1. Principles of the spectrophotometric methods mirka.rovenska@lfmotol.cuni.cz

2. Interaction of light with matter When a beam of light impinges upon a sample: a) some photons may have no interaction with a sample and betransmitted b) some photons may be absorbed by a sample c) some photons may be scattered d) some photons may be reflected The extent of a) – d) depends on the material of the sample and on the wavelength of the radiation In spectrophotometric measurements, c) and d) should by kept to a minimum The intensity of the transmitted radiation (I) is lower than the intensity of the incident radiation (I 0 ) incident radiation reflection s c a t t e r transmitted radiation absorption of radiation I0I0 I

3. Electromagnetic spectrum λ [m]λ [m] 10 -12 10 -10 10 -8 10 -3 10 -1

4. Absorption of radiation Molecules of the sample absorb the photons of a suitable wavelength ( λ ) and change their energy level (state): Molecules of the sample absorb the photons of a suitable wavelength ( λ ) and change their energy level (state): 1) in the microwave and far infrared region, the photons have such a low energy that, if absorbed, can cause only the changes of the rotational energy states 1) in the microwave and far infrared region, the photons have such a low energy that, if absorbed, can cause only the changes of the rotational energy states 2) absorption of photons of the infrared radiation can bring about the changes of the vibrational energy states 2) absorption of photons of the infrared radiation can bring about the changes of the vibrational energy states 3) energy of photons of UV and visible light (VIS) is sufficient to cause the transition of electron to a higher electronic energy level 3) energy of photons of UV and visible light (VIS) is sufficient to cause the transition of electron to a higher electronic energy level http://uk.video.search.yahoo.com/search/video?rd=r1&p=molecular+vibration&toggle=1&cop=mss&ei=UTF-8&fr=yfp-t-702

5. Energy levels of a molecule energy electronic levels 1st excited state of electron vibrational levelsrotational levels UV/VIS IR ΔEr far IR Thus, the change of the electronic state is accompanied by changes of vibrational as well as rotational states!!! ΔE = ΔE e + ΔE v + ΔE r ; ΔE = h ν = hc/ λ ΔE e >> ΔE v >> ΔE r ΔEeΔEe ΔEvΔEv

6. Colour Only those substances appear coloured that absorb VIS radiation Only those substances appear coloured that absorb VIS radiation The colour is then determined by the reflected light (the colour of the substance is complementary to that one which has been absorbed): The colour is then determined by the reflected light (the colour of the substance is complementary to that one which has been absorbed):

7. Chromophores Absorption of visible light can cause transitions of π or n electrons (for transition of σ electrons, UV absorption is necessary); thus, only substances containing π or n electrons can appear coloured Absorption of visible light can cause transitions of π or n electrons (for transition of σ electrons, UV absorption is necessary); thus, only substances containing π or n electrons can appear coloured Groups containing unsaturated centres ( π electrons) and non-bonding electrons are called chromophores – e.g..: Groups containing unsaturated centres ( π electrons) and non-bonding electrons are called chromophores – e.g..: (absorption maximum then moves to a longer λ, i.e. from the UV to the visible region): A compound will absorb in the visible region (and thus appear coloured) if it contains at least several chromophores (absorption maximum then moves to a longer λ, i.e. from the UV to the visible region): The compounds that absorb only in the UV region are NOT coloured (saturated hydrocarbons) The compounds that absorb only in the UV region are NOT coloured (saturated hydrocarbons)

8. Absorption of UV and visible light Routinely in biochemistry, absorption of UV and VIS light (that causes electronic transitions) is measured Routinely in biochemistry, absorption of UV and VIS light (that causes electronic transitions) is measured The UV/VIS absorption is the principle of all the methods that will be discussed from now on The UV/VIS absorption is the principle of all the methods that will be discussed from now on

9. The Beer-Lambert law This decrease of the radiation intensity can be expressed as: T = I/I 0 T is called transmittance and varies from 0 to 1 (0 – 100%); it is the ratio of the transmitted to the incident radiation intensity Compounds with T (in the VIS region) approaching 100%...transparent; 0%......opaque  due to absorption, the intensity of the transmitted light is lower than the transmitted light is lower than the intensity of the incident light the intensity of the incident light incident radiation reflection s c a t t e r transmitted radiation absorption of radiation I0I0 I l

10. Absorbance is then defined as: A = - log T = log I 0 /I Absorbance is then defined as: A = - log T = log I 0 /I The Beer-Lambert law states that the absorbance (at a given λ ) is directly proportional to the thickness of the absorbing layer (l) and to the molar concentration (c) of the absorbing substance: The Beer-Lambert law states that the absorbance (at a given λ ) is directly proportional to the thickness of the absorbing layer (l) and to the molar concentration (c) of the absorbing substance: A = ε λ c l A = ε λ c l ε …molar absorption coefficient [dm 3 mol -1 cm -1 ] = [M -1 cm -1 ] ε …molar absorption coefficient [dm 3 mol -1 cm -1 ] = [M -1 cm -1 ] The law is only true for monochromatic light !!! The law is only true for monochromatic light !!! ε depends on λ and this dependence characterizes the substance ε depends on λ and this dependence characterizes the substance

11. Absorption spectrum Spectrum is the dependence of the intensity of radiation on its wavelength or frequency ( ν = 1/ λ ) Spectrum is the dependence of the intensity of radiation on its wavelength or frequency ( ν = 1/ λ ) Absorption spectrum can be acquired by analysis of radiation (emitted from the source) that has passed through the analyzed substance (by comparing the intensities of the incident and transmitted light) Absorption spectrum can be acquired by analysis of radiation (emitted from the source) that has passed through the analyzed substance (by comparing the intensities of the incident and transmitted light) Absorption spectrum of a given substance is often depicted as the dependence of absorbance or ε on the wavelength of the radiation Absorption spectrum of a given substance is often depicted as the dependence of absorbance or ε on the wavelength of the radiation Spectrophotometry deals with acquiring and analyzing the absorption spectra Spectrophotometry deals with acquiring and analyzing the absorption spectra KMnO 4 A

12. Characteristics of an absorption band Absorption band is characterized by the: wavelength(s) λ max of its peak(s) – usually, A is measured at this λ max by the corresponding ε max λ [nm] Absorbance (relative units) λ 1 max λ 2 max

13. Example 1: the Beer-Lambert law If we know ε (for the compound and given λ ) and the thickness of the absorbing layer (i.e. the width of a cuvette), we can calculate the concentration of the absorbing compound (using the B-L law) from the absorbance measured If we know ε (for the compound and given λ ) and the thickness of the absorbing layer (i.e. the width of a cuvette), we can calculate the concentration of the absorbing compound (using the B-L law) from the absorbance measured E.g.: How many grams of vitamin D 2 are solved in 1 liter of solution, if its absorbance measured in the 2-cm wide cuvette is A 264 = 0,4 and ε for vit. D 2 at this λ is 18,4 [dm 3 mol -1 cm -1 ]. M r of vitamine D 2 is 396. E.g.: How many grams of vitamin D 2 are solved in 1 liter of solution, if its absorbance measured in the 2-cm wide cuvette is A 264 = 0,4 and ε for vit. D 2 at this λ is 18,4 [dm 3 mol -1 cm -1 ]. M r of vitamine D 2 is 396. A = ε λ.c.l  c = A / ( ε λ.l) = 0,4 / (18,4. 2) = 0,01 M A = ε λ.c.l  c = A / ( ε λ.l) = 0,4 / (18,4. 2) = 0,01 M m = c. M. V = 0,01. 396. 1 = 4,3 g m = c. M. V = 0,01. 396. 1 = 4,3 g

14. Calibration graph In practice, it can often be more precise to determine the concentration of the absorbing compound not by calculation according to the Beer-Lambert law, but by construction of the calibration graph: In practice, it can often be more precise to determine the concentration of the absorbing compound not by calculation according to the Beer-Lambert law, but by construction of the calibration graph: we prepare a series of standards of known concentration of the absorbing compound, measure absorbance for each of them and plot the absorbance values against their individual concentrations we measure absorbance of the „unknown“ sample; its we measure absorbance of the „unknown“ sample; its concentration can then be read from the calibration graph

15. Example 2: determination of concentration using calibration graph To determine the protein conc. in the sample, the Lowry protein assay can be used: by the reaction of proteins with the reagent, a coloured complex is formed that absorbs light at λ = 750 nm To determine the protein conc. in the sample, the Lowry protein assay can be used: by the reaction of proteins with the reagent, a coloured complex is formed that absorbs light at λ = 750 nm We prepare several samples of a pure protein, e.g. BSA (bovine serum albumin) so that the final conc. of BSA is 5, 10, 20, 40, 60, and 80 µg/ml. The reagent is added. We prepare several samples of a pure protein, e.g. BSA (bovine serum albumin) so that the final conc. of BSA is 5, 10, 20, 40, 60, and 80 µg/ml. The reagent is added. We measure the absorbance values of these standards and plot them against conc. of BSA (protein): We measure the absorbance values of these standards and plot them against conc. of BSA (protein): Then, we add the reagent to the sample in which we want to determine the protein concentration, measure its A 750 and read the corresponding protein conc. from the calibration graph (e.g. for A = 0,3, the conc. is 50 µg/ml) Then, we add the reagent to the sample in which we want to determine the protein concentration, measure its A 750 and read the corresponding protein conc. from the calibration graph (e.g. for A = 0,3, the conc. is 50 µg/ml) protein concentration

16. Example 3: monitoring enzymatic reactions Using spectrophotometry, we can monitor the increase of the product concentration or the decrease of the substrate concentration, respectively Using spectrophotometry, we can monitor the increase of the product concentration or the decrease of the substrate concentration, respectively Often, reactions using NAD + /NADH+H + as coenzymes are monitored, based on the difference between the absorption spectra of these two coenzymes: Often, reactions using NAD + /NADH+H + as coenzymes are monitored, based on the difference between the absorption spectra of these two coenzymes: E.g: determination of acetoacetate and  -hydroxybutyrate: their ratio in arterial blood reflects the intramitochondrial redox state and is used to assess the energy state of the liver after transplantation or failure: E.g: determination of acetoacetate and  -hydroxybutyrate: their ratio in arterial blood reflects the intramitochondrial redox state and is used to assess the energy state of the liver after transplantation or failure: molární [M -1 cm -1 ] The increase/decrease of NADH concentration per a fixed period is measured at 340 nm and compared with calibration  -hydroxybutyrate + NADH + H + acetoacetate + NAD + molar absorption coefficient wavelength [nm]

17. How can we measure absorbance? Most often by a spectrophotometer that consists of: Most often by a spectrophotometer that consists of: The source usually emits a polychromatic radiation (contains various λ ) from which monochromator (prism or diffraction grating + slit) separates the monochromatic light (one λ ) The source usually emits a polychromatic radiation (contains various λ ) from which monochromator (prism or diffraction grating + slit) separates the monochromatic light (one λ ) As a source of the visible light, the tungsten lamp is used; the deuterium lamp can serve as a source of UV As a source of the visible light, the tungsten lamp is used; the deuterium lamp can serve as a source of UV source prism monochromator sample detector readout system PC slit

18. Spectrophotometers Before we start to analyze the samples, we have to measure the A value of „blank“, i.e. solution containing the same components as the samples (the same buffer, coenzymes…) except for the absorbing substance; its absorbance value must be subtracted from the A values of the samples The solvent should not absorb at the wavelength used for measurement The solutions must not be turbid, must not contain bubbles… The error of measurement is acceptable for A values ranging from 0,1 to 1; if A > 1, we have to dilute the sample and measure A once again!

20. Cuvettes Cuvettes must by made of a material that does not absorb the radiation used for measurement  cuvettes used for measurement in the VIS region can be made of glass, cuvettes used for measurement in the UV region must be quartz Cuvettes must by made of a material that does not absorb the radiation used for measurement  cuvettes used for measurement in the VIS region can be made of glass, cuvettes used for measurement in the UV region must be quartz

21. Measuring absorbance in microplates If we want to measure A values of more samples at once, we can pipette all the samples into the wells of a microplate that can be analyzed in the microplate reader: If we want to measure A values of more samples at once, we can pipette all the samples into the wells of a microplate that can be analyzed in the microplate reader: http://www.moleculardevices.com/pages/instruments/readers_main.html

22. ELISA – sandwich assay ELISA – sandwich assay ELISA=enzyme-linked immunosorbent assay ELISA=enzyme-linked immunosorbent assay Wells of the plate are coated with the antigen (Ag) Wells of the plate are coated with the antigen (Ag) We add the sample in which the (primary) antibody (Ab) against the antigen is to be determined; if the antibody is present, it binds the antigen We add the sample in which the (primary) antibody (Ab) against the antigen is to be determined; if the antibody is present, it binds the antigen After the unbound components have been washed away, the secondary antibody, specific to the primary antibody, is added After the unbound components have been washed away, the secondary antibody, specific to the primary antibody, is added The secondary antibody is enzyme-linked and after addition of a substrate (S), the enzyme (E) catalyzes formation of a coloured product that is measured using spectrophotometer The secondary antibody is enzyme-linked and after addition of a substrate (S), the enzyme (E) catalyzes formation of a coloured product that is measured using spectrophotometer ELISA can also be performed other way round: the wells are coated with the antibody that binds the antigen present in the sample; then, another, enzyme-linked antibody against the antigen is added ELISA can also be performed other way round: the wells are coated with the antibody that binds the antigen present in the sample; then, another, enzyme-linked antibody against the antigen is added positive sample negat. control Ag Ab against Ag secon- dary Ab wash → measurement of A Ab against Ag enzyme-linked Ab E S coloured product E

23. Example: HIV infection screening Presence of anti-HIV antibodies in patient's serum is assessed: Presence of anti-HIV antibodies in patient's serum is assessed: the wells are coated with the antigen – viral protein (e.g. protein of the viral envelope…commercially available) and patient's serum is added; after infection, the serum contains antibodies against the viral proteins and these antibodies bind the antigen in the well the wells are coated with the antigen – viral protein (e.g. protein of the viral envelope…commercially available) and patient's serum is added; after infection, the serum contains antibodies against the viral proteins and these antibodies bind the antigen in the well after washing, the enzyme-linked secondary antibody is added that binds the patient's antibodies; the enzyme then catalyzes formation of a coloured product after washing, the enzyme-linked secondary antibody is added that binds the patient's antibodies; the enzyme then catalyzes formation of a coloured product HIV protein Ab against HIV secondary Ab positive negative

24. Turbidimetry and nephelometry Turbidimetry measures the decrease of the intensity of the TRANSMITTED light compared to the intensity of the incident light that has been caused by scatter on the particles of a SUSPENSION (e.g. precipitate) Turbidimetry measures the decrease of the intensity of the TRANSMITTED light compared to the intensity of the incident light that has been caused by scatter on the particles of a SUSPENSION (e.g. precipitate) Turbidance is defined by analogy with absorbance: Turbidance is defined by analogy with absorbance: Under certain conditions and when using the monochromatic light, turbidance is directly proportional to the concentration of suspended particles and thickness of the layer (analogy of the B-L law) Under certain conditions and when using the monochromatic light, turbidance is directly proportional to the concentration of suspended particles and thickness of the layer (analogy of the B-L law) Turbidance can be measured by spectrophotometers; the detector is thus aligned with the cell and the source of radiation: Turbidance can be measured by spectrophotometers; the detector is thus aligned with the cell and the source of radiation: S = log I 0 /I detector I0I0 transmitted radiation cuvette source

25. Nephelometry measures the intensity of the SCATTERED radiation (scattering is again caused by the suspended particles) that is emitted from the cuvette in the direction perpendicular to the path of incident radiation: Nephelometry measures the intensity of the SCATTERED radiation (scattering is again caused by the suspended particles) that is emitted from the cuvette in the direction perpendicular to the path of incident radiation: The intensity is measured by nephelometers: The intensity is measured by nephelometers: source I0I0 cuvette scaterred radiation detector

26. Applications of turbidimetry and nephelometry These methods can be used to assess clinically relevant proteins in blood: These methods can be used to assess clinically relevant proteins in blood: an antibody against the protein (antigen) is added and formation of immunocomplexes antigen-antibody is monitored an antibody against the protein (antigen) is added and formation of immunocomplexes antigen-antibody is monitored e.g.: e.g.: quantification of CRP (C-reactive protein): its concentration quantification of CRP (C-reactive protein): its concentration increases with all invasive bacterial infections, but rarely with viral infections  it‘s determination is useful to the clinician in evaluating acute phase response quantification of IgG, IgA a IgM: can be helpful in differential diagnosis of chronic liver diseases and in diagnosis of immuno- deficiencies and autoimmune diseases quantification of IgG, IgA a IgM: can be helpful in differential diagnosis of chronic liver diseases and in diagnosis of immuno- deficiencies and autoimmune diseases