Hi all,
I would like to know if the data, for a given drug, obtained from the UV/VIS Spectrometer obey Beer's law or not.
Is there a general rule?
Also, why an Absorbance Reading above one is not reliable?
Cheers,
Beer's Law is a simple linear proportionality between concentration and absorbance. All you have to do is plot the absorbance of a set of different concentrations of the drug and see if that relationship holds. The slope of the plot of absorbance versus concentration is the extinction coefficient.
It is not a general rule that absorbances above 1 are unreliable. The limitation has to do with the quality of the spectrophotometer. An absorbance of 1 represents a decrease in light intensity of 90%. An absorbance of 2 represents a decrease in light intensity of 99%. The useful range of the absorbance measurement depends on how accurately the instrument can measure low light intensities compared to the unattenuated light intensity, i.e. the dynamic range. This range can also be dependent on the wavelength. Inexpensive spectrophotometers may only be accurate up to absorbances of 1, but higher quality ones may be capable of accurately measuring absorbances of 3.
Bear in mind that the absorbance also depends directly on the path length through the sample, which is determined by the width of the liquid space in the cuvette parallel to the light beam. Most cuvettes have a 1-cm path length, but if you want to measure absorbances of solutions that are too concentrated without diluting them, you can use a cuvette with a shorter path length.
The material of which the cuvette is made can also be a factor. Quartz cuvettes should be used for ultraviolet measurements because cheaper glass or disposable plastic cuvettes are opaque to UV light.
Finally, deviation from Beer's law may only be apparent. If the compound has poor solubility in the solvent, it may form a turbid solution at the concentrations you are testing. Light scattering by the turbid solution will add to the apparent absorbance, causing the deviation. To test for this, measure an absorption spectrum at each concentration. Then normalize all the spectra to 1 at the peak wavelength (that is, divide all the absorbance measurements by the highest absorbance) and overlay them. They should all overlap if there is no turbidity. If there is turbidity, the spectra of the higher concentrations will look different.
Beer's Law is a simple linear proportionality between concentration and absorbance. All you have to do is plot the absorbance of a set of different concentrations of the drug and see if that relationship holds. The slope of the plot of absorbance versus concentration is the extinction coefficient.
It is not a general rule that absorbances above 1 are unreliable. The limitation has to do with the quality of the spectrophotometer. An absorbance of 1 represents a decrease in light intensity of 90%. An absorbance of 2 represents a decrease in light intensity of 99%. The useful range of the absorbance measurement depends on how accurately the instrument can measure low light intensities compared to the unattenuated light intensity, i.e. the dynamic range. This range can also be dependent on the wavelength. Inexpensive spectrophotometers may only be accurate up to absorbances of 1, but higher quality ones may be capable of accurately measuring absorbances of 3.
Bear in mind that the absorbance also depends directly on the path length through the sample, which is determined by the width of the liquid space in the cuvette parallel to the light beam. Most cuvettes have a 1-cm path length, but if you want to measure absorbances of solutions that are too concentrated without diluting them, you can use a cuvette with a shorter path length.
The material of which the cuvette is made can also be a factor. Quartz cuvettes should be used for ultraviolet measurements because cheaper glass or disposable plastic cuvettes are opaque to UV light.
Finally, deviation from Beer's law may only be apparent. If the compound has poor solubility in the solvent, it may form a turbid solution at the concentrations you are testing. Light scattering by the turbid solution will add to the apparent absorbance, causing the deviation. To test for this, measure an absorption spectrum at each concentration. Then normalize all the spectra to 1 at the peak wavelength (that is, divide all the absorbance measurements by the highest absorbance) and overlay them. They should all overlap if there is no turbidity. If there is turbidity, the spectra of the higher concentrations will look different.
Mr. Alzidan, The callibration and validity of BLB law within the linear range are students exercises (BSc grad) studying the following methods: UV, Fs, NMR, EPR, IR, AAS, AES, XRS, etc. This law is nothing other than a linear relationship between the response of the analytical instumentation (signal) and the analyte concentration. Depending on the instrumental method there are different linear ranges of applicability of the BLB law. In other words: basic knowledge from BSc grad in the Chemistry.
Dear Adam B Shapiro,
Thank you for the valuable information.
To be honest, I was just looking for a simple clarification on the sentence "...the obtained data obey Beer's law" because recently I have read a number of articles, like the one I have attached, mentioned this sentence without further explanation of its meaning. However, your reply was of great help in explaining the point of Absorbance reading above 1.
Cheers,
Hi Bojidarka,
I feel sorry you missed the point of my inquiry.
I am neither looking for the principle of Beer's law nor its applications; because I have came across this topic during the 2nd year of my undergraduate study in the College of Pharmacy.
My question was specific; what is the meaning of the following sentence "...the obtained data obey Beer's law"-- which is commonly used in many published articles without emphasizing its meaning.
Regarding my second question, Dr Adam's reply was of great help in explaining this point.
Cheers,
Hi Radhwan,
since Beer's law is a limiting law (see e.g.Article Beer's Law – Why Absorbance Depends (Almost) Linearly on Concentration
), i.e. valid only for vanishing concentrations, I would interpret this sentence in the way that in the concentration range investigated, the increase of absorbance was proportional to the increase of concentration within experimental errors.- Badges
- Science method
- Similar topics
- Methodology
- Absorption Spectroscopy
- UV-Visible Spectroscopy