1. 1901 Application of Spectrophotometry
Chemical Analysis
Problem:

2. Organic Compounds
Organic compounds with single bonds absorb in the UV region because electrons from single bonds are tightly held. Electrons in double or triple bonds absorb in the UV and visible region are called chromophores. Chromophores are unsaturated organic functional groups that red shifts if conjugation of two or more chromophores are in a compound.

3. Inorganic Compounds
Inorganic compounds involve transition between d-orbitals. Crystal field theory explains the nature of the transition. Absorption spectra of ion of the lanthanide and actinide series differs substantially from transition metals.

4. Charge Transfer Transitions
An important class of absorption is the charge-transfer which originates from electron-donor group bonded to an electron acceptor group. The excited state results in an internal oxidation/reduction process.
Examples are phenolic complex of iron, pyridyl complexes, the 1,10-phenanthroline complexes, the iodide complex of molecular iodine and the ferro/ferricyanide complex . Some of these transition originate from metal to ligand charge transfer and others are ligand to metal charge transfer.
In some charge transfer process, the excited complex dissociate and produce photochemical oxidation/reduction products.
Iridium bipyridine complexes

5. UV-Vis Spectral Limits
UV-Vis spectra do not have sufficient fine structure to permit unambiguous identification. UV-Vis must be supplemented with other physical or chemical evidence.
Effect of slit width. The spectra shows cytochrome C under four spectral bandwidth (1) 20 nm, (2) 10 nm, (3) 5 nm, and (4) 1 nm.
For qualitative analysis, it is important that the solvent is transparent in the region of the chromophores. The table shows the window for various solvents.
Scattering should also be corrected for. In some instances, false peak will be observed because of operation of the spectrometer to its wavelength extreme. Shown is the spectra of cerium(IV) obtained in glass optical (A) and in quartz (B).

6. Versatility of UV-Vis spectroscopy
Most important characteristics of spectrophotometric and photometric methods :
1. Wide applicability - Inorganic, organic and biochemicals can be analyzed using this technique. 90% of analysis in lab are based on UV-Vis spectroscopy.
2. High Sensitivity - Analysis can be performed with dilute solutions, 10-4 to 10-5 M and at times 10-7M
3. Moderately high selectivity - Can perform analysis without interference of solvent or other chemicals.
4. Good accuracy - The relative error in the technique is in the order of 1% - 5% which can be improved to 0.1 %
5. Ease of convenience - One of the easiest technique to use with minimal setup. The analysis can also be automated.

7. Application to Absorbing and nonabsorbing species
Absorbing Species - Ideal technique if the chemical being analyzed absorbs in region that can be detected.
Nonabsorbing speices - Chemicals that do not absorb, can be reacted with chromophoric reagents to yield products that can be analyze using UV-Vis spectroscopy.
Typical chelating reagent for absorption
(a) diethyldithicarbamante
(b) Diphenylthiocarbazone

8. Procedural Details
Things to consider towards reliable results in absorption spectroscopy:
Wavelength selector - Maximum sensitivity yield good results if wavelength is used in which the absorbance is the largest.
Absorbances factors - Factors such as solvent, pH, temperature, ionic strength... must be controlled to minimize complication of spectra.
Concentration relationship - Standardization plots must match as close as possible the condition of chemical being analyzed.
Standard addition - Matrix effect of can be minimized by introducing into the standards species that approximate the amounts found in the sample via standard addition.
Analysis of mixture - Total absorbance features overlap linearly for two or more components. In principle, the components can be separated to their individual components.

9. Instrumental Uncertainties
Accuracy and precision of spectrophotometric analysis limited by indeterminate errors.
These errors are summarized in the following table. A B C
Error in the measurements of T (transmittance) is constant and independent of the magnitude of T. The type of instrument may give rise to error in the meter read out. Above are graphs showing the error curves for various categories of instrument uncertainties.

10. Error in Transmittance for Different Instruments
The RSD analysis for the two instruments show that absorbance lower than 0.1 are not reliable and should be avoided. The reason is the concentration is directly proportional to the difference in intensity of Po and Pi, through A = log Po -log Pi. At low concentrations, log Pi is nearly large as Po and a small difference in large numbers lead to big errors. Note as well that at absorbance higher than 1.2 is also not reliable and that is because the power of the beam is so low that it cannot be measured accurately.
Experiment curves relating relative concentrations uncertainties to absorbance for two spectrometers. Data obtained with (a) Spectronic 20 and (b) a Cary 118 spectrophotometer.

11. Photometric and Spectrophotometric Titrations
Equivalent points in titration can be determine by spectrophotometric analysis. Analysis requires that one or more of the reactant or product shows absorbance features. Molar absorptivites of the analyte, the product, and the titrant are A, P and T respectively.
Photometric titration curve at 745 nm to 100mL of a solution that was 2.0•10-3M in Bi3+ and Cu2+.

12. Analysis of Complex ions
Composition of a complex in solution can be determined without actually isolating the complex as a pure compound. The techniques used for such studies are (1) method of continuous variations, (2) the mole-ratio method, and (3) the slope-ratio method.
Continuous variation: Method in which the cation and ligand in solutions have identical analytical concentrations with total volume and moles of reactant constant but mole ratio of reactants systematically varied. In the graph shown, the curve maxima is the result of incompleteness of the complex-formation reaction.
Mole-ratio: Method in which analytical concentration of one reactant (usually cation) is held constant with variation of other reactant.
Slope-ratio : Method linear regression is used with Beer’s Law. The method assumes (1) complex-formation reaction is complete with excess of either reactant. (2) Beer’s Law is obeyed. (3) Complex absorbs at desired wavelength.

13. Infrared Spectroscopy
Versatile technique that provides molecular structure information. This makes the technique optimal for qualitative analyses but poor for quantitative analyses. The energy of infrared can excite vibration and rotational transitions, but are not sufficient for electronic transitions. Samples in the solid, liquid and gas phase can be analyzed.

14. IR Instrumentation
There are three types of IR instruments: dispersive spectrometers, Fourier-Transformed (FTIR), and filter photometers. The first two provide complete spectral analysis with the filter photometer designed for quantitative analysis.
Filter Photometers: General use to monitor air particulates and can measure amounts of particles in the gas phase.
Dispersive Instruments: Double beam in which samples are placed between light source and monochromator. The radiation source is generally thermal with the detectors sensitive to the heat rather hand photons.
Fourier Transformed: High speed with great resolution because all wavelengths are detected and measured simultaneously using a Michelson interferometer. The basic idea of this techniques is that the source signal is modulated and passed through the source sample. The resulting signal is an interferogram that is Fourier Transformed to yield all the frequencies that was absorbed by the sample.
Infrared light transmission photometry
Main article: Infrared spectroscopy
Spectrophotometry in infrared light is mainly used to study structure of substances, as given groups give absorption at defined wavelengths. Measurement in aqueous solution is generally not possible, as water absorbs infrared light strongly in some wavelength ranges. Therefore, infrared spectroscopy is either performed in the gaseous phase (for volatile substances) or with the substances pressed into tablets together with salts that are transparent in the infrared range. Potassium bromide (KBr) is commonly used for this purpose. The substance being tested is thoroughly mixed with specially purified KBr and pressed into a transparent tablet, that is placed in the beam of light. The analysis of the wavelength dependence is generally not done using a monochromator as it is in UV-Vis, but with the use of an interferometer. The interference pattern can be analyzed using a Fourier transform algorithm. In this way, the whole wavelength range can be analyzed simultaneously, saving time, and an interferometer is also less expensive than a monochromator. The light absorbed in the infrared region does not correspond to electronic excitation of the substance studied, but rather to different kinds of vibrational excitation. The vibrational excitations are characteristic of different groups in a molecule, that can in this way be identified. The infrared spectrum typically has very narrow absorption lines, which makes them unsuited for quantitative analysis but gives very detailed information about the molecules. The frequencies of the different modes of vibration varies with isotope, and therefore different isotopes give different peaks. This makes it possible also to study the isotopic composition of a sample with infrared spectrophotometry.

15. Spectra Interpretation
IR frequencies for various functional groups

16. Quantitative Applications of IR
IR spectra consist of bands that correspond to infrared absorption. Bands in the shorter wavelength, mm ( wavenumbers, cm-1), are characteristics of functional groups in the structure of the chemical. To truly identify chemical the whole spectrum mm must be analyzed. It may be possible to compare the spectrum to published results in the literature.