# carotenoid assay

Jonathan B. Marder marder at agri.huji.ac.il
Wed Oct 8 10:26:56 EST 1997

```Laurie Zettler u wrote in message ...
+AD4-I am a 4th year biology student at Wilfrid Laurier.  I am looking for
+AD4-some information on how to measure carotoid concentrations from leaf
+AD4-discs.  I have a method where leaf discs are placed in a test tube and
+AD4-incubated in DMSO for 1 hour at 60 degrees celcius.  The absorbance of
a
+AD4-specific volume of the sample is then taken at 450 nm.  The problem I
+AD4-have now is there being no formula to plug this value into.  This
method
+AD4-is also used for chlorophyll a and b but I have formulas to derive the

Laurie, the 470 nm extinction coefficient for carotenoid
mixtures is usually taken as about 2000 to 2500. That
means in the absence of chlorophyll, all you would need
to do is multiply your A470 by about 4.5 for a carotenoid
concentration in microgram per ml. Probably, the 450 nm
measurement is close enough - anyway, there is considerable
leeway unless we are talking about a particular carotenoid.
The biggest problem is the STRONG absorbance by chlorophyll
in the blue. 450 nm is right in the sloping part of the chl peak.
470nm  is slightly better since it is a chl b absorbance peak,
with not much chl a absorbance. However, this leaves a pretty
huge correction factor. I calculated the following multipliers
from some tables (source unknown) lying around in my lab.
though I'm not sure about its veracity.
A664       A647       A470
Carotenoids        +-1.5       -6.8       +-4.7
Chl A             +-12.64      -2.99       0
Chl B              -5.6      +-23.26       0

You probably recognize the values in the bottom two rows for
calculating the chlorophyll.
The top row is what you need to calculate the carotenoid content
i.e. car +AD0- 4.7+ACo-A4770 +- 1.5+ACo-A664 - 6.8+ACo-A647
BTW, this is designed to give you micrograms/ml in the cuvette.
You will have to plug in the appropriate dilution factor.
I'm not terribly confident about these numbers,
so you should continue to ask around. I've never had a good enough
reason
to verify this further. I should add that typically we get a chl to
carotenoid
ratio around 5:1 with these numbers.

Also, using A450 instead of A470 will make quite a difference since
you would correct more for chl A and less for chl B at this wavelength.
Good luck+ACE-
If you can find a reliable source of coefficients, please let me know.
I would appreciate it.