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Instrument 2:

• Collimate
• Monochromator
• Wavelength
• Spectroscopy
• Spectrometry
• Photometry
• Spectrum
• Slit
• Cuvette
• Absorb
• Transmit

• 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.
• Spectroscopy
• The science of studying the interaction between matter and radiated
• Spectrometry
• Method used to acquire a quantitative measurement of the spectrum.
• Spectrum
• Band of colours, as seen in a rainbow, produced by separation of the
components of light by their different degrees of refraction according
to wavelength.
• Photometry
• The science of the measurement of light
• Spectrophotometer
• Instrument for measuring the intensity of light in a part of the
spectrum, especially as transmitted or emitted by particular
Band of colours, as seen in a rainbow, produced by separation of the
components of light by their different degrees of refraction according
to wavelength.
The distance between successive crests of a wave, especially
points in a sound wave or electromagnetic wave.
Spectrometry & Photometry

Spectrometry Photometry
Spectrometer: It produces a desired range of wavelength of light. Photometer: After the desired range
First a collimator (lens) transmits a straight beam of light of wavelength of light passes through
(photons) that passes through a monochromator (prism) to split it the solution of a sample in cuvette,
into several component wavelengths (spectrum). Then a the photometer detects the amount
wavelength selector (slit) transmits only the desired wavelengths of photons that is absorbed and then
sends a signal to a galvanometer or a
digital display
Component of Spectrophotometry
Spectrophotometer consists of three main parts:

1. Emission source which produces the spectrum

2. Optical system which collimates and disperses the
3. Detecting device to measure the emitted lines
• A cuvette (French: cuvette = "little
vessel") is a small tube-like
container with straight sides and a
circular or square cross section.
• It is sealed at one end, and made
of a clear, transparent material
such as plastic, glass, or fused
absorbs: Red, Green
transmits: Blue

• many solutions have distinctive colours

– absorb/transmit different wavelengths of visible light

• intensity of colour is an indication of the

solution’s concentration
– more concentrated = darker colour
• a quantitative method of studying matter
• studies amount of light transmitted/absorbed by
Electromagnetic Spectrum
400 nm 700 nm
UV-Vis and IR Spectrophotometer
• Depending on the range of wavelength of light
source, it can be classified into two different

• UV-visible spectrophotometer: uses light over the

ultraviolet range (185 - 400 nm) and visible range
(400 - 700 nm) of electromagnetic radiation
• IR spectrophotometer: uses light over the
infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.
• measures absorbance or
transmittance of light, as a
function of wavelength
a cuvette

General procedure:
– sample is placed into cuvette
– light of selected wavelength (λmax)
is passed through sample
– instrument measures the amount
of light absorbed by the sample
• Reference blank
– contains everything found in the sample solution,
except the substance being analyzed
– calibrates the absorbance reading to A=0.000


Monochromic light

• Tungsten halogen • Prism

lamps (VIS, visible).
• Diffraction grating
• Deuterium lamps (UV)

Let’s watch a

an optical component with a An optical element with flat,

periodic structure that splits polished surfaces that refract light.
and diffracts light into At least two of the flat surfaces
several beams travelling in must have an angle between
different directions. them.
• Io = Original intensity of light
• It = Intensity of transmitted light

Monochromator separates and Two ways of Reporting:

transmits a particular 1. Transmission, T = It/Io
wavelength (colour) of light
or %T = It/Io × 100%

2. Absorbance, A = -log(%T)
Absorbance and wavelength
Choosing a Wavelength
• use wavelength at which most light is absorbed

a) What is λmax for this

b) What colour light would
you use to analyze this
c) What colour does this
solution appear to the

Absorption spectrum for a sample

Beer-Lambert Law
• Beer-Lambert Law (also known as Beer's Law) states that
there is a linear relationship between the absorbance and
the concentration of a sample. Beer's Law can be applied
when there is a linear relationship. Beer's Law is written as:

(or molar extinction


• Transmittance is the fraction of light that passes

through the sample. This can be calculated using the
• Absorbance stands for the amount of photons
that is absorbed.
What does this tell us?
• There is a direct relationship between absorbance
and concentration
• Empirical evidence: prepare solutions of known
concentration and analyze them at λmax: plot of
absorbance vs. concentration is linear.
 Since A is a linear function of c,
equation of line: y = mx + b
A = εLc

…slope of best-fit line = εL

Calculation 1
• Guanosine has a maximum absorbance of 275
nm. ϵ275 = 8400 L mol-1 cm-1, and the path
length is 1 cm. Using a spectrophotometer,
you find the that A275=0.70. What is the
concentration of guanosine?
• Answer:
• A = ϵlc
• 0.7 = (8400 L mol-1 cm-1)x(1 cm)x(c)
• c = 8.33x10-5 mol/L
Calculation 2
• Using a cuvette with a length of 1 cm, you
measured the absorbance of a solution with a
concentration of 0.05 mol/L. The absorbance
at a wavelength of 280 nm was 1.5. What is
the molar absorptivity of this solution?
• Answer:
• ɛ280 = A/lc = 1.5/(1 x 0.05) = 30 L mol-1 cm-1
Calculation 3
• There is a substance in a solution (4 g/liter). The
length of cuvette is 2 cm and only 50% of the
certain light beam is transmitted. What is the
absorption coefficient?
• Answer:
• Absorbance (A) = -log(It/Io) = -log(50/100)
• -log(50/100) = 0.30102999
• A = 0.30102999
• A = eLc = e(2)(4)
• e = 0.30102999/8 = 0.0376
Calculation 4
Information given:
• Concentration of compound M = 8 g/liter
• Cuvette length = 2 cm
• The value of molar extinction coefficient for compound M =

Based on information above, how much is the percentage beam of

light is transmitted through compound M?


• -log(It/Io) = eLc
• -log(It/Io) = (0.0376)(2)(8) = 0.6016
• It/Io = ?
• 25%
Calculation 5
• The absorption coefficient of a glycogen-
iodine complex is 0.20 at light of 450 nm.
What is the concentration when the
transmission is 40 % in a cuvette of 2 cm?

• Answer
• -log (0.4) = 0.20 x C x 2
• C = 0.9948
• Matter interacts with light by absorbing and
transmitting different wavelengths.
– The specific nature of the interaction depends on a
compound’s molecular structure.

• Spectrophotometry quantitatively describes either

the transmittance or absorbance of light by a
– the optimal light wavelength to use for analysis is
denoted λmax, and is the wavelength at which maximal
light absorbance occurs
• Beer’s Law states that a substance’s
absorbance value is directly proportional to
its concentration.
– if absorbance can be measured, it can be used to
determine the concentration of an unknown

• familiarize yourself with the three methods

by which Beer’s Law can be used to
determine the concentration of an unknown
Precautions When Handling a
• Allowing the lamps and electronics to warm up
• Using the correct wavelength
• Wiping fingerprints and spilt sample off the outside of
the cuvette before measuring.
• Avoid to make any scratch on the surface of cuvette.
• Carrying out the set-up procedure in the correct order
• Performing calibration checks after set up
• Closing the door to the cuvette compartment before
reading the result
• Cleaning up any spills inside the cuvette compartment

Turn on the spectrophotometer. Most spectrophotometers need to

warm up before they can give an accurate reading. Turn on the machine
and let it sit for at least 15 minutes before running any samples.
However, this is still depending on the unit/brand/model that you are

Clean the cuvettes or test tubes. Rinse each cuvette thoroughly with
deionized water.

Load the proper volume of the sample into the cuvette. Some cuvettes
have a maximum volume of 1 milliliter (mL) while test tubes may have a
maximum volume of 5 mL. As long as the laser producing the light is
passing through the liquid and not an empty part of the container, you
will get an accurate reading. If you are using a pipette to load your
samples, use a new tip for each sample to prevent cross-contamination.

Prepare a control solution. Known as a blank, the control solution has

only the chemical solvent in which the solute to be analyzed is dissolved
in. For example, if you had salt dissolved in water, your blank would be
just water. If you dye the water red, the blank must also contain red
water. The blank is the same volume as the solution to be analyzed and
kept in the same kind of container.

Wipe the outside of the cuvette. Before placing the cuvette into the
spectrophotometer you want to make sure it is as clean as possible to
avoid interference from dirt or dust particles. Using a lint free cloth,
remove any water droplets or dust that may be on the outside of the
cuvette. Avoid from making any scratch on the surface of cuvette.

Choose and set the wavelength of light to analyze the sample with.

Calibrate the machine with the blank. Place the blank into the cuvette
holder and shut the lid.

Remove the blank and test the calibration.


Place the test sample, then make the reading


Calculate the transmittance and absorbance of the sample, in order to

determine the amount of species/compound solution. Using current
spectrophotometer models, everything is calculated for you.
Thank you