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Inventi Rapid: Pharm Analysis & Quality Assurance Vol. 2012, Issue 4[ISSN 0976-3813]

2012 ppaqa 515, CCC: $10 Inventi Journals (P) LtdPublished on Web 17/09/2012, www.inventi.in

REVIEW ARTICLE

INTRODUCTION

Chromatography is a non-destructive procedure forseparation and extraction of individual analyte from themixture. For many years, researchers have been findingfast LC method. Smaller columns and faster flow rates(amongst other parameters) have been used to check theseparation speed. Analysis at increased temperature is alsochecked, it can increase diffusivity of analyte due toreduction of viscosity. Using normal pressure and particlesize we can achieve high resolution 3. So High performanceliquid chromatography (HPLC) is introduced 30 years ago.We cannot get full advantage of small particle size in simpleHPLC. UPLC can be considered as new invention for liquidchromatography. UPLC refers to Ultra Performance Liquid

Chromatography. Using UPLC technique we can getincreased sensitivity, resolution and speed. This systemoperates at higher pressure than HPLC ,uses smaller particlesize(less than 2.5m) and mobile phase at higher linearvelocity. Simultaneously Super critical fluid chromatographyis also introduced since about 1982.To take the advantagesof both the technology Waters introduce AQUITY based newproduct Ultra Performance Convergence Chromatograph (UPC2), which expands the boundaries of reversed phaseliquid chromatography (LC) and of gas chromatography (GC)separations and offers a replacement option for normalphase chromatography applications 4, 5.

UPC combines SFC with UPLC technology and expertisein fluidics, kinetics, and thermodynamics to deliver fast,reproducible separations. Supercritical CO2 has very lowviscosity, so it increases mass transfer, and hence columnefficiency, even compared to hexane or water when used asa mobile phase in this technique. Along with sub-2 mparticle column chemistries, UPC2 System gives the abilityto precisely vary mobile phase strength, pressure, andtemperature. With this ability to fine-tune the resolvingpower and selectivity of the system, one can exercise bettercontrol over the retention of analytes for separating,

1Post Graduation Department of Pharmaceutical Sciences, Sardar PatelUniversity, Vallabh Vidhyanagar- 388120, Gujarat, India.E-mail: [email protected]*Corresponding author

detecting and quantifying Whether analyzing natural

products, traditional medicines, drugs, food additives orcontaminants, pesticides, surfactants, polymer additives, orbiofuels - all compounds that are often difficult to separateby any other means. The UPC2 System now takes its placealongside LC and GC as a powerful complementary techniquefor taking on the laboratory's toughest separationschallenges. UPC 2 can be coupled with a range of Waterssolutions previously considered incompatible with normalphase chromatography and SFC. It is also best complementto MS with low solvent load, high resolution, narrow peaksand fast separation. Key benefit of system is use of CO2 as amobile phase instead of toxic and expensive organic solventas a mobile phase. UPC 2 can analyze diverse range of analyte

with faster separation and superior resolution 1, 47.

HISTORY AND NEED

UPLC 13

As demonstrated by Knox, Giddings and other authors,small particle diameters induce an increase in efficiency,optimal velocity and mass transfer. Therefore, fastseparations can be performed with relatively shortcolumns. However, only since 2004 columns packed withsub-2 m porous particles are commercially available andyield reliable performance. The use of columns packed withsub-2 m particles while operating at high pressures wasdeveloped ten years ago by Jorgenson and co-workers and and Lee and co-workers. This strategy allowsa significant decrease (up to 20) in analysis time becausethe latter is directly proportional to the maximal systembackpressure, as demonstrated elsewhere. Today,numerous manufacturers commercialize analytical devicesthat are able to handle pressure higher than 400 bar, suchas UPLC, for Ultra Performance Liquid Chromatography.Columns compatible with extreme pressures arecommercially available. The packing material is a bridgedethyl hybrid (BEH) technology, which improves mechanicalresistance.

SFCKlesper, Corwin and Turner first demonstrated SupercriticalFluid Chromatography (SFC) in Communications to the

Ultra Performance Convergence Chromatography (UPC2)

Paradigm Changed: SFC Based UPLC Technique

Divan Tanvi A1*, Parmar Shraddha J1, Patel Bhavana A1

Abstracts: Ultra Performance Convergence Chromatography (UPC2) is a new category of separations science in chromatographythat makes the complex analysis routine. It combines the potential of SFC technique with available UPLC technique. Use ofcompressed carbon dioxide CO2 (green solvent) and co-solvent as a mobile phase makes the technique inexpensive and nontoxic and it avoids use of toxic, expensive and volatile organic solvents. Along with sub-2 m particle column chemistries, systemgives the ability to precisely vary mobile phase strength, pressure, and temperature. With the ability to fine-tune the resolvingpower and selectivity of the system, one can exercise better control over the retention of analytes like natural products,traditional medicines, drugs, food additives or contaminants, pesticides, surfactants, polymer additives, or biofuels-allcompounds that are often difficult to separate by any other means. It is also best complement to MS with low solvent load, highresolution, narrow peaks and fast separation. Waters Corporation has been awarded the prestigious 2012 Pittcon Editors GoldAward for the Waters ACQUITY UPC2System as the best new product at the 2012 Pittsburgh Conference on Analytical Chemistryand Applied Spectroscopy.

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editor entitled High Pressure Gas Chromatography aboveCritical Temperatures in 1962. Sie, Beersum andRijnders published a series of articles on High-PressureGas Chromatography with Supercritical Fluids inSeparation Science (2-5) in 1966 and 1967. In thesearticles, they extensively used carbon dioxide as mobilephase. Since the 1980s, supercritical carbon dioxide

became the most preferred fluid in SFC and inSupercritical Extraction (SFE) because it is inexpensive,non-toxic, non-flammable and it has relatively low

critical temperature and pressure. In 2008, ultrafast SFCalso introduced 6.

Super critical fluid extraction and ultra performanceliquid chromatography combination with MS detection isalso performed earlier 29, 41.

In order to reduce the analysis time, maintain goodefficiency in liquid chromatography (LC) and to take

advantage of both above techniques, several solutions arecurrently being investigated. For this purpose, the differentapproaches were discussed.

Figure 1:Block diagram of ultra performance convergence chromatography

Figure 2:Normal phase HPLC separation of Anthralin and o-nitroaniline.17 Figure 3:UPC2

separation of Anthralin and o-nitro aniline.17

Figure 4: Normal phase HPLC analysis of Tolbutamide andTolazamide12.

Figure 5: Acquity UPC2 analysis of Tolbutamide andTolazamide12.

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REVIEW ARTICLE

For this previously super critical fluid chromatographywith different particle size (3 m, 1.5 m pellicularparticles and 1.8 m, 5 m) packed column is tried 2.Although many users have had success using thisapproach, limitations in analytical SFC instrument designand column availability have prevented the adoption ofsmall particle pSFC. Earlier attempts at sub-2 m pSFChave resulted in many false starts due to the lack ofcommercially available dedicated instrumentation. As foranalytical pSFC, many systems identified in publicationutilize instrument configurations limited by large systemvolume and extra column volumes affecting the ability to

achieve optimal chromatographic performance. The scopeof this work explores the impact of a low dwell volumeand low extra column volume instrument configurationwith intent to determine any benefits in chromatographicefficiency, resolution, sensitivity for sub 2 m particlesstationary phases. The advantages of overcoming theseinstrumental performance limitations will allow a trueassessment of pSFC for compatible sub-2 m stationaryphases. 36

It appeared that columns packed with sub-2 mparticles under high-pressure conditions (UPLC) were welladapted and this option represents an attractive alternativeto conventional LC.

To overcome the expanding need, challenge to get moreand better information faster, cost effectiveness newtechnology based on existing technology is introduced byWaters in 2011.

WATERS designed UPSFC system combining theadvantage of 2 m particle size and the efficiency of SFC 30,46. This new system provides shorter run times, reducessolvent usage &

Cuts the cost of analyses. Later, this system with MSdetection and other features is introduced as UltraPerformance Convergence Chromatograph (UPC2).

PRINCIPLEUPC2 is based on principle of ultra performanceconvergence chromatography 1.

The UPC2 is based on the principal of use of stationaryphase consisting of particles less than 2 m and supercritical solvent CO2as a mobile phase. By the van Deemterequation, which is an empirical formula that describes therelationship between linear velocity (flow rate) and plateheight (HETP or column efficiency), we can understand theuse of small particle size in column. The Van Deemtercurve, governed by an equation with three componentsshows that the usable flow range for a good efficiency withsmall diameter particles is much greater than for largerdiameters 8.

H=A+B/v+CvWhere A, B and C are constants and v is the linear

velocity, the carrier gas flow rate. The A term isindependent of velocity and represents "eddy" mixing. It issmallest when the packed column particles are small anduniform. The B term represents axial diffusion or thenatural diffusion tendency of molecules. This effect isdiminished at high flow rates and so this term is divided byv. The C term is due to kinetic resistance to equilibrium inthe separation process. The kinetic resistance is the timelag involved in moving from the gas phase to the packingstationary phase and back again. The greater the flow ofgas, the more a molecule on the packing tends to lag behindmolecules in the mobile phase. Thus this term isproportional to v. So if we decrease the particle size itincreases the column efficiency. Super critical fluids(compressed CO2) can be used as a mobile phase because itprovides greater diffusivity, lower viscosity and lowersurface tension than other organic solvents. Solvatingpower of super critical fluid is based on its density. Aspressure increases in system, solvating power of fluidincreases. So it decreases elution time 39.

INSTRUMENTATION

UPC2 system contain binary solvent manager, sample

manager, column manager and column heater withtemperature range of 20 to 90C ,Acquity upc2manager,Acquity upc2 PDA detector and Acquity upc2 instrument

Figure 6.Normal phase HPLC chromatogram (A) and UPC2 chromatogram (B) of BINOL 18.

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control. System is provided with 200 prepacked, pre-measured ready to use standards and reagents. Systemuses compressed CO2 and co-solvent as a mobile phasewhich is stable, non flammable green solvent, inexpensiveand nontoxic solvent 10.

Column Chemistry 15The reduction and control of silanol activity on AQUITYUPC2particles delivers, under SFC conditions, excellentpeak shapes, without use of mobile phase additives evenfor well retained basic achiral compound. Four types ofcolumns with dimension of 2.1 to 4.6 mm I.D. upto 150 mmin length are available which extends the selectivity rangeof SFC separation.BEH 2-EP column provides goodretention ,peak shape and selectivity.BEH column providesheightened interaction with polar group. CSH fluro-phenylprovides good retention of weak bases and alternateelution order for acidic and neutral compounds. Using HSSC18 SB column, glyceride analysis (Figure 1).

ADVANTAGES 1

Over Simple LC and HPLC

Instead of other organic liquid solvents, this system usescompressed CO2 gas as a mobile phase which is nonflammable green solvent, inexpensive and nontoxic solvent.Low viscosity of mobile phase achieves higher diffusionrate and enhanced mass transfer in UPC2.

Achiral analysis can be done with low solvent usage andat lower cost than HPLC. Chiral analysis can be done atmuch faster rate than normal phase HPLC.

With this system, analysis of diverse range of

compounds like hydrophobic and chiral compounds, lipids,thermally-labile samples and polymers which cannotanalysed by other technique.

Over GC

Analysis can be done at much lower temperature with CO2.

Over SFC

In UPC2SFC is combined with UPLC, so advantage of lowerparticle size is also involved in it. Column efficiencyincreases than SFC column.

APPLICATIONS

Enantiomeric Impurity AnalysisMost of the drugs are available in their enantiomeric form.Only single enantiomeric form has high efficacy and betterpharmacological effect. So manufacturer of the enantiomerhave to remove other stereoisomer from the product. UsingUPC2, detection and quantification of low levelenantiomeric impurities in drug substance can be donewith high sensitive detection and repeatability. The highdetection sensitivity of the ACQUITY UPC2 System enablesthe identification and quantification of enantiomericimpurities in drug substances. 9

Analysis of TabletAssay of tablet using normal phase HPLC takes more timeand consumes more solvents in separation. The UPC2

System is an ideal solution for laboratories looking formore efficient and cost effective methods for the analysis ofDapsone tablets while enhancing health, safety, andenvironmental concerns. ACQUITY UPC2 System enablesseven time faster analysis of Dapsone tablet withsignificant reduction in use of organic solvents 14.

Assay of Drug Substance

UPC2technique also performs the assay of drug substanceswith high quality results. A USP compendial HPLC methodfor assay of drug substance Anthralin was successfullyconverted to a high quality UPC2method at a cost of $0.05per run (compared to $0.90) and was 1.6 times faster. Inthis, each normal phase HPLC run used 16.4 ml of hexaneand 1.2 ml of dichloromethane. In contrast UPC2 methodonly uses 1.05 ml of methanol. This demonstrates thereduction in use of organic solvents 17.

Normal Phase HPLC separation of Anthralin and o-nitroaniline is shown in Figure 2. 17 and UPC2 separation ofAnthralin and o-nitro aniline shown in Figure 3.17

Purity Estimation

The UPC2 method used for the estimation of thechromatographic purity of estradiol was three times fasterthan the current normal phase HPLC method and reducedcost per analysis by more than 100 times 11.

Pesticide and Insecticide Analysis

Twenty five percent of pesticide currenty used are chiralcompounds. So there is increasing the demand ofstereoselective separation techniques and analytical assays

for evaluation of enantiomeric purity of pesticide.Compared to chiral HPLC methods, UPC2offers a completebaseline resolution of all permethrin isomers withsignificantly shorter run time; ideal for pesticidemanufacturers who routinely perform diastereomeranalyses45.

Chromatographic Assay

This new Ultra Performance Convergence Chromatography(UPC2) method produced data of equal or better qualitythan the current HPLC method, was 10 times faster, andconsumed less solvent. eg. Tolbutamide assay with useof tolazamide as an internal standard (Figure 4 and

Figure 5) 12.

Organic Compound Analysis

Separation of organic compounds like binol can be doneby UPC2. Ultra Performance ConvergenceChromatography (UPC2) demonstrated a faster separationof BINOL enantiomers (by 9 times) compared to normalphase HPLC as well as appreciable cost savings peranalysis (Figure 6) 18.

Enantiomeric Separation

The Waters ACQUITY UPC2 System is ideal for laboratories

pursuing high throughput enantioselective analyses,enantiomeric excess determination in a complex mixture ormatrix, and impurity profiling 7.

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Fat Soluble Vitamin Analysis

The use of Ultra Performance Convergence Chromatography(UPC2) in fat-soluble vitamin analysis provides a singleviable technique that is cost-effective, sustainable, and agreen technology alternative that lowers the use of organicsolvents, provides fast analysis19.

Analysis of Formulated Lithographic Material

Lithographic material like photo resist and anti reflectivecoatings is formulated for electronics industry. Manyproducts utilize various Dyes. Rapid analysis of dyeimpurity, dye concentration can be done in less than twominutes with UPC2system48.

CONCLUSIONFrom this review we can consider UPC2 as a best analyticalseparation technique for the separation of diverse range ofcompounds. UPC2 System expands the boundaries ofreverse phase liquid chromatography (LC) and of gaschromatography (GC) separations and offers a replacementoption for normal phase chromatography applications.Employing the principle of Ultra Performance ConvergenceChromatography, UPC2 System is a novel analyticalsystem that gives laboratories an indispensable tool fortackling tough-to-analyze compounds includinghydrophobic and chiral compounds, lipids, thermally-labilesamples and polymers. System uses CO2as a mobile phasewhich is a green solvent and this technique provides betterselectivity, resolution and faster analysis than othertechniques. Waters Corporation has been awarded for theWaters Acquity UPC2system in 2012.

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Cite this article as: Divan Tanvi A, Parmar Shraddha J,Patel Bhavana A. Ultra Performance ConvergenceChromatography (UPC2) Paradigm Changed: SFC Based

UPLC Technique. Inventi Rapid: Pharm Analysis &Quality Assurance, 2012(4): 1-6, 2012.

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