Experiment-2

Electrophoresis of Amino Acids

Dr. Joost Luecker

Course: BIOL 4150

Name: Heli Patel

Student ID: T00703039

Partner’s Name: Ayushi Patel

Date of Experiment: Sept 18, 2025

Date of Submission: Sept 25, 2025

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Abstract
Electrophoresis is a technique that separates charged biomolecules based on their migration in an

electric field, which is strongly influenced by pH and isoelectric point (pI). In this experiment,

glutamate, alanine, lysine, and an unknown mixture were subjected to horizontal paper

electrophoresis at pH 6.5 and pH 1.9. At pH 6.5, glutamate migrated 3.90 cm toward the anode,

alanine 1.95 cm toward the anode, and lysine 5.10 cm toward the cathode, which is consistent

with their predicted charges. The unknown mixture migrated 2.50 cm toward the anode, which

indicated the presence of amino acids with pI values near 6. At pH 1.9, all amino acids were

expected to have net positive charges and migrate toward the cathode, with lysine showing the

greatest displacement (1.47 cm). These results confirm the relationship between pH, pI, and

electrophoretic mobility, demonstrating that small charge differences yield distinct separations at

near-neutral pH, whereas extreme acidity reduces resolution.

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Introduction
Electrophoresis encompasses techniques that enable the separation of molecules based on

their electric charge and size in an electric field of direct current. Electrophoresis has become one

of the most widely used techniques in biochemical analysis since the majority of biomolecules

are charged. The electric charge on a biomolecule is extremely pH-dependent on its surrounding

environment. Thus, electrophoretic separations are always performed in well-established buffer

systems.

Electrophoresis provides a solid support to prevent the charged molecules from diffusing

freely in the buffer by restricting the volume of electrolyte. Cellulose chromatography paper is a

very good medium for entrapping electrolytes within its fibres, and it can be employed in the

separation of small biomolecules such as amino acids. However, larger biomolecules such as

proteins or chromosomal DNA must be separated on polyacrylamide or agarose gels that have

larger pore sizes (Wilson & Walker, 2000).

The objective of this experiment was to separate and compare the electrophoretic

behaviour of glutamic acid, an unknown mixture, alanine, and lysine. These amino acids were

subjected to horizontal paper electrophoresis in two different pH media: an alkaline / near-neutral

buffer (pH 6.5) and an acidic buffer (pH 1.9). The relationship between net charge, molecular

size, and electrophoretic mobility was discovered by measuring both direction and distance of

migration. The ionization state was dictated by their respective pKa values. Consequently, the net

charge of each amino acid and unknown was determined by the ionization state of the α-carboxyl

(α-COOH), α-amino (α-NH₃), and any ionizable R-group functional groups. (Luecker, 2025).
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Methods
Preparation of Electrophoresis System

A basic horizontal paper electrophoresis system was employed (Luecker 2025). The phosphate

buffer with a pH of 6.5 was used for the main separation. The buffer was poured into both sides

of the electrophoresis bath and flowed over the center ridge to create equal liquid levels in both

electrode compartments. This step equalized and prevented siphoning of liquid across the filter

paper during the run. The electrodes were confirmed to be fully submerged in the buffer. This

operation was repeated using a formic acid buffer with a pH of 1.9 (50 mL 1 M formic acid +

150 mL glacial acetic acid per litre).

Sample Application

Chromatography strips of paper were handled only at the edges to avoid contamination (Luecker

2025). Across the long axis, a pencil line was drawn in the middle, and four application points

(G, M, A, L) were marked equally along the line, and outer points were marked around 1 cm

from the edges. The strip was labelled with positive (+) ends, negative (−) ends, and the pH of

the buffer. A drop of each of the amino acids was applied: glutamic acid at point G, alanine at

point A, lysine at point L, and a mixture at point M. Various pipettes were used to avoid cross-

contamination. Small (about 1 cm) and tight spots were created, and strips were left for a few

minutes to dry thoroughly.

Electrophoresis Run and Visualization

The dried paper was placed at both ends of the strip and allowed to migrate toward the center

until buffer fronts met, which minimized pre-run sample migration. The lid of the apparatus was

closed and connected to a 200 V d.c. source for exactly 35 minutes. Power was turned off at the
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end of a run, and the paper strip was removed, drained horizontally, and then placed on a clean

paper towel. Strips were dried in an oven at 70-80 °C for about 30 min. Visualization was

performed by spraying strips in a fume hood with 0.2% ninhydrin in 95% ethanol. Then, the strip

was re-heated until spots turned colour (Luecker, 2025). Spots were outlined by pencil before the

colour faded, and the distance from the center to the spot was measured without getting

ninhydrin on the skin.

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Results
The pKa values of amino acids correspond to the ionization of their functional groups,

such as the α-carboxyl, α-amino, and the side chain (R-group). The isoelectric point (pI) is the

pH at which the amino acid carries no net charge, which was calculated by averaging the two

closest pKa values for known amino acids, as shown in Table 1. Alanine without ionizable side

chains has a pI value near neutral, while glutamate with acidic side chains has a lower pI value,

and lysine with basic side chains has a higher pI value (Sonagra AD, 2025).

Table 1. The pKa and pI values for the ionizable groups of the amino acids were used to predict

their net charge (Z) at the buffer pH of 6.5 and 1.9.

Amino acid pKa (⍺- pKa (⍺- pKa (R- pI Net Charge at Net Charge at

COOH)1 N+H3)1 group)1 value1 pH 6.5 pH 1.93

Glutamate (G) 2.19 9.67 4.25 3.22 Negative Positive

Alanine (A) 2.34 9.69 - 6.02 Slight negative Positive

Lysine (L) 2.18 8.95 10.53 9.74 Positive Highly positive

3
Atfield & Morris, 1961, Analytical separations by high-voltage paper electrophoresis.
1
Luecker J. 2025. BIOL 4150: Biochemical Techniques I laboratory manual.

Pre-dominating ionic forms of amino acid at pH 6.5:

• Glutamate (pI 3.22): Side-chain carboxyl group is deprotonated, which gives it a negative

charge.

⁻OOC–CH2–CH2–CH(NH3⁺)–COO⁻

• Alanine (pI 6.02): Alanine is mainly zwitterionic (little excess of COO⁻ over NH3⁺)

⁻OOC–CH(NH3⁺)–CH3

• Lysine (pI 9.74): side-chain amine still protonated, which gave lysine a net positive charge

⁻OOC–CH(NH3⁺)–(CH2)4–NH3⁺
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Pre-dominating ionic forms of amino acid at pH 1.9:

• Glutamate (pI 3.22): all carboxyl and amino groups are protonated, which gives glutamate a

positive charge

HOOC–CH2–CH2–CH(NH3⁺)–COOH

• Alanine (pI 6.02): carboxyl and amino groups are protonated, making the net charge positive

HOOC–CH(NH3⁺)–CH3

• Lysine (pI 9.74): both α-amino and side-chain amino protonated, carboxyl protonated, which

makes lysine highly positive

HOOC–CH(NH3⁺)–(CH2)4–NH3⁺

Table 2. The data for electrophoretic mobilities (distance moved in cm) and movement direction

of amino acids and the unknown.

Amino acid Mobility at pH Mobility at pH Direction of Direction of

6.5 (cm) 1.9 (cm)3 movement(pH 6.5) movement (pH
1.9)3
Glutamate (G) 3.90 0.67 Anode (+ve) Cathode (-ve)
Mixture (M) 2.50 - Anode (+ve) -
Alanine (A) 1.95 1.00 Anode (+ve) Cathode (-ve)
Lysine (L) 5.10 1.47 Cathode (-ve) Cathode (-ve)
3
Atfield & Morris, 1961, Analytical separations by high-voltage paper electrophoresis.

Analysis at pH 6.5

The isoelectric point (pI) of Alanine (pI 6.02) is only slightly lower than the buffer pH.

Thus, Alanine carried a minimal net negative charge and migrated slowly toward the anode,

resulting in the smallest positive mobility (+1.95 cm). In contrast, Glutamate (pI 3.22) was

significantly deprotonated at pH 6.5 and possessed a strong net negative charge, which caused it

to move rapidly toward the anode (+3.90 cm). Conversely, Lysine (pI 9.74) was heavily
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protonated at pH 6.5, carried a strong positive charge and migrated rapidly toward the cathode (-

5.10).

The correlation between the pI value and net charge is described as an amino acid with as

low as a pI value(<7) moves as fast as towards the anode (+), whereas an amino acid with as

high as a pI value(>7) moves as fast as towards the cathode(-) at pH 6.5. The mobility of an

unknown mixture was +2.50 cm, which depicts that the pI value of the mixture falls between the

pI values of glutamate and alanine. The predictable amino acids for the mixture are leucine (pI

5.98), serine (pI 5.68), methionine (pI 5.74), cysteine (pI 5.07), and glycine (pI 5.97) obtained

(Luecker, 2025; Atfield & Morris, 1961).

Analysis at pH 1.9

At the highly acidic pH of 1.9, all amino acids were expected to be positively charged, as

the buffer pH was lower than the pI of all three amino acids. However, the primary separation

was not observed at this pH due to the faster movement of ions compared to pH 6.5. The

separation is predicted to be based on the magnitude of this positive charge. Lysine (pI 9.74),

being far from its pI, carried the highest positive charge due to three protonated groups, and

migrated the furthest toward the cathode (-1.47 cm). Glutamate (pI 3.22) and Alanine (pI 6.02)

carried less positive charge than lysine at this pH. Nonetheless, glutamate (-0.67 cm) and

alanine(-1.0 cm) still migrated towards the cathode (Atfield & Morris, 1961; Awapara, 1949).

The unknown was also predicted to move toward the anode (-0.89 cm) (Hames, 1998).

The mobility of charged molecules is also influenced by their size. However, size

differences are generally minimal compared to charge differences for small biomolecules like

amino acids. The use of larger proteins would make a more significant difference to the

separation.
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Discussion

The results at pH 6.5 strongly supported the idea that the electrical mobility of amino

acids depends on the relationship between buffer pH and the isoelectric point (pI). Glutamate

with a negative charge and neutral alanine moved toward the anode. In contrast, lysine, which

has a much higher pI of 9.74, was positively charged and migrated quickly toward the cathode.

These findings confirm published studies showing that amino acids with pI values lower than the

buffer pH move toward the anode and those with pI values above the buffer pH move toward the

cathode (Atfield & Morris, 1961; Sonagra et al., 2025). The unknown mixture migrated to an

intermediate position, suggesting it contained amino acids like serine, methionine, leucine, or

glycine, all of which have pI values around 6 (Luecker, 2025; Atfield & Morris, 1961).

However, the results at pH 1.9 were unexpected. At this low pH, all amino acids were

thought to be positively charged and should have moved toward the cathode. However, there

were no clear or distinct spots after ninhydrin staining. This may have happened because the

heavy protonation at pH 1.9 caused amino acids to move too fast, leaving the strip during the

run. Also, strong ionization masked charge differences and decreased resolution (Awapara, 1949;

Hames, 1998). Additionally, the ninhydrin reaction is less effective in highly acidic conditions,

which may have reduced visibility (Wilson & Walker 2000).

Several errors affected the quality of the results. Mistakes in buffer preparation could

change the effective pH and alter amino acid ionization. Uneven hydration of the paper,

excessive sample volume, pouring the excess buffer on the strip, and variations in drying times

might have caused diffusion. These (Atfield & Morris 1961; Dreyer & Bynum 1967). Other

possible reasons include poor buffer performance, decreased current flow, or heating effects,

which can affect migration (Dreyer & Bynum, 1967).

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Future improvements such as using freshly prepared buffers with accurate pH, use of an

ice bath tray to reduce heat generation, applying smaller and more concentrated sample volumes,

and adjusting the voltage or run time to keep amino acids from moving off the strip. Running

replicate strips with additional known amino acid standards would also help in identifying

unknowns. Using gel-based methods (Wilson & Walker, 2000) could also result in better

resolution and consistency compared to paper electrophoresis.

Overall, the experiment showed the expected principles at near-neutral pH while

highlighting significant challenges in acidic conditions. The lack of spots at pH 1.9 highlighted

the sensitivity of electrophoresis to buffer composition and sample handling. These results

highlight the need to improve experimental conditions to overcome challenges using high-

voltage paper electrophoresis for separating amino acids.

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References

1. Luecker J. 2025. BIOL 4150: Biochemical Techniques I laboratory manual. Fall 2025 ed.

Kamloops (BC): Thompson Rivers University; 152 p.

2. Wilson K, Walker J. 2000. Principles & techniques of practical biochemistry. 5th ed.

Cambridge (UK): Cambridge University Press. 784 p. ISBN 0-521-65873X.

3. Atfield G.N., Morris C. 1961. Analytical separations by high-voltage paper electrophoresis.

Amino acids in protein hydrolysates. Biochem J. 81(3):606-614.

[Link]

4. Awapara J. Application of paper chromatography to the estimation of some free amino acids

in tissues of the rat. J Biol Chem. 1949;179:243–253. [Link]

9258(18)56939-8

5. Dreyer WJ, Bynum E. 1967. High-voltage paper electrophoresis. In: Hirs CHW,

editor. Methods in Enzymology. Vol. 11. New York: Academic Press. p. 32–39.

[Link]

6. Hames, B. D., editor. Gel Electrophoresis of Proteins: A Practical Approach. 2nd ed. Oxford:

Oxford University Press; 1998.

7. Sonagra AD, Zubair M, Dholariya S. Electrophoresis. In: StatPearls [Internet]. Treasure

Island (FL): StatPearls Publishing; 2025 Jan, Available from:

[Link]
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Appendix

Figure 1. The resultant chromatogram strips typically illustrate the origin line and the final

position of the colored spots, with movement towards the anode (+ve) or cathode (-ve) indicated

at pH 6.5 for the phosphate buffer solution.

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Figure 2. The resultant chromatogram strips illustrate the expected origin line and the final

position of the colored spots, with movement towards the cathode (-ve) indicated at pH 1.9 for

the formic acid buffer solution (Atfield & Morris, 1961).

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Additional Lab Questions

Question-1

Using the data below, calculate the approximate charge on each of the following amino acids at

the stated pH. For each line, indicate the chosen charge by marking the appropriate box.

Amino acid pKa of ⍺-COOH pKa of ⍺-NH2 pKa of R-group

Glycine 2.3 9.6 -

Histidine 1.8 9.2 6.0

Tyrosine 2.2 9.1 10.1

Aspartate 2.0 9.9 3.9

Box 1 2 3 4 5 6 7

Charge +1½ +1 +½ 0 -½ -1 -1½

Line 1: Glycine at pH 12.0 → 6

Line 2: Histidine at pH 6.0 → 3

Line 3: Tyrosine at pH 6.0 → 4

Line 4: Aspartate at pH 6.0 → 6

Line 5: Histidine at pH 4.0 → 2

Line 6: Glycine at pH 6.0 → 4

Line 7: Aspartate at pH 7.4 → 6

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Question -2

Indicate which of the following are true or false:

Line 1: The pH meter tells us the concentration of free protons in solution: False

Line 2: The pKa value is a useful index of a functional group's ability to interact with changing

pH: True

Line 3: Each ⍺-amino acid has at least two reversible proton acceptors: True

Line 4: The calibration of pH meters means setting them to pH 7: False

Line 5: A solution that has a concentration of 0.1 M means 0.1 moles in a litre: True

Line 6: A solution which is 10% (w/v, weight/volume) contains 10 g in a 1000 ml: True