Expt1 Final
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Transcript of Expt1 Final
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EXPERIMENT 1:
AMINO ACID AS AMPHOLYTES
Baranda, Coronel, Galingana
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INTRO
Changes in pH results in adverse effects on
an organism.
suppress enzyme action
affect the side chains of amino acids.
alter the conformation and structure of proteins
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INTRO
Buffer
solutions that resist changes in pH upon addition
of small amounts of acids and bases.
composed of weak acid/base and the salt ofconjugate base/acid.
ex. Phosphate buffer, Carbonate buffer, proteins
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INTRO
Henderson-Hasselbach equation:
pH = pKa log [A-]
[HA]Ampholytes
Molecules that contain both acidic and basic
groups
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INTRO
Objectives:
Understand the acid/base properties of amino
acids
To prepare a buffer
To construct and interpret the titration curve of
amino acids.
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METHODOLOGY: PREPARATION OF BUFFER
Choose an appropriate weak acid.
Calculate the concentrations of salt andacid needed in buffer. Mix.
Dissolve in enough water then transferto 250mL vol. flask
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METHODOLOGY: PREPARATION OF BUFFER
Adjust pH by addingNaOH or HCl.
Transfer to a
reagent bottle
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METHODOLOGY: TITRATION
Unknown liquid + 0.1 M NaOH (0.5mLincrements)
Keep pH meter submerged in solution.
Record pH readings and construct titrationcurve.
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RESULTS
Amount of buffer needed:
500mL of 0.25M pH 6.7 phosphate buffer
6.7 = 7.12 + log [HPO42-
]/[H2PO4-
]0.38 = [HPO4
2-]/[H2PO4-]
0.38[H2PO4-] = [HPO4
2-]
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RESULTS
0.25 = [H2PO4-
] + [HPO42-
]0.25 = 0.38[H2PO4
-] + [H2PO4-]
0.25 = 1.38[H2PO4-]
[H2PO4-
] = 0.181153 M[HPO4
2-] = 0.0688 M
g NaH2PO4 = (0.181153M)(0.500L)(156.01)
= 14.13 g
g Na2HPO4 = (0.0688M)(0.500L)(177.99)
= 6.13 g
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RESULTS
0
2
4
6
8
10
12
14
0 5 10 15 20 25 30 35 40
pH
Volume of NaOH (mL)
pH
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BUFFERS
HA(aq) + H2O(l) H3O+(aq) + A(aq) Le Chateliers Principle
To prepare the buffer -> Henderson-
Hasselbach equation was used:pH = pKa log [A-]
[HA]
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TITRATION OF BUFFER SOLUTION
Buffering action is easily seen using a
titration curve.
Slow pH change regions -> buffering regions
half-equivalence point -> maximum buffering
capacity
Equivalence point -> average of two pKa
values
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PHOSPHATE BUFFER
Phosphoric acid has three ionizable
hydrogen atoms = 3 equivalence points
pKa1 = 1.97
pKa2 = 7.0
pKa3 = 12.5
Only two species exist at a time.
Titrated with NaOH
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AMINO ACIDS
Ampholytes: -COOH andNH2 group and R
side chain.
Has both acidic pKa and basic pKa
Act as a base in acidic conditions
Act as an acid in basic conditions
Zwitterions ampholyte wherein its positive
and negative charges are equal.
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AMINO ACIDS
pH level lower that pKa1 -> fully
deprotonated
As pH increases, amino acid gets
deprotonated.
Isoelectric point> point in the titration curve
where the amino acid is electrically neutral
Main buffering constituent in blood.
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CONCLUSION
pH affects biomolecules
Amino acids can act as buffers due to the
presence of both acidic and basic groups.
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RECOMMENDATIONS
Use CO2-free water
Wash glasswares with chromic acid.
Preferably store the buffer in plastic bottles
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GUIDE QUESTIONS How would you prepare a 0.1M buffer using 0.5M stock
solution of the acid and its salt?
If 1L of 0.1 M phosphate buffer was used at pH 7.4
7.4 = 7.0 + log([A-]/[HA])
0.4 = log([A-]/[HA])
100.4 = x/(0.1-x)
100.4(0.1-x) = x
X = 0.07122 M
0.1-x = 0.0288 M
Vsalt = (0.07122 M)(1 L)/(0.5 M) = 142.44mL
Vacid = (0.0288 M)(1 L)/(0.5 M) = 57.5 mL
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GUIDE QUESTIONS
What is the biological significance of pH? How
does it affect biological activities and functions?
Certain enzymes responsible for biological
activities in the body, work at certain pH levelsonly. Enzymes are either inactive or are
denatured if the surrounding solution does not
have the right pH. Some biological activitiesmight not occur because the enzymes are not
active.
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GUIDE QUESTIONS
Why is the observed pH different from the actualpH?
There was a visible discrepancy between the
observed pH and the actual pH and this iscaused by the high temperature of thesurroundings. The conditions are not ideal andso the pH was different. Another possible
reason for the discrepancy is the calibration ofthe pH meter. The pH meter might not havebeen calibrated properly and so the pH readingwas off.
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GUIDE QUESTIONS
What is the significance of the leveling
shown in your diagram in terms of the
buffering action of your amino acid?
The leveling areas show the buffering
regions, where the acid acts as a buffer at its
maximum capacity. In these areas, the
concentration of the weak acid is equal to theconjugate base.
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GUIDE QUESTIONS
What is the significance of the titration curve ofyour acid?
Amino acids are similar to other polyprotic
acids since it can release more than one protonand have different pKa values. The titrationcurve of the given acid is comparable to thetitration curve of most amino acids. The titration
curve also implies that the species present inthe solution varies as the titrant is continuouslyadded to the analyte
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GUIDE QUESTIONS
How does it compare with amino acids?The titration curve of the unknown liquid has a
total of two inflection points and three pKas whereasmost of the amino acids have only two pKas. This
titration curve should have been similar to someamino acids, but the pKas of the unknown acid aretoo low as compared to most of the amino acids. Thesecond pKa of the unknown acid is approximately 7.0whereas the usual second pKa of amino acid is
somewhere between 9.0 and 10. The third pKa ofthe unknown is almost 12 as compared to the thirdpKa of amino acid which is about 11.
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REFERENCES
Matthews, C. K. et al. (1996). Biochemistry 2nd
edition. Menlo Park, CA: The
Benjamin/Cummings Publishing Company, Inc.
Nicholas, M. G. (2002). Modules inBiochemistry.
Stryer L. Biochemistry. 4th ed. NY: W.H
Freeman and Company; 1995:36-41 Cox MM, Lehninger AL, Nelson DL. Principles of
Biochemistry. 2nd ed. 33 Irving place, NY: Worth
Publishers. 1993:111-129