expt1 final

7/27/2019 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

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