Drug Isolation processes:

Distillation,

Fractional distillation has been traditionally used for separation of the components of volatile
mixtures; in phytochemistry it has been widely used for isolation of components of volatile
oils. Steam distillation is much used to isolate volatile oils and hydrocyanic acid from plant
material

Steam distillation/hydro distillation: Usually the volatile oil, which is less affected by heat, is
isolated by this method. In this method plant material is either macerated with H2O or
directly passed with steam and the essential oil obtained in distillate is extracted with organic
volatile solvent like petroleum ether.

Crystallization,

Sublimation

sublimation may sometimes be possible on whole drug, as in the isolation of caffeine from
tea or for the purification of materials present in a crude extract. Modern equipment employs
low pressures with a strict control of temperature.Freeze-drying or lyophilization requires a
first step where the temperature of the extract (solutes and solvents) is reduced until they are
frozen, followed by a sublimation step carried out via a very high vacuum which sometimes
accompanied by heating.Freeze drying is widely applied whenever a thermolabile substance
is of interest. It forms part of extraction process of several products, for example; antibiotics,
antioxidants, hormones, etc

At certain critical pressure and temperature the gasses behaves like a liquid, which diffuse
well through solids and acts as good solvent. The gasses like CO2 are chemically inert, non-
inflammable, nontoxic, easy to eliminate, selective, readily available and do not cause any
hydrolysis or rearrangement of component to be extracted. Hence, in spite of high cost this
method is enormously spreading for supercritical fluid to liquid-liquid extraction. Now days
this method is used for production of decaffeinated coffea, nicotinless tobacco products and
terpenless oils.

Centrifugation, and

Chromatography

Classification: Morphologic, Taxonomic, Therapeutic, Chemical and Alphabetical

1. SEPARATION AND ISOLATION OF CONSTITUENTSThe instrumentation for the
structure for the structureelucidation of organic compounds becomes effective
andallows the use of increasingly.The most difficult operation in phytopharmacetical
researchis the isolation and purification of plant constituents.The physical methods
used are chromatographic techniquesand methods such as fractional crystallisation,
fractionaldistillation, fractional liberation.Chemical method is based on groups or
moieties present inthe compound and chemical reactions.

2. 27. FRACTIONAL CRYSTALLISATIONIt is an important method for the

purification ofcompounds from mixture.It depends upon the compound which form
crystals atthe point of super saturation in the solvent in which it issolubleMany natural
products are crystaline nature even inmixture, process such as concentration,
slowevaporation, refrigeration are using for crystalisation

3. 28. FRACTIONAL DISTILLATIONThis method is used for the separation ofthe

components from volatile mixturesLargely using in the separation ofhydrocarbons
from oxygenated volatileoil eg citral, eucalyptolFRACTIONAL LIBERATIONIn this
proces the groups of compoundshaving the tendency of precipitation fromthe
solution.Incertain cases the compounds maymodified by converting to its salt
form.This proces is often used in separation ofcinchona alkaloids, morphine etc.

4. 29. SUBLIMATIONHere the compound isheated the solid statechanges to gaseous

statewithout passing via liquidstate. Such compounds getdeposited in form ofcrystals
or cake.This method is traditionallyused for the separation ofcamphor from chips
ofcinnamomum camphora.

5. 30. CHROMATOGRAPY Chromatography is widely used for the separation &

identification of components of a mixture. Separation of chemical compounds is
carried out by mobile phase and stationary phase.Chromatography can be classified
according to mechanism ofseparation as:adsorption chromatography, partition
chromatography, ion exchange chromatography,size exclusion chromatography and
affinity chromatography.

6. 31. PAPER CHROMATOGRAPHYThe principle is partitionMainly the stationary

phase ismoisture present in the cellulosefibers and mobile vary as we using.The
components separated basedon their solubilityThe ratio between the distance travelled
on the paper by acomponent of the test solution & the distance travelled by thesolvent
 is termed the RF value. Under standard conditions, thisis a constant for the particular
compound.In practise, however, variations of the RF value often occur & itis best to
run a reference compound alongside the unknownmixtures.

7. 32. ADVANTAGESi. Simple & inexpensiveii. Sensitive – gives good separation of

very small amounts, of especially water-soluble compounds, e.g.
sugars.DISADVANTAGESi. Fragile – chromatogram may be destroyed by chemicals
used for visualizationii. May be time-consuming.

8. 33. THIN LAYER CHROMATOGRAPHY (TLC)TLC is an e.g. of adsorption

chromatography, the stationaryphase being a thin layer adsorbent held on a suitable
backing.Separation of the compounds present in the plant extractdepends on the
differences in their adsorptive/desorptivebehaviour in respect of the stationary
phase.TLC involves a thin layer of adsorbent, mixed with a bindersuch as CaSo4,
which is spread on a glass plate & allowed todry.The plant mixture to be separated is
applied as a spot near thebase of the plate, which is then placed in a closed glass
tankcontaining a layer of developing solvent.

9. 34. ADVANTAGES OF TLC OVER PAPER CHROMATOGRAPHY Separation of

compounds can be achieved more rapidly & with less plant material. -The separated
spots are more compact & clearly demarcated from one another -Reagents such as
concentrated H2SO4 would destroy a paper chromatogram, but ma be used to locate
the separated substances on a TLC plate.

10. 35. COLUMN CHROMATOGRAPHYIt is a method used to purifyindividual

chemicalcompounds from mixtures ofcompounds the principle ofseparation is
adsorption. The classical preparativechromatography column, is aglass tube with a
diameter from5 mm to 50 mm and a height of5 cm to 1 m with a tap and somekind of
a filter (a glass frit orglass wool plug – to prevent theloss of the stationary phase) atthe
bottom.

11. 36. GAS CHROMATOGRAPHY (GC)It is an analytical technique for separating

compounds based primarily ontheir volatilities. GC provides both qualitative and
quantitative information for individualcompounds present in a sample. Compounds
move through a GC column as gases, either because thecompounds are normally
gases or they can be heated and vaporized into agaseous state. The differential
 partitioning into the stationary phase allows the compoundsto be separated in time and
space.

12. 37. APPLICATIONSQuality controlcontamination of plant and plant based products

with pesticides, herbicides and manyother materials that are considered a health risk,
all such products on sale today mustbe carefully assayedIdentification of Source
/OriginThe source of many plants (herbs and spices) can often be identified from
thepeak pattern of the chromatograms obtained.Technique of fingerprint could really
identify the false herbal products.The fundamental reason of quality control of herbal
medicines is based onthe concept of phytoequivalence of herbs, and then to use this
conception toidentify the real herbal medicine and the false one, and further to
doquality control.Qualification and Quantification of
PhytoconstituentsAlkaloidsCapillary gas chromatography (GC), often coupled with a
mass spectrometeras a detector (GC-MS), is a well established technique for
analyzing complexmixtures of alkaloids.

13. 38. TerpenesA qualitative comparative study was performed for terpenesfrom volatile
oils by GC and GC-MS techniqueFlavanoids and FlavonesFlavonoids receive
considerable attention in the literature,specifically because of their biological and
hysiologicalimportance. Gas Chromatography Coupled to MassSpectrometry GC-MS
is established as a routine techniquefor the analysis of flavonoid aglycones.Essential
Oils /Volatile oilsMany pharmacologically active components in herbalmedicines are
volatile chemical compounds. Thus, theanalysis of volatile compounds by gas
chromatography isvery important in the analysis of herbal medicines

14. 39. High-performance liquid chromatography (HPLC)High performance liquid

chromatography is a powerful tool inanalysis. This page looks at how it is carried out
and showshow it uses the same principles as in thin layer chromatographyand column
chromatography.

15. 40. Application of HPLC1- Isolation and purification of biologically active natural
products2- Control of synthetic reactions Identification of intermediates and target
compound.3- Biosynthesis study Detection of biogenetic intermediates and enzymes
involved.4-Control the microbiological processUsed for separation of antibiotic from
broth mixture5- Pharmacokinetics study Pharmacokinetic study comprises the
measurement of drug metabolitesconcentration in body fluids, absorption,
bioavailability and elimination ofdrugsHPLC determines the drug and its metabolites
in one step.6- Stability test Rapid method of analysis in stability test.7- Quality
control HPLC is used to know the identity, purity and content of the
ingredients(drugs, raw and pharmaceutical products,8- Drugs metabolisms