Mohini patil

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Mohini patil

  1. 1. ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) Presented By, Guided By, Miss. Mohini Patil Dr. Rajesh J Oswal Prof.Sandip Kshirsagar Department of Pharmaceutical Chemistry 6/13/2012 1
  2. 2.  INTRODUCTION PRINCIPLE INSTRUMENTATION EXAMPLE OF UPLC SEPRATION COMPARISON BETWEEN HPLC & UPLC SEPRATION APPLICATIONS 6/13/2012 2
  3. 3.  INTRODUCTION: UPLC provides improved resolution, speed, and sensitivity By using conventional particle sizes, speed, pressures and peak capacity can be extended to new limits and compromises with sacrificing retention time. As the particle size is 1.7µm there is a significant gain in efficiency along with resolution and without diminish efficiency. (Figure 1) 6/13/2012 3
  4. 4. Figure1. Van Deemter plot, illustrating the evolution ofparticle According to figure 1 , column efficiency (N) is inversely proportional to particle size (dp). Thus, smaller particles provide higher resolution and lower retention time. 6/13/2012 4
  5. 5. PRINCIPLE:The UPLC is based on the principal of HPLC. It involves stationary phase consisting of particles sub 2μm, as HPLC columns are typically filled with particles of 3to 5 μm. Efficiency is the primary separation parameter behindUPLC since it relies on the same selectivity and retentivity asHPLC. In the fundamental resolution equation resolution isproportional to the square root of N. 6/13/2012 5
  6. 6. Where, N is number of theoretical plates, α is Selectivity factor and k is mean retention factor.But since N is inversely proportional to particle size (dp); N α 1/dpAs the particle size is lowered by thrice i.e. from 5 mm to1.7mm, N is increased by three and the resolution by square rootof three i.e 1.7 6/13/2012 6
  7. 7. 1. Binary SolventManager2. Sample Manager3. Column Heater4. Optional Sample Organizer5. Optical Detectors 6/13/2012 7
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  9. 9. 4. Optional Sample Organizer: Sample organizer storesmicro titer or vial plates & transfer them to & from samplemanager.5. Optical Detectors: The system can be configured with anTUV (Tunable Ultraviolet), PDA (Photodiode Array) or ELS(Evaporative Light Scattering) optical detector or anycombination of the three. 6/13/2012 9
  10. 10. Column: 2.1 by 30mm 1.7 mm ACQUI-TY UPLC BEH C18at 358C.A 20–40% B linear gradient over 1.0 minute, at a flow rate of0.86mL/min was used. 6/13/2012 10
  11. 11. Mobile phase: A was 0.1% formic acid, B was Acetonitrile.UV detection: at 254 nm and 40pts/sec.Peaks are in order: 1: 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, 4-hydroxy coumarin, coumarin, 7- methoxycoumarin, 7-ethoxycoumarin, and 4-ethoxycoumarin. 6/13/2012 11
  12. 12. Figure shows an HPLC versus UPLC separation comparisonof a ginger root extract sample where both speed andresolution are improved, as well as an increase in sensitivity. 6/13/2012 12
  13. 13. 1. UPLC with MS: UPLC coupled to quadrupole tandem mass spectrometry which operates with rapid, generic gradients and shows increase in analytical throughput and also shows sensitivity in high throughput pharmacokinetics or bioanalysis studies, the rapid measurement of potential p450 inhibition, induction, and drug-drug interactions had been studied by UPLC/MS/MS.2. UPLC in Pharmaceutical Development: Now a day’s UPLC is a very attractive tool for the pharmaceutical development laboratory because of high resolution obtained in extremely short period of analysis times, as UPLC provides high throughput, high productivity and high resolution 6/13/2012 13
  14. 14. 3. UPLC used in Identification of Metabolite: Biotransformation ofnew chemical entities (NCE) is necessary for drug discovery. When acompound reaches the trial stage, metabolite identification is requiredand it is necessary for lab to successfully detect and identify allcirculating metabolites of a contender drug.4. UPLC used. in Bioanalysis / Bioequivalence Studies: ForPharmacokinetic, Bioequivalence and toxicity studies, the quantitativeanalysis of a drug in biological samples is an important part of drugdevelopment process and this is carried out by UPLC5. UPLC used in stressed degradation Studies: The most commonanalytical technique for monitoring forced degradation experiments isHPLC with UV and/or MS detection for peak purity, mass balance, andidentification of degradation products but these HPLC-basedmethodologies are time-consuming and provide only mediumresolution 6/13/2012 14
  15. 15. to ensure that all of the degradation products are accurately detected.PDA/MS (photodiode array and MS) used along with UPLC, whichallows for faster and higher peak capacity separations of complexdegradation product profiles.7. UPLC used in Impurity Profiling: UPLC PDA detector involvestwo analytical flow cells with maximum flexibility and according toapplication requirements, as one for maximum chromatographicresolution and a second for high sensitivity. UPLC also ensure thelatest peak detection algorithms and custom calculations to optimizedata processing and reporting. It also assertively detects impurities incompounds even at trace levels. To characterize impurities, it is oftennecessary to perform several analytical runs to obtain the necessaryMS and MS/MS data. 6/13/2012 15
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