# Determining concentrations with spectrophotometer There are numerous ways to calculate the concentration of unknown samples through spectrophotometry, however using reference standards gives the best accuracy.

If you input the formula, your spreadsheet program will do the calculation for you. IR spectrophotometer: uses light over the infrared range - nm of electromagnetic radiation spectrum.

### How to calculate concentration from absorbance and wavelength

Using the spreadsheet program, fit a linear equation to this graph. In other words, unknown 1 will contain 0. Figure 5 illustrates transmittance of light through a sample. Moreover, the amount of absorbed light is related to the amount of sample, and thus, quantitative analysis is possible by optical spectroscopy. Standards are known concentrations and are used to calibrate equipment and check accuracy. Set the wavelength on the spectrophotometer. If all five fall in the same range as the standards, you can use all five instead, but you need to use at least three. Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications. This instrument measures Transmittance which is the ratio of the transmitted intensity I to the original intensity of light. Any application that deals with chemical substances or materials can use this technique. To calculate the concentration, you need to compare your reading with readings for standards of known concentration. Each unknown sample you inserted into the spectrophotometer was diluted by a different amount. New York: Oxford University Press, Since compounds absorb light in different spectral ranges, the right wavelength must be set for the analysis. If not, then samples will need to be diluted or the concentration of standards increased.

For now, assume nm, although remember that you will need to change this for different experiments. Choose five concentrations for your standards. Spectrophotometry is widely used for quantitative analysis in various areas e.

## Determining concentrations with spectrophotometer

Add your results together, and divide them by the number of results. Tips This procedure may sound complicated, but it's actually fairly straightforward once you've started. Figure 4 shows an example of an isosbestic point. All samples should fall within the working range of the standards. Mark another five test tubes as "Unknown To calculate the concentration, you need to compare your reading with readings for standards of known concentration. Calculating through Straight Line Equations Analyze reference standards at the beginning of the analysis. For instance, a solution sample that absorbs light over all visible ranges i. Figure 2: A single wavelenth spectrophotometer You need a spectrometer to produce a variety of wavelengths because different compounds absorb best at different wavelengths. As always when working in a lab, put on your goggles, gloves and long-sleeved coat to ensure your own safety. Take the equation for the best-fit line from your spreadsheet program and solve it for y by subtracting b from both sides and dividing both sides by m. These procedures may vary depending on the kind of machine you are using, so consult the manufacturer's instructions for details.

Figure 1: Basic structure of spectrophotometers illustrated by Heesung Shim A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer. Photo Credits. Cap each of the standards tubes with parafilm and invert them to mix.

Try watching the two videos under the Resources section to familiarize yourself with the procedure. Rated 8/10 based on 39 review