University:
Boston University
Course:
CAS CH 214 | Organic Chemistry with Qualitative Analysis
Academic year:

2023
Views:

499

Pages:

21
Author:

Melina Hobbs

Buffers and Titrations Procedures • Calibrate pH meter and measure pH of different solutions • Titrate histidine solution and deionized water to obtain a corrected histidine titration curve • Perform a pH stat experiment (hydrolyze BSA with trypsin) to calculate the number of Lys and Arg residues in BSA pH Meter ● Measures the difference in electric potential between a pH electrode and a reference electrode ● ● ● ● ● ● Difference in electric potential related to acidity (pH) of the solution. Use standards to set the pH values in meter Glass‐electrode sensitive to hydrogen ions Electrode somewhat sensitive to other alkali metals (Na+ vs K+) Complete system contains: ● Electrometer – 5 ● Reference electrode – 6 ● Solution to be measured – 1,4 ● pH glass electrode – 2,3 Always protect your pH meter! Titration Curves in Non‐buffered Solutions Weak Acids: mediocre H+ donors (e.g. acetic acid) [HA] = CH3COOH [A‐] = CH3COO‐ ● ● ● ● Strong acids: excellent H+ donors (e.g. hydrochloric acid) [HA] = HCl [A‐] = Cl‐ Equivalence Point ● ● Point at which reaction is neutralized Vertical‐like inflection point in titration curve Weak acids are good for buffering solutions Strong acids are good for titrating solutions Table of pKa values Buffered Titration Curve ● ● Empirically, Henderson‐ Hasselbalch equation is useful for buffering range Buffers most effective near pKa ← Henderson‐Hasselbalch equation pH = pKa when ‐ [A ] = [HA] Buffering Capacity ● ● ● Ability of buffer to resist changes in pH with addition of acid or base Highest buffering capacity obtained when [A‐] = [HA] (when pH = pKa) At any given pH, the buffer capacity increases proportionally as the concentration of the buffer increases Evidence that Buffer Capacity is highest at pH near pKa 𝑏𝑢𝑓𝑓𝑒𝑟 𝑐𝑎𝑝𝑎𝑐𝑡𝑖𝑦 𝐻𝐴 𝐴 2.303 𝐻𝐴 𝐴 𝐻𝐴 1 𝐻𝐴 2.303 𝐻𝐴 𝐻𝐴 1 2.303 𝐻𝐴 𝐻𝐴 1 2 Procedure A: Calibration and Measurements ● Calibrate your pH meters ● ● ● Measure the following solutions ● ● ● ● ● ● YouTube video uploaded tonight! Immediately approach your TF is you encounter problems (don’t waste precious lab time) Tap water Deionized water (take measurement in first 10 seconds, pH fluctuates, why?) 0.01 N HCl 0.01 N KOH 0.01 N NaOH Pay attention to all the solutions and concentrations throughout the lab Procedure B: Titration ● Make His Buffer ● ● ● ● Starting pH? Titration of a triprotic acid ● Titrate Acid Group of His (1.82) ● Titrate the Two Basic Groups of His ● Titrate imidazole (6.00) ● Titrate amino group (9.17) Histidine Water corrections ● Titrate Water with Acid ● Titrate Water with Base Subtract water titration values from histidine titration values to obtain corrected histidine curve Procedure B: Titration ● Make His Buffer – 0.4 M His‐HCl = 0.4 M [HA] ● Deprotonated His‐HCl = [A‐] = [H+] ‐log[H+] = -log[Ka] + log[H+] - log [HA] 2(-log[H+]) = -log[Ka] - log [HA] 2pH = pKa - log[HA] • Substituting pKa of His‐HCl = 6.04 pH = (6.04 ‐ log [0.4])/2 = 3.22 Problem #9 in Lab Manual Procedure B: Titration ● Make His Buffer ● ● Four Titrations ● Titrate acid group of His ● Titrate the two basic groups of His ● ● ● Starting pH = 3.22 (show your work for pre‐lab) Histidine Titrate di‐water with acid (count drops of titrant instead of adding certain volumes of titrant) Titrate di‐water with base (count drops of titrant instead of adding certain volumes of titrant) Subtract water titration values from histidine titration values to obtain corrected histidine curve in post‐lab write‐up Part C: Digestion of BSA with Trypsin Proteolytic Hydrolysis (Cleavage) of Proteins Trypsin (enzyme): Cleaves c‐terminus of the amino acids lysine or arginine, except when either is followed by proline Trypsin Procedure C ● ● Denature BSA at 80‐90 °C until cloudy ‐> Use a 50 mL beaker (not 25 mL) Digest BSA with Trypsin ● Titrate during reaction to maintain pH value 8.5 ● ● ● Do not overshoot titration! You’ll ruin your trypsin enzyme Record volume KOH added and the time elapsed Calculate the Number of Peptide Bonds Cleaved When Reaction is Complete ● Calculate mmols KOH added at endpoint ● Calculate number of Arg + Lys per molecule BSA Relating the Titration to Arg + Lys Residues ● ● How much H+ is actually produced? pH of reaction is only slightly greater than the pKa of N‐terminus ● Not every amino group will gain a proton Relating the Titration to Arg + Lys Residues ● Problem 10, p. 47: What is ratio of [A‐]/[HA] for the protonation of an amine with a pKa = 8.2, at pH 8.5? 8.5 = 8.2 + log [R-NH2]/[R-NH3] = 8.2 + log [A-]/[HA] pH of reaction Amino group pKa 0.3 = log [A-]/[HA] [A-]/[HA] = 100.3 = 2/1 2/3 depronated [A‐], 1/3 protonated [HA] But what does this 2:1 ratio actually mean? (pages 44-45 in lab manual) N’-Gly-Lys-Ala-Arg-Val-Lys-Leu-C’ > Water molecules are incorporated into peptide hydrolysis Nterm-Gly-Lys-COOH HHN-Ala-Arg-COOH HHN-Val-Lys-COOH HHN-Leu-Cterm > At pH 8.5, carboxyl is completely deprotonated, and one out of every three amino groups accepts these protons Nterm-Gly-Lys-COO- H+ H+ HHN-Ala-Arg-COOHHN-Val-Lys-COO- HHHN-Cterm > You are titrating the other two hydrogen ions ( H+ and H+) Relating the Titration to Arg + Lys Residues ● ● The trypsin digestion alters the buffer capacity of the solution ● As more amino groups are formed, some accept a proton ● Other protons are neutralized by KOH titration Total # of peptide bonds cleaved = (mmol of KOH added)(3 peptide bonds cleaved/2 mmol KOH added) ● ● This 3/2 correction ratio is highly dependent on the starting and ending pH of your experiment. If your pH did not begin exactly at 8.5, then you need to recalculate this ratio for your experiment. Total # of Lys + Arg per molecule of BSA = (# of peptide bonds cleaved)/(mmol of BSA used) ● Calculate mmol of BSA using MW (66,000 g/mol) Chapter 2 pre-lab checklist In your data collection sheets, you should have:  5 self-prompts for the pHs of your various solutions  4 tables for the pH titrations (you will record a lot of points!)  A table for time, volume of KOH added, and corresponding pH for proteolysis experiment - Don’t forget to record the starting pH of your experiment Chapter 2 laboratory plan To maximize in-lab efficiency:  One person calibrates the meter and measures pH of solutions; another person prepares the histidine buffer and prep the burets  During histidine titrations, one person controls the buret and calls out volumes/drops, another person records the data and checks the pH  One person does the histidine titrations, another person prepares and denatures the BSA solution  Work together on the BSA proteolysis experiment (~35 min) A few lab notes moving forward At the end of lab, you should have:  pH recordings for tap water, deionized water, 0.01 N HCl, 0.01 N KOH, and 0.01 N NaOH  Titrated histidine solution to pH 1.0 with 0.5 N HCl, and recorded pH readings and volumes.  Titrated a fresh aliquot of histidine solution to pH 12.5 with 0.5 N KOH, and recorded pH readings and volumes  Repeated titration method with deionized water between pH 2 and 12-13. Recorded pH readings and volumes.  Completed proteolytic hydrolysis with BSA & Trypsin. Recorded volumes and corresponding timepoints.  Cleaned out burets and leave filled with diH2O. Properly stored pH meter. Cleaned bench. Lab notes moving forward Meet with your lab partners ahead of time to be better organized in lab No need to rinse tubes or plastic tip/liquid waste buckets with extra water. Washing/rinsing the plastic soup cups at the sink is okay if you dumped as much as possible in the appropriate waste! (Trace amounts is okay down the drain!) Be conscious of all the reagents, and put them back where you found them!

Buffers and Titrations Lecture Note

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