SPECTROPHOTOMETRY

Dr. Kazi Hanium Maria

Course teacher
NE4109
 SPECTROPHOTOMETRY
◆ 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.
 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.

How it does this?

1. Diffracting the light beam into a spectrum of
wavelengths
2. Direct it to an object
3. Receiving the light reflected or returned from the object
4. Detecting the intensities with a charge-coupled device
5. Displaying the results as a graph on the detector and
then the display device.
 SPECTROPHOTOMETRY
1. Compounds absorbs light radiation of a specific wavelength.
2. The amount of light radiation absorbed by a sample is measure.
3. The light absorption is directly related to the concentration of the compound in
the sample.
4. As concentration increases, light absorption increases linearly. As concentration
increases, light transmission decreases consequently.
 TYPE OF SPECTROPHOTOMETER
Depending on the range of wavelength of the 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 the electromagnetic radiation spectrum.
IR spectrophotometer: uses light over the infrared range (700 – 1000 nm) of
the 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 overall visible ranges (i.e.,
transmits none of the 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 wavelengths (to filter out other wavelengths) so that the particular
beam of light is passed through a solution sample.
 PURPOSE OF SPECTROPHOTOMETER
✓ Measure the concentration of the solution:
1. A spectrophotometer optically determines the absorbance or
transmission of characteristic wavelengths of radiant energy (light)
by a chemical species in solution.
2. Each molecule absorbs light at certain wavelengths in a unique
spectral pattern because of the number and arrangement of its
characteristic functional groups, such as double bonds between
carbon atoms.
3. According to the Beer-Lamberts law, the amount of light absorbed
at these wavelengths is directly proportional to the concentration of
the chemical species.

✓ Identify organic compounds by determining the absorption maximum:

Spectrophotometers are used to identify organic compounds by
determining the absorption maxima (which for most compounds and
groups of compounds have very distinct fingerprints that’s what the
absorption curves and peaks are called)
PURPOSE OF SPECTROPHOTOMETER
✓ Used for color determination within the spectral range:
If one is working in the range of 380 to 700 nm, the spectrophotometers
can also be used for color determination within this spectral range.
✓ Example:
In the figure below the red part of the spectrum has been almost
completely absorbed by CuSO4 and blue light has been transmitted.
Thus, CuSO4 absorbs little blue light and therefore appears blue.
We will get better sensitivity by directing red light through the solution
because CuSO4 absorbs strongest at the red end of the visible spectrum.
But to do this, we have to isolate the red wavelengths.
 HISTORY OF SPECTROPHOTOMETER
✓ In older times, it took two weeks for the results to come out and, most of
the time, there was only 25 percent accuracy.
✓ It was rather depressing for these scientists because quick results are very
important to them, there wasn’t any good laboratory equipment at that time
to make the process faster.
✓ A scientist named Arnold J. Beckman and his colleagues at the National
Technologies Laboratory (NTL) invented the Beckman DU
spectrophotometer in 1940.
✓ Results come through simple process within few minutes which is 99.99%
accurate.
 PRINCIPLE OF SPECTROPHOTOMETER
Spectrophotometer is based on the photometric technique which states that when a beam of
incident light of intensity I0 passes through a solution, a part of the incident light is
reflected (Ir), a part is absorbed (Ia) and rest of the light is transmitted (It)
Thus, I0 = Ir + Ia + It

In spectrophotometer, (Ir) is eliminated because

the measurement of (I0) and it is sufficient to
determine the (Ia). For this purpose, the amount
of light reflected (Ir) is kept constant by using
cells that have identical properties. (I0) & (It) is
then measured.
Transmittance (T) = It/Io
Where It is the light intensity after the beam of light passes through the cuvette and Io is
the light intensity before the beam of light passes through the cuvette. Transmittance is
related to absorption by the expression:
Absorbance (A) = −log(T)= −log(It/Io) = log(I0/It)
Where absorbance stands for the amount of photons that is absorbed. With the amount of
absorbance known from the above equation, you can determine the unknown concentration
of the sample by using Beer-Lambert Law. Figure illustrates transmittance of light through
a sample. The length l is used for Beer-Lambert Law described below.
 PRINCIPLE OF SPECTROPHOTOMETER
The mathematical relationship between the amount of light absorbed and the
concentration of the substance can be shown by the two fundamental laws of
photometry on which the Spectrophotometer is based.
Beer’s Law
⇒ This law states that the amount of light absorbed is directly proportional to the
concentration of the solute in the solution.
Log10 I0/It = asc
where,
as = Absorbency index
c = Concentration of Solution
Lambert’s Law
⇒ The Lambert’s law states that the amount of light absorbed is directly proportional to
the length and thickness of the solution under analysis.
A = log10 I0/It = asl
Where,
A = Absorbance of test
as = Absorbance of standard
l = length / thickness of the solution
 PRINCIPLE OF SPECTROPHOTOMETER
The mathematical representation of the combined form of Beer-Lambert’s law is as
follows:
Log10 I0 / It = ascl
If l is kept constant by applying Cuvette or standard cell then,
Log10 I0/It = asc
The absorbency index as is defined as
as = A/cl
Where,
c = concentration of the absorbing material (in gm/liter).
l = distance traveled by the light in solution (in cm).
In simplified form,
The working principle of the Spectrophotometer is based on Beer-Lambert’s law which
states that the amount of light absorbed by a color solution is directly proportional to the
concentration of the solution and the length of a light path through the solution.
A ∝ cl or, A = ∈cl
Where,
A = Absorbance / Optical density of solution
c = Concentration of solution
l = Path length
∈ = molar extinction coefficient or molar absorptivity or Absorption coefficient
The molar extinction coefficient is given as a constant and varies for each molecule. Since
absorbance does not carry any units, the units for ∈ must cancel out the units of length
and concentration. As a result, ∈ has the units: L·mol-1·cm-1.
 EXAMPLES OF BEER-LAMBERT’S LAW
◆ Guanosine has a maximum absorbance of 275 nm. ϵ275=8400 M−1cm−1 and the path
length is 1 cm. Using a spectrophotometer, you find that A275=0.70. What is the
concentration of guanosine?
Solution: To solve this problem, you must use Beer's Law. A = ∈cl
 0.70 = (8400 M-1 cm-1)(1 cm)(c)
 c = 8.33x10-5 mol/L
◆ 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? how much is
the beam of light is transmitted when 8 g/liter ? what is the molar absorption coefficient
if the molecular weight is 100?
Solution: Using Beer-Lambert Law, we can compute the absorption coefficient.
Thus, log(I0/It) = log (0.5/1.0) = A = ∈cl
 log (0.5/1.0) = (4 g/liter) (2 cm) ∈
 ∈ = 0.0376
 EXAMPLES OF BEER-LAMBERT’S LAW
the transmission, A= log(I0/It) = log(1)−log(It) = 0 − log(It) = 0.0376 x 8 x 2 = 0.6016
 log(It) = -0.6016,
 It = 0.2503 = 25%
∈ can simply obtained by multiplying the absorption coefficient by the molecular
weight. Thus, ∈ = 0.0376 x 100 = 3.76 L·mol-1·cm-1
◆ 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?
Solution
It can also be solved using Beer-Lambert Law.
log(I0/It) = log (0.4/1.0) = A = ∈cl
 −log(It)=−log(0.4)=0.20×c×2
 c = 0.9948
 DEVICES AND MECHANISM
Figure illustrates the basic
structure of
spectrophotometers. It
consists of a light source,
a collimator, a
Monochromator, a
wavelength selector, a
cuvette for sample
solution, a photoelectric
detector, and a digital
display or a meter.

Figure shows a sample

spectrophotometer
(Model: Spectronic 20D).
 DEVICES AND MECHANISM
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.
Spectrometer: It produces a desired range of wavelength of light. First a collimator (lens)
transmits a straight beam of light (photons) that passes through a monochromator (prism)
to split it into several component wavelengths (spectrum). Then a wavelength selector
(slit) transmits only the desired wavelengths.
Photometer: After the desired range of wavelength of light passes through the solution of
a sample in cuvette, the photometer detects the amount of photons that is absorbed and
then sends a signal to a galvanometer or a digital display.
Spectrophotometers come in a variety of shapes and sizes and have multipurpose uses to
them. The different types of spectrophotometers available are all different from one
another, based on their application and desired functionality. The most popular
spectrophotometers are 45 degrees, sphere and multi-angle spectrophotometers.
Basically a spectrometer is needed to produce a variety of wavelengths
because different compounds absorb best at different wavelengths. For
example, p-nitrophenol (acid form) has the maximum absorbance at
approximately 320 nm and p-nitrophenolate (basic form) absorb best at 400nm,
as shown in Figure.

Figure :Absorbance of two different compounds

From graph that absorbance and wavelength can be measured. An isosbestic point can also
be observed. An isosbestic point is the wavelength in which the absorbance of two or more
species are the same. The appearance of an isosbestic point in a reaction demonstrates that
an intermediate is NOT required to form a product from a reactant. Figure shows an
example of an isosbestic point.

The amount of photons that goes through the cuvette and into the detector is dependent on
the length of the cuvette and the concentration of the sample. Once we know the intensity of
light after it passes through the cuvette, we can relate it to transmittance (T). Transmittance
is the fraction of light that passes through the sample. This can be calculated using the
equation:
 STANDARDIZATION GRAPH

Standards (solutions of known concentration) of the compound of

interest are made, treated and their absorbance (ABS) and
concentration values are used to create a standardization graph.
INTERNAL COMPONENTS OF
SPECTROPHOTOMETER
 PARTS OF SPECTROPHOTOMETER
There are 6 essential parts of a spectrophotometer
1. Light source – In a spectrophotometer, three different sources of light are commonly
used to produce light of different wavelengths. The most common source of light used in
the spectrophotometer for the visible spectrum is a tungsten lamp. For Ultraviolet
radiation, commonly used sources are the hydrogen lamp and the deuterium lamp. Nernst
filament or globar is the most satisfactory source of IR (Infrared) radiation.

Tungsten lamp
Tungsten Halogen lamp – It is the most common light source used in spectrometer. This
lamp consists of a tungsten filament enclosed in a glass envelope, with a wavelength range
of about 330 to 900 nm, are used for the visible region. They are generally useful for
measuring moderately dilute solutions in which the change in color intensity varies
significantly with changes in concentration. It has long life about 1200h.
 PARTS OF SPECTROPHOTOMETER
Hydrogen/Deuterium lamps – For the ultraviolet region, hydrogen or deuterium lamps
are frequently used. Their range is approximately 200 to 450 nm. Deuterium lamps are
generally more stable and has long life about 500h. This lamp generates continuous or
discontinuous spectral.

Xenon flash lamps – Xenon flash lamps have several advantages as the following:
1. Their range between (190 nm – 1000 nm)
2. Emit both UV and visible wavelengths
3. Long life
4. Do not heat up the instrument
5. Reduce warm up time.
 PARTS OF SPECTROPHOTOMETER

2. Dispersion devices:
A special plate with hundreds of
parallel grooved lines. The grooved
lines act to separate the white light
into the visible light spectrum. The
more lines the smaller the wavelength
resolution.

Monochromator – To select the particular

wavelength, prism or diffraction grating is used to
split the light from the light source. It accepts
polychromatic input light from a lamp and outputs
monochromatic light. Monochromator has entrance
slit, collimating lens or mirror, dispersion element,
focusing lens or mirror and exit slit.
 PARTS OF SPECTROPHOTOMETER
Types of dispersion devices:
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.
Filter
Filters separate different parts of the electromagnetic spectrum by absorbing or
reflecting certain wavelengths and transmitting other wavelengths.
Absorption filters are glass substrates containing absorbing species that absorb certain
wavelength. A typical example is a cut on color filter, which blocks short wavelength
light such as an excitation source, and transmits longer wavelength light such as
fluorescence that reaches a detector.
Interference filters are made of multiple dielectric thin films on a substrate. They use
interference to selectively transmit or reflect a certain range of wavelengths. A typical
example is a Bandpass interference filter that transmits a narrow range of wavelengths
and can isolate a single emission line from a discharge lamp.
 PARTS OF SPECTROPHOTOMETER
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.
3. Focusing devices:
Combinations of lenses, slits and mirrors. It relay and focus light through the instrument.
Variable slits also permit adjustments in the total radiant energy reaching the detector.
PARTS OF SPECTROPHOTOMETER
Optical materials
Mirrors

Types of rays Mirror materials

X-rays- Ultraviolet (UV) Aluminum
Visible Aluminum
Near infrared Gold
Infrared (IR) Copper or gold

Lenses

Types of rays Mirror materials

X-rays- Ultraviolet (UV) Fluid silica, sapphire
Visible Glass
Infrared (IR) Glass
 PARTS OF SPECTROPHOTOMETER
4. Sample holder – Test tube or Cuvettes are used to hold the colored solutions. They
are made up of glass at a visible wavelength.
Cuvettes:
• It is designed to hold samples for spectroscopic
experiments made of plastic, glass or optical
quartz.
• It should be as clear as possible, without
impurities that might affect a spectroscopic
reading.
• It is usually a small square tube sealed at one end. Like a test-tube, a cuvette may be open
to the atmosphere on top or have a glass or Teflon cap to seal it shut.
• Cuvettes are chosen for transparency in the spectral wavelengths of interest. For
measurement in the visible region cuvettes of optical glass are sufficient; however, optical
glass absorbs light below 350 nm, and more expensive quartz or fused silica must be used
for these wavelengths. The sample cuvettes are placed in a darkened analysis chamber;
some chambers have rotating carousels that can hold several cuvettes.
 PARTS OF SPECTROPHOTOMETER
5. Detectors:
• it can convert radiant energy (photons) into as electrical signal. The photocell and
phototube are the simplest photodetectors, producing current proportional to the
intensity of the light striking them. When light falls on the detector system, an electric
current is generated that reflects the galvanometer reading.
• Any photosensitive device can be used as a detector of radiant energy.

There are two types of detectors.

Silicon pin photodiodes photovoltaic V-series: blue
enhances for spectral range from 350 nm to 1100 nm;
designed for low-noise, D.C. to medium bandwidth Photomultiplier tube Detectors

applications. Active areas range from 0.31 mm2 to 100 mm2.

applications include: low light level measurements, particle
counting, chemical and analytical measurement and detection.
 PARTS OF SPECTROPHOTOMETER
Gallium Nitride (GaN) UV detectors: This family of
Gallium Nitride (GaN) UV detectors are Schottky processed
fully passivated U.V. photodiodes. Spectral range from 200
nm to 365 nm and is ideal for UVA(315-400 nm) or
UVB(280-314 nm) sensing applications and is packaged with
a quartz window.

Beam splitter – It is present only in double beam spectrophotometer. It is used to split the
single beam of light coming from the light source into two beams.
Bandpass filter – It is a device that passes frequencies within a certain range and rejects
frequencies outside that range.
Mirror – It is also present only and double beam spectrophotometer. It is used to the right
direction to the splitted light from the beam splitter.
 PARTS OF SPECTROPHOTOMETER
6. Display devices – The data from a detector are displayed by a readout device, such
as analog meter, a light beam reflected on a scale, or a digital display, or liquid crystal
display (LCD). The current from the detector is fed to the measuring device – the
galvanometer. The meter reading is directly proportional to the intensity of light. The
output can also be transmitted to a computer or printer.
 HOW DOES A SPECTROPHOTOMETER WORK?
• First put the sample into a cuvette, then the light source generates light at a
specific wavelengths and the light passes through the dispersion devices that
separate the light into its components wavelengths.
• Slits then isolate the wave lengths needed for measurement with a bandpass filter
to improve its purity.
• The light passes through the
sample and a portion of
radiant energy absorbed. The
remaining light is transmitted
to the photometer, which
convert light energy to
electrical energy can be
registered on a meter.

• The amount of light absorbed depends on the nature of the concentration of the
sample.
 TYPES OF SPECTROPHOTOMETER

Spectrophotometer can be classified into two different types:

Single beam spectrophotometer operates between 325 nm to 1000 nm
wavelength using the single beam of light. The light travels in one direction
and the test solution and blank are read in the same. To measure the intensity
of the incident light the sample must be removed so that the reference can be
placed each time. This type of spectrophotometer is usually less expensive and
less complicated.

Single beam spectrophotometer

 TYPES OF SPECTROPHOTOMETER
Double beam spectrophotometer operates between 185 nm to 1000 nm wavelength. It

has two photocells. This instrument splits the light from the Monochromator into two

beams before it reaches the sample. One beam is used for reference and the other passes

through the sample for reading. This gives an advantage because the reference reading

and sample reading can take place at the same time. It eliminates the error which occurs

due to fluctuations in the light output and the sensitivity of the detector.

Double beam spectrophotometer

 WORKING PRINCIPLE OF THE
SPECTROPHOTOMETER

When using a Spectrophotometer, it requires being calibrated first which is done by

using the standard solutions of the known concentration of the solute that has to be
determined in the test solution. For this, the standard solutions are filled in the Cuvettes
and placed in the Cuvette holder in the spectrophotometer.

⇒ There is a ray of light with a certain wavelength that is specific for the assay is directed
towards the solution. Before reaching the solution the ray of light passes through a series
of the diffraction grating, prism, and mirrors. These mirrors are used for navigation of the
light in the spectrophotometer and the prism splits the beam of light into different
wavelength and the diffraction grating allows the required wavelength to pass through it
and reaches the Cuvette containing the standard or Test solutions. It analyzes the reflected
light and compares with a predetermined standard solution.
 WORKING PRINCIPLE OF THE
SPECTROPHOTOMETER
⇒ When the monochromatic light (light of one wavelength) reaches the Cuvette
some of the light is reflected, some part of the light is absorbed by the solution
and the remaining part is transmitted through the solution which falls on the
photodetector system. The photodetector system measures the intensity of
transmitted light and converts it into the electrical signals that are sent to the
galvanometer.

⇒ The galvanometer measures the electrical signals and displays it in the digital
form. That digital representation of the electrical signals is the absorbance or
optical density of the solution analyzed.
 WORKING PRINCIPLE OF THE
SPECTROPHOTOMETER
⇒ Absorption of the solution is higher means more light absorbed by the solution and
if the absorption of the solution is low, that means more lights will be transmitted
through the solution which affects the galvanometer reading and corresponds to the
concentration of the solute in the solution. By putting all the values in the formula we
can easily determine the concentration of the solution.

⇒ In double beam spectrophotometers, the beam splitters are present which splits the
monochromatic light into two beams one for the standard solution and the other for
test solution. In this, the absorbance of Standard and the Test solution can be measured
at the same time and any no. of test solutions can be analyzed against one standard. It
gives more accurate and precise results, eliminates the errors which occur due to the
fluctuations in the light output and the sensitivity of the detector.
 IMPORTANT FORMULA
To determine the concentration of a substance in the Test solution, the following
formula is used: 𝐴 = ∈ 𝑐𝑙
for two solutions (test and standard)
∈ = constant and 𝑙 = constant (because both using the same cuvette)
𝐴𝑇
AT = ∈ 𝑙 cT  =∈𝑙 ….. (i)
𝑐𝑇

𝐴
AS = ∈ 𝑙 cS  𝑐 𝑆 = ∈ 𝑙 ….. (ii)
𝑆

From (i) & (ii), AT × CS = AS × CT  CT = (AT/AS) × CS

Where,
CT = Concentration of the Test solution
AT = Absorbance/ Optical density of the test solution
CS = Concentration of the standard
AS = Absorbance / Optical density of the standard solution
 DIFFERENCE BETWEEN SPECTROMETER AND
SPECTROPHOTOMETER
A spectrometer is used by scientists to gather details of a substance based on the light
it projects, be it visible, ultraviolet, or infrared. It is applicable in different fields of
science. In astronomy, astronomers used spectrometers to check the object’s
temperature while in space. They also use spectrometer to measure the speed it travels
and estimate the weight of the object. In a scientific study, scientists use spectrometer
to find out the composition of things on earth and/or in space including the elemental
components. In a laboratory setting, spectrometers can identify toxins in the
bloodstream, contaminants, and diseases.
The spectrophotometer is a tool designed to measure the intensity of electromagnetic
radiation at different wavelengths. It measures the absorbency of the wavelength in a
given solution, transparency or transmittance of solids, and reflectance of solutions. In
an electromagnetic radiation spectrum, the spectrophotometer can assess the diffusivity
of the light range, especially those with various calibrations and controls.
Spectrophotometers have two basic classifications too – double beam and the basic.
The double beam compares the intensity of light between the reference light path and
the substance being measured. The basic measures the relative light intensity of the
beam before and after introducing the sample.
A spectrometer is a component of spectrophotometer but a spectrophotometer is a
complete system consists of a light source that gathers light that interacted with the
subject and the spectrometer for measurement.
 APPLICATIONS
• Detection of concentration of substances
• Detection of impurities
• Structure elucidation of organic compounds
• Monitoring dissolved oxygen content in freshwater and marine
ecosystems
• Characterization of proteins
• Detection of functional groups
• Respiratory gas analysis in hospitals
• Molecular weight determination of compounds
• The visible and UV spectrophotometer may be used to identify
classes of compounds in both the pure state and in biological
preparations.
 PROBLEMS
1. A solution of Tryptophan (trp) has an absorbance at 280 nm of 0.54 in a 0.5 cm length cuvette.
Given the absorbance coefficient of trp is 6.4 × 103 LMol-1cm-1. What is the concentration of
solution?

2. A solution of thickness 2 cm transmits 40% incident light. Calculate the concentration of the
solution, given that ε = 6000 dm3Mol-1cm-1.

3. A solution shows a transmittance of 20%, when taken in a cell of 2.5 cm thickness. Calculate its
concentration, if the molar absorption coefficient is 12000 dm3Mol-1cm-1.

4. Calculate the molar absorptivity of a 1 x 10-4 M solution, which has an absorbance of 0.20, when
the path length is 2.5 cm.

5. The concentration of yeast t-RNA in an aqueous solution is 10 M. The absorbance is found to be

0.209 when this Solution is placed in a 1.00 cm cuvette and 258 nm radiations are passed through it.
a) Calculate the specific absorptivity, including units, of yeast t-RNA. b) What will be the
absorbance if the solution is 5 M? c) What will be the absorbance if the path length of the original
solution is increased to 5.00 cm?
 PROBLEMS
6. Calculate the molar absorptivity of a 0.5 x 10-3 M solution, which has an absorbance of 0.17,
when the path length is 1.3 cm.

7. A CaCO3 solution shows a transmittance of 90%, when taken in a cell of 1.9 cm thickness.
Calculate its concentration, if the molar absorption coefficient is 9000 dm3Mol-1cm-1.

8. Extinction coefficient of NADH at 340 nm is 6440 Lmol-1cm-1whereas NAD does not absorb at
340nm. What absorbance will be observed when light at 340 nm passes through a 1cm cuvette
containing 10uM NADH and 10 uM NAD.

9. A 1.00 × 10–4 M solution of an analyte is placed in a sample cell with a path length of 1.00 cm.
When measured at a wavelength of 350 nm, the solution’s absorbance is 0.139. What is the
analyte’s molar absorptivity at this wavelength?

10. The absorbance of a Cu sulphate solution containing 0.500 mg Cu/mL was reported as 0.3500 at
440 nm. a) Calculate the specific absorptivity, including units, of Cu sulphate on the assumption
that a 1.00 cm cuvette was used. b) What will be the absorbance if the solution is diluted to twice its
original volume?