PS

6. Absorption 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 , i.e. electromagnetic waves in the 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 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 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

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 =

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 .

– 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. What kind of colour impression do you get?

 Think about what wavelengths (“colours”) are contained in the light.

 Can you state the wavelength unambiguously by means of the colour impression? (Remember that colours can be mixed...)

The subjective colour impression is primarily due to the absorption spectrum of the different viewing colourants in the cones of the retina. Such absorption spectra can be calculated with transmission spectra, which are measured with spectrometers. In the following part you will ex- tend the experimental set-up, such that quantitative measurements of transmission spectra become possible. (However not with viewing colourants, but on the basis of other examples).

6.2.2 Quantitative measurements

The detector

In the preliminary tests you could evaluate the spectrum only qualitatively by eye. To be able to perform quantitative measurements, meaning to measure the intensity of the light for all wavelengths (or as small wavelength intervals as possible), one needs a more objective measuring tool than the eye: In our case it is the camera, which is already mounted on slider no. 8.

• Start the computer. Important: Do not turn on the camera (power supply unit), before the computer is fully launched and a dialogue box with the user identification appears!

• Connect the camera via the attached power supply unit to a socket.

• Log in to the computer with the usual user ID.

• Execute the programme Spektrometer on the user interface by double clicking on the symbol (or from the start menu: Start/Programme/Praktikum/Spektrometer).

2They are called interference filters - in contrast so called stained glasses absorb parts of the light and appear to be coloured. Absorption spectroscopy 7

• As soon as the programme is running, click on Start Versuch Spektrometer and chose Monitor on the appearing window.

A diagram should appear on the screen, for which the ordinate is denoted as “Intensitt (Skt)” (meaning in arbitrary units, in german: in beliebigen Skalenanteilen) and the abscissa as “Pixel”. These pixels are the measuring points of the camera: The light with its spectrum goes through a slit in the front panel into the camera where it hits a so-called CCD–row 3. There, distributed over the width of the panel, 256 measurement points (pixel4) for the intensity measurement are located. Those CCD-pixel work in a similar manner like the rod cells in the human eye: They absorb the incoming light and convert it via charge separation into an electric signal. Just like the rod cells, also the pixels of the CCD-row can distinguish only between different intensities but wavelengths (only the relative sensitivity of the pixels changes for different wavelengths). Therefore the different wavelength have to be separated spatially by the spectrometer before.

• Click on the start button. The measurement can be interrupted at all times by clicking on the stop button. Hold the left mouse button longer (around 1 second), if the programme doesn’t react. Due to the high data traffic between the camera and the computer, the mouse is requested less frequently.

• Furthermore, avoid sudden movements and vibrations during the measurement.

The intensities for the individual pixels are now being displayed as a curve in the diagram. The resulting intensity distribution is however still referring to pixels and not to wavelengths.

• Make sure that the spectrum lies as close to the middle of the aperture as possible. This should ensure, that the CCD-row is oriented ideally and captures the whole visible spectrum.

• Move a piece of cardboard or a white piece of paper slowly from the red and blue end into the spectrum and observe the influence on the intensity distribution respectively. As soon as the red or the blue end of the spectrum is being shielded by the cardboard, one should immediately observe the effects in the spectrum. Otherwise, the CCD-row does not capture the whole visible spectrum of light.

• If this is not the case, you can move the second lens (7) on its slider perpendicular to the light beam, by loosening both screws from the slider. Should it be necessary, one can turn the prism just a tiny bit. If you do so, please contact an assistant.

3CCD stands for “charge-coupled device” (Explanation in text). 4PixEL is short for “picture element” 8 Absorption spectroscopy

 At which pixel do you find the blue or the red end of the spectrum respectively?

red end at pixel no.

blue (violet) end at pixel no.

• Check, whether the intensity lies above 3-5 Skt and under 95 Skt (but in general as high as possible) in the whole visible range of the spectrum.

• If this is not the case, you can

– increase or decrease the intensity overall by shifting the light bulb slightly – (if necessary) change the intensity distribution slightly by turning the prism (without shifting the boundaries of the visible spectrum outside the measurement range) – ask an assistant to lend aid.

• Since the calibration depends very delicately on the position of the lamp, the lenses and the prism as well as on the angle between the optical benches, carefully check, that all sliders are screwed firmly on the optical bench. Do not try to twist the two benches against each other, because the angle is important and should remain as it is. The pivot is fixated.

In the current state, the display of the intensity distribution on the screen is not yet usable for a quantitative analysis: One has to assign wavelength dimensions to the pixel entities on the x-axis (wavelength calibration). To begin with a transition measurement is reasonable. In the following, both will be done,

Dark measurement

Due to the open set-up of the spectrometer, the influence of disturbing can not be prevented. To minimize the error in the measurement, one should darken the room (empirically it is sufficient to lower the roller blinds to the level of the tabletop).

• Turn of the light source of the spectrometer.

• Start the measurement and click on the button Dunkelspektrum. The y-axis should be displayed only in the range from 0 to 20 Skt. A line at 5 Skt indicates the value of the stray light, that is barely acceptable.

• Make sure, that the intensity lies beneath this line and darken the room with the help of the assistant, if necessary. Especially in the blue range of the spectrum, this influence should be very small.

• If this has been achieved, stop the measurement (the mapping should change the scale auto- matically) and turn on the lamp again. Absorption spectroscopy 9

Incidental remark: It wouldn’t make any sense, to subtract this dark spectrum from all further measurements, since the stray light rates could change drastically during the experiment. If so, redo the dark measurement.

Transmission measurement

Obviusly, the measured intensity is not equal over the whole spectrum.  Which factors could lead to this behaviour? In which range of the spectrum does the intensity appears the largest to you (see spectrum on the front panel)?

Due to the wavelength dependence intensity in the measured spectrum, one can hardly compare the influence of different filters with each other and even less describe it quantitatively.

• Examine this issue by putting two filters into the beam path one after the other.

It is more sensible to perform a relative measurement without filters on the intensity distribu- tion. This is done by saving the existing intensity distribution (without filters) as a reference and refer the further measurements on this reference measurement:

• Start the measurement.

• Under Messung chose the subitem Referenz. Consequently the current intensity distri- bution is being saved as a reference and being displayed as a additional blue curve to the current measurement respectively.

• Again, put an arbitrary filter into the beam path. With the help of the reference, its effect should be way easier to evaluate by now.

• Inside Anzeige, chose the subitem Transmission. For each measurement point the quotient of the currently measured intensity (red) and the saved reference intensity (blue) is being calculated and indicated as transmission in %. (Check, if the caption of the y-axis has been changed appropriately.) 10 Absorption spectroscopy

 What value should the transmission take on, as long as you don’t put any filter or suchlike into the beam path? What effects can be responsible for variations of this value?

In-between the measurements check regularly if the transmission without any filters continuously holds the correctly expected value. If variations occur, you have to – as described above – settle a new reference. Thereafter, the transmission should hold the expected value again.

Wavelength calibration of the x-axis

A wavelength calibration is necessary, so that a wavelength instead of a number of a pixel is assigned to the x-axis, that is then related to a pixel. With help of the CCD-row one can not measure the wavelength itself, you have to perform this assignment with the aid of several known wavelengths yourself. You have already done a rough qualitative assignment when you did the adjustment of the apparatus, to determine, at which pixels the red and blue ends of the spectrum lie.

• Review your current reference through a transmission measurement without filter.

• Mount the filter no. 3 on the slider (4). This filter should show two distinct edges at known wavelengths in the spectrum. These wavelengths are preset in the programme.

• Record a transmission spectrum of the filter.

• Stop the measurement.

 Set the two cursors on the transmission spectrum at both places, where the transmission adds up 50% of the maximal transmission in the spectrum (if this is not exactly possible, chose the measurement points, that are closest) and the corresponding pixel numbers. If the cursors are not placed on the curve, you put them there by holding the left mouse button and dragging it into place (small round resp. cornered symbol with hairline cross). The respective position of the cursors is displayed in the left part of the screen. You should examine this calibration carefully.

Number of pixel of the left increasing edge:

Number of pixel of the right decreasing edge: Absorption spectroscopy 11

• Click on the button Wellenl¨angenkalibrierung.

• A dialogue box appears, in which you shall enter the number of pixels.

• After putting in the values, confirm the input by clicking on OK. The number of pixels are now assigned to the predefined wavelengths.

With this information the programme calculates a complete wavelength calibration and labels the axis appropriately. The assignment of the wavelengths by this method is accurate to approximately 5 %. For the measurements we perform, this is sufficient, a more accurate calibration is only possible by adding further known wavelength.

Filter transmission curves

By now, you can measure the transmission spectra of the filters quantitatively.

• Again, one after the other, mount all the filters on the filter holder. At the same time, compare the changes in the spectrum on the front panel and on the screen respectively.

 From the transmission curve you can read out, how much percent of the incoming light of the particular wavelength the filter does transmit. In the following table, fill in, in which wavelength ranges the respective filter transmits more than 50 %. (From time to time, check, whether the reference has to be set again!)

Filter transmissions spectra Filter Transmission Colour Impression Wavelength range(s) (> 50% transmission) No. 1 short-waved light No. 2 long-waved light No. 3 medium wave lengths

 The decreasing edge in the blue part of the spectrum of filter no. 1 should lie at 475 ± 10 nm (50% transmission), the increasing edge in the spectrum of filter no. 2 at 600 ± 10 nm. If this is not the case, you should redo the calibration.  Compare the transmission spectra of filter no. 1 with the (qualitative) result, you obtained in the first part 6.2.1 of the experiment. What disagreements do you make out? Can you explain them? (Hint: Think about, where the visible part of the spectrum ends and what part links to that.) 12 Absorption spectroscopy

6.2.3 Fundamentals of spectrometry

In biochemical lab diagnostics, spectrometry and photometry are almost exclusively done in a solution. The containers of the solution, one wants to analyse, are called cuvettes. Cuvettes usually posses two (sometimes four are needed) polished lateral surfaces, through which the light enters. These surfaces are tagged with a small triangle near the opening. They should touch the cuvettes only on the dull sides, to avoid errors during the measurement due to fingerprints and scratches on the polished flats. Furthermore, cuvettes have a precisely prescribed pitch between the polished surfaces. This distance is termed as the layer thickness – it specifies the path distance, which the light travels inside the solution and has to be known exactly for the respective concentrations. For the cuvettes in this experiment, it amounts to 10 mm.

• Mount the cuvette holder (Fig. 6.2) on slider no. 5, such that the aperture of the holder points towards the prism.

• With the help of a sheet of paper, check whether the prism is still fully illuminated, the aperture must not cut off the light strip on the prism on the upper or lower end. If this happens, adjust the height of the cuvette holder.

• Take one of the narrow, empty (“smal”) acrylic glass cuvettes, which are at your disposal in the lab (only touch on the dull sides!) and put them inside the provided notches in the cuvette holder (see Fig. 6.2), such that the polished surfaces face away from the light path.

• The light should hit the surfaces concentrically – as the circumstances require, turn the cuvette holder, until this is ensured.

Large cuvette

Light incidence

Slot for small plastic cuvette

Aperture

Abbildung 6.2: Scheme of the cuvette holder; the polished surfaces of the cuvettes has to be situated inside the light path.

Hints: Absorption spectroscopy 13

– Due to the fact that from now on you will work with coloured solutions, please perform all experiments calmly and carefully! We can’t take any responsibility for blotches on clothes or other personal items!

– Do not pour any solutions on the optical elements or computers, keyboards, etc.!

– The cuvettes should be filled sufficiently, such that no light can enter the detector from above the fluid through the cuvette. However don’t fill them up to the rim, to avoid an overflow of the fluid.

– Don’t fill the cuvettes at the experimental workstation, but rather at the designated table.

– Apart from pure distilled water, don’t pour the solutions away after the measurements, but fill them back into the bottles and return those to the assistant.

– Bottles, pipettes and funnels are characterised in terms of colour, to avoid any possibility of confusion and mixing of different solutions. Hence, always memorize the current solution that is inside the pipette...

– Although that the solutions are about vitamins: Do not take them in – they pro- bably won’t do you any good. Most probably. Just saying.

The reference spectrum

As you have seen in the previous experiments, transmission measurements always has to be per- formed in relation to a reference spectrum. As distinct to before, additionally a cuvette is placed inside the beam path now. While the cuvette appears to be completely transparent and colourless to us, it can absorb strongly which often doesn’t happen in the visible range of light (there exist special materials for studies with infra-red and radiation respectively).

• Take the cuvette (not the cuvette holder!) out of the ray path and start the measurement.

• Settle the measurement as a reference spectrum by clicking on the button Referenz.

• Fill the cuvette with distilled water which is located in the spray bottle.

• Measure the transmission spectrum of the water-filled cuvette relative to the one without a cuvette.

 Are there major differences in the spectra? Are there areas with a very low transmission? How big is the average transmission now?

Transmission of the water-filled cuvette in the visible range:

Apparently the intensity of the light and therefore the measured transmission of the light changes 14 Absorption spectroscopy when passing the (colourless) cuvette and the (colourless) solution. The light, which is not paralle- lized, can be bunched together a little bit at the boundary layers due to refraction. This leads to a small increase of intensity. Lacking intensity is mainly due reflections at the interface acrylic glass – air (similar effects had been observed in the experiment Ultraschall/Sonographie. The reflectivity at a boundary layer depends on the proportion of the refraction indices – glass and water have refraction indices of about 1.5, air of about 1. For each boundary the reflectivity therefore accounts for 4%. In addition to it, there is a further reason, that the reference spectrum has to be the one of a cuvette with solution, which we can’t demonstrate due to temporal limitations: Solutions, just like cuvette materials, can absorb light in certain spectral ranges and therefore sophisticate the measurement. Of course, this also applies to further reagents like enzymes or special stains which are inside the solution. Therefore it always holds that:

Reference spectrum = Spectrum with solvent !

Transmission and extinction

For photochemical precesses, i.e. processes that are induced by light, the determining physical quantity is the absorption of a substance. The dependence of the absorption on the wavelength one denotes as the absorption spectrum of a substance. The spectrum depends on the chemical composition and the microscopic structure of the substance and is therefore specific for the respec- tive substance. The Measurement of the absorption is only possible indirectly, by counting back from the transmission spectrum to the absorption spectrum, For precise measurements one has to include measurements for the the spectra of the reflected and scattered light respectively. In photometry first of all one wants to determine the presence and concentration of a substance inside a solution. Here it is sufficient to measure the weakening of light when it passes the solution. Since the weakening is not exclusively due to absorption, one talks about extinction. For diluted solutions the extinction gets dominated by absorption.

In this part of the experiment you will determine the extinction of a solution of vitamin B12 (Cyanocobalamin) in water as a function of the wavelength. The vitamin plays an important role for the blood formation (haematosis), but is not responsible for its colour as one could suppose based on its colour. The latter is due to the light absorption of the haem (this is an important point for the determination of the oxygen content in the blood with the help of optical methods, s Part 6.3, Physikalische Grundlagen (German version)).

• Keep the cuvette with distilled water from the last experiment – in the following you will need it frequently.

• Take another acrylic glass cuvette and pipet a sufficient amount of vitamin B12–solution into the cuvette.

• Measure the transmission spectrum of distilled water and declare it as your reference. Absorption spectroscopy 15

 Based on the colour impression of the solution, in which wavelength range will the solution absorb light most likely?

 Implant the cuvette with the solution into the holder and record the spectrum. Switch the display on transmission, stop the measurement and print the spectrum (button Drucken). Then glue it on at the bottom.  In which wavelength ranges does the solution absorb?

 Chose a point in this range and measure the wavelength λ and transmission T with the help of the cursors.

Wavelength λ =

Transmission T = Transmission spectrum of vitamin B12. 16 Absorption spectroscopy

When passing through an absorbing medium, the intensity of light decreases exponentially with the travelled distance. This is relation is denoted as the Lambert-Beer–law. To get more practical magnitudes for measurements and calculations, one defines the extinction E at the wavelength λ as the negative logarithm (basis 10) of the transmission T at λ : 5

 I(λ)  E(λ) = − log(Tλ) = − log , IRef(λ) where I(λ) and IRef(λ) is the intensity of the light after transition and the reference respectively. Note that E , as the logarithm of a dimensionless quantity, is dimensionless itself and you can not utilise the value in percent (division by 100).

 From the transmission value above, determine the extinction of vitamin B12 by clicking Ex- tinktion in the menue point Anzeige. Now the extinction is being calculated out of the transmission for the whole spectrum and displayed. Determine the value at the chosen wave- length with the cursor.

Extinction E = − log(T ) =

Monochromatic light sources and filters

To perform photometric analyses it is mostly sufficient to measure the extinction at a specific wavelength. In this case, a wide spectrum as it is being send out from the lamp does often have a distorting influence: In the region of interest, as in this case, the intensity is relatively low, whereas in other wavelength ranges (in our case for long-waved light) the detector almost reaches its saturation. It is therefore of advantage to restrict the spectrum of the light source to the region of interest a priori. This can be done in two ways:

Monochromatic light sources emit light of one wavelength (or in a very narrow range). Mo- nochromatic light can be generated comfortably by electroluminescent diodes or lasers. A disadvantage of this method is though, that the measurement apparatus is kind of inflexible in its applications.

Filters, which have their maximum transmission in the region of interest and filter out all other wavelengths are easy and flexible in application.

 To get a sensitive measurement, one has to restrict the white spectrum of the lamp to a region in that the solution strongly absorbs. Which of the three filters would be qualify for

the vitamin B12 solution?

Filter no.

5Because the transmission is smaller than 1 (=100%), the logarithm is negative – to obtain positive values for the extinction, one multiplies the logarithm by (-1). Absorption spectroscopy 17

• Mount the filter on the slider (4).

• Since the light source, i.e. the lamp plus filter, emit a different spectrum now, you have to reset your reference.

• Use the cuvette with the distilled water and start a measurement (Displaying mode: Spektren).

• Increase the sensibility of the detector in the region of interest up to about 80-95 Skt, by varying the length of exposure with the arrow keys on the left side of the screen. The length of exposure can be altered in steps of 1.25 ms. The maximum intensity should not exceed 95 Skt because otherwise the detector gets saturated. If you reach saturation in the long-waved red range , you can cover the appropriate side of the detector with a sheet of paper.

• Control the height of the background signal, by turning of the lamp shortly and switch on dark measurement.

• As soon as the background measurement and intensity if the signal are satisfactory, do another reference measurement of the solution.

• Replace the cuvette by the cuvette with solution.

 Start a measurement and determine the transmission and extinction at a chosen wavelength.

Transmission T =

Extinction E =

 Does the obtained value matches the one found before?

Even if the improvement of the signal does not appear to be significant for you in this particular case, the advantage of high sensibility for strongly absorbing solutions or in wavelength ranges with low intensities (of the light source) can be tremendous.

• Tip the solution of vitamin B12 back into the bottle and throw it away together with the reference cuvette.

6.2.4 The extinction’s dependence on the concentration

We are going to examine one of the most important properties of the extinction: the dependence on the concentration of the substance one wants to investigate on in the solution. The sample is a diluted solution of vitamin B2 (Riboflavin) in distilled water. The solution is yellowish, hence it will probably absorb light of the complementary colours (blue). 18 Absorption spectroscopy

• The extinction shall be determined at a wavelength from about 400 to 450 nm, that means it is reasonable to use the filter no. 1. Mount it on the slider (4).

• Due to the sensitivity of the following measurement, the very expensive glass cuvettes are going to be used. You’ll find one at your workstation. Please treat them carefully and do not throw them away after the experiment has been done!

• Maximise the intensity in the blue range by adjusting the exposure time, as you did in the preceding part. Again, make sure that the detector doesn’t get saturated completely (max. 95 Skt).

• Acquire the reference spectrum of distilled water in the glass cuvette.

 The following steps shall be done for all the four different concentrations, which are available, starting from the lowest in an increasing manner. Please use only one cuvette for the whole series of experiments: a

1. Pipette the the solution in the cuvette until it is filled up to about 3/4.

2. Write down the concentration of the solution (not the should-be concentration!) in the following table. A list is on display with the solutions.

3. Record a spectrum and measure the extinction (mode Anzeige/Extinktion) with the cursor at an arbitrary, but for all solutions fix, wavelength of 400 - 450 nm, chosen by you. Notate the wavelength in the table.

4. Repeat these steps for all further solutions with increasing concentration.

aAlternatively cuvettes can be prepared for all concentrations. The cuvettes will circulate amongst the groups. Don’t forget to measure reference spectra regularly!

 With the given mass concentrations in µg/ml, calculate the molar concentrations cM (unit 1 M = 1 mol/l) and fill in the values into the third column of the table. The

conversion happens due to division of the mass concentration by the molar mass MM of the substance:

c [g/l] molar concentration: cM [mol/l] = . MM [g/mol]

molar mass of vitamin B2: 376.4 g/mol. Absorption spectroscopy 19

Extinction of vitamin B2 at λ = Solution no. Concentation [µg/ml] Concentration [mol/l] Extinction 1 2 3 4

 In the following graph, plot the extinction against the molar concentration - do to so you first have to set the scales. Fill out the graph with your measurement data as widely as possible (see Appendix, “Auswertung von Messdaten”). You should obtain a straight line.

Extinction

Concentration (mol/l)

Indeed, the extinction depends linearly on the concentration:

E = ε · cM · d, (6.1) where cM is the molar concentration of the solution, d is the thickness of the irradiated fluid (layer thickness) and ε is the so-called (decadal) extinction coefficient. The unit of the extinction coefficient usually refers on the concentration indications in units of 1 M = 1 mol/l and on a layer thickness in cm: 20 Absorption spectroscopy

E 1 1 Extinction coefficient ε = ; Unit [ε] = 1 = 1 . cM · d M cm mol/l cm

 Now, the extinction coefficient shall be calculated. For that, lay a linear fit on your data and determine its slope (units!):

Slope of the linear fit α = ∆E = ∆cM

 With the slope, calculate the extinction coefficient according to Eqn. (6.1). The layer thickness adds up to d = 1 cm (units!):

ε = E = α = cM ·d d

at λ =

• Modify as little as possible on the experimental apparatus, since it’s still going to be used in the next experiment.

6.2.5 Measurement of an unknown concentration by means of photometry

You will perform a determination of concentration on the basis of photometry. The sample is once more a solution of vitamin B2 in water, this time, with unknown concentration. The measurement shall be performed at the wavelength you have chosen in the last measurement. For the calculation of the concentration we’re going to use the value of the extinction coefficient, found by you. This procedure, i.e. the determination of an unknown concentration relative to a standard (solution of a known concentration), measured under the same circumstances, follows the common method in biochemistry.

• Insert the glass cuvette with the solution (distilled water) into slider no. 5 (Fig. 6.2 and specify the measured spectrum as a new reference spectrum.

• Now, fill the cuvette with the solution of unknown concentration and record the transmission spectrum. Absorption spectroscopy 21

 Switch for the display of the extinction and determine the extinction at wavelength you had chosen:

Extinction E =

for wavelength λ =

 Therewith, determine the concentration in mol/l graphically with the help of the reference fit on page 19. Convert the molar concentration into the mass concentration c in g/l (molar

mass MM = 376.4 g/mol).

cM =

c = MM · cM =

 Compare your results with the declaration on the list in the laboratory as well as with the results of other groups – everything correct?

Please clear up you workstation:

• Return the glass cuvettes with the solutions to the assistant.

• Put the lens no. 2 (f = 150 mm), the cuvette holder and the filters back into the metal rack.

• Terminate the Programme “Spektrometer” in the main interface by clicking on Beenden.

• Turn off the computer, the screen, the camera (pull the power supply) and the lamp.

6.3 Physics behind this experiment

This section only exists in the German version of the lab manuals.