Learning Outcomes
1. To identify the R group in amino acids.
2. To determine the pH of various amino acids in water
3. To use chromatography to separate amino acids.
4. To calculate Rf value for amino acids
5. Use Rf values to identify amino acids
A. STRUCTURE OF AMINO ACIDS AND DIPEPTIDES
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B. pH OF AMINO ACIDS
Table 5.1 pH of amino acids
Sample pH
Glutamic Acid (1%) 4
Cystine 6
Alanine 6
Aspartic Acid 4
Lysine 5-6
The results of the pH determination showed that the amino acids tested exhibited different
pH values depending on the nature of their R groups. Acidic amino acids such as aspartic
acid and glutamic acid showed pH values around 4, indicating their acidic character. This
behavior is due to the presence of an additional carboxyl group (–COOH) in their side
chains, which ionizes easily and releases hydrogen ions (H⁺) into the solution.
Neutral amino acids such as alanine and cysteine showed pH values closer to neutrality,
around 6. Alanine has a nonpolar methyl group (–CH₃) as its side chain, which does not
donate or accept protons, resulting in little ionization. Cysteine, on the other hand,
contains a weakly polar sulfhydryl group (–SH) that does not ionize significantly under
neutral conditions, keeping its pH near neutrality.
Lysine and cystine showed pH values of about 5–6, which appeared slightly acidic.
However, theoretically, they would yield a basic pH around 8–10 due to the presence of
basic side chains containing amino groups (–NH₂) that can accept protons, increasing the
overall basicity of the solution
basic side chains containing amino groups (–NH₂) that can accept protons, increasing the
overall basicity of the solution.
C and D. PAPER and THIN-LAYER CHROMATOGRAPHY
The separation of amino acids depended on the polarity and ionization of their R groups.
Nonpolar side chains migrated farther with the mobile phase, while polar, acidic, or basic side
chains interacted more with the stationary phase and moved less, following the principle of
“like dissolves like.” The solvent system (butanol–acetic acid–water, 4:5:1) provided moderate
polarity, allowing both polar and nonpolar amino acids to separate based on their solubility
differences. In paper chromatography, cellulose acted as the stationary phase and separation
occurred by partition, while in thin-layer chromatography (TLC), silica gel acted as the
stationary phase and separation occurred by adsorption. Since amino acids are colorless,
ninhydrin was used to produce visible purple spots that allowed measurement of the migration
distance and calculation of Rf values.
Table 5.2 Chromatography Data of Glutamic Acid
Sample Chromatography Solvent Sample Rf
Type Distance Distance (Observed)
(mm) (mm)
Glutamic Acid Paper 59 12 0.203
TLC 37 22 0.594
Glutamic acid showed Rf values of 0.203 in paper chromatography and 0.594 in thin-
layer chromatography. The low Rf value in paper chromatography indicated limited migration
due to its acidic side chain containing an additional carboxyl group (–COOH), which increases
polarity and enhances attraction to the polar stationary phase. In TLC, the higher Rf value
reflected weaker interaction with the silica surface, allowing greater movement. The moderate
polarity of the solvent system supported partial migration of this highly polar amino acid.
Theoretically, glutamic acid should still yield a low Rf value because of its strong polarity.
Table 5.3 Chromatography Data of Cysteine
Sample Chromatography Solvent Sample Rf
Type Distance Distance (Observed)
(mm) (mm)
Cysteine Paper 50 30 0.600
TLC 29 25 0.862
Cysteine exhibited Rf values of 0.600 in paper chromatography and 0.862 in thin-layer
chromatography, indicating relatively high mobility with the solvent front. This behavior can
be explained by the presence of a weakly polar sulfhydryl (–SH) group in its side chain.
Although the –SH group can form hydrogen bonds, it is much less polar than hydroxyl (–OH)
or carboxyl (–COOH) groups, which allows cysteine to interact less with the polar stationary
phase and more with the mobile phase. As a result, cysteine traveled farther compared to amino
acids with more polar or ionizable side chains. The moderate polarity of the butanol–acetic
acid–water solvent system also enhanced its solubility and facilitated its migration.
Theoretically, cysteine should exhibit a moderate Rf value, since its polarity lies
between that of alanine and the more polar amino acids such as aspartic acid and glutamic
acid. However, the experimentally observed higher Rf value suggests that cysteine was less
retained than expected, possibly due to stronger solubility in the solvent mixture or reduced
adsorption to the stationary phase.
Table 5.4 Chromatography Data of Alanine
Sample Chromatography Solvent Sample Rf Theoretical
Type Distance Distance (Observed) Rf
(mm) (mm)
Alanine Paper 22 12 0.545 0.38
TLC 39 26 0.667 0.30
Alanine showed Rf values of 0.545 in paper chromatography and 0.667 in thin-layer
chromatography. These moderate Rf values indicate balanced interaction between the
stationary and mobile phases. Alanine’s nonpolar methyl group (–CH₃) does not form
hydrogen bonds, reducing its attraction to the polar stationary phase and allowing moderate
travel with the solvent. The butanol–acetic acid–water solvent system, with its moderate
polarity, supported this migration. Theoretically, alanine should yield a medium to high Rf
value, consistent with its weak polarity and nonpolar nature.
Table 5.5 Chromatography Data of Aspartic Acid
Sample Chromatography Solvent Sample Rf Theoretical
Type Distance Distance (Observed) Rf
(mm) (mm)
Aspartic Acid Paper 46 30 0.652 0.24
TLC 32 7 0.218 0.24
A spartic acid displayed Rf values of 0.652 in paper chromatography and 0.218 in thin-
layer chromatography. The lower Rf in TLC indicated strong retention on the silica surface due
to its high polarity and acidic nature. The additional carboxyl group (–COOH) in its side
chain allowed hydrogen bonding and ionic attraction to the stationary phase, limiting
movement. In paper chromatography, partial solubility in the moderately polar solvent allowed
greater movement. Theoretically, aspartic acid should show a low Rf value, as its strong
polarity keeps it closer to the origin
Table 5.6 Chromatography Data of Lysine
Sample Chromatography Solvent Sample Rf Theoretical
Type Distance (mm) Distance (Observed) Rf
(mm)
Lysine Paper 36 22.5 0.625 0.14
TLC 43 15 0.349 0.12
Lysine showed Rf values of 0.625 in paper chromatography and 0.349 in thin-layer
chromatography. These results indicated stronger retention on the silica surface compared to
paper, consistent with its basic amino side chain (–NH₂), which can accept protons and
increase polarity. The strong electrostatic attraction to the polar stationary phase reduced its
mobility, while the solvent’s moderate polarity allowed only partial migration. Theoretically,
lysine should exhibit a low to moderate Rf value, aligning with its basic and polar
characteristics.
Based on the TLC results, the amino acids ranked from highest to lowest mobility as
cysteine > alanine > glutamic acid > lysine > aspartic acid. Theoretically, according to
polarity and expected chromatographic behavior, the order should be alanine > cysteine >
glutamic acid > aspartic acid > lysine, since alanine and cysteine are less polar and therefore
travel farther than the more polar acidic and basic amino acids. Cysteine, containing a weakly
polar sulfhydryl (–SH) group, interacts less with the silica surface and moves moderately with
the solvent, while glutamic acid, aspartic acid, and lysine migrate shorter distances due to
stronger attraction to the polar stationary phase.
The difference between the observed and theoretical rankings may be due to solvent–
stationary phase interactions under the experimental conditions. The butanol–acetic acid–water
solvent may have enhanced cysteine’s solubility or reduced its adsorption, resulting in greater
mobility. Minor experimental factors such as uneven spotting, solvent contamination, or
incomplete chamber saturation could also have contributed to the deviation.
