Lecture 2: Water, pH, pKa, and Buffers (Cont.)

General Course Information

• Campus Wire and iClicker: Enrollment should be automatic, facilitating communication and participation tracking.

• Gradescope: Enrollment is also automatic, streamlining assignment submissions and grading.

• iClickers and Test: iClickers are postponed; the test is moved to Monday, with a review on Tuesday.

• Excused Lectures: The same number of lectures will be excused, providing flexibility.

• Upcoming Quiz and Homework: Be prepared for the next quiz on Wednesday and homework submission to stay on schedule.

Polyprotic Acids

• Phosphoric Acid ($H_3PO_4$ ): Can lose up to three protons with different $pKa$ values: $2.1$, $7.2$, and $12.3$. These values are critical for understanding its behavior.

• Hydroxyl Groups: The three $OH$ groups are identical, but the $pKa$ values differ. Removing subsequent protons becomes increasingly difficult due to the molecule becoming more negatively charged; each removal requires more energy.

• Proton Removal:

  • Removing the first proton ($pKa=2.1$) creates a negative charge, hindering the removal of the second proton.

  • Stabilizing two negative charges requires more energy due to increased electrostatic repulsion.

• Resonance Stabilization: Resonance in the carbonyl group helps stabilize negative charges by distributing the charge across multiple atoms, reducing the intensity of the negative charge.

Reactions of Phosphoric Acid

• Three Reactions:

  • $H_3PO_4\rightarrow H_2PO_4^{-}+H^{+}$

  • $H_2PO_4^{-}\rightarrow HPO_4^{2-}+H^{+}$

  • $HPO_4^{2-}\rightarrow PO_4^{3-}+H^{+}$

• Conjugate Acid-Base Relationship: The conjugate acid of one reaction is the conjugate base of the previous reaction, which is key to predicting reaction direction.

• pH Increase Effect: As pH increases, the molecule shifts from being the base of the previous reaction to having its proton removed, which is fundamental to acid-base chemistry.

Titration Curve

• Illustration: The titration curve shows the predominant forms of phosphoric acid at different pH levels. There are four forms, each predominant in a specific pH range.

• Process:

  • Starting with pure $H_3PO_4$ at low pH, where $H_3PO_4$ predominates until the first half equivalence point.

  • At the first half equivalence point, equal amounts of $H3PO4$ and $H_2PO_4^{-}$ are present, providing optimal buffering capacity.

  • Past the first half equivalence point, $H2PO4^-$ becomes predominant. At the first equivalence point, $H2PO4^-$ dominates.

  • Moving past the first equivalence point, $HPO4^{2-}$ starts to form. At the second half equivalence point, equal amounts of $HPO4^{2-}$ and $H2PO4^-$ are present.

  • $HPO4^{2-}$ is predominant between the second and third half equivalence points.

  • Past the second equivalence point, $PO4^{3-}$ starts to form as the third proton is removed.

• Equivalence Points: The curve shows three equivalence points and three half equivalence points, corresponding to the removal of each proton.

• Deprotonation Regions:

  • The region between zero and one equivalent of $OH^-$ corresponds to the deprotonation of the first proton.

  • The region between one and two equivalents corresponds to the deprotonation of the second proton.

  • The region between two and three equivalents corresponds to the deprotonation of the third proton.

Buffer Regions

• Existence: Three buffer regions exist where pH changes are minimal relative to the amount of $OH^-$ added. These regions show the coexistence of a conjugate acid and its conjugate base.

• Flatlining Regions: The titration curve shows flatlining regions within the buffer regions, indicating effective buffering.

• Importance: Understanding predominant forms at different pH levels is crucial, especially for amino acids. The principles of the phosphoric acid titration curve apply to amino acids, which undergo multiple protonation and deprotonation steps. Titration curve principles applicable to amino acids.

Amino Acids

• Major Parts:

  • Alpha amino group

  • Alpha carbon

  • Alpha carboxyl group

• R-Group: The side chain (R-group) attached to the alpha carbon differentiates amino acids, dictating their unique properties.

• Amino pKa: Ranges between 9 and 11. Side chains influence the pKa values of other groups through inductive effects, electronic drawing, and size/charge properties.

General Amino Acid Structure

• Structure: Amino group, alpha carbon with side chain, and carboxyl group.

• Homework Chart: The chart in homework one unifies alpha carboxy and alpha amino PKAs, aiding in understanding protonation states at different pH values.

• Neutral Form: The typical representation of amino acids in a neutral form is incorrect.

  • Amino pKa ranges between 9 and 11.

  • Carboxyl pKa ranges between 1.9 and 2.5.

• Zwitterionic Form: The amino $pKa$ ranges between $9$ and $11$ and the carboxyl $pKa$ ranges between $1.9$ and $2.5$, it is impossible for it to ever be in a neutral state.

  • The zwitterionic form is the actual neutral form, with balanced charges and is the predominant form at physiological pH.

  • The amino acid content sheet shows the zwitterionic structure.

pKa Values

• Provision: pKa values will always be provided; memorization is unnecessary. Focus on understanding how to apply them.

Acid and Base Behaviors

• Carboxylic Acids: Go from neutral to negative when deprotonated.

• Hydroxyl Side Chains: Go from neutral to negative when deprotonated.

• Amines: Go from positive to neutral when deprotonated.

Titration with Tyrosine

• Process: Proceed through various pHs to determine the structure depending on the pKa chart. At a pH of 1, all groups are protonated.

• Chart Usage:

  • Lines 3 and 4 of the chart are used for free amino acids.

  • Lines 1 and 2 are used for amino acids in a polypeptide chain.

  • Use 2 and 9.5 for alpha carboxyl and alpha amino groups in free amino acids.

Ranges of Behavior Based on pKa Values

• Below 2: All groups are protonated, and the charge is +1.

• From 2 to 9.5: The zwitterionic form exists.

• Between 9.5-10: The overall charge is -1.

• Above 10: All groups are deprotonated, and the charge is -2.

• Examples: Use aspartic acid and lysine.

Titration Curves

• Application: The process for polyprotic titration is applicable here as well. Understanding the similarities will help.

Isoelectric Point

• Objective: To find the pH at which the molecule has no net charge. Zwitterions are found at this point, which is crucial for understanding protein behavior.

Method for Determining Isoelectric Point Practically

• Setup: Set up electrophoresis gel and place compounds in wells in the center of the gel.

• Charge Application: Apply a negative charge (cathode) on one end and a positive charge (anode) on the other end.

• Migration: Molecules migrate based on their charge at pH 6.

  • Arginine migrates to the cathode because it is positively charged.

  • Aspartic acid migrates to the anode because it is negatively charged.

  • Alanine and isoleucine do not migrate significantly because they are neutral.

• Distance: The distance traveled indicates the isoelectric point, but migration length is not needed to calculate this theoretically in this course.

Isoelectric Point Math

• Alanine pKa's: 2.34 (carboxylic acid) and 9.69 (amine).

• Charge States:

  • Below 2.34, alanine has a charge of +1 because both groups are protonated.

  • Between 2.34 and 9.69, alanine is zwitterionic (neutral).

  • Above 9.69, alanine has a charge of -1 because the amine group is deprotonated.

• Calculation: To find the isoelectric point, take values above and below the zwitterionic range and find the average; it’s the midpoint of the neutral range.

Isoelectric Point for Alanine

• Isoelectric Calculation: $(2.34 + 9.69) / 2 = 6.015$

Net Molecular Charge

• Relationship: The plot shows the relationship between pH and net charge per molecule.

• Charge Distribution:

  • At the first pKa, 50% of molecules are protonated, and 50% are zwitterionic, resulting in a net charge of +0.5.

  • At the isoelectric point, most molecules are in the zwitterionic form, and the net charge is close to zero.

  • Beyond the isoelectric point, the population forms a more anionic population. At a higher pH, there's 50% zwitterionic and 50% anionic, with a charge of -0.5.

• General Math: The math will remain the same independent of the number of pKa's.

Polypeptide Chains

• Formation: Ribosomes catalyze the formation of peptide bonds. The alpha amino group of one amino acid attacks the alpha carboxyl group of another.

• Reaction: A condensation or dehydration reaction occurs, releasing water: $(OH + H = H_2O)$

Forming Peptide Bonds

• Drawing: Drawing polypeptides of an arbitrary length is a useful skill for the midterm. Draw the backbone first, then add side chains later.

• Amino Acid Residues: When in a chain, amino acids are referred to as amino acid residues. The N-terminus is at the beginning, and the C-terminus is at the end.

• Amide Group: The amide group (i.e., peptide bond) links one amino acid with the next amino acid.

Amino Acid Content Sheet

• Zwitterionic Form: All amino acids are in their zwitterionic form at physiological pH.

• Basic Information to Memorize:

  • Given a name you should know the category, the 3 letter code, the one letter code, the structure

  • Given a structure, conversely you should know the aforementioned name categories and codes