Ultraviolet Visible Spectrophotometer What is UV Vis spectroscopy

Ultraviolet – Visible Spectrophotometer

What is UV – Vis spectroscopy? Ultraviolet – visible spectroscopy is a measure of the absorbance of radiation (light) by a sample in the ultraviolet – visible regions in the electromagnetic spectra. It is a type of spectroscopy in which ultraviolet or visible radiation is used to induce electronic transitions (electrons go from ground state to excited state) in the molecules within a sample in order for analysis. It is a very quick, accurate and reliable technique. • UV-Visible spectroscopy measures between 200 nm – 800 nm of the electromagnetic spectra. • Ultra-violet region is between 200 nm – 400 nm. • Visible region is between 400 nm – 800 nm. This the region of the spectra is visible to the naked eye.

What is UV-Vis spectroscopy used for? • For the identification and quantification of a broad range of chemical and biological samples. Including – ØPharmaceuticals – drugs ØFertilisers ØProteins

Principal of UV – Visible Spectroscopy • It is a measure of the intensity of the light (the amount of radiation) after the light has passed through a sample and compares it to the intensity of the light before it has passed through the sample. This is done in order to find the amount of light that is actually absorbed by the sample, i. e. absorbance value. Light entering the sample Light leaving the sample A slight decrease in the colour intensity, which indicates some of the light was absorbed by the sample.

UV – Vis Spectrophotometer A basic spectrophotometer consists of 5 main components – 1. 2. 3. 4. 5. Radiation source Monochromator Sample cell compartment (sample holder) Detector Output display device

1. Radiation Source • For measurements >320 nm (visible region) a tungsten-halogen light source is used. • For measurements <320 nm (UV region) deuterium arc light source is used. 2. Monochromator • Is like a diffraction grating, it is used to select a narrow range of wavelengths to pass through the sample.

3. Sample cell compartment (sample holder) • • • Compartment within the instrument used to ‘hold’ the sample cell. Different types of sample holders can be used, depends on the type of sample being analysed. The cell/container in which the sample is placed is called a cuvette. If measuring in the UV region (200 – 400 nm) a silica/quartz cuvette is used. If measuring in the visible region (400 – 800 nm) a glass/plastic cuvette is used. The cuvette windows must be flat, polished (2 sides frosted 2 clear) and parallel in order for the radiation to pass through.

4. Detector • A detector is used in order to sense the radiation that passed through the sample and than arrives at the sensing surface of the detector. It then provides/converts to an electrical signal which is proportional to the intensity of that radiation. • The most common detectors used in a UV-Vis spectrophotometer are a photomultiplier tube, silicone diode and diode array. 5. Output display device • The results from the detector are passed onto the computer programme for analysis and interpretation. A graph also appears which illustrates the wavelength(s) of light a which the sample absorbs.

How does the spectrophotometer work? • The radiation source i. e. the light bulb, provides the source of light for the absorbance to occur. • The beam of light from the bulb ‘hits’ the diffraction grating (monochromator) and this causes the light to ‘slit’ or separate into its different wavelength. • Only the desired wavelength of light (pre-programmed) reaches the exit slit which than comes in contact with the sample. • This specific wavelength of light than interacts with the sample and the light that is not absorbed by the sample is directed onto the detector.

• The detector than measures the absorbance and transmittance values of the sample. • Absorbance is a measure of the amount of light absorbed by the sample. • Transmittance is a measure of the amount of light that passes through the sample and hits the detector, i. e. the light not absorbed by the sample. • The detector senses the light that is being passed through the sample (transmittance value, light not absorbed) and converts this amount of light into a reading on the digital output display.

UV – 200 – 400 nm Visible - 400 – 800 nm UV-Vis spectroscopy measures the amount of light absorbed by a sample in order to find its specific wavelength. The specific wavelength found allows for the identification of the solution, as every sample/solution absorbs at a specific wavelength. What have I learned? Radiation source, monochromator, sample holder, detector and output display device are the main components of the instrument. Radiation source >320 nm a tungstenhalogen light <320 nm deuterium arc light Samples are held in containers called cuvettes.