Buffer Analysis Laboratory Report

Categories: Biology

Analysis, Pages 5 (1020 words)

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Abstract

Buffers are mixtures of weak acids and conjugate bases that play a crucial role in maintaining stable pH levels in various biochemical processes. This experiment aims to investigate and analyze the functionality of different buffers commonly used in biological applications.

Introduction

Buffers are aqueous solutions known for their ability to resist significant pH changes when exposed to acids or bases. A typical buffer consists of a weak acid and its conjugate base [1]. Buffers function primarily through neutralization, where added hydrogen ions react with the base, while hydroxide ions react with the acid [2].

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Maintaining the pH within a specific range is essential for proper biochemical reactions. Historically, inorganic substances like phosphate, carboxylic acids, and bicarbonate were used as buffers. However, they had limitations such as enzyme interactions, lack of inertness, and the formation of precipitates, particularly the phosphate buffer's tendency to react with ions like Ca2+ and Mg2+. To overcome these limitations, zwitterionic buffers like Tris were introduced. Tris offers advantages like not forming metal complexes, but it can produce radicals under certain conditions and is sensitive to ion concentration and temperature fluctuations.

Organisms and cells must maintain specific pH values to ensure their proper functioning. Different buffers are chosen based on the specific requirements of the system. Criteria for selecting a biological buffer, as per the lab manual, include buffering at the optimal pH for the process, high solubility, minimal salt effects, chemical stability, and low light absorption in the ultraviolet and visible spectra.

Experimental

Experiment 1: Preparation of Sodium Phosphate Buffer

In this experiment, 13.

81g of 1M NaH2PO4 (monobasic) solution and 14.2g of 1M Na2HPO4 (dibasic) solution were weighed separately and added to two 100mL beakers. For the monobasic solution, approximately 60% of the beaker was filled with distilled water, and a magnetic stir bar was added. The beaker was placed on an electric stirrer until the chemicals completely dissolved. Once dissolved, the solution was transferred to a 100mL graduated cylinder and diluted with distilled water to the 100mL mark, and the pH was recorded. The same procedure was followed for the dibasic solution, with the addition of heat for proper dissolution.

Experiment 2: Preparation of "Resolving Buffer"

To prepare the Resolving Buffer, 18.17g of 1.5M Tris (hydroxymethyl) amino methane was added to a 100mL beaker with 80% distilled water. The solution was stirred until completely dissolved, and the pH was adjusted using 1N HCl and 6N HCl to achieve a final pH of 8.82. The solution was then diluted to 100mL and stored for future experiments.

Experiment 3: Preparation of "Stacking Buffer"

The Stacking Buffer was prepared by adding 6.05g of 0.5M Tris to a 100mL beaker with 60% distilled water. After complete dissolution, the pH was adjusted to 6.86 using 6N HCl. The solution was diluted to 100mL and stored for future experiments.

Experiment 4: Preparation of 10X SDS Running Buffer

For this buffer, 3.03g of Tris, 14.41g of Glycine, and 1g of SDS were added to a 100mL beaker with a magnetic stir bar. 70mL of distilled water was added, and the chemicals were stirred until completely dissolved. The pH was adjusted to 8.30 using 6N HCl. The solution was diluted to 100mL and stored for future use.

Experiment 5: Preparation of 10X Native Running Buffer

The 10X Native Running Buffer was prepared by adding 3.03g of Tris and 14.41g of Glycine to a 100mL beaker with a magnetic stir bar. 70mL of distilled water was added, and the mixture was heated until complete dissolution. The pH was adjusted to 8.34 using 6N HCl. The solution was diluted to 100mL and stored for future experiments.

Results

Table 1: Composition of Solutions and Buffers

Sample	Concentration	Volume (mL)	Molecular Weight (g/mol)	Mass (g)
Solution 1: 1M NaH2PO4	1M	100	137.99	13.8
Solution 2: 1M Na2HPO4	1M	100	142	14.2
Buffer 1: 100 mM Sodium Phosphate buffer, pH 7.0	100 mM	100	N/A	N/A
Buffer 2: "Resolving Buffer"	1.5M	100	121.14	18.2
Buffer 3: "Stacking Buffer"	0.5M	100	121.14	6.1
Buffer 4: 10X SDS Running Buffer	10X	100	N/A	N/A
Buffer 5: 10X Native Running Buffer	10X	100	N/A	N/A

Table 2: Initial and Final pH Readings

Sample	Initial pH	Final pH
Solution 1: 1M NaH2PO4	4.02	No adjusting of pH
Solution 2: 1M Na2HPO4	8.91	No adjusting of pH
Buffer 1: 100 mM Sodium Phosphate buffer	6.87	No adjusting of pH
Buffer 2: "Resolving Buffer"	11.0	8.82
Buffer 3: "Stacking Buffer"	10.35	6.86
Buffer 4: 10X SDS Running Buffer	9.25	8.30
Buffer 5: 10X Native Running Buffer	8.45	8.34

Sample Calculations

Calculation for Solution 1: 1M NaH2PO4

Mass of NaH2PO4 = (1 mol/L) x (137.99 g/mol) x 0.100 L = 13.799g NaH2PO4

Calculation for Solution 2: 1M Na2HPO4

Mass of Na2HPO4 = (1 mol/L) x (142 g/mol) x 0.100 L = 14.2g Na2HPO4

Calculation for Buffer 2: "Resolving Buffer"

Mass of Tris = (1.5 mol/L) x (121.14 g/mol) x 0.100 L = 18.171g Tris

Calculation for Buffer 3: "Stacking Buffer"

Mass of Tris = (0.5 mol/L) x (121.14 g/mol) x 0.100 L = 6.057g Tris

Calculation for Buffer 4: 10X SDS Running Buffer

Mass of Tris = (0.25 mol/L) x (121.14 g/mol) x 0.100 L = 3.029g Tris

Mass of Glycine = (1.92 mol/L) x (75.07 g/mol) x 0.100 L = 14.413g Glycine

Calculation for Buffer 5: 10X Native Running Buffer

Mass of Tris = (0.25 mol/L) x (121.14 g/mol) x 0.100 L = 3.029g Tris

Mass of Glycine = (1.92 mol/L) x (75.07 g/mol) x 0.100 L = 14.413g Glycine

Discussion

The sodium phosphate buffer prepared in experiment 1 falls within the typical pH range (6.5-7.5) for such buffers [1]. It consists of both monobasic and dibasic components, providing stability to cellular pH.

Experiments 2 and 3 involved the preparation of zwitterionic Tris buffers, known for their utility in separating proteins by size in electrophoresis. The Resolving Buffer has a pH of 8.82, while the Stacking Buffer has a pH of 6.86. These pH differences facilitate the separation of proteins and nucleic acids based on their charge and size.

The 10X SDS Running Buffer in experiment 4 contains Tris, glycine, and SDS, enabling protein denaturation. Similarly, the 10X Native Running Buffer in experiment 5, lacking SDS, allows the study of natural cell extracts and target enzyme presence without protein denaturation.

Conclusion

In conclusion, this experiment successfully prepared various buffers and demonstrated the use of a pH meter to measure their pH levels accurately. The results indicate the appropriateness of the buffers for their intended applications. It is crucial to select the right buffer and maintain precise pH conditions for successful biochemical experiments.