Chapter 2 Diluting part 1

Introduction

In the lab, you often need to work with dilutions. The concentration of the analyte must lie
between the concentrations of the reference solutions in order to obtain measurements as
accurate as possible. In addition, the concentrations being measured must fall within the
measuring range of the equipment used. If the concentration of a sample solution is too high,
the sample solution needs to be diluted. Dilutions are therefore common when working in a
lab.

Theory

Diluting
When preparing a dilution, additional solvent (such as demineralised water) is added to the
solution. The concentration of the solute will therefore decrease.

The basic principle of diluting is that the amount of solute (for example in g or in
number of molecules) is equal before and after diluting, while the volume after
diluting has increased. The concentration of solute (in g/L of molecules/L) will
therefore be lower after diluting.

In figure 1, the principle of diluting is illustrated. The total number of molecules of acetic acid
present (depicted as stars in the figure) remains the same after diluting. The concentration of
acetic acid is reduced by diluting. This is demonstrated by the number of particles within the
square in the figure.

Figure 1. The solution in beaker 1 is mixed with solvent, and thereby diluted. Source: Albers, F.J.M & Geurts,
F.A.J.G., 1998. Chemisch rekenen MLO, Educatieve Partners Nederland BV, Houten, ISBN 9040108668

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Example
Imagine that you had 50.0 mL of household vinegar (40.0 g/L of acetic acid) and added an
equal volume of demineralised water. The amount of acetic acid (in g) originally present, will
not change. The demineralised water that you add does not contain any acetic acid. Nor is any
acetic acid lost by adding demineralised water. The volume increases causing the
concentration to decrease. In this case the volume is doubled, resulting in a decrease of the
concentration by half.

Calculating with dilutions

When calculating with dilutions it is important to remember the basic principles of diluting.
The amount of solute is equal before and after diluting, the volume increases and as a result
the concentration decreases.
In this chapter the mass concentration (g/L), in other words the mass of solute per volume,
will be used. The same principle holds for the molar concentration (mol/L), which will be
explained in a later chapter.
If we multiply the volume of the solution (L) with the mass concentration (g/L), this results in
the mass of the solute (g). Because the mass of the solute doesn’t change, the diluted solution
contains the same amount of solute.
This means that (using the figures from the acetic acid example in brackets):

𝑔
𝑉𝑏𝑒𝑓𝑜𝑟𝑒 𝑑𝑖𝑙𝑢𝑡𝑖𝑛𝑔 ∙ 𝑐𝑏𝑒𝑓𝑜𝑟𝑒 𝑑𝑖𝑙𝑢𝑡𝑖𝑛𝑔 = 𝑚𝑠𝑜𝑙𝑢𝑡𝑒,𝑏𝑒𝑓𝑜𝑟𝑒 (50.0 ∙ 10−3 𝐿 ∙ 40.0 = 2.00 𝑔)
𝐿

and

𝑔
𝑉𝑎𝑓𝑡𝑒𝑟 𝑑𝑖𝑙𝑢𝑡𝑖𝑛𝑔 ∙ 𝑐𝑎𝑓𝑡𝑒𝑟 𝑑𝑖𝑙𝑢𝑡𝑖𝑛𝑔 = 𝑚𝑠𝑜𝑙𝑢𝑡𝑒,𝑎𝑓𝑡𝑒𝑟 (100.0 ∙ 10−3 𝐿 ∙ 20.0 = 2.00 𝑔)
𝐿

The mass of the solute is the same in both situations (msolute, before= msolute, after). Therefore:

𝑽𝒃𝒆𝒇𝒐𝒓𝒆 𝒅𝒊𝒍𝒖𝒕𝒊𝒏𝒈 ∙ 𝒄𝒃𝒆𝒇𝒐𝒓𝒆 𝒅𝒊𝒍𝒖𝒕𝒊𝒏𝒈 = 𝑽𝒂𝒇𝒕𝒆𝒓 𝒅𝒊𝒍𝒖𝒕𝒊𝒏𝒈 ∙ 𝒄𝒂𝒇𝒕𝒆𝒓 𝒅𝒊𝒍𝒖𝒕𝒊𝒏𝒈

This is generally represented as:

𝑉𝑏𝑒𝑓𝑜𝑟𝑒 ∙ 𝑐𝑏𝑒𝑓𝑜𝑟𝑒 = 𝑉𝑎𝑓𝑡𝑒𝑟 ∙ 𝑐𝑎𝑓𝑡𝑒𝑟

or

𝑉1 ∙ 𝑐1 = 𝑉2 ∙ 𝑐2

This formula will be used frequently for preparing dilutions in practice as well as to answer
the questions (at the end of the chapter).

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Dilution factor
In the lab, we sometimes work with a for example 20-fold diluted solution. This means that
the final concentration is 20 times lower than the original concentration. This is what we call a
dilution factor.
A 20-fold diluted solution can be made by mixing a certain volume of a solution with 19
times that volume of solvent in order to create a final volume that is 20 times the original
volume.
Another way to express this is diluting 1 : 19 (or 1 : 19), so 1 volume of the original solution
with 19 volumes of solvent.

The dilution factor can thus be calculated as cbefore / cafter or as Vafter / Vbefore.

Calibration series
A calibration series is a series of solutions containing the same substance, but in different
concentrations (figure 2). These series are prepared by diluting different volumes of the stock
solution with solvent to form solutions with the same final volume. The solutions in the
calibration series can then be used to create a calibration curve (figure 3). If an unknown
sample is measured using the same analysis method, the concentration of the sample can be
calculated or read off the calibration curve.

Figure 2. Calibration series (http://www.exo.science.ru.nl/bronnen/biologie/smart.html, 10-10-2017)

10

8
Absorbance [a.u.]

0
0 1 2 3 4 5
Concentration [g/L]
Figure 3. Example of a calibration curve

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To prepare a calibration series, you need to calculate the volume of the stock solution and
solvent that must be added together. As support in the calculations a table is often created (see
example below).

Final concentration Final volume (mL) Volume stock solution Volume demi
BSA (mg/mL) 10 g/mL BSA (mL) water (mL)
0 5.00
200 5.00
400 5.00
600 5.00
800 5.00
1000 5.00

Each dilution is calculated using the formula 𝑉1 ∙ 𝑐1 = 𝑉2 ∙ 𝑐2, where V1 and c1 refer to the
stock solution, and V2 and c2 refer to the diluted solution. Note: ensure that the same units are
used for the volume and concentration of the stock and diluted solution!

Example (calibration series example table)

𝑉1 ∙ 𝑐1 = 𝑉2 ∙ 𝑐2
V1 is unknown and needs to be calculated.

𝑉2 ∙ 𝑐2
𝑉1 =
𝑐1
𝑚𝑔
5.00 𝑚𝐿 ∙ 200 𝑚𝐿
𝑉1 = 𝑔
10.0 𝑚𝐿
Note: c1 and c2 have a different unit! So first convert the unit!

𝑔
5.00 𝑚𝐿 ∙ 0.200 𝑚𝐿
𝑉1 = 𝑔 = 0.10 𝑚𝐿
10.0 𝑚𝐿

Therefore, 0.10 mL of BSA stock solution is needed to prepare a 200 mg/mL BSA solution.
The final volume is 5.00 mL, so 5.00 – 0.10 = 4.90 mL of demineralised water needs to be
added.

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The completed table looks like this:
Final concentration Final volume (mL) Volume stock solution Volume demi
BSA (mg/mL) 10 g/mL BSA (mL) water (mL)
0 5.00 0.00 5.00
200 5.00 0.10 4.90
400 5.00 0.20 4.80
600 5.00 0.30 4.70
800 5.00 0.40 4.60
1000 5.00 0.50 4.50

Dilution series
A dilution series is a series of solutions containing the same substance, but in declining
concentrations. A dilution series is prepared by diluting a certain volume of the solution with
a certain volume of the solvent. The diluted solution will then be diluted again. Usually, the
same dilution factor is used for each step, for example a factor of 10. In that case, it would be
referred to as a ten-fold dilution series (figure 4).

Figure 4. A ten-fold dilution series (http://biology.kenyon.edu/courses/biol09/tetrahymena/serialdilution3.htm,

23-11-2017)

Ten-fold dilution series are among others used for samples where the number of bacteria
needs to be determined. By diluting the sample by a factor of 10 each time, this will
ultimately produce a sample which allows the number of bacteria per mL to be determined.
To calculate the concentration (or number of bacteria) in the original sample, the determined
concentration (in one of the dilutions) has to be recalculated to the concentration in the
undiluted sample. This is done by multiplying the determined concentration by the dilution
factor.

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Example
A bacterial culture is diluted in a ten-fold dilution series (i.e. by a factor of ten each time).
Each dilution is prepared by mixing 1.0 mL of the previous dilution with 9.0 mL of
physiological saline. The dilution 10-5 appears to contain 132 bacteria per mL. The number of
bacteria per mL of undiluted culture is therefore 132 ∙ 105 = 1.32 ∙ 107 bacteria per mL.
The number of bacteria per mL of a sample is also known as the plate count.

Assignments level 1 (warming-up)

1. 50.0 mL of household vinegar (40.0 g/L acetic acid) is diluted with water to 200.0 mL.
a. Calculate the concentration of acetic acid in g/L after diluting.
b. Calculate the concentration of acetic acid in g/mL.
c. Calculate the concentration of acetic acid in μg/cL.
2. The concentration of a copper sulfate solution is 1.0105 g/L. 2.00 mL of this solution
is pipetted into a 100.0 mL volumetric flask. The flask is topped up to 100.0 mL.
a. Calculate the concentration of copper sulfate (in g/L) after topping up.
b. Calculate the concentration of copper sulfate in mg/L.
c. Calculate the concentration of copper sulfate in kg/m3.
3. A sugar solution of 100.0 g/L is diluted 25-fold.
a. Calculate the new concentration of sugar in g/L.
b. Calculate the new concentration of sugar in g/mL.
c. What volume of water needs to be added to 10 mL of the solution in order to
make a 25× dilution?
d. What volume of water needs to be added to 25 mL of the solution in order to
make a 25× dilution?
4. A sodium carbonate solution has a concentration of 75.0 mg/mL and a diluted solution
has a concentration of 5.00 mg/mL.
a. What is the dilution factor of the diluted solution?
b. What volume of water needs to be added to 15.0 mL of the 75.0 mg/mL
solution to make the 5.00 mg/mL solution?

Assignments level 2 (advanced)

5. A sample is tested for the presence of bacteria. A count of the 10-7 dilution reveals that
there are 258 bacteria/mL present in the dilution. What is the plate count of the
undiluted sample?
6. The number of yeast cells in a home-made beer is counted. A count of the 10-4 dilution
reveals that there are 148 yeast cells present per mL. What is the number of yeast cells
per mL in the undiluted beer?
7. Fill in the table below;
Stock solution: 200 mg/L potassium permanganate
Solutions required: 100 mL each of 0, 2, 4, 6, 8 and 10 mg/L

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Final concentration Final volume (mL) Volume 200 mg/L Volume demi
(mg/L) stock solution (mL) water (mL)

8. Make a table for the following calibration series;

Stock solution: 10.0 g/mL BSA (bovine serum albumin)
Solutions required: 1.00 mL each of 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 g/mL.

Assignments level 3 (exam level)

9. In the lab we need 100 mL of a 5.00 g/L sugar solution. How do you dilute an 8.00
%(m/V) sugar solution to the desired concentration?
10. In the lab we need 1.00 L of physiological saline. This is a 0.900 %(m/V) NaCl
solution. How do you make this solution from a stock solution of 50.0 g/L?
11. A student pipettes 25.00 mL of hydrochloric acid (7.060 %(m/m); ρ = 1.032 g/mL)
into a 500.0 mL volumetric flask. Then, he tops up and homogenises the solution.
Calculate the concentration of the solution in g/mL.
12. A student pipettes 50.0 mL of a salt solution (12.0 %(m/m); ρ = 1.83 g/mL) into a
250.0 mL volumetric flask. Then, he tops up and homogenises the solution. Calculate
the concentration of the solution in g/L.