# How to identify the wavelength to compare absorption at in spectrophotometry?

I boiled spinach solution (ground spinach leaves homogenized in 90% methanol) in solutions of varying pH, and am using spectrophotometry to determine chlorophyll content. My results show changes in lambda max, and all my trials seem to end up with varying wavelengths. I was planning on picking one wavelength to compare absorption between my samples, but due to these variances, I am unsure of how to choose a wavelength. How should I do this? I will attach examples in the changes of lambda max. (Lambda max is 403.0 nm in sample 1 and 410.5 nm in sample 2.) Also, a community member suggested that I use a wavelength at which both chlorophyll a and b absorb light (where they overlap on the absorption spectrum), which is at around 450 nm and 650 nm. The peaks in my results do not match these wavelengths, but would it be okay to compare the absorption levels even if they are not peaks?

• Are you sure that your two spectra are different form one another ? If you repeat the measurement of the first spectrum, are you sure to obtain a maximum at $403$ nm ? If you repeat the second spectrum, are you sure to obtain the maximum at $410$ nm ? Don't you think that both maximums may be identical ? As a scientist, you must know than repeating a measurement never gets exactly the same result. This is part of the so-called statistical analysis. Aug 15, 2021 at 10:21
• Those peak shapes suggest you might be saturating your detector. compare abs at a couple different dilutions to ensure that you are in the linear range for both samples. You might also see a more consistent max wavelength Aug 15, 2021 at 11:26
• What type of spectrophotometer are you using? Don't forget that spinach extract is a complex mixture. You cannot do simple spectrophotometry to follow its degradation. Other pigments besides chlorophyll are interfering. Aug 15, 2021 at 13:26
• Are you following a tested protocol or lab procedure or is this ad hoc? I hope it is not the latter because there are many ways things could go awry. It might help to measure the absorbance spectrum of something that has a known absorbance spectrum, e.g. a potassium permanganate solution or holmium chloride solution.
– Ed V
Aug 15, 2021 at 15:52
• Looks ok to me. Chl does have a Soret band at 400nm ish and characteristic transitions at 680-700nm ish so that is ok. Additionally there are carotenes/xanthophylls that absorb in this region that will confuse things. The size of the absorption is ok (an OD of 2 is ok but not 3) but you do expect variations due to amount extracted. As you have a mixture take absorption as $\pm 3$ nm ish as being the same. Find the carotene absorption spectrum to check against your spectrum and/or remove carotenes somehow from your sample. Aug 17, 2021 at 6:44