A spectrophotometry is an experimental technique used to measure the concentration of a solute in a specific solution, calculating the amount of light absorbed by it. This technique is robust because certain compounds will absorb different wavelengths of light with different intensities. By analyzing the light that passes through the solution, it is possible to identify the specific substances dissolved in that solution and what the concentration of these substances is. A spectrophotometer is the apparatus used to analyze solutions in a research laboratory.
Steps
Part 1 of 3: Prepare the samples
Step 1. Turn on the spectrophotometer
Most spectrophotometers need to warm up before they can give an accurate reading. Turn on the device and let it run for at least 15 minutes before using it to read samples.
Take advantage of the warm-up time to prepare your samples
Step 2. Clean the cuvettes or test tubes
If you are doing a lab for school, you may be using disposable test tubes that do not need to be cleaned. If you are using reusable cuvettes or test tubes, make sure they are thoroughly clean before each use. Rinse each cuvette carefully with deionized water.
- Handle the cuvettes with care as they can be quite expensive, especially if they are made of glass or quartz. The quartz cells are designed for use in ultraviolet-visible spectrophotometry.
- When handling a bucket, avoid touching the sides that the light will pass through (usually the transparent sides of the bucket). If you accidentally touch these sides, wipe the bucket with a delicate-task cloth (which is designed not to scratch the glass).
Step 3. Pour the appropriate amount of sample into the cuvette
Some cuvettes have a maximum volume of 1 mL, while test tubes can have a maximum volume of 5 mL. As long as the laser that produces the light is passing through the liquid and not an empty part of the container, you will get an accurate reading.
If you are pouring the samples with a pipette, be sure to use a new one for each sample to avoid cross contamination
Step 4. Prepare the control solution
The control solution contains only the chemical solvent in which the solute to be analyzed is dissolved. For example, if you had salt dissolved in water, the control solution will be just water. If you had stained the water red, the control solution should also contain red water. The control solution must have the same volume as the solution to be analyzed and be stored in the same type of container.
Step 5. Clean the outside of the bucket
Before placing the cuvette in the spectrophotometer, make sure it is as clean as possible to avoid interference from dust or dirt particles. Using a lint-free cloth, wipe off any liquid or dust droplets that may be on the outside of the bucket.
Part 2 of 3: Do the Experiment
Step 1. Choose and determine the wavelength of light with which to analyze the sample
Use a single light wavelength (monochrome color) to make the experiment more effective. The color chosen should be one that is known to absorb one of the chemicals believed to be in the sample solute. Set the desired wavelength according to the spectrophotometer specifications.
- If you are in the school lab, they will most likely tell you what wavelength to set.
- Since the sample will reflect all the light of the same color, the wavelength of the experiment should always be a different color than the sample.
- Objects look certain colors because they reflect light of certain wavelengths and absorb all other colors. Grass is green because the chlorophyll it contains reflects green light and absorbs everything else.
Step 2. Calibrate the instrument using the control solution
Place the control solution in the sample compartment and close the lid. In an analog spectrophotometer, there will be a screen with a needle that moves depending on the intensity of light detection. When the solution is in, you should see the needle move to the right. Record that value, in case you need it later. With the control solution still in the machine, adjust the needle to zero using the set knob.
- Digital spectrophotometers can be calibrated in the same way, only they will give a digital reading. Adjust the control solution to 0 using the adjustment buttons.
- When you remove the solution, the calibration should be maintained. When measuring the rest of the samples, the absorbance of the control solution will be subtracted automatically.
- You should only use one control solution per session, so each sample is calibrated with it. For example, if you apply a control solution to the spectrophotometer, only run a few samples and put back a control solution, the remaining samples will be inaccurate. You would have to start over.
Step 3. Remove the control solution and check the calibration
After the solution is removed, the needle should remain at zero or the digital reading should remain zero. Put the control solution back into the device and make sure there are no changes to the needle or the reading. If the instrument is properly calibrated with the control solution, everything should zero.
- If the needle or reading does not indicate zero, repeat the steps of the calibration process with the control solution.
- If you continue to have problems, seek help or have the device serviced.
Step 4. Measure the absorbance of your sample
Remove the control solution and place the experiment sample in the apparatus. Wait about 10 seconds, until the needle stabilizes or until the digital numbers stop changing. Record the percent transmittance or absorbance values.
- The absorbance is also known as the optical density.
- The more light that is transmitted, the less light the sample absorbs. As a general rule, it is convenient to record absorbance values, which are usually decimal (for example, 0.43).
- If you get an out-of-the-box result (like 0.900 when the others get 0.400), dilute the sample and measure the absorbance again.
- Repeat the reading for each individual sample at least three times and calculate the average of the readings. This way you can get a more accurate reading.
Step 5. Repeat the test with successive light wavelengths
The sample may have multiple unknown compounds, the absorbance of which will vary according to wavelength. To eliminate uncertainties, repeat the readings at 25 nm intervals across the spectrum. This will allow you to detect other chemicals that are suspected to be present as well.
Part 3 of 3: Analyze the absorbance data
Step 1. Calculate the transmittance and absorbance of the sample
Transmittance measures how much light that has passed through the sample has reached the spectrophotometer. Absorbance measures how much light has been absorbed by one of the chemicals present in the solute. Many modern spectrophotometers show both transmittance and absorbance, but if you have recorded intensity, you can calculate those values.
- The transmittance (T) is found by dividing the intensity of the light that has passed through the sample solution by that that has passed through the control solution. It is usually expressed as a decimal or a percentage. T = I / I0 where l is the intensity of the sample and l0 is the intensity of the control solution.
- The absorbance (A) is expressed as the negative of the base 10 logarithm (exponent) of the transmittance value: A = -log10T. For a T value of 0.1, the value of A is 1 (0.1 is 10 raised to -1), indicating that 10% of the light is transmitted and 90% is absorbed. For a value of 0.01, the value of A is 2 (0.01 is 10 to the power of -2); that is, 1% of the light is transmitted.
Step 2. Plot the absorbance versus wavelength values on a graph
The absorbance value is presented on the vertical y axis, versus the light wavelength given for a particular experiment on the horizontal x axis. Graphing the maximum absorbance values for each light wavelength tested displays the absorbance spectrum of the sample and identifies the compounds that make up the sample substance and their proportions.
The absorbance spectrum often has peaks at certain wavelengths that can help identify specific elements
Step 3. Compare the representation of the absorbance spectrum with those known for specific elements
Each compound has a unique absorbance spectrum and will always produce a peak at the same wavelength each time it is measured. By comparing the graph of unknown elements with known compounds, you can identify the solutes in the solution.