UV-VIS SPECTROSCOPY (Handout 2) (CAPE CHEMISTRY - UNIT 2: MODULE 2) Origin of UV-VIS Absorption Molecular absorption of ultraviolet and visible

wavelengths usually forms one or more electronic absorption bands, each of which consists of closely spaced discrete lines. Each line results from the transition of an electron from the ground state to one of the many vibrational and rotational energy levels associated with each excited electronic energy level. The wavelength at which the sample shows maximum absorption is denoted by max, this is the tallest part of the peak for an absorption band. Organic functional groups can be identified by their typical max values while the concentration of a solution can be determined from the amount of light absorbed by a solution. The amount of light absorbed depends on the colour intensity of the solution which is directly related to its concentration. For example a bright red solution has a higher concentration of a particular molecule as compared to a light red solution of the same molecule. Absorption by Organic Molecules Only some types of organic molecules absorb in the UV-VIS region of the spectrum. The wavelengths at which an organic molecule absorbs depend on how tightly its electrons are bonded. Electrons in unsaturated bonds and nonbonded pairs absorb in the UV-VIS region because they are relatively loosely held and are easily excited at these wavelengths. When atoms combine to form molecules, the electrons in the atomic orbitals form molecular orbitals. Electrons may occupy sigma ( ) orbitals, pi ( ) orbitals or non-bonding (n) orbitals. When a sigma bond or a pi bond is formed a higher unfavorable energy level called an anti-bonding orbital is formed along with the bonding orbital. Anti-bonding orbitals are given the symbols * and *. Atomic orbitals with lone pairs or non-bonding orbital are given the symbol n. Molecules in the ground state generally have electrons in the bonding and non-bonding orbitals. Absorption of energy can promote an electron from one of the filled , or n orbital to an anti-bonding * or *orbital. Of all the possible transitions only *, n *and n* normally produce absorption in the UV-VIS region, the others require more energy. Thus only molecules with and/or n electrons give characteristic UV-VIS spectra. Molecules like alkanes will show no absorption in these regions since electrons are only in orbitals and transitions from * and * require energy greater than that of ultraviolet radiation. The structural features of organic molecules which cause absorption of ultraviolet and visible wavelengths are called chromophores. Increasing the extent of delocalization in a system containing double bonds increases the intensity of the absorption and shifts the position of absorption to a longer wavelength. Conjugated compounds have alternating double and single bonds which result in more delocalized electrons, therefore less energy is required for a * transition. If the degree of conjugation is sufficient, absorption can take place in the visible region of the spectrum and the compound may appear coloured. For example, the C=C double bond in ethane absorbs at 175 nm whereas -carotene which has eleven double bonds absorbs at 450 nm causing it to appear bright orange.

Absorption by Transition Metal Complexes Transition metal ions show a wide range of colours. Most of them form complexes when surrounded by coordinating groups called ligands. The interaction between the ligands and the d orbitals of the metal ion causes the d orbitals to have different energies.

The energy E is required for d-d transitions and is in the visible region of the spectrum. Therefore coloured complexes are formed when electrons move from one d orbital to another. The colour of the complex is a complement of the colours absorbed from the visible light. It must be noted that ions with d 0 and d10 arrangement have no d-d transition possible and hence appear colourless. Colourless Compounds Colour intensity is used to determine the concentration of a sample in UV-VIS spectroscopy. However colourless compounds do not absorb visible light and cannot be quantified by direct measurement. In many cases the colourless sample is reacted with a colouring agent to form a highlt coloured complex which absorbs visible light. The colouring agent contains a group which can be attached to the chromophore to modify both the wavelength and intensity of its absorption. This group is called an auxochrome. Many auxochromes contain N or O which possess n electrons which may be promoted into the * anti-bonding orbital of the chromophore. A colouring agent used in the analysis of iron is 1, 10 phenanthroline. The iron in the sample is dissolved in acid and reduced to Fe2+. It is then added to 1, 10 phenanthroline to form an intense red complex which absorbs at 512nm. Similarly ammonium molybdate [(NH 4)6Mo7O24.4H2O] can react with colourless phosphate to form a blue complex in SnCl2 solution, thus allowing phosphate to be determined by UV-Visible spectroscopy. Analyzing Samples by UV-VIS Absorption A UV-VIS spectrophotometer or colorimeter is used to quantitatively measure the amount of light absorbed by a solution. The basic components of a double-beam ultraviolet-visible spectrophotometer are a light source, a monochromator, a beam splitter, sample and reference cells, a detector and an amplifier. The light source is a deuterium or tungsten lamp which supplies a constant amount of light to the sample. The monochromator selects the required wavelength to be absorbed by the sample. The cell or curvetter is a transparent plastic or glass container for the sample or reference. The beam splitter divides the light into two paths, one passes through the sample cell and the other passes through the reference cell. Both paths meet at the detector which compares the two beams and records it. To find the concentration of an unknown sample, the instrument is first set at zero absorbance using a blank sample. Then a series of standard solutions of known concentrations are prepared. The absorbance of each solution and the unknown sample are then measured at a known maximum wavelength. A graph of absorbance versus the known concentration of the series of standards is plotted to generate a calibration curve. The calibration curve is then used to determine the concentration of the unknown sample.

Beer-Lambert Law The intensity of light exiting a solution, (I), is less than the intensity entering the solution, (I 0), because solute molecules absorb some of the energy. The amount of energy absorbed can be expressed in terms of either the transmittance (T) or the absorbance (A). The transmittance is simply the ratio of the exiting to the incoming radiation. T= It is often expressed as a percentage %T = Unfortunately, transmittance is not proportional to the concentration of the absorbing species, thus absorbance which is proportional to the concentration is used. A = - log T = log If there is no absorption of light at a given wavelength, the percentage transmittance is 100 and the absorbance is 0. However, if the sample absorbs all of the light, the percentage transmittance is 0 and absorbance is infinity. A relationship between the light absorbed by a compound and its concentration was derived by Beer and Lambert, which is extremely convenient for quantitative analysis of concentration. This relationship is referred to as BeerLambert law which states that the degree of absorption at a given wavelength of an absorbing compound in a nonabsorbing solvent depends on the concentration of compound and the path length of the radiation. This can be written as: A = cl Where A is the absorbance, is the molar absorptivity constant in dm3cm-1mol-1, c is the concentration in moldm-3 and l is the cell length in cm. Since the intensity of the incident light is decreased after passing through the sample, absorbance can also be expressed as: A = log = cl Where Io is the intensity of the incident light and I is the intensity of the emerging light. When the cell length is 1cm, a plot of absorbance versus concentration shows that the amount of light absorbed is directly proportional to the concentration of the compound absorbing it. The slope is and its value is an indication of the sensitivity of the method. Once has been determined for a particular species, the concentration can be calculated directly from the absorbance. Beer-Lambert law is generally obeyed for dilute solutions. It is assumed that absorbing species behave independently of the solvent and neighboring molecules. Concentrations that are greater than 0.01 moldm-3 can lead in interactions between neighboring molecules resulting in a change in the absorbance characteristics of molecules. In addition, the law only holds for light of a single wavelength or narrow band of wavelengths.

Standard Addition Method (Spiking) This method is usually used to determine the concentration of an analyte in a complex mixture such as biological fluids or soil samples. These mixtures may contain other components that interfere with the analyte signal which can give an inaccurate value for the concentration. The technique involves adding a set of a standard solution containing the analyte to equal volumes of the sample (spiking the sample) and monitoring the change in the instrument signal. The signal is measured for each spiked sample and the data is used to plot a calibration curve. The slope ( ) and the yintercept (absorbance of the sample) are used to calculate the concentration of the analyte in the sample using BeerLambert law. It is assumed that the change in the signal between the sample and the spiked samples is due only to the change in the analyte concentration. Applications of UV-VIS Spectroscopy Qualitative applications of UV-VIS spectroscopy are useful in detecting chromophoric groups. Unkown groups in a compound can be identified by comparing the spectrum with those of molecules containing various chromophoric groups. However sufficient details for it to be used alone to identify a compound is not evident and data must be supplemented with other information such as infrared or mass spectra. Quantitative analysis is widely used to determine the concentration of both organic and inorganic molecules such as glucose and urea in blood, iron in iron tablets and cyanide in water. This method is highly sensitive and can detect substances in the range of parts per million. Very often a wavelength can be found at which the analyte alone absorbs making the method moderately selective. In addition, measurements have good accuracy and are easily performed with modern instruments. Clinical Applications: UV/Vis molecular absorption is one of the most commonly employed techniques for the analysis of clinical samples, several examples of which are listed in Table 10.7. The analysis of clinical samples is often complicated by the complexity of the sample matrix, which may contribute a significant background absorption at the desired wavelength. Industrial Analysis: UV/Vis molecular absorption is used for the analysis of a diverse array of industrial samples, including pharmaceuticals, food, paint, glass, and metals. For example, the iron content of food can be determined by bringing the iron into solution and analyzing using the o-phenanthroline method. Many pharmaceutical compounds contain chromophores that make them suitable for analysis by UV/Vis absorption. Products that have been analyzed in this fashion include antibiotics, hormones, vitamins, and analgesics. One example of the use of UV absorption is in determining the purity of aspirin tablets, for which the active ingredient is acetylsalicylic acid. Salicylic acid, which is produced by the hydrolysis of acetylsalicylic acid, is an undesirable impurity in aspirin tablets, and should not be present at more than 0.01% w/w. Samples can be screened for unacceptable levels of salicylic acid by monitoring the absorbance at a wavelength of 312 nm. Acetylsalicylic acid absorbs at 280 nm, but absorbs poorly at 312 nm. Conditions for preparing the sample are chosen such that an absorbance of greater than 0.02 signifies an unacceptable level of salicylic acid. Forensic Applications: UV/Vis molecular absorption is routinely used in the analysis of narcotics and for drug testing. One interesting forensic application is the determination of blood alcohol using the Breathalyzer test. In this test a 52.5-mL breath sample is bubbled through an acidified solution of K 2Cr2O7. Any ethanol present in the breath sample is oxidized by the dichromate, producing acetic acid and Cr3+ as products. The concentration of ethanol in the breath sample is determined from the decrease in absorbance at 440 nm where the dichromate ion absorbs. A blood alcohol content of 0.10%, which is the legal limit in most states, corresponds to 0.025 mg of ethanol in the breath sample.

Key Terms Measurements Precision: The capacity that analytical method has of giving similar results when it is applied to a sample. There are three main ways of determining precision: 1. Repeatability: repeating the technique under the same working conditions. 2. Intermediate Precision: repeating the technique under different working conditions (different equipment, days and analysts) 3. Robustness: making replicas of the procedure, introducing small changes in operational conditions. These small changes are chosen by the analyst (applied fundamentally in chromatographic techniques). Sensitivity: The parameter through which we can demonstrate the amount of the analyte capable of being detected or quantified by the analytical technique being employed. Detection Limit/Lower Limit of Detection (LOD): The lowest quantity of a substance that can be distinguished from the absence of that substance. Specificity: Parameter that demonstrates that the technique responds only to the analyte and that there are no interferences (in this case it is extremely important to know the possible interferences to determine the specificity). Exactness/Accuracy: The capacity of an analytical method to give results as close as possible to the true value.