Question

Beer’s Law

Objective : We will explore an application of absorption spectroscopy using calibration curves and Beer’s Law.

Use the “LAB : HOW TO…” link from the class website if you need help with how to use balance, Bunsen burner… and such.

Introduction:

You may write this information in your lab notebook for your own reference. It can’t be cut and pasted.

Different solutions have different spectral properties. In this portion of the experiment those properties will be utilized to determine the concentration of an unknown sample. In looking at the absorbance of a solution it can be noted that the absorbance of a solution is directly proportional to the concentration of the solution and the length of solution the light has to pass through. This relationship is known as Beer’s Law and can be expressed as follows: A = εlc where A is absorbance, ε is the molar absorptivity (in L/mol*cm), l is the path length (in cm), and c is the concentration (in mol/L).

A calibration curve can be generated by measuring the absorbance of a series of standard solutions, and plotting Absorbance (Abs) versus concentration, with Abs on the y-axis and concentration on the x-axis. The linear regression for this plot will yield a familiar equation of the line y = mx + b. In this case, we can relate this to the equation above and think of it as A = εl * c + b. Then by measuring the absorbance of an unknown solution, we can plug it into our equation and solve for the concentration of our unknown.

The primary objective of lab is to determine the concentration of an unknown cobalt (II) chloride, CoCl2, solution using a calibration curve. You will first measure the absorbance of a standard solution over the visible light spectrum and select the wavelength of maximum absorbance (λ max). You will prepare five cobalt (II) chloride solutions of known concentration (standard solutions) and measure the absorbance of the standard solutions at the λ max. The graph of absorbance vs. concentration for the standard solutions will describe a direct relationship, known as Beer’s law. Finally, you will determine the concentration of an unknown CoCl2 solution by measuring its absorbance with a spectrometer and using the best-fit line equation of the Beer’s law curve to calculate the unknown’s concentration.

_____________________ CUT HERE __________________________________

Procedure :

1. Obtain ~40 mL of 0.20 M CoCl2 solution and distilled water in separate beakers.

2. Label five clean, dry, test tubes C1–C5. Use pipets to prepare four standard solutions according to the chart below (the fifth standard is the stock 0.20 M CoCl2 solution). Thoroughly mix each solution with a stirring rod. Clean and dry the stirring rod between uses.

Test Tube number	0.20 M CoCl2 (mL)	Distilled H2O (mL)	Concentration (M)
C1	10	0	0.20
C2	8	2	0.16
C3	6	4	0.12
C4	4	6	0.08
C5	2	8	0.04

3. In Test Tube 6 and 7, obtain ~ 5 mL of two different unknown samples. Report the unknown #’s.

4. Use a USB cable to connect a Vernier Spectrometer or a Vernier SpectroVis to a computer.

5. Start the Logger Pro 3 program on your computer.

6. Calibrate the spectrometer.

   1. Prepare a blank by filling an empty cuvette ¾ full with distilled water.

   2. Open the Experiment menu and select Calibrate → (Spectrometer:1). The following message appears in the Calibrate dialog box: “Waiting … seconds for the device to warm up.” After the allotted time, the message changes to: “Warmup complete.”

   3. Place the blank in the spectrometer; make sure to align the cuvette so that the clear sides are facing the light source of the spectrometer. Click “Finish Calibration”, and then click
      ОК
      .

7. Determine the λ max for CoCl2 (aq) and set up the data collection mode.

   1. Rinse and fill a cuvette ¾ full with the 0.20 M CoCl2 solution and place it in the spectrometer.

   2. Click . A full spectrum graph of the CoCl2 solution will be displayed. Note that one area of the graph contains a peak absorbance (λ max). Click to complete the analysis.

   3. To save your graph of absorbance vs. wavelength, select Store Latest Run from the Experiment menu.

   4. Click the Configure Spectrometer icon, $-Evnr0nzZgGy0_bC02ki_nAAfAaNDW2Cish872ht$, on the toolbar. A dialog box will appear.

   5. Select Abs vs. Concentration under Set Collection Mode. The λ max will be automatically selected. Click to proceed.

8. Collect absorbance-concentration data for the five standard solutions.

   1. Leave the cuvette in the spectrometer. Click . When the absorbance reading stabilizes, click $jX8tOZc3-Jxr5SdLRY3TXdH_5yXywkMDpDKjuBvJ$. Enter “0.20” (the solution concentration). Click .

   2. Using the solution in Test Tube 2, rinse and fill the cuvette ¾ full. Wipe the cuvette and place it in the spectrometer. When the absorbance reading stabilizes, click $jX8tOZc3-Jxr5SdLRY3TXdH_5yXywkMDpDKjuBvJ$. Enter “0.16” and click .

   3. Repeat Step 8b for the remaining test tubes of the standard CoCl2 solution. And your unknown solution. When you have finished testing the standard solutions, click .

9. To export your data open the file menu and select Export As → CSV (Excel, InspireData, etc.)…

   1. You may save this to a USB drive or email the file to yourself to work up your data in excel later.

_____________________ CUT HERE __________________________________

Prelab Questions :

PL1) Using the sample data below, make a plot of Absorbance vs [CoCl2] and determine the equation for the calibration curve.

Test Tube	[CoCl2] (mol/L)	Absorbance
1	0.20	1.50
2	0.16	1.25
3	0.12	1.00
4	0.08	.75
5	0.04	.50

PL2) Using the calibration curve from PL1), what is the concentration of [CoCl2] if your unknown solution has absorbance of 0.88? Show this on the calibration curve directly.

PL3) Using the calibration curve from PL1), what is the concentration of [CoCl2] if your unknown solution has absorbance of 103.66? Explain how you got this answer.

PL4) Draw a data table you will be filling in during the lab. Include all the measurements that will be made in this lab and qualitative observation if needed.

Analysis

A1) Create a graph showing the data and y=mx+b equation for C1-C5 solutions.  

A2) Calculate/find the molar concentration of your unknown CoCl2 solutions. Clearly show directly on the graph how it was calculated/found.

Post Lab Questions :

Q1) In your own words, what is the purpose of calibration curve? What information do you need to construct a calibration curve?

Summary :

Site the page number for the calibration curve. Make sure it includes y=mx+b and unknown information directly on it.

Abstract:

No more than 100 words stating why the lab was done, what was done, the major result(s), and conclusion(s).

Reflection Statement:

One thing I might do differently next time is …… because…….

Answer 1

J _ E F G H I Concentration vs Absorbance A B C 1 Test Tube (CoCl2] (mol/L) Absorbance 0.2 1.5 0.16 1.25 0.12 0.08 5 0.04 0.75 absorbance y= 6.25x +0.25 R2 = 1 13 o 0.05 Bentration 0.15 0.2 0.25 Concentration mol/L PL2) Given, absorbance = y = 0.88, then Draw a parallel line to the x-axis from 0.88 absorbance unit till it crossections the calibration line. From the crossection point again draw perpendicular line to the x-axis and see where it cuts the x- axis, that will be your concentration. corrosponding concentration x = 0.10 mol/L PL3) Given, absorbance = y = 103.66, then corrosponding concentration x = (103.66-0.25)/6.25 = 16.55 mol/L 25 Please note that your experimental data is not provided hence may not be possible to answer PL4, 26 A1 and A2. 27

Post lab Q1) The calibration curve is used to minimize most of the instrumental errors in the measurement of the data. It is used to determine the unknown concentration from unknown sets of data points such as absorbance with respect to known concentration. The calibration curve is a graph of concentration vs absorbance (in this experiment). The data is plotted for known concentration. From that unknown sample is measured for the absorbance and corresponding concentration is calculated using either equation or plotting on the graph itself. If the absorbance is pout of the range of calibration curve then corresponding calculated concentration may have some error.