UPLC NEW

PowerPoint Presentation: 

Devang patel 1 Presented By: Devang D Patel Guided by:Dr.Ujash shah M.Pharm (sem-1) Associate professor of Department of Quality Assurance . Q.A Department Nootan pharmacy college

Contents: 

Devang patel 2 Contents Introduction History Ultra Performance Liquid Chromatography Principle Instrumentation Comparison between HPLC and UPLC Applications References

Introduction: 

Devang patel 3 Introduction History : Started in late 1990’ s. Pioneered by Jorgenson and Lee groups. The Water Company has been working on UPLC.

How UPLC differs From HPLC: 

Devang patel 4 How UPLC differs From HPLC Efficiency Analysis Time Sample Throughput Sensitivity Pressure

Difference between HPLC and UPLC: 

Devang patel 5 Difference between HPLC and UPLC HPLC UPLC Particle size 3-10  m 1-2  m Pressure 4000-6000 psi 15000-100000 psi Flow rate 100  L /min 300-800  L /min Separation time High Low Sensitivity Low Higher Resolution Less High Method development cost High Low

Comparison Of HPLC and UPLC: 

Devang patel 6 Comparison Of HPLC and UPLC

Principle : 

Devang patel 7 Chromatographic resolution is described by Where Rs=Resolution N=Separation efficiency (plate number) α =Selectivity factor k=Retention factor (Capacity factor) Principle

PowerPoint Presentation: 

Devang patel 8 Separation efficiency (N) where L=Column length H = Plate height h = reduced plate height and dp= particle diameter Therefore

UPLC Method : 

Devang patel 9 UPLC Method Example of Naproxen and Ketorolac Isocratic condition BEH C 18 Column -2.1 X 50 mm, 1.7 µ m) Mobile phase water/acetonitrile/formic acid (65/30/0.1;v/v/v/) Flow rate 0.1 – 1.2 mL/min Pressure -13,500psi

Factors Affecting Performance of UPLC: 

Devang patel 10 Factors Affecting Performance of UPLC

Effect of Temperature: 

Devang patel 11 Effect of Temperature To improve protein separation. Modify properties of column surface alter protein structure. Column efficiency increase. Reduces mobile phase viscosity decrease in backpressure. Reduced run times in UPLC experiments

PowerPoint Presentation: 

Devang patel 12 Effect of pressure – Higher the pressure higher is the resolution. Effect of column – Column used for UPLC are specially prepared for withstanding high pressure Effect of particle size of column – Lesser particle size can withstand higher pressure applied in UPLC

Instrumentation: 

Devang patel 13 Instrumentation Autosampler – Reduced Cycle Time and niglisible carryove Flow cell Van guar column – for protecting column performance Tubings – Special for UPLC, reduced volume, optimized connection Mobile Phase Filters – To prevent system damage column Detectors

Schematic Presentation of UPLC: 

Devang patel 14 Schematic Presentation of UPLC Gradient Controller Pump • Column Detector Injector Mobile Phases

Ultra Performance Liquid Chromatography: 

Devang patel 15 Ultra Performance Liquid Chromatography

Tubing and Fitting : 

Devang patel 16 Tubing and Fitting Tubing and fittings are carefully designed to provide leak-free connections, with low dead volume, and minimal band spreading. For best performance, these components must be clean, and designed to work together.

Mobile Phase Filters: 

Devang patel 17 Mobile Phase Filters It is good practice to always filter your solvents to prevent system damage. Filters should be changed periodically depending on usage and mobile phase. Buffers pH 3 ammonium formate pH 10 ammonium bicarbonate

UPLC Particles : 

Devang patel 18 UPLC Particles Ethylene Bridged Hybrid (BEH) Particles Wide pH range Five chemistries High Strength Silica (HSS) Particles Only UPLC certified 100% silica particles

Ethylene Bridged Hybrid(BEH) Chemistries Of UPLC : 

Devang patel 19 Ethylene Bridged Hybrid(BEH) Chemistries Of UPLC

High Strength Silica (HSS) Chemistries of UPLC: 

Devang patel 20 High Strength Silica (HSS) Chemistries of UPLC HSS T3 – Design for maximum retntivity HSS C18 –Silica particle performance HSS C18 SB – Selectivity for Bases

Detectors: 

Devang patel 21 Detectors Evaporative Light Scattering (ELS) Detector Fluorescence (FLR) Detector Photodiode Array (PDA) Detector Tunable UV (TUV) Detector Single Quadrapole Detector (SQD)

UPLC - MS: 

Devang patel 22 UPLC - MS Water acquity UPLC-MS

What is UPLC –Q- TOF: 

Devang patel 23 What is UPLC –Q- TOF Highly sophisticated High resolution mass spectrometry system, which employs a time-of-flight mass spectrometry technology. Used to analyze compounds in samples with a mass accuracy of typically 5 ppm (or better) between the found and the theoretical molecular weight of the analyte.

Q-TOF Source : 

Devang patel 24 Q-TOF Source Uses very low particle size column packing (i.e.1.7 um) Excellent chromatographic separation at the cost of high operating system pressure. Overcome by a high-precision plumbing and fittings system design, in which exotic materials, such as gold, etc., are used to efficiently resist high liquid pressure (i.e., 14,000 psi or more) and provide a leak-free environment.

Advantages Of UPLC-MS: 

Devang patel 25 Advantages Of UPLC-MS

Applications of the UPLC/Q-TOF: 

Devang patel 26 Applications of the UPLC/Q-TOF High resolution mass spectrometry Accurate mass determinations Bioanalytical method of validation Application to a pharmacokinetic study Comprehensive screening and quantification of veterinary drugs in milk Peptide and Protein Research (MW approx. <12 kDa) with Accurate Mass

ADVANTAGES OF UPLC: 

Devang patel 27 ADVANTAGES OF UPLC Decreases run time and increases sensitivity Provides the selectivity, sensitivity, and dynamic range of LC analysis Maintaining resolution performance. Expands scope of Multiresidue Methods UPLC’s fast resolving power quickly quantifies related and unrelated compounds Faster analysis through the use of a novel separation material of very fine particle size Operation cost is reduced Less solvent consumption Reduces process cycle times, so that more product can be produced with existing resources Delivers real-time analysis in step with manufacturing processes Assures end-product quality, including final release testing

PowerPoint Presentation: 

Devang patel 28 Disadvantages OF UPLC Due to increased pressure requires more maintenance and reduces the life of the columns of this type.

Applications: 

Devang patel 29 Applications For separation of multicomponent drugs Separation of antitubercular drugs For analysis several metabolites Hair analysis of histamine in rabbits Analysis of drugs in human plasma Analysis of Levofloxacin For quantification of Palonsetron in human plasma and its application to a pharmacokinetic study Bioanalysis for measurement of plasma amrubicin and its metabolite Analysis of preservatives Analysis of bromine containing preservatives Pharmacokinetic study Analysis of Baicalin in CSF of rabbits

PowerPoint Presentation: 

Devang patel 30 Analysis of Natural Products and Traditional Herbal Medicine Study of Metabonomics / Metabolomics ADME (Absorption, Distribution, Metabolism, Excreation ) Screening Dissolution Testing Forced Degradation Studies Method Development / Validation Manufacturing / QA / QC Impurity Profiling Bioanalysis / Bioequivalence Studies (cont….

For Separation of Antitubercular Drugs: 

Devang patel 31 For Separation of Antitubercular Drugs Isoniazid (ISN), Pyrazinamide (PYR) and Rifampicin (RIF) were separated Less than 2 min was necessary for the complete separation of antituberculosis drugs, while the original USP method was performed in 15 min A large number of samples per day can be analyzed due to the short analysis times .

Analysis of Levofloxacin in human plasma: 

Devang patel 32 Analysis of Levofloxacin in human plasma Internal standard used – Niacin Column - BEH C18 Mobile Phase –acetonitrile Buffer - 0.4%triethylamine buffer(pH3) Pressure – 11000 psi Flow rate – 0.3 mL/min

Conclusion: 

Devang patel 33 Conclusion UPLC is properly design system provide significantly more resolution while reducing run times. This technique in both chemistry and instrumentation boosts productivity by producing more information per unit work as UPLC fulfils promise of increased resolution, speed sensitivity predicted.

References: 

Devang patel 34 References SSkoog, Holler,Nieman. Principles of Instrumental Analysis ,5 th ed.1992 ; 716 -721:828-851. Dae-Jin Park, Prasad B. Phapale, In-Jin Jang, Song Cui.etal ‘An improved UPLC Method for Rapid Analysis of Levofloxacin in Human Plasma” Chromatographia, 2008,68,187-192. Shao L.XinZhong Li,etal ‘UPLC-MS-MS Analysis Of Baicalin in Cerebrospinal Fluid of Rabbits:Application to a pharmacokinetic study”Chromatographia,2008,68,463-466. Lars Bendahl , Steen Honoré Hansen,Bente Gammelgaard,Camilla Nielsen ‘Hyphenation of ultra performance liquid chromatography (UPLC) with inductively coupled plasma mass spectrometry (ICP-MS) for fast analysis of bromine containing preservatives', Journal of Chromatography2006,68,169-173. Nguyen D.T.,Davy G.,etal ‘ Validation of an ultra fast UPLC-UV method for separation of antituberculosis tablets’Separationscience redifined,may2005,36-40.

PowerPoint Presentation: 

Devang patel 35 Hiroki K.,Kenichi Ito,etal, Hair analysis of histamine and several metabolites in C3 H/HeNCri mice by UPLC with Electro spray ionization time of flight mass spectrometry ;Influence of hair cycle and age,Bio anal Chemistry,jun2005,135-141. www.pubmedcentral.nih.gov www.sciencedirect.com www. chromatographyonline.com www.lib.bioinfo.pl www.waters.com

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Devang patel 36 Thank You