Operating the bbe BenthoTorch
bbe BenthoTorch Manual Page 22
After incubation, approximately 3ml of the extract is filtrated with a syringe filter into a cuvette.
The sample is measured with a spectrophotometer first at a wavelength of 665nm and then at
750nm both against the offset of reference 90% ethanol solution. The second measurement at
750nm is used for the compensation of the sample’s turbidity.
In the second step, 3M HCl (30µl for 10ml of extraction volume) is added to the filtrated extract
and the solution is shaken. The solution is incubated for 10 minutes in darkness. This converts
the chlorophyll-a to pheophytin-a. After this process, the extract is measured again in the
spectrophotometer at 665nm and 750nm over the 90% ethanol reference sample also treated
with 3M HCl.
Calculation of the chlorophyll-a concentration:
The concentration of chlorophyll in µg/L can be calculated from a formula:
Equation 1
‘Chlorophyll concentration in µg/L’
A
v665
‘Absorbtion at 665nm before acidification’
A
v750
‘Absorbtion at 750nm before acidification’
A
n665
‘Absorbtion at 665nm after acidification’
A
n750
‘Absorbtion at 750nm after acidification’
V
E
‘Extract volume’
V
p
‘Sample volume’
d ‘Cuvette wideness in cm, typical 1cm’
The concentration of phaeophytin in µg/L can be calculated from a formula:
Equation 2
‘Phaeophytin concentration in µg/L’
To Next Page
Loading...