Absorbation Photometry

							Laboratory practice in Physics___________________________________________________


                             Absorbation Photometry:


Experiment : 6


                                                                    Date:16.02.06
                                                                    Place:Biophysics lab

Theoretical background:
       If light of suitable wavelength is passed through a sample, part of the energy is
transmitted to the molecules. As a result, the emergent beam Fex has less energy than the
incident beam Fin.




                                                          Absorption


The amount of light absorbed generally follows the Lambert-Beer Law and is therefore
proportional to the number of absorbing molecules and the path length traversed (d).

Lambert-Beer Law

Named after the two scientists Johann Heinrich Lambert (1728-1777) and August Beer (1825-
1863), this law states the correlation between the absorbance A, the path length traversed, and
the concentration of the absorbent substance:

A= ·c·d
where the concentration c is stated in mol/l and the path length d in cm. The proportionality
constant, the relative spectral absorption coefficient is a substance-specific function of the
wavelength.

By this law the concentration of a substance dissolved in liquids or gases can be determined by
measuring the absorbance A.




By Marte A Sørensen 2005 / 2006________________________________________________
Laboratory practice in Physics___________________________________________________




Absorbation Photometry

The absorbation spectra of molecules ability to reversible donate or take up protons(s) are
depended on the change of the hydrogen ion concentration.
This change can be observed in the absorbtion spectrum only if the n- and/ or - electrons of
the acid, or alkali group ( wich is involved in the protonation /deprotonation processes) are
part of the conjugated electron system of the molecule.
The spectral changes is caused by changes in the enviroment of the molecules or chemical
reaction wich can appear as a wavelength shift (shorter/longer wavelenght) or as an intensity
shift (increase/decrease)

The ratio of protonated/deprotonated molecules at a given PH or the PH necessary to maintain
a given protonated state can bi determinded from the acid- base dissociation constant Ks

Equation 1:

Ks = H+ D         D and P = consentration of the deprotonated and protonated
       P                           molecule

Equation 2:
                      H+ = proton concentration
pKs = PH- log D
              P

Isobestic point :
A certain wavelength were all the spectrums have the same absorbation value, regardless to
the actual PH.
If use of strong acids or alkali solution is not possible measurments carried out at this
wavelength is an solution to the problem. Though it very often differs from the maximum
absorbation wavelength, wich might lead to a low valueof the absorbation.




By Marte A Sørensen 2005 / 2006________________________________________________
Laboratory practice in Physics___________________________________________________


Determination of the acid –base dossociation constant:

Equation 2:

pKs = PH- log D
              P


The PH values are selected to cover the total PH range characteristic to the dissociation range
of the substance.
Then the specra of the solutions are recorded, and one or two wavelength are selected at wich
the protonated or deprotonaded substance has the maximum absorbation.
If the range of the PH was choosen appropriatly the plots of absorbation verses PH should
give an sigmoid shape.

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


Short description of experiment:
Sample preparation:
   1. Pipette 2 – 2 ml-s of the buffer solutions ( with different PH values ) into each glass
      tubes.
   2. Transfer 20 – 20 l from the fluorescein stock to each tube.

   Measurment
   Record the absorbation spectrum om each solution from 350 to 550 nm. The back ground
   solution is always the appropriate buffersolution. Use the same cuvette for the reference
   and as wll as the sample solution.

   Data evaluation:
   1. Determind the absorbation values at 490 nm as a function of PH, and summarize the
      data in a table format
   2. Plot the 490 nm absorbation values agains PH
   3. Determind the value of the acid – base dissociation constant from the inflexion point
      of the curve, What can be the atomic group involved in attaining/releasing the proton
      in this PH- range?




By Marte A Sørensen 2005 / 2006________________________________________________
Laboratory practice in Physics___________________________________________________


Result:




By Marte A Sørensen 2005 / 2006________________________________________________