Aminoacid Titration

 

AIM 

The aim of the experiment of this week was the determination of the unknown aminoacid (histidine, arginine, aspartic acid, alanine) by using the pH values obtained whenever a specific amount of the base NaOH was added to plot the titration curve and then to determine the pI value by using this graph.  

DISCUSSION 

During this laboratory hour, unknown aminoacid samples had to be identified by using the titration by  strong base ( NaOH)  and acid (HCl). Initially, the aminoacids were protonated with HCl and afterwards, NaOH was added into aminoacid solution to ensure a buffer system. Due to this procedure, the plot of titration curve for each aminoacid were drawn. The pKa values of aminoacids were demonstrated, where each pKa value is specific for a specific aminoacid. Also, the pI values for each aminoacids were calculated using given pKa values, then they were compared with their theoretical calculations. Due to graph, the first sample was determined to be Aspartic acid due to the acidic activity it shows. The second graph was estimated to be Histidine, while the third and the forth one were determined to be Glycine and Arginine respectively[1]. According to the graph obtained by the data of our experiment, the approximate pI value of Aspartic acid is 7, while the theoretical one found in the database is 5.86. The experimental pI value of Histidine is 3, while the theoretical one is 4.35. The experimental pI value of Glycine is approximately 6, while the theoretical one is 4. The experimental pI value of  Arginine according to the graph is approximately 5.5, while the theoretical one 6.05. The calculated pI value found by using the pKa values of the ionizable groups of the hexapeptide “DENISA” is 3.15, while the one found in the database is 3.67. The calculated pI value was found by using the pKa values of the ionizable groups of the pentapeptide “NAYCI” is 5.345, while theoretical one obtained from the database is 5.52. The calculated pI value found by using the pKa values of the ionizable groups of the tetrapeptide”RHAN” is 10.735. while the theoretical one obtained from the database is 9.76 [2]. The reasons why there is a mismatch between the theoretical and the experimental pI value can be because of some of the side chains which can be buried or located in salt bridges. The environment where the aminoacids are located affects the aminoacids leading therefore  to changes in pKa and in the pI value as well. Additionally the presence of the ionic lipid headgroups near the aminoacids lead to a change in the pKa values due to the ionic activity [3]. The changes in pH during titration can affect the ionizable groups and this can also be the reason about the changes in each specific pKA and the pI value as well. Also it had to be mentioned that the values found in the database are calculated in the optimum conditions [4]. 

 

REFERENCES 

1.http://www.laney.edu/wp/cheli-fossum/files/2012/01/Amino-Acid-Sidechains.pdf retrieved 01.11.2016 from Internet source 

2.http://web.expasy.org/cgi-bin/compute_pi/pi_tool retrieved 01.11.2016 from Internet source 

3.https://www.researchgate.net/post/What_is_the_significance_of_the_theoretical_pI_of_a_protein_calculated_using_bioinformatics_tools_when_it_is_expressed_in_the_body retrieved 01.11.2016 from Internet resource 

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911520/retrieved 01.11.2016 from Internet resource 

Spectrophotometric Measurements

 

AIM 

The aim of this experiment concluded of the preparation of some specific buffers by using the Handerson-Hasselbach`s equation and some solutions, such as glucose and SDS. Additionally, the arrangement of pH values of the buffers by adding either a certain base or acid and the glucose/SDS solutions by adding distilled water was aimed in that experiment.  

 DISCUSSION 

 In this experiment, we’ve aimed the preparation of buffers including Z and pH7 phosphate buffer and other solutions including glucose and SDS solutions which are used in various biological processes. We’ve understood that the meaning of buffer and solutions. Buffer solution is the aqueous solution which composed of a mixture of a weak acid and its conjugate base or inversely and used to keep the pH level at an approximately constant value for usage in various applications. Also the 4th group used the Handerson-Hasselbalch Equation method with Na2HPO4 and NaH2PO4 and according to equation between these chemicals which gave below: 

H2PO4 ↔H+ + HPO4 

They calculated the molarity of H2PO4 and HPOby using a formula as pH= pKa + log [conjugate base] / [acid] and so they calculated the amount of each chemical which will be used. Thereby, we’ve learned the applying the Handerson-Hasselbalch Equation method. Actually, we’ve referred as the pH of a solution is based on the concentration of H3O+ ions which are present in the solutions and so weak acids don’t completely dissociate into ions, when they react with water. This case can change different weak acids. In addition to this, the Handerson-Hasselbalch Equation shows us the balance pH calculation of a solution including weak acid and its conjugate base which is given below equation : 

pH = pKa + log ([base])/([acid]) 

According to this equation, pKa is referred as the weak acid’s ionization meaning that dissociation of weak acids into ions even including the H3O+ ions. So the Handerson-Hasselbalch Equation is a common method which used in preparation of buffers for usage in the several laboratory applications. The reason of adding the HCl into the buffer was to decrease the pH value. If we could added the NaOH instead of HCl into the buffer, gradually, the pH value could be larger than 6 because of increasing the OH- ions in the buffer. For the pH 8 Z buffer, we’ve measured the first value lower than 8 and so we’ve tried to increase this value until pH 8 by adding NaOH basic solution slowly. The reason of adding the NaOH into the buffer was to increase the pH value. If we could added the HCl instead of NaOH into the buffer, gradually, the pH value could be lower than the first measured pH value because of increasing the H+ ions in the buffer. The reason of this can be mistakes in weighing of the chemicals or not completely dissociation of ions, so the weak acid for this buffer may not be dissociated into its ions completely. Temperature is another factor that affects the ionizations, because when the temperatures increase, chemicals`s ionizations increase, leading thus to a decrease in the pH values. Additionally, the amount of dioxide carbon which is dissolved on the water affects in the lowering of pH as it causes acidification.  

 

Buffer and Solution Preparation

 

AIM 

The aim of this experiment concluded of the preparation of some specific buffers by using the Handerson-Hasselbach`s equation and some solutions, such as glucose and SDS. Additionally, the arrangement of pH values of the buffers by adding either a certain base or acid and the glucose/SDS solutions by adding distilled water was aimed in that experiment.  

 DISCUSSION 

 In this experiment, we’ve aimed the preparation of buffers including Z and pH7 phosphate buffer and other solutions including glucose and SDS solutions which are used in various biological processes. We’ve understood that the meaning of buffer and solutions. Buffer solution is the aqueous solution which composed of a mixture of a weak acid and its conjugate base or inversely and used to keep the pH level at an approximately constant value for usage in various applications. Also the 4th group used the Handerson-Hasselbalch Equation method with Na2HPO4 and NaH2PO4 and according to equation between these chemicals which gave below: 

H2PO4 ↔H+ + HPO4 

They calculated the molarity of H2PO4 and HPOby using a formula as pH= pKa + log [conjugate base] / [acid] and so they calculated the amount of each chemical which will be used. Thereby, we’ve learned the applying the Handerson-Hasselbalch Equation method. Actually, we’ve referred as the pH of a solution is based on the concentration of H3O+ ions which are present in the solutions and so weak acids don’t completely dissociate into ions, when they react with water. This case can change different weak acids. In addition to this, the Handerson-Hasselbalch Equation shows us the balance pH calculation of a solution including weak acid and its conjugate base which is given below equation : 

pH = pKa + log ([base])/([acid]) 

According to this equation, pKa is referred as the weak acid’s ionization meaning that dissociation of weak acids into ions even including the H3O+ ions. So the Handerson-Hasselbalch Equation is a common method which used in preparation of buffers for usage in the several laboratory applications. The reason of adding the HCl into the buffer was to decrease the pH value. If we could added the NaOH instead of HCl into the buffer, gradually, the pH value could be larger than 6 because of increasing the OH- ions in the buffer. For the pH 8 Z buffer, we’ve measured the first value lower than 8 and so we’ve tried to increase this value until pH 8 by adding NaOH basic solution slowly. The reason of adding the NaOH into the buffer was to increase the pH value. If we could added the HCl instead of NaOH into the buffer, gradually, the pH value could be lower than the first measured pH value because of increasing the H+ ions in the buffer. The reason of this can be mistakes in weighing of the chemicals or not completely dissociation of ions, so the weak acid for this buffer may not be dissociated into its ions completely. Temperature is another factor that affects the ionizations, because when the temperatures increase, chemicals`s ionizations increase, leading thus to a decrease in the pH values. Additionally, the amount of dioxide carbon which is dissolved on the water affects in the lowering of pH as it causes acidification.