Lab #02: Buffers & pH, Titration of Amino Acids and Thin Layer Chromatography

Question 1

Consequences of Fluctuating [H+] In Blood

Answer 1

• Any fluctuations in the hydrogen ion concentration in blood can result in serious consequences such as illness and death. Thus there must be a thorough regulation system on their concentration.

Question 2

Autoionization of Water

Answer 2

○ Water can act as a Bronsted Acid (proton donor) or a Bronsted base (proton acceptor). Water can actually be in a reaction with itself and play both roles resulting in H3O+ and OH-.
○ The equilibrium of the ionization of water by far favors the side with the two water molecules.
○ In pure water the concentration of H+ and equal to that of OH-.
§ Ion Product of Water = 1.0 x 10^-14 at 25 degrees Celsius.

Question 3

What is pH?

Answer 3

○ pH is a convenient way to expression hydrogen ion concentration.
○ pH = -log[H+]

Question 4

pH Paper

Answer 4

○ pH paper has specific chemicals (acid-base indicators). The indicators are dyes that change color at specific pH values. Example it litmus paper.

Question 5

Hydrion Universal Paper

Answer 5

Another type of pH paper is called Hydrion universal paper. Will indicate if a solution is neutral, and the extent of acidity and basicity. Contains two types, wide and narrow range.
□ Narrow range covers 2 pH units.
□ Wide range covers 12 pH units.

Question 6

pH Meter

Answer 6

pH meter is a more quantitative way to measure pH. It has a glass electrode connected to a meter. When electrode is put in a solution, a voltage that is dependent on the pH will be produced. The electrical potential produced is a linear function of pH. A pH meter MUST be standardized by buffer solutions whose pH has been accurately determined prior to measuring the pH of an unknown solution.

Question 7

Dissociation of a Weak Acid

Answer 7

○ Strong acids like HCl dissociate completely in water and exist as ions in the solution. But, most acids are weak, and only partially ionized.
○ HA <–> H+ + A-
○ According to law of mass action the value Ka (acid dissociation constant) can reflect strength of an acid.
§ Ka = [H+][A-]/[HA}
○ The more hydrogen ions released upon dissociation, the higher the Ka will be and thus stronger the acid.

Question 8

What is a buffer?

Answer 8

○ Consists of a mixture of a weak acid and its conjugate base. Tend to resist changes in pH upon addition of acid or base.

Question 9

Buffer Mechanics

Answer 9

CH3COOH <–> H+ + CH3COO-

Consider the weak acid acetic acid and its conjugate base acetate. If a strong acid is added, the added hydrogen ions are going to react with acetate and more acetic acid will be produced. So equilibrium will shift to the left (Le Chatelier’s Principle). If a strong base is added, the hydroxyl ions will react with the weak acid (acetic acid) to form water and the conjugate base (acetate). Equilibrium shifts to right. So addition of strong acids and bases will result in slight change in pH until buffering capacity is exceeded.

Question 10

Le Chatelier’s Principle

Answer 10

Le Chatelier’s Principle states that if stress is applied to equilibrium, the system will shift in direction that will relieve the stress.

Question 11

Buffering Capacity

Answer 11

Buffering capacity is maximum amount of acid or base that can be added to buffer before pH begins to change significantly. Dependent of concentration of weak acid/conjugate base pair and volume of buffer.

Question 12

pKa

Answer 12

Buffers have a pKa which is a numerical measure of acid strength of the buffer.
§ pKa = -log Ka

Question 13

Henderson-Hasselbalch Equation

Answer 13

The Henderson-Hasselbalch Equation is used to calculate pH of buffers. Equation is derived as follows.

1. Dissociation of weak acid. HA <–> H+ + A-
   2. Acid Dissociation Constant Expression: Ka = [H+][A-]/[HA]
   3. Solve this expression for [H+]. [H+] = Ka x [HA]/[A-]
   4. Take -log of both sides: -log[H+] = -log Ka [HA]/[A-]
   5. Rearrange the equation. -log[H+] = -logKa - log[HA]/[A-]
   6. pH is equal to -log[H+] and pKa is equal to -log[Ka]: pH = pKa - log[HA]/[A-]
   7. To use the positive log, equation is represented as: pH = pKa + log[A-]/[HA]

Question 14

Applying H-H Equation

Answer 14

Based on the H-H equation, when the concentration of the weak acid is equal to that of the conjugate base, the pH of solution is equal to the pKa of weak acid. The maximum buffering capacity is near the pKa, so when choosing a buffer, it’s good to choose with a pKa close to the desired pH.

Question 15

Calculation of the pH of a Buffer

Answer 15

• Calculation of the pH of a Buffer
○ The H-H equation is used frequently to calculate pH of buffer.
○ The concentrations of weak acid and conjugate base refer to the equilibrium concentrations of the conjugate acid-base pair.
○ This equation is best applied to weak acids because the amount of acid that dissociates is assumed to be minimal. But if pKa is less than 2.0, the H-H equation should not be used since it’s likely the dissociation is not small.

Question 16

Role of Amino Acids

Answer 16

○ Excess dietary amino acids are converted to common metabolic intermediates like pyruvic acid. So not only are amino acids building blocks or proteins, they’re also fuel because they’re precursors of glucose, fatty acids, and ketone bodies.

Question 17

Significane of Amino Acid Sequence

Answer 17

○ The linear amino acid sequence in a protein determines the structure and function of protein.

Question 18

Amino Acid Structure

Answer 18

○ All amino acid alpha Carbons are binded to four different groups and are chiral EXCEPT glycine, which has hydrogen as an R group. All other amino acids cannot be superimposed on its mirror image, are optically active, and thus capable of rotating the plane of polarized light.

○ The alpha carbon of amino acids has a tetrahedral arrangement of bonding orbitals, thus the four different groups occupy two different spatial arrangements which are called enantiomers. For historical reasons, each one is referred to either D or L.
§ If amino group is left of alpha carbon, L-form. If right, D-form. All amino acids in proteins are in the L form.

Question 19

Amino Acid Classes

Answer 19

○ Five Main Classes:

1. Nonpolar, aliphatic R groups
2. Nonpolar, aromatic R groups
3. Polar, uncharged R groups
4. Positively charged R groups
5. Negatively charged R groups

Question 20

Nonpolar Aliphatic Amino Acids

Answer 20

○ The R groups of glycine, alanine, proline, valine, leucine, isoleucine, and methionine are all nonpolar and hydrophobic. They all stabilize protein structure by hydrophobic interactions.
§ Glycine’s side chain does not make a significant contribution since it is simply a hydrogen atom, but nonetheless is nonpolar.

Question 21

Nonpolar Aromatic Amino Acids

Answer 21

○ The R groups of phenylalanine, tyrosine, and tryptophan are all comparatively nonpolar and hydrophobic cause of their aromatic side chain. Tyrosine also has power to participate in hydrogen bonding via its hydroxyl group.

Question 22

Polar Uncharged Amino Acids

Answer 22

○ Amino acids with polar uncharged R groups are serine, threonine, cysteine, asparagine, and glutamine. These amino acids all contain R groups which are polar and thus hydrophilic. Both serine and threonine have polar hydroxyl groups. Cysteine has a polar sulfhydryl group, and asparagine and glutamine have a polar amide group.

Question 23

Polar Positively Charged Amino Acids

Answer 23

○ Three amino acids have positively charged/basic R groups: lysine, histamine, and arginine. R group in all three are positively charged at or near neutral pH.
§ Histidine, its R group imidazole group has a pKa of 6, near physiological pH 7.4. So, in proteins histidine can exist in the protonated and unprotonated from depending on the pH, and the “state” of this imidazole group can significantly influence the properties of many proteins.

Question 24

Polar Negatively Charged Amino Acids

Answer 24

○ Only two amino acids with negatively charged R groups are asparate and glutamate. Or acidic. Both have a carboxyl group in their side chain.

Question 25

Zwitterion

Answer 25

○ At pH 7, an amino acid is a zwitterion, or a dipolar ion where both the amino group and the carboxyl group are charged. If amino acid lacks a charged group on R group, at neutral pH the zwitterion has an equal number of positive and negative charges, and is therefore electrically neutral. So it can act as both a acid by donating one from amino group, or a base and accept one with carboxyl group.

Question 26

What Titration Involves

Answer 26

○ Titration of a specific substance involves adding or removing protons. When an amino acid is titrated with a base, a characteristic titration curve is made.

Question 27

Titration with Ionizable R Groups

Answer 27

○ For amino acids with ionizable R groups, titration curves reveal 3 pKa values. In order to determine pI for these amino acids, the correct pKas must be averaged.

§ Consider titration of glutamate. At low pH, glutamate has a +1 charge due to positive charged amino group. As pH increases, carboxyl group loses a proton and becomes negatively charged, which occurs at pH of 2.19 (pK1). Increase pH more results in removal of proton from carboxyl group in side chain of glutamate. pH this occurs is 4.25 and is called pKR. At this pH there is an equimolar concentration of the zwitterion and the anion. As pH goes up to 9.67, amino group is deprotonated and glutamate has a -2 charge. In order to determine pI for glutamate the two pKas that must be averaged are pK1 and pKR.

Question 28

Thin Layer Chromatography

Answer 28

○ This is a technique used to separate components of mixture by using a liquid mobile phase and solid stationary phase.

Question 29

Thin Laer Chromatography: Two Phases

Answer 29

○ Separation is based on relative affinity a substance has for stationary and mobile phase.
§ Substances that interact strongly with mobile phase will migrate further.
§ Substances that interact strongly with solid phase will not travel as rapidly.

Question 30

Thin Layer Chromatography: Basis of Separation

Answer 30

○ Separation of solutes is based on polarity. Also there are additional factors like type of plate used, molecular weight, temperature, and relative miscibility of solvent components.

○ Nonpolar amino acids will preferentially associate with mobile phase because it is more nonpolar than the stationary phase. Polar amino acids will preferentially associated with polar stationary phase via hydrogen bonding. Thus amino acids with nonpolar side chains will migrate further.

Question 31

Retention Factor Expectations

Answer 31

○ Polar amino acids expected to have Rf values close to 0, and nonpolar ones close to 1.0.