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Plotting The Titration Of Amino Acids Biology Essay

The purpose of this experiment is to plot the titration curves of three different amino acids with different pH values; basic, acidic and neutral and compare their curves. Also to calculate the Pi values of the amino acids; glycine, lysine and aspartic acid.

Proteins (polypeptides) are organic compounds that are made of amino acids. The amino acids joined together by peptide bonds which are between the carboxyl and amino groups of each amino acid. in the reaction of forming peptide bonds between amino acids a water molecule is eliminated and an amine bond is formed. This amine bond is the peptide bond.

in amino acid structure there are carboxyl groups, amino groups and R groups. R groups are different in every amino acid in size, shape, charge, hydrogen bonding capacity and chemical reactivity.

Zwitterions are molecules which have both positive and negative charges, but the net charge of the molecule is zero. Acids and bases may exist as ions, or as uncharged molecules. At neutral pH levels, both acids and bases exist as ions. Most zwitterions are ampholytes which contain both acidic and basic groups, for example amino acids; they have both acidic and basic groups in a single molecule. Because both the acid and the base are charged at neutral pH, the net charge of the amino acid is zero. Because of its electrical charges, zwitterions are highly soluble in water, insoluble in organic solvents. Water molecules are polar because of the electrical differences between the hydrogen and oxygen molecules in water because the oxygen atoms in water are attracted to cations and the hydrogen atoms in water are attracted to anions. When a zwitterion is dissolved in water, the hydrogen atoms in water surround the negatively charged group of the zwitterion and the oxygen atoms in water surround the positively charged group of the zwitterion, and thus dissolve it. [2]

2.3) isoelectric point:

isoelectric point is the point where the pH at which a protein’s net charge is zero. Below this point proteins have a net positive charge and above it a net negative charge. The isoelectric point is important in protein purification because it is the pH at which solubility is often minimal and the point at which the protein will accumulate. [3]

For an amino acid with only one amine and one carboxyl group, the pi can be calculated from the mean of the pKa's of this molecule.

For amino acids with more than two ionizable groups, such as lysine, the same formula is used, but this time the two pKa's used are those of the two groups that lose and gain a charge from the neutral form of the amino acid. Lysine has a single carboxylic pKa and two amine pKa values (one of which is on the R-group), so fully protonated lysine has a +2 net charge. To get a neutral charge, lysine must be deprotonated twice. [4]

3) EQUiPMENT AND CHEMiCALS

3.1) equipments:

Beaker

Laboratory scale

Weighing dish

Spatulas

Burette

Stand

Erlenmeyer flask

pH meter

Latex gloves

Laboratory coat

3.2) chemicals:

Distilled Water

Glycine

Lysine

Aspartic acid

NaOH

HCl

4) PROCEDURE

Every group prepared one of the three amino acids glycine, aspartic acid and lysine 100 ml 0.1 M.

500 ml 0.1 M NaOH was prepared.

The burette was rinsed with NaOH solution and the burette was filled with NaOH.

1 M HCl was added on to the each amino acid until the pH was measured around 1.5.

The solution with the amino acid and HCl was put into Erlenmeyer flask .

NaOH solution was started to run from the burette into the flask with 2ml at the beginning and after each addition of 2 ml NaOH the pH of the solution was measured.

When the pH of the solution was around 3 the addition of NaOH was done by adding 0.1 ml and then it was increased again until pH became 11.

Titration curve was plotted and Pi value was calculated.

5) RESULTS

5.1) Observations:

in this experiment we observed three different amino acid’s titrations and their titration curves. it is observed that at different pH values the titration curves of amino acid were also different. Lysine which is a basic amino acid, glycine a neutral amino acid and aspartic acid an acidic amino acid had different titration curves. it is also observed that as the pH increased during the titration the temperature was also increasing.

5.2) Calculations:

Calculation of grams for lysine and NaOH:

Structure of aspartic acid

Structure of lysine

Calculating the Pi of lysine from the titration curve:

Theoretic curve

DATA:

volume

pH

2.5 ml

1.66

2.5 ml

1.89

2.5 ml

1.68

2.5 ml

1.67

2.5 ml

1.66

4 ml

1.69

2 ml

1.71

2 ml

1.80

2 ml

1.93

1 ml

1.95

2 ml

1.94

2 ml

1.95

2 ml

1.97

2 ml

2.03

1 ml

2.04

1 ml

2.04

1 ml

2.10

1 ml

2.05

1 ml

2.14

2 ml

2.10

2 ml

2.13

2 ml

2.18

2 ml

2.23

2 ml

2.24

2 ml

2.29

2 ml

2.33

2 ml

2.38

2 ml

2.44

2 ml

2.49

2 ml

2.54

2 ml

2.60

2 ml

2.67

2 ml

2.75

2 ml

2.86

2 ml

2.93

1 ml

3.07

1 ml

3.12

1 ml

3.22

1 ml

3.32

1 ml

3.46

0.5 ml

3.65

0.5 ml

3.80

0.5 ml

4.06

2 drop

4.61

2 drop

4.94

1 drop

5.91

1 drop

6.42

1 drop

6.78

1 drop

6.98

2 drop

7.12

2 drop

7.28

1 drop

7.38

3 drop

7.46

0.5 ml

7.57

1 ml

7.86

1 ml

8.12

1 ml

8.30

1 ml

8.46

1 ml

8.60

1 ml

8.72

2 ml

8.76

1 ml

8.93

2 ml

8.97

2 ml

9.10

2 ml

9.20

2 ml

9.31

3 ml

9.46

2 ml

9.53

2 ml

9.80

3 ml

9.94

3 ml

10.09

3 ml

10.28

4 ml

10.63

3 ml

10.75

4 ml

10.93

1.5 ml

11.00

Values read from the titration curve we plotted:

Pi1 = 9.80 if we choose the point straight from the curve but if we use the formula below;

pK2 = 9.64

pK3 = 10.63

pi = (9.64 + 10.63) / 2

pi2 = 10.13

Error Calculation:

Error for Pi1 ═

Error % = % 4.4

Error for Pi2 ═

Error % = % 1.17

6)DiSCUSSiON

in this experiment our purpose was to plot the titration curves of three different amino acids; glycine, aspartic acid and lysine. These three amino acids were expected to have different titration curves since glycine is a neutral amino acid, aspartic acid is acidic and lysine is basic amino acids. Functional groups of these amino acids determine whether they are acidic, basic or neutral. For example lysine has two positive charges in its structure one from the functional group and one from the amino group. That’s why the isoelectric point of lysine is 10.25 theoretically. Lysine is deprotonated twice, that’s why after the pH 9 we see two small peaks. And between these peaks we calculate the Pi value which is lysine is at zwitterion form and thus it is the Pi value.

During the titration when the pH was about 4 we started to add NaOH solution as drops because we didn’t wanted to miss the point where the graph makes a sharp increase. So we calculated how much NaOH is in one drop, and it is found that it was 0.07 ml. Because of this there might be error and it might not be an accurate measurement but if we didn’t add NaOH as drops and instead if we tried to add NaOH as 0.1 ml it would also cause error. Reading 0.1 or 0.2 ml NaOH from the burette is not very accurate either.

While we were titrating the lysine when the solution was at 2.03 pH we needed to change our flask into a bigger one. After we poured the solution into the new flask we read the pH of the same solution as 1.96, it was decreased. The reason for that is because when we changed the flask the temperature was also changed. Since pH and temperature are related pH was decreased as the temperature decreased.

As a result we found the Pi value of the lysine as 10.13 by taking the mean values from before and after the small peak in the graph when the pH is around 9-10, in this way we used the formula we took the two pKa values but there is another way of finding the Pi by just taking the peak value in the graph which was 9.80. When we look at the error percentage of the two Pi values we see that the value which is found by using the formula is more accurate. The points that we took may not be accurate because in the graph it wasn’t possible to select the exact points. But according to the points we choose the error was calculated as %1.7 for the value which is found by using the formula since the theoretical Pi value is 10.25 and the other error that found by taking the point on the graph was % 4.4.

When we look at the titration curve we see that at the beginning there is a small increasing from the initial pH this is where the buffering happens.

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