COMPARE SPECTROPHOTOMETRY,

SPECTROFLUORIMETRY AND APPLICATION IN

AGRICULTURE AND MEDICINE.
UNIVERSITY OF NIGERIA, NSUKKA
AYOLOTU, M. A.
INTRODUCTION
SPECTROPHOTOMETRY
In chemistry, spectrophotometry is the quantitative measurement of the
reflection or transmission properties of a material as a function of wavelength. It is
more specific than the general term electromagnetic spectroscopy in that
spectrophotometry deals with visible light, near-ultraviolet, and near-infrared, but
does not cover time-resolved spectroscopic techniques. Spectrophotometry is a
method to measure how much a chemical substance absorbs light by measuring
the intensity of light as a beam of light passes through sample solution. The basic
principle is that each compound absorbs or transmits light over a certain range of
wavelength. This measurement can also be used to measure the amount of a known
chemical substance. Spectrophotometry is one of the most useful methods of
quantitative analysis in various fields such as chemistry, physics, biochemistry,
material and chemical engineering and clinical applications. Every chemical
compound absorbs, transmits, or reflects light (electromagnetic radiation) over a
certain range of wavelength. Spectrophotometry is a measurement of how much a
chemical substance absorbs or transmits. Spectrophotometry is widely used for
quantitative analysis in various areas (e.g., chemistry, physics, biology,
biochemistry, material and chemical engineering, clinical applications, industrial
applications, etc). Any application that deals with chemical substances or materials
can use this technique. In biochemistry, for example, it is used to determine enzymecatalyzed reactions. In clinical applications, it is used to examine blood or tissues for
clinical diagnosis. There are also several variations of the spectrophotometry such
as atomic absorption spectrophotometry and atomic emission spectrophotometry.
A spectrophotometer is an instrument that measures the amount of photons (the
intensity of light) absorbed after it passes through sample solution. With the
spectrophotometer, the amount of a known chemical substance (concentrations)

can also be determined by measuring the intensity of light detected. Depending on

the range of wavelength of light source, it can be classified into two different types:
UV-visible spectrophotometer: uses light over the ultraviolet range (185 400 nm) and visible range (400 - 700 nm) of electromagnetic radiation
spectrum.
IR spectrophotometer: uses light over the infrared range (700 - 15000 nm)
of electromagnetic radiation spectrum.
In visible spectrophotometry, the absorption or the transmission of a certain
substance can be determined by the observed color. For instance, a solution sample
that absorbs light over all visible ranges (i.e., transmits none of visible wavelengths)
appears black in theory. On the other hand, if all visible wavelengths are transmitted
(i.e., absorbs nothing), the solution sample appears white. If a solution sample
absorbs red light (~700 nm), it appears green because green is the complementary
color of red. Visible spectrophotometers, in practice, use a prism to narrow down a
certain range of wavelength (to filter out other wavelengths) so that the particular
beam of light is passed through a solution sample.

Figure 1: Basic structure of spectrophotometers (illustrated by Heesung Shim)

A spectrophotometer, in general, consists of two devices; a spectrometer and a
photometer. A spectrometer is a device that produces, typically disperses and
measures light. A photometer indicates the photoelectric detector that measures the
intensity of light.
SPECTROFLUOROMETRY
Fluorescence spectroscopy
(also
known
as fluorometry or
spectrofluorometry) is a type of electromagnetic spectroscopy which analyzes
fluorescence from a sample. It involves using a beam of light, usually ultraviolet
light, that excites the electrons in molecules of certain compounds and causes them

to emit light; typically, but not necessarily, visible light. A complementary technique
is absorption spectroscopy.
Both fluorescence and phosphorescence are types of photoluminescence
(luminescence)
Luminescence: It is the process of reemission of previously absorbed light
When the molecules in the ground state absorb UV light, they are transferred to the
excited state, then, reemission of the previously absorbed light takes place and the
molecules return to the ground state where fluorescence or phosphorescence takes
place
Molecular Emission:
After the absorption of UV Visible light, the excited molecular species are extremely
short-lived and deactivation occurs due to:
a- Internal collision (internal conversion)
b- Cleavage of chemical bonds initiating photochemical reactions
c- Re-emission of light (luminescence)
d- Heat
e- Interaction between the solute and the solvent
Molecules on excitation normally posses higher vibrational energy than they had in
the ground state. This extra vibrational energy is lost by collision after which the
molecules return to the ground electronic state with the emission of light as
fluorescence. Deactivation as fluorescence is a rapid process occurring within 10-6 10-9 seconds of the excitation. Figure 1 shows the energy transfer during the
absorption, fluorescence and phosphorescence of UV-Visible radiation. The lowest
set of energy levels represents the ground electronic level and its associated
vibrational levels. The upper set of energy levels represents the first excited
electronic state and its associated vibrational sub-levels.
Photoluminescence should involve both photo-excitation and emission processes
1- Photoexcitation process:
It may occur by absorption of one of the following forms of radiant energy:
Sunlight, visible radiation, UV, X-rays etc.
2- The emission process:
It is the emission of radiant energy from an excited electronic state.
Photoluminescence is called fluorescence when the spin of the excited electron does
not change as the photoexcited species undergoes a transition from the excited state
to the ground state.
Instrumentation:
When both the excitation and emission spectra are to be recorded, two
monochromator are essential, one for the light source (excitation monochromator)

and one for the fluorescence (emission monochromator). The light source must
provided a high level of UV and Visible radiation and a compact high pressure Zenon
arc lamp is used.

The production of ozone by the photochemical conversion of atmospheric oxygen in

the lamp compartment presents a toxic hazard unless the ozone is thermally
decomposed or removed by adsorption onto charcoal. As many experiments will
almost certainly entail the measurement of very weak fluorescence. The detector
must be a highly sensitive photomultiplier tube of low dark current.
If the main interest lies in the fluorescence emission spectra, one monochromator
may be dispensed with a suitable light source and filter used instead. The rather
poor luminosity associated with the monochromator even with a xenon arc lamp is
replaced by the much more intense light from a source such as a mercury vapour
lamp, from which a suitable activation beam is isolated by means of the filter. This
arrangement partially overcome, one of the difficulties inherent in
spectrofluorimetry, i.e., that so much of the available light is lost.
COMPARISM BETWEEN SPECTROPHOTOMETRY AND SPECTROFLUORIMETRY
1.
High sensitivity: Substances that are reasonably fluorescent may be
determined at concentration up to 1000 times lower than those required for
absorption spectrophotometry. In spectrofluorimetric measurement, the
photomultiplier tube measures a single light intensity (relative to a zenon light
intensity) which may be amplified electronically many times without introducing
significant noise. In UV-Vis absorption spectroscopy, the photomultiplier tube
measure two intensities I0 and IT. at very low absorbance, the small difference
between I0 and IT approach the noise of the signal and cannot be measured with
satisfactory precision. The high sensitivity offered by spectrofluorimetry may be of
no advantage if the sample contains a sufficient quantity of the analyte for assay by

absorption spectrophotometry, the latter being generally the more precise

technique. For example, the highly fluorescent substance quinine sulphate may be
assayed with good accuracy and precision in Quinine sulphate tablets (300 mg) by
measuring the absorbance at 348 nm of the filtered extract of the tablet powder in
0.1 M HCl. However, low dosage drug formulations containing less than 1 mg per
dose unit and biological samples (blood, urine, etc....) containing low concentration
of the drugs, may require the high sensitivity of spectrofluorimetry, thus the
spectrofluorimetric method is of choice for the determination of many hormones,
alkaloids and vitamins in formulations and biological fluids.
2.
Selectivity: Two factors confirm on spectrofluorimetry a greater selectivity
than that given by UV-Visb. Absorption spectrophotometry. First, not all the
substances that absorb in the UV-Vis region fluoresce. In non fluorescent molecules,
absorbed energy is lost by alternative radiationless pathways, principally by
internal conversion. Molecules require in addition to a chromophore, a degree of
rigidity in their structure to reduce the dissipation of the absorbed energy by
internal conversion.
Substances that are fluorescent are characterized by their wavelengths of maximum
excitation and emission. Different fluorescent species may show different
wavelengths of maximum excitation and /or emission. The facility to vary
independently the wavelength of excitation and the wavelength of fluorescence
allows the analyst to select the optimum combination of wavelength for the analyte
and to reduce interference from other fluorescing species in the sample.
3.
Adsorption: The extreme sensitivity of the method requires very dilute
solution, 10-100 times, weaker than those employed in absorption
spectrophotometry. Adsorption of the fluorescent substance on the container walls
may therefore presents serious problems and strong stock solutions must be kept
and diluted as required. Quinine is a typical example of a substance which is
adsorbed onto cell walls.
4.
Photodecomposition: In absorption spectrophotometry, the intensity of the
radiation passing through solution is weak by photochemical standards, although
adequate for measurements; decomposition of the solute is therefore, not very
likely. Spectrofluorimetry, on the other hand, requires high intensity illumination
for irradiation, and the risk of photochemical change is thereby increased. An error
up to 20% could quite easily arise. It may be possible in unfavourable cases to select
radiation of a wavelength which is not strongly absorbed so that the extent of
photochemical change is reduced, at the same time adequate sensitivity is retained.

1.

APPLICATIONS OF SPECTROFLUORIMETRY
In Agriculture:

Single substances which are in themselves, non-fluorescent may be determined as a

result of chemical change. This method is useful for both inorganic and organic
compounds, and many inorganic compounds, form highly fluorescent complexes by
combination with organic reagents. The determination of selenium illustrates the
increase in the sensitivity which can be obtained with fluorimetry as as compared
with that for absorption. Thus 0.3 gml-1 of selenium may be determined by
measurement of the absorbance of its complex with 3,3- diaminobenzidine, but by
using the fluorescence of the complex ,0.04 g of selenium can be measured. The
sensitivity is further increased to 0.002 g of selenium with 2,3 diaminonaphthalene
as reagent. e.g.: Thiamine HCI in pharmaceutical preparations such as tablets and
elixirs and in food stuffs such as flour is relatively easily determined by oxidation to
highly fluorescent thiochrome. The product is soluble in 2-methyl-propan-1-ol and
hence is easily extracted from the reaction mixture for measurements

For mixture of two components, it may be possible to select the exciting radiation of
appropriate wavelengths, such that only one compound fluoresces at any time. Even
if there is not possible, measurements of the fluorescence at two wavelengths may
be sufficient to determine the composition of the mixture.
2. In Medicine:
The highly fluorescent substance quinine sulphate may be assayed with good
accuracy and precision in Quinine sulphate tablets (300 mg) by measuring the
absorbance at 348 nm of the filtered extract of the tablet powder in 0.1 M HCl.
However, low dosage drug formulations containing less than 1 mg per dose unit and
biological samples (blood, urine, etc....) containing low concentration of the drugs,
may require the high sensitivity of spectrofluorimetry, thus the spectrofluorimetric

method is of choice for the determination of many hormones, alkaloids and vitamins
in formulations and biological fluids.
APPLICATIONS OF SPECTROPHOTOMETRY
1.
In Agriculture:
Traditional techniques for analyzing physical, chemical and biological properties
of soil as described above are renowned for being costly and time consuming.
Advances in spectrometer hardware, computing and statistical software mean that
Near-Infrared (NIR) spectra of soil samples have the potential for fast, nondestructive, accurate and cheap soil analysis, particularly for applications in the
field and where a high spatial density of sampling is needed. It is now routinely used
for analyses of a wide range of materials in laboratory and process control
applications in agriculture, food and feed technology, geology, biomedicine, and
space exploration, and has been a key technology in enabling the development of
soil health surveillance systems by providing a rapid and reliable tool for soil health
screening in the AfSIS project.
2. In Medicine:
Pharmaceutical companies must monitor and control production of the small
molecules they manufacture and they often rely on spectrophotometers for many of
these procedures.
It s no surprise that the ingredients used in making pharmaceuticals are highly
regulated, requiring a series of tests and quality control steps to ensure that patients
are receiving safe and correct dosages of sometimes dangerous compounds.
Manufacturers take these controls seriously, but the toll they can take on a
pharmaceutical plant s output can be high. Thankfully, technology in particular,
bench top spectrophotometers are helping pharmaceutical makers find a way to
improve testing and save valuable time and money.
Pharmaceutical companies use bench top spectrophotometers for color
measurement in a variety of applications. One use is to ensure that the color is
consistent in the dosage. Pills, for example, need to have unique colors and shapes
so they are not mistaken for other medicines. Bench top spectrophotometers are
used in the measurement of solids, liquids, powders, pastes and creams. The CM-5
spectrophotometer is in widespread use in pharmaceutical companies due to its
versatility in sample measuring.
Many pharmaceutical labs also use bench top spectrophotometers to evaluate the
effect of different dosages of ingredients in a medication. Assays are created on a
sectioned plate, and each section is administered with a different amount of the

active product ingredient (API). A dye that activates when cells in the assay
incorporate the ingredient is also administered to each section of the plate. Using
the bench top spectrophotometer, scientists can easily measure numerically the
effect the ingredient has on a cell by measuring the color of the dye in each section.
Since pharmaceutical labs have so many uses for bench top spectrophotometers,
these instruments must be versatile, accurate and easy to use in a variety of ways.
The Konica Minolta CM-5 spectrophotometer uses 3 easy steps to measure color in
laboratory samples. Basically the user turns it on, employs a wizard to adjust the
settings, positions the sample to be measured and presses a button to start. All the
color data, spectral graphs and colorimetric plots, are displayed on its LCD screen,
so that the user does not have to use a separate computer to see the data. It has two
ways of positioning the object to be measured. There is a top port for measuring
pills, granules, and pastes, and a large transmittance chamber with no sides, to
measure liquids, films or plates up to 60mm thick. Results can be evaluated in terms
of industry specific color scales such as Gardner, Iodine, Hazen (APHA), European
Pharmacopoeia, and US Pharmacopeia. The instrument also has the ability to
interface with PC software which can be FDA 21 CFR Part 11 compliant.
REFERENCE
Allen, D., Cooksey, C., and Tsai, B. (2010). Spectrophotometry. Retrieved from
http://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm
Eisinger, J., Flores J. (1979). "Front-face fluorometry of liquid samples". Analytical
Biochemistry 94 (1): Retrieved 2009-02-06.
Hisham E . (2000). Instrumental Analysis pp, 90-111
http://sensing.konicaminolta.us/2012/08/how-food-companies-measure-color-toproduce-superior-products/
http://www.fao.org/soils-portal/soil-survey/sampling-and-laboratorytechniques/en/
James H., Douglas A. S., and Stanley R. C. (2006). Principles of instrumental analysis
Rendell, D. (1987). Fluorescence and Phosphorescence. Crown
Schwedt, Georg. (1997). The essential guide to analytical chemistry. (Brooks
Haderlie, trans.). Chichester, NY: Wiley. pp. 16-17