SPECTROSCOPY
Spectroscopy
is the branch of science dealing with the study of interaction of
electromagnetic radiation with matter. Due to such interaction is that energy
is absorbed or emitted by matter in discrete amounts called quanta.
Electromagnetic radiation:
Light or electromagnetic radiation is a form of energy that
is transmitted through space at a constant velocity of 3 × 108 ms-1.
The term electromagnetic radiation is made up of an electrical and a
magnetic wave.
Wave theory of
electromagnetic radiation:
According to this theory, the electromagnetic radiations
travel in the form of waves. This wave motion consists of oscillating electric
and magnetic fields directed perpendicular to each other and perpendicular to
the direction of propagation of the wave.
The points X, Y, P and Q on the wave represent the maximum
disturbances in the electrical field. The distance from the mean position is
known as the amplitude of the wave. The distance from the crest X to crest Y is
the wavelength λ. The number of
complete wavelength units passing through a given point per second is called frequency.
These two quantities are related to each other, and given,
C
V = ----- --------------
(1)
λ
λ
Where, c → velocity of the
electromagnetic wave. Since c is constant (3 × 108 ms-1)
for all types of electromagnetic radiations.
So the above relation may be expressed as,
1
V α --------- ----------------
(2)
λ
Reciprocal of wavelength i.e.,
1/ λ is called wave number, v. Hence equation 1 may be written as,
V = cv -----------------
(3)
The wavelength is expressed in terms of centimeter (cm),
metre (m), micron (μ) or micrometer (μm) or angstrom (A˚) units. The other
commonly used unit is nanometer (nm) where 1 nm = 10-9.
Frequency is measured as cycles per second (cps) called
hertz (Hz) or kilocycles per second (kHz) or mega cycles per second (mHz).
1 kHz = 103 Hz
1 mHz = 106 Hz or cps
The wave number is the number of waves per unit distance
and is expressed in the units of cm-1 called Kaysers (K).
Sometimes kilokayser (kK) is also used.
1 kK = 1000 K = 1000 cm-1.
Quantum theory of electromagnetic radiation:
The quantum theory describes the electromagnetic radiation
as one consisting of a stream of energy packets called photons or quanta, which
travel in the direction of propagation of the beam with the velocity of light.
The energy E of photon is proportional to the frequency of radiation and is
given by the equation,
E = hv --------------------
(4)
Where,
h → Planck’s constant (6.6.25 ×10-27
erg/second)
By substituting c/ λ, for v as expressed in equation 1,
equation 4 can be alternatively expressed as,
E = hv = hc/ λ
By substituting cv for v as expressed in equation 3,
equation 4 can be alternatively expressed as,
E = hcv
TYPES OF SPECTROSCOPY:
Absorption of photons by the molecules may change its internal
energy (electronic, vibrational or rotational energy) or may cause transitions
between different spin orientations of nuclei in the magnetic field. Almost all
parts of the electromagnetic spectra are used for the studying matter; the
following types of spectroscopy are in common use,
1.
Colorimetry
2.
Ultraviolet and visible spectroscopy (uv – vis)
3.
Flame photometry
4.
Raman spectroscopy
5.
Infrared spectroscopy
6.
Atomic spectroscopy
7.
Mass spectroscopy
8.
Nuclear magnetic resonance (NMR) spectroscopy
9.
X – ray spectroscopy
10.
Fluorimeter
11.
Electronic Spin Resonance (ESR)
UV – VISIBLE SPECTROSCOPY
Ultraviolet-visible
spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or
UV/Vis) involves the spectroscopy of photons in
the UV-visible region. This means it uses light in the visible and adjacent
(near ultraviolet (UV) and near infrared
(NIR)) ranges. The absorption in the visible ranges directly affects the color
of the chemicals involved.
Principle:
This
instrument works based on the principle of Beer - Lambert’s Law. This is
also known as Beer’s Law or Lambert’s Law. Two basic laws proposed by Beer and Lambert
explain the absorption of light by a substance in solution.
Beer’s Law states that “when a parallel beam of monochromatic light
passes through a light absorbing medium, the amount of light that is absorbed
is directly proportional to the number of light
absorbing molecules in that medium (in other words, the concentration of
the substance in that medium).
i.e., A α C
where, A→ the absorbance or
optical density
C → the concentration of the light absorbing substance in
the medium.
Lambert’s Law states
that “when a parallel beam of monochromatic light passes through a light
absorbing medium, the amount of light absorbed is directly proportional to the
length of the medium through which the light passes.
i.e., A α L
where, A→ the absorbance
L
→ the length of the medium
Since the measurement of light absorption depends on both
the laws, it is popularly known as Beer – Lambert’s Law.
Thus, A
α C× L
A
= e CL
Where e is known as the
extinction co-efficient.
A = log
Ii / It
A = log 100 / T
= log
100 – log T
= 2 –
log T
Where,
Ii → intensity of the incident light
It → intensity of the transmitted light
T → transmittance
Instrumentation:
The instrument used in ultraviolet-visible spectroscopy is called a
UV/Vis spectrophotometer. It measures the intensity of light passing
through a sample (I), and compares it to the intensity of light before
it passes through the sample (Io). The ratio I / Io are called the transmittance, and are usually
expressed as a percentage (%T). The absorbance, A, is based on the transmittance:
A = − log (%T
/ 100%)
Parts of a spectrophotometer:
·
A light source,
·
A monochromator
·
Transparent vessel
·
A photosensitive
detector
·
Amplifier &
recorder
1. Light source:
Most commonly
used sources of ultraviolet radiation are the hydrogen lamp and the dueterium
lamp. Both the systems consist of a pair of electrodes enclosed in a glass tube
provided with a quartz window. The glass tube is filled with hydrogen or
deuterium gas at low pressure. When a stabilized voltage is applied they emit
radiation in UV region. Xenon lamp is also used but the emission of light is
not stable as hydrogen lamp.
2. Wavelength selectors:
The light
from the source is composed of a wide range of wavelengths. This light is
called polychromatic or heterochromatic. The polychromatic light
reflected back using a plane mirror, passes through an entrance slit and
through a condensing lens and falls onto a monochromator. The monochromator
disperses the light and the desired wavelength is focuses on the exit slit
using the wavelength selectors.
The Wavelength selectors are of two types. They are filters
and monochromators.
i) Filter:
Filters operate
by absorbing light in all other regions except for one, which they reflect.
Gelatin filters are made of a layer of gelatin. Colored with organic dyes and
sealed between glass plates. Most modern filter instruments use tinted glass
filters. They are capable of transmitting light over a limited portion of the
spectrum only.
ii) Monochromators:
A
monochromators resolves the polychromatic radiation into its individual
wavelengths and isolates these wavelengths into very narrow bands. The
essential components of a monochromator are,
·
An entrance slit → which admits polychromatic light from the source
·
A collimating
device such as lens or a mirror → collimates
the polychromatic light on to the dispersion device.
·
A wavelength resolving
device like a prism or a grating → which breaks the radiation into component wavelengths.
·
·
A focusing lens or
mirror
·
Exit slit → which allows the monochromatic beam to escape.
The entire assembly is mounted in a light tight box.
The effective band width of
the light emerging from the monochromator depends mostly upon the dispersing
element (prism or grating) and the slit widths of the both the entrance and the
exit slits.
Prism:
A prism
disperses polychromatic light form the source into its constituent wavelengths
to a different extent. The shorter
wavelengths are diffracted most. The degree of dispersion by the prism depends
upon,
·
The apical angle of
the prism (usually 60°)
·
The material of which
it is made.
Two types of
prisms, namely 60° Cornu quartz prism and 30° Littrow prism are usually
employed in commercial instruments. For ultra violet region fused silica or
quartz prism are used.
Grating:
Gratings are
often used in the monochromators of spectrophotometers operating in UV, visible
and IR. They possess a highly aluminized surface with a large number of
parallel grooves which are equally spaced. These grooves are also known as
Lines. A grating may have between 600 to 2000 lines per mm.
Very often, the monochromator consists of both the prism
and grating. The prism placed before the grating is known as foreprism.
Single beam spectrophotometer:
The
monochromator disperses the light into its component wavelengths. Using the
wavelength selector, the desired wavelength is selected. Now the selected beam
of monochromatic light passes again through a lens to a light tight compartment
where the sample, the transmitted light falls on a photomultiplier tube (PMT).
The PMT converts the light energy into electrical energy, which is amplified,
measured and recorded on the analog/digital read out.
Double – beam spectrophotometer:
In Double –
beam spectrophotometers, the monochromatic light coming out form the lens is
split into two halves by placing a half silvered mirror (HSM) on its path. Now
50% of the light passes directly through the mirror and falls on the reference
cuvette and 50% of the light reflected onto a second silvered mirror and then
allowed to fall on the sample cuvette. At any given time, the intensities of
the transmitted light from the reference and sample cuvettes are measured,
amplified, the difference in intensities computed and sent to the read out.
Cuvettes:
The optically
transparent cells (cuvette) are made up of glass, plastic, silica or quartz.
Glass and plastic absorb UV light below 310 nm. Hence they cannot be used for
light measurements in UV region. Silica and quartz do not absorb UV light and
hence they are used for both UV and visible light measurements. Since quartz
absorbs light below 190 nm, cuvettes of lithium fluoride can be used which
transmit radiation down to 110 nm. Oxygen also absorbs light at wavelengths
less than 200 nm. The standard cuvettes are made up of quartz, have an optical
path of 1 cm and hold a volume of 1-3 ml. The rectangular shaped cells are
widely used.
Detection devices:
Most
detectors depend on the photoelectric effect, where the incident light (photon)
liberates electrons from a metal or other material surface. Some external
circuits collects the electrons and measures their number as current. The
current is then proportional to the light intensity and measure it. The
detectors include,
a)
High sensitivity to
detect the low levels of radiant energy
b)
Short response time
c)
Long term stability
d)
An electronic signal
to amplify.
There are 3 basic kinds of
detectors in UV region. They are photocells, phototubes and photomultiplier
tubes.
1. Photovoltaic or barrier layer cells:
It employs
semiconductor materials. They are crystalline and a number of materials are
used in photocells (cadmium sulphide, silicon and selenium). Selenium based
photocells are most common. Photocells have a long life and are inexpensive and
reliable.
2. Phototubes or photo emissive tubes:
The
components of a phototube includes,
·
An evacuated glass
envelope with a quartz window
·
A semi cylindrical
cathode (inner surface is coated with alkali )
·
A centrally located
metal wire anode.
The quartz window allows the passage of
radiation which strikes the photo emissive surface of the cathode. The energy
of the photon is transferred to the loosely bound electrons of the cathode
surface. The electrons become excited and finally leave the surface and travel
towards the anode causing current to flow in the circuit. If the electron
collection is 100% efficient, the phototube current should be proportional to
the light intensity. This current is amplified electronically and measured.
3) Photomultiplier tube:
These
detectors are designed to amplify the initial photoelectric effect and are suitable
for use at very low light intensities. A photomultiplier consists of,
a)
an evacuated glass
tube which is sealed the cathode and the anode
b)
additional intervening
electrode – Dynodes
This
photomultiplier tube has a cathode with photo emissive surface (a selenium
layer) and a wire anode. In addition to the photo emissive cathode, it also
contains a circular array of nine additional cathodes called dynodes.
The electrons
emitted from the photosensitive cathode strike dynode 1, which emit several
additional electrons. The electrons are accelerated towards dynode 2, which
again emit several electrons. The amplified electrons flow to the anode
generating a much larger photoelectric current than in a photocell.
General Procedure:
§
When the sample kept
in the sample cell, the light source is passed through the silvered mirror.
§
The reflected light
from the mirror passes through an entrance slit and a condensing lens.
§
The lens renders the
light rays into parallel beams and the parallel beams of light now fall on a
monochromator.
§
The monochromator
disperses the light into its component wavelengths.
§
By using the
wavelength selector, the desired wavelength is selected.
§
Now the selected beam
of monochromatic light passes again through a lens to a light-tight compartment
where the sample is kept in a cuvette.
§
After passing through
the sample, the transmitted light falls on a photomultiplier tube (PMT).
§
The PMT converts the
light energy into electrical energy, which is amplified, measured and recorded
on the analog/digital read out.
Applications:
UV – Vis spectrophotometer is
a more refined instrument and it gives a far better precision and resolution
than a colorimeter. It has a wide range of applications in biological research.
Qualitative
analysis: This technique may be used to
identify the biological compounds in both the pure state and in biological
preparation. This is done by plotting the absorption spectrum curves.
Quantitative analysis: to determine the unknown concentration of a given species
by absorption spectrometry. By using the known concentration of a sample, one
can calculate the concentration of an unknown sample.
Enzyme assay: The quantitative assay of enzyme activity is carried out
most quickly and conveniently.
Molecular weight
determination: This is a very reliable
method to determine the molecular weight of a particular compound.
To study the Cis-Trans
isomerism: The compound in Cis form will
have a particular absorption spectra and same for the trans form compounds. But
the trans isomer is usually more elongated than its cis counterpart. It is
usual therefore for the trans isomer have a higher wavelength of maximum
absorption.
To study the physiochemical
characters: it has been used to study such
physiochemical phenomena as heat formation by the addition of compounds,
assosciation of constants of weak acids and bases in organic solvents, protein
dye interactions, chlorophyll protein complexes etc.
Control of purification: This is one of the most important uses of this technique.
Impurities in a compound can be detected very easily by this method. For E.g.
carbon disulfide impurity in carbon tetra chloride can be detected easily by
measuring absorbance at 318 nm where carbon disulfide absorbs.
It is used to estimate the
concentration of both colored as well as colorless solutions.
Even very small volume of
sample can be used for estimation of precious sample.
It usually does not degrade or
modify the materials studied and hence the materials can be recovered and
reused.
It is also used to find out
the absorption maxima of compounds with a wide range of wavelengths.
It is also used to measure the
growth of bacteria and yeasts and to determine the number of cells in a
culture.
COLORIMETER
A Colorimeter
is a device used in Colorimetry. In scientific fields the word generally refers to the
device that measures the absorbance of
particular wavelengths of light
by a specific solution. This device is most commonly used to determine the concentration of a
known solute in a given
solution by the application of the Beer-Lambert law,
(which states that the concentration of a solute is proportional to the
absorbance).
Colorimetry:
Colorimetry
is a form of photometry which deals with measurement of light absorption by
colored substances in solutions. The instrument which measures the intensity of
the color is known as colorimeter.
Principle:
This is based
on the principle that when a beam of incident light passes through a colored
solution, the colored substances in the solution absorb a part of light and
hence the amount of the transmitted light (the light comes out) is always less
than that of the incident light. As the number of light absorbing molecules
increases, the intensity of light coming out of the medium decreases
exponentially and vice versa. The difference in intensities between the
incident and transmitted light, in turn reflects the number of absorbing
molecules or in other words, the concentration of the absorbing molecules in
that solution.
Beer – Lambert’s Law:
This
instrument works based on the principle of Beer - Lambert’s Law. This is
also known as Beer’s Law or Lambert’s Law. Two basic laws proposed by Beer and
Lambert explain the absorption of light by a substance in solution.
Beer’s Law states that “when a parallel beam of monochromatic light
passes through a light absorbing medium, the amount of light that is absorbed
is directly proportional to the number of light
absorbing molecules in that medium (in other words, the concentration of
the substance in that medium).
i.e., A α C
where,
A→ the absorbance or optical density
C → the concentration of the light absorbing substance in
the medium.
Lambert’s Law states
that “when a parallel beam of monochromatic light passes through a light
absorbing medium, the amount of light absorbed is directly proportional to the
length of the medium through which the light passes.
i.e., A α L
where, A→ the absorbance
L → the length of the medium
Since the measurement of light absorption depends on both
the laws, it is popularly known as Beer – Lambert’s Law.
Thus, A
α C× L
A = e CL
Where e is known as the
extinction co-efficient.
A = log Ii/It
A = log 100/T
= log
100 – log T
= 2 –
log T
Where,
Ii → intensity of the incident light
It → intensity of the transmitted light
T → transmittance
Molar Extinction
Coefficient:
In the above equation, when concentration is 1 M, i.e., one
mole per litre and path length of light is 1 cm, and then e is known as Molar
Extinction Coefficient (∑M). (∑M is constant for a
particular compound at particular wavelength and has a maximal value when the
compound is in its purest state).
Deviation of Beer –
Lambert’s Law:
§
In case of high sample
concentration the molecules may dimerize.
§
If the spectra differ
the absorption coefficient will also change, which leads to a positive or
negative deviation.
§
The temperature of the
sample also affects the result.
§
Sample instability
also leads to variations in result.
§
Some solutes may
fluoresce. For such kind of samples a considerable deviation will occur.
§
Turbidity solution
always ends up giving higher absorbance than the actual absorbance.
§
Thus the Beer –
Lambert’s Law has many deviations.
Instrumentation:
Parts of a colorimeter:
·
A light source
·
A condensing lens
·
A filter
·
A sample holder
(cuvette)
·
A photo cell
·
A galvanometer
1. Light source:
·
The light is an
ordinary low-voltage filament lamp or a tungsten lamp (wave length
between 400 and 900 nm).
·
It is inexpensive and
emits continuous radiation in the region between 350 and 2500 nm.
·
Carbon arc is
commercially used light source which provides more intense visible radiation.
2. Wave length selectors:
Wavelength selectors are of two types. They are filters and
monochromators.
i) Filter:
Filters operate by absorbing light in all other regions
except for one, which they reflect. Gelatin filters are made of a layer of
gelatin. Colored with organic dyes and sealed between glass plates. Most modern
filter instruments use tinted glass filters. They are capable of transmitting
light over a limited portion of the spectrum only.
Incase of colorimeter the instrument is provided with a set
of replaceable filters marked “V, B, G, Y, O, and R”.
Instead of the above mark a number may be
written on each, which indicates the wavelength of light that the filter
transmits. The filter is not removable and they are fixed on to a disc, which
can only be rotated to bring the appropriate filter in the light path. Filters
are limited specificity i.e., it is designed to transmit 540 nm may actually
transmit light between 520 and 560 nm with a peak transmittance at 540 nm.
Visible Spectrum
|
Colour of the
Solution
|
Range of wavelength
|
Complementary of
Subtraction color
|
|
Violet
|
400 – 465
|
Greenish yellow
|
|
Blue
|
465 – 482
|
Yellow
|
|
Green
|
498 – 530
|
Red purple (magenta)
|
|
Yellow
|
576 – 580
|
Blue
|
|
Orange
|
587 – 610
|
Greenish blue
|
|
Red
|
617 – 660
|
Bluish green
|
|
Purple red
|
670 – 720
|
green
|
ii) Monochromators:
A monochromators resolves the polychromatic
radiation into its individual wavelengths and isolates these wavelengths into
very narrow bands. The essential components of a monochromator are,
·
An entrance slit → which admits polychromatic light from the source
·
A collimating
device such as lens or a mirror → collimates
the polychromatic light on to
the dispersion device.
·
A wavelength resolving
device like a prism or a grating → which breaks the radiation into component wavelengths.
·
A focusing lens or
mirror
·
Exit slit → which allows the monochromatic beam to escape.
·
The entire assembly is
mounted in a light tight box.
3) Sample holder:
§
Samples are taken in a
small sample holder called Cuvette. A round glass cells are used.
§
The standard path
length is 1 cm. However cuvettes of path length of 1mm to 10 mm are available
for special cases.
§
They are usually made
up of either ordinary glass or quartz.
§
The surface of the
cuvettes must be kept clean.
§
There should not any
finger prints or traces of previous samples. Because it may interfere the
optical length and causes errors.
§
Rinsing with water is
sufficient for normal cuvettes. If the dirt is abnormally tenacious, sulfonic
detergents or nitric acid may be used.
4. Detection devices:
Most detectors depend on the
photoelectric effect, where incident light liberates electrons from a metal or
other material surface. Some devices convert the transmitted light energy into
electrical energy. This is amplified and measured by the Galvanometer.
The Galvanometer is calibrated to read the absorbance/transmittance directly.
It is necessary to standardize
the instrument or set it at zero using the blank, change cuvettes and read the
absorbance.
General procedure:
§
The light from a
tungsten lamp passes through a slit.
§
The sample should be
in room temperature and the cuvette should be clean. Before and after
keeping the cuvette in the holder it should be wiped thoroughly.
§
There should not very
low amount of sample and also it should not overflow.
§
Before keeping the
sample the optical density (O.D) must be set up with a blank as zero.
§
A condensing lens and
a filter emerge as a parallel beam of monochromatic light.
§
The monochromatic
light passes through the sample solution.
§
And the transmitted
light falls on the photocell.
§
The photocell converts
the transmitted light energy into electrical energy, which is amplified and
measured by the galvanometer.
§
The optical density
will appear in the output device, and based on the standard the concentration
of the unknown can be calculated.
Applications:
§
Colorimetry has
perhaps the widest application in biological sciences.
§
The concentration of
any unknown substance can be determined using a colorimeter.
§
If the substance by
itself is colorless it can be chemically converted to a colored substance
(stoichiometrically by adding a chromophoric group), and the concentration
measured.
§
By comparing the
standard curve the concentration of unknown can be calculated.
§
Thus colorimeter plays
an important role in the biological field.
FLUORESCENCE SPECTROSCOPY
Introduction
Molecular fluorescence is the
optical emission from molecules that have been excited to higher energy
levels by absorption of electromagnetic
radiation. The main advantage of fluorescence
detection compared to absorption measurements is the greater sensitivity achievable because
the fluorescence signal has in principle a zero background. Analytical
applications include quantitative measurements of molecules in solution and
fluorescence detection in liquid chromatography.
Basic Design
As stated in the introduction, the purpose
of a spectrophotometer is to chracterise how molecules react with photons of
varying wavelengths. This easiest way to do this is to design an instrument
that can control what type of light is in contact with the sample and the
amount of that light that makes it through. Shown below is a simple
spectrophotometer that does just what was described. A light source produces
light that can be separated and controlled by the monochrometer which then
allows only the desired light to impinge upon the sample. The light that
travels through the sample is detected and sent to a recorder. Keep this
overall picture in mind when thinking about the components and what each of
their parts are in whole experiment.
Sources
The first piece of equipment needed is
a source of photons (light). Since we are interested in the UV-Vis region of
the electromagnetic spectrum, we need to assure that this source is able to
produce photons with the correct wavelengths (180-780 nm) that are
characteristic to the spectrums of interest so that the experiment can be
completed. Typical sources come in two forms, either in the form of a lamp, or
in the form of a laser. Lamps can provide a wide spectrum of light that can be
used for almost any purpose at a very reasonable cost, while lasers provide
very intense monochromatic light and tend can be used for fluorescence and
other specialized experiments due to their high costs.
Filters and Monochrometers
Unless a laser is used, a device is
needed to restrict and isolate the wavelengths that our sources provide in
order to control the experiment. If a isolated wavelength is needed or isn't
needed, interference or absorption filters can be used. If a spectrum of
wavelengths needs to be passed through the sample, monochrometers provide the
ability to separate and control the light passed through the sample while
preserving the available spectrum.
Filters
Intereference filters are designed
to provide constructive or destructive interference of light by taking
advantage of the refraction of light through different materials. As light
passes from one medium to the other the direction and wavelength of light can
be changed based on the index of refraction of both mediums involved and the
angle of the incident and exiting light. Due to this behavior, constructive and
destructive interference can be controlled by varying the thickness (d) of a
transparent dielectric material between two semi-reflective sheets and the
angle the light is shined upon the surface. As light hits the first semi-reflective
sheet, a portion is reflected, while the rest travels through the dielectric to
be bent and reflected by the second semi-reflective sheet. If the conditions
are correct, the reflected light and the initial incident light will be in
phase and constructive interference occurs for only a particular wavelength.
Absorption filters work on the
premise of being able to filter light by absorbing all other wavelengths that
aren't of interest. They are normally constructed from a colored glass that
absorbs over a wide range. Although normally cheaper than interference filters,
absorbance filters tend to be less precise at filtering for a selected
wavelength and also have the added penalty of absorbing some of the selected
light thus lowering the intensity.
Monochrometers
Monochrometers are used to control
and separate light so that a sample can be subjected to a span of
wavelengths. Light entering a monochrometer is filtered by a thin slit.
The filtered light is then focused by a mirror onto a dispersing element
that separates the light into its different wavelengths. The separated light is
then focused again and angled toward an exit slit which then filters against
all wavelengths except the desired one. The wavelength selected can then easily
be changed by rotating the dispersing element to support the transmission of
the new desired wavelength. Though monochrometers nearly all have the same
practical design, the difference normally is determined by the type of
dispersing element.
One type of dispersing element is a prism which disperses
light by refraction through two angled surfaces. In order to separate the light
into different wavelengths, the prism needs to be made of material that has a
change in the index of refraction with respect to wavelength so that each
wavelength is bent at a different angle. The larger the difference in the index
of refraction the better the separation is between wavelengths. For the
UV-Vis range, a typical prism is cut from left handed quartz at a thirty degree
angle and attached to another piece of quarts cut the same way except from
right handed quartz to make a Conru Prism. The pitfall of prism
based dispersing elements is the fact that the index of refraction for
most materials varies nonlinearly when compared to wavelength which results in
a smaller degree of separation at longer wavelengths.
More commonly used due to less
expensive fabrication costs and its ability to separate light in a linear
fashion are grating dispersing elements. Gratings are designed to diffract
light which is a separation based on the angle of the incident light to the
grating normal and the spacing between groves. When beams of light impinge upon
the grating some beams travel farther than others and this causes an effect
akin to a interference filter which allows for constructive and destructive
interference and provides the means to reflect a specific wavelength based on
the angle of incidence. The most common type of grating is the echellette
grating in which the grooves are angled in order to provide maximum reflection
of the incident light into a single order of reflected light.
Sample Cell
Care must be taken when considering
a sample container to avoid unwanted absorption in the range of interest. While
glass might be perfect for the visible range, it absorbs in the UV range.
Quartz can be used for both but most likely would cost considerably more.
Disposable Plastic cuvettes created from polystyrene or polymethyl methacrylate
are in common use today as they are cheap to purchase and eliminate the need
for cleaning cuvettes in order to analyze multiple samples. The shape of the
sample holder is also very important as unwanted scattering of light should be
minimized and pathlength through the cell should remain constant. Square (1 cm)
cuvettes with frosted sides have been designed to minimize scattering and
provide a surface to hold the cuvette, thereby reducing smudges or smears of
the optical window all the while keeping a fixed path length. Cuvettes for
fluorescent experiments cannot allow the frosted sides due to the 90° angle of
most instruments and have to be carefully cleaned as bodily oils have the
ability to fluoresce. Placement of the sample within either a absorption or
fluorescence device is imperative and is normally fixed by a sample holder to
insure reproducible results.
Detectors
After the light has passed through
the sample, we want to be able to detect and measure the resulting light. These
types of detectors come in the form of transducers that are able to take energy
from light and convert it into an electrical signal that can be recorded, and
if necessary, amplified. Photomultiplier tubes are a common example of a
transducer that is used in a variety of devices. The idea is that when a photon
hits the top of the tube electrons are released, which are pulled toward the other
end of the tube by an electric field. The way the electrons are multiplied is
due to the fact that along the length of the tube there are several dynodes
that have a slightly less negative potential than surface before it which
causes the electrons traveling down the tube to hit each surface which then in
turn produces more electrons until at the very end there is a large amount of
electrons (~1,000,000) representing the one photon that started the cascade.
Charge
injection device (CID)
This device works by having a p-type
and n-type semiconductor next to each other in which the n-type material is
separated from the anodes by a silica layer that acts as a capacitor. As a
photon interacts with the n-type layer, an electron migrates to the p-type semiconductor
and a positive charge is generated that migrates toward the silica capacitor.
This process continues to happen until the potential is measured by means of
comparing the more negative of the anodes to ground (Vi) . The
charge is then transferred to the other anode and measured (Vf). The
difference between Vf and Vi is directly proportional to
the amount of photons that collided with the n-type layer. The positive charges
are then repelled when the anodes switch momentarily to having a positive charge
and then the cycle can be repeated. Thousands of these little detectors can be
aligned and used to describe the light that interacts with it and can rival the
performance of the photomultiplier tube.
Instrumental Designs
Single Beam Spectrometer
This
device can measure the transmittance or absorbance of a particular analyte at a
given wavelength provided by the source and monochrometer. Background
subtraction in these machines needs to be done separately before the analyte is
inserted and can be stored and subtracted by the signal processor attached.
For fluorescence spectroscopy the
equivalent to a single beam spectrometer has a few slight modifications in that
the detector is perpendicular to the source, and that there is an additional
monochrometer that can be used to vary the wavelength detected. In this
fashion, the source light is unable to interfere with the fluorescence light
being measured, and the fluorescent light produced can be separated and
described as the resulting wavelengths of fluorescence as a function of
incident light.
Double
Beam Spectrometer
A double beam spectrometer takes a
more analytical approach to the design. Because of fluctuations in the source
intensity and inconsistantcies in the transducer, the source light is split
into two beams, one which travels through the sample, and another that is sent
through a blank or standard solution. Both beams are then read by separate
transducers and the difference between the two is recorded as the corrected
transmittance. This allows for quick screening of analytes and negates the need
for two separate scans to complete a background subtraction. The same idea can
be applied to a fluorometer as well to obtain the same benefit.
The latest instrument designs use a
multichannel detector, such as an array of CID's, that allow for the spectrum
of an analyte to be gathered in seconds due to the fact that the light
transmitted through the sample can be split and a spectrum of wavelengths can
be monitored simultaniously instead of individually. Also fiber optic cables
can be used to transfer the light from the source to the sample or from the
sample to the dispersion grating and negate the need to consider the slit when
thinking about resolution.
Applications
§ Fluorescence spectroscopy is used in biochemical, medical
and chemical research fields for analyzing organic compounds. There has also
been a report of its use in differentiating malignant, bashful skin tumors from
benign.
§ Atomic Fluorescence Spectroscopy (AFS) techniques are useful
in other kinds of analysis or measurement of a compound present in air, water,
or other media.
§ Fluorescence can also be used to redirect photons.
§ Fluorescence spectroscopy can be adapted to the microscopic
level using microflurimetry.
§ In analytical chemistry, fluorescence detectors are used
with HPLC.
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