Spectroscopic techniques can be used to estimate herbal drugs through qualitative and quantitative analysis. Various spectroscopic methods are described such as UV-Vis spectroscopy, IR spectroscopy, NMR spectroscopy, mass spectrometry, and fluorescence spectroscopy. These techniques can be used to detect, identify, and quantify unknown phytochemicals in herbal extracts. Specific examples are provided on using fluorescence spectroscopy to analyze extracts of Digitalis purpurea and detect components like Digoxin.

1. APPLICATIONS OF SPECTROSCOPIC
TECHNIQUES FOR ESTIMATION OF
HERBAL DRUGS
SURBHI SHARMA
17BPH097
SEMESTER 5

2. INTRODUCTION
❑Spectroscopy is the study of the interaction of light with matter. There are two
distinct aspects of this interaction that can be used to learn about atoms and molecules:
✔ Identification of the specific wavelengths of light that interact with the atoms and
molecules.
✔ Measurement of the amount of light absorbed or emitted at specific wavelengths.
❑Both determinations require separating a light source into its component wavelengths.
Thus, a critical component of any spectroscopic measurement is breaking up of light into
a spectrum showing the interaction of light with the sample at each wavelength.
❑Light interacts with matter in many ways. Two of the most common interactions are :
1. Light that is absorbed by the atoms and molecules in the sample
2. Light that is emitted after interacting with the atoms and molecules in the sample.
❑ Techniques can advantageously be employed for qualitative and quantitative analysis
of plant extracts.
❑A crude drug may be assayed for a particular group of constituents. Alternatively,
it may be necessary to evaluate specific components /phytoconstituents of the drug.
So, for this quality control parameter spectroscopic techniques are used.

3. ❑A spectrometer is a spectroscope with a meter or detector so it can measure the
amount of light (number of photons) at specific wavelengths. It is designed to provide a
quantitative measure of the amount of light emitted or absorbed at a particular wavelength.
❑Using a spectrometer, it is possible to measure which wavelengths of light are present and
in what relative amounts.
WORKING:-
1. Light enters the spectrometer via the entrance slit and then passes through several parts: an
objective lens, a grating, and an exit slit. This combination of parts functions as a
monochromator, a device that selects only one color from all of the wavelengths/colors
present in the source.
2. A particular wavelength is selected, using the wavelength control, the selected wavelength
is at the center of the narrow band of wavelengths passing through the slit.
3. The light then strikes a detector that generates a voltage in proportion to the intensity of
the light hitting it. That voltage is then used to drive a read-out device that is designed to
provide data in a format such as intensity.

4. Types of spectrometer
Working

5. •Absorption spectroscopy is the study of light absorbed by molecules. For absorbance
measurements, white light is passed through a sample and then through a device (prism)
that breaks the light up into its component parts or a spectrum. White light is a mixture
of all the wavelengths of visible light. When white light is passed through a sample,
under the right conditions, the electrons of the sample absorb some wavelengths of light.
This light is absorbed by the electrons so the light coming out of the sample will be
missing those wavelengths corresponding to the energy levels of the electrons in the
sample. The result is a spectrum with black lines at the wavelengths where the absorbed
light would have been if it had not been removed by the sample.
ABSORPTION SPECTROSCOPY

6. ❑ Ultraviolet (UV) and Visible Spectroscopy:
The absorption spectra of plant constituents can be measured in very dilute
solution against a blank solvent by using automatic recording spectrophotometer.
The colorless compounds absorb in the range of 200 to 400 nm i.e. UV range and
the colored compounds absorbs in the range of 400 to 700 nm i.e. the visible
region. Different natural products are analyzed by this technique
e.g.reserpine(268 nm), morphine (286 nm) and colchicine (360 nm).
❑Infrared (IR) Spectroscopy IR spectroscopy:
It is the valuable tool for the characterization of plant constituents. It is the study
of reflected, absorbed or transmitted radiant energy in region of electromagnetic
spectrum wavelength ranging from 0.8 to 500 nm. IR is a function of wave
number. It can be near IR region(1250 to 4000 cm−1 ), mid
I.R.(4000-400cm−1)and far IR (400-20 cm−1 ). The alkaloidal components can
be identified by
the application of IR spectroscopy.

7. ❑Nuclear Magnetic Resonance Spectroscopy (NMR):
NMR spectroscopy involves the absorption of radio frequency radiation by the
substances which are kept in magnetic field. Deuterated analogues of water,
chloroform, acetone etc. are used as solvent for this purpose. It can be proton
NMR -1 H-NMR or 13C-NMR.1 H-NMR is used for determining the structure
of an organic compound by measuring chemical shift values of different types
protons present in it. The 13C-NMR spectroscopy gives direct information
on the nature of the carbon skeleton of the molecule.

8. EMISSION SPECTROSCOPY
Emission spectroscopy is the opposite of absorption spectroscopy. The electrons
of the sample are promoted to very high energy levels by any one of a variety of
methods .As these electrons return to lower levels they emit light. By collecting
this light and passing through a prism, is separated into a spectrum. In this case,
we will see a dark field with colored lines that correspond to the electron
transitions resulting in light emission.
Mass Spectrometry (MS) :
Mass spectroscopy involves the conversion of sample into gaseous ions with
or without fragmentation which are then characterized by their mass to charge
ratio(m/z). It is used to identify chemical species by measuring a spectrum and
comparing it with spectra for known chemical species to find match.Qualitative
spectroscopy is used to identify chemical species by measuring a spectrum
and comparing it with spectra for known chemical species to find a match.

9. APPLICATIONS
✔A crude plant extract may contain upto hundreds of different secondary
metabolites of different chemical nature and spectroscopic study. So it is
used for detection, identification and quantification of unknown
phytoconstituents in crude drug.
✔Used for the standardization of phenolics for its antioxidant activities
✔To determine the nature of contaminants in a sample.U.V spectroscopy
is one of the best methods for determination of impurities in crude
drug. Additional peaks can be observed due to impurities in the sample
and it can be compared with that of standard raw material.
✔The technique is used to detect the presence or absence of functional
group in the compound. Absence of a band at particular wavelength is
regarded as an evidence for absence of particular group.
✔Molecular weight of the compound can be detected.
✔UV Spectroscopy is beneficial in qualitative analysis as we get spectra
with specific solvent extraction.Spectra got can be used as fingerprint of
the sample extract. Adulterant can be found out by spectral analysis.

10. •When radiation is passed through a layer of solution containing absorbing substance, part
of the radiation is absorbed. The magnitude of absorption is expressed in terms of
absorbance A.
A= abc
a=absorbance
b=path length
c=concentration
PROCEDURE:-
✔Accurately weigh 0.15gm of the drug, in fine powder and mix with 50ml water. Place on
water bath and heat under reflux condenser for 15 minutes.
✔ Allow to cool. Centrifuge it. Transfer 20 ml supernant liquid to a 150 ml separating
funnel.
✔Add o.1ml HCl .Shake well .Add CHCl3
at three times in 15 ml quantities. Discard the
CHCl3 extract. Add 0.1gm of Sodium bicarbonate and shake for 3 min.
✔Centrifuge the aqueous layer and transfer 10.0 ml of the supernant liquid to a 100ml
round bottom flask fitted with a ground glass stopper.
✔Add 20ml of ferric chloride solution and mix well. Heat in a water bath under a reflux
condenser for 20 min .Add 1 ml HCl and continue heating for 20 minutes with frequently
shaking until the precipitate is dissolved. Cool, transfer the mixture to a separating funnel
and shake with three quantities, each of 25 ml, of ether. Combine the ether extracts and
wash with two quantities, each of 15 ml, of water.
SENNA

11. ✔ Dilute the ether extracts to 100.0 ml with ether, evaporate 10.0 ml
carefully to dryness and dissolve the residue in 10.0 ml of a 0.5% w/v
solution of magnesium acetate in methanol.
✔Measure the absorbance of the resulting solution at the maximum at
about 515 nm,using methanol as the blank. Calculate the percentage
content of hydroxyanthracene glycosides expressed as Sennoside B
taking 240 as the value of A(1%, )at the maximum at about 515nm.

13. Digitalis Pupurea:
✔ Digitalis purpurea ,extract of foxglove leaves with aqueous ethanol was
conducted to detect the presence of Digitalis purpurea , Digoxin, or
Digoxin-like substance in serial ultradilutions using IR spectroscopy or
fluorescence and cyclic voltametry for comparison of the components
of Digitalis purpurea, which has intrinsic fluorescence.
✔ The fluorescence is likely to be due to unsaturated lactone present in the
compound. The fluorescence spectrum of the compound is taken at
different dilutions in aqueous ethanol.

14. • Digoxin (B) is pure component showing emission maxima at 318 nm,
showing a relative intensity of 4.5 × 106
.
• Digitalis purpurea 6(C6) having emission maxima at 357 nm and 374 nm
showing a relative intensity of 13 ×106
and 12 × 106
.
• Digitalis purpurea 30 (D30)having emission maxima at 357 nm and
374 nm showing a relative intensity of 16 × 106
and 14 . 5 × 106
.
• E200 = Digitalis purpurea 200 having emission maxima at 357 nm and
374 nm, showing a relative intensity of ∼ 10 × 106
and 9 ×106
.

15. ✔ In all the emission studies, the samples were excited at 255 nm. The
emission spectra of different dilutions are shown as C, D, and E, and
purest medicinally active ingredient Digoxin is shown as B.
✔ These fluorescence spectra revealed that Digoxin component shows a
structure less fluorescent with emission maxima at 318 nm with a
relative intensity of 4.8x106.
✔ The emission spectra of Digitalis purpurea are shown in graph as C6.
In this case the structure less emission is found to be modified with
a semi structured emission with maxima at 357 nm and 374 nm and
further a shoulder at about 318 nm remained, which could be due to
the original B (Digoxin).
✔The appearance of additional bands at 357 and 374 nm may be
due to presence of impurities or dissociation of component
substance due to serial dilution, and these bands showed an increase in
intensity with subsequent dilutions up to a certain level.
✔RESULT: It is revealed from the emission spectra that Digoxin or
Digoxin-like product is present in the commercially available
serially ultra diluted Digitalis purpurea up to 200.

16. ❑Caralluma nilagiriana
✔Caralluma nilagiriana is a plant known to be rich in phytoconstituents that have
biological activities like antibacterial, treatment of rheumatism, diabetes, leprosy,
tumor, fungal diseases, snake and scorpion bites.
✔UV-VIS(UV-visible light spectroscopy) analysis showed the presence of phenolic
compound and flavonoids; while the peaks at 227.6nm, 401.6nm and 664.6nm of the
UV-VIS profile corresponded to those of the Flavonoids, Terpenoids, and
Chlorophylls.
✔About 1 g of the powdered material was then subjected to extractions using Methanol
for a duration extending up to 6 hours.The extracts were finally filtered and concentrated
in rotary evaporator under reduced pressure to result in thick green crude extracts.
✔The extracts obtained as above were scanned in the wavelength range of 190-900nm
UV spectrophotometer and the characteristic peaks were detected.

17. ❑Spectroscopic analysis by FT-IR(Infrared spectroscopy):
The extracted plant samples of Caralluma nilagiriana were scanned at
room temperature within a spectral range of 4000-400 cm-1. In the
present work it is possible to directly relate intensities of absorption
bands to the concentration of the corresponding functional groups.
The FTIR spectrum was used to identify functional groups of the
active components present in plant samples based on the peaks
values in the region of IR radiation. The results of FTIR analysis
had confirmed the presence of alcohol, phenol, Terpene group of
compound .
❑ Quantitative spectrophotometric analysis:
The UV-VIS profile of plant extract was studied over the 190 to 900
nm wavelength due to the sharpness of the peaks and proper
baselineThe profile showed the peaks at 227.6 nm, 401.6 nm and
664.6.

20. ❑ Discussion and Conclusion:
✔Spectroscopic technique has become a powerful analytical tool
for the qualitative and quantitative analysis of pharmaceutical
and biological materials.
✔ An IR spectrum of Caralluma nilagiriana plant extract shows the presence of OH
group, Phenol & Alcohol, Carboxylic Acids, Nitro Groups, Esters and Ethers
group of compounds, while its UV-VIS spectrum has absorption bands at 227.6, 401.6
and 664.6 nm those which are characteristic of flavonoids and its derivatives.
✔The Flavonoids and Terpenoids spectra typically consist of first two absorption
maxima, the first in the range 230-290 nm (band I) and the second in the range 400-550
nm (band II).
✔ The chlorophyll spectra typically consist of two absorption maxima in the range
600-700 nm (band III).
✔The precise position and intensities of these maxima give valuable information on the
nature of the flavonoids.

21. SALICORNIA BRACHIATA
✔ Salicornia brachiata is a euhalopytic plant belonging to the family Chenopodiaceae. Salicornia
possess antibacterial and antihypertensive properties and relief of toothache and chronic
rheumatism , constipation, obesity, diabetes and cancer.
✔ The plant powder was extracted with 500 mL of methanol. The sample was stirred in
temperature-controlled shaker at 30 ± 2 ºC for 48 h. After incubation, the solution was filtered.
✔The qualitative analysis for the phytoconstituents such as Tannins, Flavonoids, Saponins, Cardiac
glycosides, Steroids, Phlobatannins and Terpenoids.
❑Ultraviolet Visible Spectrophotometer(UV-VIS):
1g of plant powder was boiled with 10 mL of distilled water and then filtered. Extract of the filtered
sample was scanned using UV- Visible Spectrophotometer , at a range of 200 - 800 nm, to detect the
characteristic wavelength of the plant extract.
❑Fourier Transform Infra Red Spectroscopy(FT-IR):
The plant sample was dried at 40°C and ground to fine powder through mortar and pestle. The sample
was mixed with KBr at a ratio of 1:100 and pressed to a pellet. The pellet was immediately put into
the sample holder of Perkin Elmer Spectrophotometer and operated in the range 4000 – 480 cm–1.

22. ✔The active constituents of plants are the major source for the development of new
chemotherapeutic agents.
✔Flavonoids possess a wide range of biological activities such as antioxidant,
antimicrobial, anti-inflammatory and anticancer activities. The presence of tannins
and flavonoids in methanolic extract of S. brachiata suggests the potential isolation
of the phytochemical and their use in industries.
✔ UV-Vis spectroscopic analysis :Plant extract was studied at a wavelength range of
200 to 800 nm. Two major bands were recorded at 280 and 290 nm with absorbance
values of 0.28 and 4 respectively. The spectra for phenolic compounds (tannins) and
flavonoids typically lie in the range of 230-290 nm .The result of UV-VIS
spectroscopic analysis confirms the presence of tannins and flavonoids in the
methanolic extract of S. brachiata.

23. ❑Spectroscopic analysis- FT- IR:
✔ The FT-IR spectrum was performed to identify the functional groups present in S.
brachiata based on the peak values in the region of infrared radiation. FTIR studies
enable the identification of the chemical constituents and elucidation of the
structures of compounds .The major bands were observed at 3438.6, 1637.4, 1404 and
1319.59 cm-1 .
✔ The peak at 3448.4 cm-1
indicates the O-H stretch that might be due to the presence of
phenols and alcohols.
✔The bands at 1637.4 cm-1
and 1404 cm-1
corresponds to the C-C stretch, confirming
the presence of aromatic compounds.
✔The peak at 1319.59 cm-1
represents C-O stretch which shows the presence of alcohols,
carboxylic acids, esters and ethers. In addition, some weak absorption bands were
also recorded in the spectra.
CONCLUSION :
✔ FT-IR spectra represented the existence of phenolic compounds, alcohols and aromatic
compounds in the plant. The methanolic extract of the plant was found to be inactive
against the bacteria tested. This might be due to the selection of the solvent system.

24. 230-290nm:The peak indicates presence of tannins and flavonoids.

25. REFERENCES
✔https://www.alamy.com/stock-photo/corallium-nigrum.html
✔https://www.hindawi.com/journals/jamc/2012/109058/fig2/
✔https://www.arcjournals.org/pdfs/ijarcs/v2-i8/5.pdf
✔http://globalresearchonline.net/journalcontents/v43-1/31.pdf
✔https://www.researchgate.net/publication/235373882_Application
_of_spectroscopy_in_herbal_metabolomics
✔https://pharmacognosy.in/tag/infrared-spectroscopy/
✔http://www.authorstream.com/Presentation/vijay_radadiya-59815
0-evaluation-of-crude-drug-by-uv-technique/

26. THANK YOU