Studies on Stationary Phase Selectivity for Solid-Core Particles

Transcription

1 Studies on Stationary Phase Selectivity for Solid-Core Particles Richard A. enry, Wayne K. Way, Carmen T. Santasania, and David S. Bell Supelco, Div. of Sigma-Aldrich, Bellefonte, PA 6823 USA T4060 sigma-aldrich.com

2 Three Factors Control PLC and UPLC Resolution* R s = k k+ 4 α- α α = 6 (t R /w) 2 or = 5.5 (t R /w) 2 k = (t R t 0 )/t 0 Resolution (R) k α = k 2 /k All factors are important, but selectivity is considered the most powerful term K 0K 5K 20K 25K * Yun Mao, PhD Dissertation, University of Minnesota, 200. α k 2

3 LC Particle Innovation Chronology Particle Size and Architecture (porosity) More speed, efficiency 5 µm 3 µm <2 µm Solid-Core monoliths Particle Composition (and stationary phases) expanded p range and selectivity silica polymers ybrids ther oxides 3

4 Fused-Core Particle Particle properties: 90 Å and 60 Å 2.7 µm pure silica particle.7 µm solid core 0.5 µm 0.5 µm porous shell 90 Å pores (50 m 2 /g) and 60 Å pores (80 m 2 /g) 2.7 µm.7 µm Solid Core Density.3 g/cc* Extremely uniform particle size distribution Retains 75% of the surface area and sample capacity of porous particles * Typical porous silica made by a sol-gel process is.0 g/cc. 4

5 Ascentis Express Fused-Core - igher Efficiency 20-30,000 plates can be generated and maintained at moderate pressure. 4 Agilent Ascentis Express C8, 5 cm x 4.6 mm, 2.7 µm.0 ml/min, 254 nm, RT, 0 µl inj. 3. Uracil (void marker) 2. Acetophenone = 33, Benzene = 3, Toluene = 30,738 Pressure = 83 bar (2690 psi) min 5

6 Retention and Selectivity are Stationary Phase (Column) and Mobile Phase Related Current Ascentis Express Fused-Core Surface ptions C8 ES-C8 (60Å) C8 RP-Amide (RPA) Phenyl-exyl F5 (RP or ILIC) ILIC (Si) Stationary and mobile phase conditions define the operating mode which is usually reversed-phase. 6

7 LogP Values* as Guides to Initial Column/Mode Selection ydrophilic interaction dominates ydrophobic interaction dominates igh organic igh aqueous For P value of 00 (00: distribution in favor of n-octanol), logp = 2. For P value of (equal distribution between n-octanol and water), logp = 0. For P value less than (distribution favors water), logp is negative. * LogP (octanol:water partition coefficients) scale adapted from LCGC CRMacademy webinar, Understanding ILIC, presented by est Group, Inc. and Crawford Scientific (20). 7

8 Samples Studied with Different LogP Ranges Rules for C8 columns in Reversed Phase mode: LogP values of 2 or more will often be retained in 50% organic or greater. LogP values in the range of 0-2 usually require less than 50% organic to be retained. A wide range of logp values (>) indicates that gradient experiments may be needed. egative logp values (P favors water) may require a mode change to ILIC, Ion-Exchange or Ion-Pair. Compound Type logp Value Range* onpolar Steroids 2.86 to 4.5 Polar Steroids.57 to.76 Furocoumarins.52 to 2.04 Anti-ulcer drugs (gradient) -.65 to 2.36 * LogP estimated (within ca. 0%) using ACD Labs software. 8

9 onpolar Steroids with Estimated logp Values logp = 4. C 3 C C 3 C logp = 2.86 Ethinylestradiol Monoisotopic Mass = Da C 3 orethindrone Monoisotopic Mass = Da C 3 logp = 3.62 logp = -.04 Estrone [*{I}; *{USA}] Monoisotopic Mass = Da Estradiol [*{I}; *{USA}] Monoisotopic Mass = Da logp = 4.5 Two polar groups in each planar steroid structure. Uracil [*{USA}] 9

10 Ascentis Express C8: Acetonitrile (50%) onpolar steroids BP= 2300 psi. Uracil 2. Estradiol 3. orethindrone 4. Ethinylestradiol 5. Estrone 2 3,4 5 Instrument: Waters 2690 with MS detection column: Ascentis Express, 5 cm x 4.6 mm I.D., 2.7 µm particles mobile phase: 60:40 methanol: water or 50:50 acetonitrile:water flow rate:.0 ml/min. temp.: 35 C det.: UV at 220 nm injection: 0 µl sample: 0 µg/ml in mobile phase Retention and selectivity may require use of polar stationary phases Time (min) 0

11 Ascentis Express C8: Methanol (60%) Retention increases in Me but usually poor choice to improve selectivity with C8 2 3,4 onpolar steroids 5 BP= 4400 psi. Uracil 2. Estradiol 3. orethindrone 4. Ethinylestradiol 5. Estrone Time (min)

12 Ascentis Express RPA: Acetonitrile (50%) ote stronger retention and elution order change vs C onpolar steroids BP= 2300 psi. Uracil 2. Estradiol 3. orethindrone 4. Ethinylestradiol 5. Estrone Time (min) Amide phase attracts stronger -bonding donors Estradiol and Ethinylestradiol 2

13 Ascentis Express RPA: Methanol (60%) Retention increases even more in Me and elution order changes again to group the diol structures onpolar steroids BP= 4200 psi. Uracil 2. Estradiol 3. orethindrone 4. Ethinylestradiol 5. Estrone Time (min) Ternary mobile phases could be useful 3

14 Ascentis Express Phenyl exyl: Acetonitrile (50%) 3 onpolar steroids BP= 2480 psi. Uracil 2. Estradiol 3. orethindrone 4. Ethinylestradiol 5. Estrone Time (min) Retention favors ketone structures similar to C8 with slightly better selectivity. 4

15 Ascentis Express Phenyl exyl: Methanol (60%) onpolar steroids BP= 4250 psi. Uracil 2. Estradiol 3. orethindrone 4. Ethinylestradiol 5. Estrone Time (min) More retention in Me and better separation between diol and ketone pairs 5

16 Ascentis Express F5: Acetonitrile (50%) 3,4 Lightest retention and selectivity like C8 onpolar steroids 2 5 BP= 2400 psi. Uracil 2. Estradiol 3. orethindrone 4. Ethinylestradiol 5. Estrone Time (min) Virtually overlays the C8 separation suggesting no interaction between polar groups 6

17 Ascentis Express F5: Methanol (60%) onpolar steroids BP= 4200 psi. Uracil 2. Estradiol 3. orethindrone 4. Ethinylestradiol 5. Estrone Time (min) Also closely resembles results with C8 in Me and shows only weak selectivity 7

18 Polar Steroids with Estimated logp Values logp = logp=.76 logp =.57 logp=.57 C 3 C 3 ydrocortisone [*{BA}; *{I}; *{JA}] logp =.63 logp=.63 C 3 Prednisolone [*{BA}; *{I}; *{JA}] C 3 C 3 C 3 Prednisone [*{BA}; *{I}] Five polar groups in each planar steroid structure. PLC Conditions: Mobile phase: as indicated Flow Rate: 0.4 ml/min Column Dimensions: 0 cm x 3.0 mm Detection: UV at 240 nm Injection volume: 5 µl Temperature: ambient Sample: 00 µg/ml each in 30% methanol 8

19 Ascentis Express C8: Methanol vs. AC Stronger retention for polar steroids on C8 in Me. Prednisone (logp.57) 2. Prednisolone (logp.63) 3. ydrocortisone (logp.76) mix/ac 45%/C8 A : Diode Array 240 Range: 8.508e-2 AU.6e-.4e-.2e-.0e- 8.0e-2 6.0e-2 4.0e % Acetonitrile (peak changes with %AC) e A : Diode Array e- Range:.689e-.4e-.2e-.0e- 50% Methanol ,3 AU 8.0e-2 6.0e-2 4.0e-2 2.0e Time

20 Ascentis Express F5: Methanol vs. AC Stronger retention and better selectivity for polar steroids on F5 in Me. Prednisone (logp.57) 2. Prednisolone (logp.63) 3. ydrocortisone (logp.76) 50% Methanol 3 2 mix of three A : Diode Array Range: 8.424e-2 7.0e e-2 5.0e AU 4.0e-2 3.0e-2 2.0e-2.0e A : Diode Array Range: 9.89e-2 8.0e-2 6.0e % Acetonitrile AU 4.0e-2 2.0e Time

21 Furocoumarins with Estimated logp Values Angelicin logp =.97 Psoralen logp = 2.04 Xanthotoxin logp =.52 C 3 C 3 Isobergaptan logp =.97 Bergaptan logp = 2.04 C 3 2

22 Furocoumarins on Ascentis Express C8 Elution order:. Angelicin 2. Psoralen 3. Xanthotoxin 4. Isobergaptan 5. Bergaptan 2,3 column: Ascentis Express, 0 cm x 4.6 mm I.D., 2.7 µm particles mobile phase: 40:60 water:methanol flow rate: 0.8 ml/min. pressure: 70 bar temp.: 35 C det.: UV at 254 nm injection: 2 µl sample: 25 µg/ml in 90:0 water:methanol Peaks 2 and 3 do not resolve on C8 in methanol and peaks cluster into two groups Time (min) 22

23 Furocoumarins on Ascentis Express RP-Amide Elution order:. Angelicin 2. Psoralen 3. Xanthotoxin 4. Isobergaptan 5. Bergaptan 3 column: Ascentis Express, 0 cm x 4.6 mm I.D., 2.7 µm particles mobile phase: 40:60 water:methanol flow rate:.5 ml/min, pressure: 285 bar temp.: 40 C det.: UV at 254 nm injection: 2 µl sample: 25 µg/ml in 90:0 water:methanol All peaks resolve and shift in elution order with more even spacing on RP-Amide in methanol.0 Time (min) 23

24 Anti-ulcer Drugs 2 2 S S 2 S 2 Famotidine logp = -.65 Pirenzepine logp = 0.2 S - + Ranitidine logp = S Cimetidine logp = 0.56 S meprazole logp = 2.36 S S + - izatidine logp = S F F F Lansoprazole logp =

25 Anti-Ulcer Drugs on Ascentis C8 Current method: two-slope 5-55% AC gradient column: Ascentis C8, 5 cm x 2. mm I.D., 5 µm particles (58304-U) mobile phase A: 0 mm ammonium phosphate (p 6.2 with phosphoric acid) mobile phase B: acetonitrile flow rate: 0.2 ml/min. temp.: 35 C det.: UV at 220 nm injection: 5 µl sample: as indicated in 95:5 water:methanol gradient: Min %A %B Famotidine (40 µg/ml) 2. Ranitidine (40 µg/ml) 3. Cimetidine (40 µg/ml) 4. izatidine (40 µg/ml) 5. Pirenzepine (40 µg/ml) 6. meprazole (40 µg/ml) 7. Lansoprazole (40 µg/ml) 4 logp -.65 logp Min 25

26 Anti-Ulcer Drugs on Ascentis RP-Amide A reversal of peaks and 2 was noted on Ascentis RP-Amide column: Ascentis RP-Amide, 5 cm x 2. mm I.D., 5 µm particles ( U) mobile phase A: 0 mm ammonium phosphate (p 6.2 with phosphoric acid) mobile phase B: acetonitrile flow rate: 0.2 ml/min. temp.: 35 C det.: UV at 220 nm injection: 5 µl sample: as indicated in 95:5 water:methanol gradient: Min %A %B Famotidine (40 µg/ml) 2. Ranitidine (40 µg/ml) 3. Cimetidine (40 µg/ml) 4. izatidine (40 µg/ml) 5. Pirenzepine (40 µg/ml) 6. meprazole (40 µg/ml) 7. Lansoprazole (40 µg/ml) Min 26

27 Ascentis Express C8 0-50% AC screening gradient Anti-Ulcer Drugs:. Famotidine 2. Ranitidine 3. Cimetidine 4. izatidine 5. Pirenzepine 6. meprazole 7. Lansoprazole column: Ascentis Express, 0 cm x 4.6 mm I.D., 2.7 µm particles mobile phase A: 25 mm ammonium phosphate (p 6.2 with phosphoric acid) mobile phase B: acetonitrile flow rate:.5 ml/min (initial pressure bar) temp.: 35 C det.: UV at 220 nm injection: 5 µl sample: 25 µg/ml in 90:0 water:methanol gradient: Min %A %B Faster separation in same elution order as Ascentis C Time (min) 27

28 Ascentis Express RP-Amide 0-50% AC screening gradient 3 Faster separation in same elution order as Ascentis RP-Amide 6 7 Anti-Ulcer Drugs:. Famotidine 2. Ranitidine 3. Cimetidine 4. izatidine 5. Pirenzepine 6. meprazole 7. Lansoprazole Time (min) 28

29 Ascentis Express Phenyl exyl 0-50% AC screening gradient 2,3 Similar elution order to C Anti-Ulcer Drugs: Elution order. Famotidine 2. Ranitidine 3. Cimetidine 4. izatidine 5. Pirenzepine 6. meprazole 7. Lansoprazole Time (min) 29

30 Ascentis Express F5 0-50% AC screening gradient Anti-Ulcer Drugs: 3 More retention and change in elution order for polar drugs (i.e. ranitidine) 6 7 Elution order. Famotidine 2. Ranitidine 3. Cimetidine 4. izatidine 5. Pirenzepine 6. meprazole 7. Lansoprazole Time (min) 30

31 Ascentis Express (Peptide) ES-C8* 0-50% AC screening gradient Anti-Ulcer Drugs: 2 3 Comparable retention and selectivity to C8 6 7 Elution order. Famotidine 2. Ranitidine 3. Cimetidine 4. izatidine 5. Pirenzepine 6. meprazole 7. Lansoprazole Time (min) * 60 Å, no end-capping; optimized for separating peptides and small proteins using TFA gradients. 3

32 Conclusions Ascentis Express columns are excellent choices for developing selective, high speed separations. igher efficiency observed at lower pressure vs porous packings; comparable efficiency to sub-2 µm particles. Comparable retention to popular porous packings including Discovery, Ascentis and other columns. Range of very selective surface modifications available; phase selectivity correlates closely with Discovery, Ascentis, and other porous silica columns. LogP values can be very useful early in the column selection and mode selection process for any column. Correlation is not perfect because only part of the PLC retention process is due to liquid-liquid partition. 32

33 Selectivity References. D. Benhaim and E. Grushka, Amide Phase for LogP Values, JCA, 27 (200) M. R. Euerby, et. al., Classification of Phenyl Columns, JCA, 54 (2007), L.R. Snyder, J.W. Dolan and P.W. Carr, ydrophobic Subtraction Model for Classification of Reversed-Phase Columns, JCA, 060 (2004), J. W. Dolan and L. R. Snyder, Selecting an rthogonal Column, JCA,26 (2009), M. R. Schure, et. al., Molecular Level Comparison of Alkyl and Polar- Embedded Systems, Anal. Chem. 2008, 80, M. Yang, et. al., Impact of Methanol and Acetonitrile on Phenyl Selectivity, JCA, 097 (2005), D.. Marchand, et. al., Phenyl Column Selectivity, JCA, 062 (2005) Y. Kazakevitch, et. al., Surface Studies of Phenyl Modified Adsorbents, JCA, 082 (2005), Mao, Y., Selectivity ptimization in Liquid Chromatography Using the Thermally Tuned Tandem Column (T3C) Concept, Ph.D. Thesis (P. Carr), The University of Minnesota,