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.

1. High Performance Centrifugal Partition Chromatography (HPCPC) is a
practical and suitable method, particularly on the preparative scale, for the
separation of biomolecules such as proteins, enzymes, etc.
Prof. Ravisankar
Vignan Pharmacy college
Valdlamudi
Guntur Dist.
Andhra Pradesh
India.
banuman35@gmail.com
00919059994000

2. What is HPCPC?
High Performance Centrifugal Partition Chromatography, HPCPC, is a
new technique of Liquid Chromatography, LC, which is support free,
and which finds its place among the several Liquid Chromatographic
methods as shown on the following chart .

3. Centrifugal force (from Latin word
centrum, meaning "center",
and fugere, meaning "to flee")
represents the effects of inertia that arise in connection with
rotation and which are experienced as an outward force away
from the center of rotation

5. Around 50years ago, the concept of partitioning solutes between two liquids gave
birth 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 Instruments
Company Ltd.) opened the way to high performance centrifugal partition
chromatography (HPCPC), which was taking the best of the two first techniques
The versatility of a true liquid- liquid process combined with the quickness and advanced
technology of chromatography.
The HPCPC is gaining more and more interest as a semi-prep and preparative scale
chromatographic method.

6. There are four main advantages of the HPCPC as follows:
1) No loss of sample: Both mobile and stationary phases are liquids
and can be collected for total recovery.
2) The volume ratio of stationary to mobile phase is definitely much
higher, which leads to higher capacities and better resolution with no
requirement of a high number of theoretical plates.
3) The extreme flexibility of biphasic system: The mixtures of the
two, three or four solvents, which allows to modify the selectivity of a
system in order to get a pure compound in the HPCPC polarities of
both phases can be smoothly modified.
4) The reduced solvent consumption is ten times less than preparative
scale chromatography for the same throughput, it should be
environmentally considered. Separation accomplished with laboratory
HPCPC can be directly scaled up to production scale HPCPC.
Without a solid stationary phase support, The HPCPC system effectively
separate, isolate and purify milligrams to multigrams.

9. The 4main advantages of the HPCPC over its parent prepscale
column chromatography are :
1.No loss of sample since both mobile and stationary phases are liquids and can be
collected for total recovery.
The volume ratio of stationary to mobile phases is definitely much higher, which leads to
higher 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), which
allows to modify the selectivity of a system in order to get a pure compound, in the HPCPC the
polarities of both phases can be smoothly modified.
3.The reduced solvent consumption, ten times less than for preparative scale chromatography
for the same throughput, which is of interest for environmental considerations. Separation
accomplished 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 added
materials like chiral selectors or complexing agents can be recovered with no loss. Several
publications in international journals bring valuable information.
The new innovated HPCPC is getting more involved in many fields of chemistry, for purification
of antibiotics, pePtides, tannins, saponins, lipids, drugs, ...... its future development will see
the production of bigger HPCPCTM units, and it will incorporate crucial fields of chemistry,
such as chiral separations.

10. Superior to Conventional Preparative LC
HPCPC is fast !
Since stationary phase solvents are retained in the partition channels by centrifugal force, high
mobile phase flow rates may be used without appreciable loss of resolution.
Great advantages over conventional preparative LC
Since a solid support is not used with HPCPC, irreversible retention of valuable sample
components is completely eliminated, denaturation and decomposition, often encountered
with conventional LC column packings, are virtually eliminated. And this is generally
accomplished with retention of biological activity.
Moreover, the capacity of an HPCPC column (rotor) is significantly greater than an
HPLC column of the same total volume. Consequently, overload effects are rarely
encountered with HPCPC. Purification of large quantities is routine, always with
100% material balance...there is no adsorption or irreversible retention.

11. No columns or Packings to replace
The problems of formation of voids, contamination of fractions with silica and with
components of previous runs, and the cost of replacing expensive HPLC columns are gone.
Fewer Theoretical Plates are Needed
To achieve a given level of resolution between two peaks with HPCPC than with
HPLC, 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) with
HPLC....whereas only 2,200 plates are needed to do the job with HPCPC. This is a
direct consequence of the standard resolution equation for liquid chromatography.
Normal phase and reversed phase chromatography can be done in the same run
Use dual mode HPCPC to accomplish even the most demanding separations of
samples containing complex mixtures of polar/non-polar, hydrophilic/hydrophobic
substances. Even chiral substances are resolved with suitable chiral HPCPC phases.
Work at any pH
With HPCPC, there is no need to concern yourself with unwieldy pH limitations that
are often associated with solid stationary phase supports.
Innovations overcome previous weakness of the CPC

12. 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 the
separation executed files (solvents, rotations, flow rates, etc.) so that the most suitable
conditions can be considered sequentially.
3) Sequential separations with same executed file and sequential injections into same
stationary phase (reproducibility is very good by means of using same condition for
separation of samples) can be performed easily. Automation can be materialized by
connection of Autosampler and Fraction Collector.
4) Data and file handlings will become easy by linking with computer in which data
acquisition 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.

15. 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 of
complex mixtures of chemical substances.
The HPCPC is an alternative to packed-bed columns for preparative HPCPC and operates by
classical liquid-liquid partitioning in a high performance centrifugal system.
A solid stationary phase is not used. Instead, stationary phase liquid is retained by centrifugal
force 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 its
capacity.
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 mobile
phase liquid pass continuously through the stationary phase liquid. Any two-phase solvent
mixture can be used at any pH to perform normal and reversed phase chromatographic
separations.
An injected sample, carried by the mobile phase, moves sequentially through the partition
channels, 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.

19. Simple scheme used by the pioneers of HPCPC to represent the
arrangement between channels and ducts, with the droplets of mobile
phase flowing in the stationary phase.

20. Schematic representatin of the HCPC apparatus.

21. The HPCPC rotor is constituted by the
superposition of disks engraved with small
cells connected by head / tail ducts. These
cells, where the chromatographic separation
takes place, can be compared to lined-up
separate funnels. The rotor is filled with the
stationary phase, which stays inside the rotor
thanks to the rotation speed, while the mobile
phase is pumped through. CPC can be
operated in either descending or ascending
mode, where the direction is relative to the
force generated by the rotor rather than
gravity. According to the fast and permanent
evolution of the cells design, the efficiency and
flow rate with low back pressure are improved.
The CPC offers now the direct scale up from
the analytical apparatuses (few milliliters) to
industrial apparatuses (some liters) for fast
batch production

23. rotor (=column

25. Single HPCPC Unit Introduces Dual Applications
Separation by either normal-phase or reversed-phase elution is accomplished with a single two-phase solvent system. Dual-mode
HPCPC 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 strong
affinity 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 within
the stationary phase.
(3)Recycle (4)Reversed Phase Elution
After c, d & e have eluted, the Upper Mobile Phase is recirculated for a time sufficient for compete partition of components a & b.

26. 1) Washing
2) Injection
3) Ascending Mode (Mobile Phase : Upper)
4) Descending Model (Mobile Phase : Lower)
Flow Diagram of CPC System

32. Applications
Alkaloids (1-Strychnine, Quinine, Cinchonidine)
Alkaloids
(Quinine, Strychinine, Brucine, Berberine)
Angiotensin II
Assay of Environmental Pollutant
Bale Acids (Taurocholic
Acid, Taurochenodeoxycholic Acid, Cholic
Acid, Lithocholic Acid, Dexoycholic Acid)
Beta-Carotene
Bioreactor
Chlorophyll
Chromomycine
Diterpene Esters from Daphne extract
Egg Yolk Lecithin
Enzymes from Yeast Extract
Fatty Acid Esters
Fatty Acids (1) ? Saturated Fatty Acids (Stearic
Acid, Palmitic Acid, Myristic Acid, Lauric Acid)
Fatty Acids (2) ? Unsaturated Fatty Acids
(Stearic Acid, Linolenic Acid, Linoleic Acid)
Fish Oil Hydrolysate

33. Fungous Toxin Nivalenol
Herb Medicine (Isolation of Baicalin from
Scutellariae radix)
Herb Medicine (Isolation of Glycyrrhizin from
Glycyrrhiza Galbra)
Herb Medicine (Isolation of Secologanin form
Symphoricarpos Albus)
Herb Medicine (Oridonin, Enmein)
Hydrophobicity Parameters of Drugs
Indole Acetic Acids
L-Leucine Dehydrogenase
Lipid A from Cell Membrane of Salmonella
Metal Ions
Modified Oligo-Peptides
Monosaccharide
N-propyl-p-hydroxybenzoate & Prednisolone
Nucleoside (Adenine, 2-Deoxyadenine, 2-
Deoxycytidine, 2-Deoxyguanosine, Cytidine)
Oligo-Saccharides
Olive Oil with Lipase

34. Selection of Two-Phase Partition Solvents
A two-phase solvent system is used as separation medium in Centrifugal Partition
Chromatography.
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.

36. Amphotericin B (CIO4Salt) Separation

37. Ginsenosides from Panax quinquefolium L
Separation

38. APPLICATIONS
Bio Polymers Rare Metal and Earth
Beta-Carotene Metal Ions
Bioreactor Modified Oligo-Peptides
Chlorophyll Monosac Charide
Chromomycine N-propyl-p-hydroxybenzoate & Prednisolone
Diterpene Esters from Daphne extract Cavanocobalamin
Egg Yolk Lecithin L-Ascorbic Acid
Enzymes from Yeast extract Fatty Acid Esters
Fatty 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 Hydrolysate
Fungous Toxin Nivalenol
Herb 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

39. L-Leucine Dehydrogenase Lipid A from Cell Membrane of Salmonella
Nucleoside (Adenine, 2-Deoxyadenine, 2-Deoxycytidine, 2-Deoxyguanosine and Cytidine)
Oligo-Saccharides Olive Oil with Lipase
Paeoniflorin from Peony extract Paeoniflorin from Peony extract (Large scale)
Phthalic Acid Isomers from Dilute Aqueous Solution Pigments from Gardenia
Pigments in Sea Squirt Proteins in Aqueous two phase system 1
Proteins in Aqueous two phase system 2
Quinones (Anthraquinone, p-Benzoquione and Alpha-Naphthoquione)
Saiko Sapoins 1 Saiko Sapoins 2
Salmon Sperm DNA Saponins from Ginseng
Serum Proteins Synthetic Lipid A
Tannins Terpendoids in Tobacco Leaves
Tocopherols Tocopherols (Large scale)
Toxic Substance in Azalea Ubiquinone
Vegetable Oil Hydrolysate
Vitamins (A, D and E) Vitamins (Nicotinamide)
Riboflavine Thiamine
Fermentation Products Amino Acids
Fine Chemicals Alkaloids
Foods and Additives Co-enzymes
Genetically Engineered Substances Fatty Acids
Natural Organic Compounds Iridoid Glucosides
Petrochemicals Natural Products
Physiological Activated Substances Peptides
Pharmaceuticals Phospholipids
Proteins Steroidal Saponins

41. A novel and simple method has been
developed for the mutual separation
of such metal ions as Cu(II), Mn(II),
Co(II), Ni(II) by high performance
centrifugal partition chromatography
(HPCPC) with di-2-
methylnonylphosphoric acid/heptane
as a stationary phase. The HPCPC
system was operated with 2,136
partition channels, at a rotational
speed of 800 rpm, and at a flow rate
of 2.0 cm3/min. The four transition
metal ions could be eluted separately
by changing pH of the chloroacetic
acid mobile phase at two steps. The
elution curves were obtained by
monitoring the absorbance of each
metal complex post-labeled with 4-
(2-pyridylazo)resorcinol.