APPLICATION OF SPECTROSCOPY IN
IDENTIFICATION OF
HERBAL DRUGS
MAHARSHI ARVIND INSTITUTE OF
PHARMACY
Mansarovar , jaipur
GUIDED BY PREPARED BY :
Mr Radheshyam kumavat. Jasmin modi
Mr sandip kataria
APPLICATION OF SPECTROSCOPY IN
EVALUATION OF HERBAL DRUGS.
Onces the plant constituent is isolated and purified next task is to determine
which class of compound is present in it .
After that we have to find out the particular substances is it
The class of compound is clear from its response to color test
i.e. colored or colorless,
its solubility ,
Rf properties, and
U.V spectral characteristics ,
Biochemical test can also be perform
For complete identification within the class depend upon the
measuring the properties and than comparing these data with those in
the literature.
these propertied includes,
Melting point (for solids)
Boiling point (for liquids)
Optical rotation (for optically active compounds)
Some informative data can also be done with the help of
spectroscopic methods.
likes,
Ultra violet (U.V),
infra red (I.R),
Nuclear magnetic resonance(N.M.R),
Mass spectroscopy(MS) ,
X- ray crystallography.
For final conformation these data are directly compare with authentic
materials.
U.V-visible spectroscopy
Fig UV visible spectrophotometer
Principle of seperation :
if compound is colored or colorless in both type the absorption depend
upon the nature of electron present.
Molecule has three type of electron n,π, and σ or combination of
these .
These electron absorb the radiation and undergoes transition from
ground to excited state.
Range:-For ultraviolet:200nm – 400nm
For visible 400nm – 800nm.
If any colored compound present in sample it will absorb in the above
range and if any colored less compound present than it will absorb in
the ultra violet range.
so we can get the absorption curve and than compound can be
evaluated (by plotting the graph of absorbance v/s wavelength.)
solvent widely used is 95% ethenol commercial absolute ethenol
should be avoided as it contain the tracs of [Link] absorb in
u.v region.
solvent such as pyridine and chloroform should be avoided as these
are absorb strongly in u.v visible region.
Table1:Spectral properties of the different classes of plant pigments.
Pigment class Visible spectral range(nm) Ultraviolet range(nm)
chlorophylls (green) 640-660 and 430-470nm Intense short u v absorption
due to protein atttachment.
Phycobilins (red and blue) 615-660 or 540-570nm
Cytochromes (yellow) 545-605(minor band
sometimes at 415-440)
Anthrocyanins (mauve or 475-550 Ca.275
red)
Betacynanins (mauve) 530-534 250-270
Carotenoids(yellow to orange) 400-500 -
Anthraquinones (yellow) 420-460 3-4 intense peak between 220-
290
Yellow flavonols 365-390 250-270
Table1:Spectral properties of the different classes of plant pigments.
If a substance shows absorption and between 250 and 260 nm ,it could be any of
considerable number compound b(phenol, a purine, or pyrimidine, an aromatic
acid )
This can be known by table1 data
The utility of spectral measurement for identification purpose can greatly
enhanced by repeating measurement in neutral solution either at a range of different
pH value or in presence of particular inorganic salts.
Ex. When alkali is added to alcoholic solution of phenolic compound the spectra
shift towards longer wavelength (batho chromic shift) by increasing absorbance..
(fig )
By contrst ,if alkali is added to neutral solution of aromatic carboxylic acid ,the
shifts is in the opposite direction to wards shorter wave length
Infra red spectroscopy :
Principle of seperation:
Every compound has
molecule
Atom or group connected by bond .
Assume bond as a spring and not rigid in nature.
because of contineous motion the molecule maintain some
vibration with some frequency.
This is characterstic to every molecule this call as natural frequency .
When I.R radiation is applied .
When applied energy= natural energy
I.R peak observed.
(which is characteristic for particular compound)
I.R spectra can be measured by using either in solution of chloroform
or ccl4 or as a Mull oil or as a solid state mix with KBr.
In mull technique or KBr disc method a thin disc is prepared under
anhydrous condition from 1 -10 mg of sample and 10 -100 mg of KBr.
Range:4000- 667cm-1
in I.R the region above 1200cm shows spectral band or peak due to
vibration due to individual bond or functional group in molecule.
The region below 1200cm shows spectral band or peak due to
vibration of whole molecule and because of this complexity this is
called as fingerprint region
Intensity of the various band are recorded subjectively on a simple
scale as being either strong(S), medium(M),or weak (w).
Many functional group can be identified by their characteristics
vibration frequencies( as given in table) which makes the I R
spectrum simplest and often most reliable method of detecting
compound to its class.
Class of compounds Approximate position of characteristic band
above 1200cm-1
Alkanes. 2940(S),2860(M)
Alkenes. 3050(W-M),1850(W),1650(W-M),1410(W).
Aromatics. 3050(W-M),2100-1700(W)
Acetylenes. 3310(M) , 2225(W),2150(W-M), 1300(W).
Alcohols and phenols. 3610(W-M) 3600-2400(BROAD), 1410(M)
Aldehydes and ketones . 2750(W)2680(W), 1820-1650(W), 1420(W-M)
Esters and lactones. 1820-1680(S)
Carboxylic acid. 3520(W) 3400-2500(broad,W), 1760(S)
Amines. 3500(M), 3400(M), 3400-100(VARIABLE)
Cyanides. 2225(W-S)
Isocynates. 2270(VS)
Characteristic I.R frequency for some class of natural compounds
in spite of this IR spectroscopy is most frequently used in phyto
chemical studies as a fingerprinting device for comparising the natural
with synthetic materials.(as shown in fig)
I R spectra for alkaloids is given in the fig two traces components of
tobacco smoke are identified as the bases harmane and nor
harmane , using the KBr disc procedure .
here it is notice that some detail in the finger prient region of both
allaloids is absent from the natural sample ,may be due to impurity .
Fig: Infra red spectrum of two alkaloids obtain from tobacco smoke (a) harmane
natural(i) and synthetic(ii), b norharmane,natural(i),and synthetic(ii)
Mass spectroscopy :
MS , sinces it is recent introduction , (1960).
one of the adv is that only micro gram quantity of material is
required , that can provide the accurate molecular weight and that may
yield a complex fragmentation pattern which is of characteristics of
(particular) .
But unlike UV and IR spectrophotometer the mass spectrophotometer
can not be operated by the phytochemist , it is more expensive and
sophisticated so they are operated by the train person only.
MS it consist of
Degrading the trace amount of an organic compound and recording
the fragmentation pattern according to mass.
The sample vapour diffuse in to the low pressure system of the mass
spectrometer where it is ionized with sufficient energy to causes
fragmentation of the chemical bonds.
The positively ions are accelerated in a magnetic field which
disperses and permits relative abundances measurement of of ions of
given mass to charge ratio.
The resulting record of ion abudance versus mass constitutes the mass
spectral graph ,
Which thus consists of a series of lines of varying intensity at different
mass units (as in fig)
The technique worth success with every low molecular weight plant
constituents, and has been also used for peptides .
Those compound which are too involatile to vaporized in MS
instrument are converted to trimethylsilyl ether, methyl ester or
similar derivatives. ex gibberellines.
MS is frequently used with the GLC , and the combine operation
provide the qualitative and quantitative identification of the many
structurally complex components, that may be present in a particular
plant extract.
Ex: zeatin the first naturally occuring cytokinin growth regulator to be
isolated from higher plant .
New technique in the development continue to emerge in mass
spectroscopy and modern spectrometers may be provided with a
FAST ATOM BOMBRDMENT, sources and are capable of analysing
the fragile and in volatile organic compound , including salt and
higher molecular weight materials.
previously , when using the MS analysis of the plant glycosides the O-
glycosides sugar were lost in the process and escaped detection but it
is now possible wit h the FAB-MS obtain the molecular ions for the
original glycosides.
References:
Phytochemical methods. By [Link],
Third edition.
