Module 2 Video 2 static slides

1. Module 2 Video 2

1.1 Welcome to Biochemistry

1.2 Learning Outcomes

Published by Articulate® Storyline [Link]

Notes:

1.3 Water as a solvent: ionic compounds

Notes:

Water is an amazing substance and is often referred to as a universal solvent. A solvent,

by definition, is a substance that dissolves a solute resulting in a solution. For example,
let’s say you want to take these green cubes of salt (the solute) and dissolve them in
water. To do this you would combine the salt cubes in a beaker of water (water being
the solvent). Stir and you end up with a solution which is, by definition, a homogenous
mixture composed of two or more substances.

Water has many unique properties that make it very special but why does this matter?
Well because the chemical and physical processes of life require that molecules be able
to move about and interact with one another. Our cells are aqueous (or water
containing) environments so all our biochemical reactions occur in an environment
surrounded by water (well that’s not exactly true, but let’s be overly general for now).

Published by Articulate® Storyline [Link]

1.4 Water

Notes:

Water is amphiprotic: meaning that it can act as either an acid or base. Remember that
acids are proton donors and bases are proton acceptors.
So one water molecule acts as a base and accepts a proton from the second water
molecule that acts as an acid and donates the proton. Pure water will dissociate to form
equal concentrations (in our case molar concentrations) of hydronium and hydroxide
ions.
Of course, the resulting hydronium and hydroxide ions are some of the strongest acids
and bases around so if we follow the equilibrium arrow we will see that the equilibrium
lies far to the left. These strong acids and bases REALLY want to act as acids and bases so
they will quickly reform the water molecules. So at any one time there is an incredibly
small amount of hydronium and hydroxide ions present.

Published by Articulate® Storyline [Link]

1.5 Ionization of water

Notes:

Now that we know the amazing property of water to undergo ionization, reversible
ionization that is, we can start quantifying some of this. We already know that the
degree of ionization of water into hydronium (or hydrogen) and hydroxide ions is very
small. Maybe we can actually quantify just how much is present. We can describe this
equilibrium using an equation looking at Keq - the equilibrium constant of water which
is a product of the concentration of hydrogen ion and hydroxide ion concentrations or
molarities. These are actually activities, so in your textbook you might see them referred
to as a. the activity is roughly equal to the effective concentration of the chemical
species. In our practical biochemistry world we will refer to these as molar
concentrations (so moles of solute per liter of solution, M).

Pure water at 25 degrees Celsius has a concentration of ~ 55.5 M (moles/L). How did we
come up with this number? Super simple, we used the periodic table of elements to
calculate the molecular weight of water: let’s see one oxygen at 16g/mol plus 2
hydrogens at 1.008g/mol each equals 18.016g/mol.

We then re-worked the units to end up with g/mol, knowing that 1000 g equals 1L. We
can then plug in this number in our earlier equation and we end up with the following.
Rearranging for this a bit we introduce a new term, Kw, which is the new equilibrium
constant of water and therefore described as the ion product of water.

Published by Articulate® Storyline [Link]

Given that we can take electrical conductivity measurements of water (pure water
carries electrical current as H+ ions migrate toward the cathode and OH- ions toward
the anode in an electric field) and we can measure the Keq of water to be 1.8x10 -16M at
25oC. Plugging this into our equation we now know the ion product of water, which is a
constant, and it is 1.0x10-14.
This opens up a whole bunch of things that we can calculate. For example, we now
know that the product of H+ and OH- in aqueous solutions at a specific temperature
always equals the same value. We also know that in pure water the concentrations of
hydronium and hydroxide ions are equal and the solution is said to be at neutral pH. At
this pH the concentrations of the two ions can be calculated form the ion product of
water as follows. Therefore, in a sample of pure water, the hydrogen ion concentration
is 1×10−7 moles per liter at room temperature. Since we’ve already established that the
concentration of hydrogen ions is equal to the concentration of hydroxide ions, then the
concentration of hydroxide ions is also 1×10−7 moles per liter at room temperature.

As the ion product of water is constant, whenever the [H+] is greater than 1x10-7M, the
[OH-] must be less than this number and vice versa. Additionally, using this relationship,
we can calculate the [H+] if we know the [OH-] and vice versa. Pretty neat.

Untitled Layer 1 (Slide Layer)

Published by Articulate® Storyline [Link]

1.6 The pH scale

Notes:

Working with complex numbers can be rather mind boggling and as scientists we do try to keep
things simple whenever possible. Enter Soren Sorensen, a chemist from Denmark. He devised a
scale to measure acidity and basicity of substances. He called it the pH scale, which most
scientists define as the “power of hydrogen” or the “potential of the hydrogen ion” scale.

Simply put, the pH scale converts all these negative powers of 10 used to describe equilibrium
constants to much easier to digest numerical values.
pH is defined as the negative log of the activity of hydrogen ion, which is roughly equal to the
negative log of the molar concentration of hydrogen ion.
So for water, we have a Kw of 1x10-14, which can be converted to a pKw of 14. This sets the
limit of our pH scale in our aqueous environment. Of course, this is only our working definition
as pH doesn’t really have a limit since it is, by definition, an indicator of the concentration of
hydrogen ions. However, for our biochemistry purposes, the upper limit can be though to be 14.
Now we can calculate the pH and pOH as well.
Keep in mind: The pH scale is logarithmic, meaning that an increase or decrease of an integer
value changes the concentration by a tenfold. For example, a pH of 3 is ten times more acidic
than a pH of 4. Likewise, a pH of 3 is one hundred times more acidic than a pH of 5.

The other thing to note is that the higher the concentration of H+ in a solution, the lower the pH
based on this relationship. So a low pH describes an acidic solution.

Published by Articulate® Storyline [Link]

1.7 pKa and pKb – the weak world

Notes:

Let’s now delve into the weak acid/base world because this is the meat and potatoes
for biochemistry. Weak acids and bases dictate a huge number of reactions in
biochemistry (both in the cell and in the test tube). Let’s look at a general weak acid, HA,
and its dissociation in an aqueous environment. We can write this as follows:
The equilibrium constant for this dissociation reaction is:
The larger the Ka the stronger the acid (that’s because the hydronium ion concentration
is in the numerator of this equation). Now all we need to do is to express this in terms of
pKa to keep in line with the pH scale and be able to compare everything. So we write is
as:
Remember that log is an inverse relationship so the larger the Ka, the stronger the acid
and the smaller the pKa
Now that you know the relationship you can just look up pKa values from a table.

And after some proper mathematical rearrangements (which you do not need to know)
you arrive at the following key relationship: pKa + pKb = 14

Published by Articulate® Storyline [Link]

1.8 The physiological pH range

Notes:

This is our playground: the physiological pH range. The blue portion indicates basic
substances which usually range from a pH 7-14 with the closer to 14 the more basic the
solution. Ammonia is around pH 12 and therefore quite basic. Conversely, the red
portion indicates acidic substances which range from 0-7, the closer to zero the more
acidic the substance. HCl, for example has a pH of around zero which is not surprising
given that it is a strong acid.

Measurement of pH is actually paramount in biochemistry. pH affects the structure and

activity of most macromolecules, for example the catalytic activity of an enzyme is
extremely dependent on the proper pH.
Other applications of pH include the measurement of pH in blood and urine as medical
diagnoses.

Published by Articulate® Storyline [Link]

1.9 Weak acids and bases

Notes:

Acids and bases behave differently in an aqueous environment depending on whether

or not they are strong or weak acids and bases. Strong acids and bases typically ionize
fully in aqueous environments, but weak acids and bases are much more interesting to
biochemists as they play very important roles in metabolism and regulation. This is why
we need to take a moment and revisit weak acids and bases a bit more closely.
Here we have the equation of a weak acid, RCOOH where R can be a number of groups,
let’s say CH3 and we have acetic acid in an aqueous solution. Remember that acids are
defined as proton donors and bases as proton acceptors. When acetic acid loses a
proton it becomes its conjugate base, in this case the acetate anion, CH3COO-
A proton donor and its corresponding proton acceptor make up a conjugate acid-base
pair.

This is a reversible reaction, and each acid has its own characteristic tendency to lose its
proton in an aqueous solution. Typically, the stronger the acid the higher the tendency
to lose its proton. We can revisit the equation as defined by the equilibrium constant for
the weak acid reversible reaction and once again revisit Ka, which is the equilibrium
constant for this ionization reaction, typically called the acid dissociation constant.
Remember that the stronger the acid the larger the Ka.
If, however, you convert this relationship to pKa, we now have a -log relationship exactly
like pH so the stronger the acid, the stronger the tendency to dissociate a proton in an

Published by Articulate® Storyline [Link]

aqueous solution, the lower the pKa. This figure depicts the Ka and pKa values for some
well known acids. Please take a moment to review this. You do not need to memorize
these values.

Untitled Layer 1 (Slide Layer)

1.10 The Henderson Hasselbalch equation

Published by Articulate® Storyline [Link]

Notes:

The HH equation which describes a direct relationship between the pH of a solution and
the ratio of the concentrations of the deprotonated (so A-) form and protonated (so HA)
form of an ionizable group. This equation is really powerful when describing or using
weak acids and their conjugate bases. It is equally as useful for weak bases and their
conjugate acids.
Let’s take our biomolecule example. We know that the biomolecule changes its surface
charge over a range of pH and that these changes are due primarily to the carboxylic
acid and amine groups in the biomolecule. Let’s look at a carboxylic acid (RCOOH) , in
this case formic acid (HCOOH), whereby we have (A-…)

Now we can rewrite the HH equation to reflect this specific example…

The cool thing about this equation is that we can now calculate the pH of the formic
acid solution for any ratio of conjugate acid and base. So for example, we can see how
the pH of this solution changes as we add base to this solution. This is called a titration.

1.11 Titrating a weak acid with a strong base

Notes:

Published by Articulate® Storyline [Link]

Titration is the slow addition of one solution of a known concentration (called a titrant)
to a known volume of another solution of unknown concentration (the analyte) until the
reaction reaches neutralization, which is often indicated by a color change.
So let’s say that we will perform a titration of our weak acid, formic acid, with a strong
base such as sodium hydroxide. To do this in the lab we add the sodium hydroxide to
the formic acid using a buret. This reaction between a weak acid and a strong base
involves the transfer of a hydrogen ion from formic acid to the hydroxide ion of the
strong base to create the conjugate base and water.

We can actually measure the pH of a solution as we add an increasing amount of titrant.

This information can be plotted on a graph.

1.12 Titration curve of 1.0M formic acid with NaOH

Notes:

The x-axis depicts the amount of OH ions added ( in this case we would graph the volume of NaOH) and the y-axis
depicts the pH of our solution.

So in our particular example, we want to titrate a 1M solution of formic acid with sodium hydroxide. We also know
the pKa of formic acid by looking it up. The pKa is 3.75

Published by Articulate® Storyline [Link]

Let’s look at our initial time point zero (no NaOH added) as it will tell us the pH of formic acid at initial pH (no
addition of base). Assume: ionization of water negligible. If we write the initial situation under our equation (read
initial setup).

The change corresponds to the loss of the hydrogen ion and is denoted by -x for formic acid. However, given the 1:1
stoichiometry, this x value is added to the right side of the equation. At equilibrium we have 1M-x for formic acid
and plus x for both species on the right side of the equation. Plugging these values in our Ka equation, along with
the Ka of formic acid we end up with the following equation. Now we can solve for x using a quadratic equation and
obtain the following value for x (1.33 x 10-2M). This is a very small number and subtracting this number from our
initial concentration of 1M we obtain 0.9867M which is approximately 1M when rounded up. So really, this entire
quadratic equation calculation was unnecessary and we could have stated that 1-x is roughly equal to 1. To figure
out when you can make this assumption all you need to do is to take the Ka of the acid and divide into the initial
molar concentration (in this case 1M). If you get a value greater than 1000 than you can simply state that 1-x ~ 1

Plugging this hydrogen ion molar concentration into the equation of pH we get a pH of formic acid at ~ 1.9.

Now let’s see what happens upon addition of NaOH, which is an extremely strong base so it immediately dissociates
into Na+ and OH-. However, since we are in an aqueous environment which was originally in an equilibrium, and
the equilibrium constant in this milieu states that the concentration of hydrogen and hydroxide ions together must
equal the Kw = 1x10-14, it means that a sudden increase in the OH- concentration must result in an equal decrease
of H+ to maintain this relationship. This means that the [HCOO-] increases, while that of [HCOOH] decreases. Going
back to the HH equation, it tells us that the ratio increases as NaOH is added. As a consequence of solving this
equation, the pH also increases upon addition of NaOH. This is why we see a relatively sharp increase in pH at the
initial stages of NaOH addition.

At the midpoint of this titration, exactly half of the formic acid has been neutralized, this means that half of the
original formic acid is present in its acid form and the other half is present in its conjugate base form. It also means
that the HH ratio is equal to 1. At this midpoint pH = pKa

time = 0 (Slide Layer)

Published by Articulate® Storyline [Link]

time = > 0 (Slide Layer)

midpoint (Slide Layer)

Published by Articulate® Storyline [Link]

1.13 Titration curve actual data

Notes:

Here is an actual data set of three titration curves of weak acids. You will also notice
that for acetic acid the initial pH (prior to addition of NaOH) is ~ 2, then you see a sharp
increase in the curve due to an imbalance in the equilibrium as hydroxide ions are
flooding the solution, followed by a plateau. At the midpoint of this plateau, the
concentration of acetic acid is equal to its conjugate base. Now, we've just talked about
sharp increases in the curve, which is true, but it you look at the pH range between the
initial time point and the midpoint, you will notice that the change in pH for this specific
example is only about 2.
This is certainly an interesting observation. In fact, if we look at the area around the pKa
(blue lines) we can see that addition of strong base does not seem to affect the pH at all.
This is the principle of BUFFERING of solutions using weak acid-base mixtures. These
resulting buffer solutions are the base of just about every biochemical experiment.

Published by Articulate® Storyline [Link]

1.14 Buffer solutions

Notes:

By definition, a buffer solution is a solution that resists changes in pH. Suppose a

biochemist wants to study a reaction at pH 4.0. The best buffer for this situation is one
which has a pKa close to 4.0 because it is most effective at buffering near its pKa. This
will ensure that the pH will not drift too much during the experiment. Recall from the
HH equation that the pH = pKa when [acid] = [conjugate base]. For our specific example,
the formic acid/formate ion buffer would actually be a good buffer to use as the pKa of
formic acid is 3.75.
Now all we need to do is calculate the ratio of formic acid/formate ion required to make
this buffer, which is also easy because we can plug our 2 knowns in the HH equation,
such that our desired pH = 4.0 and the pKa is 3.75. You will get a ratio of 1.78, which
means that you need to add your reagents in this specific ratio. You can also use a pH
meter and prepare your formic acid buffer solution by titrating your formic acid solution
to pH 4 using NaOH (the same way we obtained the curve).

Published by Articulate® Storyline [Link]

1.15 Molecules with multiple ionizing groups

Notes:

Glycine is an ampholyte, meaning that it has 2 pKas due to its two ionizable groups. It
has an amino group that is a great proton acceptor and it has a carboxyl group that is a
proton donor. So what do you think would happen if we place glycine in a solution at pH
1? Well, I like to think of this as having lots and lots of free hydrogen ions so any group
that can be protonated will be protonated at this point. The glycine molecule will
therefore look like this and have a net positive 1 charge. Both molecules are protonated
and the net charge on glycine is +1.

Published by Articulate® Storyline [Link]

Untitled Layer 1 (Slide Layer)

Published by Articulate® Storyline [Link]