Transferring Yesterdays Methods to Tomorrows Technology: The

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<ul><li><p>Transferring Yesterdays Methodsto Tomorrows Technology: </p><p>The Evolution from HPLC to UPLCTM</p><p>Eric S. Grumbach</p><p>Thomas E. Wheat</p><p>Chuck Phoebe</p><p>Joe Arsenault</p><p>Jeffrey R. Mazzeo</p><p>Diane M. Diehl</p></li><li><p>2005 Waters Corporation</p><p>UPLC TECHNOLOGY</p><p> A new class of separation science Based on chromatography columns with very small particles Based on instruments designed to take advantage of the small </p><p>particles</p><p> Provides improved Resolution, Speed, and Sensitivity without compromise</p><p> Suitable for chromatographic applications in general Appropriate for developing new methods Appropriate for improving existing methods</p></li><li><p>2005 Waters Corporation</p><p>Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00</p><p>HPLC vs. UPLCTMSpeed, Sensitivity and Resolution</p><p>2.1 x 150 mm, 5 mRs (2,3) = 4.29</p><p>12 3</p><p>HPLC</p><p>20.00</p><p>0.26</p><p>Abs</p><p>orba</p><p>nce </p><p>at 2</p><p>70 n</p><p>m</p><p>0.00</p><p>Minutes0.40 0.80 1.20 1.60 2.00 2.50</p><p>2.1 x 50 mm, 1.7 mRs (2,3) = 4.281</p><p>23</p><p>8X Speed3.4X SensitivitySame Resolution</p><p>0.26</p><p>Abs</p><p>orba</p><p>nce </p><p>at 2</p><p>70 n</p><p>m</p><p>0.00</p><p>UPLCTM</p><p>Faster, More Sensitive Methods</p><p>Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00</p><p>3</p><p>2.1 x 100 mm, 1.7 mRs (2,3) = 6.38</p><p>1</p><p>2</p><p>4.5X Speed2X Sensitivity1.5X Resolution</p><p>4.50</p><p>0.26</p><p>Abs</p><p>orba</p><p>nce </p><p>at 2</p><p>70 n</p><p>m</p><p>0.00</p><p>UPLCTM</p><p>Faster, More Sensitive, Higher Resolution Methods</p></li><li><p>2005 Waters Corporation</p><p>What Does Ultra Performance LC Bring to the Chromatographic Laboratory? </p><p>Choices available: Increased speed and sensitivity </p><p>with the same resolution</p><p> Increased sensitivity and speed with resolution</p><p> Increased resolution and sensitivity at the same speed</p><p>Speed Resolution</p><p>Sensitivity</p></li><li><p>2005 Waters Corporation</p><p>HPLC to Optimized UPLC</p><p>Abso</p><p>rban</p><p>ce 2</p><p>54 n</p><p>m</p><p>Minutes0 10 20 30</p><p>1</p><p>23</p><p>Abso</p><p>rban</p><p>ce 2</p><p>54 n</p><p>m</p><p>Minutes0 2.6 5.2</p><p>1</p><p>23</p><p>HPLC</p><p>Optimized UPLC</p><p>Magnification ofOptimized UPLC</p><p>Abso</p><p>rban</p><p>ce 2</p><p>54 n</p><p>m</p><p>Minutes0 10 20 30</p><p>1</p><p>23</p><p>An Example of aSuccessful Method Transfer</p></li><li><p>2005 Waters Corporation</p><p>Methods Transfer Considerations</p><p> Classes of methods transfer Replace a column brand with another Replace an instrument with another Replace both the instrument and the column</p><p> The benefits of UPLC can only be realized with a new column on a new instrument</p><p> 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</p></li><li><p>2005 Waters Corporation</p><p>UPLCTM Column SelectionInternal diameter and length</p><p> Internal diameter2.1 mm or 1.0 mm</p><p> Length If primary goal is speed, choose 50 mm length If primary goal is resolution, choose 100 mm length </p></li><li><p>2005 Waters Corporation</p><p>Ratio of Column Length to Particle Size</p><p>If you keep the L/dp ratio the SAME for 2 columns, you will obtain the SAME Resolution. With smaller particle sizes in shorter columns, you will achieve the same separation, but in LESS TIME!</p><p>100mm1.7m</p><p>300mm10 m = 30,000</p><p>30,000150mm =5m100mm =3m 33,300</p><p>50mm1.7m</p><p>= 29,500</p><p>= 58,820</p><p>1970s ~ 30+ min.</p><p>1990s ~ 5-10 min.</p><p>2004 ~1- 2 min.</p><p>1980s ~10-15 min.</p><p>L/dp RATIO Typical Run Times</p><p>2x Maximum Resolution Capability</p></li><li><p>2005 Waters Corporation</p><p>UPLCTM Column SelectionLigand Selection</p><p>Available Ligands: ACQUITY UPLCTM BEH C18</p><p> Straight chain alkyl C18 ACQUITY UPLCTM BEH Shield RP18</p><p> Embedded polar group (carbamate)</p><p> ACQUITY UPLCTM BEH C8 Straight chain alkyl C8</p><p> ACQUITY UPLCTM BEH Phenyl</p><p>Why Multiple Ligands?: Changes in hydrophobicity</p><p> Changes in silanol activity</p><p> Changes in hydrolytic stability</p><p> Changes in ligand density </p><p> Changes in selectivity</p></li><li><p>2005 Waters Corporation</p><p>Reversed-Phase Column Selectivity Chart</p><p>Retentivity(ln [k] acenaphthene)</p><p>012005</p><p>SunFire C18</p><p>YMC-Pack PolymerC18</p><p>Hypersil CPS Cyano</p><p>YMC-Pack CN</p><p>Waters Spherisorb S5 P</p><p>Hypersil BDS PhenylNova-Pak Phenyl</p><p>YMC-Pack Phenyl</p><p>Hypersil PhenylInertsil Ph-3</p><p>YMC-Pack Pro C4</p><p>YMCbasic</p><p>Symmetry C8YMC-Pack Pro C8</p><p>Nova-Pak C8</p><p>XTerra MS C18 Symmetry C18</p><p>YMC-Pack Pro C18</p><p>Inertsil ODS-3</p><p>YMC-Pack ODS-A</p><p>Nova-PakC18</p><p>YMC J'sphereODSL80 Nucleosil C18</p><p>Waters Spherisorb ODS2</p><p>Waters Spherisorb ODS1Resolve C18</p><p>Bondapak C18</p><p>YMC-Pack ODSAQ</p><p>YMC J'sphere ODSH80YMC J'sphere ODSM80</p><p>Inertsil CN-3</p><p>Waters Spherisorb S5CN</p><p>Nova-Pak CN HP</p><p>SymmetryShield RP8</p><p>SymmetryShield RP18</p><p>XTerra RP8</p><p>XTerra RP18</p><p>-0.6</p><p>-0.3</p><p>0</p><p>0.3</p><p>0.6</p><p>0.9</p><p>1.2</p><p>1.5</p><p>1.8</p><p>2.1</p><p>2.4</p><p>2.7</p><p>3</p><p>3.3</p><p>3.6</p><p>-1.5 -0.5 0.5 1.5 2.5 3.5</p><p>Sele</p><p>ctiv</p><p>ity(ln</p><p>[] a</p><p>mitr</p><p>ipty</p><p>line/</p><p>acen</p><p>apht</p><p>hene</p><p>)</p><p>XTerra MS C8</p><p>Luna C18 (2)</p><p>ACQUITY UPLC BEH C18</p><p>XTerra Phenyl Luna </p><p>Phenyl Hexyl</p><p>ChromolithTMRP-18</p><p>Atlantis dC18</p><p>Zorbax XDB C18ACT Ace C18</p><p>Zorbax SB C18</p><p>SunFire C8</p><p>LunaC8 (2)</p><p>ACQUITY UPLC Shield RP18</p><p>ACQUITY UPLC BEH C8</p><p>ACQUITY UPLC BEH Phenyl</p></li><li><p>2005 Waters Corporation</p><p>Method Transfer Consideration</p><p> Solvent delivery Scale flow rate appropriate to column dimensions Scale linear velocity appropriate to particle size Adjust all segments of method, including equilibration</p><p> Sample injection Scale injection volume</p><p> Detection Ensure proper data sampling rate Ensure proper time constant</p></li><li><p>2005 Waters Corporation</p><p>Original Gradient Profile</p><p>1</p><p>1</p><p>6</p><p>*</p><p>Curve</p><p>9551.5152</p><p>5951.5304</p><p>9551.5203</p><p>5951.50Initial</p><p>%B%AFlow RateTime Since </p><p>InjectionGradient </p><p>Step</p><p>4.6 x 150 mm, 5 m HPLC Column</p></li><li><p>2005 Waters Corporation</p><p>Target Conditions Gradient Profile</p><p> Express gradient duration in % change per column volume (cv) units</p><p> Calculate each segment as a number of column volumes</p><p> Calculate time required to deliver the same number of column volumes to the UPLCTM column at the chosen flow rate.</p></li><li><p>2005 Waters Corporation</p><p>Express Gradient Segments (Steps) in Units of Column Volumes </p><p>For 15 min at 1.5 mL/min on a 4.6 x 150 mm column</p><p>Gradient Volume = Flow Rate x Time = 1.5 mL/min x 15 min = 22.5 mL</p><p>Column Volume = x r2 x L = 3.14 x 2.32 x 150 = 2.49 mL</p><p>Gradient Duration (cv) = Gradient VolumeColumn Volume</p><p>Gradient Duration = 22.5 mL2.49 mL</p><p>= 9.03 cv</p></li><li><p>2005 Waters Corporation</p><p>Original Gradient Profile for Scaling</p><p>1</p><p>1</p><p>6</p><p>*</p><p>Curve</p><p>10</p><p>5</p><p>15</p><p>0</p><p>Segment Duration </p><p>(min)</p><p>9.039551.5152</p><p>6.025951.5304</p><p>3.019551.5203</p><p>05951.50Initial</p><p>Segment Duration (Col.Vol.)</p><p>%B%A</p><p>Flow Rate</p><p>Time Since Injection</p><p>GradientStep</p><p>4.6 x 150 mm, 5 m HPLC Column</p></li><li><p>2005 Waters Corporation</p><p>Estimate Optimum UPLC Flow Rate </p><p>Consider 1.7 m target particle (2.1 mm i.d. column)Assume temperature and viscosity transferredAdjust flow rate based on van Deemter curve and </p><p>approximate molecular weight</p><p>~0.6 mL/min for average 500 dalton (molecular weight) molecules</p><p>~0.1 mL/min for larger molecules because diffusion is slower, e.g., ~2,000 dalton peptides</p></li><li><p>2005 Waters Corporation</p><p>Scaling Gradient Step Time for UPLC Flow Rate - Maintain Duration (cv)</p><p>Original Step 2: 15 min @ 1.5 mL/min with Duration of 9.03cv</p><p>Calculate Target Step 2: (keeping duration @ 9.03cv) </p><p>Target Column Volume (2.1 x 50) = 0.17 mL</p><p>Gradient Step Volume = Duration (cv) x Target Column Volume</p><p>= 9.03cv x 0.17 mL = 1.54 mL</p><p>Gradient Step Time = Gradient Step Volume / UPLC Flow Rate </p><p>= 1.54 mL / 0.60 mL/min. = </p><p>2.6 min.</p></li><li><p>2005 Waters Corporation</p><p>Optimized Gradient Profile</p><p>0015950.60InitialHold</p><p>1</p><p>1</p><p>6</p><p>*</p><p>Curve</p><p>1.74</p><p>0.87</p><p>2.61</p><p>0</p><p>Segment Duration </p><p>(min)</p><p>9.039550.62.612</p><p>6.025950.65.224</p><p>3.019550.63.483</p><p>05950.60Initial</p><p>Segment Duration (Col.Vol.)</p><p>%B</p><p>%A</p><p>Flow Rate</p><p>Time Since </p><p>Injection</p><p>Gradient</p><p>Step</p><p>2.1 x 50 mm, 1.7 m UPLCTM Column</p></li><li><p>2005 Waters Corporation</p><p>Comparing Results:Does the new method meet resolution criteria?</p><p>4.6 x 150 mmResolution 1/2 = 12Resolution 2/3 = 28</p><p>Abs</p><p>orba</p><p>nce </p><p>254 </p><p>nm</p><p>Minutes</p><p>12</p><p>3</p><p>0 10 20 30</p><p>Abs</p><p>orba</p><p>nce </p><p>254 </p><p>nm</p><p>Minutes0 10 20 30</p><p>1</p><p>23 UPLC optimized 2.1 x 50 mm</p><p>Resolution 1/2 = 11Resolution 2/3 = 26</p></li><li><p>2005 Waters Corporation</p><p>Comparing Results:Does the new method meet resolution criteria?</p><p>4.6 x 150 mm, 5mResolution 1/2 = 12Resolution 2/3 = 28</p><p>Abs</p><p>orba</p><p>nce </p><p>254 </p><p>nm</p><p>Minutes</p><p>12</p><p>3</p><p>0 10 20 30UPLC optimized 2.1 x 50 mm</p><p>Resolution 1/2 = 11Resolution 2/3 = 26</p><p>Abs</p><p>orba</p><p>nce </p><p>254 </p><p>nm</p><p>Minutes0 2.6 5.2</p><p>1</p><p>23</p></li><li><p>2005 Waters Corporation</p><p>Original HPLC Method:Caffeic Acid Derivatives in Echinacea Purpurea</p><p>Chromatographic Conditions :Columns: XTerra MS C18 4.6 x 150 mm, 5.0 mMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 1.0 mL/min Gradient: Time Profile Curve</p><p>(min) %A %B0.0 92 8 62.0 92 8 732.0 50 50 635.0 10 90 636.0 92 8 641.0 92 8 6</p><p>Injection Volume: 10.0 LWeak Needle Wash: 0.1% CF3COOH in 8% ACNSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 g/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 10 pts/secTime Constant: 0.1Instrument: Alliance 2695 Separations Module with </p><p>2996 PDA detector</p><p>Analyte1. Caftaric acid2. Chlorogenic acid3. Cynarin4. Echinacoside5. Cichoric acid</p><p>AU</p><p>0.00</p><p>0.10</p><p>0.20</p><p>0.30</p><p>0.40</p><p>Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00</p><p>1 24</p><p>5</p><p>3</p><p>AU</p><p>-0.001</p><p>0.000</p><p>0.001</p><p>0.002</p><p>0.003</p><p>0.004</p><p>0.005</p><p>Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00</p><p>Enhanced BaselineE.G.</p><p>35 min.</p></li><li><p>2005 Waters Corporation</p><p>HPLC-to-UPLCTM Method Transfer:Chromatographic Conditions</p><p>Chromatographic Conditions :Columns: XTerra MS C18 4.6 x 150 mm, 5.0 mMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 1.0 mL/min Gradient: Time Profile Curve</p><p>(min) %A %B0.0 92 8 62.0 92 8 7</p><p>32.0 50 50 635.0 10 90 636.0 92 8 641.0 92 8 6</p><p>Injection Volume: 10.0 LSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 g/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 10 pts/secTime Constant: 0.1Instrument: Alliance 2695 Separations Module with </p><p>2996 PDA detector</p><p>Chromatographic Conditions :Columns: ACQUITY UPLCTM BEH C18 2.1 x 50 mm, 1.7 mMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 0.5 mL/min Gradient: Time Profile Curve</p><p>(min) %A %B0.0 92 8 60.1 92 8 7</p><p>4.45 50 50 64.86 10 90 65.0 92 8 66.0 92 8 6</p><p>Injection Volume: 1.0 LWeak Needle Wash: 0.1% CF3COOH in 8% ACNSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 g/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 40 pts/secTime Constant: 0.1Instrument: Waters ACQUITY UPLCTM, with TUV detector</p><p>Original HPLC Method Final UPLCTM Method</p></li><li><p>2005 Waters Corporation</p><p>AU</p><p>Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00</p><p>HPLC-to-UPLCTM Method Transfer:Caffeic Acid Derivatives in Echinacea Purpurea</p><p>AU</p><p>Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00</p><p>Final UPLCTM Method = 5 minACQUITY UPLCTM BEH C182.1 x 50 mm, 1.7 m</p><p>Original HPLC Method = 35 minXTerra MS C184.6 x 150 mm, 5 m</p><p>1</p><p>1</p><p>2</p><p>2</p><p>3</p><p>3</p><p>4</p><p>4</p><p>5</p><p>5</p><p>35.00</p><p>5.00</p></li><li><p>2005 Waters Corporation</p><p>HPLC-to-UPLCTM Method Transfer:Caffeic Acid Derivatives in Echinacea Purpurea</p><p>AU</p><p>-0.001</p><p>0.000</p><p>0.001</p><p>0.002</p><p>0.003</p><p>0.004</p><p>0.005</p><p>Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00</p><p>au</p><p>-0.001</p><p>0.000</p><p>0.001</p><p>0.002</p><p>0.003</p><p>0.004</p><p>0.005</p><p>Minutes</p><p>0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00</p><p>Original HPLC Method = 35 minXTerra MS C184.6 x 150 mm, 5 m</p><p>Final UPLCTM Method = 5 minACQUITY UPLCTM BEH C182.1 x 50 mm, 1.7 m</p><p>35.00</p><p>5.00</p></li><li><p>2005 Waters Corporation</p><p>Conclusions</p><p> Methods can be moved directly from HPLC to ACQUITY UPLC Improved resolution Improved speed Improved detectability</p><p> Many parameters can and must be transferred to preserve results</p><p> Attention to detail leads to success</p></li></ul>