Week 2: Solutions and pH

This week the laboratory exercise will focus on three activities:

1. Recording the results from last week’s laboratory

2. Preparing Solutions from Dr. N. Mills
3. Solution pH: From Experimental Cell & Molecular Biology by John S. Choinski,
Jr.

Today you will be working in groups of 4. In order to complete all the experiments, each group
will start with a different activity.

Preparing Solutions
The preparation of solutions is a basic and fundamental aspect of working in the
laboratory. We generally follow several rules when we prepare our solutions for an experiment.
1. The quality of chemicals is an important consideration when we plan to conduct an
experiment. (The quality needs to match the requirements of the reaction or procedure).
2. We plan to prepare and store our solutions in clean glassware or plastic ware.
3. Unless otherwise permitted or directed, all solutions are prepared using deionized or
better water for the solvent. Deionized or distilled water should be free of most general
contaminants.
4. Do not contaminate your solution with your hands, or dirty tools, and do not contaminate
yourself. (Generally, wear gloves and protective eye wear etc., and use clean spatulas
and stirring bars etc).

5. LABELING SOLUTIONS
a. Use tape on the storage bottle. If you wrap the tape around the bottle and overlap
the tape from 1/2 to 1.0 inch in order that the tape will adhere to the bottle in extreme
heat or cold.
b. Use a permanent marker to write on the tape or plastic tube. If you write on a
plastic tube, cover the writing on the tube with scotch tape and again overlap the tape a
bit.

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 c. Label the storage bottles with your initials and last name and current date on the
bottle.
d. List clearly on the label, the compound or contents of the solution,
concentration, any analyses or checks on the solution such as pH, conductivity, density,
or refractive index.
e. If you autoclave the solution either list this after autoclaving or preferably use
autoclave tape.

SOLUTION PREPARATION:
1. Get a beaker or flask that is about 50% greater volume than the volume of the solution to
be prepared.
2. Find a stirring bar that is about 1/4 to 1/2 the diameter of the beaker bottom.
3. Locate magnetic stirring plates.
4. Determine the amount of compound required to make the volume of solution selected.
5. Substract that amount of weight from the volume of total solution in milliliters to be
prepared.
6. Measure out the remaining amount of volume as deionized water or purified water and
place this in the container (beaker).
7. Turn on the magnetic stirring plate to create a mild vortex in the solvent (water). Do not
whip air into the liquid.
8. Weigh out the required amount of compound to be dissolved (solute). Slowly add this
material to the volume of solvent. Do not add so rapidly as to block the stirring bar.
9. Once the material is completely dissolved, place the solution in a graduate cylinder that is
large enough to measure to final volume. QS (quantity sufficient) the solution to the
final volume with deionized water.
10. If you are going to adjust the pH of the solution then you would pH the solution first,
then qs to volume.
11. If you are reading conductivity or refractive index then you would want the volume qs’ed
to the final volume before measurements are taken.

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Information needed to understand preparing solutions.
A. Determination of Mass to be weighed to prepare a specific solution.
Background Information
1. The value of one is assigned as the mass of a neutron and the value of one is assigned as
the mass of a proton and the mass unit is known as a Dalton.
For example: carbon has 6 protons and 6 neutrons and an atomic mass of 12 (6+6) and
this is known as the atomic weight. A few atoms have 7 or 8 neutrons so that the
average for the atomic weight of carbon in the earth’s crust is slightly more than 12.
2. A mole is a mass of material equal to the sum of atomic weights of the elements times
their subscripts in the compound weighed out as grams. Ex. NaCl (table salt) Na =23
daltons and Chlorine = 35.5 daltons, hence the molecular weight of NaCl is 58.5. To
have a mole of NaCl we would weigh out 58.5 grams of salt. Hence, the gram•mol.wt.
of any molecule or compound is one mole of that material by definition.
3. Once the weight of a gram was arbitrarily assigned to the mol. wt. in daltons, the
question was asked – how many molecules of a compound are in one mole since the
amount of mass might be quite different in two different compounds but the total number
of molecules would still be constant.. Hence, the number of molecules in a mole of any
compound was determined to be 6.023x1023,. (Avagadro number).
4. When we prepare a solution, we want to be able to deliver a certain number of molecules
by measuring a selected volume of solution. Thus, we prepare these solutions as moles
per liter of volume. Moles/liter = molarity . A big M is used to express or represent
concentration of a solution. Ex. 1.0 M NaCl would be 58.5 grams of NaCl dissolved in
one Liter volume in H2O. There is actually less than a liter of water in the one liter
volume since part of the volume was produced from the NaCl added to the solution.
Hence, in 100 ml (0.1 Liters) of a 1.0 M solution of NaCl, we would have 5.85 grams of
NaCl and is equal to 6.023x1022 molecules of NaCl.
Likewise, table sugar or sucrose has a molecular weight of 342 daltons. To
prepare a 2.0 molar solution of sucrose we would weigh 2 x 342 = 684 grams of sugar
and dissolve this in a small volume of H2O (not more than 316 ml). Once the sugar is in
solution then we would qs the volume to 1.0 liter to make the concentration exactly 2.0

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 molar sucrose. (Karo syrup is about 3.0 Molar, how much sucrose is in one liter of Karo
syrup?)
5. The difference between the formula weight (F.W.) and the molecular weight (mol.
wt.) is that in many situations where chemicals are prepared and purified, molecules of
water are incorporated into the lattice structure of crystals when the material is purified
by crystallization in preparation for sale. This water of crystallization will add to the
mass when weighed, but does not contribute to the number of molecules of the
compound. Hence, when preparing solutions, we use the formula weight not the
molecular weight when we determine the amount to weigh out in preparing a solution.
Of course, if there is no extra molecules incorporated into the crystal structure then the
Formula weight an the Mass weight are the same thing.

B. pH, what is it, what does it mean, and how do we use it.

In 1909, Sorenson presented the idea of expressing acidity in whole positive numbers. The term
pH is an expression used to relate the hydrogen ion concentration in positive whole numbers.
The hydrogen ion concentration is usually much less than 1.0 molar. (For example, pure water
has a hydrogen ion concentration of 0.0000001 M or 1.0x10-7 moles per liter of water. With
values this small, only the exponent to the base 10 are whole numbers, ie. –7 and therefore,
Sorenson presented the idea of using the log of the H+ concentration to talk about hydrogen ions
in a solution (acidity). If you are going to manipulate numbers anyway to make them easier to
deal with, why not multiply the log value which is always negative by a negative one which will
make the number positive, hence you do not have to write the sign. Hence, we write

pH = (-1)log [H+]
pH = positive hydrogen concentration
[ ] = concentration
H+ = hydrogen ion
(-) log = (-1) x log the numerical value + the exponent to the base 10
ex. [H+ ] = 2.0 x 10-3 M or 1.0 mM. 2.0 is a number and log of 2.0 is 0.301 and the exponent to
the base 10 is a log (-3) Hence, pH = [0.301 + (-3)] (-1) = -2.699(-1) = 2.699.

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pH as we use it assumes that materials are in a water system. When a water molecule ionizes
(forms charged particles) both a hydronium ion and a hydroxyl ion are formed.

(2H2O —>H3O+ + OH- )

This happens only at the rate 0.0000001 moles per liter of H2O or 1.0x10-7 M. Clearly from the
equation about every time you produce a hydronium ion you also produce a hydroxyl ion and if
the hydronium ion concentration is 1.0x10-7 M then the hydroxyl ion concentration is 1.0x10-7
M.
When the hydronium and hydroxyl ions are equal then the solution is said to be neutral.
Obviously the pH = 7 . You can write the same equation for the hydroxyl ion. That is:

pOH = (-1) log [OH-]

and at pH 7.0 the pOH is 7.0. In water pH + pOH = 14, therefore if pH is 4 then pOH is 10 etc.
On a practical basis however, we generally use only the term pH, recognizing that values below
7 are acidic and values above 7 are basic or alkaline (have more OH ions than H ions).

We use two general methods for detecting pH.

(1) One method is to use dye or color indicators that change color in the presence of specific
concentrations of hydrogen ions (pH indicators or litmus type papers).
(2) pH meters – instruments that measure conductivity of solutions through membranes in
which only H+ can pass. Thus the amount of conductance is a direct reflection of the H+
concentration in the solution. This amount of conductance is then indirectly registered on the
meter, indicating pH.

C. Conductance - the ability of a solution or material to conduct a current of electricity

(amperes). The units of conductance are Siemens and conductance is the reciprical of resistance
(ohms).

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 On a practical basis, pure H2O has very poor conductance since the ion concentrations
(H+ and OH-) is in the 1.0 x 10-7 M range. If you add salts or other compounds that ionize in
water when it dissolves the conductivity goes up rapidly and in direct proportion to the number
of ions added to the water solution. Hence, we can use conductivity to check concentration of
materials in a solution. A conducivity electrode is set up to measure ability to conduct a current
(electrons, amperes) through a given volume of solution over a defined distance. The more
current conducted the greater the ion concentration.

D. Refraction – the ability of a solution to bend light waves as they travel through a solution or
medium.
Compounds such as sucrose (table sugar) glucose, and urea to mention a few are polar
enough to dissolve in H2O. However, these materials do not ionize (dissolve as charged particles)
in H2O. However, as the concentration increases, the refractive index of the solution will also
increase. This property can be used to determine the concentration of uncharged compounds
dissolved in H2O as well as charged compounds dissolved in water. Refractive index is used in
the candy industry to determine sugar content and the determination of salinity in salt water
aquariums.
E. Normality - This is a term used to talk about the number of moles of hydrogens in a
compound. For example, a solution containing 1.0 mole of HCl has one mole of hydrogen ions
that can be donated to a water solution. One mole of H2SO4 or sulfuric acid has 2 moles of
hydrogens that can be donated to a water solution, therefore, one molar sulfuric acid is said to be
2.0 normal. Hence, in acid base reactions, has two equivalents for each one of the compound.

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Procedure A. Solutions

1. Each group will be given a different solution to prepare; however, you will be expected to be
able to calculate a recipe for each solution.

COLOR SOLUTION VOLUME

RED 0.1M NaCl ph=8.0 50 ML

BLUE 0.05 M EDTA ph=8.0 100 ML

GREEN 1 M NaoAC ph=5.2 50 ML
YELLOW 1M Sodium Carbonate, 50 ML
check pH
ORANGE 0.5% sucrose 50 ML

2. Calculate the amount of each reagent in the solution. Show your work below.

What will you pH your solution with?

3. Have your instructor check calculations.

4. Following the directions in the preparing solutions handout, weigh out the appropriate
amount of reagent.
5. Place the chemicals in a labeled beaker with deionized water and a stir bar. DO NOT FILL
THE BEAKER WITH THE FINAL VOLUME OF WATER. For example, if you are
making a 100 ml solution, only place ~50 mls of water in the beaker.
6. Stir the solution until all reagents are dissolved.
7. pH the solution as instructed.
8. Using a graduated cylinder, measure the volume of your solution. Adjust (qs) the volume of
your solution with deionized water to the appropriate final volume.
9. Place your solution in a labeled glass bottle.

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 A. The Titration of Acetic Acid

1. Add 100 ml of 0.2M Acetic Acid into a 250 ml beaker.

2. Lower the pH electrode gently into the solution and record the pH.
3. Add 1ml of 1.0 M Sodium Hydroxide (NaOH) to the beaker, swirl the beaker
gently and record the pH in the data sheet below.
4. Repeat step 3, until the pH is ~8.0.
5. Plot a graph of pH (dependent variable) vs ml of 1M NaOH added.

Table 1: Titration of Acetic Acid

1M NAOH Added (ml) pH
0 (initial pH)
1
2
3

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B. How To Make An Acetate Buffer:
One type of acetate buffer is a mixture of acetic acid and sodium acetate. You have 0.2M
acetic acid and 0.2M sodium acetate. In this experiment you will create a series of buffers using
the chart below.

1. Label 6 vials a-f.

2. Add the appropriate volume of acetic acid and sodium acetate from the table below.
3. Record the actual pH of each buffer.
4. Calculate the final concentration of acetic acid and sodium acetate in each buffer.
5. Calculate the pH of each buffer using the Henderson-Hasselbalch equation.

Table 2: Creation of an Acetate Buffer

Buffer Buffer Acetic Sodium Final Conc. Final Conc. Calculated Actual
Sample Acid Acetate [Acetic Acid] [Sodium pH pH
(0.2M) (0.2M) Acetate]=
=0.2X
10 0.2 (10-X)
10
a X=0 0 ml 10 ml
b X=2 2 ml 8 ml
c X=4 4 ml 6 ml
d X=6 6 ml 4 ml
e X=8 8 ml 2 ml
f X=10 10 ml 0 ml
X= ml of acetic acid; 10-X = sodium acetate

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 pH and Solutions Assignment (20 points)
Due Next Week
Titration of Acetic Acid
1. Completed Table showing data (1 points)
2. GRAPH pH vs. ml of NaOH added (2 points)
3. What does the graph show you? Briefly explain the conclusion you can drive from the graph.
(3 points)
4. What is the pKa of acetic acid based on this experiment? (1 point)

How to make an acetate buffer

5. Completed Table showing data. (1 point)
6. Graph pH vs concentration of acetic acid [X]. (2 points)
7. Calculate pH of each buffer using the Henderson-Hasselbalch equation.
(4 points, 1 point each)
8. Are the calculated pH(s) different then actual pH(s) of the buffer? If so, explain why?
(2 points)
9. What is the buffering capacity of acetate buffer? Why? (2 point)
10. Calculate the expected pH of 10 ml of 0.2 M acetic acid after the addition of 1.0 ml of 0.5 M
HCL (2 point)

Microbiology Techniques assignment is also due next week (10 points)

It should include:

a) Raw data: ‘Count the colonies in each dilution’ table on page 13 of week-1 handout. (1
point)

b) Show an example of how you calculated the average cells in a dilution using your data.
(1 point/calculation)

c) Answer to the question: Why do we plate more than 1 dilution and multiple plates of each
dilution? (1 point)

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