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ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY

(UPLC)

Presented By, Guided By, Miss. Mohini Patil Dr. Rajesh J Oswal

Prof.Sandip Kshirsagar

Department of Pharmaceutical Chemistry

JSPM’S Charak College Of Pharmacy & Research

Gat no.720/1&2,Wagholi, Pune -Nagar Road, Pune -412207

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INTRODUCTION PRINCIPLE INSTRUMENTATION EXAMPLE OF UPLC SEPRATION COMPARISON BETWEEN HPLC & UPLC

SEPRATION APPLICATIONS

CONTENTS:

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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)

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Figure1. Van Deemter plot, illustrating the evolution of particle

According to figure 1 ,column efficiency (N) is inversely proportional to particle size (dp). Thus, smaller particles provide higher resolution and lower retention time.

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PRINCIPLE: The UPLC is based on the principal of HPLC.

It involves stationary phase consisting of particles sub 2 μm, as HPLC columns are typically filled with particles of 3 to 5 μm.

Efficiency is the primary separation parameter behind UPLC since it relies on the same selectivity and retentivity as HPLC.

In the fundamental resolution equation resolution is proportional to the square root of N.

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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 to1.7 mm, N is increased by three and the resolution by square root of three i.e 1.7

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INSTRUMENTATION:

1. Binary Solvent Manager

2. Sample Manager

3. Column Heater

4. Optional Sample Organizer

5. Optical Detectors

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1. Binary Solvent Manager: It provides steady solvent flow at analytical flow rate. It can pump two solvents simultaneously. It deliver solvent at flow rates of 1mL/min at 103421 kPa & Up to 2 mL/min at reduced pressure to 62053kPa.

2. Sample Manager: It will help to maintain samples at any temperature between 40to 400 C.

3. Column Heater: It will heat the column compartment to any temperature from 50C to 650C.

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4. Optional Sample Organizer: Sample organizer stores micro titer or vial plates & transfer them to & from sample manager.

5. Optical Detectors: The system can be configured with an TUV (Tunable Ultraviolet), PDA (Photodiode Array) or ELS (Evaporative Light Scattering) optical detector or any combination of the three.

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Example of UPLC Separation of Seven Coumarin

Column: 2.1 by 30mm 1.7 mm ACQUI-TY UPLC BEH C18 at 358C. A 20–40% B linear gradient over 1.0 minute, at a flow rate of 0.86mL/min was used.

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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- methoxy coumarin, 7-ethoxycoumarin, and 4-ethoxycoumarin.

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COMPARISON BETWEEN HPLC & UPLC SEPRATION (in Ginger Root Extract)

Figure shows an HPLC versus UPLC separation comparison of a ginger root extract sample where both speed and resolution are improved, as well as an increase in sensitivity.

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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

APPLICATIONS

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3. UPLC used in Identification of Metabolite: Biotransformation of new chemical entities (NCE) is necessary for drug discovery. When a compound reaches the trial stage, metabolite identification is required and it is necessary for lab to successfully detect and identify all circulating metabolites of a contender drug.

4. UPLC used in Bioanalysis / Bioequivalence Studies: For Pharmacokinetic, Bioequivalence and toxicity studies, the quantitative analysis of a drug in biological samples is an important part of drug development process and this is carried out by UPLC

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5. UPLC used in stressed degradation Studies: The most common analytical technique for monitoring forced degradation experiments is HPLC with UV and/or MS detection for peak purity, mass balance, and identification of degradation products but these HPLC-based methodologies are time-consuming and provide only medium resolution

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to ensure that all of the degradation products are accurately detected. PDA/MS (photodiode array and MS) used along with UPLC, which allows for faster and higher peak capacity separations of complex degradation product profiles.

7. UPLC used in Impurity Profiling: UPLC PDA detector involves two analytical flow cells with maximum flexibility and according to application requirements, as one for maximum chromatographic resolution and a second for high sensitivity. UPLC also ensure the latest peak detection algorithms and custom calculations to optimize data processing and reporting. It also assertively detects impurities in compounds even at trace levels. To characterize impurities, it is often necessary to perform several analytical runs to obtain the necessary MS and MS/MS data.

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