Spectroscopy is an experimental technique used to measure the concentration of solutes in a specific solution by calculating the amount of light absorbed by the solutes themselves. This is a very effective procedure because certain compounds absorb different wavelengths of light at different intensities. By analyzing the spectrum that crosses the solution, you can recognize the specific dissolved substances and their concentration. The spectrophotometer is the instrument that is used in a chemical research laboratory for the analysis of solutions.
Steps
Part 1 of 3: Prepare the Samples
Step 1. Turn on the spectrophotometer
Most of these devices need to warm up before they can give accurate readings. Start it and let it prepare for at least 15 minutes before putting the solutions in it.
Use this time to prepare your samples
Step 2. Clean the tubes or cuvettes
If you are running a laboratory experiment for the school, you may have disposable material on hand that does not need to be cleaned; if you are using reusable materials, make sure they are perfectly washed before proceeding. Rinse each cuvette thoroughly with deionized water.
- Be careful while handling this material as it is quite expensive, especially if made of glass or quartz. The quartz cuvettes are designed for use in UV-visible spectrophotometry.
- When using the cuvette, avoid touching the edges where the light will pass (usually the clear side of the vessel). If you accidentally touch them, clean the cuvette with a cloth specially designed for cleaning laboratory instruments to avoid scratching the glass.
Step 3. Transfer the appropriate amount of solution to the vessel
Some cuvettes can hold a maximum of 1ml of liquid, while tubes typically have a capacity of 5ml. As long as the laser beam passes through the liquid and not the empty space of the container, you can get precise results.
If you are using a pipette to transfer the solution into the vessel, remember to use a new tip for each sample to avoid cross-contamination
Step 4. Prepare the control solution
It is also known as an analytical blank (or simply blank) and consists of the pure solvent of the analyzed solution; for example, if the sample is composed of salt dissolved in water, the blank is represented by water alone. If you dyed the water red, the white must also be red water; furthermore, the control sample must have the same volume and be stored in an identical container to the one subject to analysis.
Step 5. Dry the outside of the cuvette
Before putting it in the spectrophotometer, make sure it is as clean as possible to prevent dirt particles from interfering. Use a lint-free cloth, wipe off any water droplets, and remove any dust that may have accumulated on the exterior walls.
Part 2 of 3: Run the Experiment
Step 1. Choose a wavelength with which to analyze the sample and set the device accordingly
Opt for monochromatic light (with only one wavelength) to proceed with a more effective analysis. You should choose a color of light that you know for sure can be absorbed by any of the chemicals you think are in the solution; prepare the spectrophotometer following the specific instructions for the model in your possession.
- Typically, during laboratory lessons at school, the problem statement or the teacher provide information about the wavelength to use.
- Since the sample always reflects all the light of its own color, you must choose a different wavelength than the color of the solution.
- Objects appear of a certain color because they reflect particular wavelengths of light and absorb all the others; the grass is green because the chlorophyll it contains reflects all the green light and absorbs the rest.
Step 2. Calibrate the machine with white
Put the control solution in the cuvette compartment and close the lid. If you are using an analog spectrophotometer, you should see a graduated scale on which a needle moves according to the intensity of the light being detected. When the blank is in the tool, you should notice that the needle moves all the way to the right; write down the value indicated in case you need it later; without removing the control solution, return the indicator to zero using the appropriate adjustment knob.
- Digital models can be calibrated in the same way, but they should have a digital display; set the white to zero using the adjustment knob.
- When you remove the control solution, the calibration is not lost; while you measure the rest of the samples, the machine automatically subtracts the white absorption.
- Make sure you use a single blank per run so that each sample is calibrated to the same blank. For example, if after calibrating the spectrophotometer with blank you only analyze a part of the samples and then calibrate it again, the analysis of the remaining samples would be inaccurate and you would have to start over.
Step 3. Remove the cuvette with the analytical blank and verify the calibration
The needle should remain at zero on the scale or the digital display should continue to show the number "0". Re-insert the control solution and verify that the reading does not change; if the spectrophotometer is well adjusted, you shouldn't notice any variation.
- If the needle or display indicates a number other than the zero number, repeat the above procedure with white.
- If you continue to have problems, ask for help or have your device checked by a technician.
Step 4. Measure the absorbance of the sample
Remove the blank and insert the cuvette with the solution into the machine by sliding it into the appropriate recess and making sure it is in a vertical position; wait about 10 seconds until the needle stops moving or the numbers stop changing. Write down the percentage values of transmittance or absorbance.
- Absorbance is also known as "optical density" (OD).
- The greater the transmitted light, the smaller the portion absorbed by the sample; in general, you have to write down the absorbance data which are expressed in decimal numbers, for example 0, 43.
- If you get an abnormal result (for example 0, 900 when the remainder is around 0, 400), dilute the sample and measure the absorbance again.
- Repeat the reading at least three times for each sample you have prepared and calculate the average; this way, you are sure to get accurate results.
Step 5. Repeat the test with the next wavelengths
The sample may have several unknown substances dissolved in the solvent, whose light absorption capacity depends on the wavelength. To eliminate this uncertainty, repeat the readings by varying the wavelength by 25 nm at a time; by doing so, you can recognize the other chemical elements suspended in the liquid.
Part 3 of 3: Analyzing the Absorbance Data
Step 1. Calculate the transmittance and absorbance of the sample
Transmittance indicates the amount of light that has passed through the solution and reached the sensor of the spectrophotometer. Absorbance is the amount of light that has been absorbed by one of the chemical compounds present in the solvent. Many modern spectrophotometers provide data for these quantities, but if you have noted the intensity, you need to calculate them.
- The transmittance (T) is detected by dividing the intensity of light that has passed through the sample by that of the light that has passed through the white and is generally expressed as a decimal number or percentage. T = I / I0, where I is the intensity relative to the sample and I0 that referred to the analytical blank.
- The absorbance (A) is expressed with the negative of the logarithm in base 10 of the value of the transmittance: A = -log10T. If T = 0, 1 the value of A is equal to 1 (since 0, 1 is 10-1), which means that 10% of the light was transmitted and 90% absorbed. If T = 0.01, A = 2 (since 0.01 is 10-2); as a result, 1% of the light was transmitted.
Step 2. Plot the absorbance and wavelength values in a graph
Indicates the first ones on the ordinate axis and the wavelengths on that of the abscissa. By entering the values of the maximum absorbency for each wavelength used, you get the graph of the absorption spectrum of the sample; you can then identify compounds by gathering the substances present and their concentrations.
An absorption spectrum typically has peaks at certain wavelengths that allow specific compounds to be recognized
Step 3. Compare the sample chart with those known for certain substances
Compounds have an individual absorption spectrum and always produce a peak at the same wavelength each time they are tested; from the comparison you can recognize the solutes present in the liquid.
