# Relationship between absorbance and cuvette pathlength

### Lambert's Law / Path Length // Confused! - The Student Room

If a sample is measured to have an absorbance of , at a concentration M and in a cuvette with a pathlength of cm, determine the molar extinction. There is a relationship between concentration and absorbance. l = the sample path length measured in centemeters (i.e. the width of the cuvette—almost. I was wondering if measuring fluorescence of DOM on larger cuvette was . Think about the relationship between Absorption and path length as per Beer's law.

What is the extinction coefficient? Suppose you have got a strongly colored organic dye. 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.

## The Beer-Lambert Law

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 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. 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. Generating and Using a Calibration Graph How to Work with Plots You have two different plots, your absorbance spectrum on the left, and your calibration plot on the right. The calibration plot is like taking a vertical slice through the all the absorbance spectra at the specific wavelength nm.

The wavelength nm was a very good choice for the calibration plot, but how do you know which wavelengt is the best wavelength, based on the absorbance spectrum? Choosing Your Wavelength Look at the images above. The left is an absorbance spectrum of 0. Is the slope of the calibration line at nm greater than, less than, or equal to the slope at nm? You can choose any wavelength to create a calibration plot, the only differerence will be the slope of the line.

### Chem - Experiment II

When you actually choose your wavelength to create your calibration graph, you would generally like to choose a wavelength where there is room for the concentration to decrease.

Look at the spectrum above. Do you think nm would be a good wavelength to use for a calibration graph? You would not choose that wavelength because when you lower the concentration, you would not be able to see much of a difference in the absorbance, and the calculations would be inaccurate.

You would most likely want to choose wavelengths like nm or nm where there is a lot of room for absorbance change.

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