LECTURE 1

Spectrophotometer and pH Meter

DR. SARAVANA KUMAR JAGANATHAN

 About me

1. Open Chemistry, (Publisher: De

1) PhD ( IIT Kharagpur, 2009)
2) M.Sc (RWTH Aachen, 2005)
3) BE (University Madras, 2002)
1) Professional member – IOM3,
Grutyer) 2. International Journal of UK
Polymer Analysis and Characterization, 2) C. Eng
(Publisher: Taylor and Francis)

EXPERIENCE AWARDS

EDUCATION EDITOR PROFESSIONAL

1) Lecturer – Hull University, June 2019 MEMBERSHIP
2) Senior Lecturer, UTM – Since March 2014
3) Professor and Head (PSNACET, 2 year)
4) Professor and Dean Research “Best Researcher award”
(PSNACET, 1 year) for four successive years
(2015, 2016, 2017, 2018) -
UTM
Publication Profile

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https://scholar.google.co.in/citations?user=Ia2hFysAAAAJ
 Spectrophotometry

• Spectrophotometry is the most important of all the instrumental methods of

analysis in clinical chemistry.
• This method is based on the absorption of electromagnetic radiation in the visible,
ultraviolet and infrared ranges.
• The defined energy states of an atom or molecule are changed from one state to
another, would, therefore, require a definite amount of energy.
• The exact quantity of energy required to bring about a change from one given
state to another will be provided by photons of one particular frequency, which
may thus be selectively absorbed.
• The study of the frequencies of the photons which are absorbed would thus
provide information about the nature of the material
 Types of energy

• Molecules possess three types of internal energy—electronic, vibrational and

rotational.
• When a molecule absorbs radiant energy, it can increase its internal energy in a
variety of ways.
• The quantized and amount of energy necessary to cause any change corresponds
to the specific regions of the electromagnetic spectrum.
• A) Electronic transitions correspond to the ultraviolet and visible regions,
B) Vibrational transitions corresponds to the near infrared and infrared regions and
C) Rotational transitions corresponds to the infrared and far-infrared regions.
 Interaction of radiation with matter (1)

• When a beam of radiant energy strikes the surface of a substance, the radiation
interacts with the atoms and molecules of the substance.
• The radiation may be transmitted, absorbed, scattered or reflected, or it can excite
fluorescence depending upon the properties of the substance.
• The interaction however does not involve a permanent transfer of energy.
• The velocity at which radiation is propagated through a medium is less than its
velocity in vacuum.
• It depends upon the kind and concentration of atoms, ions or molecules present in
the medium.
Interaction of radiation with matter (2)
 Interaction of radiation with matter (3)

• The various possibilities which might result when a beam of radiation strikes a
substance are:
(a) The radiation may be transmitted with little absorption taking place, and
therefore, without much energy loss.
(b) The direction of propagation of the beam may be altered by reflection,
refraction, diffraction or scattering.
(c) The radiant energy may be absorbed in part or entirely by the substance.
 Spectrophotometer type instrument

The essential components of the spectrophotometer are:

• A source of radiant energy, which may he a tungsten lamp, a xenon-mercury arc,
hydrogen or deuterium discharge lamp, etc.
• Filtering arrangement for the selection of a narrow band of radiant energy.
• It could be a single wavelength absorption filter, an interference filter, a prism or a
diffraction grating.
• An optical system for producing a parallel beam of filtered light for passage
through an absorption cell (cuvette). The system may include lenses, mirrors, slits,
diaphragm, etc.
• A detecting system for the measurement of unabsorbed radiant energy, which
could be the human eye, a barrier-layer cell, phototube or photo-multiplier tube.
• A readout system or display, which may he an indicating meter or a numerical
display.
Various components of a spectrophotometer type instrument
 Radiant sources (1)

• The function of the radiation source is to provide a sufficient intensity of light

which is suitable form making a measurement.
• The most common and convenient source of light is the tungsten lamp.
• This lamp consists of a tungsten filament enclosed in a glass envelope.
• It is cheap, intense and reliable.
• A major portion of the energy emitted by a tungsten lamp is in the visible region
and only about 15 to 20% is in the infrared region.
 Radiant sources (2)

• When using a tungsten lamp, it is desirable to use a heat absorbing filter

between the lamp and the sample holder.
• This filter will absorb most of the infrared radiation without seriously
diminishing energy at the desired wavelength.
• For work in the ultraviolet region, a hydrogen or deuterium discharge lamp is
used.
• For fluorescent work, an intense beam of ultraviolet light is required. This
requirement is met by a xenon arc or a mercury vapour lamp.
 Optical filters

• A filter may be considered as any transparent medium which by its structure,

composition or colour enables the isolation of radiation of a particular wavelength
• For this purpose, ideal filters should be monochromatic, i.e. they must isolate
radiation of only one wavelength
• A filter must meet the following two requirements:
(a) high transmittance at the desired wavelength and
(b) low transmittance at other wavelengths.
Optical properties of a light filter
 Absorption filter

• The absorption type optical filter usually consists of colour media: colour glasses,
coloured films (gelatin, etc.), and solutions of the coloured substances.
• This type of filter has a wide spectral bandwidth, which may be 40 to 50 m in width
at one-half the maximum transmittance.
• Their efficiency of transmission is very poor and is of the order of 5 to 25%
 Interference filter

• These filters usually consist of two semi-transparent layers of silver, deposited on

glass by evaporation in vacuum and separated by a layer of dielectric (ZnS or
MgF2).
• In this arrangement, the semi-transparent layers are held very close.
• The spacer layer is made of a substance which has a low refractive index.
• The thickness of the diaelectric layer determines the wavelength transmitted.
 Working of interference filter

• Some part of light that is transmitted by the first film is reflected by the second
film.
• The light was again reflected on the inner face of the first film, as the thickness of
the intermediate layer is one-half a wavelength of a desired peak wavelength.
• Only light which is reflected twice will be in-phase and come out of the filter, other
wavelengths with phase differences would cause destructive interference.
• Constructive interference between different pairs in superposed light rays occurs
only when the path difference is exactly one wavelength or some multiple thereof
 Monochromators

• Monochromators are optical systems, which provide better isolation of spectral

energy than the optical filters
• Monochromators are preferred to isolate narrow bands of radiant energy
• Monochromators usually incorporate a small glass of quartz prism or a diffraction
grating system as the dispersing media
• The radiation from a light source is passed either directly or by means of a lens or
mirror into the narrow slit of the monochromator
• Then it is allowed to fall on the dispersing medium, where it gets isolated
• The efficiency of such monochromators is much better than that of filters and
spectral half-bandwidths of I nm or less
 Prism monochromators

• Isolation of different wavelengths depends upon the fact that the refractive index
of materials is different for radiation of different wavelengths.
• If a parallel beam of radiation falls on a prism, the radiation of two different
wavelengths will be bent through different angles.
• The greater the difference between these angles, the easier it is to isolate the two
wavelengths.
• The materials are selected whose refractive index changes sharply with
wavelength.
 Diffraction grating

• A diffraction grating consists of a series of parallel grooves ruled on a highly

polished reflecting surface.
• When the grating is put into a parallel radiation beam, so that one surface of the
grating is illuminated, this surface acts as a very narrow mirror.
• The reflected radiation from this grooved mirror overlaps the radiation from
neighbouring grooves
• The waves would, therefore, interfere with each other.
• By changing the angle at which the radiation strikes the grating, it is possible to
alter the wavelength reflected.
Dispersion phenomenon in diffraction gratings
 Holographic grafting

• The holographic or interference gratings possess superior performance in reducing

stray light as compared to diffraction gratings.
• Holographic gratings are made by first coating a glass substrate with a layer of
photo-resist.
• It is exposed to interference fringes generated by the intersection of two
collimated beams of laser light.
• When the photo-resist is developed, it gives a surface pattern of parallel grooves.
• When coated with aluminum, these become the diffraction gratings.
 Optical components

• Several different types of optical components are used in the construction of

analytical instruments based on the radiation absorption principle.
• They could be windows, mirrors and simple condensers.
• The material used in the construction of these components is a critical factor and
depends largely on the range of wavelength of interest.
• Normally, the absorbance of any material should be less than 0.2 at the
wavelength of use.
 Photosensitive detectors

• After isolation of radiation of a particular wavelength in a filter or a

monochromator, it is essential to have a quantitative measure of its intensity.
• This is done by causing the radiation to fall on a photosensitive element, in which
the light energy is converted into electrical energy.
• The electric current produced by this element can be measured with a sensitive
galvanometer directly or after suitable amplification.
 Sample holder

• Liquids may be contained in a cell or cuvette made of transparent material such as

silica, glass or perspex.
• The faces of these cells through which the radiation passes are highly polished to
keep reflection and scatter losses to a minimum.
• Solid samples are generally unsuitable for direct spectrophotometry.
• It is usual to dissolve the solid in a transparent liquid.
• Gases may be contained in cells which are sealed or stoppered to make them air-
tight.
• The sample holder is generally inserted somewhere in the interval between the
light source and the detector.
• For the majority of analyses, a 10 mm path-length rectangular cell is usually
satisfactory.
 Applications

In my research,

– Honey was characterized for

biomaterial applications

– Based on spectroscopy
 Color of honey

• The color of the honey varies according to the floral source and its mineral content (Amoit
et al, 1989)

• It usually ranges from water white to dark amber. (Baltrusaityte et al, 2007)

• According to the National honey Board, USA, color of the four honey types were classified
as follows

Honey Colour
A B (at 560 nm grading)
Sample A Extra light amber

Sample B white

Sample C Light amber

C D Sample D Extra light amber

 Flavonoids content by AlCl3 - method

• Sample A : 8.34 QE
15
(QE: Quercetin Equivalents/100 g of honey)
mg QE/ 100g of honey

Sample B : 4.23 QE
10
Sample C : 8.84 QE
Sample D : 10.10 QE
5

• These are higher than the reported values of French (1 QE )

0
and Burkin Faso (6.41 QE) honey. (Amiot et al, 1989); (Meda
A B C D et al, 2005)
e e e e
pl pl pl pl
m m m m
Sa Sa Sa Sa

Sample D > Sample C > Sample A > Sample B

Phenolics content by folin-ciocalteau method

• Sample A 60.45 GAE (mg Gallic Acid Equivalents/ 100 g of

honey)
Sample B 29.96 GAE
Sample C 65.08 GAE
Sample D 47.10 GAE

• Average phenolic content poses almost similar values as

French, Greek and Slovenian honeys. (Amiot et al, 1989);
(Kefalas et al, 2001); (Bertoncelj et al, 2007)
 Blood gas analyser

• Blood gas analyzers are used to measure the pH, partial pressure of carbon dioxide
(pCO2) and partial pressure of oxygen (pO2) of the body fluids with special
reference to the human blood.

• The measurements of these parameters are essential to determine the acid-base

balance in the body.

• A sudden change in the pH and pCO2 could result in cardiac arrhythmias,

ventricular hypotension and even death.

• This shows the importance of the maintenance of physiological neutrality in blood,

and consequently the crucial role that the blood gas analyzers play in clinical
medicine.
 Acid-base balance (1)

• The normal pH of the extracellular fluid lies in the range of 7.35 to 7.45,
indicating that the body fluid is slightly alkaline.
• When the pH exceeds 7.45, the body is considered to be in a state of
alkalosis.
• A body pH below 7.35 indicates acidosis. Both acidosis or alkalosis are
disease conditions widely encountered in clinical medicine.
• Any tendency of the pH of blood to deviate towards these conditions is
dealt with by the following three physiological mechanisms:
– (i) buffering by chemical means,
– (ii) respiration,
– (iii) excretion, into the urine by kidneys.
 Acid-base balance (2)

• The blood and tissue fluids contain chemical buffers, which react with
added acids and bases and minimize the resultant change in hydrogen
ions.
• They respond to changes in carbon dioxide concentration in seconds.
• The respiratory system can adjust sudden changes in carbon dioxide
tension back to normal levels in just a few minutes.
• Carbon dioxide can be removed by increased breathing and therefore,
hydrogen concentration of the blood can be effectively modified.
• The kidney requires many hours to readjust hydrogen ion concentration by
excreting highly acidic or alkaline urine to enable body conditions to
return towards normal.
 Introduction to pH

• pH is a unit of measure which describes the degree of acidity or alkalinity (basic) of

a solution.
• It is measured on a scale of 0 to 14.
• The formal definition of pH is the negative logarithm of the hydrogen ion activity.
• pH = -log[H+]
 pH value

• The pH value of a substance is directly related to the ratio of the hydrogen ion and
hydroxyl ion concentrations.
• If the H+ concentration is higher than OH- the material is acidic.
• If the OH- concentration is higher than H+ the material is basic.
• 7 is neutral, < is acidic, >7 is basic
 pH scale

• The pH scale corresponds to the concentration of hydrogen ions.

• If you take the exponent of the H3O+ concentrations and remove the negative sign
you have the pH of the solution.
• For example pure water H+ ion concentration is 1 x 10-7 M, therefore the pH would
then be 7.
 pH

• The addition of acid to water increases the concentration of hydrogen ions and
reduces the concentration of hydroxyl ions
• The addition of a base to water would increase the concentration of hydroxyl ions
and decrease the concentration of hydrogen ions
 pH measurement

• A pH measurement system consists of three parts: a pH measuring electrode, a

reference electrode, and a high input meter.
• The pH measuring electrode is a hydrogen ion sensitive glass bulb.
• The reference electrode output does not vary with the activity of the hydrogen ion.
 Glass electrode (1)

• The key feature of the pH-sensitive electrode is a thin glass membrane whose
outside surface contacts the solution to be tested.
• The inside surface of the glass membrane is exposed to a constant concentration
of hydrogen ions (0.1 M HCl).
• Inside the glass electrode assembly, a silver wire, coated with silver chloride and
immersed in the HCl solution, is called an Ag/AgCl electrode.
 Glass electrode (2)

• This electrode carries current through the half-cell reaction.

• The potential between the electrode and the solution depends on the chloride ion
concentration, but, since this is constant (0.1 M), the electrode potential is also
constant.
• The potential difference between the inside of the glass electrode and the outside
is caused by the oxides of silicon in side the glass:
Si.O- + H3.O+ = Si.O.H+ + H2.O
 Reference electrode

• A reference electrode is needed to complete the electrical circuit.

• The calomel reference electrode consists of a glass tube with a potassium chloride
(KCl) electrolyte which is in intimate contact with a mercuric chloride element at
the end of a KCL element.
 Working of pH meter (1)

• A sample is placed in a cup and the glass probe at the end of the retractable arm is
placed in it.
• The probe is connected to the main box.
• There are two electrodes inside the probe that measure voltage.
• One is contained in liquid with fixed pH.
• The other measures the acidity of the sample through the amount of H+ ions.
 Working of pH meter (2)

• A voltmeter in the probe measures the difference between the voltages of the two
electrodes.
• The meter then translates the voltage difference into pH and displays it on the
screen.
• Before taking a pH measurement the meter must be calibrated using a solution of
known pH.
 Calibration (1)

• The pH meter should be standardized each time it is used with a buffer of

known pH
• To calibrate the pH meter, rinse the electrode with deionized water
• Place the electrode in a standard solution, e.g., pH 7
• Turn the selector to "pH"
• Adjust the pH meter to the appropriate pH
• Rinse the electrode with second standard
 Calibration (2)

• Standard pH buffers used should be 7and 4 or 10, depends on the final pH

• Turn the selector to "pH".
• Adjust the temperature knob to the second standard pH.
• Rinse the electrode with deionized water, and return the electrode to the
soaking solution
 Precautions

• The glass membrane must be thoroughly hydrated to work properly.

• Do not allow the electrode to remain out of water any longer than necessary.
• When the electrode is not in use, keep it immersed in the pH 4.00 buffer.
• Do not put the electrode down on the desk.
• The pH-sensitive glass membrane is very thin and very easily broken.
• Do not touch the membrane with anything harder than a Kim-Wipe and do that
very gently.
• Do not drop the electrode or bump it on the bottom of the beaker when
immersing it in a solution.
 Temperature and buffers

• Temperature compensation is contained within the instrument because pH

electrodes are temperature sensitive.
• Temperature compensation only corrects for the change in the output of the
electrode, not for the change in the actual solution.
• Buffers are solutions that have constant pH values and the ability to resist changes
in pH.
• They are used to calibrate the pH meter.