UPLC

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UPLC

  1. 1. Ultra Performance LiquidChromatography Prepared by: SHAIMAA OBAID AHMADEEN Supervised by: DR / NOuRAH AL ZOMAN
  2. 2. Presentations outlines:• Introduction• Definition• UPLC vs HPLC• efficiency of UPLC• Switching from HPLC to UPLC• Examples showing different UPLCinstruments• Applications of UPLC in pharmacy• Conclusion 9/23/2012 2
  3. 3. Introduction : The pharmaceutical industry is under intense pressure to increaseproductivity and put new drugs onto the market in a shorter period of time. Analytical chemists are challenged to find faster ways of delivering quality data across arange of project needs. A number of approaches are being employed to 9/23/2012 3
  4. 4. (TLC) Gravity Flash Chrom. Chrom. 1978 HPLC UPLC 1903 1952 2004 9/23/2012 4
  5. 5. Ultra : means not within reasonable limits or far beyond the normalUltra high pressure• Equal to or more than 1500psi (1034 pa =1000 ba )Ultra small packaging material• Particle size 1.7 , 1.8µm9/23/2012 5
  6. 6. Definition : UPLC is a chromatographic technique that can separate a mixture of compounds, and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture. Utilizing a smallpackaging particle sizes columns and ultra high pack pressure 9/23/2012 6
  7. 7. UPLC Vs HPLC9/23/2012 7
  8. 8. HPLC vs UPLC HPLC UPLC• id (3-10) µm • id (0.75-1.8)µm• inlet pressure 400 ba • inlet pressure more• Lower comparative than1000ba precision in sample • Higher precision in sample introduction introduction• Detectors that use larger • Detectors that use small flow rates and larger flow rates and low detection cells detection limits• Some are equipped with • Most of equipments with automated sampling automated sampling devices 9/23/2012 devices 8
  9. 9. Why is UPLC more efficient• Peak capacity (P) is the number of peaks that can be resolved in a specific amount of time.• P is proportional to the inverse of the square root of the Number of theoretical plates (N): N = L/H• Lower plate heights generate a greater number of plates (N : efficiency )• Plate heights are correlated through the Van Deemter equation 9/23/2012 9
  10. 10. Van Deemter Eqn.• H = A +B/µ +C x µ• A is related to the mobile phase movement through paths in the stationary phase.• B describes longitudinal diffusion• C relates the analyte to mass transfer between the pores of the stationary phase9/23/2012 10
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  12. 12. Chemistry of Small ParticSo as the particle size decreases toincrease N and subsequently Rs, anincrease in sensitivity is obtained, sincenarrower peaks are taller peaks.Narrower peaks also mean more peak 9/23/2012 12
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  15. 15. UPLC columns9/23/2012 15
  16. 16. Scaling the SeparationThe gradient is scaled from HPLC to UPLCusing: L2/L1 x tg1 = tg2Where L1and L2 are the lengths of the HPLC and UPLCcolumns, and tg1 and tg2 are the times of each gradient steprespectively.Flow rate is scaled taking into account thedifference in the diameter of the two columns: (d2)2/(d1)2 x F1 = F2Where d2 and d1 are the column diameters and F1 and F2 theflow rates.The injection volume is scaled taking intoaccount the volumes of the two columns: 9/23/2012 16 1 2 2 2 1 2
  17. 17. Differences in instrumentation between HPLC & UPLC9/23/2012 17
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  19. 19. Nano-UPLC SYSTEMFEATURES:•Maximum resolution to yield maximum peak capacityfor complex mixtures.•Maximum reproducibility – Direct nano-flow controland a novel, moveable heating and trapping module for< 0.25 minute standard deviation run-to-runreproducibility.•Maximum throughput –with faster gradientseparations for mixtures•Robustness – Nano-scale columns and fittingsprovide reliable operation for up to 10,000 psi, withnovel high pressure trapping.•Ease-of-use – Direct gradient control enables 9/23/2012 19
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  21. 21. Nano-ACQUITY UPLC System with 2D 9/23/2012 21
  22. 22. Hydrogen Deuterium Exchange (HD-x) is an experimentaltechnique to obtain structural data on proteins. It relies onthe accurate measurement of the degree of labeling of theprotein by deuterated hydrogen. 9/23/2012 22
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  24. 24. Contrasting HPLC and • UPLC gives UPLC with better faster results resolution • UPLC uses less of valuable solvents like acetonitrile which lowers cost • The reduction of solvent use is more environmentally friendly • Increased sensitivity 9/23/2012 24
  25. 25. Applications of UPLC1.Higher Speed separation of caffeic HPL acid derivatives from C Echinacea Purpurea UPL C 9/23/2012 25
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  27. 27. 2.Higher performance 100 %Intensity 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 Time (minutes) Total ion chromatogram of rat urine obtained with a 2.1 × 150 mm, 1.7 μm Acquity BEH C18 column and a gradient over 60 minutes at 90°C at 0.8 mL/min. Gradient: 0-95% B; A = 0.1% formic acid in water, B = acetonitrile with 0.1% formic acid 9/23/2012 27
  28. 28. 3.Complex Separation in a Short Time 9/23/2012 28
  29. 29. 4.Bio-Separations with high resolutionGain in separation performance and in analysis time when switching from a 25 cm 3 μmcolumn to a 15 cm 1.7 μm column. Calculation for a typical protein digest. 9/23/2012 29
  30. 30. 5.Baseline enhancement (HPLC-top) and 3 (UPLC-Bottom) showing increased UPLC resolution and signal to noise (sensitivity). 9/23/2012 30
  31. 31. Conclusion :(UPLC) is a relatively new techniquegiving new possibilities in liquidchromatography :• Decreasing time of analysis.• Decreasing solvent consumption.• Increasing the resolution.• Increasing the sensitivity.UPLC chromatographic system isdesigned in a special way:• to withstand high system back-pressures• Special analytical columns packed with 1.7 μm particles are used• The quality control analyses of various 9/23/2012 31
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