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Spectrofluorimetry as an Analytical Tool

Article  in  Pharmaceutica Analytica Acta · October 2011

DOI: 10.4172/2153-2435.1000107e

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 Pharmaceutica Nahata, Pharm Anal Acta 2011, 2:7
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Analytica Acta

Editorial Open Access

Spectrofluorimetry as an Analytical Tool

Alok Nahata*
Department of Pharmaceutical Sciences, Doctor Hari Singh Gour Vishwavidyalaya, SAGAR – 470003 (M.P.) INDIA

Although some information about molecular structure may be ground state. Electron withdrawing groups containing –Cl,
derived from excitation and emission spectra, qualitative application -Br, -I, -NHCOCH3, -NO2 or -COOH decrease or quench
of spectrofluorimetry are rare and vast majority of applications completely the fluorescence.
in pharmaceutical analysis concern the quantitative assay of
drugs, decomposition products and metabolites. Still the use of • Molecular rigidity: Fluorescence is particularly favoured in
spectrofluorimetry as an analytical tool provides a well defined identity molecules that possess rigid structures. Molecular rigidity
of the compounds present in the sample on the basis of their unique lessens the possibility of competing nonradiative transitions by
fluorescent nature decreasing vibrations; this minimizes intersystem crossing to
the triplet state and collisional heat degradation. For example,
Fluorescence fluorescein and eosin are strongly fluorescent, but a similar
When an illuminating system emits light of wavelength different compound phenolphthalein, which is non rigid and where the
from the incident light, the phenomenon is termed as fluorescence and conjugate system is disrupted, is not fluorescent.
it takes place as soon as the light is absorbed and ceases as soon as the
light is stopped. Fluorescence usually is seen at moderate temperature • Polarity of the solvent: The polarity of the solvent also affects
in liquid solution. the fluorescence and phosphorescence. Solvents containing
heavy atoms or other such atoms in their structures decrease
The intensity of the photoluminescence spectrum depends on the the fluorescence.
excitation wavelength, although its spectral position does not. The
photoluminescence spectrum appears at longer wavelengths than the • Presence of dissolved oxygen: The presence of dissolved
excitation spectrum. This phenomenon arises because the excitation oxygen often reduces the emission intensity of a fluorescent
process requires an amount of energy equal to electronic energy change solution probably due to photochemically induced oxidation
plus a vibrational energy increase; conversely each deexcitation yields of the fluorescent material. Quenching also occurs as a result of
the electronic excitation energy minus a vibrational energy increase [1]. paramagnetic properties of molecular oxygen.
High sensitivity and high specificity • Changes in pH: pH also exhibits a marked effect on the
Fluorescence spectroscopy has assumed a major role in analysis, fluorescence of compounds. For example aniline shows a
particularly in the determination of trace contaminants in our blue fluorescence in the range pH 5-13, when excited at 290
environment, industries, and bodies, because for applicable compounds nm. At lower pH, aniline exists as the aniline cation and in
fluorescence gives high sensitivity and high specificity. High sensitivity highly alkaline media as the anion. Neither anion nor cation
results from a difference in wavelength between the exciting and is fluorescent. Fluorescence is more commonly associated with
fluorescence radiation. These results in a signal contrasted with π−π∗ states than with n−π∗ states because π−π∗ states possess
essentially zero background. High specificity results from dependence shorter average lifetime and because deactivation process that
on two spectra: the excitation and the emission spectrum. compete with fluorescence are much less likely to take place.
In analytical work, fluorescence is important because of the fact that • Quenching: Quenching is the reduction in the intensity
the intensity of light emitted by a fluorescent material depends upon of fluorescence due to specific effect of constituents of the
the concentration of that material. In this manner, the measurement of solution itself. Quenching may be caused in several ways. For
fluorescence intensity permits the quantitative determination of traces example, concentration quenching may be caused by excessive
of many inorganic species. absorption of either primary or fluorescent radiation by the
In florescence analysis, the amount of light emitted characteristically solution. This is also called the inner filter effect. If this effect
under suitable excitation is used as a measure of the concentration of occurs as a result of absorption by the fluorescent substance
the responsible species. itself, the phenomenon is called self-quenching [1,2].
Factors affecting fluorescence
The intensity of exciting light is proportional to both the intensity *Corresponding author: Alok Nahata, [Link]., Ph.D., Department of
Pharmaceutical Sciences, Doctor Hari Singh Gour Vishwavidyalaya, SAGAR
of exciting light and the concentration of the fluorescing material at
– 470003 (M.P.) INDIA, Tel: +919827544352; E-mail: aloknahata@[Link]
low concentration (10-4-10-7M) and with in narrow limits.
Received September 30, 2011; Accepted October 01, 2011; Published October
Structural factors: 03, 2011

• Substituents: Substituents strongly affect fluorescence. A Citation: Nahata A (2011) Spectrofluorimetry as an Analytical Tool. Pharm Anal
Acta 2:107e. doi:10.4172/2153-2435.1000107e
substituent that delocalizes electrons, such as –NH2 –OH,
-F, -OCH3, -NHCH3 and N(CH3)2 groups, often enhances Copyright: © 2011 Nahata A. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
fluorescence because they tend to increase the transition use, distribution, and reproduction in any medium, provided the original author and
probability between the lowest excited singlet state and the source are credited.

Pharm Anal Acta

ISSN: 2153-2435 PAA, an open access journal Volume 2 • Issue 7 • 1000107e
Citation: Nahata A (2011) Spectrofluorimetry as an Analytical Tool. Pharm Anal Acta 2:107e. doi:10.4172/2153-2435.1000107e

Page 2 of 2

Internal conversion It is well evident from (4) fluorescent intensity is a linear function
of concentration.
The term is used to describe intermolecular processes by which a
molecule converts to lower energy electronic state without emission Calculation of Results
of radiation. Internal conversion is very efficient when two electronic It is very important for the analyst to run a blank and two standards
energy levels are sufficient close for an overlap of vibrational levels to of known composition that cover the range of concentration expected.
exist. The internal conversion through overlapping vibrational levels is The blank must be kept relatively low and blank reading should be
usually more probable than the loss of energy by fluorescence from a subtracted from all other readings.
higher exited state [3].
Fs = KCs (5)
Relation between intensity of fluorescence and concentration
Fu = KCu (6)
The use of fluorescence in quantitative analysis is based on the fact
that there should be a definite relationship (preferably linear) between s and u are subscripts for standard and unknown respectively. Standard
concentration and intensity of fluorescence. On the basis of theory as should be very close to the unknown in composition.
well as experiment such a linear relationship has actually been found to Dividing (6) by (5)
exist and it is related to the familiar Lambert-beer law, from which it
can be derived as under: C u Fu C s .Fu
= or Cu =
The intensity of light absorbed by a solution is (I0 - I) C s Fs Fs
Thus unknown concentration can be determined.
Where I = I0 e-acl
Factors affecting fluorescence intensity
Hence the intensity of light absorbed is
Fluorescence intensity of a substance is proportional to
I0 - I0 e-acl or I0 (I - e-acl) (1)
concentration only when absorbance in a 1 cm cell is less than 0.02. If
Where I0 = Intensity of incident light the concentration of fluorescent substance is so great that all incident
radiation is absorbed, then
I = Intensity of transmitted light
F = I0φ
a = Absorptivity (or extinction coefficient) multiplied by 2.303.
i.e. fluorescence is independent of concentration and proportional to
c = Concentration
the intensity of incident radiation only.
l = length of optical path.
A further problem ensues if the emission and excitation spectra
The intensity of fluorescence (F) emitted can be obtained by overlap, which results in the reabsorption of fluorescence and a
multiplying the amount of light absorbed by quantum yield (φ), where negative dependence of fluorescence on concentration.
φ is the ratio of light emitted to the light absorbed. Thus
Research based on this approach
F = φ X I0 (I – e–acl) (2)
We have developed a spectrofluorimetric method to determine
Reabsorption and scattering of light have not been taken into coumarins in a well known traditional Indian drug ‘Shankhpushpi’.
consideration in the above equation. In this study we estimated the amount of total coumarins calculated
as scopoletin in different varieties of Shankhpushpi viz., Evolvulus
It is also assumed that both absorption and emission occur only
alsinoides and Convolvulus pluricaulis [4]. Further simultaneous
due to single molecular species and degree of dissociation does not vary
estimation of scopoletin and mangiferin in a methanolic extract of
with concentration. Now e-acl can be exponentially expressed as
Canscora decussata was performed and a successful interpretation
about the content of scopoletin and mangiferin in the drug was
(acl)2 (acl)3 (acl)n
e-acl =1- acl + - +...........+ done [5]. Another attempt in this direction was the development of
2 6 n!
a spectrofluorimetric method for the determination of curcumin
If the magnitude of acl is small, all terms after first two can be [6]. Recently we have developed a spectrofluorimetric method for
neglected. Hence the determination of testosterone in biological fluids. The method is
specific, precise, accurate and validated [7].
e–acl = 1 – acl (3)
Putting in equation (2) we have
Conclusion
The method is less tedious and less cumbersome as compared
F = φ X I0 (1 – 1 + acl) = φ X I0 acl
to HPLC and other methods which require long run time and suffer
For a given fluorescent compound, solvent and temperature, and from tedious operation procedures. Moreover the sensitivity offered
in a cell of definite dimensions, all other factors are constant giving by this method is far higher than that of HPLC. The compounds can
simply, be analysed upto the levels of nanograms. Hence high sensitivity and
specificity are real advantages of this method. Further quantitative
F = Kc (4)
estimation is possible which can easily measure the amount of the
Where K is proportionality constant. analyte in the mixture.

Pharm Anal Acta

ISSN: 2153-2435 PAA, an open access journal Volume 2 • Issue 7 • 1000107e
Citation: Nahata A (2011) Spectrofluorimetry as an Analytical Tool. Pharm Anal Acta 2:107e. doi:10.4172/2153-2435.1000107e

Page 3 of 2

References 5. Sethiya NK, Nahata A, Dixit VK (2008) Simultaneous Spectrofluorimetric

Determination of Scopoletin and Mangiferin in a Methanolic Extract of Canscora
1. Willard HH, Merritt LL, Dean JA, Settle FA (1986) Instrumental Methods of decussata Schult. Asian Journal of Traditional Medicines 3: 224-229.
Analysis. 6th Edition. CBS Publishers and Distributors, New Delhi. p. 105-111.
6. Gupta NK, Nahata A, Dixit VK (2010) Development of Spectrofluorimetric
2. Beckett AH, Stenlake JB (2002) Practical Pharmaceutical Chemistry.4th Edition, Method for the determination of curcumin. Asian Journal of Traditional
Part II. CBS Publishers and Distributors, New Delhi. p. 358-366. Medicines 5: 12-18.
3. Sharma BK (2002) Instrumental Methods of Chemical Analysis. 21st Edition, 7. Nahata A, Gupta NK, Dixit VK (2011) A simple and rapid spectrofluorimetric
Goel Publishing House, Meerut. p. 360-373. method for the determination of testosterone in biological fluids. Oriental
Pharmacy and Experimental Medicine In Press.
4. Nahata A, Dixit VK (2008) Spectrofluorimetric Estimation of Scopoletin in
Evolvulus alsinoides Linn. and Convulvulus pluricaulis Choisy. Indian J Pharm
Sci 70: 834-837.

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