HPTLC PROFILING OF THE VARIOUS EXTRACTS
The powdered drug (5.0) is refluxed hexane (50ml x 3) for 2 hrs. The extract is
filtered and the solvent removed in a under reduced pressure. The residue (150mg) is
dissolved in 10ml hexane and used for analysis
3 mg of Alpa amyrin dissolved in 10 ml ethanol (0.3 mg/ml)
10 l each of the test solution and standard solution on pre coated silica gel 60 F254
TLC plate (E.Merck) of uniform thickness of 0.2 mm. Develop the plate in the solvent
system in twin tough chamber to a distance of 8 cm, scan it densitometrically at 200
nm, record the peak area against the concentration of Alpa amyrin applied.
HPLC PROFILING OF THE HEXANE EXTRACT
The HPLC system consist of Shimadzu LC -10 ATVP pump, a valve type Indicator,
Shimadzu SPDM 10 AVP model photo diode array detector (Shimadzu, Tokyo,
Japan) phenomemex Luna C-18(250*4.6mm) column with a particle size of 5mm .
Hexane and water (HPLC Grade, E Merck India) were used as eluent for the analysis
Sample: Hexane extract of Ficus microcarpa was used for HPLC profiling .Extract
was filtered through 0.22 m filters and used for HPLC analysis .
Standard solution was prepared by dissolving the Ficus microcarpa in hexane.
Chromatographic condition: Hexane : water (40:60) mixture was used as the
mobile phase. The column was equilibrated for half an hour and the mobile phase was
pumped at the rate of 1.0 ml/min with a backpressure of 200 psig. The injector and
detector were flushed with the mobile phase and the injection volume was 20ml for
the analytical wok. The chromatogram was observed at 298 nm.
ISOLATION AND CHARECTERISATION
Using Preparative TLC
A TLC Plate was Prepared on a (20X20cm) size glass plate. A slurry was prepared by
50gm of silica gel G and 100ml distilled water and blended thoroughly in a conical
flask. It was poured in to an applicator placed on a glass plate and drawn to obtain
layer of 0.2mm thick layer. Four such plate were prepared. It was kept at room
temperature for 30 minutes, and then activated at 100 degree Celsius for 1 hour. These
plates were used for preparative TLC .Samples applied at a one side one centimeter
above the margin. The plate developed in a solvent system chloroform:
hexane(6:4).after the development each plate was monitored in UV 254 nm. and blue
fluorescence band was scraped out and dissolved in methanol.(100ml).The methanol
extract thus obtained was concentrated on a water bath to 10ml.This was kept aside
the residue obtained was recrystallised from methanol and the yield of this colorless
.powder was about 10mg.this was subjected to UV and IR analysis.
ULTRA-VIOLET VISIBLE SPECTROPHOTOMETER
Shimadzu UV 1700 model double spectrometer, Quartz cuvettes.
A small quantity of the extract is dissolved in small quantity of the solvent and
background correction or base line correction was done with the used solvents. And UV
spectrum was recorded. And we get peaks particular nano meter according to the
absorption of deferent compounds.
Each compounds have a particular absorption in UV region. From that reading we can
find out the identity of the compound isolated.
FOURIER TRANSFORM INFRARED SPECTROSCOPY
Shimadzu-8400 Spectrometer (shimadzu, Tokyo Japan) NaCl crystal
FTIR spectrum of the extracts were taken was done in Shimadzu spectrometer using
8000DRS reflectance attachment and integrated using kubelka munk conversion.
Background correction was done with appropriate solvent. And the extract mixed with
the solvent was introduced in it and spectrum recorded. The spectrum of one isolated
compounds were taken.