Extended Temperature Range Impact on HPLC Column Stability and Performance

Transcription

1 Extended Temperature Range Impact on HPLC Column Stability and Performance Stephanie J. Marin Dale Felix, Jody Clark Selerity Technologies, Inc. Salt Lake City, UT

2 The Selerity Series 9000 Total Temperature Controller Forced air oven and chiller Isothermal and thermal operation Sub-zero to 200 C Flow rates up to 10.0 ml/min Thermal up to 30 C/min Mobile phase pre-heating Peltier effluent cooling Vapor sensor Compatible with any HPLC system

3 The High Temperature HPLC Advantage Temperature Programming Speed Efficiency

4 Better Chromatography with Temperature Gradient Programming Change retention through temperature programming Replace solvent s with temperature s Water less polar and more like methanol so less organic modifier needed

5 Faster and More Efficient Separations Speed Flatter van Deemter curves allow operation at flow rates many times optimal velocity Higher efficiency - better resolution Increased diffusion rates provide lower plate heights at higher flow rates Lower viscosity and back pressure permits higher flow rates with smaller particle size packings

6 The Real Power of Temperature in HPLC is Temperature Programming but what about columns? Evaluated several different types of columns under programmed HTLC conditions Compared thermal and solvent results

7 Column Characteristics Selerity Blaze C 8 Polydentate silica, 3µm, 100 x 4.6 mm Stable to 100 C, ph 2-8 Hamilton PRP -1 PSDVB, 5µm, 100 x 4.1 mm Stable to 150 C, ph 0-14 Thermo Hypersil-Keystone Hypercarb Graphitic carbon, 7µm, 100 x 4.6 mm Stable to 200 C, ph 0-14 Zirchrom PBD, Diamondbond and CARB

8 Traditional Silica Columns Can t Take the Heat Water attacks siloxane bond or behind point of phase attachment Backside Attack H 2O: R Degradation of the Silica Support CH 3 Si + OH OH HO :OH O CH 3 Si O Si O Si H O + Si

9 The Selerity Blaze Silica Column Can Take the Heat Selerity polydentate phase protects the silanol groups Polymer attaches to backbone at several points R Si Si R Si R O Si O OH Si O O Si O O Si O

10 Blank Thermal Gradients 50:50 Acetonitrile:Water at 254 nm Thermo Hypersil-Keystone Hypercarb Selerity Blaze C8 Hamilton PRP-1 Zirchrom Diamondbond Zirchrom CARB Zirchrom PBD 40 to 200 C at 15 /min hold 5 min 40 to 100 C at 15 /min hold 5 min 40 to 150 C at 15 /min hold 5 min 40 to 200 C at 15 /min hold 5 min 40 to 200 C at 15 /min hold 5 min 40 to 150 C at 15 /min hold 5 min mv Minutes

11 Column Evaluation Conditions Seven component mix of acidic, basic and neutral compounds: acetophenone amitriptylene aniline Ibuprofen salicylic acid styrene glycol 2-phenyl-2-propanol

12 Column Evaluation Conditions Three mobile phases: acetonitrile:water acetonitrile:water with 0.1% TFA ph 2 acetonitrile:ammonium hydroxide ph 10 Analyzed using a solvent and a thermal to give similar retention times. 50:50 acetonitrile:aqueous 50% to 100% ACN over 10 minutes, hold five minutes 50 C to 100 C, 150 C or 200 C at 15 /min, hold five minutes

13 Column Evaluation Conditions retention times collected individually Eight solvent s and eight temperature s per column column was evaluated initially and between each mobile phase change with two component test mix uracil and phenol 40 C 50:50 ACN:Water retention time, peak area and efficiency were recorded

14 150 RT vs C and RT vs %ACN for PRP -1 ACN:Water C or %ACN salicylic acid styrene glycol 2-phenyl-2-propanol (1) aniline 2-phenyl-2-propanol (2) acetophenone amitriptylene RT > 25 min Thermal Solvent Retention Time (min)

15 C or % ACN RT vs C and RT vs %ACN for PRP -1 ACN:Water with 0.1% TFA aniline styrene glycol amitriptylene salicylic acid 2-phenyl-2-propanol (1) 2-phenyl-2-propanol (2) acetophenone Ibuprofen Thermal Solvent Retention Time (min)

16 C or %ACN RT vs C and RT vs %ACN for PRP -1 ACN:Water ph 10 salicylic acid/ibuprofen styrene glycol 2-phenyl-2-propanol (1) aniline 2-phenyl-2-propanol (2) acetophenone amitriptylene Thermal 75 Solvent Retention Time (min)

17 C or %ACN RT vs C and RT vs %ACN for Hypercarb Column ACN:Water with 0.1%TFA aniline styrene glycol 2-phenyl-2-propanol amitriptylene acetophenone salicylic acid Ibuprofen Thermal Solvent Retention Time (min)

18 150 RT vs C and RT vs %ACN for Hypercarb Column ACN:water ph 10 C or %ACN salicylic acid styrene glycol Ibuprofen aniline 2-phenyl-2- propanol acetophenone Thermal Solvent Retention Time (min)

19 C or %ACN RT vs C and RT vs %ACN for Blaze C 8 ACN:Water styrene glycol salicylic acid aniline 2-phenyl-2-propanol (1) 2-phenyl-2-propanol (2) acetophenone Ibuprofen Thermal Solvent Retention Time (min)

20 C or % ACN RT vs C and RT vs %ACN for Blaze C 8 ACN:TFA aniline styrene glycol salicylic acid 2-phenyl-2-propanol (1) 2-phenyl-2-propanol (2) amitriptylene acetophenone Ibuprofen Thermal Solvent Retention Time (min)

21 Peak Capacity Comparison for Thermal and Solvent Gradients Blaze TM C 8 PRP-1 HyperCarb Mobile Phase Thermal Solvent Thermal Solvent Thermal Solvent Acetonitrile:Water Acetonitrile:Water with 0.1% TFA Acetonitrile: 20 mm Ammonium Hydroxide ph Comparable peak capacities were obtained for the solvent and thermal runs.

22 Efficiency Comparison of Solvent and Temperature Gradient for PRP -1 Column 2,500 styrene glycol H2O TFA ph10 2-phenyl-2-propanol H2O TFA ph10 H2O TFA ph10 acetophenone H2O TFA ph10 2,000 Theoretical Plates 1,500 1, thermal solvent

23 Efficiency Comparison of Solvent and Temperature Gradient for Hypercarb Column 8,000 7,000 styrene glycol H2O TFA ph10 2-phenyl-2-propanol H2O TFA ph10 acetophenone H2O TFA ph10 Theoretical Plates 6,000 5,000 4,000 3,000 2,000 1,000 0 thermal solvent

24 Efficiency Comparison of Solvent and Temperature Gradient for Blaze Column 14,000 12,000 styrene glycol H2O TFA 2-phenyl-2-propanol H2O TFA H2O TFA acetophenone H2O TFA Theoretical Plates 10,000 8,000 6,000 4,000 2,000 0 thermal solvent

25 Comparison of Retention and Efficiency of PRP -1 During High Temperature Evaluation Analyte RT plates area uracil initial ,234 2,916,552 uracil after initial runs before TFA ,300 3,061,639 uracil after TFA before ammonia ,357 3,118,760 uracil after ammonia ,603 2,931,591 phenol initial , ,020 phenol after initial runs before TFA , ,543 phenol after TFA before ammonia , ,936 phenol after ammonia , ,660

26 Comparison of Retention and Efficiency of Hypercarb During High Temperature Evaluation Analyte RT plates area uracil initial ,827 3,152,477 uracil after initial runs before NH ,227 2,698,373 uracil after NH3 before TFA ,658 2,991,397 uracil after TFA ,298 2,909,248 phenol initial , ,970 phenol after initial runs before NH , ,160 phenol after NH3 before TFA , ,921 phenol after TFA , ,484

27 Comparison of Retention and Efficiency of Blaze C 8 During High Temperature Evaluation Analyte RT plates area uracil initial ,898 2,637,763 uracil after initial runs ,826 3,112,145 uracil after TFA ,006 3,071,317 phenol initial , ,090 phenol after initial runs , ,308 phenol after TFA , ,861

28 Conclusions Column bleed can be an issue with zirconia columns during temperature programming Several columns do give good performance under high temperature conditions without evidence of stationary phase degradation Similar retention characteristics are observed with solvent s and temperature programming Comparable or better peak quality is observed with temperature programming when compared to solvent s for the columns evaluated

29 Acknowledgements Thermo Hypersil-Keystone Steve Kozel and Rick Ludwig Hamilton Company Dan Lee and Mike Benning

30 Turn up the Heat! Selerity Technologies Inc W. Custer Rd. Salt Lake City, UT Booth #208