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
a) LIQUID –LIQUID EXTRACTION.
b) 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.

6. 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.

7. PRINCIPLE
Distribution coeffecient:
At certain temperature the ratio of
concentrations of a solute in each of the solvents
is always constant. This ratio is known as the
distribution coefficient.
Kd =Concentration of solute in phase 1/
Concentration of solute in phase 2

8. 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.

10. 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.

11. INSTRUMENTATION

12. COLUMNS OF CCC
A. 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
 They enclose geometrical volumes ,tubes, channels, or
locules that repeat themselves through connecting tubes
forming a pattern.

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

14. 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.

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

16. 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

17.  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.

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

19. 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 After 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

20. 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.

21. 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.

23. 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 :-
a) Liquid-solid extraction
b) Bonded phase extraction
c) Column phase extraction
d) Digital chromatography

24. 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.

25. STEPS INVOLVED IN SOLID PHASE
EXTRACTION

26. 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.

27.  96 Well Plate:
Simultaneous sample
processing allows 96 samples
to be extracted in
approximately in 1 hour or
less.
 Syringe Barrels
 Pipette Tip

28. 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

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

30. SOLVENT ELUTION

31. 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 .

32. 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.

33. 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.

34. 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

35. 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.

37. 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.

38. 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.

39. 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.

41. 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}

43. 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

44. 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

45. 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.

46. 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

47. 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

48. PROCESS
 Buffer selection.
 Sample preparation and loading.
 Flow rate.
 Elution.

50. 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

51. 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.

52. 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.

53. APPLICATIONS
 Used 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.

54. REFERENCE
 http://www.authorstream.com/Presentation/shilpabhat1089-
1543269-gel-filtration-chromatography/
 http://epgp.inflibnet.ac.in/epgpdata/uploads/epgp_content/S0
01174BS/P001204/M011038/ET/1479291507P9M16eTextO
ct12.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

55.  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-

56. THANK YOU