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: Quantitative Analysis Remember from the measurement lab, concentration is the amount of a solute, also called an analyte, in a given amount of solution. We need a way to measure the concentration of an analyte in solution. In order to measure the concentration we need an instrument that can detect the solute, and a standard of a known concentration. In the measurement laboratory we learned how to make known concentrations – standards, by weighing or measuring the correct amount of the solute and solvent to obtain the correct concentration. We can use this standard to determine the concentration of an unknown. In this laboratory we will use a spectrometer to detect the solute. The Spect 21 is a relatively simple instrument consisting of a light source, a sample compartment and a detector. The desired wavelength is selected through a grating. We can use the Beer- Lambert relationship between the concentration and the absorbance of the analyte to determine the concentration of our analyte. The Beer- Lambert relation is: A = elc, where A is the absorbance, e is the extinction coefficient, l is the path length of the sample cell, and c is the concentration of the analyte. The units for l is cm, for c is mol/liter, and for the extinction coefficient: liters/(mol*cm).
There are many ways we can determine the concentration of the analyte using a standard solution, one is known as the single point calibration where only one concentration of standard is used. This is an easy way to measurement to give a ballpark estimation, but you do not know if the relationship between the concentration and the
absorbance is linear. You need to perform a multi point calibration curve where dilutions are made of the standard to produce series of concentrations. This will determine the linear range of your analyte for your instrument. To determine the concentration of an unknown you need to perform linear regression analysis: y = mx+b, by plotting the absorbance on the y-axis and the concentration on the x-axis. In this equation y is the absorbance, m is the slope, x is the concentration, and b is y-intercept.
Single Point Calibration:
Multi point calibration cure
Calculate the following dilutions :
A. 1.5 ml of 100 mg/dl
B. 1.2 ml of 75 mg/ dl
C. 1 ml of 60 mg/ dl
D. 1 ml of 45 mg/ dl
E. 0.9 ml of 25 mg/dl
Desired concentration Volume (μl) of stock or previous standard dilution
Volume of distilled water
100 mg/dl
75 mg/dl
60 mg/dl
45 mg/dl
25 mg/dl
Our “blank” will contain distilled water. Set the wavelength to 600 nm. We will use the same test tube, cuvette, for the blank and for each standard and the unknown. You will need to rinse the cuvette in between each reading and wipe the outside of the cuvette with chem.-wipes. Follow the instructions given by the instructor on how to perform a blank.
Creating the “standard curve”.
Use 2 00 microliters of each standard dilution into 3 mls of distilled water.
Be sure to use a different pipette tip for adding each dilution.
Make sure there are no “extra” drops on the outside of the pipette tip when adding your dilutions to the water.
Gently mix the contents of each tube to create a homogeneous solution.
Record the absorbance values of each solution in the table below. Don’t forget your control (blank). Start with the blank, then the lowest concentration of standard, working your way up to the highest concentration of standard. Then use 1 00 microliters of the “unknown” into 2 mls of distilled water and record the absorbance in the table below.
Concentration Absorbance 0 mg/dl = blank
2 5 mg/dl
4 5 mg/ dl
6 0 mg/ dl
75 mg/ dl
100 mg/ dl
Unknown
After you have your results, enter the data in the computer, and use linear regression to determine the concentration of the unknown.
