Characteristics and Advantages of Zirconia-Based Stationary hases for Use in Multi-Dimensional LC Dwight Stoll and Clayton V. Mceff ZirChrom Separations, Inc. Multi-Dimensional Chromatography Workshop Eastern Analytical Symposium, 2003
utline 1. Review of theory and requirements Why bother with multidimensional chromatography? 2. Why use zirconia for two-dimensional chromatography? 3. ZirChrom -CARB and DiamondBond-C18 TM Very unique phases for RLC 4. ZirChrom -BD, ZirChrom -EZ and ZirChrom -MS hases with mixed mode retention characteristics for ionizable analytes 5. Selectivity comparisons using ZirChrom -CARB 6. Selectivity comparisons using ZirChrom -BD 7. An example two-dimensional LC separation of ten triazine herbicides using ZirChrom -BD and ZirChrom -CARB 8. Conclusions
A Common roblem in LC Sample composed of 20 components with randomly distributed k values 150 mm x 4.6 mm i.d. column AU 0.6 0.5 0.4 0.3 0.2 0.1 0 = 10,000 plates/column n c = 38, 12 unresolved components 0 5 10 15 20 25 30 Time (min.) Even with state-ofthe-art LC, only 50% of the components in this sample can be resolved!!! AU 1 0.8 0.6 0.4 0.2 0 = 25,000 plates/column n c = 60, 9 unresolved components 0 5 10 15 20 25 30 Time (min.)
Requirements and Advantages in Two- Dimensional LC *Two conditions must be met for the technique to be considered two-dimensional 1. rthogonality of separation mechanisms This is a requirement imposed on the stationary phase chemistry 2. Separation gained in one dimension cannot be diminished by separation in the other dimension If these two conditions are satisfied, the maximum total peak capacity of the two-dimensional system is: n = n ctotal n c1 c2 Giddings, J. C. Multidimensional Chromatography: Techniques and Applications; Marcel Dekker: ew York, 1990
What Can We Expect From a Two- Dimensional Separation Based on Known ne-dimensional Data? Condition A' Condition A is the same as Condition A except that the retention has been varied randomly by 5% 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0.0 2.0 4.0 6.0 8.0 10.0 Condition A This scenario is ineffective in twodimensional LC Condition B Condition B assumes no relationship to Condition A 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0.0 2.0 4.0 6.0 8.0 10.0 Condition A This scenario has a higher probability of success in two-dimensional LC
Comparison of Variables Affecting Selectivity 30% AC vs. 50% AC 2.00 1.00 R 2 =0.989 SD=0.05 ZirChrom -CARB vs. ZirChrom -BD 2.00 1.00 Me vs. TF R 2 =0.896 SD=0.17 0.00 0.00 1.00 logk' (50/50 AC/ 2 ) 80 o C vs. 30 o C 2 R 2 =0.995 SD=0.03 1.00 0.00 R 2 =0.385 SD=0.42 0.00 0.00 1.00 logk' (TF/ 2 ) DS vs. ZirChrom -BD 1.00 R 2 =0.973 SD=0.09 1-1.00 0.00 logk' (BD-Zr 2 ) 0.00 0 0 1 2 3 logk' (C18, 30 o C) -2.00-1.00 0.00 1.00 logk' (BD-Zr 2) Stationary phase type can have a very large effect on selectivity
Why Zirconia-Based hases - Advantages for Multi-Dimensional RLC 1. Stability - Enables the use of otherwise extreme conditions for adjustment of selectivity 2. Stationary phase chemistry Allows the user to explore a wide range of chemistry to obtain the largest changes in selectivity A. Carbon-clad zirconia phases B. olymer coated phases with mixed mode charateristics I. Reversed-phase II. Ion-exchange 3. Speed Thermal stability allows for faster multi-dimensional separations
ZirChrom LC Columns art # acking Mode DB01 DiamondBond -C18 Reversed-hase EZ01 ZirChrom -EZ Reversed-hase (Lewis Acid Deactivated) MS01 ZirChrom -MS Reversed-hase (Lewis Acid Deactivated) ZR01 ZirChrom -CARB Reversed-hase ZR02 ZirChrom -ASE ormal hase and SEC ZR03 ZirChrom -BD Reversed-hase ZR04 ZirChrom -WCX Weak Cation-Exchanger ZR05 ZirChrom -WAX Weak Anion-Exchanger and Sugar Analysis ZR06 ZirChrom -SAX Strong Anion-Exchanger ZR07 ZirChrom -SAX Strong ydrophilic Anion-Exchanger ZR08 ZirChrom -EZ Cation-Exchanger for roteins ZR09 ZirChrom -S Reversed-hase
ZirChrom -CARB and DiamondBond -C18 (C 2 ) 16 C 2 (C 2 ) 16 C 2 (C 2 ) 16 C 2 C C C C C C C C C C C C C C C C C C C C C C C C C C C C Zirconia Substrate ZirChrom -CARB Zirconia Substrate DiamondBond-C18 TM
Retention of Different Solutes on DS, ZirChrom -BD and ZirChrom -CARB 1.50 ZirChrom -CARB 1.00 0.50 Log k' 0.00 DS -0.50 ZirChrom -BD -1.00-1.50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1. -benzyl formamide 6. Acetophenone 11. Benzene 16. Bromobenzene 21. ropylbenzene 2. Benzylalcohol 7. Benzonitrile 12. p-chlorotoluene 17. aphthalene 22. n-butylbenzene 3. henol 8. itrobenzene 13. p-nitrobenzyl chloride 18. Ethylbenzene 4. 3-phenyl propanol 9. methyl benzoate 14. Toluene 19. p-xylene 5. p-chlorophenol 10. Anisole 15. Benzophenone 20. p-dichlorobenzene
p-xylene Selectivity and Shape: Isomeric Analytes α DS =1.03 ethylbenzene α C-Zr =1.58 C-C-C-C-C-C-C-C-C-C-C-C C-C-C-C-C-C-C-C-C-C-C-C
Selectivity of ZirChrom -CARB and SB-C18 for 18 Substituted henols 1.50 1.00 Log k' - SB-C18 0.50 0.00-0.50-1.00-1.50-0.50 0.00 0.50 1.00 Log k' - ZirChrom-CARB LC Conditions: Mobile phase, 45/55 AC/10mM phosphoric acid, p 2.4; Flow rate, 2.0 ml/min.; Temperature, 40 o C Data courtesy of Adam Schellinger
ZirChrom -BD and ZirChrom -S C 2 C C 2 C C C 2 C 2 C C C 2 C 2 C C 2 C C C 2 C 2 C C C 2 ZirChrom -BD ~20 Zirconia Substrate ZirChrom -S Zirconia Substrate
ZirChrom -EZ EDTA - BD - C 2 2 C 2 C 2 C C 2 C 2 - - - 2-2 2 Zr Zr Zr Zr Zr Zr Zr Zr 1. Coat bare zirconia with polybutadiene (BD) 1 2. Crosslink BD chains together using dicumyl peroxide as initiator 3. Reflux BD-Zr 2 in Ethylenediamine-,,, -tetra(methylenephosphonic)acid (EDTA) solution 4. Wash to remove residual EDTA 1) Li, J. W.; Reeder, D..; McCormick, A. V.; Carr,. W. Journal of Chromatography A 1998, 791, 45-52
ZirChrom -MS zirconia - + + - EDTA Chemisorptions BD/ dicumyl peroxide Crosslinking - + + - 1 Chemisorb Ethylenediamine,,, -tetra(methylenephosphonic)acid (EDTA) to the zirconia surface. 2 Quaternize amines on the zirconia surface with allyl iodide. B D B D n Allyl iodide quaternization 3 Coat polybutadiene (BD) on the chelator-modified zirconia surface and crosslink BD with allyl group and BD itself using dicumyl peroxide as initiator. n n
Selectivity Study of Eleven Antidepressants Cl + - S S Chlordiazepoxide Desipramine ortriptyline Doxepin Imipramine S Cl S Cl Amitriptyline Buclizine ydroxyzine Thioridazine erphenazine Dai, J.; Yang, X.; Carr,. W. Journal of Chromatography, A 2003, 1005, 63-82
Selectivity for Antidepressant Compounds on DS Brand A vs. Brand B log k' - Silica Brand A 1.0 0.8 0.6 0.4 0.2 0.0-0.2-0.4-0.6-0.8-1.0 R 2 = 0.84 Std. Dev. = 0.15-1.00-0.50 0.00 0.50 1.00 1.50 log k' - Silica Brand B LC Conditions: Mobile phase, 72/28 Me/25 mm ammonium phosphate, p 6.0; Flow rate, 1.0 ml/min; Temperature, 35 C; UV detection at 254 nm.
Selectivity for Antidepressant Compounds on ZirChrom -BD vs. DS log k' - Silica Brand A 1.0 0.8 0.6 0.4 0.2 0.0-0.2-0.4-0.6-0.8-1.0 R 2 = 0.09 Std. Dev. = 0.66-1.00-0.50 0.00 0.50 1.00 1.50 log k' - ZirChrom-BD LC Conditions: Mobile phase, 72/28 Me/25 mm ammonium phosphate, p 6.0; Flow rate, 1.0 ml/min; Temperature, 35 C; UV detection at 254 nm.
Significantly igher Ion-Exchange Retention of Amines on ZirChrom -BD Leads To Selectivity Differences k ' IEX = k ' k ' R k'iex/k' of p-propylbenzylamine 100% 80% 60% 40% 20% 0% Ace Alltima BD-Zr2 15mM 25mM 35mM 50mM [ + 4 mm]
Retention of Basic Analytes on ZirChrom -BD Adsorbed Lewis base anion - - A + A + A + A + BD coating - 2 2 2 Zr Zr Zr Zr Zr Zr Zr Zr BD 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.
ZirChrom -BD is Very Different Compared to All DS hases 0.8 0.7 Standard Deviation 0.6 0.5 0.4 0.3 0.2 0.1 BD-Zr2 0.0 ACE IER EX EC RX SB ALLTIMA IER EC SB The very large s.d. for ZirChrom -BD vs. all other phases indicates a dramatic difference in selectivity from DS (Antidepressant solute set)
ZirChrom -MS Compared to DS ln k' ZirChrom-MS R 2 = 0.340 4.00 3.50 3.00 2.50 2.00 1.50 1.00-1.50-1.00-0.50 0.00 0.50 1.00 ln k' Silica C18 Basic Compounds are much more retained on ZirChrom-MS than on Silica C18 and have very different chromatographic selectivity. LC Conditions: Mobile hase, 72/28 Me/25mM Ammonium phosphate, p 6.0; Flow Rate, 1.0 ml/min.; Temperature, 35 o C; Injection Volume, 5 µl; Detection by UV at 254 nm; Solutes from left to right: Methapyrilene, yrilamine, Tripelennamine, Brompheniramine, Desipramine, ortryptyline, Doxepin, and Amitryptyline.
An Example 2DLC Separation - Ten Triazine erbicides 3 C Cl 3 C 3 C Cl Cl C Atrazine Cl Simazine Cl ropazine 3 C S 3 C 3 C S Terbuthylazine 3 C Cyanazine 2 Ametryne S Simetryne S rometryne Terbutyrne 2-nitroxylene 3 C 3 C Atraton rometon
2DLC Separation of Ten Triazine erbicides mau 100 80 60 40 20 0 0 5 10 15 20 T im e (m in.) 3 C Cl Simazine 1 st Dimension Conditions: Column, 50 mm x 2.1 mm i.d. ZirChrom -BD; Mobile phase, 20/80 AC/Water; Flow rate, 0.08 ml/min.; Injection volume, 20 µl; Temperature, 40 o C 2 nd Dimension Conditions: Column, 50 mm x 2.1 mm i.d. ZirChrom -CARB; Mobile phase, 20/80 AC/Water; Flow rate, 7.0 ml/min.; Injection volume, 15 µl; Temperature, 150 o C; 1 st dimension sampling frequency, 0.1 z
Conclusions 1. The importance of differences in selectivity between conditions selected for different dimensions in multidimensional chromatography cannot be emphasized enough. 2. The most dramatic changes in selectivity are most easily brought about by changing the stationary phase. 3. Zirconia-based reversed phases (there are 5 of them) offer dramatically different selectivity relative to conventional silicabased phases for several classes of analytes
Acknowledgements ZirChrom Separations, Inc. Bingwen Yan University of Minnesota Xiqin Yang Jun Dai Adam Schellinger