NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Module 2 PDF

NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Module 2 Spectroscopic techniques Lecture 3 Basics of Spectroscopy Spectroscopy deals with the study of interaction of electromagnetic radiation with matter. Electromagnetic radiation is a simple harmonic wave of electric and magnetic fields fluctuating orthogonal to each other (Figure 3.1A). Figure 3.1: An electromagnetic wave showing orthogonal electric and magnetic components (A); a sine wave (B); and uniform circular motion representation of the sine function (C). A simple harmonic function can be represented by a sine wave (Figure 3.1B): 𝑦 = 𝐴 sin 𝜃 ································· (3.1) Sine wave is a periodic function and can be described in terms of the circular motion (Figure 3.1C). The value of y at any point is simply the projection of vector A on the y-axis, which is nothing but A sinθ. Equation (1) can therefore be written in terms of angular velocity, ω. 𝑦 = 𝐴 sin(𝜔𝑡) ························· (3.2) 𝑦 = 𝐴 sin(2𝜋ν𝑡) ························· (3.3) 𝑧 𝑦 = 𝐴 sin(2𝜋ν 𝑐 ) ························· (3.4) where, z = displacement in time t and c is the velocity of the electromagnetic wave If the wave completes ν cycles/s and the wave is travelling with a velocity c metres/ 𝑐 sec, then the wavelength of the wave must be ν metres. 2𝜋𝑧 𝑦 = 𝐴 sin( 𝜆 ) ··························(3.5) Joint initiative of IITs and IISc – Funded by MHRD Page 1 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Energy of electromagnetic radiation: Energy of an electromagnetic radiation is given by 𝑐 𝐸 = ℎν = ℎ 𝜆 ·································· (3.6) where h is Planck’s constant and has a value of 6.626 × 10-34 m2·kg·s-1. Based on the energy, electromagnetic radiation has been divided into different regions. The region of electromagnetic spectrum human beings can see, for example, is called visible region or visible spectrum. The visible region constitutes a very small portion of the electromagnetic spectrum and corresponds to the wavelengths of ~400 – 780 nm (Figure 3.2). The energy of the visible spectrum therefore ranges from ~2.5 × 10-19 to ~5 × 10-19 Joules. It is not convenient to write such small values of energy; the energies are therefore written in terms of electronvolts (eV). One electronvolt equals 1.602 × 10-19 Joules. Therefore, the energy range of the visible spectrum is ~1.6 – 3.1 eV. Spectroscopists, however, prefer to use wavelength (λ) or frequency (ν) or wavenumber (ν ) instead of energy. Figure 3.2 Electromagnetic spectrum Quantization of energy: As put forward by Max Planck while studying the problem of Blackbody radiation in early 1900s, atoms and molecules can absorb or emit the energy in discrete packets, called quanta (singular: quantum). The quantum for electromagnetic energy is called a photon which has the energy given by equation 3.6. A molecule can possess energies Joint initiative of IITs and IISc – Funded by MHRD Page 2 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics in different forms such as vibrational energy, rotational energy, electronic energy, etc. Introduction to the structure of an atom in a General Chemistry course mentions about the electrons residing in different orbits/orbitals surrounding the nucleus, typically the first exposure to the discrete electronic energy levels of atoms. In much the same way, rotational and vibrational energy levels of molecules are also discrete. A molecule can jump from one energy level to another by absorbing or emitting a photon of energy that separate the two energy levels (Figure 3.3). Figure 3.3 Transitions of a molecule between energy levels, E1 and E2 by absorbing/emitting the electromagnetic radiation Electromagnetic spectrum and the atomic/molecular processes: Molecules undergo processes like rotation, vibration, electronic transitions, and nuclear transitions. The energies underlying these processes correspond to different regions in the electromagnetic spectrum (Figure 3.4): i. Radiofrequency waves: Radiofrequency region has very low energies that correspond to the energy differences in the nuclear and electron spin states. These frequencies, therefore, find applications in nuclear magnetic resonance and electron paramagnetic resonance spectroscopy. ii. Microwaves: Microwaves have energies between those of radiofrequency waves and infrared waves and find applications in rotational spectroscopy and electron paramagnetic resonance spectroscopy. iii. Infrared radiation: The energies associated with molecular vibrations fall in the infrared region of electromagnetic spectrum. Infrared spectroscopy is therefore also known as vibrational spectroscopy and is a very useful technique for functional group identification in organic compounds. Joint initiative of IITs and IISc – Funded by MHRD Page 3 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics iv. UV/Visible region: UV and visible regions are involved in the electronic transitions in the molecules. The spectroscopic methods using UV or visible light therefore come under ‘Electronic spectroscopy’. v. X-ray radiation: X-rays are high energy electromagnetic radiation and causes transitions in the internal electrons of the molecules. Figure 3.4 The range of atomic/molecular processes the electromagnetic radiation is involved in. Mechanisms of interaction of electromagnetic radiation with matter: In order to interact with the electromagnetic radiation, the molecules must have some electric or magnetic effect that could be influenced by the electric or magnetic components of the radiation. i. In NMR spectroscopy, for example, the nuclear spins have magnetic dipoles aligned with or against a huge magnetic field. Interaction with radiofreqency of appropriate energy results in the change in these dipoles. ii. Rotations of a molecule having a net electric dipole moment, such as water will cause changes in the directions of the dipole and therefore in the electrical properties (Figure 3.5A and B). Figure 3.5B shows the changes in the y- component of the dipole moment due to rotation of water molecule. Joint initiative of IITs and IISc – Funded by MHRD Page 4 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics iii. Vibrations of molecules can result in changes in electric dipoles that could interact with the electrical component of the electromagnetic radiation (Figure 3.5C). iv. Electronic transitions take place from one orbital to another. Owing to the differences in the geometry, size, and the spatial organization of the different orbitals, an electronic transition causes change in the dipole moment of the molecule (Figure 3.5D). Figure 3.5 Panel A shows the rotation of a water molecule around its centre of mass (A). The change in the dipole moment as a result of rotation in plotted in panel B. Panel C shows the change in dipole moment of water due to asymmetric stretching vibrations of O—H bond. Panel D shows an electronic transition from π to π* orbital and the geometry of the two orbitals. The above examples suggest that a change in THINK TANK?? either electric or magnetic dipole moment in a molecule is required for the absorption or Will there be a change in the dipole moment if there is emission of the electromagnetic radiation. symmetric stretching of O―H bond in water molecule? Absorption peaks and line widths: Absorption of radiation is the first step in any spectroscopic experiment. Absorption spectra are routinely recorded for the electronic, rotational, and vibrational spectroscopy. It is therefore important to see how an absorption spectrum looks like. Joint initiative of IITs and IISc – Funded by MHRD Page 5 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics As we have already seen, a transition between states takes place if the energy provided by the electromagnetic radiation equals the energy gap between the two ℎ𝑐 states i.e. ∆𝐸 = ℎ𝜐 = 𝜆. This implies that the molecule precisely absorbs the radiation of wavelength, λ and ideally a sharp absorption line should appear at this wavelength (Figure 3.6A). In practice, however, the absorption lines are not sharp but appear as fairly broad peaks (Figure 3.6B) for the following reasons: i. Instrumental factors: The slits that allow the incident light to impinge on the sample and the emerging light to the detector have finite widths. Consider that the transition occurs at wavelength, λt. When the wavelength is changed to λt + ∆λ or λt – ∆λ, the finite slit width allows the radiation of wavelength, λt to pass through the slits and a finite absorbance is observed at these wavelengths. The absorption peaks are therefore symmetrical to the line at λ = λt. ii. Sample factors: Molecules in a liquid or gaseous sample are in motion and keep colliding with each other. Collisions influence the vibrational and rotational motions of the molecules thereby causing broadening. Two atoms/molecules coming in close proximity will perturb the electronic energies, at least those of the outermost electrons resulting in broadening of electronic spectra. Motion of molecules undergoing transition also causes shift in absorption frequencies, known as Doppler broadening. iii. Intrinsic broadening: Intrinsic or natural broadening arises from the Heisenberg’s uncertainty principle which states that the shorter the lifetime of a state, the more uncertain is its energy. Molecular transitions have finite lifetimes, therefore their energy is not exact. If ∆t is the lifetime of a molecule in an excited state, the uncertainty in the energy of the states is given by: ℎ ∆𝐸 × ∆𝑡 ≥ 4𝜋 ·································· (3.7) ℏ ∆𝐸 × ∆𝑡 ≥ 2 ·································· (3.8) ℎ where, ℏ = 2𝜋 Two more features worth noticing in the Figure 3.6B are the fluctuations in the baseline and the baseline itself, which is not horizontal. The small fluctuations in the baseline are referred to as noise. Noise is the manifestation of the random weak Joint initiative of IITs and IISc – Funded by MHRD Page 6 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics signals generated by the instrument electronics. To identify the sample peaks clear of the noise, the intensity of the sample peaks has to be at least 3-4 times higher than the noise. A better signal-to-noise ratio is obtained by recorded more than one spectra and averaging; the noise being random gets cancelled out. Instrumental factors are responsible for the non-horizontal baseline observed in Figure 3.6B: The light sources used in the instruments emit radiations of different intensities at different wavelengths and usually the detector sensitivity is also wavelength-dependent. A reasonable horizontal baseline for the samples can easily be obtained by subtracting the spectrum obtained from the solvent the sample is dissolved in. Figure 3.6 An idealized spectrum for a single wavelength transition (A) and an experimentally obtained spectrum (B) Other features of the spectroscopy and the spectra obtained will be discussed as and when they arise in the following lectures. Joint initiative of IITs and IISc – Funded by MHRD Page 7 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Lecture4 UV/Visible Absorption Spectroscopy-I We see a lot of colorful things around us. What exactly is the color and what make the things exhibit these colors? We know that the color we see is the visible region of the electromagnetic spectrum. We also know that matter can absorb the electromagnetic radiation of different energy (or wavelengths). The region of electromagnetic energy that is not absorbed is simply reflected back or getstransmitted through the matter. The colored compounds are colored because they absorb the visible light. The color that is perceived is called the complement color to the absorbed wavelength and is represented by a color wheel (Figure 4.1). Figure 4.1 A simplified color wheel showing complementary colors. Green is interesting as it can arise from the absorption of radiation to either end of the visible spectrum. Joint initiative of IITs and IISc – Funded by MHRD Page 8 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Absorption of ultraviolet (UV) and visible radiation is one of the most routinely used analytical tools in life sciences research. The simplest application of UV/Visible radiation is to quantify the amount of a substance present in a solution.UV region of electromagnetic radiation encompasses the wavelengths ranging from ~10 nm – ~400 nm while visible region encompasses the wavelengths from ~400 nm – ~780 nm. For the sake of convenience in discussing the observations, UV region is loosely divided into near UV (wavelength region nearer to the visible region, λ ~ 250 nm – 400 nm), far UV region (wavelength region farther to the visible region, λ ~ 190 nm – 250 nm) and vacuum UV region (λ< 190 nm). The wavelength ranges defined for these regions are not strict and people use slightly different ranges to define these regions. We shall, however, stick to the wavelengths defined here. As has been discussed in the previous lecture, the absorption of UV and visible light is through the transition of an electron in the molecule from lower to a higher energy molecular orbital. The various electronic transitions observed in organic compound are shown in Figure 4.2. Figure 4.2 Schematic diagram showing energy levels of different orbitals and possible absorption transitions As shown in figure 4.2, σ → σ* transition is a high energy process and therefore lies in the vacuum UV region. Alkanes, wherein only σ → σ* transition is possible show absorption bands ~150 nm wavelength. Alkenes haveπ and π* orbitals and can show several transition; the lowest-energy transition, π → π* gives an absorption band ~170-190 nm for non-conjugated alkenes (effects of conjugation on electronic transitions are discussed later). The presence of nonbonding electrons in a molecule Joint initiative of IITs and IISc – Funded by MHRD Page 9 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics further expands the number of possible transitions. The entire molecule, however, is not generally involved in the absorption of the radiation in a given wavelength range. In an aliphatic ketone, for example, the absorption band around 185 nm arises due to the π → π* transition in the carbonyl group. Atoms that comprise the molecular orbitals involved in the electronic transitions constitute the molecular moiety that is directly involved in the transition. Such a group of atoms is called a chromophore. A structural modification in a chromophore is generally accompanied by changes in the absorption properties. Instrumentation: Figure 4.3A shows a schematic diagram of a single-beam spectrophotometer. The light enters the instrument through an entrance slit, is collimated and focused on to the dispersing element, typically a diffraction grating. The light of desired wavelength is selected simply by rotating the monochromator and impinged on the sample. The intensity of the radiation transmitted through the sample is measured and converted to absorbance or transmittance (discussed later). Double beam spectrophotometers overcome certain limitations of the single beam spectrophotometers and are therefore preferred over them. A double beam spectrophotometer has two light beams, one of which passes through the sample while other passes through a reference cell (Figure 4.3B). This allows more reproducible measurements as any fluctuation in the light source or instrument electronics appears in both reference and the sample and therefore can easily be removed from the sample spectrum by subtracting the reference spectrum. Modern instruments can perform this subtraction automatically. The most commonly used detectors in the UV/Visible spectrophotometers are the photomultiplier tubes (PMT). Modern instruments also use photodiodes as the detection systems. These diodes are inexpensive and can be arranged in an array so that each diode absorbs a narrow band of the spectrum. Simultaneous recording at multiple wavelengths allows recording of the entire spectrum at once. The monochromator in these spectrophotometers is placed after the sample so that the sample is exposed to the entire spectrum of the incident radiation and the transmitted radiation is dispersed into its components. Joint initiative of IITs and IISc – Funded by MHRD Page 10 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Figure 4.3 Schematic diagram showing a single beam (A) and a double beam (B) spectrophotometer Beer-Lambert Law It is quite intuitive that a higher concentration of the absorbing species in a sample would lead to higher absorption of the light. Furthermore, the higher thickness of the sample should result in higher absorption. Consider a cell (also called cuvette) of length, l, containing a solution of an absorbing molecule. The absorbing species in the sample can be represented by discs of cross-sectional area, σ. Now, let us consider a slab of infinitesimal thickness, dx and area, A (Figure 4.4). If an incident radiation of the resonance frequency (the frequency that causes maximum transition) having intensity Io enters the sample cell, its intensity decreases as it penetrates the sample. Let us suppose that the intensity of the radiation before entering the infinitesimal slab is Ix. Joint initiative of IITs and IISc – Funded by MHRD Page 11 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Figure 4.4A diagrammatic representation of light absorption by sample molecules in an infinitesimal thin slab within the sample 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠 If the concentration of the absorbing molecules = 𝑛 𝑐𝑚3 , the fraction of the area 𝜎 × 𝑛 × 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑙𝑎𝑏 𝜎 × 𝑛 × 𝐴 × 𝑑𝑥 occupied by the molecules in the slab = 𝐴 = 𝐴 = 𝜎 × 𝑛 × 𝑑𝑥 𝑑𝐼 Therefore, the fraction of the photons ( 𝐼 ) absorbed is proportional to 𝜎 × 𝑛 × 𝑑𝑥. 𝑥 Assuming the probability of absorption if a photon strikes the molecule to be unity, 𝑑𝐼 𝐼𝑥 = − 𝜎 × 𝑛 × 𝑑𝑥 ························ (4.1) The negative sign represents a decrease in intensity Integrating equation 4.1 from x = 0 to x = l ln𝐼|𝐼𝐼𝑜 = − 𝜎 × 𝑛 × 𝑥|𝑙0 ························· (4.2) ln 𝐼 − ln 𝐼𝑜 = −𝜎 × 𝑛 × 𝑙 ························· (4.3) 𝐼 − ln 𝐼 = 𝜎 × 𝑛 × 𝑙 ························· (4.4) 𝑜 Now, the molar concentration of the molecules, c can be given by: Joint initiative of IITs and IISc – Funded by MHRD Page 12 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics moles molecules 1 mole cm3 𝑐 litre = 𝑛 cm3 × 6.022 ×1023 molecules × 1000 litre Substituting for n in equation 4.4 and converting natural logarithm, ln into log10 gives: 𝐼 − 2.303 × log 𝐼 = 𝜎 × {𝑐 × 6.022 × 1020 } × 𝑙 ·························· (4.5) 𝑜 𝐼 6.022 ×1020 −log 𝐼 = 𝜎 × 𝑐 × × 𝑙 ·························· (4.6) 𝑜 2.303 𝐼 6.022 ×1020 −log 𝐼 is defined as the absorbance and 𝜎 × is defined as the molar 𝑜 2.303 absorption coefficient, denoted by the Greek alphabet, ε. Therefore, equation 4.6 can be written as: Absorbance, A = εcl ························· (4.7) This equality showing linear relationship between absorbance and the concentration of the absorbing molecule (or chromophore, to be precise) is known as the Beer- Lambert law or Beer’s law. Transmittance is another way of describing the absorption of light. Transmittance (T) is simply the ratio of the intensity of the radiation transmitted through the sample to that of the incident radiation. Transmittance is generally represented as percentage transmittance (%T): 𝐼 %𝑇 = 𝐼𝑜 × 100 As is clear from the definition of absorbance and transmittance, both are dimensionless quantities. Absorbance and transmittance are therefore represented in arbitrary units (AU). The quantity of interest in an absorption spectrum is the molar absorption coefficient, ε which varies with wavelength (Figure 4.5). The wavelength at which highest molar absorption coefficient (εmax) is observed is represented as λmax. Area of cross-section of the absorbing species puts an upper limit to the molar absorption coefficient. Joint initiative of IITs and IISc – Funded by MHRD Page 13 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Figure 4.5 An absorption spectrum of N-acetyl-tryptophanamide Deviations from Beer-Lambert law: Beer-Lambert law can be used to determine the ε values of a compound by recording its absorption spectra at known concentrations. Alternatively, knowledge of ε enables the user to calculate the concentration of a compound in a given solution. It is, however, not uncommon to observe deviations from the Beer-Lambert law. Three major reasons that are responsible for the breakdown of linear relationship between absorbance and the concentration of the absorbing molecule are: i. High sample concentration: The Beer-Lambert law generally holds good only for dilute solutions. At higher concentrations, the molecules come in close proximity thereby influencing their electronic properties. Although introduced as a constant at a particular wavelength for a compound, ε depends on the concentration of the compound and therefore results in deviation from linearity. At lower concentrations, however, ε can practically be assumed to be a constant. ii. Chemical reactions: If a molecule undergoes a chemical reaction and the spectroscopic properties of the reacted and unreacted molecules differ, a deviation from Beer-Lambert law is observed. Change in the color of the pH indicator dyes is a classical example of this phenomenon. Joint initiative of IITs and IISc – Funded by MHRD Page 14 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics iii. Instrumental factors: As ε is a function of wavelength, Beer-Lambert law holds good only for monochromatic light. Use of polychromatic radiation will result in deviation for linearity between absorbance and concentration. For practical purposes, the samples giving absorbance values between 0.05 – 0.5 are considered highly reliable. At lower concentrations, the signal to noise ratio is small while at higher concentrations, absorbance values underestimate the concentration of the compound as increase in absorbance no longer matches the increase in concentration. If the absorbance values are higher, a sample can be diluted or a sample cell with smaller path length can be used; usually dilution of sample is preferred. In the following lecture, we shall discuss the various factors that influence the absorption spectra of molecules and look at the applications of UV/Visible absorption spectroscopy for studying the biomolecules. Joint initiative of IITs and IISc – Funded by MHRD Page 15 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Lecture 5 UV/Visible Absorption Spectroscopy-II In the previous lecture, we studied that UV/Visible radiation is absorbed by the molecules through transition of electrons in the chromophore from low energy molecular orbitals to higher energy molecular orbitals. We are interested in the transitions that lie in the far UV, near UV, and visible regions of the electromagnetic spectrum. The molecules that absorb in these regions invariably have unsaturated bonds. Plants are green due to unsaturated organic compounds, called chlorophylls. A highly unsaturated alkene, lycopene, imparts red color to the tomatoes (Figure 5.1). Figure 5.1 Structure of lycopene, the pigment that imparts red color to the tomatoes As can be seen from its structure, lycopene is a highly conjugated alkene. As compared to the simple non-conjugated alkenes that typically absorb in vacuum UV region, absorption spectrum of lycopene is hugely shifted towards higher wavelengths (or lower energy). There can be factors that could shift the absorption spectra to smaller wavelengths or can increase/decrease the absorption intensity. Before understanding how conjugation causes shift in the absorption spectra, let us look at some important terms that are used to refer to the shifts in absorption spectra (Figure 5.2): Bathochromic shift: Shift of the absorption spectrum towards longer wavelength Hypsochromic shift: Shift of the absorption spectrum towards smaller wavelength Hyperchromic shift: An increase in the absorption intensity Hypochromic shift: A decrease in the absorption intensity Joint initiative of IITs and IISc – Funded by MHRD Page 16 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Figure 5.2 Terminology for shifts in absorption spectra Conjugation: Conjugation brings Energy levels of conjugated alkenes’ about a bathochromic shift in the molecular orbitals: The energy levels of the absorption bands. The higher the orbitals increase as the number of vertical extent of conjugation, the more is nodes increase. The lowest energy π orbital has no nodes while the highest energy π* the bathochromic shift. Such shift orbital has n–1 nodes where n is the number in absorption spectra can easily be of p–orbitals combined. explained using molecular orbital theory. Figure 5.3 shows the molecular orbitals drawn for ethylene; 1,3-butadiene; and 1,3,5-hexatriene on a qualitatively same energy scale for comparing their energies. As is clear from the figure, the energy differences between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) decreases as the conjugation increases. This provides an explanation as to why an electronic transition is possible at lower energy (higher wavelength) as the conjugation increases. Joint initiative of IITs and IISc – Funded by MHRD Page 17 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Figure 5.3 Molecular orbitals of ethylene; 1,3-butadiene; and 1,3,5-hexatriene. Notice the decrease in the energy gap of HOMO and LUMO as the conjugation increases. Auxochrome: Auxochromes are the chemical groups that result in a bathochromic shift when attached to a chromophore. The strongest auxochromes like –OH, –NH2, – OR, etc. possess nonbonding electrons. They exhibit bathochromism by extending conjugation through resonance. The auxochrome modified chromophore is a new chromophore in real sense. The term auxochrome is therefore rarely used these days, and the entire group (basic chromophore + auxochrome) can be considered as a chromophore different from the basic chromophore. Alkyl groups also result in the bathochromic shifts in the absorption spectra of alkenes. Alkyl groups do not have non-bonded electrons, and the effect is brought about by another type of interaction called hyperconjugation. Joint initiative of IITs and IISc – Funded by MHRD Page 18 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Solvents: The solvents used in any spectroscopic method should ideally be transparent (non-absorbing) to the electromagnetic radiation being used. Table 5.1 shows the wavelength cutoffs (the lowest working wavelength) of some of the solvents used in UV/visible spectroscopy. Table 5.1 Solvents commonly used in UV/visible spectroscopy Solvent Wavelength cutoff Water 190 nm Acetonitrile 190 nm Cyclohexane 195 nm Methanol 205 nm 95% ethanol 205 nm Water, the solvent of biological systems, thankfully is transparent to the UV/visible region of interest i.e. the regions above λ > 190 nm. Solvents also play important role on the absorption spectra of molecules. Spectrum of a compound recorded in one solvent can look significantly different in intensity, wavelength of absorption, or both from that recorded in another. This is not something unexpected because energies of different electronic states will depend on their interaction with solvents. Polarity of solvents is an important factor in causing shifts in the absorption spectra. Conjugated dienes and aromatic hydrocarbons are little affected by the changes in solvent polarity. α,β-unsaturated carbonyl compounds are fairly sensitive to the solvent polarity. The two electronic transitions π → π* and n → π* respond differently to the changes in polarity. Polar solvents stabilize all the three molecular orbitals (n, π, and π*), albeit to different extents (Figure 5.4). The non-bonding orbitals are stabilized most, followed by π*. This results in a bathochromic shift in the π → π* absorption band while a hypsochromic shift in n → π* absorption band. Shift to different extents of the two bands will result in the different shape of the overall absorption spectrum. Joint initiative of IITs and IISc – Funded by MHRD Page 19 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Figure 5.4 Differential stabilization of molecular orbitals in polar solvents Biological chromophores Amino acids and proteins: Among the 20 amino acids that constitute the proteins, tryptophan, tyrosine, and phenylalanine absorb in the near UV region. All the three amino acids show structured absorption spectra. The absorption by phenylalanine is weak with an εmax of ~200 M-1cm-1 at ~250 nm. Molar absorption coefficients of ~1400 M-1cm-1 at 274 nm and ~5700 M-1cm-1 at 280 nm are observed for tyrosine and tryptophan, respectively. Disulfide linkages, formed through oxidation of cysteine resides, also contribute to the absorption of proteins in near UV region with a weak εmax of ~300 M-1cm-1 around 250-270 nm. The absorption spectra of proteins are therefore largely dominated by Tyr and Trp in the near UV region. In the far UV region, peptide bond emerges as the most important chromophore in the proteins. The peptide bond displays a weak n → π* transition (εmax ≈ 100 M-1cm-1) between 210- 230 nm, the exact band position determined by the H-bonding interactions the peptide backbone is involved in. A strong π → π* transition (εmax ≈ 7000 M-1cm-1) is observed around 190 nm. Side chains of Asp, Glu, Asn, Gln, Arg, His also contribute Joint initiative of IITs and IISc – Funded by MHRD Page 20 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics to the absorbance in the far UV region. Figure 5.5 shows an absorption spectrum of a peptide Figure 5.5 Absorption spectrum of a peptide. The absorption band ~280 nm is due to aromatic residues. Absorption band in the far UV region arises due to peptide bond electronic transitions. Nucleic acids: Nucleic acids absorb very strongly in the far and near UV region of the electromagnetic spectrum. The absorption is largely due to the nitrogenous bases. The transitions in the nucleic acid bases are quite complex and many π → π* and n → π* transitions are expected to contribute to their absorption spectra. A 260 nm wavelength radiation is routinely used to estimate the concentration of nucleic acids. Though the molar absorption coefficients vary for the nucleotides at 260 nm, the average εmax can be taken as ~104 M-1cm-1. It is important to mention that nucleotides show hyperchromicity when exposed to aqueous environment. The absorbance of the free nucleotides is higher than that of single stranded nucleic acid which is higher than that of the double stranded nucleic acid (assuming equal amount of the nucleotides present in all three). Other chromophores: Nucleotides like NADH, NADPH, FMN, and FAD; porphyrins such as heme, chlorophylls and other plant pigments; retinal (light sensing molecule); vitamins; and a variety of unsaturated compounds constitute chromophores in the UV and visible region. Joint initiative of IITs and IISc – Funded by MHRD Page 21 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Having studied the principles of the UV/visible absorption spectroscopy and various factors that influence the electronic transitions, we can now have a look at its applications, especially the applications for analyzing the biological samples. Applications: i. Determination of molar absorption coefficient: From Beer-Lambert law, A = εcl. It is therefore straightforward to calculate the molar absorption coefficient of a compound if the concentration of compound is accurately determined. ii. Quantification of compounds: This is perhaps the most common application of a UV/visible spectrophotometer in a bioanalytical laboratory. If the molar absorption coefficient at a wavelength is known for the compound, the concentration can easily be estimated using Beer-Lambert law. The compounds can still be quantified if their molar absorption coefficients are not known. Estimation of total protein concentration in a given solution is an important example of this. As the given solution is a mixture of many different proteins, the ε is not available. There are, however, dyes that specifically bind to the proteins producing colored complex. The color produced will be proportional to the amount of the protein present in the solution. Performing the experiment under identical conditions using known concentrations of a protein gives a standard graph between absorbance of the dye and the amount of protein. This standard graph is then used to estimate the concentration of the given protein sample. iii. Quality control: A given organic compound such as a drug can be studied for its purity. Comparison of spectrum with the standard drug will detect the impurities, if any. UV/Visible absorption is often used to detect the nucleic acid contamination in the protein preparations. Aromatic amino acids as well as the nucleotides show 𝐴260 ratio is not useful in detecting protein absorption band in the near 𝐴280 contaminations in DNA preparations. This is UV region and there is a because of the large difference in molar considerable overlap in the absorption coefficients of these molecules. 𝐴260 absorption spectra of To cause an appreciable change in the 𝐴280 aromatic amino acids and ratio, there should a large amount of protein the nucleotides. A nucleic present. acid contamination in a protein, however, can be determined by measuring Joint initiative of IITs and IISc – Funded by MHRD Page 22 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics absorbances at 260 and 280 nm. A typical nucleic acid containing all four bases shows an absorption band centered ~260 nm while a protein having aromatic amino acids shows absorption band centered ~280 nm. It is possible to determine the purity of protein preparations by recording absorbances at both 260 and 280 nm. A ratio of the absorbance at 260 nm to that at 280 nm 𝐴260 i.e. is a measure of the purity. 𝐴280 iv. Chemical kinetics: UV/visible spectroscopy can be used to monitor the rate of chemical reactions if one of the reactants or products absorbs in a region where no other reactant or product absorbs significantly. v. Detectors in liquid chromatography instruments: UV/visible detectors are perhaps the most common detectors present in liquid chromatography systems. Modern instruments use photodiode array detectors that can detect the molecules absorbing in different spectral regions (Figure 5.6). Figure 5.6 Diagram of a photodiode array detector Joint initiative of IITs and IISc – Funded by MHRD Page 23 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics vi. Determination of melting temperature of DNA: A double stranded DNA molecule can be denatured into the single strands by heating it. Melting temperature, Tm is the temperature at which 50% of the DNA gets denatured into single strands. Denaturation of DNA is accompanied by hyperchromic shift in the absorption spectra in the near UV region. A melting curve (plot between temperature and absorbance at 260 nm) is plotted and Tm is determined (Figure 5.7). Figure 5.7 Thermal denaturation of a DNA sample; a plot of absorbance at 260 nm against the temperature allows determination of the melting temperature (Tm). vii. Microbial growth kinetics: A UV/visible spectrophotometer is routinely used to monitor the growth of microorganisms. The underlying principle behind this, however, is not absorbance but scattering. As the number of microbial cells increase in a culture, they cause more scattering in light. The detector therefore receives less amount of radiation, recording this as absorbance. To distinguish this from actual absorbance, the observed value is referred to as the optical density. Joint initiative of IITs and IISc – Funded by MHRD Page 24 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics QUIZ Q1: The molar absorption coefficient of tyrosine in water is 1280 M-1cm-1 at 280 nm. Calculate the concentration of a tyrosine solution in water if the absorbance of the solution is 0.34 in a 1 cm path length cell. Ans: Given: λ = 280 nm ε280 nm = 1280 M-1cm-1 l = 1 cm A = 0.34 From Beer-Lambert Law: A = εcl 𝐴 0.32 c= = = 0.00025 M = 250 μM 𝜀𝑙 1280M−1 cm−1 × 1 cm Q2: Calculate the concentration of a tryptophan solution that gives an absorbance of 0.25 at 280 nm in a 1 mm path length cell (Given ε280 nm = 5690 M-1cm-1). Ans: The concentration of the given sample can be estimated using Beer-Lambert law: 𝐴 = 𝜀𝑐𝑙 𝐴 𝑐 = 𝜀𝑙 0.25 𝑐= 5690 𝑀−1 𝑐𝑚−1 ×1 𝑚𝑚 0.25 𝑐= 5690 𝑀−1 𝑐𝑚−1 ×0.1 𝑚𝑚 𝑐 = 4.39 × 10−5 𝑀 = 43.9 𝜇𝑀 Q3: Concentration of a pure compound in solution can easily be determined by taking absorbance at any wavelength in a given spectral region if ε at these wavelengths is known. Why then absorbance is generally recorded at λmax? Ans: This is done for the following reasons: a) At λmax, the ε value is maximum, therefore reliable absorbance i.e. A between 0.05 – 0.5 can be obtained at lower concentrations of the compound. 𝑑𝐴 𝑑𝜀 b) At λmax, the slope of the absorption spectrum, 𝑑𝜆 or 𝑑𝜆, is zero. This ensures that for a given bandwidth of the incident radiation, the ε is relatively constant in this region as compared to the regions of non-zero slopes. If ε is not constant, the linearity of the Beer Lambert law is compromised. Joint initiative of IITs and IISc – Funded by MHRD Page 25 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Lecture 6 Fluorescence Spectroscopy-I This lecture is a very concise review of the phenomenon of fluorescence and the associated processes. Let us move a step forward from the absorption of the UV/visible radiation. What happens to the electrons that absorb UV/visible light and occupy the high energy molecular orbitals? In a UV/visible absorption experiment, the samples continue absorbing light. This means that the higher energy molecular orbitals never get saturated. This further implies that after excitation, the molecules somehow get rid of the excess energy and return back to the ground state. The electrons can return back to the ground state in different ways such as releasing the excess energy through collisions or through emitting a photon. In fluorescence, the molecules return back to the ground state by emitting a photon. The molecules that show fluorescence are usually referred to as the fluorophores. Various electronic and molecular processes that occur following excitation are usually represented on a Jablonski diagram as shown in Figure 6.1. Figure 6.1 Jablonski diagram showing various processes following absorption of light by the fluorophore Joint initiative of IITs and IISc – Funded by MHRD Page 26 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics S0, S1, and S2 represent the singlet electronic states while the numbers 0, 1, 2 represent the vibrational energy levels associated with the electronic states. T1 depicts the first triplet electronic state. Let us go through the processes shown in Figure 6.1: Absorbance: S0 state with 0th vibrational level is the state of lowest energy and therefore, the highest populated state. Absorption of a photon of resonant frequency usually results in the population of S1 or S2 electronic states; but usually a higher vibrational state. Transition of electrons from low energy molecular orbital to a high energy molecular orbital through absorption of light is a femtosecond (10-15 s) phenomenon. The electronic transition, therefore, is too quick to allow any significant displacement of the nuclei during transition. Internal conversion: Apart from few exceptions, the excited fluorophores rapidly relax to the lowest vibrational state of S1 through non-radiative processes. Non-radiative electronic transition from higher energy singlet states to S1 is termed as internal conversion while relaxation of a fluorophore from a higher vibrational level of S1 to the lowest vibration state is termed as vibrational relaxation. The terms ‘internal conversion’ and ‘vibrational relaxation’, however, are often interchangeably used. The timescale of internal conversion/vibrational relaxation is of the order of 10- 12 seconds. Fluorescence: Fluorescence lifetimes are of the order of 10-8 seconds, implying that the internal conversion is mostly complete before fluorescence is observed. Therefore, fluorescence emission is the outcome of fluorophore returning back to the S0 state through S1 → S0 transition emitting a photon. This also explains why emission spectra are usually independent of the excitation wavelength, also known as Kasha’s rule (However, there are exceptions wherein fluorescence is observed from S2 → S1 transition). The S1 → S0 transition, like S0 → S1 transition, typically results in the population of higher energy vibrational states. The molecules then return back to the lowest vibrational state through vibrational relaxation. Intersystem crossing: Intersystem crossing referes to an isoenergetic non- radiative transition between electronic states of different multiplicities. It is possible that a molecule in a vibrational state of S1 can move to the isoenergetic vibrational state of T1. The molecule then relaxes back to the lowest vibrational state of the triplet state. Joint initiative of IITs and IISc – Funded by MHRD Page 27 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Phosphorescence: The molecule in the triplet state, T1, can return back to the S0 state emitting a photon. This process is known as phosphorescence and has time scales of several orders of magnitudes higher than that of fluorescence (10-3 – 10 s). Characteristics of fluorescence: Figure 6.2 shows absorption and fluorescence emission spectrum of a hypothetical fluorophore. The important characteristics of the fluorescence emission can be briefly summarized as follows: Figure 6.2 Absorption and fluorescence emission spectrum of a hypothetical fluorophore Stokes shift: A fluorescence emission spectrum is always shifted towards longer wavelengths with respect to the absorption spectrum. This shift is known as Stokes shift and is expected as excited molecules lose energy through processes like internal conversion and vibrational relaxation. The emitted radiation is therefore expected to be of lower energy i.e. higher wavelength. Kasha’s rule: As fluorescence emission is observed from S1 → S0 transtions (except a few exceptions), fluorescence absorption spectrum is independent of the excitation wavelength. Joint initiative of IITs and IISc – Funded by MHRD Page 28 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Franck-Condon principle: The Franck-Condon principle states that the positions of the nuclei do not change during electronic transitions. The transitions are said to be vertical. This implies that if the probability of 0th → 2nd vibrational transition during S0 → S1 transition is highest, the 2nd → 0th transition will be most probable in the reciprocal transition (Figure 6.3). Figure 6.3 Potential energy diagrams showing the Franck-Condon principle This results in an emission spectrum that is a mirror image of the S0 → S1 transition in terms of the shape. There are several exceptions to the mirror image rule that arise largely due to the excited state reactions of the molecule. Joint initiative of IITs and IISc – Funded by MHRD Page 29 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Quantum yield: As has been mentioned earlier, an excited molecule can come back to the ground state through non-radiative pathways. Fluorescence quantum, 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝ℎ𝑜𝑡𝑜𝑛𝑠 𝑒𝑚𝑖𝑡𝑡𝑒𝑑 𝛤 𝑄 = = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝ℎ𝑜𝑡𝑜𝑛𝑠 𝑎𝑏𝑠𝑜𝑟𝑏𝑒𝑑 𝛤 + 𝑘𝑛𝑟 where, Γ is the rate of radiative process i.e. fluorescence knr is the rate of all the non-radiative processes bringing molecule to the S0 state Fluorescence lifetime: Lifetime of a fluorophore is defined as the average time it spends in the excited state before returning to the S0 state. It is therefore the reciprocal of the rate of processes de-exciting the molecule. 1 Fluorescence lifetime, 𝜏 = 𝛤 + 𝑘𝑛𝑟 Fluorescence quenching, resonance energy transfer and anisotropy Fluorescence spectroscopy comprises of experiments exploiting various different phenomena related to it. Discussion of all these experiments is beyond the scope of this course, but we shall have a quick look at a few important phenomena related to fluorescence. Fluorescence quenching: A decrease in fluorescence intensity is referred to as quenching. A molecule that quenches the fluorescence of a fluorophore is called a quencher. A quencher can be either a collisional quencher or a static quencher. A collisional quencher brings about decrease in fluorescence intensity by de-exciting the excited fluorophore through collisions. Addition of another non-radiative process to the system leads to lower quantum yield. A static quencher forms a non-fluorescent complex with the fluorophore. It effectively leads to a decrease in the concentration of the fluorophore thereby decreasing the fluorescence emission intensity. Joint initiative of IITs and IISc – Funded by MHRD Page 30 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Resonance energy transfer: Resonance energy transfer (RET), also known as fluorescence resonance energy transfer (FRET) is an excited state phenomeneon wherein energy is transferred from a donor molecule (D) to an acceptor molecule (A). The prerequisite for the energy transfer is that there should be an overlap between the emission spectrum of the D and the absorption spectrum of the A (Figure 6.4). Figure 6.4 Diagrammatic representation of spectral overlap between donor’s emission and acceptor’s absorption spectrum. The efficiency of energy transfer depends upon i. the distance between D and A ii. the relative orientation of the transition dipoles of D and A iii. the extent of the overlap between D’s emission spectrum and A’s absorption spectrum 𝑅06 Efficiency of energy transfer 𝐸 = 𝑅06 + 𝑟 6 where, r is the distance between D and A. R0 (also called the Förster distance) is the distance (r) between D and A at which the efficiency of energy transfer is 50%, and is characteristic of a D-A FRET pair. Resonance energy transfer can be used to determine the distances between D and A, and is therefore also termed as molecular ruler. Joint initiative of IITs and IISc – Funded by MHRD Page 31 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Fluorescence anisotropy: The radiation emitted by a sample following excitation with polarized light can be polarized. Polarization is measured in terms of anisotropy. Zero anisotropy implies Transition dipole moment: The transition dipole isotropic/non-polarized moment represents the transient dipole moment radiation while non-zero generated from the charge displacement during a anisotropy implies some transition. The transition dipole moments are defined vector quantities for the transitions of a degree of polarization. Figure particular molecule. 6.5 shows how fluorescence anisotropic measurements are made. Figure 6.5 A schematic diagram showing the measurement of fluorescence anisotropy The sample is excited with the linearly polarized light and emission is recorded at 90°. A polarizer is placed before the detector that allows intensity measurement of the light polarized parallel (𝐼∥ ) and perpendicular (𝐼⊥ ) to the direction of excitation radiation. The anisotropy (r) is given by 𝐼⊥ − 𝐼∥ 𝑟= 𝐼⊥ + 2𝐼∥ Joint initiative of IITs and IISc – Funded by MHRD Page 32 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Molecular tumbling before emission changes the orientation of the transition dipole moment, resulting in the loss of polarization (Figure 6.6). As rotational diffusion of the molecules depends on their sizes, fluorescence anisotropy can be used to measure the diffusion coefficient and therefore the sizes of the molecules. Figure 6.6 Depolarization of radiation as a result of molecular tumbling We shall, in the next lecture, discuss the biological fluorophores and the applications of fluorescence in understanding the biomolecules. Joint initiative of IITs and IISc – Funded by MHRD Page 33 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Lecture 7 Fluorescence Spectroscopy-II Biological fluorophores Amino acids: Aromatic amino acids tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) are perhaps the most important intrinsic biological fluorophores. Proteins harboring these amino acids become intrinsically fluorescent. Proteins: Proteins are fluorescent due to the presence of aromatic amino acids that fluoresce in the near UV region. Certain proteins, however, do fluoresce in the visible region. Green fluorescent protein (GFP), for example, fluoresces in the green region of the electromagnetic spectrum. The discovery of green fluorescent protein has revolutionized the area of cell biology research. It is therefore important to see what green fluorescent protein is and why it fluoresces in the visible region (See Box 1). Box 7.1: Green Fluorescent Protein (GFP) Green fluorescent protein, abbreviated as GFP was discovered by Shimomura and coworkers in 1962. The protein was isolated from the jellyfish, Aequorea victoria, that glows in the dark. GFP is a 238 amino acid long protein that folds into an 11- stranded β-barrel structure wherein an α-helix passes through the barrel. Joint initiative of IITs and IISc – Funded by MHRD Page 34 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics The fluorophore of the GFP, p-hydroxybenylideneimidazolinone is formed by the residues 65-67 (Ser-Tyr-Gly) and is present in the α-helix passing through the barrel. The excitation spectrum of GFP exhibits a strong absorption band at 395 nm and a weak band at 475 nm. Emission is observed at ~504 nm i.e. in the green region. GFP is an excellent fluorophore with a molar absorption coefficient of ~30000 M- 1 cm-1 at 395 nm and fluorescence quantum yield of 0.79. GFP has been engineered through extensive mutations to remove the undesirable properties that could affect its use as a potential fluorophore. For example, a Ser65 → Thr65 mutant has improved quantum yield and its major excitation band shifted to 490 nm. GFP has the tendency to form oligomers, seriously questioning its use as a fluorescent probe. The aggregation tendency has also been removed through extensive mutations. GFP can easily be tagged to a protein by expressing the fusing gene (GFP gene fused with the gene expressing the desired protein). The GFP then acts as a reporter for all the processes the linked protein is involved in. Several color variants of GFP have been generated through modifications in the residues that constitute the fluorophore. Development of the GFP variants with varying excitation and emission characteristics has made it possible to label the proteins differentially. This is a huge breakthrough and allows easy monitoring of the biological processes using fluorescence microscopy as discussed in lectures 15 and 20. Nucleotides: Nicotinamide adenine dinucleotide in its reduced form, NADH and the flavin adenine dinucleotide in its oxidized form, FAD are fluorescent in the visible region of the electromagnetic spectrum. It is not necessary for all the biomolecules to have an intrinsic fluorophore to perform fluorescence experiments. Fluorescent groups can be covalently incorporated into the molecules making them fluorescent with desirable fluorophore. Such externally incorporated fluorophores are called extrinsic fluorophores. Joint initiative of IITs and IISc – Funded by MHRD Page 35 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Applications of fluorescence Protein folding: High sensitivity of tryptophan fluorescence to the polarity of solvent makes it an interesting intrinsic fluorescent probe for studying protein folding. In the proteins having Phe and Tyr, Trp can be selectively excited at 295 nm. In water and other aqueous solutions, tryptophan fluoresces with an emission maximum, λmax around 350 nm. A tryptophan present in the hydrophobic environment usually displays a blue shift in the emission spectrum and an increase in quantum yield. Due to the hydrophobic nature of the indole side chain, tryptophans are usually buried inside the core of the proteins. The folding can therefore be studied by monitoring the Trp fluorescence as protein folds burying the water-exposed Trp residues inside the protein. Peptide-lipid interactions: Interaction of the peptides having Trp residues with lipid bilayers can easily be studied using fluorescence spectroscopy. Interaction of the peptide with lipids brings the tryptophan in relatively hydrophobic environment causing a blue shift in emission spectrum (Figure 7.1). Figure 7.1 Spectral changes in tryptophan fluorescence upon binding to lipid bilayers Joint initiative of IITs and IISc – Funded by MHRD Page 36 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Binding studies: Binding of small fluorescent molecules to the biomacromolecules can be studied using fluorescence anisotropy. Binding of the fluorophore to a macromolecule will reduce its tumbling (increase its rotational correlation time) thereby resulting in higher fluorescence anisotropy. FRET: i. The distance between two sites in a biomacromolecule such as a protein can be calculated by labeling these sites with suitable donor-acceptor FRET pair. FRET can also be used to study the intermolecular interaction if the interacting molecules comprise of the fluorophores making a FRET pair. ii. Interactions of peptides and other molecules with lipid bilayers comprising fluorophore labeled lipids. If the interacting molecule makes a FRET pair with the fluorescent lipid, the distance between them can be calculated providing information about the insertion of the molecule in the lipid bilayer. iii. FRET has been utilized to study the kinetics of enzymatic reactions. For example, a DNA molecule, tagged with the fluorescence donor at one end and an acceptor at the other end can be used as a substrate to study the restriction endonuclease activity and cleavage reaction kinetics (Figure 7.2). A similar assay can be used to study the proteases using peptides as the substrates. Figure 7.2 Decrease in fluorescence intensity of the acceptor following cleavage of DNA molecules. Joint initiative of IITs and IISc – Funded by MHRD Page 37 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Fluorescence quenching: i. Interaction of a fluorophore with another molecule(s) may provide it protection against a collisional quencher. For example, interaction of a Trp containing peptide with lipid bilayers can be studied using iodide (I − ) as the collisional quencher. The peptide sample in the presence of lipid vesicle is titrated with the potassium iodide (KI) and fluorescence spectra recorded at each quencher concentration. The collisional fluorescence quenching is described by a plot of ‘the ratio of quantum yield in the absence of quencher to that in the presence of quencher’ against ‘the quencher concentration’. Such a plot is known as the Stern-Volmer plot. The Stern-Volmer equation is given by 𝐹0 = 1 + 𝑘𝑞 𝜏0 [𝑄] = 1 + 𝐾𝑠𝑣 [𝑄]········································· (7.1) 𝐹 where, F0 = Fluorescence intensity in the absence of quencher F= Fluorescence intensity in the presence of quencher kq = Bimolecular quenching constant τ0 = Fluorescence lifetime in the absence of quencher [Q] = Quencher concentration Ksv = Stern-Volmer constant A normalized accessibility factor (NAF) is defined as the ratio of ‘the Ksv in the presence of the binding partner of the fluorophore’ to ‘that without the binding partner’. ii. The fluorescence intensity of a sample increases with an increase in the fluorophore concentration. Beyond certain concentration, however, the fluorescence intensity decreases due to self collisional quenching. This property is often used to study the membranolytic activities of a compound. A fluorescent dye at self-quenching concentrations is trapped inside a lipid vesicle. A membranolytic compound results in the release of the fluorescent dye causing increase in fluorescence emission intensity (Figure 7.3). Joint initiative of IITs and IISc – Funded by MHRD Page 38 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Figure 7.3 Membranolytic activity of a compound monitored through dye release assay. Release of dye from the lipid vesicle diminishes the self-quenching resulting in enhanced fluorescence emission. iii. Fusion of lipid vesicles can also be studied using the same approach. Vesicles that contain self-quenching concentrations of the fluorescent dye are titrated with the vesicles without fluorophores. A fusion will result in the dilution of fluorophores; the consequent decrease in self-quenching is exhibited as an increase in the fluorescence intensity (Figure 7.4). Figure 7.4 Fusion of fluorescent dye-containing lipid vesicles with vesicles without dye results in dilution of dye. The dilution results in lesser self-quenching thereby increasing the fluorescence intensity. Joint initiative of IITs and IISc – Funded by MHRD Page 39 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics QUIZ Q1: Shown below are the absorption and emission spectra of a fluorophore. The fluorescence emission for this fluorophore is not the mirror image of the absorption spectrum. How do you explain this? Ans: The low wavelength absorption band is likely to be arising from S0 → S2 transition. As fluorophore relaxes back to S1 state prior to emission, the fluorescence band is the mirror image of the band arising from S0 → S1 transition, not the entire absorption spectrum. Q2: If the efficiency of energy transfer between a donor and acceptor is 80%. Calculate the distance between them if the Förster distance between them is 40 nm? Ans: The efficiency of energy transfer, E is given by: 𝑅06 𝐸= 𝑅06 + 𝑟 6 Given: E = 80% = 0.8, R0 = 40 nm Rearranging the expression for the efficiency of energy transfer 1 𝐸= 𝑟 6 1+ 𝑅0 1 0.8 = 𝑟 6 1+ 40 𝑟 6 1 1 + 40 = 0.8 = 1.25 Joint initiative of IITs and IISc – Funded by MHRD Page 40 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 𝑟 6 40 = 1.25 − 1 = 0.25 1 𝑟 40 = (0.25)6 = 0.7937 𝑟 = 0.7937 × 40 = 31.748 nm ≈ 31.75 nm Joint initiative of IITs and IISc – Funded by MHRD Page 41 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Lecture 8 Circular Dichroism Spectroscopy-I Introduction Before going ahead to see what circular dichroism (abbreviated as CD) means, let us have a quick revisit on the polarized light. Light, as we have discussed in lecture 3 is electromagnetic radiation where electric field and the magnetic field are always perpendicular to each other. From now on, we shall mention only electric field; it is implicit that at all points in time and space, the magnetic field vector is perpendicular to both the electric field vector and the direction of the propagation of light. Unpolarized light is comprised of several electromagnetic waves with their electric field vectors (and therefore magnetic field vectors also) pointing in all possible directions, but perpendicular to the direction of light propagation. If the vectors in all, but one, directions are cut off, the resulting radiation is a plane polarized light as the electric field vector is confined to one plane (Figure 8.1). Looking towards the light source will exhibit electric field fluctuations in one line; the plane polarized light is therefore also referred to as the linearly polarized light. Figure 8.1 Plane polarized light produced by a linear polarizer Joint initiative of IITs and IISc – Funded by MHRD Page 42 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Superposition of polarized waves Two electromagnetic waves can be superposed through vector addition of their electric field vectors. The properties of the resultant waves depend on the wavelength, polarization, and the phase of the superposing waves. In-phase superposition of two waves of same wavelength that are linearly polarized in two perpendicular planes results in a linearly polarized light with its electric field vector oscillating in a plane that is inclined at an angle of 45° to the polarization planes of both the waves (Figure 8.2). Figure 8.2 Superposition of linearly polarized waves Let us see what happens when the two plane polarized waves, polarized in two perpendicular planes meet each other out of phase. Suppose the two waves have a phase difference of 90°. As the two waves have same wavelength, a 90° phase difference implies that when one of the wave is at maximum amplitude, the amplitude of the other one is minimum and vice versa. If the amplitudes of the two waves are equal, their superposition with a 90° phase difference results in a wave wherein electric field vector traverses a circular path (Figure 8.3). The electric field of the resultant wave is never zero but a vector of constant length. When looked at the travelling wave from the direction of propagation, the electric field appears to be Joint initiative of IITs and IISc – Funded by MHRD Page 43 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics rotating in a circle. The resulting light is therefore termed as circularly polarized light (Figure 8.3). Figure 8.3 Superposition of waves linearly polarized in mutually perpendicular plain and that meet together 90° out of phase. Joint initiative of IITs and IISc – Funded by MHRD Page 44 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics The direction of rotation depends on phase difference; a -90° phase difference would result in a circularly polarized light where the electric field rotates in opposite direction. When looked towards the light source, the electric field vector of a right circularly polarized wave appears to rotate counterclockwise in space while that of a left circularly polarized wave rotates clockwise. What happens when the right circularly polarized light (RCPL) and the left circularly polarized light (LCPL) superpose? The resultant wave is a linearly polarized wave (Figure 8.4). A linearly polarized light can therefore be considered as being composed of a right circularly polarized light and a left circularly polarized light. Figure 8.4 Superposition of left and right circularly polarized light resulting in plane polarized light. Circular Dichroism Circular dichroism, abbreviated as CD, is a chiroptical spectroscopic method. A chiral molecule or an achiral molecule in asymmetric environment interacts differently with the LCPL and the RCPL. The literal meaning of dichroism is ‘two colors’. In chiroptical spectroscopy, dichroism means differential absorption of the lights with different polarizations. Circular dichroism, therefore, refers to the differential absorption of the left and right circularly polarized light and is defined as: 𝐶𝐷 = ∆𝐴 = 𝐴𝑙 − 𝐴𝑟 ···························(6.1) where, Al and Ar are the absorbances for the left and right circularly polarized lights, respectively. Joint initiative of IITs and IISc – Funded by MHRD Page 45 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics We can therefore say that the molar absorption coefficients for the two lights are different and can write the equation 6.1 can be written as: 𝐶𝐷 = (𝜀𝑙 − 𝜀𝑟 )𝑐𝑙 ······················································(6.2) 𝐶𝐷 = ∆𝜀𝑐𝑙 ······················································(6.3) The preferential absorption of LCPL over RCPL (or vice versa) results in elliptical polarized light (Figure 8.5). Figure 8.5 Differential absorption of the left and right circularly polarized light resulting in elliptically polarized light. Notice that if one component is completely absorbed, the resultant wave will be circularly polarized. CD is historically represented in terms of ellipticity (θ) which is the tangent of ratio of minor to major axis of the ellipse. The relationship between CD and θ is give by: 2.303 𝜃 (radians) = 4 × 𝐶𝐷 ··················(6.4) 2.303 180 𝜃 (degrees) = 4 × 𝐶𝐷 × 𝜋 ·········(6.5) 𝜃 (degrees) ≈ 33.0 × 𝐶𝐷 ··················(6.6) A plot between ∆A or ∆ε or θ against the wavelength of light represents a CD spectrum. In this lecture, we shall be discussing only electronic CD. That means that we shall be looking at the electromagnetic region that causes electronic transition, which of course is UV/Visible region. Circular birefringence If a sample reduces the velocity of the LCPL and RCPL to different extents, the sample is said to be circularly birefringent and the phenomenon circular birefringence. Let us see what happens when the linearly polarized light (having two components, LCPL and RCPL) traverses a circular birefringent medium: the velocities of the two components are reduced to different extents i.e. they have different wavelengths in the Joint initiative of IITs and IISc – Funded by MHRD Page 46 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics sample. After emerging from the samples, the wavelength is restored but two components can be out of phase. This results in the rotation of the polarization axis. If the material is not circularly dichroic, the plane of the linearly polarized light is rotated (Figure 8.6A). If the material is both circularly dichroic and birefringent, the plane polarized light will become elliptically polarized light with the major axis of the ellipse tilted with respect to the polarization axis of the incident polarized light (Figure 8.6B). Figure 8.6 A linearly polarized light passing through a circular birefringent but not circular dichroic material (A) and through a material that is both circular birefringent and circular dichroic (B). Circular dichroism results in elliptically polarized light while circular birefringence causes change in the polarization axis. Instrumentation As CD is simply Photoelastic modulator: A photoelastic material is the one the difference in that exhibits birefringenece under mechanical stress. The the absorbance of photoelastic modulator in a CD instrument comprises of a quartz crystal fused to a piezoelectric material. Oscillations the LCPL and in the piezoelectric material drive the quartz crystal to RCPL lights, a CD oscillate at the same frequency. The crystal optical axis is at spectrometer, also 45° to the linearly polarized light. The crystal retards one known as a CD component of the light more than the other when compressed. When expanded the velocity of the two spectropolarimeter, components gets reversed. A PEM, therefore gives is basically an alternating LCPL and RCPL. absorption spectrophotometer (Figure 8.7). The instrument has a light source, usually a Xenon lamp. The polychromatic light from the source is converted to monochromatic radiation which is further converted to linearly polarized light by a polarizer. The linearly polarized light passes through a photoelastic modulator that alternately converts the linearly polarized light into LCPL and RCPL. The LCPL and the RCPL, therefore pass through the sample alternately and their absorbance gets recorded. Absorbance is recorded at various wavelengths to obtain a CD spectrum. Joint initiative of IITs and IISc – Funded by MHRD Page 47 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Single wavelength CD values are also important in studying the fast reactions such as protein folding/unfolding (discussed in the next lecture). Figure 8.7 Schematic diagram of a CD spectropolarimeter. Joint initiative of IITs and IISc – Funded by MHRD Page 48 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Lecture 9 Circular Dichroism Spectroscopy-II CD of biomolecules Most biomolecules are chiral and the biomacromolecules are composed of chiral components. Folding of biomacromolecules into higher order structures further imparts them the asymmetry. CD has not been used as much to study other biomolecules probably, as it has been used to study proteins. CD of proteins Proteins are usually composed of 20 amino acids, 19 of which (except glycine) are chiral. This chirality also reflects in the higher order structures that the polypeptides adopt; α-helix, for example, is a right handed helix. If a polypeptide adopting α- helical structure is synthesized using D-amino acids, it folds into the left-handed α- helix under identical conditions. The other structural features of a polypeptide backbone include β-sheets, that are comprised of extended polypeptide chains; β- turns, that usually, but not essentially, link the β-strands in an antiparallel β-sheet; and unordered conformation. CD spectra of the proteins contain information about the asymmetric features of the polypeptide backbone. Furthermore, it can provide information about the orientation of the side chains. CD, therefore, is capable of providing information about the structure of proteins which in turn helps understanding their function. The chromophore that provides information about the conformation of the peptide backbone is the peptide bond (Figure 9.1); the spectra are therefore recorded in the far UV region, the region where peptide bond absorbs. Figure 9.1 The peptide bond showing molecular orbitals involved in electronic transitions Joint initiative of IITs and IISc – Funded by MHRD Page 49 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Let us have a look at the CD spectra characteristic of the different structural components of the proteins (Figure 9.2). α-helix: The right handed α-helix displays two negative absorption bands centered around 222 nm (n → π* transition) and 208 nm (a part of the π → π* transition) and a strong positive band around 192 nm (a part of the π → π* transition). β-sheet: β-sheets are characterized by the presence of a negative band centered around 216-218 nm (n → π* transition) and a positive band of comparable intensity at around 195 nm (π → π* transition). β-turn: A β-turn comprises of a four residue protein motif that causes the polypeptide backbone to take an approximately 180° turn. The CD spectrum for a β-turn is not well defined. A typical β-turn, however, shows a weak negative band around 225 nm (n → π* transition), a strong positive band between 200 – 205 nm (π → π* transition), and a strong negative band (π → π* transition) between 180 – 190 nm. Random coil: Random coil or unordered conformation shows a weak positive band around 218 nm (n → π* transition) and a strong negative band (π → π* transition) below 200 nm. Figure 9.2 Far UV circular dichroism spectra of α-helix (red), β-sheet (blue), and unordered conformation (green) Joint initiative of IITs and IISc – Funded by MHRD Page 50 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics The CD spectrum of a protein can be written as a linear combination of the spectra of all the structural components: CD (protein) = a CD(α-helix) + b CD(β-sheet) + c CD (Random coil) As the CD spectra of different structural components are quite distinct, it is possible to estimate the fraction of different structural components in a protein from its CD spectrum. As discussed in lecture 5, proteins also have chromophores that absorb in the near UV region. These include the aromatic amino acids and disulfide linkages. The CD of aromatic amino acids is highly dependent on their environment and therefore near UV CD of proteins can provide the information about the environments these residues reside in as well as their orientations in the structure. As it provides information about the tertiary region, near UV CD is also referred to as tertiary CD in the context of the proteins. CD of nucleic acids As mentioned in lecture 5, nitrogenous bases constitute the chromophores of nucleic acids in the near and far UV region. The CD of the stacked bases is larger in magnitude as compared to that of the isolated bases. As the double helical nucleic acids have stacked bases, what we measure essentially is the CD that arises due to coupling of the chromophores. As the stacking geometries are different for different forms of nucleic acids such as B-DNA, Z-DNA, and A DNA; CD can help in determining which DNA form is present in a given sample. Applications in biomolecular analysis i. Determination of protein/peptide structure: As has already been discussed earlier, far UV CD spectroscopy provides information about the secondary structural elements in a protein. A mixture of structures can be deconvoluted to obtain the fraction of different structural elements. Furthermore, near UV CD provides information about the tertiary structure of the protein. ii. Comparison of structures: Mutants of proteins are often required for understanding the functions of the proteins. It, however, needs to be ascertained that the mutation does not cause any significant change in the overall structure of the protein. CD spectroscopy happens to be a fast and extremely reliable tool to compare the conformations of the wild type proteins with their mutants. Joint initiative of IITs and IISc – Funded by MHRD Page 51 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics iii. Stability of proteins: Stability of the proteins to denaturants or heat can be studied using CD spectroscopy. In such studies CD is usually monitored at a single wavelength, typically around 220 nm. Plotting the change in ellipticity against increasing denaturant concentration/temperature provides the denaturation curve. Figure 9.3 shows the denaturation curves for three related proteins. The denaturation curves suggest that the protein indicated with the blue trace is most stable while the one indicated with red trace the least. Figure 9.3 Comparison of thermostability of three related proteins. The blue trace represents the most stable protein. iv. Binding of ligands to proteins: Binding of a ligand to a protein usually does not affect the secondary structural elements significantly. However, such a binding can cause changes in the local tertiary structure. Binding of ligands accompanying such conformational changes can be studied using tertiary CD if the binding region happens to have one or more aromatic residues. Short peptides, on the other hand, can undergo large scale structural changes sometime involving completely switching from one secondary structure to another. Such changes can easily be observed using far UV CD. v. DNA structure: CD in the 200 – 300 nm region can be used to identify which structural isoform of DNA is present in the given sample. The left-handed helical DNA form, the Z-DNA was indeed identified using CD spectroscopy. The typical CD signatures of the B, Z, and A form of DNA are: B-DNA: In its most common form i.e. B-DNA with ~10.4 bases per turn, a positive band ~275 nm, a crossover ~258 nm, and a negative band at ~240 nm are observed. Z-DNA: A negative band ~290 nm and a positive band ~260 nm; a crossover between 180-185 nm. A-DNA: A positive band ~260 nm, a negative band ~210 nm. Joint initiative of IITs and IISc – Funded by MHRD Page 52 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics vi. Protein folding/unfolding: CD is used for studying the folding and unfolding of proteins. For monitoring the fast reactions such as protein folding, a single wavelength CD is recorded in a stopped flow experiment wherein the protein solution is mixed with a denaturant and CD is recorded as a function of time. Modern instruments take ~1 millisecond time between mixing and recording data allowing the understanding of the folding/unfolding events that occur on milliseconds to seconds timescale. A diagrammatic unfolding experiment is shown in figure 9.4 Figure 9.4 A diagram showing the kinetics of unfolding of a hypothetical protein. The protein is unfolded with different concentrations of a denaturant. Protein and denaturant are mixed in a stopped flow apparatus (mixing time typically ~1 ms) and changes in ellipticity are monitored over time. vii. Molecular self-assembly: Self-assembly into structural and functional superstructures is integral to biomolecules and therefore to living systems. Inspired by the naturally occurring superstructures, short peptides have attracted considerable attention as the monomers for designing superstructures with novel properties and applications in biomedicine. Circular dichroism has been central in elucidating the conformations of the peptides in superstructures as well as the interactions that drive this assembly. Circular dichroism, therefore, is a powerful tool in studying the conformations of biomolecules as well as the processes these molecules are involved in. Joint initiative of IITs and IISc – Funded by MHRD Page 53 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Lecture 10 Infrared Spectroscopy Introduction Infrared (IR) region of the Conventions for IR radiation electromagnetic spectrum lies between visible and microwave Wavelength: The wavelength of IR region ranges from ~780 nm – 250000 nm. regions and therefore spans the Writing such big number is avoided by wavelengths from 0.78 – 250 μm. expressing the wavelengths in micrometers The energies associated with (0.78 – 250 μm). molecular vibrations are smaller Wavenumber (𝝂 ): Wavenumber means the than those associated with electronic number of wavelengths per unit distance. Therefore, 100 cm-1 implies there are 100 transitions and fall in the IR region. wavelengths per cm. 𝜈̅ in cm-1 is given by: IR spectroscopy, therefore, is used 1 to probe the vibrations in molecules 𝜈̅ (𝑐𝑚−1 ) = × 104 𝜆 (𝜇𝑚) and is also known as vibrational spectroscopy. Infrared region is usually divided into three regions: near infrared, mid- infrared, and far infrared (Figure 10.1). IR spectroscopists use wavenumbers (𝜈̅ ) to represent the IR spectra and we shall be following the same convention. Mid-IR region (λ = 2.5 -25 μm; 𝜈̅ = 4000 – 400 cm-1) is the region of interest for studying molecular vibrations. Figure 10.1 Infrared region of the electromagnetic spectrum Joint initiative of IITs and IISc – Funded by MHRD Page 54 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Degrees of freedom and molecular vibrations At non-zero temperatures, i.e. temperatures above 0 K, all the atoms in a molecule are in motion. The molecule itself also is in translational and rotation motion. In a three dimensional space, an atom in isolation has 3 degrees of freedom, corresponding to the motion along the three independent coordinate axes. A molecule composed of N atoms has a total of 3N degrees of freedom (Figure 10.2). Figure 10.2 Degrees of rotational freedom for a diatomic (A) and a triatomic (B) molecule For a non-linear molecule, three of these 3N degrees of freedom correspond to translational motion, three correspond to rotational motion while rest 3N-6 are the vibrational degrees of freedom. For a linear molecule, there are only two rotational degrees of freedom that correspond to the rotation about the two orthogonal axes perpendicular to the bond (Figure 10.2). A linear molecule, therefore, has 3N-5 vibrational degrees of freedom. Let us have a look at the degrees of freedom of a diatomic molecule. A diatomic molecule has a total of 3 × 2 = 6 degrees of freedom. Three of these six degrees of freedom correspond to translational motion of the molecule; two of them define rotational degrees of freedom; while one corresponds to the vibration of the atoms along the bond. The 3N-6 vibrational degrees of freedom (3N-5 for linear molecules) represent the true/fundamental modes of vibration of a molecule. The different types of vibrations are shown in Figure 10.3. Joint initiative of IITs and IISc – Funded by MHRD Page 55 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics Figure 10.3 Stretching and bending vibrations in molecules Hooke’s law and frequency of vibration We have seen that the bonds are not static but vibrating in different ways. A vibrating bond can therefore be considered a spring with its ends tethered to two atoms (Figure 10.4). Figure 10.4 Spring analogy of a bond vibration If the masses of the atoms are m1 and m2, the frequency of stretching vibration of the diatomic molecule can be given by the Hooke’s law: 1 𝑘 𝜈= ····················································· (10.1) 2𝜋 𝜇 where, ν is the frequency of vibration, k is the spring constant, and μ is the 𝑚1 𝑚2 reduced mass i.e. 𝑚 1 + 𝑚2 Dividing equation 10.1 by λ gives: 𝜈 1 𝑘 = ······················································(10.2) 𝜆 2𝜋𝜆 𝜇 Joint initiative of IITs and IISc – Funded by MHRD Page 56 of 99 NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics 1 1 𝑘 = ·················································· (10.3) 𝜆 2𝜋(𝜆𝜈) 𝜇 1 𝑘 𝜈̅ = 𝜇 ······················································(10.4) 2𝜋𝑐 The spring constant, k is Anharmonic oscillator the measure of the bond Real molecules are anharmonic oscillators. Unlike strength. The stronger harmonic oscillator wherein energy levels are equally the bond, the higher the spaced; energy levels in an anharmonic oscillator are k, and consequently the more closely spaced at higher interatomic distances. A treatment for anharmonicity is beyond the scope of our higher is the frequency discussion. of vibration. This treatment implies that the diatomic molecule is a simple harmonic oscillator. The energy of a quantum harmonic oscillator is given by: 1 𝐸 = 𝑛 + 2 ℎ𝜈 ···················································· (10.5) where, n = 0, 1, 2, …… and h is the Plancks’s constant Absorption of infrared radiation A molecular vibration is IR active i.e. it absorbs IR radiation if the vibration results in a change in the dipole moment. A diatomic molecule, that has one mode of vibration, may not absorb an IR radiation if the vibration does not accompany a change in the dipole moment. This is true for all the homonuclear diatomic molecules such as H2, N2, O2, etc. Vibration of carbon monoxide (C=O), on the other hand, causes a change in dipole moment and is therefore IR active. Vibration of a bond

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