Broad absorption maximum
Absorption of light by any molecules or atoms is controlled by Beer’s law, whose equation is:
A = a b C (g/l) or A = ε b C (mol/l)
Where (a) is the absorpitivity
(ε) is the molar absorpitivity
(b) path length (i.e. the length that the light goes through in the solution).
λ υ = C
A = a b λ υ
From the relation, we find that Beer’s law is applicable for monochromatic radiation which has single frequency and hence single wavelength
But the spectrophotometers – used in clinical labs – uses a light source that emits a visible light with many frequencies and hence many wavelengths and it is impossible to extract a monochromatic radiation from light source emits continuum radiation of the visible light but you can extract certain band from the visible light and this band is consists of a certain range of wavelengths and frequencies.
So, to make Beer’s law applicable, you should use the band that contains a range of wavelengths of the almost same frequencies and hence same wavelength, thus you can consider as it is a monochromatic light.
But, how can I find this band?
Look at the following the picture
==> In the absorption spectrum, different wavelengths are absorbed to different degrees
In graph (A)
All wavelengths in band (A) are absorbed at the same time with the same degrees and give almost one absorbance but the amount absorbed from this band is changed by the change of the concentration (i.e. the absorbance is changed by the concentration) and you can notice that this band give one concentration reading.
In another word, we can consider this band as monochromatic light because all its wavelength are almost has the same frequency and hence has the same wavelength, therefore we can consider this range of wavelengths as one wavelength and hence monochromatic light.
But in band (B), the wavelengths are absorbed at the same time with different degrees and each wavelength gives different absorbance and hence different concentration readings.
Because photometric estimation of concentration of solutions is depended on Beer’s law and Beer’s law applicable on monochromatic light, so, the band (A) is the band of choice
Band (A) is found at the broad absorption maximum (i.e. the maximum peak of the grap) – we talk about – of the plot……and it is better to measure the concentration of any compound whose absorption vs. wavelength gives a more broad absorption peak.
Thus, when we want to measure the concentration of any biological compound in lab by spectrophotometer, you should use chemicals that react with this compound to produce a colored compound that has a broad absorption maximum to get accurate concentration reading.
In graph (B)
Also, if you measure the absorbance by the band (A) and calculate the concentration of solution then plot a curve between A vs. C, you will get a straight line pass through the origin while you get curved line if you use band (B).
What if the compound hasn’t a broad absorption maximum?
This means, that you will use a band like band (B) and get an inaccurate concentration reading as we explain before and you should try to convert the substance you want to measure into another compound that has a broad band maximum.
In Measurement of Hemoglobin concentration, Why we should convert Hb into colored compound while Hb itself is colored?
Because hemoglobin itself hasn’t broad absorption maximum, so, we should convert it to another colored compound that has a broad absorption maximum.
But, When you use a spectrophotometer in clinical labs, you set the device at a specific wavelength not at a range of wavelengths????
Suppose that the range of wavelengths in the band (A) is from 560nm to 520 nm, to determine the wavelength at which you will measure the absorbance is equal (560+520)/2 = 540nm and this is called Lamda-max (λmax).