CHMI 2227E
Biochemistry I
Refresher:
-acid-base
chemistry
-spectrophotometry
CHMI 2227 - E.R. Gauthier, Ph.D.
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Acid base chemistry
- strong acids
HCl  H+ + ClpH = - log [H+]
Where [H+] is in molar (M) concentration units.

Strong acids dissociate completely in water.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Acid base chemistry
- weak acids
HA
Ka = [H+] x [A-]
[HA]
H+
+
A-
pKa = -log Ka
Ka =
Where:
-HA is the undissociated acid
-A- is the conjugated base
of acid HA
1
10pKa

Weak acids do not dissociate completely in
water;

The extent to which the weak acid will dissociate
is indicated by the dissociation constant (Ka).
CHMI 2227 - E.R. Gauthier, Ph.D.
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Acid base chemistry
- weak acids

The pH of weak acids solutions can be
determined by first calculating the [H+], taking in
consideration the Ka;
Ka = [H+] x [A-]
[HA]

pH = - log [H+]
Alternatively, one can also use the HendersonHasselbach equation:
pH = pKa + log [A-]
[HA]
CHMI 2227 - E.R. Gauthier, Ph.D.
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Acid base chemistry
- weak acids: titration

Adding a strong
base to a weak acid
solution will
progressively
convert more and
more HA to A-.
pH
HA
H+ + A100% A-
Mid-equivalence
point:
- 50% HA
- 50% ApKa

Notice that the pH
doesn’t change
significantly near the
pKa: the solution is
said to be buffered.
pH at mid-equivalence
point = pKa
100% HA
CHMI 2227 - E.R. Gauthier, Ph.D.
0.5
NaOH
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Acid base chemistry
- weak acids

Example 1: What is the pH of a 0.1 M solution of acetic
acid (Ka = 1.76 x 10-5M).
CH3-COOH
CH3-COO- + H+
Ka = 1.76 x 10-5 M = [H+] x [A-] = [H+]2
[HA]
[HA]
[H+]2 = 1.76 x 10-5 M x [HA] = 1.76 x 10-5 M x 0.1M
[H+] = 1.33 x 10-3 M
And finally: pH = -log [H+] = 2.88
CHMI 2227 - E.R. Gauthier, Ph.D.
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Acid base chemistry
- weak acids

Example 2: What will be the pH of a solution made of 0.3
M acetic acid and 0.1 M acetate (pKa = 4.8).
CH3-COOH
CH3-COO- + H+
pH = pKa + log [A-]
[HA]
pH = 4.8 + log 0.1M
0.3M
pH = 4.8 + (-0.477)
pH = 4.3
CHMI 2227 - E.R. Gauthier, Ph.D.
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Spectrophotometry
Cuvette
Light source
Detector
Incident
light
Transmitted
light
Intensity of
transmitted light
same as incident
light
Intensity of
transmitted light
less than
incident light
Incident
light
Transmitted
light
CHMI 2227 - E.R. Gauthier, Ph.D.
In other words, the blue
solution absorbed some of
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the incident light.
Spectrophotometry

Different light sources can be
used.

In biochemistry, the two most
widely used light sources are:

Visible light (for coloured
compounds);
 Ultraviolet light: uncolored
compounds with aromatic
rings/conjugated double
bonds.

For this reason, we call this
method UV-Vis
spectrophotometry.
CHMI 2227 - E.R. Gauthier, Ph.D.
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
The wavelength to use
depends on the type of
compound you’re interested in.

Usually, preliminary
experiments have to be
performed to find the
wavelength where your
compound will absorb the
most;

increases sensitivity of the
assay.
Absorbance intensity
Spectrophotometry
400
450
500
550
600
Wavelength (nm)
CHMI 2227 - E.R. Gauthier, Ph.D.
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Spectrophotometry


The big deal with
spectrophotometry is that
is allows you to use the
amount of absorbed light
to measure things.
We use absorbance
instead of transmittance
because it’s easier to see
differences in values.

For example:


Solution A:

1M: 65% transmittance and
0.25 absorbance

2M: 70% transmittance and
0.5 absorbance
The relationship between
the transmittance and
absorbance is given by the
Beer-Lambert equation.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Spectrophotometry
- Beer-Lambert equation
-logT = log (1/T) = ecl =A
Where: T = transmittance
A = absorbance
l = light path (cuvette size)(cm)
c= concentration of analyte (M)
e = absorption coefficient (M-1cm-1)

Usually, l = 1 cm.

e is a property of the molecule studied under standardized conditions, and is
found in handbooks.

So: if you know A, l and e, you can immediately know c.

However, e is rarely known, or not valid under the conditions used in the lab.

WHAT TO DO??????????????
CHMI 2227 - E.R. Gauthier, Ph.D.
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Standard curves

Very frequently used in
biochemistry;
Absorbance of
the unknown
Pretty simple:

The absorbance of a set of solutions
of the compound of interest, of
known concentrations, is first
determined.

A graph of the absorbance vs
concentration is then made. THIS IS
YOUR STANDARD CURVE.

The absorbance of the same
compound, but of unknown
concentration, is then determined.

As long as the absorbance of the
unknown fits in the linear part of the
standard curve, you can determine
the concentration of your sample.
Absorbance intensity

Non-linear part
of the curve.
CHMI 2227 - E.R. Gauthier, Ph.D.
BEWARE!!!
Concentration of
the unknown
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Concentration(units)
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Spectrophotometry

Spectrophotometry is used all the time in biochemistry,
and not only to measure the concentration of molecules:

Enzymatic reactions:


The formation/exhaustion of a
light-absorbing molecule as a
function of time can be
determined by spectrophotometry.

This allows us to monitor the
progress of enzymatic reactions.
Sample detection:



Proteins absorb at 280 nm;
UV-Vis is often used to follow the
progress of protein purification by
monitoring the absorbance at 280
nm.
Sample purity:

Pure DNA samples absorb at 260
nm and 280 nm in such a way that
the ratio of the absorbance
A260/A280 is around 2.

A A260/A280 ratio of less than 2 tells
you that your DNA is
contaminated with proteins.
CHMI 2227 - E.R. Gauthier, Ph.D.
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