Bio 120 Lab 5: Quantitative Analysis

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1 Bio 120 Lab 5: 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

2 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

3 We will be determining the concentration of methylene blue in solution, using a known standard solution. We will make a series of dilutions from a stock (concentrated) standard solution of methylene blue. The molecular mass of methylene blue is g/mol. The stock solution has a concentration of 200 mg/dl solution. We will make a serial dilution to obtain standards with the following concentrations: 100 mg/dl, 75 mg/dl, 60 mg/dl, 45 mg/dl, and 25 mg/dl. The most accurate and user-friendly way of making many concentrations of a single solution is to perform SERIAL DILUTIONS, or making many sequential dilutions from a single stock solution. This is faster and more accurate than making every solution from scratch. You will need to use the following equation to create your dilutions: Dilution Equation: C i V i = C f V f Where: C i = Concentration of initial solution V i = Volume of initial solution to be used C f = Concentration of final dilute solution V f = Volume of final dilute solution Calculate the following dilutions, remembering that we are performing serial dilutions. Therefore the C i for the first dilution we will make is the stock solution (200 mg/dl), but the C i for the next dilution is 100 mg/dl. You will need to convert your volume to microliters.

4 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 100 mg/dl Volume (µl) of stock or previous standard dilution Volume of distilled water 75 mg/dl 60 mg/dl 45 mg/dl 25 mg/dl

5 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 200 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 100 microliters of the unknown into 2 mls of distilled water and record the absorbance in the table below. Concentration 0 mg/dl = blank Absorbance 25 mg/dl 45 mg/ dl 60 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.

6 Questions: 1. What is the linear regression equation for your data? 2. What is the concentration of the unknown? 3. What is the extinction coefficient of methylene blue? 4. Why did we use serial dilutions instead of making each dilution from the stock solution? 5. Why did we use a multi point curve instead of a single point calibration?

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