Instrumental Methods of Analysis

Section - I: Spectroscopy

1. UV Visible Spectroscopy

2. Fluorimetry

3. IR Spectroscopy

4. Flame Photometry

5. Atomic Absorption Spectroscopy

6. Nepheloturbidimetry

CHAPTER 1

UV Visible Spectroscopy

INTRODUCTION

Spectroscopy is the branch of science that deals with studying the interaction of matter with electromagnetic radiation. Due to this interaction, energy is either absorbed or emitted by the matter in discrete amounts called quanta. The measurement of this absorbed or emitted radiation by the matter forms the basis of spectroscopic techniques. Spectroscopic techniques are helpful in the study of atomic and molecular structures. Spectroscopy is one of the most powerful tools available for the qualitative and quantitative analysis of different samples.

Spectroscopy can be studied under two major headings

1. Atomic spectroscopy - deals with the interaction of electromagnetic radiation with atoms in the ground energy level (lowest energy).

E.g., Atomic absorption spectroscopy, Atomic emission spectroscopy, Flame emission spectroscopy

2. Molecular spectroscopy - deals with the interaction of electromagnetic radiation with molecules.

E.g., IR spectroscopy, Raman spectroscopy.

Spectroscopy can also be classified based on the measured mode of interaction of matter with electromagnetic radiation, which is given in the Table 1.1.

Table 1.1 Instrumental Methods based on measurement of property

PROPERTIES OF ELECTROMAGNETIC RADIATION

Electromagnetic radiation is an energy form that travels with enormous velocity in space. Electromagnetic radiation (EMR) possesses both wave and particle nature. These properties are a distinct feature of EMR, and they are inseparable.

Wave properties of Electromagnetic radiation

Electromagnetic radiation has an electrical component and a magnetic component oscillating in perpendicular planes and also perpendicular to the direction of propagation. The wave nature of EMR shows properties like refraction, reflection, constructive and destructive interferences.

Fig. 1.1 Electromagnetic wave with its components.

The electromagnetic wave parameters are

(a) Wave length, λ: Is the distance between two successive maxima or minima in an electromagnetic wave . It is denoted by the Greek letter lambda. Units of wavelength are meters (m), centimeter (cm), millimeter (mm), micrometers (μm), nanometers (nm) and angstrom (Å).

A beam of radiation with only one wavelength is called monochromatic radiation, and the beam consisting of several wavelengths is called polychromatic or heterochromatic radiation.

(b) Frequency, υ: Is the number of wavelength units passing through a given point in unit time. It is denoted by the Greek letter υ.Unit is cycles per second or Hertz.

(c) Wave number Frequency is usually a large number making its use more difficult in regular practice. To overcome this problem, frequency is usually expressed aswave number. The wave number is the number of waves in vacuum . The symbol is Unit for wave number is cm -1

Particle properties of electromagnetic radiation

When electromagnetic radiation hits a material, it emits electrons called photoelectrons. This photoelectric effect is due to the particle properties of EMR. To explain this property, it is assumed that EMR consists of a stream of discrete packets (particles) of energy called photons or quanta. The energy of the photon is proportional to the frequency of the radiation and is given by the relationship

E = hυ, where,

E is the energy of a photon in ergs, υ is the frequency of the EMR in cycles per second, and h is called Planck’s constant (6.624 × 10-³⁴ joules/second).

EMR of longer wavelength (low frequency) has lower energy than the shorter wavelength (high frequency).

Electromagnetic spectrum

The entire range of EMR is called as EMR spectrum.

Fig. 1.2 Electromagnetic spectrum showing different regions of EMR.

The various spectral regions are

γ(Gamma) ray region - lies between 0.02 to 1 angstrom. These are the shortest waves.

X-ray region -This region lies between 1 to 10 angstrom. These rays cause core electron excitation.

Visible and Ultra violet region - These rays cause valence electron excitation. This region consists of three parts

Infrared region - This region is subdivided into three regions

Microwave region - 0.1 mm - 1 cm

Radiofrequency region - 100m - 1 cm

INTERACTION OF ELECTROMAGNETIC RADIATION WITH MATTER

When electromagnetic radiation passes through matter, a variety of events may occur. Some of them are

1. Absorption of radiation: If the radiation of appropriate energy is used, they may be absorbed by the matter resulting in electronic, vibrational, and rotational changes or a combination of these changes. After absorption, the molecules become excited and go to a high energy level (low stability). They lose energy in the form of heat or re-emit EMR to come back to the ground state (high stability).

The total energy of the molecule at the ground state is given as

E0 Eelectronic + Evibrational + Erotational

The schematic energy level diagram for a simple diatomic molecule is shown in the figure.

Fig. 1.3 Energy level diagram of a molecule showing vibrational and rotational energy level.

An electronic transition due to absorption of light may include vibrational or rotational changes.

2. Scattering : Sometimes, the radiation is not absorbed completely by the matter, and a portion of it may undergo scattering or reflection

3. Emission: In some cases, the molecules become excited after absorbing the radiation, and they lose energy by re-emitting radiation either instantaneously or with a time delay.

Franck-Condon principle

Franck-Condon principle gives the interaction between the electronic and vibrational motions. It states that the timescale of electronic transitions are so rapid when compared with the nuclear motion and the mass of the nuclei are heavy compared with electrons. An electronic transition is therefore considered to be a vertical transition. The light is absorbed in femtoseconds to nanoseconds, and within this timescale, electrons move, but the nuclei cannot. The heavier atomic nuclei cannot adjust during the absorption but readjust after the absorption process, creating vibrations. Simultaneous occurrence of electronic and vibrational transitions is called vibronic transitions, which give rise to the vibrational structure of the electronic bands.

The quantum mechanical formulation of this principle is that the intensity of a vibronic transition is proportional to the square of the overlap integral between the vibrational wave functions of the two states that are involved in the transition.

Fig. 1.4 Franck-condon principle for vibronic transitions in a molecule.

The Figure illustrates the Franck-Condon principle for vibronic transitions in a molecule. Potential energy functions in both the ground and excited electronic states are shown. In the low temperature, the molecule in the v = 0 vibrational level of the ground electronic state absorbs a photon of the necessary energy, makes a transition to the excited electronic state. The electron configuration of the excited state results in a shift of the equilibrium position of the nuclei of the molecule. In the figure, this shift in nuclear coordinates between the ground and the first excited state is labeled as q 01. In a simple case of a diatomic molecule, the nuclear coordinate axis refers to the internuclear separation. The vibronic transition is indicated by a vertical arrow, assuming constant nuclear coordinates during the transition. In the electronic excited state, molecules quickly relax to the lowest vibrational level of the lowest electronic excitation state (Kasha's rule). From there, they decay to the electronic ground state through photon emission. The Franck-Condon principle is applied equally to absorption as well as fluorescence.

1.1 VISIBLE SPECTROPHOTOMETRY AND COLORIMETRY

UV-Visible Spectroscopy is one of the important absorption spectroscopic techniques, and it is most commonly employed for quantitative and qualitative analysis. It is also playing a role in the structural elucidation of organic compounds.

The wavelength range of visible light is 800-400 nm (8000-4000 Angstrom). This range of visible radiation is used for colorimetric analysis. Determination of concentration of coloured compounds in solution is called colorimetry. A colorimeter is a device used to test the concentration of a solution by measuring its absorbance at a specific wave length of light.

Coloured substances absorb in the visible region, and the colorless substances absorb in the UV region. The amount of light absorbed differs with the wavelength, and a plot of absorbance vs. wavelength is called an absorption spectrum. In the absorption spectrum, the wavelength at which maximum absorption occurs is called λmax. This λmax is characteristic or unique for every substance and is used to identify the substances (qualitative analysis). λmax is independent of concentration, meaning that the λmax will not change even if the concentration is changed.

A calibration graph is a plot of concentration vs. absorbance. This graph helps to determine the concentration/amount of the drug present in the given sample solution (quantitative analysis).

Fig. 1.5 Absorption spectrum.

Fig. 1.6 Absorption spectrum of a substance in different concentrations.

Fig. 1.7 Calibration curve.

THEORY OF SPECTROPHOTOMETRY & COLORIMETRY

(ABSORPTION LAWS)

When light is incident upon a homogenous medium, a part of the incident light is reflected, a part is absorbed, and the remainder is transmitted so

Where Io - incident light; Ia - absorbed light; It - transmitted light and Ir - is reflected light. The value of Ir is minimal (about 4%), and it is eliminated for air-glass interfaces if a comparison cell is used. So equation (1.1) becomes as

The two laws governing the absorption of light are:

1. Beer’s law (concentration-dependent)

2. Lambert’s law (Thickness/path length dependent absorbance)

Lambert’s law states that when a beam of monochromatic light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of the medium is directly proportional to the intensity of incident light.

On integration

where b is the integration constant

When thickness is zero, there is no absorbance. So It = Io

Substituting this in equation 1.4

Substituting this value of b in equation 1.4

On rearranging

Removing natural logarithm

Taking inverse on both sides

This equation is called as Lambert’s law where Io is intensity of incident radiation, It is transmitted radiation and k is a constant.

Lambert’s law shows the logarithmic relationship between transmittance and the optical path length

Beer’s law: Beer observed a similar relationship between transmittance and the concentration of a solution. Beer’s law states that the intensity of a beam of monochromatic light decreases exponentially with an increase in the concentration of absorbing species arithmetically.

Rearranging the equation

On integration

where b is the integration constant

When concentration is zero, there is no absorbance. So It = Io

Substituting this in equation 1.7

Substituting this value of b in equation 1.7

On rearranging

Removing natural logarithm

Taking inverse on both sides

This equation is called as Beer’s law equation where I

o

is intensity of incident radiation, It is intensity of transmitted radiation and k is a constant.

On combining equations 1.5 and 1.8, we get

on changing from natural logarithms to base 10,

Rearranging terms

Taking inverse on both sides

Taking log on both sides

Transmittance T = It/Io, Absorbance A = log 1/T

Substituting for T in absorbance A, the equation becomes

From equations 1.9 and 1.10

A = Kct.

If a is used instead of K, then

A = act

log Io/It = act

is the mathematical expression of Beer Lambert’s law,

Where A= absorbance, a = absorption coefficient or absorptivity, c= concentration of the substance and t = path length in cm.

Absorptivity reflects the sensitivity of the procedure. Sensitivity increases with an increase in absorptivity.

If the concentration is expressed in moles/liter, then a is replaced with ε.Then the equation becomes

where

A = absorbance, ε = molar absorption coefficient or Molar absorptivity, c = concentration of the substance in mol/lit, t = path length in cm.

A = ε, when the concentration is one mole per liter and the path length is one centimeter.

Thus Molar absorption coefficient is defined as the absorption of a one molar solution when the path length is one cm. The molar absorption coefficient is otherwise called molar absorptivity.

The value of molar absorptivity is constant even if the concentration of the solution and the thickness of the container change. But it differs with different wavelengths.

Absorbance also varies with wavelength. For this reason, only monochromatic light is used in spectrophotometry and colorimetry.

Molar absorption coefficient ε is expressed as

Where, is the specific absorbance which is the absorbance of 1% w/v solution, using a path length of 1cm. It is a constant for each drug at the given wavelength. This specific absorbance can be used for the quantitative estimation of formulations. εmax is the value of ε at λmax.

DEVIATIONS FROM BEER’S LAW

According to Beer’s law, the plot of concentration Vs. absorbance should give a straight line passing through the origin. The straight line can also be obtained by using the line of best fit or method of least squares or by joining the maximum number of points so that the positive and negative errors are balanced or minimized. The regression line obtained can also be used for determining the concentration of a solution. When a nonlinear curve (not a straight line) is obtained in a plot of concentration vs. absorbance, the system is said to have deviations from Beer’s law.

Beer’s law is normally obeyed only in a certain concentration range, and above or below it may exhibit deviation.

The reasons for the deviations may be classified as

1. Real Deviations

2. Instrumental Deviations

3. Chemical Deviations

4. Incomplete reaction

Fig. 1.8 Real deviations

1. Real Deviations: There are two types of real deviations from Beer’s law

(i) Positive deviation (concave upwards): It occurs when a small change in concentration produces a great change in absorbance.

(ii) Negative deviation (concave downwards): It occurs when a large change in concentration produces a small change in absorbance.

2. Instrumental deviations: Factors like stray radiation, improper slit width, fluctuation in a single beam, and the type of light used can influence the deviation.

•If monochromatic light is not used, deviation may occur.

•If the width of the slit is not proper and if it allows undesirable radiations to fall on the detector, these undesirable radiations might be absorbed by the impurities present in the sample leading to changes in absorbance.

•The possibility of error due to monochromatic radiation may be minimized by selecting a spectral region, where the change in absorptivity with a change in wavelength is very small.

•This dictates that wavelength selection should be from a broad band (close to λ max ) rather than a sharply rising or sharply falling section of the absorption curve.

•It is necessary to prepare a calibrated curve for the absorbance concentration relationship at the chosen wavelength to avoid errors.

3. Chemical deviations:

Physicochemical changes in solution: Factors like association, dissociation, ionization (change in pH), faulty development of colour (incompletion of reaction), and refractive index at high concentration can influence such deviation.

•Association: Methylene blue at a concentration of 10 -5 M exists as monomer and has λ max of 660nm. But methylene blue at a concentration above 10 -4 M exist as a dimer or trimer and has a λ max of 600. If the shift of this λ max is not considered during absorbance measurements, then deviations occur.

•Dissociation : Benzyl alcohol in chloroform exists in a polymeric equilibrium; 4C 6 H 5 CH 2 OH  ↔ (C 6 H 5 CH 2 OH) 4 .

Dissociation of the polymer increases with dilution. The monomer absorbs at 2.750 to 2.765 μ, where as the polymer absorbs at 3.0 μ. Hence absorption at 2.75 micron shows a negative deviation whereas at 3.0 μ gives a positive deviation.

Potassium dichromate at high concentration exists as an orange solution (λmaxof 450nm). But on dilution, dichromate ions are dissociated into chromate ions which is yellow in color (λmaxof 410nm)

If 450 nm is used for absorbance measurement for dilute solutions, then it leads to deviations from Beer’s law.

•Coloured solute ionization or dissociation in solution leads to deviation in Beer’s law. Colour change of dichromate ion on dilution can be given as an example.

•The deviation may also occur due to the presence of impurities that fluoresce or absorb at the absorption wavelength. This interference introduces an error in the measurement of absorption of the sample.

•If the solute undergoes polymerization , Ex Benzyl alcohol in carbon tetrachloride in high concentration exists in polymeric form. Dissociation of this polymer increases with dilution, which causes changes in absorbance, causing deviation.

•Beer’s law can’t be applied for suspensions, but colorimetry can be used with different known concentrations to prepare the reference curve.

4. Incomplete reaction:

•Sufficient time should be allowed before taking absorbance for the reaction to complete so that the colour will be formed completely.

•If the readings are taken after the colour fades away (instability), deviations can occur. Ex: Limit test for iron: reaction of iron with thioglycolic acid.

INSTRUMENTATION

Table 1.2 Summary of instrumentation

The common instruments used for measuring emission or absorption of radiant energy are

(i) Photometer (ii) Spectrophotometer

Photometer: These are inexpensive and less accurate instruments. Filters are employed to isolate a wave length region, and a photocell or photo tube is used as a detector. A commercial photometer with filters is called colorimeter. It measures either absorbance or transmittance. The working range of wavelength for these instruments is 400-700nm.

Spectrophotometer: These are a little more expensive than colorimeters. They are used for wider wavelength regions, i.e., 360 nm- 900nm or 1000nm. Since these instruments employ grating monochromators for scanning and photomultipliers as detectors, they are highly accurate. The modern spectrophotometers are microprocessors or computer-based for easy handling of data.

The basic components of both types of instruments are

(a) Source of light

(b) Filters or monochromators

(c) Sample cells

(d) Detectors

Now we will discuss these components

(a) Source of light /radiation sources: The visible region extends from 400-800nm

The requirements of the radiation sources are

1. It must be stable and no fluctuations should be there.

2. It should provide continuous radiation from 400-800nm.

3. The intensity should be adequate.

The common radiation sources used in colorimetry (visible region) are

1. Tungsten lamp: Most widely used. The lamp consists of a tungsten filament in a vacuum bulb. It provides sufficient intensity.

Advantages

(i) Stable, robust and easy to use.

(ii) The Emission intensity varies with wavelength.

Disadvantages

(i) The intensity at the shorter wavelength is less

(ii) Difficult to maintain a constant intensity

2. Carbon arc lamp: This lamp is used for high intensity. It provides an entire range of the visible spectrum.

(b) Filters and Monochromators: The source emits continuous spectra from 400-800nm. This is called Polychromatic light consisting of several wavelengths. A colorimeter or spectrophotometer works only with a monochromatic light consisting of a single wavelength. A filter or a monochromator is used in these instruments to convert the polychromatic light into monochromatic light.

(a) Filters: There are two types of Filters

1. Absorption filters.

2. Interference filters

1. Absorption filters: These filters are made of a solid sheet of glass coloured by dissolved or dispersed pigments. Dyed gelatin is also used as an absorption filter.

The selection of filters is based on this color wheel. If the solution is red coloured, then the green filter is used. For the green solution red filter is used (complementary colors).

Fig. 1.9 Color wheel showing