Martin Clyde G.

Paglinawan
BSMT-3B
July 23, 2014
SPECTROPHOTOMETER
A spectrophotometer is a device that measures the absorbance of light at a fixed
wavelength. Spectrophotometers are a common part of research facilities,
especially those that use dyes and colors in their work. Basically, a
spectrophotometer shines light at a certain frequency at a sample, and the intensity
of light that transmits through the sample becomes the data output on the other
side. The resulting data is a quantitative way of assessing the color (wavelength) or
intensity (absorbance) of any given sample. Spectrophotometers are also useful in
the textile and art industries for standardizing product colors.
Although different kinds of spectrophotometers exist, the most commonly used and
easily understood spectrophotometers are those that work within the visible
wavelength range. The visible wavelength range includes colors that the naked eye
is able to perceive, between 400 to 700 nanometers. Each color in the color
spectrum correlates to a wavelength within the visible region. For example, the dye
Acridine Orange is the color orange, which corresponds to between 595 and 610
nanometers. However, the transmittance wavelength for Acridine Orange does not
include the color orange. The eye perceives the color orange because the dye
absorbs all wavelengths except orange orange reflects. Therefore, the wavelength
that transmits through the sample is the opposite of orange. In fact, Acridine
Orange has an absorption wavelength of 469 nanometers.

Figure 1. The color wheel visible spectrum.

Each color corresponds to a certain
wavelength.
The visible-wavelength spectrophotometer
uses the concept of wavelength, along with
the intensity of the transmitted light, to
produce an absorbance value. The BeerLambert law quantitatively describes the
relationship between the absorbance and intensities of light in liquids.

Where:

A = Absorbance
I0 = Incident light intensity
I = Transmitted light intensity

Before light passes through the sample, it may have an incident light intensity of 1.
Once the light passes through the sample, the actual light that transmits to the
other side of the sample is the transmitted light intensity. The Beer-Lambert Law
relates the two light intensities in order to give a final absorbance value at a fixed
wavelength.
PARTS AND FUNCTIONS OF A SPECTROPHOTOMETER
Every single-beam spectrophotometer needs seven crucial parts in order to
function. The following lists each part in the order that they appear in the process of
running a spectrophotometer reading.

Figure 2. Seven crucial parts of a spectrophotometer. Hybrid diagram:

Light Source: emits white light (all frequencies). The light source transmits white
light, which contains all frequencies in the visible light spectrum. Usually the light
source is just a normal light bulb. For special wavelength readings, the light source
may be a UV bulb or some other wavelength.
Monochromator: selects one frequency (or color) of light. The word
monochromator literally means one color (mono-chrom) in Latin. This part of
the spectrophotometer filters out all other wavelengths except for the one specified
by the user.
Aperture: directs light. The aperture is a small hole that directs the specified
wavelength to a part of the sample. Some spectrophotometers have adjustable
apertures in order to capture absorbance at different spatial parts of the sample.
The aperture guarantees that each sample reading directs at the same place in the
sample, which eliminates error in uneven sample distributions.
Cuvette: holding cell for sample. Is usually plastic but may be made of other
materials. Cuvette volume capacities range from 1.5-3 ml and sizes also vary. Most
spectrophotometers allow for multiple cuvettes, but cuvettes must be the same
size.
Sample: solution user desires to quantify. A sample contains any liquid solution
which the user chooses to quantify. Liquid solutions usually contain dye, and the
resulting absorbance values correspond to the amount of dye (and therefore

product to which dye attaches) in the solution. Some spectrophotometers are able
to quantify solid or gas mixtures, but are much more expensive and use a different
process.
Photoresistor: detects transmittance intensity of light. The photoresistor is a very
sensitive electrical device that detects the intensity of light that is being transmitted
through the sample. The photoresistor measures in Ohms, the units of resistance.
However, the spectrophotometer contains a processing system that converts
resistance values to the absorbance of light.
Output Device: displays absorbance values for the user. A computer screen that
has a separating operating system from the spectrophotometer usually functions as
the output device. Both the spectrophotometer and the computer internally
connect, and the information output from the spectrophotometer saves directly on
the computer. However, some spectrophotometers have an imbedded screen on
which the output displays.
A. DIFFRACTION GRATING
Diffraction gratings Diffraction grating is an
optical component with a regular pattern, which
splits (diffracts) light into several beams
travelling in different directions. The directions
of these beams depend on the spacing of the
grating and the wavelength of the light so that
the grating acts as a dispersive element. The
diffraction grating disperses the light into a
linear spectrum of its component wavelengths,
which is then directed, in whole or in part along
the light path of the instrument
B. PRISM
Prism is used to isolate different wavelength
.If a parallel beam of radiation falls on a
prism, the radiation of two different
wavelength will be bent through different
angles. Prism may be made of glass or
quartz. The glass prisms are suitable for
radiation essentially in the visible range
whereas the quartz prism can cover the
ultraviolet spectrum also. It is found that the
dispersion given by glass is about three
times that of quartz.