UV-VIS SPECTROPHOTOMETER

INSTRUMENTATION Structure
• With the spectrophotometer, we measure a
color (wavelength) using an artificial source
instead of the sun, and a detector and a meter
instead of our eyes.

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 UV-VIS Spectrophotometer Basic
structure & component
General:
• In principle, all spectrophotometers consist of four
major sub-units:
• 1.A source that generates electromagnetic radiation
• 2.A dispersion device that selects a particular
wavelength (correctly a waveband) from the broad
band radiation of the source
• 3.A sample area
• 4.A detector to measure the intensity of radiation

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 UV-VIS Spectrophotometer Basic
structure & component

Block diagram of basic spectrophotometer

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 UV-VIS Spectrophotometer Basic
structure & component
• In addition, there are other optical
components such as lenses or mirrors that
relay the light through the instrument.
• To determine the degree of interaction of the
sample with radiation (transmittance or
absorbance) it is necessary to measure both
the intensity of the incident radiation, i.e.
without the sample, and the transmitted
intensity, i.e. with the sample.

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 UV-VIS Spectrophotometer
Basic component
• Light Sources; Two sources are commonly
used as the light sources of a
spectrophotometer:
• A tungsten lamp for measurement in the
visible and near-infrared ranges.
Properties of light source
• Type Tungsten
• Sign WI
• Wavelength range 340-1100nm
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 UV-VIS Spectrophotometer
Basic component
• A Deuterium for measurement in the
ultraviolet range
• Type Deuterium
• Sign D2
• Wavelength range 185-360nm
• Typically a lamp will have a "half-life" (the
time for the intensity to fall to half the initial
value) of about 1000 hours
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 UV-VIS Spectrophotometer
Basic component
• Dispersion Devices: A dispersion device plays
a role in selecting a monochromatic light from
a light source (white light).
It includes
• Filter type,
• Prism type, and
• Grating (diffraction grating) type.

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 UV-VIS Spectrophotometer
Basic component

Filter
• A single wavelength can be extracted with a
filter. A filter is also used in combination with
diffraction grating for filtering out stray light
• Types and materials Colored glass filter,
Interference filter

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 UV-VIS Spectrophotometer
Basic component
Monochromators can be
based on the use of
• gratings, which work on
the principle of
diffraction
or

• prisms, which work on

the principle of
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refraction 10
 UV-VIS Spectrophotometer
Basic component
• Grating (diffraction grating)

• Dispersion is homogeneous at the entire

wavelength, and a wide-range wavelength can
be obtained with a diffraction grating. In
addition, a constant spectrum featuring a
constant slit breadth can be obtained
Types and materials: Plane diffraction grating.
Concave diffraction grating
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 UV-VIS Spectrophotometer
Basic component
• PRISM

• A spectrum of 175-2700 nm can be dispersed.The

degree of dispersion varies with the wavelength.
• Types and materials: Crystal or fused quartz
• The advantage of prisms is that they are simple and
inexpensive to make but they suffer the disadvantage
that the dispersion is angularly non-linear and they
are temperature-sensitive

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 UV-VIS Spectrophotometer
Basic component
• Most modern spectrophotometers use holographic
gratings. These are made from glass blanks onto which are
ruled very narrow grooves.
• The main advantages of holographic gratings are that they
give a linear angular dispersion with wavelength, they are
also temperature insensitive.
• The disadvantage is the fact that light is reflected in
different orders which overlap. Filters must be used to
ensure that only the light from one chosen reflection order
reaches the detector.
• A concave holographic grating combines the two functions
of dispersing and focusing light at the same time.

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 SPECTRAL RESOLUTION & INSTRUMENTAL
BANDWIDTH
• Ideally – output from a wavelength selector is
a single wavelength. In practice the output is
always a band with, ideally, a symmetrical
shape.
• The width of the band at half its height is the
instrumental bandwidth (IBW).
• The narrower the band, the better the
wavelength selector, the greater the spectral
resolution attainable.

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 Instrumental bandwidth

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 SPECTRAL RESOLUTION & INSTRUMENTAL
BANDWIDTH
• The effective bandwidth is one important defining factor
of a wavelength selector’s performance, defining its
resolution & it determines the spectrometer’s
resolution. i.e. The ability to distinguish absorbances at
different wavelengths close to one another.
Spectral slit width or effective bandwidth
• Defines spectrometer resolution
• Affects the applicability of Beer’s Law
• Affects S/N; The higher the resolution the lower the S/N
• Affects the ability to acquire detailed spectral
information. The higher the resolution the greater the
information content (potentially)

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 UV-VIS Spectrophotometer
Basic component
• Sample cell: A container that contains a sample is
usually called "cell";
• Two types are available, glass and quartz cells.
• Glass cell is used for measurement in the visible
range of 340 nm or more. Light in the ultraviolet
range with a wavelength of 340 nm or less hardly
passes through a glass cell,
• On the other hand, although a quartz cell allows
passage of light in the entire wavelength in the
ultraviolet and visible ranges, it is mainly used for the
measurement in the ultraviolet range due to its high
price.

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 Types & shape of sample cell

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 UV-VIS Spectrophotometer
Basic component
• Detectors: The detector is a device which
converts a light signal into an electrical signal.
Ideally it should give a linear response, with
low noise and high sensitivity.
• Two types of device are commonly used in
spectrophotometers.
• Optical semiconductor & photoemissive
devices Types.

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 UV-VIS Spectrophotometer
Basic component
• Optical semiconductor: High speed, high
sensitivity, and a low noise are features of
optical semiconductor detector.
• A Si photodiode is a representative one.

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 UV-VIS Spectrophotometer
Basic component
• Photo emissive devices: In the UV-Vis region there are 3
types, the first 2 are very similar in principle but have very
different performance characteristics.
• Vacuum Phototube
• Photomultiplier tube
Both of these are “photoemissive” devices. Shine light on them,
and they emit electrons.
• Multichannel photon transducers. Shine light on these, and
they conduct electricity. Advantage of smaller size and can
construct instruments with a different design as a result.

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Cross section of a photomultiplier tube
 UV-VIS Spectrophotometer
Basic component
Optical system & chopper
• In addition to the basic component described above,
spectrophotometer uses some optics. Optical components are
used to relay and focus light through the instrument. They are
either lenses or concave mirrors.
• Simple lenses are inexpensive but suffer from chromatic
aberration, that is, light of different wavelength is not focused
at exactly the same point in space.
• Achromatic lenses combine multiple lenses of different
glasses with different refractive indices into a compound lens
which is largely free of chromatic aberration. They offer good
performance but at relatively high cost.

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 UV-VIS Spectrophotometer
Basic component
• Chopper /The rotating discs/:
• A "chopper" used in double beam design of
spectrophotometer. Its function is to alternately
transmit and reflects the light beam so that it travels
down the blank and the sample optical paths to a
single detector.
• Most of the time rotating disc is used as a chopper.
Each disc is made up of a number of different
segments.

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 Rotating disc chopper

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 Rotating disc chopper

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 Rotating disc chopper
• When the light coming from the diffraction grating
and slit will hit the rotating disc and one of three
things can happen.
• If it hits the transparent section, it will go straight
through and pass through the cell containing the
sample. It is then bounced by a mirror onto a second
rotating disc.
• This disc is rotating such that when the light arrives
from the first disc, it meets the mirrored section of
the second disc. That bounces it onto the detector.

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 Rotating disc chopper
• If the original beam of light from the slit hits the mirrored
section of the first rotating disc, it is bounced down along the
green path. After the mirror, it passes through a reference
cell.
• Finally the light gets to the second disc which is rotating in
such a way that it meets the transparent section. It goes
straight through to the detector
• If the light meets the first disc at the black section, it is
blocked - and for a very short while no light passes through
the spectrometer. This just allows the computer to make
allowance for any current generated by the detector in the
absence of any light.

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 Types of spectrophotometer with their
feature
1.The Conventional Scanning Spectrophotometer
• The conventional scanning spectrophotometer uses a single
detector and a "forward" optics configuration, that is, the
dispersion device comes before the sample.
• A feature of such an optical system is that the sample area
must be completely covered to prevent ambient light
reaching the detector.
• To measure at different wavelengths or to measure a
spectrum with a conventional instrument with a single
detector, it is necessary to rotate the dispersion device.
Data acquisition therefore is sequential.

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 Schematic of a conventional single-beam spectrophotometer

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 Types of spectrophotometer with their
feature
• There are many variations in designs based on
this concept but, in general, there are four
important groups:
• Single Beam,
• Double Beam,
• Split Beam, and
• Diode-Array.

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 Types of spectrophotometer with their
feature
• Single Beam: To make a measurement, the blank is first
placed in the instrument and measured to obtain Io which is
usually stored within the instrument. Then the sample is
placed in the instrument (using the same cuvette) and
measured to get I, and the instrument calculates the
transmittance or absorbance as desired

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 Types of spectrophotometer with their
feature
• The advantages of the single beam design are low
cost, high throughput, and hence high sensitivity,
because the optical system is simple.
• The disadvantage is that an appreciable amount of
time elapses between taking the reference (I) and
making the sample measurement (Io) so that there
can be problems with drift.
• This was certainly true of early designs but modern
instruments have better electronics and more stable
lamps, so stability with single beam instruments is
now more than adequate for the vast majority of
application

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 Types of spectrophotometer with their
feature
• Double Beam: The double beam instrument design
aims to eliminate drift by measuring blank and
sample virtually simultaneously.
• A "chopper" alternately transmits and reflects the
light beam so that it travels down the blank and the
sample optical paths to a single detector.
• The chopper causes the light beam to switch paths at
about 50 Hz causing the detector to see a "saw
tooth" signal of Io and I which are processed in the
electronics to give either transmittance or
absorbance as output.
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 Types of spectrophotometer with their
feature

• The advantage of the double beam design is high stability

because reference and sample are measured virtually at the
same moment in time but the disadvantages are higher cost,
lower sensitivity because throughput of light is poorer
because of the more complex optics and lower reliability
because of the greater complexity.

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 Optical system of a double-beam spectrophotometer

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 Types of spectrophotometer with their
feature
• Split Beam: The split beam spectrophotometer is
similar to the double beam spectrophotometer but it
uses a beam splitter instead of a chopper to send
light along the blank and sample paths
simultaneously to two separate but identical
detectors.
• Thus blank and sample measurements can be made
at the same moment in time. Spectra are measured
in the same way as with a double beam
spectrophotometer.
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 Types of spectrophotometer with their
feature
• The advantage of this design is good stability,
though not as good as a double beam
instrument because two detectors can drift
independently, and good signal to noise ratio,
though not as good as a single beam
instrument because the light is split so that
less than 100% passes through the sample

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 Types of spectrophotometer with their
feature
• The Diode-Array Spectrophotometer:
• The diode-array spectrophotometer uses an array of detectors and a "reverse"
optics configuration, that is, the dispersion device comes after the sample.
• An advantage of the reversed optics configuration is that only light traveling
along the axis from source to inlet slit before the dispersion device can reach
the detector; light from other angles cannot. Thus it is not susceptible to
interference from ambient light and the sample area can be left open, making
the instrument easier to use.
• Light of all wavelengths falls on the diode-array and is measured
simultaneously, that is, data acquisition is done in parallel. A spectrum is
obtained by electronically scanning the array. In principle diode-array
spectrophotometers can be designed as single-, double- or dual-beam but, in
practice, the advantages of the single beam design combine well with diode-
array detection

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 Optical system of Diode-Array
Spectrophotometer

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 Schematic of a diode-array
spectrophotometer

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 Types of spectrophotometer with their
feature
Single vs double monochromes system
• For Careful selection of wavelength a monochromator plays
the role of a sorter that picks up the light of the desired
wavelength out of the light containing various wavelengths.
• But, however carefully they are checked, wavelengths other
than the wavelength of interest may slip in (this light that was
lost or slipped in is called stray light).
• This stray light may affect the accuracy of the measurement. A
double monochrome system conducts a second check on the
light that passed the first check. The system that checks only
once is called a single monochrome system.

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 Types of spectrophotometer with their
feature

Single vs double monochromes system

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 Measuring Systems
• The primary function of spectrophotometer
ends with the provision of a signal (normally an
electrical voltage) that is proportional to the
absorption by a sample at a given wavelength.
• The signal handling and measuring systems can
be as simple as an amplifier and a meter or as
elaborate as a personal computer and printer,
depending on the application.

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44
 WHAT IS THE “BLANK” AND WHY DO WE HAVE TO USE
IT?

• All substances, clear or not, absorb some light.

• Therefore, if we wish to measure the absorbance (or the
transmittance) of a required material in solution, we must
not only consider the light absorbed by the material in
solution, but also the light absorbed by the cuvette and the
light absorbed by the solution in which the material is
dissolved.
• In order to measure only the absorbance of the required
material in the solution, we must “subtract” out the
absorbance due to the cuvette and the solvent in which the
required material is dissolved.

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 WHAT IS THE “BLANK” AND WHY DO WE HAVE TO USE IT?

• If our required material is dissolved in water, and we are

trying to assess how much material is in solution, we would
ultimately want to measure how much light is absorbed by
the required material alone and not how much light is
absorbed by either the water or the cuvette.
• In order to subtract out these factors, we prepare another
cuvette with only water in it. This is called the “blank.”
• In general, the blank is a cuvette which contains
everything that is in the sample (or experimental)
cuvette, except the one material whose absorbance
we are measuring.

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 Sequence of instrumental analysis with uv-vis
spectrophotometer: Quantitative Analysis
• Any colored solution is colored because it
absorbs some wavelengths of light and
transmits others.
• The spectrophotometer is designed to
measure the amount of light absorbed by a
solution.
• we can quantitatively measure absorbance,
and this information can be used to determine
the concentration of the absorbing molecule.

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 Sequence of instrumental analysis with uv-vis
spectrophotometer Quantitative Analysis
• Colorimetric Assays: It is a quantitative analysis which refers to an
assay to measure the content of a certain substance (in a solution
state).By Measurement of color strength.
• For example, the water contains metals, small quantities of
agricultural chemicals, microorganisms, and so forth. However, the
amounts contained cannot be determined visually.
• Generally, a coloring reagent is added to a sample to see the degree
of coloring in the reaction with the substance of interest.
• We use a method consisting of a standard solution and a sample,
adding the reagent for coloring the substance of interest, and
comparing the absorbance between the standard solution and the
sample using a calibration curve to determine the unknown sample
concentration
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 Determining concentration with a
spectrophotometer
• There are two ways we could measure the
concentration of some unknown solution using the
spectrophotometer.
1.with a standard curve
2.with calculation (Beer-Lamberts law)
A=ecl

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 Determining concentration with a standard
curve
• The first is to generate a standard curve: a graph of
absorbance vs. concentration for standard solutions whose
concentrations are known.
• We then compare the absorbance of an unknown solution
(of the same molecule) to that curve.
• Essentially, we are “calibrating” the spectrophotometer to
measure the concentration of the molecule we’re
interested in using known wavelength (the wavelength at
which the molecule absorbs best.)

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 Determining concentration with a standard
curve

• To make a standard curve, you’ll need at least

three or four different solutions with known
concentrations (called standards).
• After generating a standard curve, you can
measure the absorbance of the unknown
solution and compare it to the absorbance of
the known solutions

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 Determining concentration with a standard
curve
Controls:
• When we run an assay we must ensure that
only the substance we are assaying is
responsible for absorbance of light in the
wavelength range of interest.
Accurate results cannot be obtained when
absorption occurs at the same wavelength
for the solute and the solvent.

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 Determining concentration with a standard
curve
• All conditions under which standards and unknowns
are prepared should be kept identical. If solutes in
the sample affect absorbance, then we have a
problem.
• We won't obtain accurate results if the timing of
reading absorbance, temperature at which we keep
the materials and all other physical factors are
different. It should be kept all the same.
• we need only ensure that none of the components of
any of the solution has a significant effect on
absorbance due to such external factor.

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 Sources of Error
UV-Visible measurements are very precise
• standard deviations of 0.01% are easily
achievable.
• However, there are many potential sources of
error in quantitative analysis.
• They can be grouped under sample- related
and instrument-related.

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 Sources of Error
Sample-related sources of error include
• errors in preparation of standards or samples
• suspended particulates that cause scattering,
• dirty cuvettes, etc.
These errors can be minimized only by good
laboratory practice.

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 Sources of Error
Instrument-related errors arise from
• noise, drift, photometric accuracy,
• errors due to stray light or inadequate
resolution,
• wavelength accuracy and wavelength
reproducibility.

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 Sources of Error
Instrumental errors can be classified as follows:
Random error : Bias
Noise Insufficient resolution
Wavelength reproducibility Wavelength accuracy
Drift Stray light
Random error is not reproducible whereas bias is an error
that is reproducible.
For absolute measurements, all sources of error are
significant.
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 Sources of Error
Systematic Error
• Systematic errors are errors that produce
a result that differs from the true value
by a fixed amount.
• These errors result from biases
introduced by instrumental method, or
human factors.

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 Sources of Error /Systematic Error/
• An example of an instrumental bias is an incorrectly
calibrated meter that shows concentration values 0.5 units
lower than the true value.
• An example of a method error would be different kind of
blank used for standard and sample, in measurement.
• An example of human bias is a student who records
titration endpoints beyond the true endpoint due to color
blindness.

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 calculation (Beer-
Determining concentration with
Lamberts law)
• At a specific wavelength for a given chromophore,
( Group in a molecule which absorb light ) there is a
relationship between the absorbance and the
number of molecules whose chromophore is
absorbing at that wavelength, the path length, and
a constant called the molar extinction (absorption)
coefficient which indicates the intensity of the
absorption.

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 calculation (Beer-
Determining concentration with
Lamberts law)

A = e c l =log 1/T = log (I0/I)

• Where:- A is the measured absorbance, [no units,
because it is calculated as A = log (I0/I), where I0 is the
incident light’s intensity and “ I “ is the light intensity
after it passes through the sample]
• e is the molar extinction (absorption) coefficient
• l is the path length
• C is concentration

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Applications
 Environmental Applications
• Methods for the analysis of waters and wastewaters relying
on the absorption of UV/Vis radiation are among some of the
most frequently employed analytical methods. Many of these
methods are outlined in Table 10.6, and a few are described
later in more detail.
• Although the quantitative analysis of metals in water and
wastewater is accomplished primarily by atomic absorption
or atomic emission spectroscopy, many metals also can be
analyzed following the formation of a colored metal–ligand
complex.
• One advantage to these spectroscopic methods is that they
are easily adapted to the field analysis of samples using a
filter photometer
 Quantitative Analysis of Mixtures
• The concentrations of Fe3+ and Cu2+ in a mixture can be determined
following their reaction with hexacyanoruthenate (II), Ru(CN)6 4–, which
forms a purpleblue complex with Fe3+ (λmax = 550 nm), and a pale green
complex with Cu2+ (λmax = 396 nm).The molar absorptivities (M–1 cm–1) for
the metal complexes at the two wavelengths are summarized in the
following table.

• When a sample containing Fe3+ and Cu2+ is analyzed in a cell with a path
length of 1.00 cm, the absorbance at 550 nm is 0.183, and the absorbance
at 396 nm is 0.109. What are the molar concentrations of Fe3+ and Cu2+ in
the sample?
Solution
• Remember:
(A)λ1 = (εX)λ1 b CX + (εY)λ1 b CY
(A)λ2 = (εX)λ2 b CX + (εY)λ2 b CY
• Substituting known values into the equations
gives

• Conc Fe3+ = 1.80 x 10–5 M.

• Conc Cu2+ = 1.26 x 10–4 M.
 Scale of Operation
• Molecular UV/Vis absorption is routinely used
for the analysis of trace analytes in macro and
meso samples.
• Major and minor analytes can be determined
by diluting samples before analysis, and
concentrating a sample may allow for the
analysis of ultratrace analytes.
 Selectivity
• Selectivity is rarely a problem in molecular
absorption spectrophotometry. In many cases it is
possible to find a wavelength at which only the
analyte absorbs or to use chemical reactions in a
manner such that the analyte is the only species that
absorbs at the chosen wavelength.
• When two or more species contribute to the
measured absorbance, a multicomponent analysis is
still possible.
 Accuracy
• Under normal conditions relative errors of 1–5% are easily
obtained with UV/Vis absorption. Accuracy is usually
limited by the quality of the blank.
• Examples of the type of problems that may be encountered
include the presence of particulates in a sample that scatter
radiation and interferants that react with analytical
reagents. In the latter case the interferant may react to
form an absorbing species, giving rise to a positive
determinate error. Interferents also may prevent the
analyte from reacting leading to a negative determinate
error. With care, it may be possible to improve the accuracy
of an analysis by as much as an order of magnitude.
 Time, Cost, and Equipment
• The analysis of a sample by molecular absorption
spectroscopy is relatively rapid, although additional time
may be required when it is necessary to use a chemical
reaction to transform a nonabsorbing analyte into an
absorbing form. The cost of UV/Vis instrumentation ranges
from several hundred dollars for a simple, manually
operated, single-beam instrument equipped with an
inexpensive grating, to as much as $50,000 for a computer-
controlled, high-resolution, double-beam instrument
equipped with variable slits and operating over an
extended range of wavelengths.
 Precision
• In absorption spectroscopy, precision is limited by
indeterminate errors, or instrumental “noise,”
introduced when measuring absorbance.
• Precision is generally worse with very low
absorbances due to the uncertainty of distinguishing
a small difference between P0 and PT, and for very
high absorbances when PT approaches 0. We might
expect, therefore, that precision will vary with
transmittance.
• To obtain a relative uncertainty in concentration of ±1–2%,
the absorbance must be kept between 0.1 and 1 (curve A)
• Curve B shows that the relative uncertainty in
concentration is large for low absorbances, but is less
affected by higher absorbances. Although the relative
uncertainty reaches a minimum when the absorbance is
0.96, there is little change in the relative uncertainty for
absorbances between 0.5 and 2. This indeterminate error
generally limits the precision of high-quality UV/Vis
spectrophotometers for mid-to-high absorbances.
• Values of sT are directly proportional to transmittance for
indeterminate errors due to fluctuations in source intensity and
for uncertainty in positioning the sample cell within the
spectrometer. The latter is of particular importance since the
optical properties of any sample cell are not uniform. As a
result, repositioning the sample cell may lead to a change in the
intensity of transmitted radiation.
• As shown by curve C, the effect of this source of indeterminate
error is only important at low absorbances. This source of
indeterminate errors is usually the limiting factor for high-
quality UV/Vis spectrophotometers when the absorbance is
relatively small.
 Advantages of UV/Vis
• many organic and inorganic compounds have
strong absorption bands in the UV/Vis region
of the electromagnetic spectrum
• analytes that do not absorb UV/Vis radiation,
or that absorb such radiation only weakly,
frequently can be chemically coupled to a
species that does.
• Methods are reliable and relatively simple