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A seminar on

UPLC(Ultra Pressure Liquid

Chromatography)

Presented by: Guided by: Ms. Sonali R.Phadke Dr. Asha B. Thomas M.Pharm :sem-I(QAT) UPLC-An overview

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Comparison between HPLC and UPLC :HPLC

dp(3-10)µmInlet pressure 5000 psiLower precision

comparative in sample introduction.

Detector that uses large flow rates and larger detection cells.

Some are equipped with automated sampling devices.

Slower with lower resolution.

UPLCdp.(1.75-1.8)µmInlet pressure more than

15000psiHigher precision in sample

introduction.Detector that uses small flow

rates and low detection limits.

Most of are equipped with automated sampling devices.

Rapid with higher resolution.

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The fundamental resolution equation :

Rs= ) () efficiency selectivity retensivity

In UPLC, increasing N (efficiency) is the primary focus.

As Rs is proportional to square root of NRs

If N

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Improving particle size:Efficiency is inversely proportional to particle size so

NRs 1.7X (i.e. Rs)

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Resolution (constant column length):Optimal flow rate is inversely proportional to dp

FoptIsocratic analysis time is Inversely

proportional to FTherefore

T 3X

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Peak width and sensitivityEfficiency E, is inversely proportional to square of peak width.

N 1/W2

Peak height is inversely proportional to peak width

Height 1/w sensitivity 7 x

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Sensitivity increases:Assuming same efficiency, peak height is

inversely proportional to column length,

Height=1/LFor same length column length L decreased

proportional to particle size dp (N=1x, Rs=1x)

Sensitivity

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Backpressure increases:Backpressure (p) is directly proportional to column length

PFor constant L/dp, Backpressure is inversely proportional to the square to particle size,dp

Pdp2

P

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HPLC vs UPLC

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Why to migrate from HPLC to UPLC?Acquire results in less time and

with more resolution.More information-fasterMore robust method-more

confidenceBetter situational responseMore samples analyzed per

system, per scientist

More productivity

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UPLC stationary phases :Mechanical strength and stability of columns

were critical issues due to high pressure.Classical gel-sol method used for generation

of durable columns which are superior than silica particles

Made by incorporating carbon in form of methyl groups

Mechanically strong, better efficiency and can be used over extended range of pH.

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Ethylene Hybrid Bridge(EHB) particles :This technology increases the stability of the

1.7 µm particles by bridging the methyl group in the silica matrix.

Can withstand high pressure and pH.Efficiency of column is directly proportional

to column length and inversely proportional to particle size.

Pore diameters are 130 Å and 300Å.

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High Strength Silica (HSS) particlesOnly UPLC certified

100% silica particles having 1.8

These are tested for 15000 psi pressure

Include tribonded C18 ligand

Long column life and UPLC efficiencies

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Column chemistries:

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Charged surface Hybrid(CHB) technologyThese particles incorporate a low level surface hybrid charge improves sample loadability and peak asymmetry in low ionic strength mobile phases.

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Instrumentation :

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• Acquity UPLC system consists :

Binary solvent manager Two individual serial flow pumps to deliver a parallel binary gradient. provides steady pulse free solvent flow at analytical flow rate(1-2 ml/min).Sample manager including sample heater Injects sample draws from vial in to chromatographic flow stream. Can perform injection approx. at 15 sec. Also controls column temperature.Optional sample managerStores micro meter or vial plates and to and from the sample manages, automating their processing and increasing through put.

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Injecting the sample:

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Pumps: pumps

Constant flow pumps

Reciprocating piston

pumpsDual piston

pumps

Constant pressure pumps

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UPLC columns:

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Detectors:

detectorsOptical detector

Tunable UV detector

Evaporating light scattering

detector

Florescence detector

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Tunable UV detector: These are used for UV/visible

rangeConsists light guided flow cell

like optical fiber contains volume of 500

nanolitre and pathlength of 10 mm, and high sensitivity flow cell with volume 2.4 microliters and 25 mm pathlength.

Operates at wavelength ranging 190-700 nm

Empower and MassLynx software is used for operations

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Applications:

Identification of metabolites in in vitro discovery studies.

Bioequivalence studyDetection of impuritiesDissolution testing in relatively high potent

drug sampleMethod development/validationForced degradation studiesImpurity profiling

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References :Waters AAPS 2007 seminars, November 12-13,2007UPLC: a preeminent technique in pharmaceutical

analysis by Ashok Kumar et al, Acta Poloniae Pharmaceutica-Drug research, vol.69 No.3pp.371-380,2012

Ultra Performance Liquid Chromatography: An Indroduction And Review by T. Sunil Kumar Reddy et al,IJPRA,vol 2,issue 1, 2012,24-31

Ultra Performance Liquid Chromatography : A Review by Patil V.P. et al.IRJP2(6) 2011 39-44

Ultra Performance Liquid Chromatography: A chromatography technique by Uttam Singh Baghel et al.IJPQA.2010;2(1):19-25

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Thank you