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6. Absorption Spectroscopy Photometry and Lab Diagnostics PS

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6. Absorption Spectroscopy Photometry and Lab Diagnostics PS PS 6. Absorption spectroscopy Photometry and lab diagnostics 6.1 Medical relatedness and purpose of the experiment In this experiment the fundamentals of the procedures of spectral analysis and photometry are being acquired. This techniques usually uses visible light, i.e. electromagnetic waves in the wavelength range from 380 nm (\violet") to 780 nm (\red"). They serve as a qualitative and quantitative analysis of different materials, in particular components of blood. In some cases investigations in the range of infra-red (IR) (Wavelength from 780 nm to approx. 10 µm) and ultra-violet (UV) (10 nm to 380 nm) light are being undertaken. In the experiment you will use a spectrometer, which enables the qualitative and quantitative analysis in the whole wavelength range of visible light. Beside spectrometers, in lab diagnostics one often uses photometers for the quantitative analysis in small wavelength ranges. The chosen wavelength range has to be adjusted to the questioning, such that a photometer can be applied easier but at the same time is less flexible than a spectrometer. With the help of a spectrometer you can, for example, find out in which wavelength ranges a photometer would make more sense for certain areas of application. The basis of all procedures for spectral analysis and photometry is the fact, that the absorption behaviour of many substances give some indication to their composition. E.g. oxygenated blood appears \redder" already with the naked eye than de-oxygenated venous blood. Nevertheless, a distinct assignment of (subjective) color impression to wavelengths is not possible and does not allow a quantitative analysis. In the first part of the experiment, you shall examine the relation between color impressions and wavelengths. The spectra (Dependence of the light intensity on the wavelength) will be made visible on a screen and can be evaluated objectively. At the same time, the spectra will be measured over the whole wavelength range by a detector and presented on a PC, where the data will be further processed. Photometric analyses in medical diagnostics usually are being carried out in a solution. Light is being weakened when travelling through. Besides absorption and scattering due to the analysed substance, absorption and scattering due to the solution and losses due to reflection at boundary layers of the cuvette also contribute to the weakening of the light. One has to account for these 1 2 Absorption spectroscopy disruptive effects when doing the analysis. As a practical quantity for the weakening of light when passing through the solution one introduces the extinction, which for diluted solutions, as a logarithmic measure, is proportional to the concentration of the solute. Therefore, photometry analyses outline an easy and precise method for the determination of concentrations. In this experiment you will learn the fundamentals of absorption spectroscopy and concentration analyses employing extinction measurements using the example of various vitamins. You will often use those kinds of analyses in the frame of the biochemical lab courses later on. 6.2 Execution of the experiment 6.2.1 Preliminary tests: Spectra and Colours Emergence of a spectrum 1 2 3 4 5 6 7 8 Abbildung 6.1: \Base frame" for the set-up of the spectrometer (in the lab, some devices are mounted mirror-inverted). The numbers mean: 1 = Lamp 5 = Slider for holder of the cuvette 2 = variable gap 6 = Prism 3 = Lens 1 (100 mm, with filter) 7 = slider for lens 2 (150 mm) 4 = slider for filter 8 = camera In the first part of the experiment you shall mount a spectrometer stepwise and learn about its different components as well as their advantages against the subjective evaluation of the colour impressions. Them both provide the fundamentals for the understanding of spectrometric and photometric processes. For the build-up of the spectrometer a optical bench is available (compare Fig. 6.1). Several sliders (1-8) are mounted on the bench, of which some have to be equipped during the experiment. In addition a rack with various filters, a holder for the cuvette and a lens can be found at the workstation. Absorption spectroscopy 3 Hints: { With exception of slider 4 and 5 for the filter and the cuvette respectively, the sliders must NOT be moved. If so, the spectrometer has to be readjusted, which is connected to a great time exposure. { Mind, not to touch the lenses, the filter and the cuvette at the polished surfaces. This holds especially for the very expensive prisms. { At the beginning, there are only a lamp (1) with a gap (2) and a lens (3), a prism table (6) and a camera with a screen (8) mounted to the sliders. At the end of the experiment you have to return the device in this exact state. { Please avoid to turn the lamp on and off unnecessarily often. • Connect the power supply for the lamp to a socket and turn on the lamp. The lamp illuminates the gap, which is mounted at the end of the lamp casing. The gap therefore depicts the actual light source for the spectrometer. Since the gap is located in the focal plane of the lens (f = 100 mm), the light coming from the gap becomes parallel behind the lens. 1 This holds only for the horizontal spread of the light. As distinct from the experiment Geometrische Optik/Auge, S. ??, where a circular aperture had been used, the gap is elongated vertically. Therefore the light is not parallel in vertical direction - you can convince yourself about this by putting a sheet into the beam path, holding it vertically first and then horizontally. 1In the holder of this lens there is also a filter. His purpose is to dampen the intense infra-red and red light emitted by the lamp and make the visible spectrum of the lamp a little more regular. 4 Absorption spectroscopy Mount the filter no. 3 on to the slider (4) (Fig. 6.1). Turn the filter, until the reflected light falls on the gap (2) - the filter is now adjusted correctly, i.e. perpendicular to the beam path. Using a white sheet of paper, study the beam in front of and behind the prism. Describe your observations. Also think about the physical effects which occur at the interface between air and glass (prism). Remove the filter again and put it back into the metal rack. Again look at the beam path and describe your observations. The coloured phenomena, which you observe on the sheet behind the prism are denoted as spec- trum, more precisely as the spectrum of light, which passes the prism. It comes into existence due to the fact that the prism refracts light of different wavelengths differently. This effect is denoted as dispersion. Increase of intensity by the use of lenses To achieve a better evaluation of the effects of the filters, you have to increase the intensity of the spectrum. This is done by the use of lenses. The first lens is already mounted. Its purpose is to generate parallel light in the horizontal direction on the one hand and to collimate the light in the vertical direction to increase the intensity on the other hand. • Mount the second lens (focal length f = 150 nm) with the label in direction of the prism on slider no. 7, such that the the middle of the lens is on the same height like the prism. The spectrum on the screen should become more intensive and smaller by now. • Use lens no. 2 to center the spectrum on the slit in the screen: The lens should stand as straight as possible in the beam, the height oh the spectrum is adjustable by changing the height of the lens. • Mount the filter no. 1 on the slider (4). Absorption spectroscopy 5 Filter no.1 basically only allows short-wave light to pass. What colour impression do you have about this light in front of the prism (but behind the filter)? Write down you observa- tions for the colour impression in front of the prism and the (qualitative) composition of the spectrum for this filter in following table. This spectrum is denoted as the transmission spectrum of the filter, because it shows only those wavelength, which the filter allows to pass through (transmit). One after the other, mount the filters no.2 and 3 on the filter holder and note the colour im- pression for the filtered and unfiltered light in front of the prism, as well as the composition of the spectrum behind the prism in the table (3rd and 4th column). Filter Transmission Colour impressi- Qualitative observations on in front of the prism without no. 1 short-wave light no. 2 long-wave light no. 3 intermediate wavelengths Based on your observation, conclude which type of wave lengths are being refracted stronger or weaker. Can you tell which wavelengths the lamp is emitting, extracting the information from these spectra? What colour impression is this light giving you (in front of the prism and without any filters.)? As you have seen, one can match a certain colour impression to an appropriate wave length. The question is, whether an inverse assignment of wavelength to colours is possible as well. • Again, mount the filter no. 1 on the slider (4). • Put the filter slightly diagonal, such that its reflected light can be seen nearby the slit of the lamp. 6 Absorption spectroscopy The filters are build in a way, such that no light is being absorbed, but either gets transmitted or reflected. 2 The reflected light from the filter therefore corresponds to the light of the lamp without the transmitted parts. Put a white sheet of paper to where you can see the reflection of the filter.
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