The amount of light absorbed by the solutes in the solution is calculated by using a technique called stelephotometry.The technique is powerful because certain compounds absorb different levels of light.You can identify dissolved substances by analyzing the light that passes through the solution.In a laboratory research setting, a spectrophotometer is used to analyze solutions.
Step 1: The spectrophotometer needs to be turned on.
Before they can give an accurate reading, most spectrophotometers need to warm up.Allow the machine to sit for at least 15 minutes before running any samples.Prepare your samples using the warm-up time.
Step 2: The test tubes should be cleaned.
If you are doing a lab for school, you may be using disposable test tubes that don't need to be cleaned.Make sure the test tubes are cleaned before use.Deionized water should be used to rinse eachvette.Cuvettes can be quite expensive and should be taken care of.UV-visible spectrophotometry is possible with the use of Quartz Cuvettes.The light will pass through the clear sides of the container if you touch the sides.If you accidentally touch the sides, wipe them down with a kimwipe, which is formulated to prevent scratching the glass.
Step 3: The volume of the sample needs to be loaded.
Test tubes can have a maximum volume of 5 mL, while some Cuvettes can only have 1 mL.If the light is passing through the liquid and not an empty part of the container, you will get an accurate reading.Use a new tip for each sample if you are using a pipette.
Step 4: Prepare a solution.
The control solution only has a chemical solvent in which the solute to be analyzed is dissolved.If you had salt dissolved in water, your blank would be water.The blank must also contain red water.The blank is the same volume as the solution to be analyzed and kept in.
Step 5: It is a good idea to wipe the outside of the car.
If you want to avoid interference from dirt or dust, you need to make sure the sample is as clean as possible.Dust and water droplets can be removed using a lint free cloth.
Step 6: To analyze the sample, choose the wavelength of light.
To make the testing more effective, use a single wavelength of light.One of the chemicals thought to be in the test solute should be absorbed by the light chosen.The wavelength should be set according to the specifications of the spectrophotometer.The wavelength will be given to you in the classroom.The sample will reflect all light of the same color as it appears, so the experimental wavelength will always be a different color than the sample.The objects reflect light of particular wavelength and absorb all other colors.Grass is green because of its ability to absorb green light.
Step 7: Use the blank to Calibrate the machine.
Put the blank in the holder and close the lid.There is a screen with a needle that moves based on the intensity of light.The needle should move to the right when the blank is in.If you need it later, record this value.The adjustment knob can be used to move the needle to zero.The digital spectrophotometers will have a digital readout.Use the adjustment knobs to set the blank to 0The calibration will still be in place when you remove the blank.The absorbance from the blank will be subtracted when you measure the rest of your samples.It's important to use a single blank per session so that each sample is adjusted to the same blank.If you blank the spectrophotometer, the remaining samples would be inaccurate.You would have to start over.
Step 8: Test the calibration by removing the blank.
The needle should stay at 0 if the blank is removed.Place the blank back into the machine to make sure the needle doesn't change.Everything should stay at 0.If the needle is not 0, repeat the steps with a blank.You can either seek assistance or have the machine looked at.
Step 9: Measure the absorbance of your sample.
Place the sample into the machine.Make sure the cuvette stands upright by sliding it into the designated grooves.Wait about 10 seconds until the needle is steady.Record the values of transmittance and absorbance.The optical density is also known as the absorbance.The sample absorbs less light from the light that is transmitted.For example, if you want to record the absorbance values as a decimal, you should do so.If you get anoutlying result such as 0.900, you need to measure the absorbance again.If you want to average them together, repeat the reading for each individual sample at least 3 times.The more accurate the readout, the more this ensures.
Step 10: The test should be repeated with successive light waves.
Depending on wavelength, your sample may have multiple unknown compounds.If you want to eliminate uncertainty, repeat your readings across the spectrum.You can use this to detect other chemicals in the solute.
Step 11: Determine the transmittance and absorbance of the sample.
Themittance is the amount of light that passed through the sample.The amount of light absorbed by one of the chemicals in the solute is called absorbance.If you recorded intensity, you can calculate the values of transmittance and absorbance.The transmittance is determined by dividing the intensity of the light that passed through the sample solution with the amount that left the blank.It is usually expressed as a percentage.I/I0 is the intensity of the sample and the blank.The absorbance is expressed as a negative of the base 10 logarithm.10% of the light is transmitted and 10% is absorbed for a T value of 0.1.1% of the light is transmitted for a T value of 0.01, which is 10 to the -2 power.
Step 12: The absorbance values can be plotted on a graph.
The absorbance value is plotted against the wavelength of light used for the test on the horizontal x- axis.Plotting the maximum absorbance values for each wavelength of light helps to identify the compounds in the sample.There are peaks in the absorbance spectrum that can allow you to identify compounds.
Step 13: You can compare your absorbance spectrum plot to known plots of compounds.
Every time a compound is measured, it will produce a peak at the same wavelength.You can identify the solutes that compose your solution by comparing your plots of unknown compounds to those of known compounds.This method can be used to identify the contaminants in your sample.You know something isn't right if you get 2 peaks at different wavelengths.