Spectrophotometry is used in Biology to plot optical density curves (to determine the concentration of biochemicals) or to conduct a cell count for a suspension.

1. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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SPECTROPHOTOMETRY IN BIOLOGY
“Spectrophotometry is the quantitative measurement of the reflection or transmission
properties of a material as a function of wavelength, it deals with visible light, near-
ultraviolet, and near-infrared. Spectrophotometers are special types of photometers that
can measure a light beam's intensity as a function of its color (wavelength).”
Important features of spectrophotometers are spectral bandwidth (the range of colors it can
transmit through the test sample), the percentage of sample-transmission, the logarithmic
range of sample-absorption, and sometimes a percentage of reflectance measurement.
STRUCTURE OF A SPECTROPHOTOMETER
A modern spectrophotometer includes: (1) an optic/beam splitting unit which isolates a light
ray of the desired wavelength, (2) sample unit which holds the sample, (3) light detection and
processing unit, and (4) display and user interface.
Figure: Table top spectrophotometer
1. BEAM SPLITTING UNIT (OPTICAL UNIT)
Light Source: The common light sources of a spectrophotometer are tungsten halogen lamps
(for visible light), deuterium lamps (for UV light), and xenon lamps (visible + UV).
Light Source Switch: It is used to sequentially switch the UV light source and visible light
source.

2. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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Collimating Mirror (Reflector & Light-in Slit): Before the light been spitted into a single color
beam, the original light from the lamps can be preprocessed into a straight parallel light rays
by these optic devices.
Grating: The surface of a grating is a jagged mirror. It is used to disperse the original color-
mixed “white light” into several individual color light (it has replaced prisms).
Collimating Mirror (Light-out Slit): After the white light is dispersed into pure individual
colors, the light is unfolded like a folding fan. By rotating the angle of a collimating mirror, a
specific wavelength of the color light can be aligned on the light-out slit. After the color light
passing through the slit, the unwanted wavelengths are filtered out, and a single pure light
(of the desired wavelength) is produced.
2. SAMPLE UNIT
Cuvette: A cuvette is a container for liquid samples, with an opened square hole on the top,
for pouring into the sample. The general materials of a cuvette can be glass, quartz (for UV),
or plastic, and so on. The width of a cuvette affects the path length of the beam getting
through the sample, resulting in different absorbance. The most common cuvettes are 10mm
(1cm) wide. Some "micro cuvettes" are designed for a tiny amount of samples, such as DNA
and RNA samples. Or some long optical path, 10cm wide cuvette for low concentration
samples.
Cuvette Holder: The cuvette is placed on the cuvette holder, which is usually a four-channel
manual pull-push holder with a rod for four cuvettes. There are hollowed-out holes on the
sides of the holder, which allow the beam to pass through the sample. There are also
automatic cuvette holder in some higher-end spectrophotometer, which switches the
cuvettes one by one automatically, by the preset program.
3. LIGHT DETECTION & PROCESSING UNIT
Light Detector: The most common light detector is silicon photodiodes. The signal outputs
from the photodiode is voltage, which is an analog signal. So, it will need an analog to digital
converter (A/D converter), to digitalize the intensity of the light.
Digital Signal Processor: The processor can process the digital signals from the light sensor,
and simultaneously control the step motors and moving parts in the instrument. The result
received from the light detector is processed a series of absorbance values that the user can
read.
4. DISPLAY & USER INTERFACE
LCD Screen & Keyboard: LCD screen has different sizes and resolutions on a
spectrophotometer. On a high-end spectrophotometer, the resolution of the screen accepts
the spectrum to be displayed directly. And the operation can be down via the touch screen.

3. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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USB Connection: It allows the users to connect the spectrophotometer to a computer, or one
can insert a USB stick to export measurement results. Via this feature, one can use a computer
to draw a spectrum, or export a report to a PDF file, etc.
SINGLE BEAM & DOUBLE BEAM SPECTROPHOTOMETERS
Spectrophotometers can either use a single beam or two beams:
1. DOUBLE BEAM SPECTROPHOTOMETER: It compares the light intensity between two
light paths, one path containing a reference sample and the other the test sample.
2. SINGLE BEAM SPECTROPHOTOMETER: It measures the relative light intensity of the
beam before and after a test sample is inserted.
Figure: Single Beam Spectrophotometer
Although comparison measurements from double-beam instruments are easier and more
stable, single-beam instruments can have a larger dynamic range and are optically simpler
and more compact. Additionally, some specialized instruments, such as spectrophotometers
built onto microscopes or telescopes, are single-beam instruments due to practicality.
TRANSMISSION & REFLECTANCE MEASUREMENTS
The lights rays may be measured either after they have passed through a sample or reflected
off its surface:
1. TRANSMISSION MEASUREMENTS: The spectrophotometer quantitatively compares the
fraction of light that passes through a reference solution and a test solution, then
electronically compares the intensities of the two signals and computes the percentage
of transmission of the sample compared to the reference standard.
2. REFLECTANCE MEASUREMENTS: The spectrophotometer quantitatively compares the
fraction of light that reflects from the reference and test samples.

4. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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WORKING OF A SPECTROPHOTOMETER
Light from the source lamp is passed through a monochromator, which diffracts the light into
a "rainbow" of wavelengths through a rotating prism and outputs narrow bandwidths of this
diffracted spectrum through a mechanical slit on the output side of the monochromator.
These bandwidths are transmitted through the test sample. The transmittance or reflectance
value for each wavelength of the test sample is then compared with the transmission or
reflectance values from the reference sample. Most instruments will apply a logarithmic
function to the linear transmittance ratio to calculate the 'absorbency' of the sample, a value
which is proportional to the 'concentration' of the chemical being measured. In short, the
sequence of events in a scanning spectrophotometer is as follows:
 The light source is shone into a monochromator, diffracted into a rainbow, and split into
two beams. It is then scanned through the sample and the reference solutions.
 Fractions of the incident wavelengths are transmitted through, or reflected from, the
sample and the reference.
 The resultant light strikes the photodetector device, which compares the relative intensity
of the two beams.
 Electronic circuits convert the relative currents into linear transmission percentages
and/or absorbance/concentration values.
ABSORBANCE & OPTICAL DENSITY
Absorbance measures the ability of a refractive medium or optical component to absorb light.
It measures how much light is lost during the light's passage through the given medium. It is
given by the following formula:
Where: A is absorbance, Io is the intensity of the light entering
the specimen and I is the intensity of the light leaving the sample.
The optical density of a substance is the logarithmic ratio of the intensity of transmitted light
to the intensity of the incident light passing through the substance. It is otherwise measured
as the absorbed radiation of the corresponding wavelength. Optical density refers to the
absorbance of a substance. It is given by the following formula:
Where, OD is optical density and T is transmittance (the ratio of
the light energy falling on a body to that transmitted through it).
APPLICATIONS IN BIOCHEMISTRY
Spectrophotometry is an important technique used in many biochemical experiments that
involve DNA, RNA, and protein isolation, enzyme kinetics, and biochemical analyses. Since
samples in these applications are not readily available in large quantities, they are especially

5. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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suited to being analyzed in this non-destructive technique. Also, precious samples can be
saved by utilizing a micro-volume platform, where, as little as 1µL of sample is required for
complete analyses. A brief explanation of the procedure of spectrophotometry includes
comparing the absorbency of a blank sample that does not contain a colored compound to a
sample that contains a colored compound. This coloring can be accomplished by either a dye
such as Coomassie Brilliant Blue G-250 dye measured at 595 nm or by an enzymatic reaction
as seen between β-galactosidase and ONPG (turns sample yellow) measured at 420 nm. Some
important applications of spectrophotometry in biochemistry are as follows:
 In biochemical experiments, a chemical and/or physical property is chosen and the
procedure that is used is specific to that property to derive more information about the
sample, such as the quantity, purity, enzyme activity, etc.
 Spectrophotometry can be used for several techniques such as determining optimal
wavelength absorbance of samples, determining optimal pH for absorbance of samples,
determining concentrations of unknown samples, and determining the pKa of various
samples.
 Spectrophotometry is also a helpful process for protein purification.
 It can also be used as a method to create optical assays of a compound.
 Spectrophotometric data can also be used in conjunction with the Beer-Lambert Equation
to determine various relationships between transmittance and concentration, and
absorbance and concentration.
TURBIDIMETRY
The turbidimetric method uses spectrophotometers or colorimeters to measure the number
of bacterial cells in a solution based on the turbidity of the solution, due to their presence.
Microbiologists use turbidity as a measure of cell density within a culture sample.
Microbiologists use photometers and spectrophotometers that shine different types of light
through culture samples to determine turbidity. The general assumption is that the higher the
turbidity, the higher number of cells within the culture. It is one of the most popular methods
for estimating microbial population of a medium and also to note the rate of growth. But it
does have some limitations:
 The culture to be measured must be dense enough to register some turbidity on the
instrument.
 It may not be possible to measure cultures grown on a deeply colored media.
 It may not be possible to measure cultures that contain suspended material other than
bacteria.
 Both dead and living cells contribute to the turbidity.
UV-VISIBLE & IR SPECTROPHOTOMETRY

6. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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The details of the two most common types of spectrophotometric techniques, UV-Visible
spectrophotometry and Infrared Spectrophotometry, are as follows:
1. UV-VISIBLE SPECTROPHOTOMETRY: Many spectrophotometers are used in the UV
and visible regions of the spectrum. Ultraviolet-Visible (UV-VIS) spectroscopy involves
energy levels that excite electronic transitions. Absorption of UV-VIS light excites
molecules that are in ground-states to their excited-states. Visible region 400 – 700 nm
spectrophotometry is used extensively in colorimetry science. Scientists use this
instrument to measure the amount of compounds in a sample. If the compound is more
concentrated more light will be absorbed by the sample. Samples are usually prepared in
cuvettes; depending on the region of interest, they may be constructed of glass, plastic
(visible spectrum region of interest), or quartz (Far UV spectrum region of interest). Some
applications require small volume measurements which can be performed with micro-
volume platforms.
2. IR SPECTROPHOTOMETRY: Spectrophotometers designed for the infrared region are
quite different because of the technical requirements of measurement in that region. One
major factor is the type of photo-sensors that are available for different spectral regions,
but the infrared measurement is also challenging because virtually everything emits IR
light as thermal radiation. Another complication is that quite a few materials such as glass
and plastic absorb infrared light, making it incompatible as an optical medium. Ideal
optical materials are salts, which do not absorb strongly. Samples for IR
spectrophotometry may be smeared between two discs of potassium bromide or ground
with potassium bromide and pressed into a pellet. Where aqueous solutions are to be
measured, insoluble silver chloride is used to construct the cell.