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What is spectroscopy?
When it propagates; light exhibits wave property and energy particle during its interaction with matter. The double nature of light (being waves and particles in nature) is known as dualism: (1). Wave property EMR display the property of continuous waves and can be described by the characteristics of wave motion. Such wave motion in conveniently classified according to the wavelength.
a)Frequency [ (nu)]: is the number of waves/ second, cycles / second (CPS), or Hertz (Hz). Relation between & C= . or = C/ b) Wave number (): is the number of waves/ cm = 1/
Max Planck presented light as matter (with particulate nature) that forms of packets of energy known as (photons). The energy (E) of photons is variable. It is proportional to the frequency i.e related to C and . It can be expressed by max plank relation: E = h (h = Max Plank constant = 3.63 x 10-27 erg. sec.) i.e E or E 1/
Accordingly, energy of EMR increases as wavelength decreases. e.g. U.V range contains shorter , hence having more energy than visible range, and visible range more than I.R range.
i.e the shorter the wave length, the greater the energy of the photons and the more powerful the radiation. The regions of I.R, visible and U.V radiations are used for analytical purposes usually. Day light (visible radiation) consists of colored radiations, which are, red, orange, yellow, green, blue, indigo and violet (ROYGBIV). If we observe the visible radiations of all wavelengths, we see white light (remember the color wheel!).
A beam containing several wavelengths is called polychromatic light, while a beam single wavelength is said to be monochromatic.
The relative energies of transitional, vibrational and rotational are roughly in the order of 10000: 100: 1. When a molecule interacts with radiant energy in the visible and U.V region, it gains energy that leads to displacement of an outer electron (valence electron) and the molecule is said to be excited because electron undergoes transition from original energy level (ground state = Eg) to an excited state (Es). The energy of transition can be represented by the following equation: E = Es Eg = h Where h is Max Planck constant and is the frequency of EMR at which the transition take place, which is characteristic for each molecule.
N.B: Absorbing large amount of energy (from far U.V region), which is sufficient to exceed the energy of the formation of certain bonds, may lead to bond rupture and new compounds being formed. This phenomenon is described as photolysis. Emission of radiant energy. In all cases, absorbed energy doesn't accumulate in electronic system of a molecule. If an excited electron returns to the ground state, it may lose absorbed energy in the form of heat, light or molecular collision. If the excited electron returns directly to the ground state heat is evolved. When excited electron returns to the ground state via second excited state, light is emitted as f1uorescence or phosphorescence (see later).
The relation between spectra and chemical structure The absorbance of EMR in the U.V-VIS regions depends on structure of organic molecule (i.e. upon the number and arrangement of the electrons in organic molecule). Absorbance of EMR by organic molecule is achieved by chromophoric groups (chromophores) assisted by auxochromes, where the electron of absorbing molecule gets excited, (i.e. when it undergoes transition from the ground state to the excited state). What is a Chromophore? It is unsaturated organic group responsible for electronic absorption e.g.
What is an Auxochrome? It is a saturated organic group which when attached to a chromophore, changes both the and intensity of absorption maxima e.g. -OH, - NH2, - Cl. Notice: All auxochromes have one or more non-bonding pair of electrons. If an auxochrome is attached to a chromophore, it helps extending the conjugation by sharing of non-bonding electrons.
The outer electrons in an organic molecule may occupy one of three different energy levels; namely: Sigma () electrons: They are bonding electrons which represent valence bonds formed due to linear overlapping of electronic clouds of S or SP orbitals. They have the lowest energy level (i.e. they are the most stable). Pi () electrons: they are the bonding electrons constituting the pi bonds (double bonds) and result from lateral overlap of electronic clouds of P orbitals. They are of higher energy than sigma electrons. Non-bonding (n) electrons: they are of atomic ortbitals of hetero atoms (N, O, X or S) which don't participate in bonding. n electrons occupy the highest level of ground state.
In excited state electrons occupy an antibonding energy level denoted as * and the transition is termed - * transition. electrons occupy the antibonding * level, while n electrons occupy either * or *. The absorption spectra of organic molecules depend on the electronic transitions occurring. The following figure represents the different electronic energy levels and different types of transition which may occur:
-Absorption:
Compounds containing only -electrons are the saturated hydrocarbons which absorb at < 170nm (i.e. in the far UV region). They are transparent in the near UV (200-300nm), which makes of them ideal solvents for other compounds to be studied in this region. n-electrons absorption in saturated compounds:
For saturated compounds containing heteroatoms (S, N, O or halogens), the majority of these compounds show absorption in the near UV region e.g. Methanol at 177nm, triethylamine at 199nm and chloroform at 173nm.
Alcohols and ethers absorb at wavelength shorter than 185nm and so they are useful as common solvents at > 200nm (that will help in quantitative analysis of drugs later). However, their intense absorption usually extends to the edge of the near UV region (Cut off wavelength) in the 200220nm region.
Absorption spectra Absorption spectrum is the plot of Absorbance (A) as a function of wavelength (). Shape of absorption spectrum depends on electronic transitions that occurs in each organic molecule. Absorption spectrum characterizes each molecule. It has characteristic shape which shows the of maximum absorbance ( max). max is characteristic for each molecule, therefore it is used for identification of a chemical substance (i.e. qualitative analysis). Also max is used for quantitative measurement, in order to increase sensitivity and to minimize error of the analytical method.
N.B Sometimes, absorption spectra may show a shoulder or even no absorption characteristics.
Shifting of max
This feature usually happens due to change in configuration of chromophore or auxochrome groups.
1- Effect of pH on absorption spectrum The spectra of compounds containing acidic (phenolic-OH) or basic (-NH2) groups are dependent on the pH of the medium. Phenol and aniline are typical examples.
Phenol
The U.V spectrum of phenol in acid medium is completely different from its spectrum in alkaline medium. In acid medium the benzenoid form is the predominant species; represented as phenate anion.
The spectrum in alkaline medium exhibits bathochromic shift (red shift) due to participation of the pair of electrons of oxygen in resonance with the -electrons of aromatic ring, thus increasing the delocalization of the -electrons, leading to the formation of conjugated system (quinonoid structure); i.e. electrons become more energetic and need less energy to be excited, therefore absorb longer and hyperchromic effect is observed.
Aniline
Aniline behaves like phenol, i.e. its spectrum exhibits bathochromic shift and hyperchromic effect in alkaline medium due to its conversion to the quinonoid species, while in acid medium its spectrum exhibit hypsochromic shift and hypochromic effect due to its conversion to the benzenoid species.
Note: Generally, increase in conjugation leads to increase in absorbance of light by a compound, which appears colored and exhibits hyperchromic effect.
On running U.V spectrum of known concentration of phenol as a function of pH (i.e. at different pH). The spectrum will be shifted to different max by changing the pH, but all spectra intersect at certain which is known as isosbestic point. The isosbestic point is the wavelength at which the same absorbance is given for the same concentration at different pH (i.e. at this point absorbance is not pH dependent but concentration dependent).
When a monochromatic light having intensity (I0) is allowed to pass through absorbing medium, some is absorbed (Ia), reflected (Ir), transmitted (It), refracted (If) and scattered (Is). i.e. I0 = Ia + It + Ir + If + Is
Is = zero for clear solution, while If and Ir may be canceled by means of control cuvette containing the solvent in which the substance to be anaylsed is dissolved. Therefore, under experimental conditions: I0 = Ia + It Or Ia = I0 - It
B) Beer's Law:
When a monochromatic light enters an absorbing medium, its intensity is decreased exponentially with the increase of the concentration of the absorbing medium, when (b) is constant. i.e log I0 / It C or log I0 / It = KC
Beer-Lambert's law:
According to lamberts' law log I0 / It b (thickness or pathlength) According to Beers' law: log I0 / It C (Concentration) i.e. log I0 / It bc or log I0 / It = abc or A = abc where: A = log I0 / It = absorbance a is a constant, known as absorptivity which is the absorbance, when thickness of solution is unity (i.e. l cm) and concentration is unity. Molar absorptivity or epsilon () If the unit of concentration is 1M, a is known as molar absorptivity or epsilon () or molar extinction coefficient. (Unit of is L mol1- cm1-)
A (1% - 1cm): If unit of concentration is 1%, a is known as A (1 %, lcm). Absorptivity a, can be calculated from the slope of the curve produced on plotting (A) as function of (C) at fixed (b). When (b) is 1 cm. A = a C or a = A/C Both and A (1%, 1 cm) are characteristic for each substance and are used for qualitative purpose.
Colorimetry
Principle: in colorimetry we are measuring absorbance A of visible radiation by a colored sample under study. To be measured colorimetrically, the substance has to fit one of three categories: 1- The substance to be measured must be colored e.g CuSO4, KMnO4, organic dyes, etc. 2- If the substance to be analysed is colorless, it must be reacted first with certain reagent (known as chromogen) to produce equivalent colored product e.g. orthophenanthrolene which reacts with ferrous (Fe2+) in buffered medium (acidic pH) to produce intense red color.
3- If the substance to be analysed is colorless and there is no suitable chromogen, it must be converted to a certain derivative which has a suitable chromogen. e.g. in determination of ester, which is first converted to hydroxamic acid derivative through the reaction with hydroxylamine. Hydroxamic acid derivative gives purple color on addition of ferric (Fe3+) due to the formation of iron chelate.
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What are the requirements for the colored product? It should be of intense color, to increase the sensitivity of measurement. The reaction of color formation have to be rapid and quantitative. It should be unaffected by pH or the pH must be specified and maintained by suitable buffer or the measurement is carried out at of isosbestic point. It should be stable with time. The colored product, should obey Beer-Lambert's law, i.e. on plotting A versus C at fixed b, we obtain straight line passing through the origin.
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The full development of color must be rapid. Produces only one color of specified max. It should be selective (i.e. reacts only with the substance to be analysed).
Instrumentation
1- Visual methods: which are applied only in case of colored samples. It is also called visual colorimetric methods.
2- Photo-electric methods: which are applied in both colored and colorless samples . It is also called photo-electric colorimetric methods.
I. Visual methods Human eye is the detector in this case and its sensitivity varies with wavelength. Long observations weaken the eye sensitivity. a. Standard series method. (using polychromatic light) Principle: This method is based on the comparison of the colored sample with standard series of colors. Standard colors may be freshly prepared or a permanent colored system: For freshly prepared standards, matched Nessler tubes are used. For permanent colored system colored disc, lovibond comparator or sealed ampoules are used
i.e. we Compare a sample with a solution of known concentration of the same sample under investigation (standard solution) till the intensity of It emerging from sample and standard solution are the same, then we calculate the concentration of the unknown sample from the equation above.
It depends on using a dual matched optical system made of glass plungers (p); the depth of their immersion bring about changes in the thickness (b) of colored solutions of the sample
Intensity of emerging light (It) is, proportional to thickness (b) and concentration of the sample (C2) is given by:
C2 = Cl (b1/b2)
2- Hehner tube: designed to withdraw the solution from the more concentrated solution, until the color of the sample and that of the standard solution are matched.
This method depends on the photoelectric phenomenon, where, the intensity of EMR is measured through the intensity of electric current produced by electrons liberated from a
The instrument used consists of 5 basic components Radiant energy source (light source). Dispersing system (or monochromator).
Light source
Monochromator
Cuvette
Detector
Recorder
- It converts polychromatic light to monochromatic light, i.e. of definite range or . - Monochromatic light may be obtained by one of the following systems:
a)- Filters They function by selective absorption of unwanted and transmit the complementary color, which is needed to be absorbed by the sample to be analysed. Complementary colors can be represented by colored wheel or the following table:
Colour Violet Blue Greenish blue Bluish green Green Yellowish green Yellow Orange Red Wavelength () 400-435 435-480 480-490 490-500 500-560 560-580 580-595 595-610 610-750 Complementary colour Yellowish green Yellow Orange Red Purple Violet Blue Greenish blue Bluish green
Notes: - If a substance absorbs all visible light it will appear black, and if a substance doesn't absorb visible light, it will appear colorless. - Filters may be: gelatin, liquid and tinted glass. - These types of filter transmit a wide band of 35-50 nm which is not exactly monochromatic. - A narrower band can be obtained by using interfering filters, which consist of successive multiple layers of high and low refractive index material (e.g. MgCl2 and Ag film). Interference results in a narrower band of 10-17 nm.
b)-Prisms They act by refraction of light. Glass prism is used in visible range, while in U.V range we use prism made of quartz or fused silica.
R O Y G B I V
c)-Grating This one acts by diffraction and interference. - Grating consists of a large number of parallel lines ruled very close to each other on a highly polished surface e.g aluminum or aluminized glass (600 line/mm). - Each ruled groove functions as a scattering center for light rays falling on its edge. - Through diffraction and interference, the grating disperses the light beam into almost single .
Associated optics
3. Sample compartment (Cuvette) Cuvette is made of glass for visible range and quartz or fused silica for U.V range. Its standard path length is 1cm (10 mm) and sometimes it is 1/2 cm.
Transparent surface Obaque surface
4. Light Detector There are two types of detectors: A- photocells (Photovoltaic cell) e.g Barrier layer cell Principle: light falls on a semiconductor surface, where electrons are excited and produce EMF (current) proportional to the intensity of incident light. N.B. This type requires no external source of power.
Light falling on cell
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Metal base Plate of iron
B) Phototube (photomultiplier or photoemissive tube) In order to obtain greater sensitivity when the light signal is weak, multiplication of the initial photoelectrons by secondary emission; using several anodes arranged in gradually increasing potential is implemented. i.e. (greater sensitivity is obtained through magnification of EMF produced), N.B. this type requires external source of power.
5. Recorder {meter} The amplified electric signal produced in detector is fed to a sensitive galvanometer, its scale is graded in absorbance or/and transmittance units.
Commercial instruments
In this type, fluctuations in source intensity of EMR source are automatically cancelled due to the two cell set up.
3. Prism spectrophotometer
2) Instrumental deviations This type of deviation may be irregular or regular a. Irregular deviations, may result from: 1. Use of unclean optics (lenses, mirrors or lamp) 2. The use of unmatched cuvette (due to industrial defects) 3. Use of unclean handling (e.g finger print on the cuvette)
b. Regular deviations, may result from: 1. Error in scale. 2. Error in slit width control (maximum opening of slit leads to non-specific ). 3. Error in potentiometric reading of absorbance. 4. Non-linear response of detector which gives calibration curve that doesn't pass through the origin. 5. Stray light: - Stray light is any radiations or other than that absorbed by the sample or any light that reaches the detector without passing through the sample. - It may result from, incorrect choice of filters, the presence of fluorescent impurities, aging of mirrors and light source. N.B. Radio & T.V waves also interfere, hence avoid their presence close to the spectrophotometer..
3) Chemical deviations
These include: effect of pH (which leads to shifting of max), temperature effect and, time factor (the obtained color may fade by time due to deterioration by oxidation, reduction, hydrolysis, etc .... ).
Applications
1. 2. 3.
1- Qualitative analysis Absorptivity ( or A (1% - lcm), max., U.V and visible absorption spectrum usually give finger print of the sample to be analysed, hence could be used for its identification.
2- Quantitative analysis a. Quantitative analysis of a single component. For quantitative determination of a single component we should consider the following: - The substance to be analysed is well dissolved in a suitable solvent, e.g. methanol, ether, water, etc ... - The solvent is used as blank (to cancel its interference if any). - Absorbance readings are taken in the expected range (e.g. 200-400 nm for colorless samples and 400-800 nm for colored samples).
- Detect max of the substance to be analysed after dissolving in a suitable solvent. - Construct a calibration curve by plotting A against C at fixed b using standard series of the same chemical present in the sample, at the characteristic max. - The absorbance of the sample to be analysed is determined under the conditions adopted during construction of the calibration curve. - The concentration of the sample can be determined from the calibration curve.
A
b- Quantitative analysis of multicomponent mixture Consider a mixture of X and Y. For their quantitative analysis, the following has to be considered: 1. The absorption spectrum of X and Y should not show sever overlap. 2. X and Y must be chemically inert to each other. 3. Beer-Lambert's law must be obeyed for X and Y at their characteristic max.
c- Determination of some physical constants e.g. (Determination of pKa of weak acid or pkind. ) Consider the weak acid HB during its dissociation, the following equilibrium is established:
To determine pKa, the absorption spectra of a fixed concentration of HB as function of pH is constructed (as shown in Figure A). Then absorbance is measured as a function of pH at 1, (max of HB) and 2 (max of B) (a shown in figure B). The pH at which the two curves are intersected represents pKa of weak acid.
- The ratio of (Mn+ : L) can be detected by their ratio at the point of maximum or minimum absorbance (as shown in the figure).
2. Effect and detection of impurities Absorbing impurities have disastrous effects in spectrophotometric applications: They cause distortion of the absorption curve of the mother substance leading to over estimation (i.e. higher absorbance); They may also lead to shifting of max. Accordingly it is important to detect their presence.
Detection of impurities
a) Impurity index (I.I.) Construct the absorption spectra of tested sample and reference standard of the same substance contained in the tested sample.
A' and A refer to the absorbance of the sample and reference standard respectively. I.I = (A'1 / A'2) (A1/A2), it must be equal to zero if the sample is 100% pure as the reference.
b) Spectrophotometric Purity Index (S.P.I) S.P.I = (A' 1/ A' 2) / (A1 / A2) this value equals 1 in case of pure samples.
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