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ULTRA PERFORMANCE LIQUIDCHROMATOGRAPHY

K Naresh1*, S Bhawani1 and T Maneesh Kumar1

Review Article

Ultra Performance Liquid Chromatography (UPLC) takes the advantage of technological stridesmade in particle chemistry performance, system optimization, detector design and dataprocessing and control. Using sub 2 mm particles and mobile phases at higher linear velocitiesand instrumentation that operates at higher pressures than those used in HPLC, dramaticincreases in resolution, sensitivity and speed of analysis can be obtained. This new category ofanalytical separation science retains the practicality and principles of HPLC while creating astep function improvement in chromatographic performance. This review introduces the theoryof UPLC and summarizes some of the most recent work in the field.

Keywords: UPLC, HPLC, Sensitivity, Resolution

*Corresponding Author: K Nareshbanu.sunkara@gmail.com

INTRODUCTIONUPLC refers to high Ultra Performance LiquidChromatography. It improves in three areas:chromatographic resolution, speed and sensitivityanalysis. It uses fine particles and saves timeand reduces solvent consumption. UPLC comesfrom High Performance Liquid Chromatography(HPLC). HPLC has been the evolution of thepacking materials used to effect the separation.An underlying principle of HPLC dictates that ascolumn packing particle size decreases,efficiency and thus resolution also increases.HPLC is approved technique as it has been usedin laboratories worldwide over the past 30-plusyears. HPLC technology simply doesn’t have the

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Int. J. Pharm. Med. & Bio. Sc. 2014

1 Balaji Institute of Pharmaceutical Sciences, Laknepally, Narsampet, Warangal-506 331, Andhra Pradesh, India.

capability to take full advantages of sub-2µm particles (Michael, 2005).

UPLC can be regarded as new inventionfor liquid chromatography. UPLC brings dramaticimprovements in sensitivity, resolutionand speed of analysis can be calculated. It hasinstrumentation that operates athigh pressure than that used in HPLC and in thissystem uses fine particles (less than 2.5 µm) andmobile phases at high linear velocities decreasesthe length of column, reduces solventconsumption and saves time. According to thevan Deemter equation, as the particle sizedecreases to less than 2.5 µm, there is asignificant gain in efficiency, while the efficiency

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does not diminish at increased flow rates or linearvelocities (Swartz, 2005).

Therefore by using smaller particles,speed and peak capacity (number of peaksresolved per unit time in gradient separations)can be extended to new limits, termed UPLC. Thetechnology takes full advantage ofchromatographic principles to run separationsUsing columns packed with smaller particles (lessthan 2.5 µm) and/or higher flow rates forincreased speed, this gives superior resolutionand sensitivity .Now a days in industrial areaUPLC refers for some of  the  most recent workfield.

CHEMISTRY OF SMALLPARTICLESSmaller particles provide not only increasedefficiency, but also the ability to work at increasedlinear velocity without a loss of efficiency, providingboth resolution and speed. Efficiency is theprimary separation parameter behind UPLC,since, it relies on the same selectivity andretentivity as HPLC. In the fundamental resolution(Rs) equation: resolution is proportional to thesquare root of N.

Re

1

4 1System Efficiency Selectivity sentivity

N kRs

k

But since N is inversely proportional to particlesize (dp): as the particle size is lowered by afactor of three, from, for example, 5 µm (HPLCscale) to 1.7 µm (UPLC-scale), N is increasedby three and resolution by the square root of threeor 1.7. N is also inversely proportional to thesquare of the peak width:

2

1N

w

This illustrates that the narrower the peaks are,the easier they are to separate from each other.Also, peak height is inversely proportional to thepeak width:

1H

w

So as the particle size decreases to increaseN and subsequently Rs, an increase in sensitivityis obtained, since narrower peaks are tallerpeaks. Narrower peaks also mean more peakcapacity per unit time in gradient separations,desirable for many applications, e.g., peptidemaps. Still another equation comes into play whenmigrating toward smaller particles:

1xxxF

dpc

This relationship also is revealed from the vanDeemter plot. As particle size decreases, theoptimum flow F opt to reach maximum Nincreases. But since back pressure isproportional to flow rate, smaller particle sizesrequire much higher operating pressures, and asystem properly designed to capitalize on theefficiency gains; A system that can both reliablydeliver the requisite pressures and that canmaintain the separation efficiency of the smallparticles with tightly managed volumes. Efficiencyis proportional to column length and inverselyproportional to the particle size.

LN

dp

Therefore, the column can be shortened bythe same factor as the particle size without loss

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of resolution. Using a flow rate three times higherdue to the smaller particles and shortening thecolumn by one third (again due to the smallerparticles), the separation is completed in 1/9 thetime while maintaining resolution. Although higheff iciency, nonporous 1.5- particles arecommercially available, they suffer from poorloading capacity and retention due to low surfacearea. Silica-based particles have good mechanicalstrength but can suffer from a number ofdisadvantages, which include a limited pH rangeand tailing of basic analytes. Polymeric columnscan overcome pH limitations, but they have theirown issues including low efficiencies, limitedloading capacities and poor mechanical strength.Packing a 1.7 m particle in reproducible andrugged columns was also a challenge that neededto be overcome, however. A smoother interiorsurface for the column hardware, and re-designing the end frits to retain the small particlesand resist clogging were necessary. Packed beduniformity is also critical, especially if shortercolumns are to maintain resolution whileaccomplishing the goal of faster separations.

PRINCIPLEThe UPLC is based on the principal of use ofstationary phase consisting of particles lessthan 2.5 m (while HPLC columns are typicallyfilled with particles of 3 to 5 m). The underlyingprinciples of this evolution are governed by theVan Deemter equation, which is an empiricalformula that describes the relationship betweenlinear velocity (flow rate) and plate height (HETPor column efficiency) (Van et al., 1956) .

H=A+B/v+Cv

where;

A, B and C are constants

v is the linear velocity, the carrier gas flow rate.

*The A term is independent of velocity andrepresents “eddy” mixing. It is smallest when thepacked column particles are small and uniform.

The B term represents axial diffusion or thenatural diffusion tendency of molecules. Thiseffect is diminished at high flow rates and so thisterm is divided by v.

*The C term is due to kinetic resistance toequilibrium in the separation process. The kineticresistance is the time lag involved in moving fromthe gas phase to the packing stationary phaseand back again. The greater the flow of gas, themore a molecule on the packing tends to lagbehind molecules in the mobile phase. Thus termis proportional to v. Therefore it is possible toincrease throughput, and thus the speed ofanalysis without affecting the chromatographicperformance. The advent of UPLC has demandedthe development of a new instrumental systemfor liquid chromatography, which can takeadvantage of the separation performance (byreducing dead volumes) and consistent with thepressures (about 8000 to 15,000 PSI, comparedwith 2500 to 5000 PSI in HPLC). Efficiency isproportional to column length and inverselyproportional to the particle size (Lars andHonore, 2003). As shown in Figure 1,smaller particles provide increased efficiency aswell as the ability to work at increased linearvelocity without a loss of efficiency, providing bothresolution and speed. Efficiency is the primaryseparation parameter behind UPLC since it relieson the same selectivity and retentivity as HPLC.

UPLC Stability Indicating AssayUPLC Conditions1. Column: 2.1 x 30 mm 1.7 m ACQUITY BEH

C18

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2. Temp: 30°C.  A 45 s, 5-85% B linear gradient,

3. Flow rate: 0.8 mL/min was used.

4. Mobile phase: A was 10 mm ammoniumformate, pH 4.0, B was acetonitrile. UVdetection at 273 nm and 40 pts/s

5. Peaks in order: 5-nitroso-2, 4, 6-triaminopyrimidine,4-amino-6-chloro-1, 3-benzenesulfanamide,hydrochlorthiazide, triamterine, and methylbenzenesulfanamide; 5 µL injection, 0.1mg/mL each.

Capitalizing on Smaller ParticlesOnly small particles are not responsible for fast resolution and sensitivity, some specialinstrumentation system should be design. Sosome special kind of system capable of deliveringthe pressures required  to realize the potential ofUPLC have been reported in the literature andelsewhere (Swartz, 2005). In early 2004, the firstcommercially available UPLC system thatembodied these requirements was described forthe separation of various pharmaceutical relatedsmall organic molecules, proteins, and peptides;it is called the ACQUITY UPLCTM System. TheACQUITY UPLC System consists of a binarysolvent manager, sample manager (including thecolumn heater), detector, and optional sampleorganizer. The binary solvent manager uses twoindividual serial flow pumps to deliver a parallelbinary gradient mixed under high pressure.

InstrumentationThe UPLC System has been holistically designedto match the performance needs of innovativecolumn chemistries with robust hardware, easy-to-use software and specialized support services.It consists of:

• Small, pressure-tolerant particles

• High-pressure fluidic modules

• Minimized system volume

• Negligible carryover

• Reduced cycle times

• Last response detectors

• Integrated system software and diagnostics

The ACQUITY UPLC System’s high-pressurefluidics optimizes flow rates to make the most ofsmall particle technology. The ACQUITY UPLCSystem’s sample-handling design is designed toensure exceptionally low carryover and reducedcycle time. And when interfaced with the SampleOrganizer, it increases unattended samplecapacity by ten times. High-speed detectors, bothoptical and mass, contribute to increasedsensitivity and help manage the heightened speedand resolution requirements of UPLC.

• ACQUITY UPLC Systems are easilycontrolled, diagnosed, and monitored via agraphical system console interface. Theconsole offers:

• Quick and easy access to critical instrumentparameters.

• Simple system start-up, elegant system statusmonitoring and predictive performanceindicators to ensure maximum productivity.

• Data management capabilities that aresupported by both MassLynx™ andEmpower™ software.

• The ACQUITY UPLC System is also supportedby Intelligent Device Management technologywith our Connections INSIGHT™ service,providing instrument diagnostics.

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1. Sample injection

2. UPLC columns

3. Column manager and heater or cooler

4. Detectors

5. Softwares

6. Accessories

7. Connection insight service (if provided bymfg.company.water provided it)

Sample InjectLee et al. described the design of injection valvesand separation reproducibility (Wu et al., 2001)and the use of a carbon dioxide enhanced slurrypacking method on the capillary scale for theseparation of some benzodiazepines, herbicides,and various pharmaceutical compounds (Wu etal., 1997). Jorgenson et al. modif ied acommercially available HPLC system to operateat 17,500 psi and used 22 cm long capillariespacked with 1.5 mm C18-modified particles forthe analysis of proteins (Tolley et al., 2001). Thecalculated pressure drop at the optimum flow ratefor maximum efficiency across a 15-cm longcolumn packed with 1.7 µm particles isapproximately 15,000 psi. Therefore, a pumpcapable of delivering solvent smoothly andreproducibly at these pressures and that cancompensate for solvent compressibility, whileoperating in both the gradient and isocraticseparation modes is required . With 1.7 µmparticles, half-height peak In UPLC, sampleintroduction is critical. Conventional injectionvalves, either automated or manual, are notdesigned and hardened to work at extremepressure. To protect the column from extremepressure fluctuations, the injection process mustbe relatively pulse-free and the swept volume of

the device also needs to be minimal to reducepotential band spreading. A fast injection cycletime is needed to fully capitalise on the speedafforded by UPLC, which in turn requires a highsample capacity. Low volume injections withminimal carryover are also required to increasesensitivity. There are also direct injectionapproaches for biological samples.

UPLC ColumnsThe design and development of sub-2 µmparticles is a significant challenge, andresearchers have been active in this area forsome time, trying to capitalize on their advantages2-4. Although high efficiency, nonporous 1.5 µmparticles are commercially available, they sufferfrom poor loading capacity and retention due tolow surface area. Silica-based particles havegood mechanical strength but can suffer from anumber of disadvantages, which include a limitedpH range and tailing of basic analytes. Polymericcolumns can overcome pH limitations, but theyhave their own issues including low efficiencies,limited loading capacities, and poor mechanicalstrength. In 2000, Waters introduced XTerra®, afirst generation hybrid chemistry that tookadvantage of the best of both the silica andPolymeric column worlds. XTerra columns aremechanically strong, with high efficiency andoperate over an extended pH range. They areproduced using a classical sol-gel synthesis thatincorporates carbon in the form of methyl groups.But in order to provide the necessary mechanicalstability for UPLC, a second generation BridgedEthyl Hybrid (BEH) technology was developedcalled ACQUITY BEH, these 1.7 µm particlesderive their enhanced mechanical stability bybridging the methyl groups in the silica matrix.Packing 1.7 µm particles into reproducible and

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rugged columns was also a challenge that neededto be overcome. Requirements include asmoother interior surface of the column hardwareand re-designing the end frits to retain the smallparticles and resist clogging. Packed beduniformity is also critical, especially if shortercolumns are to maintain resolution whileaccomplishing the goal of faster separations. AllACQUITY UPLC BEH columns also includeeCord™ microchip technology that captures themanufacturing information for each columnincluding the quality control tests and certificates

Figure 1: ACQUITY UPLC BEHColumn Chemistries

of analysis.

Column Manager and Heater CoolerThe ACQUITY UPLC Column Manager, withautomatic column switching, is for highproductivity UPLC sample processing, and itsColumn Heater/Cooler enables labs to usetemperature as a method parameter. TheACQUITY UPLC Column Manager allows usersto take full advantage of the performance, rangeof stationary phases, and mechanical strengthoffered by ACQUITY UPLC BEH Columns. TheColumn Manager provides temperature regulationfrom 10°C to 90°C, automated switching for upto four columns with dimensions to 2.1 mm in

internal diameter (I.D.) and 150 mm in length, aswell as a bypass channel for flow injections. TheACQUITY UPLC System consists of a binarysolvent manager, sample manager including thecolumn heater, detector, and optional sampleorganizer. The binary solvent manager uses twoindividual serial flow pumps to deliver a parallelbinary gradient. There are built-in solvent selectvalves to choose from up to four solvents. Thereis a 15,000-psi pressure limit (about 1000 bar) totake full advantage of the sub-2 m particles. Thesample manager also incorporates severaltechnology advancements. Using pressureassisted sample introduction, low dispersion ismaintained through the injection process, and aseries of pressures transducers facilitate self-monitoring and diagnostics. It uses needle-in-needle sampling for improved ruggedness and aneedle calibration sensor increases accuracy.Injection cycle time is 25 s without a wash and60 s with a dual wash used to further decreasecarry over. A variety of microtiter plate formats(deep well, mid height, or vials) can also beaccommodated in a thermostatically controlledenvironment. Using the optional sample organizer,the sample manager can inject from up to 22microtiter plates. The sample manager alsocontrols the column heater. Columntemperatures upto 65o can be obtained.

Detectors Half-height peak widths of less than one secondare obtained with 1.7 m particles, which givessignificant challenges for the detector. In order tointegrate an analyte peak accurately andreproducibly, the detector sampling rate must behigh enough to capture enough data points acrossthe peak. The detector cell must have minimaldispersion (volume) to preserve separation

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efficiency. Conceptually, the sensitivity increasefor UPLC detection should be 2-3 times higherthan HPLC separations, depending on thedetection technique. MS detection is significantlyenhanced by UPLC; an increased peakconcentration with reduced chromatographicdispersion at lower flow rates promotes increasedsource ionization efficiencies. Waters detectorsenhance ability to analyze a variety of compounds.Its ACQUITY UPLC detectors.

• The Photodiode Array (PDA),

• Tunable UV (TUV),

Evaporative Light Scattering (ELS) areoptimized for UPLC system technology by virtueof their low dispersion characteristics, high dataacquisition rates, and reliable performance withcost-effective maintenance support and parts.

Tunable UV (TUV)The ACQUITY Tunable UV/Visible detector cellconsists of a light guided flow cell equivalent toan optical fibre. Light is efficiently transferred downthe flow cell in an internal reflectance mode thatstill maintains a 10 mm flow cell path length witha volume of only 500 mL. Tubing and connectionsin the system are efficiently routed to maintainlow dispersion and to take advantage of leakdetectors that interact with the software to alertthe user to potential problems [26].

UPLC is an ideal inlet for the sensitivity andspecificity offered by mass spectrometry. The lowdispersion, high-speed detection performance ofWaters MS Technologies, in combination with theperformance characteristics of UPLC, candramatically extend  detection capabilities.

SoftwaresACQUITY UPLC Systems can be easilycontrolled, diagnosed and monitored via agraphical system console interface withEmpower™ and MassLynx™ software. BothEmpower and MassLynx provide the dynamicdata processing and information managementtools to convert the results generated by theACQUITY UPLC System into valuable knowledge.

AccesoriesWaters is continually extending the functionalityof the ACQUITY UPLC System: eCord™technology, available on all ACQUITY UPLCcolumns, records column history. SampleOrganizer that increases system capacity bymore than 10 times. FlexCart system platformimproves usability, accessibility and convenience

Connection INSIGHTTM ServiceConnections INSIGHT™ uses Intelligent DeviceManagement technology to provide diagnosticinformation for the ACQUITY UPLC Systems.Connections INSIGHT creates a virtual technicalsupport presence in your lab, enabling Waters toprovide you with proactive and timely service, withthe highest level of support and satisfaction.

METHOD OPTIMIZATIONGUIDELINES ANDOBSERVATIONSDuring the course of optimizing the UPLCmethod, considerations to expedite future methodtransfers were developed, and the followingrecommendations were made: Ø Increase elutionsolvent strength to reduce run times takingadvantage of the high resolution potential of UPLC

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columns. Increase mobile phase flow ratesecondarily to solvent strength in order to promotelonger column lifetimes. While high mobile phaselinear velocities with good resolution are possible,as with any column, routine operation at 80%maximum rated pressure led to shortenedlifetimes. UPLC operation around 8000 psi or lessprovided comparable or lower column cost perassay than HPLC. Maintaining low flows as muchas possible also reduces solvent and wastedisposal costs, although these are already anorder of magnitude less than HPLC. Reducecolumn re-equilibration times by taking advantageof the low system dwell volume. Programmedchanges in the mobile phase take time to reachthe column. The small UPLC dwell volume(measured as 110 L, 15% of that of the HPLC)allowed in part the abbreviation of the originalassay. Column re-equilibration accomplishedduring next sample loading in the UPLC, furtherincreasing throughput. Reduce injection volumesappropriately for the column diameter to achievegood peak shapes. Peak splitting occur when toolarge of a strong sample solvent bolusoverwhelms the packing at the column head.While this assay method tolerated 5 L injections,volumes of 1–3 L are more typical starting points.Smaller injection volumes may be compensatedby enhanced peak height from use of the highresolution columns and by the low carryover fromthe UPLC injector (measured as 10% of theHPLC carryover for this analyte) to achieve anequivalent or even lower LOQ). An alternative tosmaller injection volumes might be to lowersample solvent strength to accomplish samplefocusing on the head of the column. Utilize partialloop-fill injections in preference to full loop-fill.

Partial loop-fill precision was good even atvolumes up to 80% of the loop total volume .Typical laboratory practice is to limit samplevolume injections to roughly 50% of the total loopvolume. The UPLC injection system, whichutilizes air-gap sandwiching of the sample, allowsbetter utilization of the sample loop and higherinjection precision, reducing the need for use ofthe full loop-fill mode. From a practical point ofview, full loop fill requires substantially greatersample movement considering overfill functions.This likely increases subsequent needle washing,this may impact sample throughput and increasewear of the washing hardware. Larger samplevolume transfers also increases exposure tosample particulates, lowering long-terminstrument reliability. If full loop-fill mode is utilized,perhaps for very high precision requirementsensure adequate loop overfilling. A significantlaminar flow velocity differential in the loadingsample between its wall interface and center iscreated in the very narrow bore tubing of the UPLCinjector. Overfilling the sample loop by at leastfour loop volumes was found necessary to fullydisplace wash solvent from the 5 L injector loop.For this instrument, the manufacturer hasdetermined and set as the default the optimumoverfill volume with typical sample solvents foreach sample loop size. Operators can specifyother overfill volumes for unusual samplecompositions. Choose the proper compositionand volume of weak sample wash to obtain goodpeak shape. A portion of the weak sample washsolvent will be co-injected with partial-loop filledsamples. The weak solvent wash shouldtherefore mimic the initial conditions mobile phasein solvent strength. Utilizing the weak wash

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Figure 2: Chromatograms (from top to bottom): Original HPLC, Initial Scaling to UPLC ShowingPeak Shape Improvement and Possibility for Further Method Optimization, and Final UPLC

Method. Order of Peak Elution: Internal Standard (IS) Then Cpd A

solvent as sample diluents in the sample loopmay enhance sample focusing onto the column.The volume of the weak wash must be sufficientto purge the former strong wash solvent from theloop.

ADVANTAGES1. Decreases run time and increases sensitivity

2. Provides the selectivity, sensitivity, anddynamic range of LC analysis

3. Maintaining resolution performance.

4. Expands scope of Multiresidue Methods

5. UPLC’s fast resolving power quicklyquantifies related and unrelated compounds

6. Faster analysis through the use of a novelseparation material of very fine particle size

7. Operation cost is reduced

8. Less solvent consumption

9. Reduces process cycle times, so that moreproduct can be produced with existing

10. Resources

11. Increases sample throughput and enablesmanufacturers to produce more material thatconsistently meet or exceeds the productspecif ications, potentially eliminatingvariability, failed batches, or the need to re-work material.

12. Delivers real-time analysis in step withmanufacturing processes

13. Assures end-product quality, including finalrelease testing

DISADVANTAGES1. Due to increased pressure requires more

maintenance and reduces the life of thecolumns of this type. So far performancesimilar or even higher has been demonstrated

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by using stationary phases of size around 2m without the adverse effects of highpressure.

2. In addition, the phases of less than 2 m aregenerally non-regenerable and thus havelimited use.

APPLICATIONSi. With UPLC increased resolution in shorter run

times can generate more information fasterwithout sacrifices. Higher sample throughputwith more information per sample maydecrease the time to market, an importantdriving force in today’s pharmaceuticalindustry. The corresponding HPLC separationtakes in excess of 12 min; UPLC accomplishesthe same separation in under 30 s.

ii. UPLC can also be used to significantly improvethe success of the drug discovery process.Drug discovery is heavily dependant upon theearly prediction of metabolic fate andinteractions of drug candidate molecules.

iii. Sensitivity, selectivity, and analysis time(sample throughput) are also some of thechallenges analysts face when analyzingenvironmental samples such as soil and water.Explosives residues in soil or environmentalwaters are of both forensic and environmentalinterest. These types of assays provechallenging because of the selectivity neededto resolve positional isomers.

iv. In addition, for complex samples like naturalproduct extracts, added resolution can providemore information in the form of additional

Comparision Between UPLC and HPLC

Characteristics HPLC Assay UPLC Assay

Column 150×3.2mm 150×2.1mm

Particle size 3 to 5µm Less than 2 µm

Flow rate 3.0 ml/min 0.6 ml/min

Needle wash methanol methanol

Injection volume 5µL 2 µL

Column temperature 30 0C 65 0C

Maximum back pressure 35-40 MPa 103.5 MPa

Total run time 10 min 1.5 min

Plate count 2000 7500

USP resolution 3.2 3.4

Delay volume 750 µL 110 µL

Note: Total solvent consumption (including 0.5 min of delay time in between injections). Acetonitrile: 10.5 ml, Water: 21.0 mlAcetonitrile : 0.53 ml,Water: 0.66 ml.

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peaks. HPLC versus UPLC separationcomparison of a ginger root extract samplewhere both speed and resolution are improved.

v. UPLC coupled with MS technology providedparent and fragment mass information of lipidsin one chromatographic run, thus, providingan attractive alternative to current LC methodsfor targeted lipid analysis as well as lipidomicstudies.

vi. Applications areas of UPLC specified inWaters literature include high throughputlibrary screening, metabolite identification andbioanalysis, peptide mapping, stabilityindicating analyses, and quantitative analysis.

POTENTIAL AREAS OF USEi. Analysis of complex mixtures (e.g., impurity

profiles, formulation inerts).

ii. At-line analysis in manufacturing (analysis atthe vessel) .

iii. Analysis of large amounts of samples ForLC/MS to get better spectra (improved signalto noise).

CONCLUSIONUPLC gives increased resolution, speed andsensitivity for liquid chromatography therefore due to UPLC new chemistry and instrumentationtechnology can provide more information per unitof work. UPLC has main advantage over othersis reduction of analysis time which also helps toreduce solvent consumption. A negative aspect

of UPLC could be the higher back pressure thanin conventional HPLC. This back pressure canbe reduced by increasing the columntemperature. But it seems that UPLC can offersignificant improvements in speed, sensitivity andresolution compared with conventional HPLC.

REFERENCES1. Michael E Swartz (2005), “UPLCTM:

An Introduction and Review WatersCorporation, Milford, Massachusetts, USA”,Journal of Liquid Chromatography &Related Technologies, Vol. 28, pp. 1253–1263.

2. Swartz M E (2005), “Ultra PerformanceLiquid Chromatography (UPLC):An Introduction”, Separation Science Re-Defined, LCGC Supplement, May, pp. 8-13.

3. Van Deemter J J, Zuiderweg F J andKlinkenberg A (1956), Chem. Eng. Sci., Vol.5, p. 271.

4. Lars Y and Honore H S (2003), J.Chromatogr., A, Vol. 1020, pp. 59–67.

5. Swartz M E (2005), “Ultra PerformanceLiquid Chromatography (UPLC):An Introduction,  and review”, Journal ofLiquid Chromatography & RelatedTechnologies, Vol. 28, pp. 1253–1263.

6. Wu N, Lippert J A, Lee M L (2001), J.Chromatogr. A , Vol. 911, p. 1.

7. Lippert J A, Xin B, Wu N and Lee M L (1997),J. Microcol. Sepn., Vol. 1, p. 631.

8. Tolley L, Jorgenson J W and Mosely M A(2001), Anal. Chem., Vol. 73, pp. 2985.