Research Question: What is the effect of the ratio of the the individual buffers in a combined tris and phosphate buffer solution on buffer capacity?
Research/Introduction
pH buffer solutions are aqueous solutions comprising of a mixture of a weak acid and a salt of its conjugate base, or a weak base and a salt of its conjugate acid. The amount of acid/conjugate base or base/conjugate acid determine its buffer capacity, or the amount of acid or base that can be added for it to still maintain constant pH.
The solutions resist change to pH as bases or acids are added by making use of Le Chatelier’s principle, which can be stated as: “A change in one of the variables that describe a system at equilibrium produces a shift in the position of the equilibrium that counteracts the effect of this change.” For example, in a carbonic acid buffer solution, H2CO3 is ionized by losing a H+ ion: H2CO3 ↔ HCO3- + H+. If more acid is added, equilibrium is shifted to the left, as long as there is HCO3- present for the H+ to react with, thus removing excess hydrogen ions. Hence, to protect against added acid, the buffer solution must have a high concentration of the conjugate base in the solution.
pH buffers have many applications in the medical field; they are used in storing organs and are essential in regulating hydrogen ion concentration in the tissue of living organisms. As such, buffers are important in the functioning of enzymes, which can slow down and denature if the pH is not within a certain range.
The bicarbonate buffer system would have been interesting to investigate in relation to the phosphate buffer system since it is essential in maintaining pH levels in the blood, whereas the latter has a similar function in all intercellular fluid. However, it would be difficult to control in a laboratory setting since carbonic acid is unstable and constantly decomposes into water and carbon dioxide. This would also be difficult to replicate exactly because the bicarbonate buffer works in conjunction with the respiratory system to regulate pH and absorb the carbon dioxide produced. Therefore, I opted to use another buffer system, Tris-HCl.
Tris, or tromethamine, is an acceptable substitute because its buffer range is similar to that of the pH range that is typical for most living organisms. It has a pKa of 8.07 at 25℃, which implies an effective pH range between 7.5 and 9.0. Not only is the tris buffer prevalent in biochemistry laboratories because of its pH range and relatively low cost, but it has several significant medical applications. In medicine, it is occasionally used as a drug, given in intensive care for its properties as a buffer for the treatment of severe metabolic acidosis.
As such, I am interested in investigating buffer properties under biological conditions so that buffers may be used more effectively in medicine. I want to ascertain whether there will be a significant difference in buffer capacity depending on the ratio of these two solutions: will the buffering capacity be enhanced? Is there an optimal ratio? How could this vary on a case-to-case basis? Considering my interest in biochemistry, I want to examine this relationship in order to see if this model should be more widely-adopted in medical research. I have personally found very little research relating to this topic- while the idea of mixing buffers is by no means a new one, I have yet to see an investigation on how mixing systems at the same pH could impact buffer capacity.
Hypothesis
It is predicted that the solution that will absorb the most HCl before its pH decreases by 1 will be a 5:5 phosphate-tris ratio solution. Even though buffers are traditionally single systems, by using the approach of biomimicry, “the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems,” it could be posited that buffer capacity would increase if systems were mixed. The human body makes use of a number of pH regulating mechanisms, i.e., the bicarbonate buffer, phosphate buffer, and hemoglobin are all pH buffers in human blood. Since a mixed buffer system would be more accurate in representing pH buffering as it applies to living organisms, this could possibly improve the current understanding of buffer systems and their biological models.
Buffer systems
Phosphate:
NaH2PO4 + Na+ ↔ H+ + Na2HPO4 (Ka = 6.23 ⨉ 10-8) (pKa = 7.21)
Ka = [H+] [A-][HA]= [H+] [ Na2HPO4][NaH2PO4 ]= 6.23 ⨉ 10-8
Since pH will be 7.4, [H+] = 10-7.4.
10-7.4 ⨉ [Na2HPO4]
________________ = 6.23 ⨉ 10-8
[NaH2PO4]
[Na2HPO4] 6.23 ⨉ 10-8
________________ = __________ = 6.23 ⨉ 10-0.6 = 1.565
[NaH2PO4] 10-7.4
Therefore, to prepare a buffer consisting of NaH2PO4 and Na2HPO4, the ratio of [Na2HPO4] to [NaH2PO4] is 1.565 : 1.
Tris:
C4H12ClNO3 ↔ C4H12NO3+ + Cl- (Ka = 8.32 x 10-9) (pKa = 8.08)
TrisHCl ↔ TrisH+ + Cl-
TrisH+ ↔ Tris + H+
Ka = [H+] [A-][HA]= [H+] [Tris][ TrisH+]= 8.32 x 10-8
Since pH will be 7.4, [H+] = 10-7.4.
10-7.4 ⨉ [Tris]
________________ = 8.32 x 10-8
[TrisH+]
[Tris] 8.32 x 10-8
________________ = __________ = 8.32 ⨉ 10-0.6 = 2.089
[TrisH+] 10-7.4
Therefore, to prepare a buffer consisting of Tris and TrisH+, the ratio of [Tris] to [TrisH+] is 2.089 : 1.
In addition, the solutions will need to maintain the same osmolarity as human blood in order to mimic human body conditions more accurately. The human body has a blood osmolarity of 300 mOsmol/L, which is equal to 0.300 M. The concentrations must add up to 0.300 M so that the buffer solutions are isotonic with human blood.
Phosphate buffer
Tris buffer
[Na2HPO4] + [NaH2PO4] = 0.300 M
1.565[Na2HPO4] = [NaH2PO4]
1.565[Na2HPO4] + [NaH2PO4] = 0.300 M
2.565[Na2HPO4] = 0.300 M
[Na2HPO4] = 0.300 / 2.565 = 0.117 M
[NaH2PO4] = 0.300 M – 0.117 M = 0.183 M
[Tris] + [TrisH+] = 0.300 M
2.089[Tris] = [TrisH+]
2.089[Tris] + [TrisH+] = 0.300 M
3.089[Tris] = 0.300 M
[Tris] = 0.300 / 3.089 = 0.0971 M
[NaH2PO4] = 0.300 M – 0.0971 M = 0.203 M
Independent Variable: The ratio of the phosphate buffer to the tris buffer will be mixed to seven different ratios: 10:0, 8:2, 6:4, 5:5, 4:6, 2:8, 0:10, with the mixed solution measuring 50 mL.
Dependent Variable: The volume of 1 M HCl absorbed by the buffer solution before its pH decreases by 1. It will be measured using a drop counter and recorded with the Graphical Analysis software.
Constants
The concentration of the phosphate buffer used to make the mixed buffer will be 0.117 M Na2HPO4 and 0.183 M NaH2PO4. This was derived by using a pH of 7.4 (the pH of human blood) and an osmolarity of 0.300 M in order to remain isotonic with blood.
The concentration of the tris buffer. The conc This was derived by using a pH of 7.4 (the pH of human blood) and an osmolarity of 0.300 M in order to remain isotonic with blood.
The volume of the mixed buffer solution will be 50 mL for each trial.
The concentration of the HCl solution will be 1 M throughout the experiment.
The speed of the magnetic stirrer.
The temperature of the buffer solution will be controlled by using the hotplate. The buffer solution will be heated to about 25℃.
Materials
Buffer solution of the system
Buffer solution of the system
1 M HCl solution
Vernier pH probe
Vernier drop counter
Computer with Graphical Analysis 4 software
Magnetic stirrer
Burette stand and burette
100 mL beakers
Thermometer
Hot plate
Safety
Name and Formula of Chemical
HCl – hydrochloric acid
Chemical Concentration
1 M
Scheme of Work/Procedure
Used as a titrant
Hazards
May cause irritation upon contact with skin or eyes
Steps Taken to Reduce Hazards
Wear goggles
Name and Formula of Chemical
C4H11NO3 – tromethamine, tris
Chemical Concentration
0.0971 M
Scheme of Work/Procedure
In powder form, made into buffer solution with hydrochloric acid after being dissolved in water.
Hazards
May cause irritation upon contact with skin or eyes
Throat irritant
Steps Taken to Reduce Hazards
Wear goggles
Avoid inhalation
Name and Formula of Chemical
Na2HPO4 – disodium phosphate
Chemical Concentration
0.117 M
Scheme of Work/Procedure
In powder form, made into buffer solution with monosodium phosphate.
Hazards
May cause irritation upon contact with skin or eyes
Throat irritant
Steps Taken to Reduce Hazards
Wear goggles
Avoid inhalation
Name and Formula of Chemical
NaH2PO4 – monosodium phosphate
Chemical Concentration
0.183 M
Scheme of Work/Procedure
In powder form, made into buffer solution with disodium phosphate.
Hazards
May cause irritation upon contact with skin or eyes
Throat irritant
Steps Taken to Reduce Hazards
Wear goggles
Avoid inhalation
Procedure
Mix the phosphate buffer system and the tris buffer system to a ratio of 5:5, with the combined solution measuring 50 mL. Plug in the hot plate and turn it on to its lowest setting. Using the thermometer, ensure that solution has a temperature of 25℃.
As the solution heats up, fill the burette with 1 M HCl solution and set up burette stand.
Place pH probe and magnetic stirrer in the 100 mL beaker. Connect the drop counter and pH probe to the computer. Open Graphical Analysis 4 and allow a graph to generate.
After the pH and drop recordings stabilize, turn the stopcock of the burette to allow drops of HCl in the beaker.
When the pH probe reads exactly 6.4, turn the stopcock to its original position and stop the recording.
Repeat steps 3-5 for two more trials.
Repeat steps 3-6 for the ratios of 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0.
Bibliography
Case 422 — Sudden onset of polydipsia and polyuria. (n.d.). Retrieved November 10, 2017, from http://path.upmc.edu/cases/case422.html
Gomori, G., Preparation of Buffers for Use in Enzyme Studies. Methods Enzymology., 1, 138-146 (1955).
Hoste, E. A., Colpaert, K., Vanholder, R. C., Lameire, N. H., De, J. J., Blot, S. I., & Colardyn, F. A. (n.d.). Sodium bicarbonate versus THAM in ICU patients with mild metabolic acidosis. Retrieved November 10, 2017, from https://www.ncbi.nlm.nih.gov/pubmed/16013019
Johnson, L. R., & Byrne, J. H. (2003). Essential medical physiology. Boston: Elsevier Academic Press.
(n.d.). Retrieved November 10, 2017, from http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch16/lechat.html
Phosphate Buffer. (n.d.). Retrieved November 10, 2017, from http://www.pathwaymedicine.org/phosphate-buffer
Scorpio, R. (2000). Fundamentals of acids, bases, buffers and their application to biochemical systems. Iowa: Kendall/Hunt Pub. Co.
What Is Biomimicry? – Biomimicry Institute. (n.d.). Retrieved November 10, 2017, from https://biomimicry.org/what-is-biomimicry/