30 Sepctrophotometry

Dr. Swagat K. Mohapatra

Contents

1. Introduction
2. Spectrophotometry Theory
3. Light and the Perception of Color
4. Transmittance, Absorbance, and Beer-Lambert’s Law
5. Instrumental Design
6. Use of Spectrophotometer
7. Variations in Spectrophotometry Technique
8. Important Spectrophotometry methods for measuring common air pollutants
9. References

Introduction:

First of all let us discuss “why leaves appear green to us”. Consider when a beam of light (i.e. sunlight) passes through a solution containing chlorophyll (the compound which is responsible for the color of the leaves). The chlorophyll molecules absorb only a few select photons in the blue and red regions of the visible portion of the electromagnetic spectrum. The energies of these absorbed photons cause electrons in the chlorophyll molecule to be excited, and in the plant cell the energy of these excited electrons is used to convert CO2 and H2O to glucose. In fact when the red and blue photons that chlorophyll absorbs are withdrawn from white light, the resulting beam of light leaving the solution appears to be green to our eye, and this is why leaves appear green to us.

In the above example, if we could measure the total number of photons of all colors that enter the sample and compare with that the total number of photons of all colors that leave the sample, we would see a lesser number photons exit the sample than entering. This is where the chlorophyll molecule absorbed some of the photons from the beam of light that entered the solution, and appeared to be colored.

Spectrophotometry is a technique, which is used for the above measurements. Thus it is the study of interaction of matter with light (or other electromagnetic radiations). In depth it deals with the measurement of the radiant energy transmitted or reflected by a body as a function of the wavelength. The benefits of spectrophotometry are: i) it is non-destructive (can measure and recover sample), ii) it is selective (often a particular compound in a mixture can be measured without separation, and iii) it has a short time interval of measurement (10-14 seconds). The instrument, which is used, for this technique is called Spectrophotometer.

Spectrophotometry Theory:

Light can be considered as a wave. This wave has two components, electric and magnetic which are perpendicular to each other (figure -1). The electromagnetic radiation exhibits a direction of propagation and wave-like properties (i.e. oscillations). The energy of electromagnetic radiation is defined as:

Further Light has both wave and particle nature. The conceptual particle of light is called a photon and is represented by h .

Electromagnetic radiation exhibits a wide spectrum and specific ranges of wavelengths have names (Figure – 2). The energy of electromagnetic radiation is inversely proportional to its wavelength.

Light and the Perception of Color:

Light or Electromagnetic radiation when falls on a substance, three possibilities are there:

i) it can be reflected by the substance

ii) it can be absorbed by the substance

iii) some can be absorbed and the remainder can be transmitted or reflected

Since reflection of light is not of interest in spectrophotometry, we will discuss on the absorbance and transmittance of light. The color we see in a sample of solution is due to the selective absorptionof certain wavelengths of visible light and transmittance of the remaining wavelengths. For example, if a sample absorbs all wavelengths in the visible region of the electromagnetic spectrum, it will appear black; if it absorbs none of them, it will appear white or colorless. We can detect the color only when a particular wavelength of radiant energy strikes our eyes.

For example when we shine a beam of white light at a substance that absorbs blue light, it appears to be yellow. Because the blue component of the white light gets absorbed by the substance, the light that is transmitted is mostly yellow, which is the complementary color of blue. This yellow light reaches our eyes, and we “see” the substance as a yellow colored substance.

Below in the table -1 given a list of complementary colors and their corresponding wavelengths.

A molecule or a substance which absorbs light is known as chromophore. Chromophores exhibit unique absorption spectra and can be defined by a wavelength of maximum absorption, or λmax.

However, one should remember that the visible range is only a very small portion of the electromagnetic spectrum (figure – 2). Ultraviolet (UV), and infrared (IR) spectrophotometric techniques are more suitable for many colorless substances that absorb strongly in the UV or IR spectral regions.

TABLE 1: ABSORBANCE AND THEIR COMPLEMENTARY COLORS

Wavelength (nm)	Color Absorbed	Color Observed
380-435	Violet	Yellow-green
435-480	Blue	Yellow
480-490	Greenish blue	Orange
490-500	Bluish green	Red
500-560	Green	Red-purple
560-595	Yellow-green	Purple
595-650	Orange	Greenish blue
650-780	Red	Blue-green

Transmittance, Absorbance, and Beer-Lambert’s Law:

Suppose the intensity of the light entering into the sample is I0 (incident light), and exiting the sample is I (transmitted light), then the ratio I/I0, which gives an indication of what fraction of light entering the sample is found exiting the sample, is called as transmittance T

Let’s consider two sample solutions of different concentrations (one fairly dilute, and the other concentrated) of one chemical species that absorbs light of a particular wavelength. When we pass a beam of light of the appropriate wavelength through the fairly dilute sample solution, we could see that the photons will encounter a small number of the absorbing chemical species, so we might expect a high percentage of transmittance and a low absorbance. Similarly when we pass the same beam of light through the concentrated solution, we could observe that the photons will encounter a large number of the absorbing chemical species, and we might expect a low percentage of transmittance and a high absorbance. Thus, the absorbance increase as the concentration of sample increase, i.e. absorbance A is directly proportional to the concentration c of the sample.

absorbance                    ∝ conecentration of sample

On the other hand, Let’s have two sample holders of different thickness (one with short path and the other with long path length), containing the same sample solution of same concentration (figure-4). When we pass the beam of light though both the sample holders, in the first case the light has to pass through only a short distance, whereas in the second case the light has to go through a much longer path of the sample. That means the light has to encounter the solution for a long period of time, and therefore we might see a low percentage of transmittance and a high absorbance. This implies that absorbance is also directly proportional to the path length of the beam through the sample.

Combining the two observations described above, we can deduce the relationship between absorbance and concentration of the sample, and absorbance and path length. This constructs the Beer-Lambert’s Law, which can be written as:

∝    ℎ     ℎ                                                             ×

or A ∝ c × l, or                      =                                                                                               (4)

Where,absorbance A is dimensionless number.

is the proportionality constant, called as the molar extinction coefficient or molar absorptivity. It is a constant for a given substance, provided the temperature and wavelength are constant. It has units of; L.mol-1.cm-1.

l and c have the units cm and mol.liter-1 respectively. From Eq(3);

Eq(6) is called as Beer-Lambert equation

Now the question arises why we prefer to express the Beer-Lambert equation in terms of absorbance rather than percentage of transmittance.

To start with, let’s consider Eq(4);

A =

From Eq(2);

I

% T = I0 × 100

From Eq(5): Eq(2) implies

I

% T = I0 × 100 = 100 ×  −

Let’s say a solution of CuSO4, which appear blue because it has an absorption maximum at 600 nm. Look the way in which the intensity of the light (radiant power) changes as it passes through the solution in a 1 cm cuvette. Let’s look at the reduction of every 0.2 m as shown in the below diagram. The law tells that fraction of light absorbed by each layer of solution is same. Let’s assume that this fraction is 0.5 cm for each 0.2 cm layer, and calculate the following data (table 2):

TABLE 2

Path length (cm)	% T	A
0	100	0
0.2	50	0.3
0.4	25	0.6
0.6	12.5	0.9
0.8	6.25	1.2
1.0	3.125	1.5

A = ε c l tells us that absorbance depends on the total quantity of the absorbing compound in the light path through the cuvette (figure-5). Further when we plot absorbance against concentration, we get a straight line passing through the origin (0,0) (figure-6).The linear relationship between concentration and absorbance is both simple and straightforward. This is why we prefer absorbance over % T to express the Beer-Lambert law.

Instrumental Design:

Figure-7 gives a schematic of a conventional single-beam spectrophotometer. Polychromatic light from the source is focused on the entrance slit of a monochromator, which selectively transmits a narrow band of light. This light then passes through the sample area to the detector. The absorbance of a sample is determined by measuring the intensity of light reaching the detector without the sample (the blank) and comparing it with the intensity of light reaching the detector after passing through the sample. As discussed above, most spectrophotometers contain two source lamps, a deuterium lamp and a tungsten lamp, and use either photomultiplier tubes or, more recently, photodiodes as detectors.

2. Since all compounds exhibit unique absorbance, spectrophotometry is also be used to determine the unknown compounds.
3. This technique is also used to measure the enzyme activity in cases45where the substrate and the product exhibit different absorption maximum (λmax). Either the disappearance of substrate or the appearance of product over time is measured.Variations in Spectrophotometry Technique:TABLE 3: OTHER FORMS OF SPECTROPHOTOMETRY TECHNIQUES
   

   Spectrophotometry	Comment
   X-ray absorption	Change in Electronic States
   Ultraviolet-Visible (UV/Vis)  absorption
   Infrared (IR)	Change  in  molecular  rotational and vibrational states
   Raman
   Optical Rotary Dispersion (ORD)	Polarize light
   Circular Dichroism (CD)
   Microwave Spectroscopy (Electron Spin Resonance, ESR)	Electron Spin Magnetic moments
   Nuclear Magnetic Resonance, NMR	Nuclear Spin Magnetic moments

   Looking at electromagnetic spectrum (figure-2), there are many types of spectrophotometry techniques known beside visible range (table 3). For instance, vibrations between the atoms of molecules can be analyzed using Infrared and Raman spectrophotometry. Many molecules exhibit characteristic signals under this technique and thus can be detected.

   Light can be polarized so that all of the waves are in the same orientation. The study of the absorption of polarized light generally yields more information about the structure of molecules if the chromophores have optically active centers. Circular Dichroism (CD) measures the ability of chromophores to differentially absorb left and right circularly polarized light. Optical rotary dispersion(ORD) measures the ability of an optically active chromophore to rotate plane-polarized light. Both CD and ORD are useful in structural studies or proteins and nucleic acids.

   The effects of molecules on the magnetic component of radiation can also be analyzed. Nuclear magnetic resonance (NMR) helps to elucidate the structural information of organic substances, and interactions between molecules and molecular motion. This method is based upon the principle that a spinning charge (i.e. nucleus) generates a magnetic field. Further an electron also possesses a spin magnetic moment which can be analyzed by electron spin resonance (ESR). A common use for ESR in biological sciences is to monitor the fluidity of membranes.

Important Spectrophotometry methods for measuring common air pollutants:

The commonly found air pollutants in the atmosphere are particulate matter, ground-level Ozone, CO, SO2, NOx, etc. They can harm your health, environment, and destroy property.

1. Method for measuring ground-level Ozone (O3):

Air containing O3 flows into the instrument through a long tube. The air is split into two parts. One stream passes through the O3-scrubber and acts as reference. Other stream acts as sample. Low pressure Mercury lamp acts as light source (254 nm). Light is passed through both the reference and sample cells. A silicon photodiode at the end of each cell detects the intensity reaching. By comparing the absorbance in two cells, O3 is measured.

2. Method for measuring SO2:

SO2 present in air is sucked by a pump and bubbled through an impinger into potassium tetrachloromercurate (K2HgCl4) solution to form disulfinatomercurate complex:

Subsequently, this complex is reacted with p-rosaniline dye and formaldehyde to form p-rosaniline methyl sulfonic acid (intensely colored red-violet). Concentration of red-violet species is measured by noting absorbance at 548 nm.

References:

• Spectrometric Idnetification of Organic Compounds – 5th Edn.- Silverstein, Bsslerm, Morrill.
• D.A. Skoog, D.M. West and F.J. Holler, Analytical Chemistry: An Introduction, 5th edition, Saunders college publishing, Philadelphia, 1990.
• Basic Concept of Analytical Chemistry By S. M. Khopkar, New Age International Publications, 2nd Edition 2004.
• A.K. Srivastava and P.C. Jain, Chemical Analysis: An Instrumental Approach for B.Sc. Hons. and M.Sc. Classes, S. Chand and company Ltd., Ram Nagar, New Delhi.
• Elementary Organic Spectroscopy: Principles and Chemical Applications, S.Chand and company Ltd., Ram Nagar, New Delhi, 1990.
• Websites: http://web.uni-plovdiv.bg/plamenpenchev/mag/ http://employees.oneonta.edu/kotzjc/LAB/ http://old.lf3.cuni.cz/chemie/english/materials_B/photometry.pdf http://www98.griffith.edu.au/dspace/bitstream/handle/10072/34561/62679_1.p http://www.ehp.qld.gov.au/air/