Chemical Thinking Lab Assignment 3 - 5 Guide v1.

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How do we identify and quantify substances?

In the United States, the food nearly everyone consumes has been processed to some
degree. During processing, chemical substances are often introduced to reduce spoilage
and inhibit growth of unwanted microorganisms. Other chemical additives make food
more attractive/appetizing by contributing color and aroma/flavor. Unfortunately, some
of these added substances may pose health risks; hence the importance of identifying
and quantifying such substances. For example, some studies suggest that artificial
coloring and flavors, as well as some preservatives, can adversely affect children with
ADHD.

Background
Coloring food to make it more appetizing has been practiced for at least 3500 years. Originally, all food colorants
were natural products. For example, Romans used saffron to impart a yellow color to certain foods.

In the 19th century, with the dawn of the chemical industry, many synthetic compounds became available, some of
which found use as food colorants. A number of these were subsequently discovered to be harmful. Licorice and
hard candy, for example, were commonly colored with arsenic compounds.

By 1900, roughly 80 compounds were in use to color foods. Concerns about their safety led Congress to create the
Food and Drug Administration (FDA), which was charged with assessing and certifying the safety of chemicals
added to foods and used as drugs. The FDA certifies 9 chemical dyes as being safe for use in foods but only 7 are
currently in use. Of the seven, just four account for the majority of colorants used in food products.

Dyes that have been thoroughly tested by the FDA’s Department of Food, Drugs & Cosmetics (FD&C) are given a
number, which has become the common way to identify these dyes. All the dyes are relatively small organic
molecules that strongly absorb light in the visible region and have pure colors. The seven dyes in current use are
presented in the following table.

Table LA3-1 FD&C Food Dyes Presently in Common Commercial Use

FD&C Dye Name Trade Name Molar Mass

Red #3 Erythrosine 879.86 g/mol

Red #40 Allura Red 496.43 g/mol

Yellow #5 Tartrazine 534.37 g/mol

Yellow #6 Sunset Yellow 452.38 g/mol

Green #3 Fast Green 765.89 g/mol

Blue #1 Brilliant Blue 792.86 g/mol

Blue #2 Indigotine 466.36 g/mol

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Spectrophotometry
Spectrophotometry is the measurement of light in the visible, ultraviolet (UV) and/or infrared (IR) regions of the
electromagnetic spectrum that is reflected or transmitted by a substance or substances as a function of
wavelength, λ (or frequency). Instruments used to accomplish such measurements are called spectrophotometers.

In the majority of laboratory applications, we work with liquid samples in which the substance or substances being
analyzed are dissolved in a solvent giving a dilute solution. Further, we are concerned with transmission of light
through this sample (as opposed to that reflected), and express this as the absorbance, A. Spectrophotometers for
such samples measure transmitted light over a range of electromagnetic wavelengths. Software then processes
and displays this information as an absorbance versus wavelength (A vs. λ) spectrum that graphically illustrates the
relationship of absorbance to the wavelength for the sample. Such A vs. λ graphs are referred to as absorption
spectra (singular, spectrum; plural, spectra). Figure LA3/5-1 is an absorption spectrum for a dye in the visible to
near-infra red region of the electromagnetic spectrum (the visible region spans 380 - 700 nm).

Figure LA3/5-1. Absorption Spectrum (A vs. λ) for a Dye

λmax

λmax = 660 nm

Notice the spectrum is characterized by a wavelength (λ) at which absorbance is maximal (λmax), which in the above
figure is around 660 nm (as interpolated from the wavelength or x-axis). Chemical substances typically exhibit
unique absorbance spectra and λmax values. This specificity is a differentiating characteristic that allows substances
to be distinguished from one another in a mixture and makes spectrophotometry useful as an identification tool.

But identifying substances is only one benefit, in most cases the primary value of spectrophotometry as an
analytical tool is quantification - determining the concentration of a chemical substance based on the amount of
light absorbed at the λmax of that substance.

Absorbance, A, can be quantitatively related to the amount of substance present using Beer’s Law. Beer’s Law
relates the absorbance (A) of a chemical substance to its molar concentration (C, mol/L):

A = εbC

where ε is the molar absorptivity and b the path length.

This is a linear equation of the form y = mx in which the dependent variable y is A, the independent variable x is C,
and the slope m, is εb. You should know from high school algebra that when m > 0 (positive), y = mx graphs as:

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Figure LA3/5-2. Graph of y = mx, for m > 0

Slope = m

Which immediately suggests A = εbC will graph as (given that ε and b are always positive, > 0):

Figure LA3/5-3. Graph of Beer’s Law: A = εbC

Slope = εb
A

C (mol/L)

From the equation, A = εbC, you should see that determining the absolute (as opposed to relative) molar
concentration of a chemical substance requires knowledge of the molar absorptivity ε and the path length b. The
path length b is the distance the light travels through the sample solution in centimeters (cm). For this course, the
path length is defined by the optically transparent container that holds the liquid sample in the
spectrophotometer. This container is called a cuvette, and maintains b at 1.00 cm.

The molar absorptivity ε is considered unique for the substance (and to a lesser degree the spectrophotometer
and experimental conditions). As such it can serve as another differentiating characteristic. The value for ε can be
inferred for a given substance by measuring the absorbance of that substance at different known concentrations
and plotting A vs. C as in Figure LA3/5-3. For example, if you have a solution of defined concentration for the
substance, you can prepare a set of parallel dilutions in such a way that the concentration of each dilution is
known (see Parallel Dilution Set below). Taking the absorbance of each dilution (at the substance’s λmax) and then
plotting A vs. C, should give a line with slope εb. From the above discussion you already know the value of b,
hence, ε for the substance can be determined.

The A vs. C graph is commonly referred to as a calibration curve or calibration plot, for it calibrates the instrument
response for a particular substance to known concentrations of that substance. That is, the calibration curve gives
you the constant of proportionality, εb, between A and C for that substance.

Now in theory, you might propose from A = εbC, that the absorbance A of only one known molar concentration C is
required to find ε given that b has a known value. However, in practice this is not acceptable for two reasons. First,
you don’t want to rely on a single experimental reading to determine ε, for an error in this single reading will likely
be difficult to detect and go uncorrected; it is far better to have a set of absorbance – molar concentration pairs

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from which a best-fit linear trend-line can be generated to find ε. Second, you want to confirm that Beer’s Law
holds for the range of concentrations under consideration; if the A vs. C plot is not linear, but curves, then either
you have failure of Beer’s Law, or there is something amiss in the experimental procedure and/or its
implementation.

At this point, you should realize with ε known for a substance of interest (and the path length b also known) that if
the absorbance A is taken of a solution with an unknown concentration of this substance, then by Beer’s Law the
substance’s concentration can be determined. This is the value of Beer’s Law; it gives you a mathematical
relationship between absorbance and concentration, so the concentration of a substance can be determined by
reading the solution absorbance at the substance’s λmax.

A few last words about the wavelength of maximal absorbance, λmax. Recall, each substance has characteristic λmax.
When using spectrophotometry to determine the concentration (Beer’s Law) of a substance, the absorbance
measurements must be performed at the substance’s λmax to obtain the highest sensitivity and minimize deviations
from Beer’s Law. Why? Because at λmax the absorbance is maximal and so the ability to read small changes in dilute
samples with accuracy and precision can be maximized. Further, if you are not measuring the absorbance at the
substance’s λmax, the absorbance from other species (with different λmax values) can interfere.

In summary, finding the concentration of a food dye in a sample first requires determining the λmax for the dye.
Second, a parallel dilution set for the dye from a more concentrated stock solution is prepared in such a way that
the concentration of each dilution is precisely known. Third, using a spectrophotometer, the absorbance of each
dilution is then read at the dye’s λmax and an A vs. C plot constructed. From the A vs. C plot (calibration curve) the
molar absorptivity ε for the dye can be found. Finally, the absorbance of the sample at the dye’s λmax is read and
the dye concentration for the sample is calculated via Beer’s Law.

Parallel Dilution Sets

As indicated, to build a calibration curve for a particular substance, the absorbances of different known
concentrations of the substance in solution are required. The absorbances can be read for the known
concentrations using a spectrophotometer. So, the central task becomes the preparation of the different known
concentrations. This is best accomplished by constructing a parallel dilution set for the substance in solution.

A dilution set is a group of solutions prepared by diluting a substance dissolved in a liquid. For the vast majority of
cases the objective of building a dilution set is to create dilutions of defined concentration from a single “stock” or
original solution of known concentration. In a parallel dilution the stock serves as the sole source for all the
dilutions (as opposed to serial dilutions in which each dilution serves as the source for the subsequent dilution). An
advantage of parallel dilutions over serial dilutions, is that one dilution error is less likely to affect the others.

Some definitions:

• A stock solution is the original solution of known concentration from which the dilution set is built.

• A diluent is a liquid by which the sample is diluted. If 0.55 mL of stock is added to 4.45 mL of solvent, then
the 4.45 mL of solvent is diluting the 0.55 mL of stock, and so is the diluent.

When building a parallel dilution set, you should consider what volume of each dilution (VD) is actually required
given the instrumentation involved. For example, finding the molar absorptivity from an A vs. C plot with a
spectrophotometer using a standard cuvette requires no more than 3 mL of each dilution. Hence, it would be poor
technique, and incredibly wasteful, to prepare a dilution set with 100 mL of each dilution. In this case, 97 mL of
each dilution would go to waste! A volume of 5 mL would be far more appropriate.

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VS

Parallel Vdiluent
Dilution

The diagram above represents the preparation of a parallel dilution set with each dilution (diluted volume, VD) at
5.00 mL. Here VS is the volume of stock solution and Vdiluent the volume of diluent (pure water) required to achieve
each dilution.

It is helpful to realize the diluted volume VD (sometimes called the total volume) is just the sum of the stock
volume VS and diluent volume Vdiluent:

𝑉𝐷 = 𝑉𝑆 + 𝑉𝑑𝑖𝑙𝑢𝑒𝑛𝑡 LA3.1

Please DO NOT CONFUSE the diluent volume Vdiluent with the diluted volume VD, which is the sum of the stock
volume VS and the diluent volume Vdiluent.

If we are following to above diagram, in which the objective is to create dilutions of defined concentration from a
single “stock” of known concentration. We will need to calculate the resulting concentration of each dilution. The
key to such dilution calculations is the relationship:

𝐶𝑆 𝑉𝑆 = 𝐶𝐷 𝑉𝐷 LA3.2

In this equation CS is the concentration of the stock solution, VS the volume of stock solution, CD the concentration
of the dilute sample, and VD its volume. To calculate the concentration of a diluted solution knowing the
concentration of the stock solution, solve LA3.2 for CD:
𝐶𝑆 𝑉𝑆
𝐶𝐷 =
𝑉𝐷

and then substitute in the appropriate values for the stock concentration, the volume of stock used and the
volume of diluted solution.

LA 3-5 Resources
• YouTube Excel Graphing Technical Guide video on the “Chemical Thinking” channel available via this link:
https://www.youtube.com/watch?v=d44eyHNxfFs
• Graphing in Excel. If you are unfamiliar with graphing in EXCEL, this guide should help.

CHEM 151 Lab Assignment 3-5 Guide v1.1 MY © 2020 8-4-20 Chem Think Technical Press CBC UofA