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HPCPC[HIGH PERFORMANCE CENTRIFUGAL PARTITION CHROMATOGRAPHY], WHAT IS HPCPC? CLASSIFICATION OF COUNTER CURRENT CHROMATOGRAPHY, INSTRUMENTATION ,APPLICATIONS OF HPCPC. BY P. RAVISANKAR.
 

HPCPC[HIGH PERFORMANCE CENTRIFUGAL PARTITION CHROMATOGRAPHY], WHAT IS HPCPC? CLASSIFICATION OF COUNTER CURRENT CHROMATOGRAPHY, INSTRUMENTATION ,APPLICATIONS OF HPCPC. BY P. RAVISANKAR.

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HPCPC[HIGH PERFORMANCE CENTRIFUGAL PARTITION CHROMATOGRAPHY], WHAT IS HPCPC? CLASSIFICATION OF COUNTER CURRENT CHROMATOGRAPHY, INSTRUMENTATION ,APPLICATIONS OF HPCPC. BY P. RAVISANKAR. ...

HPCPC[HIGH PERFORMANCE CENTRIFUGAL PARTITION CHROMATOGRAPHY], WHAT IS HPCPC? CLASSIFICATION OF COUNTER CURRENT CHROMATOGRAPHY, INSTRUMENTATION ,APPLICATIONS OF HPCPC. BY P. RAVISANKAR.
VIGNAN PHARMACY COLLEGE VADLAMUDI
GUNTUR, A.P, INDIA.

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    HPCPC[HIGH PERFORMANCE CENTRIFUGAL PARTITION CHROMATOGRAPHY], WHAT IS HPCPC? CLASSIFICATION OF COUNTER CURRENT CHROMATOGRAPHY, INSTRUMENTATION ,APPLICATIONS OF HPCPC. BY P. RAVISANKAR. HPCPC[HIGH PERFORMANCE CENTRIFUGAL PARTITION CHROMATOGRAPHY], WHAT IS HPCPC? CLASSIFICATION OF COUNTER CURRENT CHROMATOGRAPHY, INSTRUMENTATION ,APPLICATIONS OF HPCPC. BY P. RAVISANKAR. Presentation Transcript

    • High Performance Centrifugal Partition Chromatography (HPCPC) is apractical and suitable method, particularly on the preparative scale, for theseparation of biomolecules such as proteins, enzymes, etc.Prof. RavisankarVignan Pharmacy collegeValdlamudiGuntur Dist.Andhra PradeshIndia.banuman35@gmail.com00919059994000
    • What is HPCPC?High Performance Centrifugal Partition Chromatography, HPCPC, is anew technique of Liquid Chromatography, LC, which is support free,and which finds its place among the several Liquid Chromatographicmethods as shown on the following chart .
    • Centrifugal force (from Latin wordcentrum, meaning "center",and fugere, meaning "to flee")represents the effects of inertia that arise in connection withrotation and which are experienced as an outward force awayfrom the center of rotation
    • Around 50years ago, the concept of partitioning solutes between two liquids gavebirth to two eventual methods,one was the counter-current distribution,another was the liquid- liquid partition chromatography.30years ago, Sanki Engineering Ltd. (now absorbed by System InstrumentsCompany Ltd.) opened the way to high performance centrifugal partitionchromatography (HPCPC), which was taking the best of the two first techniquesThe versatility of a true liquid- liquid process combined with the quickness and advancedtechnology of chromatography.The HPCPC is gaining more and more interest as a semi-prep and preparative scalechromatographic method.
    • There are four main advantages of the HPCPC as follows:1) No loss of sample: Both mobile and stationary phases are liquidsand can be collected for total recovery.2) The volume ratio of stationary to mobile phase is definitely muchhigher, which leads to higher capacities and better resolution with norequirement of a high number of theoretical plates.3) The extreme flexibility of biphasic system: The mixtures of thetwo, three or four solvents, which allows to modify the selectivity of asystem in order to get a pure compound in the HPCPC polarities ofboth phases can be smoothly modified.4) The reduced solvent consumption is ten times less than preparativescale chromatography for the same throughput, it should beenvironmentally considered. Separation accomplished with laboratoryHPCPC can be directly scaled up to production scale HPCPC.Without a solid stationary phase support, The HPCPC system effectivelyseparate, isolate and purify milligrams to multigrams.
    • The 4main advantages of the HPCPC over its parent prepscalecolumn chromatography are :1.No loss of sample since both mobile and stationary phases are liquids and can becollected for total recovery.The volume ratio of stationary to mobile phases is definitely much higher, which leads tohigher capacities and better resolution with no need of a high number of theoretical plates.2.The extreme flexibility of biphasic systems (mixtures of two or three or four solvents), whichallows to modify the selectivity of a system in order to get a pure compound, in the HPCPC thepolarities of both phases can be smoothly modified.3.The reduced solvent consumption, ten times less than for preparative scale chromatographyfor the same throughput, which is of interest for environmental considerations. Separationaccomplished with laboratory HPCPC can be directly scaled up to production scale HPCPC.4.Another major advantage is the extremely low price of the stationary phase (solvents)compared to that of column packings. Moreover, stationary can be refreshed easily, and addedmaterials like chiral selectors or complexing agents can be recovered with no loss. Severalpublications in international journals bring valuable information.The new innovated HPCPC is getting more involved in many fields of chemistry, for purificationof antibiotics, pePtides, tannins, saponins, lipids, drugs, ...... its future development will seethe production of bigger HPCPCTM units, and it will incorporate crucial fields of chemistry,such as chiral separations.
    • Superior to Conventional Preparative LCHPCPC is fast !Since stationary phase solvents are retained in the partition channels by centrifugal force, highmobile phase flow rates may be used without appreciable loss of resolution.Great advantages over conventional preparative LCSince a solid support is not used with HPCPC, irreversible retention of valuable samplecomponents is completely eliminated, denaturation and decomposition, often encounteredwith conventional LC column packings, are virtually eliminated. And this is generallyaccomplished with retention of biological activity.Moreover, the capacity of an HPCPC column (rotor) is significantly greater than anHPLC column of the same total volume. Consequently, overload effects are rarelyencountered with HPCPC. Purification of large quantities is routine, always with100% material balance...there is no adsorption or irreversible retention.
    • No columns or Packings to replaceThe problems of formation of voids, contamination of fractions with silica and withcomponents of previous runs, and the cost of replacing expensive HPLC columns are gone.Fewer Theoretical Plates are NeededTo achieve a given level of resolution between two peaks with HPCPC than withHPLC, for instance, for a value of alpha (selictivity) = 1.2 and K (partition coefficient)= 1.... 1,85,000 T.P are needed to achieve baseline resolution (Rs of 1.5) withHPLC....whereas only 2,200 plates are needed to do the job with HPCPC. This is adirect consequence of the standard resolution equation for liquid chromatography.Normal phase and reversed phase chromatography can be done in the same runUse dual mode HPCPC to accomplish even the most demanding separations ofsamples containing complex mixtures of polar/non-polar, hydrophilic/hydrophobicsubstances. Even chiral substances are resolved with suitable chiral HPCPC phases.Work at any pHWith HPCPC, there is no need to concern yourself with unwieldy pH limitations thatare often associated with solid stationary phase supports.Innovations overcome previous weakness of the CPC
    • Weak Point (1)Separation tasks are intricate and need to attend all the time.Overcome the above by innovated automation system.1) Separation tasks can be preseNt on the 10 executed files.2) Capable to link multiple files which have different separation conditions among theseparation executed files (solvents, rotations, flow rates, etc.) so that the most suitableconditions can be considered sequentially.3) Sequential separations with same executed file and sequential injections into samestationary phase (reproducibility is very good by means of using same condition forseparation of samples) can be performed easily. Automation can be materialized byconnection of Autosampler and Fraction Collector.4) Data and file handlings will become easy by linking with computer in which dataacquisition program was pre-installed.Weak Point (2)Take long time to decide the most suitable separation conditions.Overcome by improved small rotor which is just 1/3 volume of the previous model LLB-M.
    • HPCPC Principle:The HPCPC is a new liquid chromatographic technique that utilises liquid-liquid partition,counter current distribution in the absence of a solid support to perform separations ofcomplex mixtures of chemical substances.The HPCPC is an alternative to packed-bed columns for preparative HPCPC and operates byclassical liquid-liquid partitioning in a high performance centrifugal system.A solid stationary phase is not used. Instead, stationary phase liquid is retained by centrifugalforce in discrete partition channels within a unique patented circular partition disk pack.A packed column generally contains only 2 to 7 percent of stationary phase, severely limits itscapacity.In an HPCPC system, the column contains between 50 and 80 percent stationary phase.The stationary phase is held in numerous discrete partition cells. Micro-droplets of mobilephase liquid pass continuously through the stationary phase liquid. Any two-phase solventmixture can be used at any pH to perform normal and reversed phase chromatographicseparations.An injected sample, carried by the mobile phase, moves sequentially through the partitionchannels, where components are partitioned between the mobile and stationary liquid phase,separated from each other on the basis of differences in their partition coefficients and eluted.
    • Simple scheme used by the pioneers of HPCPC to represent thearrangement between channels and ducts, with the droplets of mobilephase flowing in the stationary phase.
    • Schematic representatin of the HCPC apparatus.
    • The HPCPC rotor is constituted by thesuperposition of disks engraved with smallcells connected by head / tail ducts. Thesecells, where the chromatographic separationtakes place, can be compared to lined-upseparate funnels. The rotor is filled with thestationary phase, which stays inside the rotorthanks to the rotation speed, while the mobilephase is pumped through. CPC can beoperated in either descending or ascendingmode, where the direction is relative to theforce generated by the rotor rather thangravity. According to the fast and permanentevolution of the cells design, the efficiency andflow rate with low back pressure are improved.The CPC offers now the direct scale up fromthe analytical apparatuses (few milliliters) toindustrial apparatuses (some liters) for fastbatch production
    • rotor (=column
    • Single HPCPC Unit Introduces Dual ApplicationsSeparation by either normal-phase or reversed-phase elution is accomplished with a single two-phase solvent system. Dual-modeHPCPC is illustrated for a hypothetical mixture of five components (a, b, c, d and e) as below. Assuming their "partition coefficients"(ratio of concentrations in stationary / mobile phase) are in the order a>b>c>d>e.(1)Normal Phase Separation (2)Normal Phase Elution(1)(2)The components with larger partition coefficients (a & b) are primarily remained in the "Lower Stationary Phase" due to their strongaffinity for the Lower.The components with smaller partition coefficients (c, d & e) are separated from each other via "Upper Mobile Phase Elution".During upper phase elution, however, components a and b may migrate, very slowly and actually separated from each other withinthe stationary phase.(3)Recycle (4)Reversed Phase ElutionAfter c, d & e have eluted, the Upper Mobile Phase is recirculated for a time sufficient for compete partition of components a & b.
    • 1) Washing2) Injection3) Ascending Mode (Mobile Phase : Upper)4) Descending Model (Mobile Phase : Lower)Flow Diagram of CPC System
    • ApplicationsAlkaloids (1-Strychnine, Quinine, Cinchonidine)Alkaloids(Quinine, Strychinine, Brucine, Berberine)Angiotensin IIAssay of Environmental PollutantBale Acids (TaurocholicAcid, Taurochenodeoxycholic Acid, CholicAcid, Lithocholic Acid, Dexoycholic Acid)Beta-CaroteneBioreactorChlorophyllChromomycineDiterpene Esters from Daphne extractEgg Yolk LecithinEnzymes from Yeast ExtractFatty Acid EstersFatty Acids (1) ? Saturated Fatty Acids (StearicAcid, Palmitic Acid, Myristic Acid, Lauric Acid)Fatty Acids (2) ? Unsaturated Fatty Acids(Stearic Acid, Linolenic Acid, Linoleic Acid)Fish Oil Hydrolysate
    • Fungous Toxin NivalenolHerb Medicine (Isolation of Baicalin fromScutellariae radix)Herb Medicine (Isolation of Glycyrrhizin fromGlycyrrhiza Galbra)Herb Medicine (Isolation of Secologanin formSymphoricarpos Albus)Herb Medicine (Oridonin, Enmein)Hydrophobicity Parameters of DrugsIndole Acetic AcidsL-Leucine DehydrogenaseLipid A from Cell Membrane of SalmonellaMetal IonsModified Oligo-PeptidesMonosaccharideN-propyl-p-hydroxybenzoate & PrednisoloneNucleoside (Adenine, 2-Deoxyadenine, 2-Deoxycytidine, 2-Deoxyguanosine, Cytidine)Oligo-SaccharidesOlive Oil with Lipase
    • Selection of Two-Phase Partition SolventsA two-phase solvent system is used as separation medium in Centrifugal PartitionChromatography.One serves as the stationary phase, the other serves as the mobile phase.The solvents may be selected from an infinite variety of possible combinations.Followings are some frequently used solvent combinations.
    • Amphotericin B (CIO4Salt) Separation
    • Ginsenosides from Panax quinquefolium LSeparation
    • APPLICATIONSBio Polymers Rare Metal and EarthBeta-Carotene Metal IonsBioreactor Modified Oligo-PeptidesChlorophyll Monosac CharideChromomycine N-propyl-p-hydroxybenzoate & PrednisoloneDiterpene Esters from Daphne extract CavanocobalaminEgg Yolk Lecithin L-Ascorbic AcidEnzymes from Yeast extract Fatty Acid EstersFatty Acids 1 Saturated Fatty Acids (Stearic Acid, Palmitic Acid, Myristic Acid and Lauric Acid)Fatty Acids 2 Unsaturated Fatty Acids (Stearic Acid, Linolenic Acid and Linoleic Acid)Fish Oil HydrolysateFungous Toxin NivalenolHerb Medicine (Isolation of Faicalin from Scutellariae Radix)Herb Medicine (Isolation of Glycyrrhizin from Glycyrrhiza Galbra)Herb Medicine (Isolation of Secologanin from Symphoricarpos Albus)Herb Medicine (Oridonin and Enmein)Hydrophobicity Parameters of Drugs Indole Acetic Acids
    • L-Leucine Dehydrogenase Lipid A from Cell Membrane of SalmonellaNucleoside (Adenine, 2-Deoxyadenine, 2-Deoxycytidine, 2-Deoxyguanosine and Cytidine)Oligo-Saccharides Olive Oil with LipasePaeoniflorin from Peony extract Paeoniflorin from Peony extract (Large scale)Phthalic Acid Isomers from Dilute Aqueous Solution Pigments from GardeniaPigments in Sea Squirt Proteins in Aqueous two phase system 1Proteins in Aqueous two phase system 2Quinones (Anthraquinone, p-Benzoquione and Alpha-Naphthoquione)Saiko Sapoins 1 Saiko Sapoins 2Salmon Sperm DNA Saponins from GinsengSerum Proteins Synthetic Lipid ATannins Terpendoids in Tobacco LeavesTocopherols Tocopherols (Large scale)Toxic Substance in Azalea UbiquinoneVegetable Oil HydrolysateVitamins (A, D and E) Vitamins (Nicotinamide)Riboflavine ThiamineFermentation Products Amino AcidsFine Chemicals AlkaloidsFoods and Additives Co-enzymesGenetically Engineered Substances Fatty AcidsNatural Organic Compounds Iridoid GlucosidesPetrochemicals Natural ProductsPhysiological Activated Substances PeptidesPharmaceuticals PhospholipidsProteins Steroidal Saponins
    • A novel and simple method has beendeveloped for the mutual separationof such metal ions as Cu(II), Mn(II),Co(II), Ni(II) by high performancecentrifugal partition chromatography(HPCPC) with di-2-methylnonylphosphoric acid/heptaneas a stationary phase. The HPCPCsystem was operated with 2,136partition channels, at a rotationalspeed of 800 rpm, and at a flow rateof 2.0 cm3/min. The four transitionmetal ions could be eluted separatelyby changing pH of the chloroaceticacid mobile phase at two steps. Theelution curves were obtained bymonitoring the absorbance of eachmetal complex post-labeled with 4-(2-pyridylazo)resorcinol.