UV/VIS SPECTROSCOPY

Application Of Ultraviolet/Visible For IDENTIFICATION AND ESTIMATION of inorganic, organic and biomedical species Is employed primarily for QUANTITATIVE ANALYSIS The function of this instrument is relatively straightforward.

TYPE OF MEASUREMENT The ultraviolet (UV) region scanned is normally from 200 to 400nm, and the visible portion is from 400 to 800nm. Uses a light source emitting in the visible spectrum Two types :single beam and double beam Single beam : requires prior calibration using a standard control Double beam : simultaneously measures the sample and the standard control

Absorbance may be presented as TRANSMITTANCE (T=I/I0) or ABSORBANCE The most commonly used solvents are water, ethanol, hexane and cyclohexane. Absorbance may be presented as TRANSMITTANCE (T=I/I0) or ABSORBANCE (A= log I0/I) The wavelength of maximum absorbance is a characteristic value, designated as λmax The intensity of the reference beam, which should have suffered little or no light absorption, is defined as, I0 The intensity of the sample beam is defined as I

Single beam All the light passes through the sample Measure intensity of the incident light Less expensive Less complicated

Double Beam Light source split into two separate beams Passes through the sample Used for reference

Single beam

Double beam

OPERATING PRINCIPLE Measures a material light reflection, transmission or absorbtion properties. Measure the spectrum of a compound. Operates by passing a beam of light through a sample. Measuring the intensity of light reaching a detector. Spectrum is a graph of intensity of absorbed or emitted radiation by sample verses frequency or wavelength

TEST RELATED Used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.

TEST RELATED Variables must be controlled : The nature of the solvent The pH of the solution Temperature High electrolyte concentrations The presence of interfering substances can influence the absorption spectrum

TEST RELATED DMF (dimethylformamide) THF (tetrahydrofuran) ACETONE H20 ETHANOL

TEST RELATED used in the semiconductor industry to measure the thickness and optical properties of thin films on a wafer. UV-Vis spectrometers are used to measure the reflectance of light Can be analyzed via the Forouhi-Bloomer dispersion equations to determine the Index of Refraction (n) and the Extinction Coefficient (k) of a given film across the measured spectral range.

Film on water

SAMPLE OF TEST The sample compartment provides a light-tight environment that limits the addition of stray radiation. Samples are normally in the liquid or solution state, and are placed in cells constructed with UV/Vis transparent materials, such as quartz, glass, and plastic.

SAMPLE OF TEST From left to right (with path lengths in parentheses): rectangular plastic cuvette (10.0 mm), rectangular quartz cuvette (5.000 mm), rectangular quartz cuvette (1.000 mm), cylindrical quartz cuvette (10.00 mm), cylindrical quartz cuvette (100.0 mm).

SAMPLE OF TEST If we need to monitor an analyte’s concentration over time, it may not be possible to physically remove samples for analysis. Examples : Monitoring industrial production lines or waste lines Monitoring a patient’s blood Monitoring environmental systems

SAMPLE OF TEST Fibre optic probe

ADVANTAGES rapid means of analysis can provide very high precision and accuracy. It is useful for a wide variety of chemicals, and it is non-destructive. It can be used both quantitatively and qualitatively on pure substances.

DISADVANTAGES limited use in analysing mixtures, due to the addition of absorbance. It requires special equipment (a UV light source and UV-transparent sample holders, for example) it is not selective for compounds if they absorb at the same wavelength.

ADVANTAGES DISADVANTAGES System lies in the simplistic design of the instrument. The shutter is the only moving component of a UV-VIS spectrometer. LIGHT SOURCE No single lamp emits all the light wavelengths necessary for analysis. Changing the lamp is a time-consuming process. very quick process compared to other methods of sample detection, such as HPLC. This rapid analysis is achieved only through proper calibration. CALIBRATION require frequent calibrations to retain the accuracy and precision of the instrument. requires the knowledge of the type of sample being analysed. technique is non-destructive to the sample and has a high sensitivity for detecting organic compounds. SENSITIVITY user trying to detect the sample using too wide of a wavelength range or by poor instrument design, cause stray light to occur.