DPIP Standard Curve and Calculation of Rate of DPIP Reduction/Rate of Photosynthesis

 

The stock solution concentration of DPIP (2,6-dichloroindophenol) prepared according to the AP Biology Lab Manual (teacher guide) is 0.072 g per liter.  The formula (molecular) weight of DPIP is 290.1 g/mol.

 

0.072 g    X    mole        =   2.48 X 10-4 moles/l = 2.48 X 10-4 M or 0.248 mM

1                                    290.1 g

 

The volume of DPIP used in the standard reaction is 1 ml.  Therefore

 

0.248 mmole    X   0.001 liter   =  2.48 X 10-4 mmoles  or 0.248 mmoles of DPIP are

      1 liter

 

in each ml of DPIP stock solution.  The volume of liquid in each tube is approximately 5.1 ml.  Therefore the concentration of DPIP, if 1 ml of DPIP stock is used is

 

0.248 mmoles   =  48.63 mM   

0.0051 liter                                          

 

To prepare a DPIP standard curve, the DPIP can be diluted into several tubes as shown below. 

           

TUBE #

1

2

3

4

5

DPIP solution

1.5 ml

1 ml

0.75 ml

0.5 ml

0.25 ml

H2O

3.5 ml + 3 drops

4 ml + 3 drops

4.25 ml  + 3 drops

4.5 ml  + 3 drops

4.75 ml + 3 drops

DPIP, mmoles

0.372

0.248

0.186

0.124

0.062

DPIP, mM

72.95

48.63

36.47

24.32

12.16

% T   (OD)

 

 

 

 

 

Absorbance

 

 

 

 

 

                                                                                                           

Read each tube at 605 nm.  Plot the % transmittance and absorbance versus the DPIP (mmoles) and DPIP (mM).  Which graph(s) is/are linear?  See the next page for an explanation!

 

 

 

Using the standard curve of % transmittance versus DPIP (mmoles), you can calculate the rate or photosynthesis in mmoles/minute of your various reaction mixes.

 

 

Concentration, Absorbance and Extinction Coefficients

 

The concentration of an absorbing solute can be related to T through the Beer-Lambert Law, 

 

-log T = e x l x C = A

 

where e is the molar extinction coefficient of the absorbing species at a specified wavelength, l is the length of the light path in cm (centimeters), and C is the molar concentration. e is the absorbance of a molar solution when the light path is 1 cm. The units of e are (M-1cm-1).  e is characteristic of a compound at the specific wavelength under a defined set of conditions. e is also referred to as the absorption coefficient or absorptivity.

 

Since e and l are constant, concentration (C), is proportional to the negative logarithm of T or to absorbance.  For this reason, reading linearly in A is more convenient, and more accurate for samples of high absorbance.  Note that some publications and laboratories refer to A as optical density (OD).  

 

If you are adventurous, you can calculate the molar extinction coefficient for DPIP!  Just plot the absorbance versus the molar concentration of DPIP.  The slope of the line is e, the molar extinction coefficient.