10. The result of an electrophoretic separation of a mixture of amino acids X, Y and Z at pH = 5.0 is represented as (Given the isoelectric points of X, Y, and Z are 9.87, 3.22 and 5.43, respectively)
Electrophoretic Separation of Amino Acids Based on Isoelectric Point and pH
Understanding the Given Electrophoresis
The question describes the electrophoretic separation of a mixture containing three amino acids, X, Y, and Z, at pH 5.0. Their isoelectric points are given as:
pI of X = 9.87
pI of Y = 3.22
pI of Z = 5.43
The electrophoresis diagram shows the negative electrode (−) on the left and the positive electrode (+) on the right. To identify the correct separation pattern, we must determine the net charge on each amino acid at pH 5.0 and then predict the direction and relative extent of its movement.
The key relationship is simple. When the surrounding pH is lower than the isoelectric point of an amino acid, the amino acid carries a net positive charge. When the pH is higher than its isoelectric point, the amino acid carries a net negative charge. When the pH equals the isoelectric point, the net charge is approximately zero.
Using this principle, X and Z are positively charged and migrate toward the negative electrode, whereas Y is negatively charged and migrates toward the positive electrode. Since X is much more positively charged than Z at pH 5.0, X moves farther toward the negative electrode. Therefore, the correct arrangement is shown in Option (A).
Correct Answer: Option (A)
The correct electrophoretic pattern is:
Negative electrode (−) ← X — Z — Y → Positive electrode (+)
Therefore:
Correct Option: (A)
This arrangement correctly represents both the direction and relative extent of migration of all three amino acids.
What Is the Isoelectric Point of an Amino Acid?
The isoelectric point, commonly represented as pI, is the pH at which an amino acid has no net electrical charge. At its isoelectric point, the positive and negative charges present on the amino acid balance each other.
An amino acid at its pI predominantly exists as a zwitterion. A zwitterion contains both a positively charged group and a negatively charged group, but its overall net charge is zero.
The relationship between pH and pI determines the net charge of an amino acid:
When pH < pI, the amino acid is net positively charged.
When pH = pI, the amino acid has approximately zero net charge.
When pH > pI, the amino acid is net negatively charged.
This relationship provides the complete conceptual basis for solving the given question.
How Does Electrophoresis Separate Amino Acids?
Electrophoresis separates charged molecules by applying an electric field. Molecules with different net charges migrate in different directions and at different rates.
A positively charged amino acid is attracted toward the negative electrode, also called the cathode. A negatively charged amino acid is attracted toward the positive electrode, also called the anode.
The amount of migration also depends on the magnitude of the net charge. An amino acid with a greater positive charge generally moves more strongly toward the negative electrode than one with only a small positive charge, assuming the other relevant conditions are comparable.
Therefore, simply knowing whether an amino acid is positive or negative is not always enough. In this question, the relative difference between the experimental pH and the pI also helps us determine which positively charged amino acid migrates farther.
Determining the Charge and Migration of Amino Acid X
Why X Moves Far Toward the Negative Electrode
For amino acid X:
pH = 5.0
pI of X = 9.87
Since:
pH < pI
amino acid X carries a net positive charge.
The difference between its pI and the experimental pH is large:
9.87 − 5.0 = 4.87
This indicates that X is strongly on the protonated side of its isoelectric point and has a substantial positive character under the given conditions. Being positively charged, X is attracted toward the negative electrode located on the left side of the diagram.
Among X and Z, amino acid X is farther from its isoelectric point and therefore migrates farther toward the negative electrode.
This explains why X occupies the leftmost position in Option (A).
Determining the Charge and Migration of Amino Acid Y
Why Y Moves Toward the Positive Electrode
For amino acid Y:
pH = 5.0
pI of Y = 3.22
Since:
pH > pI
amino acid Y carries a net negative charge.
The surrounding pH is 1.78 units above its isoelectric point:
5.0 − 3.22 = 1.78
Therefore, Y is negatively charged and is attracted toward the positive electrode located on the right side of the electrophoresis diagram.
This is why Y appears on the right side in the correct answer.
Determining the Charge and Migration of Amino Acid Z
Why Z Moves Only Slightly Toward the Negative Electrode
For amino acid Z:
pH = 5.0
pI of Z = 5.43
Since:
pH < pI
amino acid Z carries a net positive charge.
However, the difference between the pH and pI is very small:
5.43 − 5.0 = 0.43
This means Z is close to its isoelectric point and therefore has only a relatively small net positive charge. As a result, it migrates toward the negative electrode, but its movement is much smaller than that of X.
This is why Z remains relatively close to the central loading region while X moves farther toward the negative electrode.
Comparing the Migration of X, Y and Z
At pH 5.0, the three amino acids behave differently because their isoelectric points are different.
Amino acid X has a pI of 9.87, which is much higher than the experimental pH. Therefore, X is strongly positively charged and moves far toward the negative electrode.
Amino acid Z has a pI of 5.43, which is only slightly higher than the experimental pH. Therefore, Z is only weakly positively charged and moves slightly toward the negative electrode.
Amino acid Y has a pI of 3.22, which is lower than the experimental pH. Therefore, Y is negatively charged and moves toward the positive electrode.
The final order is therefore:
(−) ← X — Z — Y → (+)
This exact pattern is represented by Option (A).
Why Option (A) Is Correct
X Moves Left, Z Remains Near the Centre, and Y Moves Right
Option (A) shows X farthest toward the negative electrode, Z closer to the central region, and Y toward the positive electrode.
This arrangement is scientifically correct.
X is strongly positively charged because the experimental pH of 5.0 is far below its pI of 9.87. Therefore, it migrates strongly toward the negative electrode.
Z is also positively charged because the pH is slightly below its pI of 5.43. However, because it is close to its isoelectric point, its net positive charge is small and its migration toward the negative electrode is limited.
Y is negatively charged because the experimental pH is above its pI of 3.22. Therefore, it migrates toward the positive electrode.
For these reasons, Option (A) is the correct answer.
Why Option (B) Is Incorrect
The Relative Positions of X and Z Are Reversed
Option (B) correctly places Y toward the positive electrode, but it incorrectly shows Z migrating farther toward the negative electrode than X.
At pH 5.0, both X and Z are positively charged. However, X has a pI of 9.87 and is much farther from the experimental pH than Z, whose pI is 5.43.
Therefore, X has a stronger positive character and should migrate farther toward the negative electrode. Z is close to its isoelectric point and should remain closer to the origin.
Since Option (B) reverses the relative migration of X and Z, it is incorrect.
Why Option (C) Is Incorrect
Y Is Shown Moving Toward the Wrong Electrode
Option (C) places Y toward the negative electrode and Z toward the positive electrode.
This is opposite to their expected behavior.
At pH 5.0, Y has a pI of 3.22. Since the pH is greater than its pI, Y is negatively charged and must move toward the positive electrode, not the negative electrode.
Z has a pI of 5.43. Since the pH is slightly below its pI, Z is positively charged and should move toward the negative electrode, not the positive electrode.
Therefore, Option (C) is incorrect.
Why Option (D) Is Incorrect
X and Z Cannot Remain Together at the Same Position
Option (D) shows X and Z together toward the negative side while Y moves toward the positive electrode.
Although both X and Z are positively charged at pH 5.0, they do not possess the same degree of net positive charge.
X has a pI of 9.87 and is far from its isoelectric point, whereas Z has a pI of 5.43 and is very close to its isoelectric point. Therefore, X should migrate much farther toward the negative electrode than Z.
Because X and Z should be separated from each other during electrophoresis, Option (D) is incorrect.
The Importance of the Difference Between pH and pI
The difference between the experimental pH and the isoelectric point helps explain the relative migration pattern.
For X, the difference is:
pI − pH = 9.87 − 5.0 = 4.87
Therefore, X has strong positive character and migrates far toward the negative electrode.
For Z, the difference is:
pI − pH = 5.43 − 5.0 = 0.43
Therefore, Z is only slightly positively charged and migrates only a short distance toward the negative electrode.
For Y:
pH − pI = 5.0 − 3.22 = 1.78
Therefore, Y has negative character and migrates toward the positive electrode.
This comparison clearly supports the separation pattern shown in Option (A).
Final Answer
At pH 5.0, amino acid X has a pI of 9.87 and is positively charged, so it migrates far toward the negative electrode. Amino acid Z has a pI of 5.43 and is only slightly positively charged, so it moves a shorter distance toward the negative electrode. Amino acid Y has a pI of 3.22 and is negatively charged, so it migrates toward the positive electrode.
Therefore, the correct electrophoretic arrangement is:
(−) ← X — Z — Y → (+)
Correct Answer: (A)
