Spectrophotometry Lecture PDF

Spectrophotometry Spectroscopy: The term spectroscopy referred to the branch of science which deals with the interaction of light energy with matter. Spectrophotometry: Spectrophotometry is instrumental method in which the absorption and or emission of light (electromagnetic radiation) is studied a...

Spectrophotometry Spectroscopy: The term spectroscopy referred to the branch of science which deals with the interaction of light energy with matter. Spectrophotometry: Spectrophotometry is instrumental method in which the absorption and or emission of light (electromagnetic radiation) is studied and measured to give qualitative information about the nature of the substance (i.e. to identify it) as well as to determine it i.e. quantitative analysis.  Spectrophotometry Light and radiation: The light or electromagnetic radiation (EMR) is a type of energy that is transmitted through space at enormous velocity. It has both particle and wave properties. It is necessary to view EMR as a stream of discrete energy particles of energy called photons which move in the form of wave Dual nature of light (dualism): Light (EMR) exhibits wave property during its propagation and energy particle during its interaction with matter which is known as the dual nature of light 1- Light as a Wave: Light (EMR) display the property of continuous wave and can be described by characteristics of wave motion.  Spectrophotometry A- Wavelength (λ Lambda): is the distance between successive maxima (crest) or minima (trough) of a wave. Different units of length are used to express wavelengths: The following units are in common use: 1 micrometer = 1 µm =10-6 m =10-4 cm, 1 nanometer = 1 nm =10-9 m =10-7 cm, 1 angstrom = 1 A˚ = 10- 10m = 10-8 cm, 1 nm = 10 A˚, 1 µm =104 A˚ B- Frequency ( nu) : which is the number of waves occurring per second and is expressed in cycles/sec(CPS) or Hertz(Hz). Wavelength is inversely proportional to frequency. c = λν= 3 x 1010 cm/s where, c is the velocity of light. C- Wavenumber (`) : which is number of waves per centimeter, which is expressed in cm-1. Wavenumber (`) = 1/λ ( cm-1) Relations between λ, and ` are given by the following equations: Wave number = 1/ wavelength (cm) = Frequency () / Velocity Of Light (C) Note that, the longer the wavelength, the lower the frequency and the smaller the wave number and vice versa.  Spectrophotometry 2- Light as an energy particle: As stated before light (EMR) can be viewed as a stream of discrete particles of energy called photons which move in the form of wave. The energy of a photon depends upon the frequency of the radiation. E = h where h is Plank`s constant (6.6 x 10-34 j.s) = c/ E = hc/ Therefore, the shorter the wavelength, the greater the energy of the photons and the more powerful the radiation.  Spectrophotometry Electromagnetic spectrum The electromagnetic spectrum includes gamma ray, X-ray, ultraviolet (UV), visible, infrared (IR), microwave and radio wave radiations. This electromagnetic spectrum ranges from very short wavelengths (including gamma and x-rays) to very long wavelengths (including microwaves and radio waves). Ultraviolet (UV) radiation has shorter wavelengths (200-380 nm) than violet light and cannot be seen by the eye. Infrared (IR) radiation has longer wavelengths than the red light and also cannot be seen by the eye. The visible region of the spectrum extends from 380 nm to about 780 nm. The eye can normally detect only the colors within this wavelength range that is why it is called visible.  Spectrophotometry White light can split to produce different colors or wavelengths of visible light which appear as different colors to our eyes this colors include red, orange, yellow, green, blue, indigo and violet. When all the wavelengths or colors of the visible light are transmitted together, the light appears as white light. However, if all the wavelengths or colors of the visible light are absorbed, it appears black. When white light is passed through an object, the object will absorb certain wavelengths, leaving the unabsorbed wavelengths to be transmitted. These residual transmitted wavelengths will be seen as color Absorbed Absorbed color Perceived color wavelength (nm) (transmitted) 400 Violet Yellow 480 Blue Orange 510 Green Red 570 Yellow Violet 600 Orange Blue 650 Red Green  Spectrophotometry Complementary colors It must be noted that, the absorbed color is different from the actually observed color. The observed color is complementary color which remained after the substance absorbed from white light as shown color weal UV-VIS spectrophotometry Orange The molecule at room temperature is usually in its Red Yellow lowest electronic energy state called ground state. When a molecule interacts with photons in the Violet Green UV-VIS region. The absorption of the energy Blue results in displacing an outer electron (valence electron) in the molecule. The molecule is said to undergo transition from the ground state of energy level (Eg) to an excited state of energy level (Es). Energy of transition is given by the equation: ∆E= Es - Eg = hv = h c/λ  Spectrophotometry The excited state is a transition one and the species soon loses the energy it gained and returns to its ground state by relaxation process. The energy is lost either as: heat, light or molecular collision 1-heat: If the excited electrons return directly to the ground state, heat is evolved. 2-Light: If the excited electrons return to the ground state via a second excited state, light is emitted as fluorescence and phosphorescence. 3- Molecular collision  Spectrophotometry Ground energy (Eg) and is given by: Etotal = Eelectronic + Evibrational + Erotational Where: E rot: By increasing the rotation of the molecule about the axis. E vib: By increasing the vibration of the constituent nuclei. E elec: By raising an electron (or electrons) to a higher energy level. N. B: E elec E vib  E rot (10.000:100:10) When a molecule interacts with photons of UV-VIS radiations excitation of electrons takes place to higher electronic energy level at any of its vibrational level.  Spectrophotometry Structure and energy: In the ground state, the outermost electrons in organic molecules may occupy one of three different energy levels σ, π or n: a) σ-electrons; they are bonding electrons, represent valence bonds and possess the lowest energy level (the most stable). (σ bonds) b) π-electrons; they are bonding electrons, forming the π-bonds (double bounds), and possess higher energy level than σ - electrons. c) n-electrons; they are nonbonding electrons, present in atomic orbital of hetero atoms (N, O, S or halogens). They usually occupy the highest energy level of the ground state. When light passes through a compound, some of the energy in the light kicks an electron from one of the bonding or non-bonding orbitals into one of the anti-bonding ones. The energy gaps between these levels determine the frequency (or wavelength) of the light absorbed, and those gaps will be different in different compounds.  Spectrophotometry So In excited state the σ -electrons occupy an anti-bonding energy level (σ *) and the transition is termed σ - σ* transition. π -electrons occupy an anti-bonding energy level (π *) and the transition is termed π - π * transition, While the n-electrons may occupy σ * or π * levels to give n- σ * or n- π * transition. Energy * n   n  Antibonding  Antibonding n Nonbonding  Bonding  Bonding 150 200 250 300 Wavelength, nm Fig. 4 : Different types of electronic levels and transitions  Spectrophotometry d → d transition: - Numerous inorganic salts containing electrons engaged in d orbitals are responsible for transitions of weak absorption located in the visible region. -These transitions are generally responsible for their colors. - That is why the solutions of metallic salts of titanium [Ti(H2O)6]3+ or of copper [Cu(H2O)6] 2+ are blue, while potassium permanganate yields violet solutions Spectrophotometry  Spectrophotometry Most of -* absorption takes place below 200 nm in the vaccum UV region (with higher energy than UV-VIS region). Thus, compounds containing just  bonds are transparent in the near UV-VIS region. which makes these compounds ideal solvents for other compounds studied in this range. -* and n-*absorption occurs in the near UV-VIS region and result from the presence of chromophores. Cut-off wavelengths of some common solvents Solvent λ, nm Solvent λ, nm Water 190 Chloroform 247 ether 205 Carbon tetrachloride 257 Ethanol 207 Benzene 280 Methanol 210 Acetone 331  Spectrophotometry Some important terms in spectrophotometry: - Chromophore: (Chrom = color, phore = carrier). They have unsaturated functional groups (double or triple bonds) such as - C=C, -C=O, -N=N, -C=N and etc..., responsible for -*and n-*electronic transitions, thus, they are capable of absorbing UV and/or visible light (200 - 800 nm) and imparting color to the molecules. Beta carotene Is a strongly colored red-orange pigment The absorption of the molecule is the sum of absorption of all its chromophores.  Spectrophotometry - Auxochrome: Auxochromes are saturated groups which possess unshared electrons such as -OH, -NH2, …..etc. An auxochrome doesn’t itself absorb radiation but if present in a molecule, it can enhance the absorption by chromophore or shift the wave length of absorption when attached to the chromophore.  Spectrophotometry Absorption spectrum It is a plot of the absorbance (A) against the wavelength (λ). Each substance has its characteristic absorption spectrum which depends on its structure. It has characteristic shape which shows λ of maximum absorbance [λmax]. There are two parameters which define an absorption band : 1) its position ( λmax) on the wavelength scale. (depend on ch. Structure) 2) Its intensity on the absorbance scale. (depend on concentration) λmaxis characteristic for each molecule according to its structure and consequently types of transitions. Therefore it is used for: - Identification of a chemical substance (N.B. structure similarity) - Quantitative measurement  Spectrophotometry Spectral shifts of the absorption spectrum Bathochromic shift: The shift of max to longer wavelength; known by red shift, due to: 1- Substitution with certain groups as OH and NH2 2- Solvent effect, e.g. polarity of solvent 3- In addition to presence of two or more chromophores in conjugation. Hypsochromic Shift: The shift of maxto shorter wavelength; known by blue shift, due to substitution and/or solvent effect or removal of conjugation. Hyperchromic effect: An increase in intensity of absorption, due to the presence of auxochrome. Hypochromic effect: A decrease in absorption intensity.  Spectrophotometry Absorption characteristics of chromophores: 1-Ethylenic chromophores: Their bands are difficult to observe in near UV region, so they are not useful analytically. However, substitution and certain structural features may cause red shift rendering the band observable in the near UV region. Examples: a) Alkyl substitution; Cause red shift due to hyper-conjugation and stabilization of excited state. b) Attachement to auxochromes, Cause red shift and increased absorption intensity due to extension of conjugation.  Spectrophotometry 2-Conjugated chromophores: Separated chromophores (by two or more single bonds) have additive effect only because there is little or no electronic interaction between separated chromophores. However, if two chromophoric groups are present in a molecule and they are separated by only one single bond (a conjugated system), a large effect on the spectrum results, more than found by more addition. The reason is that the -electron system is interacting and spread over at least four atomic centers. When two chromophoric groups are conjugated such as in dienes, the high intensity * transition is generally red shifted by about 15 - 45 nm with respect to the single unconjugated chromophore, Thus butadiene now absorbs around 215 nm instead of 190 nm for the isolated ethylenic groups.  Spectrophotometry Factors affecting the absorption spectrum: 1-Effect of pH: The spectra of compounds containing acidic or basic groups are dependent on the pH of the solution. Phenol and phenolic compounds are good examples, in acid medium the predominant species is the undissociated form, while in alkaline medium the predominant species is the phenate form. OH O O - OH H+ in acid medium in alkaline medium (Phenol) max = 270 nm (phenate anion)  max= 290 nm In acid medium In alkaline medium The predominant species The predominant species is the undissociated form is the phenate form (Benzenoid structure) (Quinonoid structure)  Spectrophotometry The spectrum of phenol in alkaline medium exhibits bathochromic shift and hyperchromic effect WHY? This is due to the formation of with conjugated double bonds, thus increasing the delocalization of the  electrons, the electrons become more energetic and need less energy to be excited, therefore absorb longer . On the other hand the UV spectrum of aniline in acid medium shows hypsochromic shift and hypochromic effect. This blue shift is due to the protonation NH2 + NH3 of the amine group; hence the pair of + H+ the electrons is no longer available for - H+ the quinonoid conjugated structure which is formed in alkaline medium. In alkaline medium in acid medium Aniline,  max= 280 nm Anilinium ion  max= 254 nm Thus to overcome this spectral changes with pH change the solutions must be buffered at specific pH.  Spectrophotometry 2- Effect of solvent: The solvents may have a strong effect on the position of λmax due to its effect on the energy of transition. Less polar solvents (e.g. hydrocarbons) interact less strongly with the solute than do polar solvents (e.g. water and alcohols). The solvents effects differ according to the structure of the compound: π-π*Transitions Two cases arise: π-π* bands of dienes (Compounds that contain two double bonds): Are not shifted by any change of solvent polarity. π-π* bands of enones (is an unsaturated compound consisting of a conjugated system of an alkene and a ketone): Are red shifted on increasing solvent polarity; due to stabilization of excited state by dipole-dipole solvent interaction. This stabilization leads to lowering the energy of the excited state (i.e.) smaller transition energy and hence longer λ (↓E→↑λ) S1 S2 G  Spectrophotometry n-π*Transitions Compounds possessing n-π* Transitions are blue shifted with increasing solvent polarity due to stabilization of the ground state by hydrogen bonding. Hydrogen bonding lowers the energy of the ground state (i.e.) increase energy of transition and hence decrease λ G1 G2  Spectrophotometry Quantitative Spectrophotometry When a monochromatic light (radiation of one wavelength) having an intensity I0 is allowed to pass through the solution having a thickness b and a concentration c, part of the radiation is reflected Ir, part is refracted If, part may be scattered Is, part is absorbed Ia and part is transmitted It Io = Ia + Ir + It + If + Is Where I0 is the total light entering. For clear solutions, Is = 0, If and Ir can be cancelled by using blank solution. So, under experimental conditions the equation become; Io = Ia + It If (Refracted) Io Ia It (Incident) (Absorbed) (Transmitted) Is (Scattered) Ir light source (Reflected) Sample  Spectrophotometry Transmittance (T) It is the fraction of incident radiation transmitted by the solution. Or the ratio of the intensity of transmitted radiation to that of incident light. T = I / I0 We often measure percent transmittance: % T = T × 100 A more useful measure of the quantity of radiation absorbed is the absorbance. Absorbance (A) A = log10 I0 / I A = log10 (1/T) = -log10 (T) A = 2 - log10 %T Spectrophotometry A = 2 - log10 %T  Spectrophotometry Laws of light absorbance 1-Lambert's Law According to this law, the intensity of transmitted light is decreased exponentially with the increase of thickness (b) of the absorbing medium at constant concentration. log I0/I (A)=Kb K is the proportionality constant; I0 and I are intensity of incident and transmitted radiations. The concentration (c) held constant  Spectrophotometry 2- Beer's Law: The intensity of transmitted radiation is exponentially decreased with the concentration (c) of the solution at constant thickness. It relates absorption capacity to the concentration of an absorbing solute. It stated that absorption is proportional to the number of absorbent molecules in the light path. log I0/I (A) = Kc K is proportionality constant (c) is the concentration while (b) is held constant  Spectrophotometry Both laws were combined in: 3-Beer´s- Lambert´s Law: Log I0/I = abc Where: (a) is a constant called absorptivity,(b) is the path length in cm and (c) is the concentration in grams/Liter. So the unit of (a) is L.g-1 cm-1 Absorptivity a: Is the absorbance of a substance when path length is 1 cm and concentration is 1 g/L. Log l0/l is usually substituted by A (Absorbance), then the equation become; A = abc  Spectrophotometry The value of (a) will clearly depend upon the method of expression of the concentration: - If (c) is expressed in moles/liter, and b in cm then: (a) is given the symbol ε epsilon (L.mol-1cm-1) and is called the molar absorption coefficient or molar absorptivity, A = εbc Molar absorptivity є: is the absorbance of a substance when path length is 1 cm and concentration 1mole/L. If (c) is expressed in g/100 mL (g %) and b in cm then: (a) is given the symbol A1%1cm (g%-1cm-1) A = A1%1cm bc The A1%1cm is valuable for natural products identification when their molecular weight is not definitely known. A1%1cm: Is the absorbance of a substance when path length is 1 cm and concentration 1g %. A1 %1Cm = ε x 10/M.wt  Spectrophotometry Visual Methods (Visual colorimetry): eye detection Visual methods are used for measuring the colored solutions only. A) Standard series method: The test solution contained in a test tube is matched with a series of standards similarly prepared. The concentration of unknown solution is equal to that of the standard solution whose color is matched with it exactly. Example1: The determination of copper with ammonia by forming an blue color. Example 2: The determination of iron (III) with thiocyanate anion by forming blood red color. To avoid path length effect use a set of matched Nessler cylinders.  Spectrophotometry Instrumental methods Spectrophotometers and colorimeters: A spectrophotometer has a much wider wavelength range than a colorimeter, having range from 200 nm in the UV region to 1000 nm in the IR region. Essential parts of spectrophotometer: All spectroscopic instruments operate on the same principle and are composed of five basic components: 1) A stable light source of radiant energy 2) A monochromator (wavelength selector) is used to change polychromatic light to monochromatic light (i.e. split the light into the different wavelengths and to isolate the wavelengths of interest). 3) A sample compartment holds the sample. 4) A detector is needed to measure the light passing through the sample and converts radiant energy to a measurable signal 5) A signal readout meter (recorder) (readout device converts the signal to a readable form.  Spectrophotometry 1- Light source: The light source provides the light that passes through the sample solution. The light source should deliver highly intense, continuous, constant and uniform radiation which covers the range required. a- For UV measurements: Hydrogen or Deuterium discharge lamp (give radiations from 190 - 375 nm) is used. b- For visible measurements: Tungsten lamp is used (give radiations from 350 - 1000 nm). b Li g h t Io It W a v el e n g t h Li g ht R e s ul t s s o ur c e S a m pl e s el e c t o r d et e ct or di s pl a y. ,  Spectrophotometry -Narrower bandwidth tend to enhance the sensitivity and selectivity of the absorbance measurements and give a more linear relationship between the optical signal and concentration of the substance to be determined. Two types of wavelength selectors are used, filters and monochromators. a) Filters: Absorption and interference filters are used for wavelength selection: Absorption filters; usually function via selective absorption of unwanted wavelengths and transmitting the complementary color. Interference filters; As the name implies, an interference filter relies on optical interference to provide a relatively narrow band of radiation. b) Monochromators: i-An entrance slit: allows light from the source to strike the dispersing element. ii- a dispersing element: It breaks the white light into its component wavelengths (colors). This is commonly accomplished by using a prism, or a grating. iii- An exit slit: The exit slit passes only a narrow band of the wavelengths to the sample compartment Prism Prisms act by refraction of light  Spectrophotometry Grating: Consists of a large number of parallel grooves ruled very close to each other on a highly polished surface. They function via refraction of light 3- The Sample Compartment (cell or cuvette) Cuvettes are used to hold liquid samples. There is a wide variety of cuvettes of different shapes and sizes, such as square cuvettes or test tubes. The first requirement of a cuvette is that it be able to transmit the wavelength. glass or silica is used only in the visible region and colored samples quartz is required in the ultraviolet region and transparent samples  Spectrophotometry 4) Light Detectors: Photoelectric detectors are the most frequently used for this purpose. They must give electrical signal ( measured by a galvanometer) , which is directly proportional to the intensity of the transmitted light. Two types of detectors are described here: a) Photocells b) Phototubes (Photomultiplier and photoemissive tubes) Give greater sensitivity. Used when light signal is very weak. 5) Recorder (meter): Electrical signal produced in the detector is fed to a sensitive galvanometer, attached to a scale of meter type (analog) or digital readout meter designed to give absorbance or transmittance percentage units.  Spectrophotometry Types of Spectrophotometers: 1- Single beam 2- Double beam 1-Single beam spectrophotometer Single beam spectrophotometers (colorimeter or filter photometer) All the light from the Monochromators goes to the sample, and then to the detector. Advantages: Relatively inexpensive and simple. Disadvantage: The reference point (blank) has to be reset frequently when measuring a series of samples over a period of time.  Spectrophotometry 2- Double beam spectrophotometer: The light is splitted by a beam splitter into two paths of equal intensity. One path is directed through the reference compartment and the other through the sample compartment. Both beams are either directed to the same detector, or each has its own detector. The signal for the absorption of the reference cell is automatically subtracted from the sample cell, giving a net signal corresponding to the absorption of the components in the sample solution. Advantages: 1- Stability of the readings over time. 2- Ability to simultaneously correct for any solvent effects. 3- Compensate for any fluctuations or irregularities in the source, the detector or the electronics. Disadvantages: The expense and the less sensitivity to measure highly absorbing samples (compared to single beams).  Spectrophotometry Deviation from Beer´s law 1- Real deviation: due to high conc. Beer´s law is applicable to dilute solutions within certain limits of concentration above which deviation occurs. In higher concentration molecular interaction and association may occur, this alters the ability of the species to absorb at definite wavelength. 2- Instrumental deviation: a) Irregular deviation due to: Unmatched cells, unclean handling and unclean optics. b) Regular deviation due to: - Slit width control where, wide opening of slit results in unspecific wavelength to pass. - Stray light is any radiation of wavelength other than those which are absorbed. Also includes any light reaches the detector without passing through the sample. 3- Chemical deviations: a- pH effects b- Solvent interactions c- Temperature effects d- Time factor of colored solutions (color may fade by time due to deterioration of the organic dye by oxidation, reduction, hydrolysis and other reactions). e- Photo effect, some substances as vitamin K undergo degradation when exposed to light. So it should be protected from light either in solid form or as solution  Spectrophotometry Applications of UV-VIS spectrophotometry I- Qualitative Analysis: It is used for the identification of new drugs and natural products. UV-Visible spectrum gives useful information about substance via examination of its max II- Quantitative Analysis: A- Quantitative analysis of a single component: 1- The substance to be analyzed is dissolved in a suitable solvent as methanol, water, ether. The solvent is used as a blank. 2- Absorbance readings are taken in the expected range (200-400 nm for colorless sample or 400-800 nm for colored sample). 3- The max of the substance to be analyzed is chosen. 5- Construct the calibration curve at the characteristic max. The curve must be a straight line passing through the origin, its slope is a (absorptivity).