10 Spectroscopic Instrumentation

Vinay Gupta

    Learning Objectives

Spectroscopy is the study of interaction of electromagnetic radiation with the matter.

• It is used for investigating molecular characteristics of the sample of interest. In this module we study about two most common spectroscopy method
• UV/Visible spectroscopy and Infra-red spectroscopy
• First we will study about the basic principle of measurement and basic of instrumentation behind the technique.

    Introduction

Spectroscopic methods are large group of analytical methods that are based on atomic and molecular spectroscopy. In general terms spectroscopy is the branch of science that deals with the interaction of various types of radiation with matter. This field of investigation started with the studies involved in interaction between electromagnetic radiation and matter, but at present the field has been broadened to include interactions between matter and other forms of energy, for example acoustic waves and beams of particles such as ions and electrons. Therefore, Spectrometry and spectrometric methods refer to the measurement of intensity of radiation with a photoelectric transducer or other type of electronic devices.

Most of the commercially available spectrophotometers are based on electromagnetic radiation. This type of energy can take several forms and is most easily recognizable as light or radiant heat. Based on the frequency and wavelength range electromagnetic spectrum is classified into various classes, gamma rays have smallest wavelength in the range of 1 pm and radiowaves can have wavelength has long as 100 Mm (Mm – Mega Meter; Mega = 10-6).

In this session, we will limit ourselves to UV/Vis and IR range. We will study about instrumentation and basic physics behind molecular absorption spectroscopy.

Ultraviolet /Visible Spectroscopy

Principle Behind UV/Vis Spectroscopy

UV/Vis spectroscopy falls into the category of molecular absorption spectroscopy. This technique primarily focuses on absorption characteristics of molecules within the electromagnetic spectral range of 160 to 780 nm. It is based on the measurement of Transmittance (T) or Absorbance (A) of solutions contained in transparent cell having path length of b cm. The concentration of an absorbing analyte is linearly related to absorbance.

Experimentally observed transmittance and absorbance can be approximated with the following equations –

   where Po is the power of radiation passing through the solvent and P is the power of radiation passing through the analyte present in solvent.

The relation between the absorbance and transmittance is given by the following equation

A = 2 – log %T ————–(3)

The extent to which absorbance will take place is mathematically explained by Beer’s law according to which –

A = bc ————-(4)

Where measured absorbance is directly proportional to molar absorbtivity with units of L mol-1 cm-1, b is the path length in cm i.e. width of the sample holder and c is the concentration of the compound in the solvent mol L-1.

A limitation to the Beer’s law is that it can describe absorption behavior at relatively low concentration of analyte. At high concentration usually greater than 0.01M, the average distance between the molecules responsible for absorption very less. At high concentration molecules start affecting charge distribution of their adjacent molecule. This alters molecules ability to absorb radiation at a given wavelength. As the extent of absorption depends on concentration, this phenomenon causes deviation in the linear relationship between absorbance and concentration. Deviation in the Beer’s law may also there due to because it depends upon the refractive index of the medium. If any changes in concentration can lead to alteration of refractive index of the solution, deviations in the Beer’s law maybe observed.

Instrumentation

Instruments for measuring absorption in UV/Visible range of electromagnetic spectrum are made up of (1) electromagnetic radiation source (2) wavelength selectors (3) sample containers (4) radiation transducers and (5) signal processors and readout devices.

For molecular absorption studies a continuum source is required whose power doesn’t change sharply over considerable range of wavelengths. Different types of sources are available in the market; they are hydrogen & deuterium lamps, tungsten filament lamps and xenon arc lamps. Cells or cuvettes required for holding the sample and solvent are constructed out of special material that also allows radiation to pass through to pass through in the spectral region of interest. They normally made up of quartz or fused silica and are transparent to UV (below 350 nm) and visible region of the spectra. For visible region of the spectra Plastic containers are also used.

Figure 1. Single Beam Spectrophotometer

  The two most commonly available spectrophotometers are Single beam and Double Beam spectrophotometer. Single beam spectrophotometer consists of radiation source, filter or monochromator, matched cells that can be interposed alternatively in the path of radiation beam, a photo detector, an amplifier and a read out device. The basic design single beam spectrophotometer is shown in figure 1. These systems have interchangeable tungsten/deuterium lamp sources and are equipped with high resolution grating monochromator with variable slits. Photomultiplier tubes are used as transducers and its output is digitized, for printing, plotting and storing data in useful form.

Figure 2. Double Beam Spectrophotometer

The Double-Beam spectrophotometer is similar to single beam but there are additional beam splitters that can direct radiation beam to reference and sample cells simultaneously. This is can be observed in figure 2. The signals from the two photodetector go to the difference amplifier and finally to readout device. They offer an advantage of compensating any short-term fluctuations from the radiation source and any drift in transducer and amplifier. They are also known to compensate for variations in the source intensity with the wavelength.

Infrared (IR) Spectroscopy

Principle Behind Infrared Spectroscopy

The infrared region of the spectrum encompasses wavelengths from 0.78 to 1000 m. From the standpoint of application and instrumentation infrared is divided into near-, mid- and far-infrared radiation. Though the most commonly used range in infrared region is between 2.5 to 15 m. A linear wavenumber scale i.e. cm-1 is preferred in infrared spectroscopy due to the directly proportionality between this quantity and both energy and frequency. In practice it is the frequency of absorption of radiation that is responsible for molecular vibrational frequency during the measurement. Absorption of IR radiation is largely confined to molecular species that have small energy difference between various vibrational states. If the radiation frequency exactly matches the natural vibrational frequency of the molecule, a net energy transfer takes place. This results change in amplitude of the molecular vibration and absorption of radiation is the consequence.

Figure 3. Types of Molecular Vibrations. Note:+ indicates motion from page towards the reader; – indicates motion away from the reader.

Similarly, any rotation of asymmetric molecules around their center of mass can lead to periodic dipole fluctuation that interact with IR radiation. There is no net change in the dipole moment occur during vibrational or rotation of homonuclear species such as N2, Cl2 or O2. Compounds like these cannot absorb IR radiation, whereas all other molecular species can. Various kinds of molecular vibrations that can take place in a simple triatomic molecule are shown in the figure 3. Broadly they are classified as Stretching and Bending Vibrations.

Quantum Treatment of Vibrations

Molecular vibrations like coupling of vibrations between atoms involving bonds to a single central atom are analogous to mechanical model used in describing simple harmonic oscillator. The natural frequency of an oscillator can then be described mathematically as

where k is the force constant that depends on the stiffness the spring, in our case it depends on the strength of the bond, is the reduced mass where two masses m1 & m2 are connected by a spring ( in our case molecular bond) can be written as =m1.m2/(m1 + m2).

Let us assume that the transitions in the vibrational energy levels are brought about by absorption of radiation, provided that the energy level of the radiation matches the difference in the energy level E between the vibrational quantum states, plus that the vibration causes fluctuation in the dipole. This difference in energy level can be written as

    where h is the Planck Constant.

The frequency of radiation v that can bring change identical to the classical vibrational frequencies of the bond vm can be derived from the equations 5 & 6 and is written as

   The Wavenumber   ̅ is reciprocal of wavelength in centimeters and is directly proportional to the frequency and the energy of the radiation.

Thus it may be written as   ̅= kv, where k is the constant dependent on the medium and wavenumber is equal to the reciprocal of velocity c of the electromagnetic radiation.

Now if we wish to express the equation 7 in wavenumber then it may be written as

     where   ̅is the wavenumber of an absorption peak in cm-1, k is the force constant of the bond in Newtons per meter (N/m), c is the velocity of light in cm/s and is the reduced mass.

IR Instruments

There 3 types of infrared instruments available from commercial sources (1) dispersive grating spectrophotometers that are used primarily for qualitative measurements (2) multiplex instruments employing Fourier transform and suited both for qualitative and quantitative measurements (3) non-dispersive photometers that have been developed for quantitative determination of a variety of organic species in the atmosphere by adsorption, emission, and reflectance spectroscopy.

Most widely used spectrophotometer for laboratory-based investigation is Fourier transform Infrared (FTIR) spectrophotometers because of their speed, reliability and convenience.

Figure 4. A single beam FTIR Spectrometer (Courtesy Perkin-Elmer)

  The basic design of Single beam FTIR spectrophotometer is given in figure 4. The majority of FTIR instruments are based on Michelson Interferometer. An interferometer uses patterns formed by waves to measure characteristics of the waves themselves or of the material that reflects, refracts or transmits waves.

Infrared sources (lamps) consist of inert solid that is heated electrically to a temperature between 1500 and 2200 K. A Continuum radiation approximating to that of a blackbody is emitted from the source. Various kind of IR source that are used in IR instruments are

• Nernst Glower – It is composed of rare earth oxides formed into cylinder of diameter 1 to 2 mm and length upto 20 mm.
• Globar Source – It is a silicon carbide rod, usually about 50 mm in length and 5 mm diameter
• Incandescent Wire Source – It is tightly wound spiral of Nichrome wire heated to about 1100 K by an electrical current.
• Mercury arc – It consists of quartz jacketed tube containing mercury vapor at pressure greater than one atmosphere.
• An ordinary Tungsten filament lamp
• A Carbon Dioxide Laser Source

    The basic design of Single beam FTIR spectrophotometer is given in figure 4.

Advantages. Optics of FTIR instruments has much larger energy throughput (one or two orders of magnitude) than dispersive instruments. They have limited throughput due to the use of narrow slit widths. It should be noted that interferometer is free the problem of stray radiation because each IR frequency is effectively chopped at different frequency. That means any radiation other than the frequency that is to be radiated onto the sample is removed by interferometer.

FTIR is a powerful instrument that has extensive application in the field of chemistry required for analysis of unknown component in the sample of interest. This instrument is particularly useful for

1. Very high-resolution work involving gaseous mixtures having complex spectra resulting from superposition of vibrational and rotational bands.
2. The study of analytes having high absorbances
3. The study of substances with weak absorption bands ( e.g. compounds absorbed on catalyst surface)
4. Fast scanning investigations involving kinetic studies or detection of chromatographic effluents
5. Collecting IR data from very small samples
6. Obtaining reflection spectra
7. IR emission studies

    Summary

In this module we studied about two most important types of spectrophotometry methods. First, we studied about UV/Visible Spectrophotometry and we briefly studied about Beer’s law that describes the absorption characteristics of molecules in the path of the UV/Visible radiation. Second, we studied about Infrared Spectrophotometry where we studied about different types of molecular bending vibrations responsible for absorption of radiation at particular wavenumber or frequency.

References :-

1. Electrical and Electronic Measurements and Instrumentation, Sawhney A. K., Dhanpat Rai & Sons, Reprint 1985
2. Measurements and Instrumentation, Bakshi U.A., Bakshi A.V., Technical Publications, 2009
3. Principles of instrumental analysis, Skoog, Douglas A., F. James Holler, and Stanley R. Crouc,. Cengage learning, Edition 2017
4. Instrumentation, measurement and analysis. Nakra, B.C. and Chaudhry, K.K., Tata McGraw-Hill Education, 2003.
5. Measurement and instrumentation: theory and application, Morris, A. S., & Langari, R. , Academic Press, 2012.