CHMI 2227E Biochemistry I

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CHMI 2227EBiochemistry I

• Refresher
• acid-base chemistry
• spectrophotometry

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Acid base chemistry- strong acids
HCl ? H Cl-
pH - log H
Where H is in molar (M) concentration units.

• Strong acids dissociate completely in water.

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Acid base chemistry- weak acids

• Where
• HA is the undissociated acid
• A- is the conjugated base
• of acid HA

Ka H x A- HA
pKa -log Ka

• Weak acids do not dissociate completely in water
• The extent to which the weak acid will dissociate
  is indicated by the dissociation constant (Ka).

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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
  Henderson-Hasselbach equation

pH pKa log A- HA
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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-.
• Notice that the pH doesnt change significantly
  near the pKa the solution is said to be buffered.

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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).

Ka 1.76 x 10-5 M H x A- H2
HA HA H2
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
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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).

pH pKa log A-
HA pH 4.8 log 0.1M 0.3M pH
4.8 (-0.477) pH 4.3
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Spectrophotometry
Cuvette
Light source
Detector
Intensity of transmitted light same as incident
light
Transmitted light
Incident light
Intensity of transmitted light less than
incident light
Incident light
Transmitted light
In other words, the blue solution absorbed some
of the incident light.
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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.

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Spectrophotometry

• The wavelength to use depends on the type of
  compound youre 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.

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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 its 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.

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Spectrophotometry- Beer-Lambert equation
Where T transmittance A absorbance l
light path (cuvette size)(cm) c concentration
of analyte (M) e absorption coefficient
(M-1cm-1)
-logT log (1/T) ecl A

• 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??????????????

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Standard curves

• Very frequently used in biochemistry
• 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.

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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.