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Learning Goals
Learning Goals
Develop a basic understanding of extracting chemicals.
Develop a basic understanding of spectroscopy and its applications, specifically obtaining a calibration curve and using that data to determine the concentration of a chemical in solution.
Apply the skills and concepts you have learned from previous experiments to design a new experiment investigating the effect of one variable on extraction efficiency.
Colored Molecules and Dyes
Colored Molecules and Dyes
Many of the colors that you encounter from day to day come from molecules that absorb or reflect different colors of light. The human eye is designed to perceive light in the "visible" range, which is from ~400 nm (violet) to ~750 nm (red). If an object only absorbs certain wavelengths while reflecting or transmitting others, it will appear a certain color based on the wavelengths of light that are reflected or transmitted. Sometimes these molecules can be extracted from sources, such as fruits and vegetables, and processed into dyes to be used elsewhere.
Visible spectrum showing the approximate relationship between colors and wavelengths of light
Absorbance, Transmission and Spectrometers
Absorbance, Transmission and Spectrometers
As mentioned earlier, molecules can absorb or transmit light. Absorption happens when photons are absorbed by a molecule, and the process of absorbing a photon from the visible range of the spectrum typically causes an electron to be "promoted" or "excited" into a higher energy state. This can only happen if the energy of the photon matches the specific energies needed to promote the electrons to the excited state. Transmission happens when a photon passes through a sample without being absorbed. Transmission can happen for a variety of reasons, most importantly if the energy of the photon does not match any of the specific energies needed to promote an electron to a higher energy state.
Spectrometers are instruments designed to measure the ability of molecules to interact with light. In this experiment, spectrometers are used to measure how much light is transmitted or absorbed through a sample at different wavelengths. Transmission can be measured quantitatively by comparing the amount of light that passes through a sample with the amount that passes through a blank (usually just the solvent used to dissolve the molecule of interest). Transmittance can be converted into absorbance using a logarithm, although the SpectroVis and LabQuest do this automatically (so no calculations for this are necessary).
Basic elements of a spectrometer measuring the absorbance of a sample. Light from a light source is directed through a sample and into a detector. Some of the light is absorbed as it passes through the sample, which reduces the intensity of the beam.
Spectrometer measuring a "blank" (a sample of only solvent). Light passes through the blank and into the detector. Less light is absorbed by the blank than by the sample (above), resulting in a lower absorbance. The final value displayed by the LabQuest (not shown) is the difference between the sample and blank readings.
Calibration Curves
Calibration Curves
In order to determine the concentration of your extract in Part 2 of this experiment, you will need to measure the absorbances of a few solutions of known concentration. Plotting the concentration vs. absorbance will result in a plot known as a calibration curve. The purpose of this data set is to determine a relationship between the concentration of curcumin in a sample and the sample's absorbance. Typically, the maximum absorbance and corresponding wavelength, referred to as λmax, are recorded from the most concentrated solution. The absorbances are measured at the same wavelength for the lower concentrations, even though the maximum absorbance might shift to a higher or lower wavelength. In other words, the absorbances measured for a calibration curve correspond to only one wavelength, λmax, even if the absorbance at another wavelength is greater.
Plot of absorbance vs. wavelength for various concentrations of curcumin calibration solutions. λmax is indicated with the vertical dashed line. As concentration decreases, the peak shifts to a higher wave length, but λmax remains constant.
Your data will be provided in a spreadsheet such that all absorbance values in the same row correspond to the same wavelength.
Data tables with the row corresponding to λmax highlighted
A best fit line can then be established using statistics, computer software, or visually (draw in the line yourself with a ruler). The best fit line can then be used to convert the absorbance from your extract from Part 1 to a corresponding concentration of curcumin. It is worth noting that the relationship between absorbance and concentration is linear, so your calibration curve should be a straight line.
Calibration curve for absorbance vs. concentration. Data points (shown with open circles) are plotted along with a best-fit line. The best-fit line can be used to mathematically or visually determine the concentration associated with the measured absorbance of an unknown.
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