MadSci Network: Chemistry

Re: How do you find the acid dissociation constant of plant pigments ??

Date: Sun Jan 23 14:39:14 2000
Posted By: John Christie, Faculty, School of Chemistry, La Trobe University, Bundoora, Victoria, Australia
Area of science: Chemistry
ID: 948458739.Ch
Message:

It is quite possible, and in fact it is the easiest way to find the acid 
dissociation constants for these substances. Here is a procedure that is 
fairly simple. It assumes that you have fairly large quantities of material 
available. It can be scaled down as required:

(1) Make up a large amount of stock solution of the anthocyanin at a fairly 
low concentration, so that when you measure its spectrum, the highest peak 
has an absorbance (optical density) of about 1 in the cells you are going to 
use.

(2) pipette 25 mL of stock solution into a 50 mL volumetric flask, and make 
up the solution with 0.1 M hydrochloric acid. This will produce a fairly 
pure acid form of the dye. Measure the spectrum of the acid form of the dye.

(3) pipette 25 mL of stock solution into a 50 mL volumetric flask, and make 
up the solution with 0.1 M sodium hydroxide. This will produce the pure 
basic form of the dye. Measure the spectrum of the basic form of the dye.

(4) examine the two spectra, and find a wavelength where one form of the dye 
has an absorption peak, and the other form has a much lower absorption. It 
is not a bad idea to use two such wavelengths as a cross-check.

(5) carefully remeasure the optical densities of the two solutions with the 
colorimeter set to this wavelength (these wavelengths).

(6) you must now try to make up a solution whose dye concentration is the 
same, and whose absorbance is equal to (Aacid + Abase)/2. To do this you 
must pipette 25 mL of stock solution into a 50 mL volumetric flask, and add 
buffer solution to make it up to the volume. Adjust for a colour halfway 
between the two forms by eye by adding bits of, say, acetic acid and 
concentrated sodium acetate solution. Then make it up to 50 mL. If the 
absorbances you measure are not quite right, you can make final (careful) 
adjustments with single drops of concentrated hydrochloric acid or caustic 
soda. You can only use a few drops, or the total volume will change 
significantly, affecting the concentrations.

(7) When you eventually have a solution whose absrobances are exactly 
halfway between those of the acidic and basic forms, measure its pH with a 
carefully calibrated pH meter. That pH will be exactly equal to the pKa of 
the dye.

(8) Some of these manipulations are not trivial. Seek advice and help from a 
teacher or trained chemist who is familiar with these sorts of measurements.



Current Queue | Current Queue for Chemistry | Chemistry archives

Try the links in the MadSci Library for more information on Chemistry.


MadSci Network, webadmin@www.madsci.org
© 1995-2000. All rights reserved.