Counter current extraction is a multi-stage liquid-liquid extraction method that separates components based on solubility in two immiscible liquids flowing in opposite directions. Utilizing Craig apparatus, it efficiently isolates substances such as antibiotics and chemical compounds while preventing thermal degradation. The study successfully separated anti-ulcer compounds eupatilin and jaceosidin from artemisia extracts using a two-phase solvent system, demonstrating superior yield and purity compared to traditional chromatography.

1. COUNTER CURRENT
EXTRACTION
ABHISHEK MAHAJAN
M. PHARMACY 1ST YEAR
CENTRAL UNIVERSITY OF PUNJAB

2. COUNTER CURRENT EXTRACTION
• Counter current extraction is a method of multiple liquid- liquid
extraction
• Separation of components having variable solubility in two immiscible
liquid phases is achieved
• In the counter current extraction two immiscible solvents flow in an
opposite direction in multiple stages, (after several stages pure A and B
solvents can be obtained)
• In liquid- liquid extraction the solvent is used to extract another liquid
phase

3. • To distribution of active principles between water and organic
solvent depends on the hydrophilic groups present in the constituent
molecules
• If hydrophilic groups are ionisable , pH will be an important factor

4. Principle of Counter Current Extraction
The distribution of a single solute between two immiscible liquids the
fraction p distribution into the upper phase is a function of the partition
coefficient k and the U of upper to lower phase volume
p=KU/KU+1
The fraction of solutes in the lower phase at equilibrium q is given by,
q=1/KU+1
since p+q=1
The greater the value of KU the larger the fraction p of solutes that
passes into the upper phase

5. Process of Counter- Current Extraction
• In these extraction wet raw material is pulverised using toothed disc
disintegrators to produce the fine slurry
• The material to be extacted is moved in the one direction (generally in
the form of fine slurry) within a cylindrical extractor where it comes in
contact with extraction solvent
• The further the starting material moves the more concentrated the
extract becomes
• Finally, sufficiently concentrated extract comes out at one end of the
extractor while the marc falls out the other end

6. Theory
• A method of multiple liquid-liquid extractions is countercurrent extraction,
which permits the separation of substances with different distribution
coefficients (ratios).
• A clever design known as Craig apparatus is used for this purpose.
• Craig apparatus consists of a series of glass tubes (r: 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. In the following
animated picture of a single glass tube the typical "extraction/transfer" cycle
is shown.

7. • The lower (heavier) phase of the two-phase solvent system (e.g. water, blue
layer in the picture) is the "stationary phase", whereas the upper (lighter)
phase (e.g. hexane, red layer in the picture) is the "mobile phase".
• It is interesting to examine the distribution of a substance A in each tube after
a given number of equilibration/transfer cycles.

8. • In the beginning, tube #0 contains the mixture of substances to be separated in
the heavier solvent and all the other tubes contain equal volumes of the same
solvent.
• The lighter solvent is added to tube #0, extraction (equilibration) takes place
and the phases are allowed to separate.
• The upper phase of tube #0 is then transferred to tube #1 and fresh solvent is
added to tube #0, and the phases are equilibrated again.
• The upper layers of tubes #0 and #1 are simultaneously transferred to tubes #1
and #2 respectively. This cycle is repeated to carry on the process through the
other tubes of the apparatus.
• Obviously, substances with higher distribution ratio move faster than those with
a lower distribution ratio.

10. Advantages
• CCE is a commonly done at room temperature which spare the thermolabile
constituent from exposure to heat which is employed in most other techniques
• As the pulverization of the drug is done under wet conditions the heat
generated during communication is neutralized by water this again spares the
thermolabile constituent from exposure to heat
• The extraction procedure has been related be more efficient and effective than
continuous hot extraction

11. • Isolation of antibiotics (penicillin G) from the aqueous fermentation broth
using immiscible solvent (amyl acetate or butyl acetate)
• Isolation of chemical compounds from the aqueous systems using small
quantities of organic solvents in the production of synthetic drugs and
intermediates.
• In petroleum industry products having different chemical structure but about
the same boiling range are separated.
• Separation of components from synthetic mixtures.
• Separation of components from plant extract.
• Purification of compounds (removal of impurities)
Application

13. • Counter Current Extraction has great merits of eliminating the
irreversible adsorption or chemical reaction that occur in solid absorbents
based conventional column chromatography
• The yield and recovery of target compounds are superior to those of solid
support based column chromatography
• In the present study, HSCCC was applied to separate two anti ulceric
compounds, eupatilin and jaceosidin from Artemisia extracts
• The aerial parts of A. princeps (600 g) were extracted with ethanol (4.5 l)
and evaporated under reduced pressure to give ethanol extract(Et-Fr)
• Et-Fr was suspended in water (1.5 l) and partitioned with n-hexane and
ethyl acetate sequentially

14. • The ethyl acetate layer was concentrated to dryness in reduced pressure by
rotatory evaporator to afford ethyl acetate soluble extract (Ea-Fr)
• Et-Fr was subjected to Sephadex LH-20 column chromatography to obtain
two subfractions (S1 and S2)
• Subfractions S2 were combined and concentrated to give of semi-purified
ethanol extract (Se-Fr)
• The coefficient (K) values of eupatilin and jaceosidin from Se-Fr were
estimated at different volume ratio of n-hexane– chloroform–methanol–
water system (1:5:5:2, 2:5:5:2, 3:5:5:2, v/v/v/v)
• Two milliliters of the each phase of equilibrated two-phase solvent systems
was prepared in a vial, and then 1 mg of Se-Fr was added

15. • The vial was shaken vigorously to achieve the equilibration of target
compounds between the two phases, and kept for 30 min
• The upper and lower phases were separated and evaporated under N2 gas
• The residues of each phase were dissolved in methanol (1ml), and
subjected to HPLC to evaluate partition coefficient (K) value
• The K value was expressed as a peak area of eupatilin and jaceosidin in the
upper phase divided by that in the lower phase

16. • Solvent for HSCCC separation was thoroughly equilibrated in a separatory
funnel, and upper and lower phases were separated before use
• The column was fully filled with upper stationary phase at a flow rate of
10 ml/min by a fluid metering pump and rotated at 800 rpm
• In the meantime, the lower mobile phase was eluted in a descending mode
at a flow rate of 1.3–2.0 ml/min
• The Et-Fr. (150 mg), Ea-Fr. (100 mg) and Se-Fr. (50 mg) were dissolved in
4 ml lower mobile phase and subjected to HSCCC
• The monitoring of HSCCC peak fractions was performed by combining
effluent line of the HSCCC apparatus to UV detector (wavelength at 340
nm)
Procedure

17. • In case of cirsilineol (5,4’-dihydroxy-6,7,3’-trimethoxyflavone), the
position isomer of eupatilin (5,7-dihydroxy-6,30 ,40 -
trimethoxyflavone, are contained in artemisin species as minor

18. Conclusion
• In the present study, two anti-ulcer flavonoids, eupatilin and jaceosidin,
were separated by HSCCC method.
• Two-phase solvent system composed of n-hexane–chloroform–methanol–
water was utilized and the optimal separation condition was evaluated to
achieve successful separation.
• The yield, recovery and purity of eupatilin and jaceosidin separated by
HSCCC were superior to those of conventional chromatography methods
• Thus, HSCCC separation of Et-Fr, Ea-Fr and Se-Fr were performed
utilising n-hexane–chloroform–methanol–water (2:5:5:2, v/v/v/v, 1.3
ml/min) conditions