Chem Lab 4

Excel sheet and lab report need to be compeleted.

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Experiment 4: Introduction to Spectrophotometry

Introduction

Spectrophotometry is an experimental technique to measure the amount of light absorbed by a
substance at a particular wavelength. In this experiment, you will use spectrophotometry to
examine a series of solutions with differing concentrations of a chemical substance, cobalt(II)
chloride hexahydrate. You will interpret these results using Beer’s law, which states that there is a
linear relationship between the absorbance of a solution at a specific wavelength and the
concentration of the absorbing species in solution. You will also determine the value of the
proportionality constant between the absorbance and the concentration, known as the molar
absorptivity or extinction coefficient, using a line of best fit. This line of best fit will also allow
you to calculate the concentration of a solution containing an unknown amount of cobalt(II)
chloride hexahydrate.

Physical and Chemical Principles

Many molecules and ions are colorful when isolated or in solution. Color is observed by the eye
because some wavelengths of light in the visible spectrum are absorbed by a material and others
are transmitted or reflected and then detected by the retina. The particular wavelengths of light that
are absorbed depend on the chemical species present. Upon absorption of a photon (a particle of
light with a discrete amount of energy), a molecule or ion is promoted to an excited state (a state
higher in energy). As this excited state is not stable, the energy absorbed will be released at some
later time when the molecule or ion relaxes to its ground, or most stable, state.

Spectrophotometry is a highly useful tool in many areas of chemistry and biology. The primary
reason for its utility is the fact that the amount of a particular wavelength of light absorbed is
related to the concentration of the absorbing species. Therefore, spectrophotometry provides a
relatively quick, straightforward, and sensitive way to determine the concentration of a species in
solution that absorbs visible light.

In a spectrophotometer, it is the intensity of light transmitted through a sample that is actually
measured. The instrument isolates a narrow beam of light at a particular wavelength. This light,
which has intensity I0, is passed through the sample. The intensity of the transmitted beam I is then
measured. If a species is present that absorbs the wavelength of light passing through the sample,
the intensity of the transmitted light will be decreased relative to the initial intensity. The ratio of
the intensity of the transmitted light to the initial intensity, I/I0, is called the transmittance T of the
sample. The transmittance T, or more often the percent transmittance %T (equation 1) is related to
the absorbance of the sample according to equation 2.

0

% 100%IT
I

 
= × 
 

(1)

( )log 2 log %A T T= − = − (2)

The unitless value A is the absorbance of the sample and is more commonly used over the
transmittance as the measurement of light absorption in chemistry, although spectrometers can
usually report both A and T. The reason that absorbance is more common (and more valuable!) in

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chemistry is because of the relationship between the absorbance of the sample and the
concentration (in units of molarity, or moles of species per liter of solution) of the absorbing
species. This relationship is given by the Beer-Lambert law, more often called Beer’s law, which
is expressed as:

0log
IA abc
I

 = =  
 

(3)

where a is the molar absorptivity (L∙mol−1∙cm−1), b is the pathlength (cm) that the light travels
through the sample, and c is the concentration (mol/L) of the substance being analyzed. The molar
absorptivity (also called the “extinction coefficient”) depends on both the substance and the
wavelength.

Colored species in solution generally have broad absorption peaks in the visible region of the
spectrum (approx. 380–740 nm). When attempting to determine concentrations, the absorbance is
usually measured at the wavelength of maximum absorbance (λmax) to obtain the most sensitive
measurements. Therefore, you will first obtain the visible absorbance spectrum of cobalt(II)
chloride hexahydrate to identify λmax. All subsequent absorbance measurements will be made at
this wavelength. Ensuring that the spectrometer is set to the correct λmax and reporting the specific
value of λmax used to collect absorbance measurements are vitally important in spectrophotometric
experiments because each species has its own wavelength-dependent value for the molar
absorptivity.

By examining the absorbance of solutions containing varying amounts of cobalt(II) chloride
hexahydrate, a plot of absorbance vs. concentration, known as a standard curve, can be constructed.
If the pathlength of the cuvette used in the spectrophotometer is held constant throughout the
experiment, Equation 3 indicates that such a standard curve can be used to determine the molar
absorptivity of a substance at a particular wavelength. With a standard curve in hand, the
concentration of an unknown solution of a substance can be determined.

The generation of a standard curve is a frequently encountered task in chemistry. Because your
line of best fit is dependent upon accurate and precise concentrations, careful and consistent use
of volumetric glassware (volumetric pipettes and volumetric flasks) is required. The accuracy and
precision of your pipetting skills will greatly affect the results of this experiment, so take care
while preparing all of the necessary solutions.

Experimental

Table 1 lists the equipment and chemicals needed for this experiment.

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Table 1: Equipment and Chemicals for Introduction to Spectrophotometry
Equipment and Chemicals Quantity

CoCl2·6H2O No more than 3.25 g

Weigh Bottle 1

Volumetric pipettes: 1.00, 2.00, 5.00 mL 1 of each

Cuvettes (one for reference, one for sample solution) 2

Spec 20 spectrophotometer 1

Volumetric flask, 50.00 mL 1

Volumetric flasks, 10.00 mL 4

Miscellaneous beakers and flasks As needed

Wash bottle, water 1

CoCl2·6H2O solution of unknown concentration 5 mL

Preparation

Notebook

It is necessary for you to come to lab prepared to do the exercise without the printed instructions.
You should have written a list of the steps to complete the experiment. This list should not be a
word-for-word re-copy of the instructions. It is better to make a “to do” list that is a reminder of
both the steps and specifics of the procedure. At times, minor changes in the procedure may be
announced at the start of lab. It is important to note these details as part of your notebook
procedure. This requirement forces you to develop your own understanding of the concepts and
procedures, and requires that you read the details of an experiment before coming to lab. The
instructor and laboratory assistant will be checking your notebook to see that sufficient detail is
present to complete the lab. If you cannot demonstrate that you are ready to start the experiment,
you will not be allowed to enter the lab.

You must come to lab with data tables already prepared in the notebook. This, too, provides you
with a valuable opportunity to understand the data and the calculations that will be part of the
assignment at the end of the experiment. It is not acceptable for you to paste or tape pages from
the printed instructions into your notebook.

Before the lab, you must calculate the mass of CoCl2·6H2O required to obtain 50.00 mL of a
0.2500 M solution of CoCl2·6H2O. Make sure this calculation and value are written in your
notebook.

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Pre-lab Assignment

You must complete a pre-laboratory quiz prior to entering the laboratory, which can be accessed
through the D2L website for the course. A thorough reading of these instructions should prepare
you to take the quiz. A score of at least 70% is required for participation in the experiment. You
may attempt the quiz as many times as you wish. The quiz must be completed (with a minimum
score of 70%) by 11:59 pm the night before the start of your lab section. You will not be allowed
to participate in lab without a completed pre-laboratory assignment.

Procedure

Be sure that the spectrophotometer is on and warmed up for 20 minutes before use. There may be
different models of spectrophotometer in the lab. Each will have a quick reference card that gives
specific instructions for each model. Review these instructions before acquiring data.

For precise work, solutions are measured in tubes (“cuvettes”) of square or circular cross-section,
made from optical-quality glass or silica plates. The bottom half of the cuvettes must be kept
scrupulously clean. Any dirt specks, smudges, fingerprints, or water droplets will reduce light
transmission, thus simulating higher sample absorbance. Accordingly, clean the cuvettes
thoroughly, dry them with Kimwipes, handle them only at the top, and make sure they do not pick
up dirt from test tube racks, beakers, etc. These cuvettes are not disposable, and should be cleaned
and returned to where they came from at the end of the experiment.

In all subsequent measurements, the reference cuvette should be approximately three-quarters full
with an appropriate solvent, and the sample cuvette approximately three-quarters full with the
sample (after rinsing it twice with approximately 1 mL of the sample solution). If your
spectrophotometer does not report the absorbance directly, then you must calculate the absorbance
of the sample solution from the percent transmittance (%T).

Preparation of Stock Solution

Before entering the lab, calculate the mass of CoCl2·6H2O required to obtain 50.00 mL of a
0.2500 M solution of CoCl2·6H2O. Make sure the calculation and value are written in your
notebook. Measure out this amount of CoCl2·6H2O to a weigh bottle using the mass-by-difference
technique (mass the CoCl2·6H2O vial, lightly tap some powder into the weigh bottle, then mass
the CoCl2·6H2O vial again and use subtraction to determine the mass of the solid added). Your
mass does not need to be exactly equal to your calculated mass–obtain a similar amount and record
the exact mass obtained.

Add a few milliliters of deionized water to your 50.00 mL volumetric flask and then quantitatively
transfer your solid to this flask. The phrase “quantitatively transfer” means all of the solid must be
transferred from the weigh bottle to the flask. You may need to use a small amount of deionized
water to ensure all of the solid has been transferred from the bottle.) Gently swirl the flask to begin
dissolving the solid. Keep adding small amounts of water and swirling until all of the solid is
dissolved. Once the solid is completely dissolved, fill the flask to the mark with deionized water.
The solid must be completely dissolved before the flask is filled to the mark with deionized
water. Use a Pasteur pipette or a wash bottle to add water dropwise when nearing the mark.

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Stopper the flask and invert it several times to sufficiently mix the solution. If you add too much
water and the meniscus is above the mark, you must discard the entire solution and start again.

Preparation of Diluted Solutions

You will generate four diluted solutions using various quantities of the stock solution. Pour your
stock solution into a clean and dry beaker. To generate these diluted solutions, first obtain four
10.00 mL volumetric flasks. Using one or more primed volumetric pipettes, add the following
volumes of the stock solution, each to a different volumetric flask: 1.00, 2.00, 5.00, and 7.00 mL.
Label each flask according to the volume of stock solution added to it. Fill the flasks to the mark
using distilled water. Mix each solution by inverting the stoppered flask several times. If you add
too much water and the meniscus is above the mark, you must discard that solution and create it
again from your stock solution.

Absorbance Spectrum for Determination of λmax.

You will use a Nanodrop One spectrophotometer to obtain the absorbance spectrum of your stock
solution. The spectrophotometer screen should say “New Experiment” at the top. Select the
“Custom” tab, and then select “UV-Vis”. Select “Done”. As requested by the instrument, load
1.2 μL of deionized water using a micropipette onto the lower pedestal and lower the arm of the
spectrophotometer. Once the instrument is initialized, you can touch “Blank” on the screen to
acquire the blank spectrum. Raise the arm of the spectrophotometer, wipe away the water sample
using a Kimwipe and load 1.2 μL of your stock solution of CoCl2·6H2O. Type a sample name
using your initials and press “Measure” to acquire the spectrum. Based on your spectrum,
determine the value of λmax and record it in your notebook.

Absorbance of Diluted Solutions for Standard Curve

Before entering the lab, prepare a table in your lab notebook with columns for volume of stock
solution, concentration, and absorbance.

Set the wavelength of the Spec 20 spectrophotometer to the value of λmax determined in the
previous step. Be sure the Spec 20 is set to read absorbance (A), not transmittance (T). Rinse the
sample cuvette twice with small (approximately 1 mL) portions of your most dilute solution.
Discard the rinses in a waste beaker. Add some of the same solution to the rinsed sample cuvette
and distilled water to the reference cuvette. The cuvettes should each be approximately three-
quarters full. Wipe the outside walls of the cuvettes with a Kimwipe to remove any solution or
fingerprints.

Before measuring the absorbance of your diluted solutions, zero the spectrophotometer at λmax
using the reference cuvette. Next, replace the reference cuvette with the sample cuvette and record
the absorbance of this solution at λmax. Repeat the rinsing and measuring steps for the other diluted
solutions and the stock solution, working in order of increasing concentration.

Absorbance of Unknown Solution

Following the instructions in the previous section, determine the absorbance for the CoCl2·6H2O
solution of unknown concentration.

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Waste Disposal

Do not dispose of any solutions before you create your own standard curve (see Calculations
section). After your plot has been reviewed by the instructor/TA, you may put all chemical waste
in the container labeled Introduction to Spectrophotometry Waste.

Calculations

This section describes the five calculations that you will need to perform in Excel to successfully
complete your assignment.

Mass of CoCl2·6H2O Used

Calculate the exact mass of CoCl2·6H2O used to make the stock solution from the initial mass of
the weigh bottle and the final mass of the weigh bottle after adding CoCl2·6H2O.

Moles of CoCl2·6H2O Used

Calculate the number of moles of CoCl2·6H2O in the stock solution from the exact mass used and
the molar mass of the compound.

Concentration of Stock Solution

Calculate the molarity of the stock solution you created, where molarity is a unit of concentration
defined as the moles of solute divided by the total volume of solution in liters.

Concentrations of Diluted Solutions

In a dilution a small amount of a stock solution is delivered to a new volumetric flask and then
filled to the line with deionized water. The number of moles of solute removed from the stock
solution and present in the diluted solution are equal to one another, but the two volumes are not.
Therefore, the concentration must also change. This process can be represented in a compact
formula:
2211 VMVM = (4)

where M1 is the molarity of the stock solution, V1 is the volume of stock solution added, M2 is the
molarity of the diluted solution, and V2 is the total volume of the diluted solution. Note how the
units cancel and work out to give you the correct units on your desired value. This equation is very
useful for dilutions, but should never be used for stoichiometry calculations or other applications.

Slope and Intercept of Standard Curve

Use the appropriate Excel formulas to calculate the slope (=SLOPE) and intercept (=INTERCEPT)
for the line of best fit for your plot of A vs. c corresponding to the data from the stock solution and
the four diluted solutions. Make sure your concentrations are all in units of molarity. The slope of
a plot of A vs. c is equal to ab according to Beer’s law (A = abc). The molar absorptivity a of your
aqueous CoCl2·6H2O solution can be calculated from this slope and the pathlength b, which is
1.00 cm in this experiment.

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Concentration of Unknown Sample

Calculate the molarity of the unknown solution from the absorbance of the unknown solution and
your line of best fit from the standard curve.

Standard Curve

Generate a plot of the five absorbance measurements (A of the stock and diluted solutions at λmax)
vs. concentration in molarity. Fit these data to a line and include it on the plot. The plot and line
must be created using Excel or a similar program.

Note: Everyone should construct a standard curve of their own.

Assignment

You must hand in a partial report and your Excel file for this experiment at the beginning of your
next laboratory meeting. In addition, you must submit both files in the appropriate submissions
folder on the D2L website for your section. You should review the Lab Report Guidelines, Sample
General Chemistry Lab Report, and Appendix E on D2L while writing to ensure that your drafts
are correctly formatted. The rubric for the lab report can be found on the D2L site for your lab
section.

The partial report must be neatly typewritten and contain the following elements:

Procedure

It is sufficient to cite the procedure provided above (remember to provide a suitable reference or
references if you used additional sources). The format for the citation can be found in the Lab
Report Guidelines posted on the D2L site. Note any changes to the procedure in a bulleted list. If
there were no changes made to the experimental procedure, please say so.

Results

The results section must contain a description of the main data, as well as any tables and figures.
A table or figure can never stand alone. There must be narrative text that accompanies each one,
describing and explaining the results in full sentences. Each table and figure must be introduced
by number before appearing in the report. The data should be presented succinctly in a clear,
organized fashion. Do not present all of the text, then all of the tables, then all of the figures.
Everything should be interspersed within the narrative in a logical manner. Refer to the Lab Report
Guidelines posted on D2L for more information.

In your results section, be sure to include the following elements specific to this experiment. You
should present sample calculations in the report by including only the equation in algebraic
symbols with clearly defined variables (similar to how it was done in this lab manual). The actual
numerical calculations, on the other hand, must be performed in Excel. The order of
presentation should be dictated by the logical flow of your narrative, not necessarily by the order
below.

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Sample calculations

For each calculation performed for the experiment, you must include the equation used in symbolic
form along with definitions for each variable in the appropriate juncture in the results section.
Equations should be logically interspersed within the results section. Equations should not include
numerical values. The numerical values will be reported in tables and figures in the results section
and in an Excel spreadsheet, as described below.

All necessary calculations must be performed in an Excel spreadsheet and submitted electronically
on D2L with your lab report electronic submission. The Excel spreadsheet calculations must be
organized and clearly labeled so that they can be followed by your instructor. You must use Excel
formulas (i.e., don’t use a calculator and then manually type in all of your calculated numbers!) to
earn full credit for the calculations. If the instructor cannot follow the sample calculations or if the
formulas are not present in the submitted Excel spreadsheet, no credit will be awarded.

Concentration of your stock solution

The concentration of the stock solution you prepared should be presented at a logical point in the
Results section with the correct significant figures and units

Identification of λmax for your stock soluion

The wavelength of maximum absorbance (λmax) for your stock solution should be presented at a
logical point in the Results section with the correct significant figures and units

Concentration table

Create a table containing the volume of the stock solution used and the concentration of each of
the dilutions along with their corresponding absorbance measurements. The table should be neat
and organized with appropriate labels for each row and column that include correct units. All
information should be reported with the correct number of significant figures. The table must also
have a descriptive title.

Standard curve

Make a figure for the standard curve. The figure caption should contain the equation of best fit for
the plot with appropriate units and the molar absorptivity of the analyte. Once again, see the Lab
Report Guidelines for examples of properly formatted figures with captions.

Identification of unknown concentration

The identity (e.g., “Unknown A”) and concentration of the unknown solution should be presented
at a logical point in the Results section with the correct significant figures and units.

Discussion Question

Discuss the quality of your standard curve, specifically the accuracy of the slope and intercept of
the line of best fit and the precision of the data overall. The known value of the molar absorptivity

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for an aqueous solution of CoCl2·6H2O is 4.8 M−1·cm−1 at 512 nm. (Hint: If your value of λmax was
not 512 nm, would you expect your molar absorptivity to be larger or smaller than the value at
512 nm?)

Experiment 4: Introduction to Spectrophotometry
Introduction
Physical and Chemical Principles
Experimental
Preparation
Notebook
Pre-lab Assignment
Procedure
Absorbance of Unknown Solution
Waste Disposal
Calculations
Assignment
Procedure
Results
Sample calculations
Concentration of your stock solution
The concentration of the stock solution you prepared should be presented at a logical point in the Results section with the correct significant figures and units
Identification of λmax for your stock soluion
The wavelength of maximum absorbance (λmax) for your stock solution should be presented at a logical point in the Results section with the correct significant figures and units
Concentration table
Standard curve
Identification of unknown concentration
Discussion Question