.0:acetonitrile ot MS Compatible Good peak shape, selectivity and retention with phosphate mobile phase S H 3 C H 3 C C H 3 CH3 H H
15 Bases without Phosphate Discovery Zr-PBD, 5cm x 2.1mm ID, 3µm particles (60:40) 10mM ammonium acetate, unadjusted : C 0.2 ml/min ms, esi (+) 40 o C 1µg/mL diltiazem, metoprolol in (60:40) water:acetonitrile Poor Retention S H 3 C Without phosphate, retention by only the RP mode can be weak unless bases are hydrophobic. H 3 C C H 3 CH3 H H
16 Bases without Phosphate Discovery Zr-PBD, 5cm x 2.1mm ID, 3µm particles (80:20) 10mM ammonium acetate, unadjusted : C 0.2 ml/min ms, esi (+) 40 o C 1µg/mL diltiazem, metoprolol in (60:40) water:acetonitrile Reducing rganic Percentage Isn t the Answer Selectivity and retention, but poor peak shape
17 Acids without Phosphate H 3 C H 3 C H Retention Too Strong H homovanillic acid H sorbic acid o elution due to strong Lewis acid-base interaction H H salicylic acid Discovery Zr-PBD, 5cm x 2.1mm ID, 3µm particles (95:5) 10mM ammonium formate, ph 3.5 : C 0.2 ml/min ms, esi (-) 40 o C 1µg/mL each in (50:50) water:methanol
18 Chelators without Phosphate Retention Too Strong Discovery Zr-PBD, 15cm x 4.6mm, 5µm (45:55) 0.1% formic acid in water: 0.1% formic acid in CH3H 1 ml/min 35 C UV at 254nm 25 µg/ml in 0.1% formic acid in water nly flavone elutes H H H H H H H H H H H H H H Flavone Quercetin Baicalein Myricetin
19 Possible Discovery Zr Solution for LC/MS Without phosphate or another strong Lewis base in the mobile phase - there is no negative character imparted to the surface to generate valuable cation exchange properties - bases are not well retained under desired acidic conditions and may exhibit poor peak shape - acids and chelators can interact strongly and may not elute A permanent endcapping agent with Lewis base character may solve these issues Current research has shown promising results
20 Proposed Lewis Acid Modification R R A reagent with phosphate groups could provide the ion-exchange properties and mask the Lewis acid sites without being a component of the mobile phase P - H P - H P - H H Zr Zr Zr Zr +
21 A ew Stationary Phase Strategy for LC/MS zirconia EDTPA Chemisorption H P P H P P H H Allyl iodide quaternization - P + P H P + P - H PBD/ dicumyl peroxide Crosslinking 1. Chemisorb Ethylenediamine,,, -tetra(methylenephosphonic)acid (EDTPA) to the zirconia surface. 2. Quaternize amines on the zirconia surface with allyl iodide. PB D PB D n - P + P H P + P - H 3. Coat polybutadiene (PBD) on the chelator-modified zirconia surface and crosslink PBD with allyl group and PBD itself using dicumyl peroxide as initiator.
22 LC/MS on Modified Zr-PBD Modified Zr-PBD, 5cm x 2.1mm ID, 3µm particles (60:40) 10mM ammonium acetate, unadjusted : C 0.2 ml/min MS, ESI (+) 40 o C 1µg/mL diltiazem, metoprolol in (60:40) water:acetonitrile Without Phosphate in Mobile Phase Good peak shape, selectivity and retention on modified Zr-PBD S H 3 C H 3 C C H 3 CH3 H H
23 Same Bases on Discovery R Zr-PBD Discovery Zr-PBD, 15cm x 4.6mm ID, 5µm (70:30) 25mM potassium phosphate, ph 3.0: acetonitrile 1.0 ml/min UV, 220nm 35 o C 25µg/mL diltiazem, metoprolol in (50:50) 25mM potassium phosphate, ph 3.0:acetonitrile ot MS Compatible Good peak shape, selectivity and retention with phosphate mobile phase S H 3 C H 3 C C H 3 CH3 H H
24 H Amphetamines on Modified Zr-PBD H CH H 3 CH 3 H Modified Zr-PBD 70% Acetonitrile Acetic Acid, 10mM amphetamine methamphetamine H 2 H H 3 C H 3 C MDA MDMA Time (min) 1. Amphetamine 2. MDA 3. Methamphetamine 4. MDMA Discovery C18 10% Acetonitrile Acetic Acid, 10mM Time (min)
25 Acidic Drugs on Modifed Zr-PBD mau 20 VWD1 A, Wavelength=254 nm (G:\HPCHEM\DATA\M101003T\ D) 17.5 (4) Ibuprofen H Lewis Acid-Base Interaction blocked By covalently attached EDTPA Chromatographic Conditions: Column Dimension: 50X4.6 MS101003T; Mobile phase, Machine-mixed 40/60 AC/10 mm ammonium acetate ph=5. Flow rate:1 ml/min, Temperature, 35 o C; Injection volume: 5 µl; Solutes eluted in order, (1) Acetaminophen, (2) Ketoprofen, (3) aproxen, (4) Ibuprofen, (5) Impurity; Detection, 254 nm. Pressure drop, 68 bar. min
26 β-blockers on Modified Zr-PBD mau VWD1 A, Wavelength=254 nm (G:\HPCHEM\DATA\M101003T\ D) H H (5) Alprenolol β-amino alcohols have Very good peak shape at near neutral phs min Chromatographic Conditions: Column Dimension: 50X4.6 Modified Zr-PBD; Mobile phase: Machine-mixed 65/35 AC/10 mm ammonium acetate ph=5; Flow rate:1 ml/min; Temperature, 35 o C; Injection volume: 5 µl. Solutes eluted in order: (1) Lidocaine, (2) Atenolol, (3) Metoprolol, (4) xprenolol, (5) Alprenolol Detection: 254 nm; Pressure drop, 59 bar.
27 Basic Drugs on Modifed Zr-PBD mau 30 VWD1 A, Wavelength=254 nm (G:\HPCHEM\DATA\M101003T\ D) (4 ) pk a 9.4 H (5) pk a Compounds elute according to IEX, not RP interactions at near neutral phs min Chromatographic Conditions: Column Dimension: 50X4.6 Modified Zr-PBD; Mobile phase, Machine-mixed 65/35; AC/10 mm ammonium acetate ph=5; Flow rate, 1 ml/min; Temperature, 35 o C; Inject volume, 1 µl; Solutes eluted in order: (1) Methapyrilene, (2) Bromphenriamine, (3) Doxpin, (4) Amtriptyline, (5) ortryptyline Detection, 254 nm; Pressure drop, 59 bar.
28 Acids on Modified Zr-PBD H 3 C H H H 3 C sorbic acid H Acids with strong Lewis base character or chelating properties are still retained too strongly. homovanillic acid H H salicylic acid Modified Zr-PBD, 5 cm x 2.1 mm, 3 µm 10 mm ammonium formate, ph 3.5: H 0.2 ml/min MS, ESI (-) 40 C 1 µg/ml each in (50:50) water: H
29 Chelators on Modified Zr-PBD Modified Zr-PBD, 15cm x 4.6mm ID, 5µm particles (45:55) 0.1% Formic Acid: 0.1% Formic Acid in H 1mL/min UV, 254nm 35C 10µL 25µg/mL of each Chelating compounds with phenolic groups are still retained too strongly Time (min) H H H H H H H H H H H H H H Flavone Quercetin Baicalein Myricetin
30 LC/MS Bleed Studies Method: Waters Micromass ZQ Mass Spectrometer coupled to a Waters 2690 Liquid Chromatograph A 2.1 mm X 15 cm Modified Zr-PBD and a 2.1 mm X 15 cm Discovery Zr-PBD Gradient elution was performed using the following buffer systems and acetonitrile - 5mM Ammonium Hydroxide, ph 10.9, unadjusted - 10 mm Ammonium Acetate, ph 7.0, unadjusted - 10 mm Ammonium Acetate, ph 5.0, adjusted with acetic acid - 10 mm Ammonium Formate, ph 3.0, adjusted with formic acid - 0.1% Formic Acid, unadjusted Column Temp: 35 o C Detection: UV Diode Array and ESI MS in both (+) and (-) ion modes.
31 LC/MS Bleed Results Expected mass response Significant bleed was only observed at high ph (>10)
32 β-blocker Drugs on Modified Zr-PBD 10mMAmAc_pH5 gradient_1_zrms_pos_vial_8 100 % 0 gradient_1_zrms_pos_vial_8 100 % pindolol atenolol : Scan ES e8 1: Scan ES e7 Modified Zr-PBD Gradient 5-100%B A: 10 mm H 4 Ac, ph 5 B: 10 mm H4Ac, ph 5, 10:90 buffer:ac Detection: MS ESI+ 0 gradient_1_zrms_pos_vial_8 100 % -0 gradient_1_zrms_pos_vial_8 100 acebutolol : Scan ES e8 1: Scan ES e8 Sample: Pindolol Atenolol Acebutolol Alprenolol % 0 alprenolol Time Retention by RP and SCX modes
33 Quaternary Amine Drugs on Modified Zr-PBD 10mMAmAc_pH5 gradient_1_zrms_pos_vial_9 100 % isopropramide : Scan ES e8 Modified Zr-PBD Gradient 5-100%B A: 10 mm H4Ac, ph 5 B: 10 mm H4Ac, ph 5, 10:90 buffer: AC Detection: MS ESI+ -0 gradient_1_zrms_pos_vial_9 100 oxyphenonium : Scan ES e8 Sample: Isopropramide xyphenonium % Time
34 Summary Research on a Lewis acid modified Zr-PBD phase has shown promising results for separating ionic analytes using LC/MS compatible mobile phases - Excellent retention of basic analytes without phosphate additives - Alternative selectivity to silica-based C18, especially for bases, due to mixed-mode retention mechanism - Minimal reagent bleed, except at high ph - Issues remain with certain acidic and chelating analytes Continued research is underway to produce LC/MScompatible Zr phases that take advantage of its unique selectivity and stability
35 References and Acknowledgements The authors gratefully acknowledge the assistance of scientists at Supelco Division of Sigma-Aldrich, Bellefonte, PA and Zirchrom Inc., Anoka, M. Copies of the paper may be requested at the Sigma-Aldrich Booth umber 4379
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