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Introduction To Spectrophotometry
Introduction To Spectrophotometry
BACKGROUND
Spectrophotometry is a method of analyzing that involves how light interacts with the atoms (or molecules) in a sample of matter. Visible light is only a small portion of the entire electromagnetic spectrum and it includes the colors commonly observed (red, yellow, green, blue and violet). The visible spectrum consists of electromagnetic radiation whose wavelengths range from 400 nm to nearly 800 nm.
BACKGROUND
white light is observed, what is actually seen is a mixture of all the colors of light
If the substance does not absorb any light, it appears white (all light is reflected) or colorless (all light is transmitted). A solution appears a certain color due to the absorbance and transmittance of visible light. For example, a blue solution appears blue because it is absorbing all of the colors except blue.
BACKGROUND
A sample may also appear blue if all colors of light except yellow are transmitted. This is because blue and yellow are complementary colors. (See the color wheel above.)
BACKGROUND
The amount of light absorbed by a solution is dependent on the ability of the compound to absorb light (molar absorptivity), the distance through which the light must pass through the sample (path length) and the molar concentration of the compound in the solution. If the same compound is being used and the path length is kept constant, then the absorbance is directly proportional to the concentration of the sample.
Spectrophotometer
A spectrophotometer is used to provide a source of light of certain energy (wavelength) and to measure the quantity of the light that is absorbed by the sample.
Prism Detector
Filter
Slit
Spectrophotometer
The basic operation of the spectrophotometer includes a white light radiation source that passes through a monochromator. The monochromator is either a prism or a diffraction grating that separates the white light into all colors of the visible spectrum. After the light is separated, it passes through a filter (to block out unwanted light, sometimes light of a different color) and a slit (to narrow the beam of light--making it form a rectangle). Next the beam of light passes through the sample that is in the sample holder. The light passes through the sample and the unabsorbed portion strikes a photodetector that produces an electrical signal which is proportional to the intensity of the light. The signal is then converted to a readable output that is used in the analysis of the sample.
Prism Detector
Filter
Slit
Spectrophotometer
The spectrophotometer displays this quantity in one of two ways:
(1) Absorbance -- a number between 0 and 2 (2) Transmittance -- a number between 0 and 100%.
The sample for a spectral analysis is prepared by pouring it into a cuvette which looks similar to a small test tube. A cuvette is made using a special optical quality glass that will itself absorb a minimal amount of the light. It is also marked with an indexing line so that it can be positioned in the light beam the same way each time to avoid variation due to the differences in the composition of the glass
Experiment
Viewing the Visible Spectrum
The spectrophotometer is designed to detect absorbances of light at different wavelengths when the light passes through a solution of some given concentration. Some compounds absorb more light at one wavelength than another, so the wavelength must be changed every time a different compound is being analyzed to achieve optimum results from a spectrophotometer. The wave-length of light is selected by adjusting the wavelength dial and read on the wavelength display. In this lab, the color of light associated with each wavelength will be observed with the eye. The visible range of light is approximately 400 to 700 nm. The very ends of the visible spectrum will also be determined in this experiment. Please note that the accepted symbol for wavelength is the Greek letter lambda ().
Spectrophotometer
A cuvette/cuvette rack
Procedure (Best results are obtained by doing this experiment in a dimly lit room) Cut or rub one end of the piece of chalk to produce a 45o angle. Place the piece of chalk in a cuvette with the angle end directed up. Set the wavelength of the spectrophotometer to 425 nm. Be sure the filter switch is set to the left. Place the cuvette in the spectrophotometer so the angle of the chalk faces to the right of the spectrophotometer. Open the light slit by turning the transmittance adjustment knob (right knob) clockwise. Look into the sample compartment and record on the data sheet the color of the light striking the chalk. Repeat Step 5 increasing the wavelength by 25 nm each time. Continue the process until reaching 675 nm. At 600 nm, move the filter lever (#11 in the diagram) to the right. While looking at the piece of chalk, slowly increase the wavelength to the point where the color is no longer seen. This is one end of the visible spectrum. Record this wavelength value. Adjust the wavelength back to 425 nm. While looking at the piece of chalk, slowly decrease the wavelength to the point where the color is no longer visible. This is the other end of the visible spectrum. Record this wavelength value.
6.7 x 1014
450
6.3 x 1014
475
6.0 x 1014
500
5.7 x 1014
525
5.5 x 1014
550
5.2 x 1014
575
5.0 x 1014
600
4.8 x 1014
625
Data Analysis: Now you will plot your data on a graph. (See separate instructions for Excel if you are using a computer to plot your data. Create a scatter graph and then choose the option that connects the dots to draw the curve). Wavelength is plotted on the x-axis and Absorbance is plotted on the y-axis. Remember to put units on your axes and to give your graph a title that tells the purpose of the graph.
Mix 5 drops of red and 5 drops of blue food coloring into a cuvette and then dilute with about 5 ml of distilled water. Mix the solution by carefully tapping the bottom of the cuvette as demonstrated by your teacher. Fill a second cuvette about halfway with distilled water. This will serve as a blank.
Obtain a spectral curve for the food coloring mixture from a wavelength of 350 nm to a wavelength of 675 nm and record the absorbance values in Data Table 1.
Absorbance
525
Wavelength (nm)
Absorbance
375
550
400
575
425
600
450
625
475
650
500
675
350
525
375
550
400
575
425
600
450
625
475
650
500
675