NSCI 115: Chemical Principles of NANO I

Lab 1: UV-Vis Spectroscopy and Beer-Lambert Law

I. Safety Instructions:
Follow all standard safety rules as demonstrated in class. Safety glasses/goggles, lab coats,
and gloves must be worn when working with chemicals. Wearing shorts or sandals inside the lab is
not permitted at anytime. Special caution should be exercised when working with chemicals and
glass products which are easily broken. If skin contact with chemicals should occur, rinse the area
affected for 15 minutes and call the emergency number x78600. Hands should be washed at the end
of the laboratory activity. If you hear the building evacuation alarm, evacuate immediately in an
orderly fashion. Chemical waste is considered hazardous should be segregated and collected in a
separate labeled hazardous waste container.

II. Objectives:
1. Learn basics Ultraviolet-Visible (UV-Vis) spectroscopy
2. Demonstrate usage of UV-Vis spectrophotometer
3. Demonstrate principle of Beer’s Law for absorption spectroscopy
4. Practice how to make serial dilutions

III. Procedure Outline

1. Review hardware and software for proficient operation of the UV-Vis spectrophotometer
2. Prepare serial dilutions of stock dye solution
3. Collect absorption spectra of the dye solutions to construct a standard curve and determine the
determine the molar absorptivity (ε) of the dye using Beer’s Law

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IV. Background:

Beer-Lambert Law (See also pp. A16-A18 in Zumdahl, 8th Edition):

The amount of light transmitted through a solution of an absorbing chemical in a transparent
solvent can be related to its concentration by the Beer-Lambert law, sometimes called Beer’s Law.
The Beer-Lambert law is expressed as
𝐼
𝐴 = −𝑙𝑜𝑔 𝐼 = 𝜖𝜆 𝑏𝑐 (1)
0

where A is the absorbance (a defined quantity, also referred to as the optical density, or OD), I is the
transmitted light and 𝐼0 is the incident light intensity. 𝜖𝜆 is the molar absorptivity(also called the
molar extinction coefficient) which has units of M-1cm-1, b is the path length the light (cm), and “c” is
the solution concentration (M). For these experiments, you will use the UV-Vis to measure the
absorbance which is a dimensionless quantity.
Notice that 𝜖𝜆 is a function of wavelength, and it is the quantity which represents the spectrum of
the solution. When its value is stated, it must be given for a particular wavelength (e.g. 𝜖532 ). The
only exception to this is when its value at the peak of the spectrum is given, in which case it may be
denoted as 𝜖𝑚𝑎𝑥 . If the molar extinction coefficient is known, absorption spectroscopy can be used to
quantify the amount of chemical present in a solution.

Serial Dilutions (See also pp. 94-95 in Zumdahl, 8th Edition)

Dilutions are an essential skill for manipulating solution concentration. The basic premise is to
reduce the concentration of the solute by adding a given volume of solvent. In the lab, dilutions are
calculated using the dilution calculation
𝑐1 𝑣1 = 𝑐2 𝑣2 (2)
For example, if you wanted to make 10 mL (𝑣2 ) of a solution with a final concentration of 1.5 µM
(𝑐2 ) and have a solution available that is 5µM (𝑐1), then you need to take
𝑣1 = 𝑐2 𝑣2 /𝑐1 (3)
1.5 µM∙10 mL
𝑣1 = (4)
5 µM

giving an initial volume of 3 mL. Thus, you need to add 7 additional mL of diluting solution to get
the final volume of 10 mL.

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 Serial dilutions are a common technique where the final solution of the first dilution is used as
the starting point for the next solution. This technique is commonly used to rapidly dilution stock
solutions to very low concentrations, but it is being used to today to create a set of standards to assay
an unknown. The major issue with serial dilutions is that early errors in measurement will propagate
throughout all subsequent measurement. Great care must also be taken to mix solutions completely
before taking any out for dilutions.

V. Detailed Procedure
UV/VIS Instrumentation
1. Lab instructor tutorial on UV-VIS Hardware/Software.
Standard curve sample preparation

2. The instructor will provide a 5 M stock solution of Crystal Violet (CV) dye in H2O to each
group.
3. Use the 5 M stock solution to prepare 10 mL of 2.5 M working solution. Measure all
volumes using an analytical balance. Assume the density of water is exactly 1.0 g/cm3.
Record all of mass measurements in your lab notebook for exact calculations of different
concentrations.
4. Use the working solution and serial dilute to get concentrations spanning 2.5-0.2 M (10 ml
of each, see Fig. 1). Water will be the reference sample (0 M sample). Record all your mass
measurements in your lab notebook as you take them for exact calculations of the crystal
violet concentration in each sample.

Figure 1. Serial Dilutions

2.5 uM ? uM ? uM ? uM ? uM 0 uM (control)

7 ml 6 ml 5 ml 4 ml

3 ml H2O 4 ml H2O 5 ml H2O 6 ml H2O 10 ml H2O

Serial dilutions are a series of simple dilutions to amplify a dilution factor quickly. In the case above,
the serial dilution is being used to create a standard curve of known concentrations of the dye.

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UV/VIS Spectrometer measurements
Handling Cuvettes:
Examine your cuvette to ensure the clear sides are free from scratches. Cuvettes should be
wiped clean and dry on the outside with a KimWipe before each measurement. Handle the
cuvettes near the top of the frosted/ribbed sides. Solutions should be free of bubbles. If you
are refilling a cuvette with a different solution, a small amount of the new solution should be
used to rinse the cuvette before filling. The light path of the spectrophotometer lines up with
the correct part of the cuvette. To avoid inconsistencies from different cuvettes, only one
cuvette should be used for the entire experiment.

5. Add 1-2 ml of water (i.e. the 0 M sample) into a cuvette, zero the absorbance, and run a
background spectrum) from 700 nm to 400 nm (at medium scan speed). This data is saved
into the software memory and is not shown on the screen.
V. After the background scan is acquired, run a “sample” spectra on the same solution
(the 0 M sample), this should show a straight line at A=0.
VI. Name your spectra with a convention similar to “Blank_[your initials]_[sample#].”
VII. Remove the cuvette, discard the solution and dry it with a Kimwipe.
6. Add 1-2 ml of sample (enough liquid that the light path is completely covered), starting with
the lowest sample concentration.
V. Acquire the spectrum from 700 nm to 400 nm, similar to the background. Name
your spectra with a convention similar to “[concentration]_[your
initials]_[sample#].”
VI. Measure each sample concentration in triplicate (discard the already measured
sample in the proper waste container and refill the cuvette with fresh sample
solution; then acquire spectrum again).
VII. After the 3rd spectra, remove the cuvette, fill it with 1 mL of the next sample
solution to rinse out the old sample (discard this sample solution), you can now
use it for the next measurement.
7. Repeat step 6 for all samples, going from lower  higher concentration.
8. In the end, you will have three spectra for five different concentrations (+ your blank spectra)
covering the 0-2.5 M concentration range.

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9. Measure the absorbance of a crystal violet sample with unknown concentration (provided),
following the same procedure as above (one spectrum is enough). Remove the cuvette, rinse
with DI-H2O, dry with N2.
10. Make sure you save all your data as .csv files.
If the spectra you want are in the software memory, you can check the “visible” box for your
samples and uncheck any spectra you do not want to save. If you “save as” .csv, all the
visible spectra will be saved in the same file.
11. When you are finished, discard all your solutions in the designated waste bottle, and the used
test tubes in the designated bag.
12. Clean up the hood area and throw away any trash that might have been generated.

VI. Lab Report Outline – Follow the lab report template

Data manipulation
1. Use your saved spectra and make a scatter plot of Absorbance vs. Concentration for both max,
and 509 nm (use the average of the triplicate measurement with added error bars (in the Y-
direction only) for the standard deviation of the three samples).
2. Based on Beer’s Law, determine the molar absorptivity (𝜖) of the crystal violet dye at both
max and 509 nm wavelengths. Using your standard curves, calculate the concentration of the
unknown sample.
Hint: use the “linear regression fit” function in Excel (or other software) to get a “best-fit”
representation of your standard curve data.

Lab Report
Make sure to report on the following in appropriate section
a. Calculation of the exact concentrations of standard curve solutions.
b. Beer’s law analysis of crystal violet standard solutions. This will include of a
graph (scatter plot - symbols only) with regression analysis (a linear curve fit) to
determine molar absorptivity of the dye at each of the two wavelengths analyzed.
Plot both curves in the same graph.

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 c. Report the absorbance of the unknown sample, where the concentration calculation
is based on your standard curve derived molar absorption coefficient.
d. Present one overlay graph between 400-700 nm showing one spectra for each
standard concentration. Be sure to label all curves and axis properly (use scatter
plot with smooth lines for these types of continuous data).
e. Calculate what the Absorbance would be for two samples where 99% and 99.5%
of the incident light does not reach the detector. Show your calculations.

Remember to use words (i.e. text) to guide the reader through your results; do not just add
a graph or table to the result section!

Give a printed version of the lab-report the following week to the instructor for grading,
and also send an electronic version (PDF, or Word format) for record keeping.