Adsorption photometry

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					Laboratory Experiments in Biophysics                                                        02.03.06

                              Absorption Photometry
Theoretical background

The adsorption spectra of molecules having the ability to reversibly donate or take up proton(s)
are shifted upon the change of the hydrogen ion concentration. Substantial change can be
observed in the adsorption spectrum only if the n- and/or π- electrons of the acid or alkali groups
are part of the conjugated electron system of the molecule. Adsorption bands of isolated atomic
groups are less phone to shift upon changing the pH of the medium.

The spectra changes due to the changes in the environment of the molecules or chemical reaction
can appear as a wavelength shift or as an intensity shift.

The ration of protonated/deponated molecules at a given pH or the pH necessary to maintain a
given protonated state can be determined from the acid-base dissociation constant Ks:

                                       pKs = pH – log [D]/[P]

where [D] and [P] is the concentration of the deprotonated and protonated forms of the molecule.

The PH values are selected to cover the total PH range characteristic to the dissociation range of
the substance. Then the spectra of the solutions are recorded, and one or two wavelength are
selected at witch the protonated or deprotonaded substance has the maximum absorption. If the
range of the PH was chosen appropriately the plots of absorption verses PH should give a
sigmoid shape.

The two plateaus correspond to the completely protonated (P) and deprotonated (D) forms, and
pints between the two plateous represent the ratio between them.
To determined the pKs you determined the inflexion point of the curve, were the concentration
of the protonated and the deprotonated form are equal (2nd term in EQ2 becomes zero).
This is the PH value where the change of absorption is exactly the half of the total absorption
change.According to the equation, the PH value of inflexion point gives the value of the pKs.

Ingvild Aarland Eide Group 3                                                                         1
Laboratory Experiments in Biophysics                                                        02.03.06


    1. Pipette 2 – 2 ml-s of the buffer solutions (with different pH values) into each glass tubes.
    2. Transfer 20 – 20 μl from the stock to each tube


    Record the adsorption spectrum of each solution from 350 to 550 nm. The background
    solution is always the appropriate buffer solution. Use the same cuvette for the reference and
    as well as for the sample solution.

Data evaluation:

    1. Determine the adsorption values at 490 nm as a function of pH, and summarize the data
       in a table format
    2. Plot the 490 nm adsorption values against pH.
    3. Determine the value of the acid-base dissociation constant from the inflexion point of the
       cuvette. What can be the atomic group involved in attaining/releasing the proton in this


Ingvild Aarland Eide Group 3                                                                      2
Laboratory Experiments in Biophysics   02.03.06

Ingvild Aarland Eide Group 3                 3

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