Transferring Yesterdays Methods to Tomorrows Technology: The Evolution from HPLC to UPLC TM Eric S. Grumbach Thomas E. Wheat Chuck Phoebe Joe Arsenault Jeffrey R. Mazzeo Diane M. Diehl
UPLC TECHNOLOGY A new class of separation science Based on chromatography columns with very small particles Based on instruments designed to take advantage of the small particles Provides improved Resolution, Speed, and Sensitivity without compromise Suitable for chromatographic applications in general Appropriate for developing new methods Appropriate for improving existing methods
HPLC vs. UPLC TM Speed, Sensitivity and Resolution 0.6 Absorbance at 70 nm HPLC. x 0 mm, µm Rs (,) = 4.9 0.00.00 4.00 6.00 8.00 0.00.00 4.00 6.00 8.00 0.00 Faster, More Sensitive Methods Faster, More Sensitive, Higher Resolution Methods 0.6 Absorbance at 70 nm UPLC TM 8X Speed.4X Sensitivity Same Resolution. x 0 mm,.7 µm Rs (,) = 4.8 0.6 Absorbance at 70 nm UPLC TM 4.X Speed X Sensitivity.X Resolution. x 00 mm,.7 µm Rs (,) = 6.8 0.00 0.40 0.80.0.60.00.0 0.00 0.0.00.0.00.0.00.0 4.00 4.0
What Does Ultra Performance LC Bring to the Chromatographic Laboratory? Choices available: Speed Resolution Increased speed and sensitivity with the same resolution Increased sensitivity and speed with resolution Increased resolution and sensitivity at the same speed Sensitivity
HPLC to Optimized UPLC Absorbance 4 nm An Example of a Successful Method Transfer 0 0 0 0 HPLC Absorbance 4 nm Magnification of Optimized UPLC 0 0 0 0 Optimized UPLC Absorbance 4 nm 0.6.
Methods Transfer Considerations Classes of methods transfer Replace a column brand with another Replace an instrument with another Replace both the instrument and the column The benefits of UPLC can only be realized with a new column on a new instrument As with any project to transfer a Liquid Chromatographic method, proper consideration of all the factors which control the separation process is essential for success
UPLC TM Column Selection Internal diameter and length Internal diameter. mm or.0 mm Length If primary goal is speed, choose 0 mm length If primary goal is resolution, choose 00 mm length
Ratio of Column Length to Particle Size L/dp RATIO 00mm 0 µ m = 0,000 Typical Run Times 970 s ~ 0+ min. 0mm = µm 0,000 980 s ~0- min. 00mm =,00 µm 0mm.7µm = 9,00 990 s ~ -0 min. 004 ~- min. 00mm.7µm = 8,80 x Maximum Resolution Capability If you keep the L/dp ratio the SAME for columns, you will obtain the SAME Resolution. With smaller particle sizes in shorter columns, you will achieve the same separation, but in LESS TIME!
UPLC TM Column Selection Ligand Selection Available Ligands: ACQUITY UPLC TM BEH C 8 Straight chain alkyl C 8 ACQUITY UPLC TM BEH Shield RP 8 Embedded polar group (carbamate) ACQUITY UPLC TM BEH C 8 Straight chain alkyl C 8 ACQUITY UPLC TM BEH Phenyl Why Multiple Ligands?: Changes in hydrophobicity Changes in silanol activity Changes in hydrolytic stability Changes in ligand density Changes in selectivity
Reversed-Phase Column Selectivity Chart.6 Waters Spherisorb S P Selectivity (ln [α] amitriptyline/acenaphthene)..7.4..8.. 0.9 0.6 0. 0-0. -0.6 Waters Spherisorb SCN Nova-Pak CN HP Hypersil CPS Cyano 000 Inertsil CN- Inertsil Ph- Hypersil Phenyl YMC-Pack Phenyl Nova-Pak Phenyl Hypersil BDS Phenyl YMC J'sphere ODS M80 YMCbasic Chromolith Nova-Pak YMC J'sphere ODS H80 XTerra TM RP-8 C8 Phenyl Nova-Pak Luna YMC-Pack ODS AQ YMC-Pack CN YMC-Pack Pro C4 C8 Phenyl Hexyl Atlantis dc8 YMC-Pack Pro C8 YMC-Pack ODS-A ACQUITY UPLC ACT Ace C8 Symmetry C8 Zorbax XDB C8 XTerra Luna BEH C8 MS C8 YMC-Pack Inertsil ODS- C8 () Pro C8 SunFire C8 XTerra Luna SunFire C8 MS C8 C8 Symmetry C8 SymmetryShield RP8 () XTerra RP8 Zorbax SB C8 SymmetryShield RP8 XTerra RP8 YMC-Pack PolymerC8 Retentivity (ln [k] acenaphthene) ACQUITY UPLC BEH Phenyl YMC J'sphere ODS L80 µbondapak C8 Waters Spherisorb ODS Resolve C8 Waters Spherisorb ODS Nucleosil C8 ACQUITY UPLC BEH C8 -. -0. 0.... ACQUITY UPLC Shield RP8
Method Transfer Consideration Solvent delivery Scale flow rate appropriate to column dimensions Scale linear velocity appropriate to particle size Adjust all segments of method, including equilibration Sample injection Scale injection volume Detection Ensure proper data sampling rate Ensure proper time constant
Original Gradient Profile 4.6 x 0 mm, µm HPLC Column Gradient Step Time Since Injection Flow Rate %A %B Curve Initial 0. 9 *. 9 6 0. 9 4 0. 9
Target Conditions Gradient Profile Express gradient duration in % change per column volume (cv) units Calculate each segment as a number of column volumes Calculate time required to deliver the same number of column volumes to the UPLC TM column at the chosen flow rate.
Express Gradient Segments (Steps) in Units of Column Volumes For min at. ml/min on a 4.6 x 0 mm column Gradient Volume = Flow Rate x Time =. ml/min x min =. ml Column Volume = π x r x L =.4 x. x 0 =.49 ml Gradient Duration (cv) = Gradient Volume Column Volume Gradient Duration =. ml.49 ml = 9.0 cv
Original Gradient Profile for Scaling 4.6 x 0 mm, µm HPLC Column Gradient Step Time Since Injection Flow Rate %A % B Curve Segment Duration (min) Segment Duration (Col.Vol.) Initial 0. 9 * 0 0. 9 6 9.0 0. 9.0 4 0. 9 0 6.0
Estimate Optimum UPLC Flow Rate Consider.7 µm target particle (. mm i.d. column) Assume temperature and viscosity transferred Adjust flow rate based on van Deemter curve and approximate molecular weight ~0.6 ml/min for average 00 dalton (molecular weight) molecules ~0. ml/min for larger molecules because diffusion is slower, e.g., ~,000 dalton peptides
Scaling Gradient Step Time for UPLC Flow Rate - Maintain Duration (cv) Original Step : min @. ml/min with Duration of 9.0cv Calculate Target Step : (keeping duration @ 9.0cv) Target Column Volume (. x 0) = 0.7 ml Gradient Step Volume = Duration (cv) x Target Column Volume = 9.0cv x 0.7 ml =.4 ml Gradient Step Time = Gradient Step Volume / UPLC Flow Rate =.4 ml / 0.60 ml/min. =.6 min.
Optimized Gradient Profile. x 0 mm,.7 µm UPLC TM Column Gradient Step Time Since Injection Flow Rate % A % B Curve Segment Duration (min) Segment Duration (Col.Vol.) Initial 0 0.6 9 * 0 0 Initial Hold 0 0.6 9 0 0.6 0.6 9 6.6 9.0.48 0.6 9 0.87.0 4. 0.6 9.74 6.0
Comparing Results: Does the new method meet resolution criteria? Absorbance 4 nm 4.6 x 0 mm Resolution / = Resolution / = 8 0 0 0 0 Absorbance 4 nm UPLC optimized. x 0 mm Resolution / = Resolution / = 6 0 0 0 0
Absorbance 4 nm Absorbance 4 nm Comparing Results: Does the new method meet resolution criteria? 4.6 x 0 mm, µm Resolution / = Resolution / = 8 0 0 0 0 UPLC optimized. x 0 mm Resolution / = Resolution / = 6 0.6.
Original HPLC Method: Caffeic Acid Derivatives in Echinacea Purpurea Chromatographic Conditions : Columns: XTerra MS C 8 4.6 x 0 mm,.0 µm Mobile Phase A: 0.% CF COOH in H O Mobile Phase B: 0.08% CF COOH in ACN Flow Rate:.0 ml/min Gradient: Time Profile Curve (min) %A %B 0.0 9 8 6.0 9 8 7.0 0 0 6.0 0 90 6 6.0 9 8 6 4.0 9 8 6 Injection Volume: 0.0 µl Weak Needle Wash: 0.% CF COOH in 8% ACN Sample: Caffeic Acid Derivatives in Echinacea Purpurea Sample Diluent: 0:0 H O: MeOH with 0.0% CF COOH Sample Concentration: 00 µg/ml Temperature: 40 o C Detection: UV @ 0 nm Sampling rate: 0 pts/sec Time Constant: 0. Instrument: Alliance 69 Separations Module with 996 PDA detector Analyte. Caftaric acid. Chlorogenic acid. Cynarin 4. Echinacoside. Cichoric acid AU AU 0.40 0.0 0.0 0.0 0.00 0.00 0.004 0.00 0.00 0.00 0.000 4 0.00.00 0.00.00 0.00.00 0.00.00 min. -0.00 E.G. 0.00.00 0.00.00 0.00.00 0.00.00 Enhanced Baseline
HPLC-to-UPLC TM Method Transfer: Chromatographic Conditions Original HPLC Method Chromatographic Conditions : Columns: XTerra MS C 8 4.6 x 0 mm,.0 µm Mobile Phase A: 0.% CF COOH in H O Mobile Phase B: 0.08% CF COOH in ACN Flow Rate:.0 ml/min Gradient: Time Profile Curve (min) %A %B 0.0 9 8 6.0 9 8 7.0 0 0 6.0 0 90 6 6.0 9 8 6 4.0 9 8 6 Injection Volume: 0.0 µl Sample: Caffeic Acid Derivatives in Echinacea Purpurea Sample Diluent: 0:0 H O: MeOH with 0.0% CF COOH Sample Concentration: 00 µg/ml Temperature: 40 o C Detection: UV @ 0 nm Sampling rate: 0 pts/sec Time Constant: 0. Instrument: Alliance 69 Separations Module with 996 PDA detector Final UPLC TM Method Chromatographic Conditions : Columns: ACQUITY UPLC TM BEH C 8. x 0 mm,.7 µm Mobile Phase A: 0.% CF COOH in H O Mobile Phase B: 0.08% CF COOH in ACN Flow Rate: 0. ml/min Gradient: Time Profile Curve (min) %A %B 0.0 9 8 6 0. 9 8 7 4.4 0 0 6 4.86 0 90 6.0 9 8 6 6.0 9 8 6 Injection Volume:.0 µl Weak Needle Wash: 0.% CF COOH in 8% ACN Sample: Caffeic Acid Derivatives in Echinacea Purpurea Sample Diluent: 0:0 H O: MeOH with 0.0% CF COOH Sample Concentration: 00 µg/ml Temperature: 40 o C Detection: UV @ 0 nm Sampling rate: 40 pts/sec Time Constant: 0. Instrument: Waters ACQUITY UPLC TM, with TUV detector
HPLC-to-UPLC TM Method Transfer: Caffeic Acid Derivatives in Echinacea Purpurea Original HPLC Method = min XTerra MS C 8 4.6 x 0 mm, µm AU 4 0.00.00 4.00 6.00 8.00 0.00.00 4.00 6.00 8.00 0.00.00 4.00 6.00 8.00 0.00.00 4.00.00 Final UPLC TM Method = min ACQUITY UPLC TM BEH C 8. x 0 mm,.7 µm 4 AU 0.00 0.0.00.0.00.0.00.0 4.00 4.0.00.00
HPLC-to-UPLC TM Method Transfer: Caffeic Acid Derivatives in Echinacea Purpurea 0.00 0.004 0.00 Original HPLC Method = min XTerra MS C 8 4.6 x 0 mm, µm AU 0.00 0.00 0.000-0.00 0.00 0.004 0.00 0.00.00 4.00 6.00 8.00 0.00.00 4.00 6.00 8.00 0.00.00 4.00 6.00 8.00 0.00.00 4.00.00 Final UPLC TM Method = min ACQUITY UPLC TM BEH C 8. x 0 mm,.7 µm au 0.00 0.00 0.000-0.00 0.00 0.0.00.0.00.0.00.0 4.00 4.0.00.00
Conclusions Methods can be moved directly from HPLC to ACQUITY UPLC Improved resolution Improved speed Improved detectability Many parameters can and must be transferred to preserve results Attention to detail leads to success