Titration of the Amino Acid 1

AMINO-ACID GLYCINE BUFFER WHEN DISSOLVED IN WATER.

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 Titration of the Amino Acid 1
Introduction

Living systems need to maintain homeostasis, equilibrium in temperature, respiration, and

pH in order to function properly. The exchange of various ions and water across cell membranes in

humans allows the cell to maintain its shape and continue to function. A buffer, solution that is

resistant to pH changes when an acid or base is added, is sometimes also used to maintain pH.

Using the buffer's pKa, buffers can assist in determining what the precise pH of a system actually

is. Some of these buffers can be observed in humans when various amino acids, like glycine, are

present.

pH, also referred to as potential Hydrogen. It is the measurement of Hydrogen ion

concentration in a solution. A negative logarithm is used to calculate pH, which is a measurement

of hydrogen ion concentration. In other words, pH measures a solution's ability to yield hydrogen

ions (H+), which are produced by acids and alkalis, respectively. (The Pennsylvania State

University, n.d.). Generally, pH level is determined by dipping an indicator into an aqueous

solution. Chioson et al., 2018 Stated that liquid can be classified as basic or acidic using a

conventional pH scale. The absolute concentration of a buffer, sometimes referred to as its

capacity, is another factor that determines whether or not it is useful. In other words, one needs

sufficient amounts of HA and A in order to react with the quantity of base or acid that is produced

by the reaction that is being researched. Now that we have these principles, we can turn our

attention to the subject of which pH value is most helpful for biochemistry. When acid or base are

added, a buffer prevents the pH of the solution from changing. (Pielak, 2021). The demonstration

of the ionization characteristics of weak acids and amino acids aids in the comprehension of the

biological buffering role of proteins. Numerous biochemicals in biological systems are

significantly affected by even minute changes in the quantities of free H+ ions, according to

research. Adding a sufficient buffer to the medium in order to maintain a steady pH in vitro is a
 Titration of the Amino Acid 1
crucial requirement.

When pH buffers are present, a solution is able to withstand a change in pH as a result of

the addition of acids and bases to pH buffers, which are classified as weak acids (HA+), and the

dissociation of these weak acids in water (A- and H+). Understanding the link between the quantity

of dissociation and weak acids is described by the equilibrium constant, Ka = [A-][H+]/[HA],

which may be used to build a formula describing the pH of a combination with a known pKa

(solute): pH = pKa+log[A-]/[HA+]. When the proportions of a conjugate acid and a buffering base

are equivalent, it is possible to describe the buffering capacity of a solution. Alternatively, pH and

pKa are equal.

Materials and Methods

We made sure the electrode was covered with liquid in the storage fluid, we then ensured

that all connections were proper then connected the pH meter to the mains. By pressing the

temperature button we selected Celsius mode and set the temperature at 20oC. It was imperative to

check the pH reading for the standard buffer.

After taking the electrode out of the storage buffer and placing it in the pH 4 buffer, the

"slope" control was used to adjust the pH reading until it reached 4. I washed the electrode and

double checked to make sure that the buffer at pH 7 hadn't shifted. After that, titration was carried

out using the acid, and finally, calibration of the base was carried out utilizing the pH 7 buffer.

30ml of glycine was place into a 50ml beaker, and a magnetic stirrer added. The pH was

calibarated using acid. 1ml of KOH was pipetted to speed up the process, and recorded the pH

values when an a minimal range was determined. The repetitive process continued until the pH fell

and stabilized below 1.5. the electrodes were washed the contents of the beaker were replace with a

fresh stock of 30ml glycine solution. The meter was then recalibrated with alkaline solution.

Results
 Titration of the Amino Acid 1
volume of pH volume pH

HCL added. of KOH

added

0 7.2 0 7

0.5 4.2 1 8.42

1 3.5 2 8.79

1.5 3.4 3 9

2 3.2 4 9.17

2.5 3.1 5 9.3

3 3 6 9.45

3.5 2.9 7 9.53

4 2.9 8 9.63

4.5 4.9 9 9.74

5 2.8 10 9.84

5.5 2.8 11 9.94

6 2.7 12 10.04

6.5 2.7 13 10.18

7 2.6 14 10.31

7.5 2.6 15 10.49

8 2.5 16 11.76

8.5 2.5 17 14.06

9 2.4 18 11.43

9.5 2.4 19 11.68

10 2.3 20 11.83
 Titration of the Amino Acid 1
10.5 2.3 21 12.02

11 2.3 22

11.5 2.3 23

12 2.2

12.5 2.2

13 2.1

13.5 2.1

14 2.1

14.5 2

15 2

15.5 2

16 1.9

16.5 1.9

17 1.9

17.5 1.8

18 1.8

18.5 1.8

19 1.8

19.5 1.7

20 1.7

20.5 1.7

21 1.7

21.5 1.7

22 1.6
 Titration of the Amino Acid 1
22.5 1.6

23 1.6

23.5 1.6

24 1.6

24.5 1.6

25 1.5

25.5 1.5

26 1.5

26.5 1.5

27

27.5

28

28.5

29

29.5

30

The vast majority of amino acids only have two groups that are capable of either accepting

or giving away electrons. The amino group, denoted by the symbol -NH2, and the carboxylic acid

(-COOH). Both groups of the amino acid will be ionized when it reaches the isoelectric point (Pi),

at which time the amino acid will have a net charge of zero. When the pH was compared to the

volume titers of HCL and KOH, a clear pattern emerged. The pka value for either the carbonyl or

amino group was determined by looking for the point in the reaction when the pH changed the

least as acid or base was introduced. The accompanying discussion will center on the changes in
 Titration of the Amino Acid 1
pH that take place throughout the course of an acid–base titration. Those changes will be the

primary topic. The pH of the solution that is now within the flask may be plotted against the total

quantity of acid or base that has been added to produce a titration curve. During the titration, the

shape of the curve communicates essential information about the activities that are going on in the

solution. Since glycine, like other amino acids in general, comprises both an amine group and a

carboxyl group, the molecule that contains glycine will be able to neutralize both acids and bases

owing to the capacity of the molecule to both give off and take in protons. This property allows the

molecule to neutralize both acidic and basic conditions. In this experiment, the Henderson-

Hasselbalch equation will be an essential instrument.

pH =pK+log [A-]/[HA],

Amino acids are the fundamental components of protein molecules; those amino acids that

are found in proteins have a carboxyl group and an amino group attached to them to form a peptide

bond. - Ionizable groups such as COOH- and NH3+ give a protein to the reaction.

Discussion and conclusion

A plot of a property, such as pH of a solution, versus the amount of titrant that was added is

an example of a titration curve. The pH of the solution is an important parameter to track when

performing acid-base titrations because its value shifts in a predictable manner in response to

changes in the solution's constituents. As a result, the solution's pH can be utilized to track the

titration's development and locate its conclusion point.

Table 1 provides a summary of pH/volume data pairings for both the strong and weak acid

titrations, and Figure 1 plots these data as titration curves. The following are the four steps of a

titration, which may best be addressed by comparing these two curves, which show numerous

fundamental concepts: starting state (titrant volume added equals 0 milliliters): The acid that is

being titrated is what determines the pH; as both acid samples are of the same concentration, the

weaker acid will show a higher initial pH pre-equivalence point (0 mL V 25 mL): solution The
 Titration of the Amino Acid 1
acid is used up as a byproduct of the reaction with the additional titrant as the pH steadily rises.

The unreacted acid is included in the composition with the reaction product, which is the conjugate

base equivalency point (V = 25 mL): It is noted that the solution's pH increases dramatically when

its composition shifts from acidic to either neutral (in the case of the sample coming from the

strong acid) or basic (in the case of the sample coming from the weak acid). (2014) According to

Ball and KeyAfter the postequivalence point (V > 25 mL), the conjugate base of the acid is

ionized, and this ionization determines the pH of the solution, which may be calculated using the

following equation: Since the same titrant was used to titrate both samples, it seems that their

titration curves are equivalent. When calculating the pH of a solution, more or less excess strong

base titrant is added till the desired pH is reached.

Conclusion

When carrying out biological studies, having the skill set to understand how various buffers

function and determine whether or not they are the appropriate ones to utilize is an incredibly vital

ability to have. We titrated a specific glycine buffer, and discovered that the two hydrogen

dissociation points in that particular buffer were either too low or too high to make it possible to

conduct studies using human blood. When doing tests on human blood, biologists require any

buffers they use to have pKa values that are somewhat near to the pH of human blood, which is

around 7.5. Biological buffers are essential in every living organism because it helps the organism

keep constant pH. Buffers help prevent the change in pH in an organism when acids and bases are

added. The use of the Henderson-Hasselbalch equation in labs helps us better understand the

relationship between pH and pKa and the substances being measured. Glycine solutions are

acceptable to be used as pH buffers in the laboratory because of its ability to neutralize acids and

alkalis.
Titration of the Amino Acid 1
 Titration of the Amino Acid 1

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Roger Gordon Bates (1973). Determination of PH. Wiley-Interscience.

Shah Purvesh (2015). Acid Base Titrations..

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