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Titration Curve of Amino Acids – Experiment, Significance, pKa

Last Updated : 20 Feb, 2024
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The Titration Curve of Amino Acid shows how the pH changes and how the amino acid looks after adding different pH values as a strong base (OH equivalents). Amino acids have different pKa values for each of their many ionizable groups, which include the amino and carboxyl groups. Titration curves offer valuable information about the pH range in which amino acids are most stable as well as their ability to function as a buffer.

In this article, we will learn about the definition of titration, its characteristics, the titration curve, how to calculate pKa from the titration curve, how the factors influenced titration curves, experimental technique and methodology, and the significance of the titration curve of amino acids.

What is the Titration Curve of Amino Acids?

Titration Definition: Titration is a process of chemical analysis that involves determining the concentration of an unknown solution using a solution of known concentration. 

A titration curve provides a visual representation of a solution’s pH during titration. In a strong acid-strong base titration, the equivalency point is achieved when the moles of an acid and a base are equal and the pH is seven. The pH is greater than 7 at the equivalency point in a weak acid-strong base titration.

The volume of the titrant is typically the independent variable and the pH of the solution is the dependent variable (since it varies based on the composition of the two solutions) on graphs known as titration curves, which are used to record titrations. The predominant ionic form of amino acids in solution is dependent on pH since they have an ionisable group.

The titration curve of an amino acid is a graph that shows how much a standard (strong) base can neutralise an acid depending on its pH, understand about the ionisation of acetic acid, or any weak organic acid, by NaOH. The weak acid is transformed into its conjugate base to a greater extent as additional strong base (titrant) is added to the aqueous solution. The Henderson-Hasselbalch equation governs the pH of the buffer system that develops throughout this process. The titration curve for the NaOH-mediated neutralisation of acetic acid will like this:

[Tex]CH3COOH(aq) + NaOH ⇌ CH3COO-Na + H2O[/Tex]

A buffer system is created when a base titrates a weak monoprotic acid. This system’s pH is determined by the Henderson-Hasselbalch equation. Several properties are experimentally defined by this curve (the specific number of each feature varies depending on the type of acid being titrated):

  • The number of ionizing groups, 
  • The pKa of the ionizing group(s)  
  • The buffer region(s).

When acetic acid is titrated, there is just one peak seen, indicating that the acid is monoprotic (i.e., possesses only one dissociable H+).


Calculating pKa from the Titration Curve

When analysing these compounds’ behaviour during a titration, the pKa values are essential. Ionisation reactions can occur between the basic and acidic functional groups found in amino acids. The individual amino acid and the ionizable group (such as carboxyl or amino groups) involved determine the pKa values, which indicate the pH at which these groups are half-ionized.

  1. Ionization of Functional Groups: It exists mostly in its protonated form (COOH) at pH values lower than the carboxyl group’s pKa. Deprotonation of the carboxyl group starts when the pH rises. It exists mostly in its deprotonated form at pH values higher than the amino group’s (NH2) pKa. Amino groups begin to protonate when pH falls.
  2. Zwitterion Formation: Zwitterion formation occurs at pH values around the isoelectric point (pI), which is the point at which an amino acid’s total charge is zero. The carboxyl group is deprotonated and the amino group is protonated in this condition.

Henderson-Hasselbalch equation

The pH of a solution containing a weak acid and its conjugate base, or a weak base and its conjugate acid, may be determined using the Henderson-Hasselbalch equation.

The Henderson-Hasselbalch equation for a weak acid (HA) and its conjugate base (A⁻) is:

pH=pKa+log ⁡([A−]/[HA])

And for a weak base (B) and its conjugate acid (BH⁺):

pH=pKa+log⁡ ([BH+]/[B])


  • pH: The negative logarithm of the concentration of hydrogen ions in the solution.
  • pKa: The weak acid’s acidity is measured by the negative logarithm of the acid dissociation constant.
  • [A⁻] and [HA]: For the weak acid equation, the conjugate base and weak acid concentrations are denoted by [A⁻] and [HA], respectively.
  • [BH⁺] and [B]: For the weak base equation, the conjugate acid and weak base concentrations are denoted by [BH⁺] and [B], respectively.

Characteristics of Titration Curve of Amino Acids

The link between a solution’s pH and the degree of ionization (protonation or deprotonation) of an amino acid molecule as basic or acidic functional groups gain or lose protons is shown by the amino acid titration curve. The amino acid titration curve’s characteristics features are as follows:

  • Multiple ionization State: Two ionizable groups are present in amino acids at least: the carboxyl group (-COOH) and the amino group (-NH2). These groups can exist in three distinct ionization states: protonated (+H), deprotonated (-H), or neutral, depending on the pH of the solution.
  • Isoelectric Point (pl): An amino acid’s pH is known as its isoelectric point (pI), which is the pH at which it is mostly found in its zwitterionic state, or without any net charge. The quantity of positively charged amino groups and negatively charged carboxyl groups are equivalent at this stage.
  • Titration Region: Usually, the titration curve shows three different regions: low pH, near the pI and high pH.
  • Buffering Regions: Buffering zones may be seen in the titration curve close to the pKa values of the amino and carboxyl groups. These are the areas where weak acid-base pairs are present and tiny inputs of acid or base result in negligible pH variations.
  • Sigmoidal Shape: Because the amino and carboxyl groups in the amino acid molecule cooperate in their protonation and deprotonation, the titration curve usually has a sigmoidal shape.

Factors Influencing Titration Curves

The form and properties of titration curves for acids, bases, and amino acids are influenced by several variables. These variables greatly influence how the titration process behaves and the curves that are produced. The following are the main variables that affect titration curves:

  1. Acid or Base Strength: The titration curve’s form is determined by the strength of the acid or base being titrated. Strong acids and bases have a steep initial slope of the curve because they dissociate in solution. Weak bases or acids only partly dissolve, giving the curve a softer slope and buffering areas.
  2. Concentration: The titration curve’s form and location are influenced by the concentration of the acid or base being titrated.
  3. Presence of Buffers: Titration curves can be impacted by buffers in the solution since they can withstand pH variations.
  4. Ionic Strength: The titration curve’s form is influenced by the solution’s ionic strength, which is based on the amount of ions present. Ion interactions and complex formation may cause solutions with higher ionic strengths to display different titration behaviour.
  5. Temperature: The form and location of the titration curve may change as a result of temperature’s effects on proton transfer rates and acid-base reaction kinetics.

Significance of Titration Curve of Amino Acids

Titration curves are essential for understanding how amino acids behave in biological systems because they provide information on pH-dependent activities including enzyme activity and protein folding.

  • Understanding Amino Acid Behavior in Biological Systems
    • pH Homeostasis: Titration curves can be used to predict the behaviour of amino acids in biological systems, which have a defined pH range.
    • Protein Charge and Solubility: Protein charge distribution at various pH values is revealed by amino acid titration curves.
  • Relevance to Protein Folding
    • Isoelectric Point (pI) and Zwitterion Formation: As zwitterions at the pI, proteins reduce electrostatic repulsions and promote structural stability.
    • pH-Dependent Conformational Changes: Variations in pH can cause conformational changes in proteins. Titration curves aid in the prediction of protein areas that are susceptible to structural changes brought on by changes in pH.
  • Relevance to Enzymatic Activity
    • Active Site Chemistry: In the active sites of enzymes, certain amino acid residues with crucial pKa values are frequently found. These residues’ state of ionisation influences the interactions between the enzyme and the substrate.
    • pH Rate Profiles: The link between pH and reaction rate is shown by pH rate profiles, which are obtained from titration curves. These profiles aid in clarifying the function of certain residues of amino acids in enzyme catalysis.

Conclusion: Titration Curve of Amino Acids

Titration curves are essential for understanding how amino acids behave in biological systems because they provide information on pH-dependent activities including enzyme activity and protein folding. Gaining an understanding of the relationship between the ionisation states of amino acids and biological activity is essential for improving our understanding of biochemistry and has applications in molecular biology, drug creation, and medicine.

Also Read:

  1. Difference Between Polar and Nonpolar Amino Acids
  2. Difference Between Essential and Nonessential Amino Acids
  3. Difference Between Glutamate and Glutamine

FAQs – Titration Curve of Amino Acids

What is the Titration method for the Estimation of Amino Acids?

Titrate amino acids in 97% alcohol and polypeptides in 40% alcohol. Start with 40% alcohol titration followed by 97% alcohol to estimate total polypeptides and amino acids in a mixture.

How do you Titrate Amino acids with NaOH?

Transfer 20 ml of amino acid solution to a beaker. Measure its initial pH. Titrate by adding 0.3 ml of 0.1 M NaOH incrementally until pH reaches 12.5. Record NaOH volume needed. Repeat for accuracy.

What is Titration Curve of Glycine?

Glycine’s titration curve resembles a weak diprotic acid’s. When titrated with NaOH, glycine follows a curve, despite being commonly noted as NH2COOH; it exists as a zwitterion, +NH3CH2COO.

How do you find the pKa of an Amino Acid from a Titration Curve?

Identify the midpoint of each buffering zone on the titration curve. This is where the pH equals the pKa value, indicating equal concentrations of the acid and its conjugate base. Record the pH values at these points as the pKa values.

What is the Henderson Hasselbalch Equation for Amino Acids?

The Henderson-Hasselbalch (HH) equation for amino acids is [H3O+] = Ka[HA]/[A].

What is the Purpose of the Titration Curve Experiment?

Titration curves graph changes in the measured property, like pH, as titrant is added. They serve as a basis for endpoint and equivalence point determination.

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