1

UNIVERSITY OF TECHNOLOGY, JAMAICA

DIVISION OF CHEMISTRY
GROUP: AS, SE, MT, BENG-C, BPHARM
MODULE: Analytical Chemistry (CHY2017, CHY3022)
______________________________________________________________________________
UNIT 5: INTRODUCTION TO INSTRUMENTATION
Assigned Reading: Chapters 8, 24, 25 Fundamentals of Analytical Chemistry, 8th edition

Instrumental Methods of Analysis

As mentioned in Unit 1, Instrumental Methods of Analysis can have certain advantages over
Classical Methods. As the phrase suggests, an instrument has to be involved. Instruments
receive a portion of your laboratory sample and measure some property or properties of that
sample – the analytical signal or response. The instrument detects the analytical signal, which is
related to the presence and amount of the sample, and converts it a value or image that the
analyst can understand and interpret. Different instruments respond to different signals or
properties of the analyte, such as, light absorption, mass, temperature, radioactivity, solubility.
In Unit 5, instruments that respond to light or electromagnetic radiation are discussed.

Introduction to Spectroscopy and Spectrochemical Methods

Spectroscopy is to a branch of science concerned with the interactions of radiation (light) with
matter. Spectrochemical methods are based on the measurement and interpretation of
electromagnetic radiation (EMR) as it interacts with chemical species. These chemical species
can absorb, scatter or emit electromagnetic radiation.
 If a beam of light is passed through a beaker of water, it remains white and the water
appears colourless.
 But, what happens if potassium permanganate was added to the water? The solution
appears purple in colour.
o This is an example of the interaction of matter with radiant energy or electromagnetic
radiation (EMR).
 Visible light is a form of EMR and the effects of the absorption of parts of visible light
are seen with the eye.
 The absorption of radiation takes place over a wide range of EMR, most of which cannot
be seen but can be measured with suitable instruments.

Properties of Electromagnetic Radiation (EMR)

 EMR is a form of energy that is transmitted through space at high velocities.

Dr. D. Gordon-Smith, UTech

 2

 It results from fluctuations of electric and magnetic fields that are perpendicular to each
other – waves.

 These waves can be described by the following properties:

o Wavelength, : linear distance between successive maxima or minima of a wave.
Unit of  = angstrom (Ǻ), nanometre (nm), micrometre (m), centimetres (cm) or
metres (m).
𝐜
o Frequency, : number of oscillations per second (Hz or sec-1),  = ,
where c = speed of light (3 × 108 m s-1)
𝟏
o Wave number, : the number of waves per unit of distance,  = 
𝟏
o Period, T: time the wave takes to complete one cycle, T = 
 Electromagnetic radiation can also be thought of as particles or packets of radiation
called photons or quanta.
 The amount of energy transferred per photon of radiation is given by Planck‟s equation:
𝐜
𝑬 = 𝒉 = 𝒉

Where E = energy of a photon in Joules
h = Planck’s constant 6.62 x 10-34 Js
 Therefore, the energy of a photon is directly proportional to the frequency and inversely
proportional to the wavelength.
 Radiation interacts with matter in various ways. Radiation can be absorbed or emitted as
chemical species transition between different energy levels.
 Electromagnetic radiation may be transmitted,
o absorbed – absorption spectrometry
o scattered– nephelometry
o rotated – polarimetry
o refracted – refractometry
o emitted – emission spectrometry or fluorescence

Electromagnetic Spectrum
 Comprises of a wide range of radiation energies (therefore, frequencies and
wavelengths).
 The range is so large that the scale is logarithmic and the spectrum is continuous over all
wavelengths.
 Interactions between radiation and matter take place throughout the entire
electromagnetic spectrum, but the effect of the radiation varies depending on the energy –
see Figure 24.3 in SWHC, 8th Ed.

Dr. D. Gordon-Smith, UTech

 3

 The spectrum is subdivided in name according to manner in which radiation interacts

with matter, i.e., the energy of the radiation. The regions of importance to us are given in
table 1.
 The visible portion of the spectrum (~ 400 nm to 700 nm) to which the human eye is
sensitive is tiny when compared with other spectral regions.
 The visible region is further subdivided as shown in table 2.

Table 1: Type of EMR and Interactions with Matter

Type of Radiation  range Type of transition

Gamma rays 0.005 – 1.4 Å Nuclear
X-rays 0.1 – 100 Å Inner electrons
UV-Visible 10 – 780 nm Bonding electrons
Infrared 0.78 – 300 m Rotation/vibration of molecules
Microwaves 0.75 – 3.75 m Rotation of molecules

Dr. D. Gordon-Smith, UTech

 4

Table 2: The Visible Region

Wavelength Region, nm Colour Complimentary Colour

400-435 Violet Yellow-green
435-480 Blue Yellow
480-490 Blue-green Orange
490-500 Green-blue Red
500-560 Green Purple
560-580 Yellow-green Violet
580-595 Yellow Blue
595-650 Orange Blue-green
650-750 Red Green-blue

Absorption Spectrometry
 Absorption is the process by which a medium attenuates the intensity of certain
frequencies of electromagnetic radiation.
 Every particle has a unique set of energy states; the lowest being the ground state.
 The particle (atom/ion/molecule) can absorb energy, such as EMR, and be promoted to or
transition to a higher energy, excited state:
M + h  M*
 The excited species then loses energy and returns to its original (ground) state by emitting
radiation or transferring its excess energy to other atoms or molecules:
M*  M + h or heat or energy transfer
 Absorption is possible if the energy of the photon (E) exactly matches the energy
difference between ground state and one of the higher energy states:

E4
energy emitted E3
E2 Excited States
Energy

E1

E0 Ground State

absorption emission
energy absorbed, e.g. EMR

 These energy differences are unique for each species.

 The type of transitions that can occur depends on the energy of the radiation, e.g.:
o UV/VIS radiation can lead to the electronic transitions of valence electrons.
o IR radiation can lead to molecular vibrations and rotations.

Dr. D. Gordon-Smith, UTech

 5

Atomic Absorption
 The atomic absorption spectrum is made up of a number of narrow absorption lines, for
example, the atomic spectrum for sodium is a line spectrum.
 The electron configuration for Na: 1s2 2s2 2p6 3s1. The electronic (UV/VIS) transitions
involve excitation of the single outer electron from 3s to 3p, 4p and 5p.
 These excitations are brought on by absorption of photons of radiation whose energy
exactly match the differences in energies between the excited states and the 3s ground
state.
 Below is a diagram of an atomic spectrum (Na atoms) and the corresponding energy
diagram:

 Question: The energy difference between 3p and 3s is 2.107 eV. Calculate the
wavelength of radiation needed for excitation. (1 eV = 1.6 x 10-19 J) Hint: E = hc/.

Molecular Absorption
 Absorption is more complicated.
 Electrons not only move from one molecular orbital to another (electronic transitions),
but atoms in the molecules also undergo vibrations and rotations requiring a discrete
amount of energy to initiate or maintain.
 Hence, each molecular energy state is comprised of an electronic, vibrational, rotational
component; E = Eelectronic + Evibrational + Erotational
 Vibrational transitions:
o molecules have many quantized energy levels (vibrational states) associated with
the bonds holding the molecules together.
 Rotational transitions:
o molecules exhibit rotational motion around its centre of gravity.
 Therefore, the spectrum is complicated – continuous spectrum obtained.

Dr. D. Gordon-Smith, UTech

 6

Absorption Laws
 Absorption of radiation follows definite physical laws.
 The amount of light absorbed can give us information about the substance.
 When light is absorbed within a medium, the radiant power of the beam decreases:
I0 or P0 = intensity of a beam of monochromatic light
passing through a medium
I1 or P = the intensity of radiation emerging from the
solution

 Transmittance, T is the fraction of incident radiation transmitted by the solution:

𝑰𝟏 𝑷
𝑻 = 𝒐𝒓
𝑰𝟎 𝑷𝟎

 Transmittance is often expressed as percentage, %T.

 The amount of light absorbed, called absorbance, A is:
𝑰 𝑷𝟎
A = - log T = log 𝑰𝟎 = log
𝟏 𝑷

Beer-Lambert law or Beer’s law

 The absorbance of a solution is directly proportional to the concentration of the solution,
c, and the pathlength, b, of the absorbing medium:
A = abc a = absorptivity (proportionality constant)

Dr. D. Gordon-Smith, UTech

 7

 When concentration is given in mol L-1 and b in cm, the constant is called the molar
absorptivity, .
A = bc  = L mol-1 cm-1
 This is widely used in spectroscopy to calculate solution concentrations from absorption
measurements.

Assumptions of Beer’s Law

 Beer‟s law is found to hold under certain conditions:
o The radiation absorbed must be monochromatic, i.e., single
wavelength/frequency/colour.
o Absorption occurs in a volume of uniform cross-section
o There is no interaction between species in the absorbing medium – no attractions or
repulsions, therefore, Atotal = 1c1l1 + 2c2l2 + 3c3l3

Deviations from Beer Lambert Law

 In practice this law applies only under ideal circumstances.
 There is usually a deviation from a linear relationship between concentration and
absorbance.

 These deviations can be categorised as:

1. Real Limitations
a. High Concentrations: Only dilute solution obey Beer-Lambert‟s law. At high
concentrations, the species are close to each other so interactions (electrostatic
effects) between them will affect the ability of these species to absorb a given
wavelength of radiation. How can this interaction cause deviations from the
linear relationship?
b. Very Low Concentrations: Similar effects are seen in solutions containing low
analyte concentrations but high concentration of other species. What do you think
would happen and what effect would dilution have on this?
c. Physical Deviations: If concentration changes or matrix effects cause alterations
in the refractive index of the solution, then molar absorptivity is affected and a
departure from Beer‟s law is observed.

Dr. D. Gordon-Smith, UTech

 8

2. Chemical Deviations
 These occur when an analyte dissociates, associates or reacts with a solvent
forming a product with different absorption spectrum.
 Also happens with some large organic molecules are at low concentrations.
3. Instrumental Deviations
 These are caused by polychromatic radiation and stray light.
 Because of these deviations, the extrapolation of the absorbance-concentration
relationships from standards with low concentrations to samples with high concentrations
will cause errors.
 This difficulty can be overcome by preparing calibration curves that show the
experimental relationship between A and the actual concentration
 Errors caused by (1) absorption by interfering molecules, or (2) variations in sample cell
width, may be eliminated if they are constant in the sample being analysed and the
calibration samples.
 This requires extra care when making up the standard solutions.

(consult your text for more information)

Optical Instruments
Instruments for measuring absorption are comprised of five basic components:
 Source
 Wavelength selectors
 Sample
 Radiation detector
 Readout Device

A schematic diagram of an optical instrument is given below:

Radiation Sources
 A good source should have a stable, high intensity output that covers a wide spectral
range.
 No single source is suitable for all of the spectral regions and an instrument designed to
make measurements over a wide range of wavelengths may contain two or even three
different sources. Examples of sources:

Dr. D. Gordon-Smith, UTech

 9

Source (lamps)  region, nm Type of Spectroscopy

Continuous sources (emits all wavelengths within region)
Xenon 250 – 600 Molecular Fluorescence
H2 & D2 160 – 380 UV molecular absorption
Tungsten 350 – 2200 VIS/near IR molecular absorption

Line Sources (emit few discrete lines)

Hollow cathode UV/VIS Atomic absorption, fluorescence
Laser UV/VIS/IR Molecular absorption, fluorescence

Wavelength Selectors
 Since radiation sources are continuous, some means is needed for selecting the particular
wavelength at which a measurement is to be made.
 It is impossible to isolate a single wavelength from a continuum, so a very narrow band
of wavelengths has to suffice.
 A narrow bandwidth improves sensitivity and selectivity.

Type of Wavelength Selector  range, nm

Monochromators
Grating 100 – 40,000
Prism 120 – 30,000
Filters
Interference 200 – 14,000
Absorption 380 – 750

Filters (UV/VIS):
 Usually limited to one wavelength.
 Absorption filters are relatively inexpensive but limited to VIS region.
 Interference filters use optical interference to provide narrow bands.
 These output smaller bandwidth than absorption filters
Monochromators (UV/VIS/IR):
 Monochromators are comprised of:
o Entrance & exit slits
o Collimator to produce a parallel beam of light.
o Prism or grating to disperse radiation
Dr. D. Gordon-Smith, UTech
 10

o Focusing element (mirror or lens) to project images unto focal plane.

 Typical bandwidth of 1-20 nm.
 Monochromator slits are important for the performance of the monochromator.
o The entrance slit is the radiation source and its image ultimately travels, via the
focal plane, through the exit slit.
o Bandwidth depends on the grating (or prism), entrance and exit slits.
o Some instruments use variable slits so that bandwidth can be changed.

 Gratings:
o Modern, less expensive than prisms
o However, prone to stray radiation and higher order spectra.
o Consist of a number of parallel grooves (blazes) on a polished surface.
 UV-VIS gratings (300–2000 grooves/mm)
 IR gratings (10–200 grooves/mm)
*Consult text for the diagram and operation of the Czerney-Turner grating
monochromator
 Prisms:
o Larger, more expensive
o Unstable in laboratory atmosphere

Sample
 Containers are called cells or cuvettes
 They have a variety of shapes and sizes appropriate for each instrument
 The cell/cuvette material must be transparent in the spectral region of interest
o UV – quartz
o VIS – quartz or fused silica glass
o IR – NaCl, AgCl or KBr
 Square or rectangular cell offer constant path length and are therefore free of distortions
(remember Beer‟s law assumptions!)
 Oval cells are marked in front for proper and constant light path orientation

Radiation Detectors (Transducers)

 Transducers or detectors convert radiant energy to an electrical signal.
 They should:
o Respond rapidly to the radiation
o Be non-selective over a wide range of wavelength
o Give constant response over all wavelengths within the range
o Have high efficiency i.e. a large electrical signal should be produced in response
to a low power of radiation
o Produce electrical signal that can be readily amplified
o Have a low noise level
Dr. D. Gordon-Smith, UTech
 11

 Also, the detector electrical signal must be directly proportional to the power of the beam
striking it.
 UV/VIS detectors are Photon Transducers (150 – 3000 nm):
o Respond to photons
o Based on interaction of radiation with a reactive surface.
 IR transducers are Heat Transducers (600 – 40,000 nm):
o Respond to heat

Transducer Type  range, nm

Phototubes* 150 – 1000
Photomultiplier tubes* 150 – 1000
Silicon diode 350 – 1100
Photoconductors 750 – 3000
Photovoltaic cells 380 – 780

*Consult the text for more information on the phototube and photomultiplier tube
detectors

Signal Processors & Readout Devices

 These display the amplified signal from the detector in some form useful to the operator.
 They may alter the signal from ac to de or the reverse.
 Examples are: voltmeters, digital meters, recorders and microcomputers

Instrument Designs: Double-beam vs. Single Beam

Single Beam Instruments:

 These contain matched cells that can be interposed alternatively in beam.
 Older, mechanical
 Advantages:
o Greater energy throughput
o Good signal-to-noise ratio
o Inexpensive, simple design
 Disadvantages
o Drift in source & detector output over time

Dr. D. Gordon-Smith, UTech

 12

Double Beam Instruments:

 These instruments are more modern
 The source radiation is either split in two or alternated between the sample and reference
cells
 This eliminates source/detector fluctuations.
 These instruments eliminate memory effects in detectors and can be used to obtain
continuous absorption spectra – up to 1200 nm/min.
 Below are diagrams showing the double-beam in space instruments (source beam is split
in two) – (b) and double-beam in time instruments (source beam is alternated between
sample and reference cells) – (c):

Calibration Methods
 Instruments only measure a property of the analyte (X) and therefore cannot tell the user
the concentration of the analyte without more information.
 Hence, instrumental analysis requires calibration.

Dr. D. Gordon-Smith, UTech

 13

 Calibration is the process of determining the relationship between a measured signal and
the concentration of analyte that generates the signal.

CA = kX where: CA = concentration of analyte

k = proportionality constant

 Calibration methods include:

o External standards (calibration curve)
o Standard additions
o Internal standards

External Standard (Calibration Curve) Method

 This entails preparation of several (3 or more) standard solutions of varying
concentrations.
 Their absorbance, emission intensity or some other response feature is measured.
 A plot of response (absorbance) versus concentration is made – calibration curve. The
least squares regression line is usually determined.
 The matrix of the sample, that is, the presence of all materials except the analyte must be
duplicated as exactly as possible in the standards or an assumption is made that that there
are no interferences or matrix effects.
Advantages:
 The procedure is straightforward simple and easy to apply.
 The concentration of the unknown sample can be determined from the calibration curve.
Disadvantage:
 Differences between the sample matrix and the matrix of the standards will often result in
inaccuracies.
o Other species in the samples may absorb or interfere with the results generated by the
analyte.
o Interference caused by matrix effects represents the most important disadvantage of
the calibration curve method.
o To counteract this, matrix matching can be done but it is almost impossible to
accurately add the minor constituents of a sample to the standard so as to ensure that
both standards and sample have the same matrix.

Dr. D. Gordon-Smith, UTech

 14

 NOTE: calibration curves are based on Beer‟s law and there tends to be a deviation from
linearity at high concentrations. Therefore, one needs to know the linear and dynamic
ranges of the method:

Standard Addition Method

 Although the calibration curve method is simpler and more easily applied for analyte
determinations, the standard additions method is often necessary when samples with
complex matrices that are difficult to match must be analysed.
 For example, consider a sample such as plasma or urine that is being analysed for sodium
or potassium by flame photometry. External standard solutions would contain a small
quantity of sodium chloride dissolved in deionised water. However, the matrices of the
samples and standards would be very different, as proteins, urea and several other
components would also be present. The denser and more viscous sample solutions would
flow more slowly, disperse less easily, evaporate more slowly in flame and in general
may give very different results from those of a standard with identical analyte
concentration. The overall results obtained for the samples are therefore likely to be
inaccurate since we would have been comparing „apples with oranges,‟ that is, samples
and standards that have different characteristics.
 In the standard addition method, different portions of the sample are “spiked” with
known amounts of standard solution.
 This is advantageous as the sample and standards are made up in the same solution and
their analyte contents are measured at the same time.
 This ensures that all the solutions have the same matrix as the sample and consequently
matrix effects are accounted for – more accurate results would therefore be obtained.

Standard addition methods and calculations:

 If a linear relationship exists between absorbance and concentration exists then a single
standard addition can be done:
1. Two aliquots of a sample are transferred to labelled volumetric flasks.
2. The sample in one of the flasks is diluted to volume with a suitable solvent.
3. A measured volume of a standard solution of the analyte is added to the other.
Dr. D. Gordon-Smith, UTech
 15

4. Both solutions are made up to the same volume and their absorbances are measured
5. The concentration of the analyte in the sample is determined using the following
equation:
𝑨 𝑪 𝑽
𝑪𝒙 = 𝑨 𝑽𝒙 −𝑨
𝒔 𝒔
𝒕 𝒕 𝑽
𝒙 𝒙

where:
Cx = concentration of Ax = absorbance of the Vx = volume of
the analyte analyte solution analyte solution
Cs = concentration of At = total absorbance Vt = volume of Vs = volume of
the added standard of the analyte and volumetric flasks standard solution
standard added

Multiple standard additions:

 Known amounts of standard are added to several portions of samples and a curve is
plotted of response vs. amount of standard added or concentration – looks similar to a
calibration curve.
 The straight-line plot is then extrapolated backwards to the negative x-axis to determine
the concentration of the analyte in the sample.
 This allows verification that linear relationship exists between response and [analyte] but
is time consuming!

NOTE:
i. The standard added is a small volume of a concentrated solution so that the total
solution volume and ionic strength do not change appreciably
ii. The standard addition method has the advantage that it measures analyte
concentration in the natural environment of the sample and compensates for
variations caused by physical and chemical interference in the sample solution
iii. Both standards and samples are exposed to the same instrument fluctuations since
both are measured at the same time
iv. The method is slow and the sample preparation involved may be more intricate.

Dr. D. Gordon-Smith, UTech

 16

Internal Standard (IS) method

 A fixed amount of a substance is added in a constant amount to all samples, blanks and
calibration standards in an analysis.
 This added internal standard (IS) should have a similar structure or behave chemically
similar to the analyte and should be high in concentration.
 The ratio of the response of the analyte (Sa) to that of the IS (SIS) is plotted vs. [analyte],
𝑆𝑎
vs. [analyte]
𝑆𝐼𝑆
 If Sa and SIS behave the same way then matrix effects are compensated for.
 This procedure increases the accuracy and precision of results.
 This method is widely used in flame photometry and chromatography.

NOTE:
 The IS must be absent from the sample matrix so the only source of it is the added
amount.

Dr. D. Gordon-Smith, UTech