If it is in a reasonably concentrated solution, it will have a very high absorbance because there are lots of molecules to interact with the light. However, in an incredibly dilute solution, it may be very difficult to see that it is colored at all. The absorbance is going to be very low. Suppose then that you wanted to compare this dye with a different compound.
Unless you took care to make allowance for the concentration, you couldn't make any sensible comparisons about which one absorbed the most light. The absorption coefficient of a glycogen-iodine complex is 0. Suppose this time that you had a very dilute solution of the dye in a cube-shaped container so that the light traveled 1 cm through it. The absorbance is not likely to be very high. On the other hand, suppose you passed the light through a tube cm long containing the same solution.
More light would be absorbed because it interacts with more molecules. Again, if you want to draw sensible comparisons between solutions, you have to allow for the length of the solution the light is passing through.
Both concentration and solution length are allowed for in the Beer-Lambert Law. Remember that the absorbance of a solution will vary as the concentration or the size of the container varies. Molar absorptivity compensates for this by dividing by both the concentration and the length of the solution that the light passes through.
Essentially, it works out a value for what the absorbance would be under a standard set of conditions - the light traveling 1 cm through a solution of 1 mol dm That means that you can then make comparisons between one compound and another without having to worry about the concentration or solution length.
Values for molar absorptivity can vary hugely. For example, ethanal has two absorption peaks in its UV-visible spectrum - both in the ultra-violet. Table 1 gives values for the molar absorptivity of a solution of ethanal in hexane.
White light is a mixture of all of the wavelengths in the visible range. When light strikes an object, it may be reflected , absorbed , transmitted , or diffracted. A prism or a diffraction grating separates white light into its various colors. If some of the light is absorbed, the reflected or transmitted light has the complementary color of the absorbed light.
A spectrophotometer uses an arrangement of prisms, mirrors, and slits to select light of a desired wavelength and to direct it toward a sample compartment and a detector. The detector electronically measures the intensity of the light striking it. A sample is placed in the light path, and the instrument compares the intensity of the light going through the sample I to the intensity observed without the sample I o.
The effect is measured either as Transmittance T , the percentage of light that goes through the sample or as the Absorbance Abs , representing the amount of light absorbed by the sample :. The Absorbance is seen to be proportional to the number of sheets of the colored material. This is Lambert's Law , the absorbance is directly proportional to the thickness or path length of the absorbing material.
At low concentration, not much of the radiation is absorbed and P is not that much different than P o. If the sample is now made a little more concentrated so that a little more of the radiation is absorbed, P is still much greater than P S.
As the concentration is raised, P, the radiation reaching the detector, becomes smaller. If the concentration is made high enough, much of the incident radiation is absorbed by the sample and P becomes much smaller. At its limit, the denominator approaches P S , a constant.
The ideal plot is the straight line. Spectroscopic instruments typically have a device known as a monochromator. There are two key features of a monochromator. The first is a device to disperse the radiation into distinct wavelengths.
You are likely familiar with the dispersion of radiation that occurs when radiation of different wavelengths is passed through a prism. The term effective bandwidth defines the packet of wavelengths and it depends on the slit width and the ability of the dispersing element to divide the wavelengths.
The important thing to consider is the effect that this has on the power of radiation making it through to the sample P o. Reducing the slit width will lead to a reduction in P o and hence P. An electronic measuring device called a detector is used to monitor the magnitude of P o and P. All electronic devices have a background noise associated with them rather analogous to the static noise you may hear on a speaker and to the discussion of stray radiation from earlier that represents a form of noise.
P o and P represent measurements of signal over the background noise. As P o and P become smaller, the background noise becomes a more significant contribution to the overall measurement.
Ultimately the background noise restricts the signal that can be measured and detection limit of the spectrophotometer. Therefore, it is desirable to have a large value of P o. Since reducing the slit width reduces the value of P o , it also reduces the detection limit of the device. Selecting the appropriate slit width for a spectrophotometer is therefore a balance or tradeoff of the desire for high source power and the desire for high monochromaticity of the radiation. It is not possible to get purely monochromatic radiation using a dispersing element with a slit.
Usually the sample has a slightly different molar absorptivity for each wavelength of radiation shining on it. The net effect is that the total absorbance added over all the different wavelengths is no longer linear with concentration.
Instead a negative deviation occurs at higher concentrations due to the polychromicity of the radiation. Furthermore, the deviation is more pronounced the greater the difference in the molar absorbtivity. As the molar absorptivities become further apart, a greater negative deviation is observed.
Therefore, it is preferable to perform the absorbance measurement in a region of the spectrum that is relatively broad and flat. The peak at approximately nm is quite sharp whereas the one at nm is rather broad. Given such a choice, the broader peak will have less deviation from the polychromaticity of the radiation and is less prone to errors caused by slight misadjustments of the monochromator.
It is important to consider the error that occurs at the two extremes high concentration and low concentration. A relatively small change in the transmittance can lead to a rather large change in the absorbance at high concentrations. At very low sample concentrations, we observe that P o and P are quite similar in magnitude. If we lower the concentration a bit more, P becomes even more similar to P o.
The important realization is that, at low concentrations, we are measuring a small difference between two large numbers. For example, suppose we wanted to measure the weight of a captain of an oil tanker. One way to do this is to measure the combined weight of the tanker and the captain, then have the captain leave the ship and measure the weight again. The difference between these two large numbers would be the weight of the captain. If we had a scale that was accurate to many, many significant figures, then we could possibly perform the measurement in this way.
But you likely realize that this is an impractical way to accurately measure the weight of the captain and most scales do not have sufficient precision for an accurate measurement.
Similarly, trying to measure a small difference between two large signals of radiation is prone to error since the difference in the signals might be on the order of the inherent noise in the measurement.
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