Counter current Extraction Solid phase extraction Gel filtration

Counter current extraction methods

1. COUNTER CURRENT EXTRACTION SOLID PHASE EXTRACTION GEL FILTRATION PRESENTED BY : G.VARSHASRI UNDER GUIDANCE OF : Mrs.K.S.L.HARIKA M PHARM , MBA , (PhD)

2. CONTENTS INTRODUCTION. COUNTER CURRENT EXTRACTION. SOLID PHASE EXTRACTION. GEL FILTRATION. REFERENCE. CONCLUSION.

3. INTRODUCTION Extraction is a separation process consisting of separation of a component from a liquid, semisolid, or solid material. It includes LIQUID –LIQUID EXTRACTION. SOLID PHASE EXTRACTION.

4. LIQUID-LIQUID EXTRACTION Liquid-liquid extraction (LLE) is also known as solvent extraction and partitioning, is a method to separate compounds based on their relative solubilities in two different immiscible liquids usually a polar solvent and a non polar solvent. There is net transfer of one or more species from one liquid into another liquid phase. The solvent that is enriched in solute is called extract. The residual feed solution is called as raffinate.

5. COUNTER CURRENT EXTRACTION Counter current extraction is an analytical technique which was developed by Lyman C. Craig in 1940s. Counter current extraction is a technique of liquid-liquid extraction which permits the separation of mixture of two immiscible liquid phases according to its relative solubility in two phases i.e one acts as stationary liquid phase and another one acts as mobile liquid phase and no solid support. It is also called as counter current chromatography .

6. PRINCIPLE Distribution coeffecient : At certain temperature the ratio of concentrations of a solute in each of the solvents is always constant. T his ratio is known as the distribution coefficient. K d =Concentration of solute in phase 1/ Concentration of solute in phase 2

7. STEPS IN EXTRACTION PROCESS There are various steps involved in this process Preparation of separating funnel with two phase solvent system. Introduction of compound mixture into the separating funnel. Vigorous shaking of the separating funnel to mix the two layers and allow for mass transfer of compounds in and out of the phases. The contents of separating funnel are allowed to settle back into two distinct phases. The two phases are separated from each other by draining out the bottom phase.

8. CRAIG’S APPARATUS Craig apparatus consists of a series of glass tubes (0 , 1, 2..) that are designed and arranged such that the lighter liquid phase is transferred from one tube to the next. The liquid-liquid extractions are taking place simultaneously in all tubes of the apparatus which is usually driven electromechanically.

9. INSTRUMENTATION

10. COLUMNS OF CCC Hydrostatic CCC column: The very first hydrostatic CCC columns used gravity to maintain the liquid stationary phase; they were called droplet CCC (DCCC) columns. They needed very long elution times (days ). Their two main characteristics are: They have single axis of rotation generating a constant centrifugal field and T hey enclose geometrical volumes ,tubes, channels, or locules that repeat themselves through connecting tubes forming a pattern.

11. It can be seen that there is quite a significant volume of connecting ducts which only contain the mobile phase .

12. B. Hydrodynamic ccc column Hydrodynamic centrifuges used in the CCC columns have two rotational axes, a main axis and a planetary one which generates a variable centrifugal force field. There can be any number of planetary axes but the most common are single, double, and triple axes. Each planetary axis has a bobbin or spool mounted on it that contains the coils of continuously wound Teflon tubing.

13. TYPES OF CCC Droplet counter current chromatography Elution extrusion current chromatography Centrifugal partition chromatography High speed counter current chromatography

14. CHOICE OF SOLVENTS Critical points in selection are : Sample solubility Partition coefficient Chloroform based system (or) Ternary phase diagram is used for the selecting the solvent system

15. Foucault,suggested three criteria to follow ternary phase diagrams: Select the best solvent in which sample can be completely dissolved . Select two solvents (one is less polar another is more polar), best solvent will partition into the two other solvents. The less polar fraction of the sample will preferentially go into the less polar phase and the more polar fraction will preferentially go into the more polar phase so that average partition coefficient stay around 1.

16. SOLVENT SYSTEMS Biphasic liquid system -octanol and water. Tertiary liquid system - chloroform-methanol-water. Quaternary liquid system - hexane-ethyl acetate- acetone-water.

17. MODES OF OPERATION Normal Phase MP: Non-polar , SP: Polar. Reverse Phase MP: Polar , SP: Non-polar. Elution-Extrusion As discussed in types. Gradient Mode Polarity of solvents is gradually increased. Dual Flow MP and SP are reversed part way , direction of flow changed. Dual Mode From starting both phases flowing in opposite direction. Recycling Mode Afte r elution , the target compounds are reintroduced into column. Ion Exchange and pH zone refining Mode Modifying both the phases after pre-equilibration. (MP and ionic displacer , SP and ionic retainer ) or by acids or bases. TYPE OF MODE DESCRIPTION

18. ADVANTAGES It is simple, rapid, and reproducible. High sensitivity. High performance . Rapid process and hence time saving . It is having a high resolution and separation capacity . Less requirement of mobile phase in developing chamber . Early recovery of separated component.

19. APPLICATIONS Countercurrent chromatography and related liquid-liquid separation techniques have been used on both industrial and laboratory scale to purify a wide variety of chemical substances . It has wide application for preparative separation of plant constituents and other natural products. It is particularly indicated for the isolation of polar compounds. It is known for its high dynamic range of scalability: milligram to kilogram quantities purified chemical components may be obtained with this technique. It also has the advantage of accommodating chemically complex samples with undissolved particulates.

20. SOLID PHASE EXTRACTION

21. INTRODUCTION Solid phase extraction(SPE) is a process of bringing a liquid or gaseous test sample in contact with a solid phase, whereby the analyte is selectively adsorbed on the surface of solid phase. In modern SPE, the adsorbent is packed between two flitted disks in polypropylene cartridges and the liquid phases are passed through the cartridge either by suction or by positive pressure. The various synonyms of SPE are :- Liquid-solid extraction Bonded phase extraction Column phase extraction Digital chromatography

22. PRINCIPLE The basic principle of Solid Phase extraction is Adsorption. Solid phase must have greater affinity for the analyte than the sample matrix. Separation is done based on the relative affinities of the compounds of a mixture to get adsorbed onto a solid phase. Compounds retained on the solid surface can be removed by eluting solvent having greater affinity for analyte.

23. STEPS INVOLVED IN SOLID PHASE EXTRACTION

24. FORMATS OF SPE Disc : 0.5mm thick membrane where the adsorbent is immobilized in a web of micro fibrils. The sorbent is embedded in a web of glass fiber. Cartridge : Small glass or plastic open ended container filled with absorptive particles.

25. 96 Well Plate : Simultaneous sample processing allows 96 samples to be extracted in approximately in 1 hour or less. Syringe Barrels Pipette Tip

26. SOLID PHASES Activated charcoal Alumina Silica gel Magnesium silicate ( Florisil) Chemically bonded silica phases and polymers E.g . styrene divinylbenzene According to chemical nature of The functional group bonded to the silica or the copolymer The resulting phases are classified as Non-polar Polar ion exchangers Other solid supports Polymeric resins, cellulose and zirconia

27. Examples of selective stationary phases Reaction of phenobarbitone with pentafluorobenzyl bromide onto the adsorbent . Amphetamine by Chiral derivatization of solid Phases . Doxorubicin by the metal-loaded phases in which metal cation is loaded onto a reagent-labelled phase.

28. SOLVENT ELUTION

29. MODES OF OPERATION The SPE process can be performed in two ways: Off-line SPE On-line SPE In offline SPE eluate from the cartridge is introduced into the chromatograph by means of an injection loop. In on-line SPE the extraction cartridge is inserted as part of chromatographic equipment. The eluate from the cartridge is introduced into the chromatograph by means of hexa -port switching valve .

30. SPE is typically performed manually ( off-line )but there are some significant disadvantages with this approach: Manual (off-line) SPE is time-consuming as well as labor-intensive and compromises productivity. Exposure to hazardous or infectious matrices (such as biofluids ) involves safety issues. The recovery of the analyte can vary from batch to batch causing reproducibility problems.

31. Automated ( on-line ) SPE provides the following benefits : Enables direct injection of untreated samples (e.g., plasma, urine, serum, vegetable oils, and surface water). Automates sample cleanup and/or analyte enrichment. Faster and better automated methods are less prone to errors resulting in better reproducibility. Reduction of health risks , for instance, when handling biologically hazardous samples. Increased workload per system and, therefore, higher returns on investment.

32. APPLICATIONS Application of SPE in various fields: Impurity profiling of pharmaceuticals Environmental applications Applications in food chemistry Analysis of wines and other alcoholic beverages Application to biological fluids Hair analysis

33. Application to biological fluids : Simultaneous qualitative and quantitative determination of Drugs of abuse opiates , cocaine, or amphetamines. Prescribed drugs tricycle antidepressants, phenothiazine, benzodiazepines in biological fluids was developed. Eg: A Weak Cation-Exchange Monolithic SPE Column for Extraction and Analysis of Caffeine and Theophylline in Human Urine.

34. GEL FILTRATION

35. INTRODUCTION Gel-filtration chromatography , also known as ' size exclusion chromatography' , ' molecular exclusion chromatography'  or ' molecular sieve chromatography'  is the simplest and mildest technique that separates molecules based on their size difference. This approach allows each polypeptide to be purified from other different sized polypeptides by passing through a gel filtration medium packed into the column.  It is used to separate proteins, peptides, and oligo-nucleotides.

36. DEFINITION Gel filtration is a technique of partition chromatography in which the partitioning is based on the molecular size of the substances to be separated.

37. PRINCIPLE The separation of molecules on the basis of their molecular size and shape is achieved by gel filtration chromatography. It uses the molecular sieve properties of various porous resins. Large molecules that are completely excluded from the pores pass through the void space, interstitial spaces between the resin particles and thus, they elute first. While smaller molecules get distributed between the mobile phase inside and outside the beads and therefore pass through the column at a slower rate. Thus, they elute in the last.

38. THEORY Explained by STERIC EXCLUSION MECHANISM/THEORY . Molecules with different sizes will differ in distribution coefficient between two liquid phases. The total volume of a column(V t) may be regarded as containing three compartments . V t = Vg+ Vi + Vo where , Vg =Volume occupied by gel matrix Vi=Inclusion/Internal volume/Volume of liquid inside pores. {determined by amino acid linked to fluorescent molecule- dinitro phenyl/ascorbic acid} Vi = V t - Vo Vo=Free solvent molecule/Volume outside beads {Determined using Blue Dextran}

39. Intermediate size molecules enter intersects of gel in some fraction(K d),require the elution volume of solute(V e) Therefore , V e=Vo + K d Vi This describes the behavior of column with reference to all solutes(which get eluted from column). For molecules that can enter the intersects of gel, K d=0 Therefore, V e=Vo. When mixing or diffusion occurs, the diffusion equilibrium and the retention volume (VR) of the given species is given by eq. VR = V int + K d V int

40. where distribution coefficient (K d) is given by eq . K d = V i (acc) / V t where ,V i (acc) = Accessible pore volume. V t = Total pore volume. V int = Interstitial volume

41. GEL FILTRATION MEDIA The commonly used media for gel filtration chromatography are dextran ,polyacrylamide, dextran-polyacrylamide and agarose. They are available for a large range of pore size for separation of macromolecules of different sizes. A gel with a smaller range of pore sizes gives higher resolution, while a gel with a wider range gives lower resolution. However, the benefit of using wider range gels is fractionation of higher range of sizes when molecular weights of analytes to be separated are unknown.

42. Sl. No. Type of media Commercial name Molecular weight range (kDa) 1 Dextran Sephadex G-50 Sephadex G-75 Sephadex G-100 Sephadex G-200 1.5-30 3-80 4-150 5-600 2 Polyacrylamide Bio-Gel P-10 Bio-Gel P-30 Bio-Gel P-100 Bio-Gel P-150 Bio-Gel P-200 1.5-20 2.5-40 5-100 15-150 30-200 3 Dextran-polyacrylamide gels Sephacryl S-200 Sephacryl S-300 Sephacryl S-400 5-250 10-1500 20-8000 4 Agarose Sepharose 6B Sepharose 4B Bio-Gel A-0.5 Bio-Gel A-1.5 Bio-Gel A-5 10-4000 60-20,000 10-500 10-1500 10-5000 Different commercially available media for gel filtration chromatography

43. A column packing can be divided into three parts- swelling the gel pouring the gel into the column equilibration of the column . PACKING OF A COLUMN

44. PROCESS Buffer selection. Sample preparation and loading. Flow rate. Elution.

45. There are numerous factors which affect the separation quality in gel filtration. The important factors include- (i) Column dimensions (ii) Selection of media (iii) Composition of buffer (iv) Sample volume and concentration (v) Sample loading (vi) Flow rate (vii) Length of tubing (viii) Fraction size FACTORS AFFECTING RESOLUTION OF GEL FILTRATION CHROMATOGRAPHY

46. ADVANTAGES Good separation of large and small molecules using minimal volume of eluate. No sample loss altogether as solute interact very less with stationary phase. Doesn’t depends upon a particular pH, temperatue, ionic strength and buffer composition, elution is carried out isocratically . High resolution can be achieved as wide range of porous gels are available commercially.

47. DISADVANTAGES For good resolution there has to be a 10% difference in molecular mass of solutes . Good expertise is required to achieve best results. Standards are needed . High Investment cost.

48. APPLICATIONS U sed in determination of protein structure particularly protein tertiary structure. In desalting of various bio-macromolecules. Purifications of enzymes and other proteins. Viruses , enzymes, hormones, antibodies, nucleic acids and polysaccharides have all been separated and purified by use of appropriate gels or glass granules. Estimation of molecular sizes of proteins. A polishing step in multistep complex purification scheme applied particularly in industrial purification of recombinant proteins.

49. REFERENCE http://www.authorstream.com/Presentation/shilpabhat1089-1543269-gel-filtration-chromatography/ http://epgp.inflibnet.ac.in/epgpdata/uploads/epgp_content/S001174BS/P001204/M011038/ET/1479291507P9M16eTextOct12.pdf https://www.sigmaaldrich.com/life-science/proteomics/protein-chromatography/gel-filtration-chromatography.html http://195.134.76.37/applets/AppletCraig/Appl_Craig2.html https://www.ncbi.nlm.nih.gov/pubmed/28071034

50. http://m.authorstream.com/presentation/zafariqbal224-1917636-solid-phase-extraction-spe/ https://www.slideshare.net/SumaSam1/counter-current-chromatography-71947193 https:// www.slideshare.net/kaberinath123/countercurrent-chromatography https://chem.pg.edu.pl/kcha/science/didactic-courses/basic-spe-principles-of-solid-phase-extraction-

51. THANK YOU