OFFICIAL REPORT OF EXPERIMENT

LABORATORY : Biochemistry Laboratory

EXPERIMENT : Biochemistry
EXPERIMENT TITLE : Determination of Amino Acids
In Sample

By:
Name: Era Melania Reg. Number: 18030194085 Class: PKU 2018

Program/Department: S1 Chemistry Education/Chemistry

DEPARTMENT OF CHEMISTRY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
SURABAYA STATE UNIVERSITY
A. EXPERIMENT TITLE
Determination of Amino Acids in Sample
B. EXPERIMENT STARTED DATE
Wednesday, 15th September, 2021 at 13.00
C. EXPERIMENT FINISHED DATE
Wednesday, 15th September, 2021 at 15.30
D. EXPERIMENT PURPOSE
To determine the amino acids in sample using paper chromatography
E. BASIC THEORY
1. AMINO ACIDS
The term amino acid can mean any molecule containing an amino
group and any type of acid group; however, the term is almost always used
to refer to a carboxylic acid. The simplest acid is aminoacetic acid, which
is called glycine. Other common amino acids have side chains (denoted by
R) substituted at the carbon atom. For example, alanine is an amino acid
with a methyl side chain.

Amino acids are one of the 20 most common types of monomers

used in the formation of proteins. In general, amino acids are soluble in
water and insoluble in non-polar organic solvents such as ether, acetone,
and chloroform. These amino acids have different properties from
carboxylic acids and the properties of amines. Both aliphatic and aromatic
carboxylic acids consisting of several carbon atoms are generally less
soluble in water but soluble in organic solvents. Similarly, amines are
generally insoluble in water, but soluble in organic solvents [ CITATION
APo94 \l 1057 ].
Amino acids are compounds with one or more carboxyl groups
(−COOH) and one or more amino groups (−NH2) in the molecule. Amino
acids are joined by peptide bonds between the carboxyl group of the amino
acid and the amino group of the adjacent amino acid. The arrangement
between amino acids and the types of amino acids that make up protein, is
very specific and unique for each type of protein [ CITATION Sla89 \l 1057 ].
The amide bond between amino acids is known as a peptide bond,
and the product of the formation of a peptide bond between two amino
acids is called a dipeptide. The peptide chain can be extended to combine
three amino acids in a tripeptide, four in a tetrapeptide, and so on.
Polypeptides contain many amino acid units. Proteins are naturally
occurring polypeptides containing more than 50 amino acid units—most
proteins are polymers of 100-300 amino acids. What is most striking about
proteins is the variety of roles they play in living systems: silk, hair, skin,
muscle, and connective tissue are proteins, and nearly all enzymes are
proteins. As in most aspects of chemistry and biochemistry, structure is the
key to functioning. We will explore the structure of proteins by first
concentrating on their fundamental building block unit, the -amino acid.
Then, having developed the principles of peptide structure, we will see
how the insights gained from these smaller molecules help our
understanding of proteins [ CITATION FAC00 \l 1057 ].
Proteins are polymers of amino acids, with each amino acid residue
joined to its neighbors by a certain type of covalent bond. (The term
"residue" reflects the loss of water elements when one amino acid
combines with another.) Proteins can be broken down (hydrolyzed) into
their constituent amino acids by various methods, and the earliest studies
of naturally occurring proteins focused on free amino acids derived from
from them. Twenty different amino acids are usually found in proteins.
The common amino acids of proteins have been assigned a three-letter
abbreviation and a one-letter symbol. which is used as an abbreviation to
denote the composition and sequence of polymerized amino acids in
proteins [ CITATION ALL821 \l 1057 ].
2. NINHYDRIN TEST
The ninhydrin test occurs when ninhydrin is heated with amino
acids, forming a colored complex. Amino acids can be determined
quantitatively by using the intensity of the color formed is proportional to
the concentration of amino acids. In this reaction CO2 and NH4 are
released so that amino acids can be determined quantitatively by
measuring the amount of CO2 and NH3 released. The colored complex
formed contains two molecules of ninhydrin which react with the ammonia
released on the oxidation of amino acids. Positive test results on the
ninhydrin test are given to amino acids containing -amino acids and
peptides having free -amino groups. The following is the reaction between
ninhydrin and amino acids:
O
O

COOH
OH
N + RCHO + CO2 + 3H2O
+ H 2N C H

OH R
O O

Only the nitrogen atom of the purple substance comes from amino
acids, the rest is converted to aldehydes and carbon dioxide. So, purple dye
is produced from amino acids with primary amino groups, the intensity of
the color is directly proportional to the concentration of amino acids
present. Proline which has a secondary amino group reacts with ninhydrin
to produce a yellow color.
 Ninhydrin and Alanine
Alanine is an -amino acid used in protein biosynthesis. It
contains an amine group and a carboxylic acid group, both of
which are attached to a central carbon atom which also carries a
methyl group side chain. The reaction between ninhydrin and
alanine is:
 O

OH
O
H
C (aq) + +
H3N C C O- (aq)
OH
CH3
O

O O

N (aq) +

O O

H 3C CHO (aq) + CO2 (g) + 3H2O (l) + H+ (aq)

Ninhydrin reacts with alanine and through a complex reaction

forms a Ruhemann purple end product which can be easily
visualized [ CITATION Nic06 \l 1057 ].
 Ninhydrin and Glycine
Glycine is an amino acid that has one hydrogen atom as its
side chain. It is the simplest stable amino acid, with the chemical
formula NH2-CH2-COOH. Glycine is an integral part of the
formation of the alpha helix in the secondary protein structure
because of its compact shape.
 O

OH
O
H2
C (aq) + NH2 C C OH (aq)
OH

O O

N (aq) +

O O

HCHO (aq) + CO2 (g) + 3H2O (l)

Ninhydrin reacts with glycine and through a complex

reaction forms a Ruhemann purple final product which can be
easily visualized [ CITATION Men66 \l 1057 ].
 Ninhydrin and Tyrosine
Tyrosine is a non-essential amino acid with a polar side
group. Tyrosine is generally classified as a hydrophobic amino
acid.
 O

OH
O
H
C (aq) + NH3+ C C O- (aq)
OH OH2

OH

O O

N (aq) +

O O

H2
HO C CHO (aq) + CO2 (g) + 3H2O (l) + H+ (aq)

The amount of product formed in a reaction depends on

several factors including the concentration of the reactants, reaction
medium, pH and temperature. In the current study, a reaction
mechanism has been proposed for the study of ninhydrin and Tyr.
The reaction takes place through the attack of the lone pair of
electrons from the amino nitrogen (tyrosine) on the carbonyl group
(ninhydrin) to produce a Schiff base [ CITATION Dil19 \l 1057 ].
3. THIN LAYER CHROMATOGRAPHY
TLC is a separation technique using an absorbent (stationary phase)
in the form of a uniform thin layer coated on a flat surface in the form of a
glass plate, aluminum plate, or plastic plate. Chromatographic
development occurs when the mobile phase of the filter passes through the
absorbent [ CITATION Muk14 \l 1057 ].
Thin Layer Chromatography (TLC) is a form of planar
chromatography, in addition to paper chromatography and electrophoretic
chromatography. In contrast to column chromatography where the
stationary phase is filled or encapsulated within it, in TLC the stationary
phase is a uniform layer on a flat surface supported by a glass plate,
aluminum plate or plastic plate. However, planar chromatography is an
open form of column chromatography [ CITATION IGG12 \l 1057 ].
The advantages of using TLC are that it requires fast analysis time,
uses few tools, is simple, inexpensive, and has good analytical power
[ CITATION LKW12 \l 1057 ]. TLC is widely used for various analyzes of
medicinal plants. The resulting chromatogram is a pattern that describes
the compounds in each medicinal plant so that it is useful in controlling the
quality of drugs for both the characterization of raw materials and final
products.
The general use of TLC is for the purposes of:
1. Qualitative, which is based on the value of Rf which is defined as a
comparison of the creepage distance achieved by the compound with
the mobile phase. The Rf value is not always the same, therefore the
Rf value is used as:
 Direction of relative migration distance
 Mobile phase selection orientation for column chromatography
 Monitoring the results of column chromatography separation
2. Quantitative is the visual determination of the size of the spots
compared to the comparison compound or by the spectrophotometer
method or carried out by scraping, elution and spectroscopic methods.
Quantitative use to show the amount of each component of a mixture
relative to other components or absolute if appropriate standard of
reference or calibration is used.
3. Preparative/Analytical is to obtain the components of the mixture in
sufficient quantities in a pure state for other needs. The stationary
phase in TLC has several adsorbents used, including:
a. Silica gel is the most widely used absorbent and is slightly
acidic, so the acid is slightly easier to separate by minimizing
 the acid-base reaction between the adsorbent and the
compound being separated.
b. Alumina is slightly basic and is often used to separate bases by
minimizing acid-base reactions.
c. Cellulose is a buffer material for the liquid layer used in
Liquid-Liquid Chromatography (KCC), used to separate polar
compounds such as amino acids, carbohydrates, nucleotides
and various other hydrophilic compounds. For the detection of
separated compounds can be used various ways. The simplest
detection is if the compound exhibits absorption in the
shortwave UV region (254 nm) or if the compound can be
excited to longwave UV radial fluorescence (365 nm). If the
compound cannot absorb UV light, the detection can be done
using a chemical reaction either with heating or without
heating [ CITATION Sug97 \l 1057 ].

F. EQUIPMENTS AND MATERIALS

Equipments
  TLC plate 1 piece
 Capillary pipe 1 piece
 Beaker glass 3 pieces
 Volumetric flask 1 piece
 Oven 1 unit
 Wool yarn and clips 1 set
 Chamber 1 piece
 Petri dish 3 pieces
Materials
 Glacial acetic acid sufficiently
 n-butanol sufficiently
 Aquades sufficiently
 Standard amino acid solution sufficiently
 Sample solution sufficiently

G. PROCEDURE
Preparation of Eluent
25 mL of n-
butanol

- Added 6 mL of glacial acetic acid and shaked

- Added 25 mL of aquades and shaked
- Put in the chamber

Eluent solution

Reaction:
CH3CH2CH2CH2OH (l) + CH3COOH (l) →
CH3COOCH2CH2CH2CH3 (aq) + H2O (l)

Determination of Amino Acids Component

TLC Plate
 - Drawn 0,5 cm of the upper border, 1 cm of botttom border, 0,5
cm left and right border
- Given a sample point using pencil with a distance between
samples A, B, C, D is 1 cm
- Oven at 105-110oC for 1 mins
- Dripped 4 type of solution (A, B, C, D) side by side with
capillary pipe
- Dried by aerating before the next drop is placed on it
- The maximum stain size id 0,4 cm
- Hung in the chamber to be saturated with eluent steam until the
eluent reaches the upper border

TLC plate that has

been treated with
eluent

- Taken out
- Oven at 105 -110oC for 5 mins
- Sprayed with ninhydrin
- Oven at 100-105oC for 3 mins
- Marked with pencil

TLC plate with

amino acid stain
- Noted the color
- Calculated the Rf value for each stain
- Determined the amino acids component in the solution
- Compared the Rf value with the Rf value of standard amino acid

Amino acids
component

Formula: Rf = the distance taken by the sample / the distance taken by eluent

Reaction:
O

COOH
OH
+ H2N C H

OH R
O
O

N + RCHO + CO2 + 3H2O

O
H. OBSERVATION RESULT
No Observation Result
Procedures Before After Hypothesis/Reaction Conclusion
.
1. Preparation of Eluent Solution  n-butanol: n butanol + Reaction: CH3CH2CH2CH2OH There is
colorless glacial acetic (l) + CH3COOH (l) → esterification
solution acid + aquades: CH3COOCH2CH2CH2CH3 (aq) + reaction
 glacial colorless H2O (l) produce. The
acetic acid: solution Properties: product of this
colorless  n-butanol: semi polar esterification
solution  glacial acetic acid: non polar reaction is
 aquades:  aquades: polar buthyl acetate.
colorless  eluent: semipolar
solution  So, polarity level is aquades
> n-butanol > glacial acetic
acid
No Observation Result
Procedures Hypothesis/Reaction Conclusion
Before After
.
2.  A solution  Dropped 4 Rf value based on theory: The sample is
(alanine): kinds of Rf alanine = 0,38 amino glycine.
colorless solution (A, Rf glycine = 0,26 Based on the
solution B, C, D): Rf tyrosine = 0,45 calculation, the
 B solution stains not Reaction of ninhydrin with sample Rf is
(glycine): visible alanine 0.26. That is,
colorless  Hanging in the Rf value of
solution the the sample is
 C solution chamber: the same as the
(tyrosine): eluent rises Rf value of
colorless to the upper glycine based
solution limit on the theory.

 D solution  Dry at 105 Reaction of ninhydrin with The Rf value of

(sample): ℃ for 1 glycine sample B is the

colorless minute: dry same as D, so it

solution TLC can be

 Ninhydrin  Spry it with concluded that

sample D is the
No Observation Result
Procedures Hypothesis/Reaction Conclusion
Before After
.
solution: ninhydrin, amino acid
colorless and dry at glycine.
solution 100-105℃
for 3
minutes:
Visible
Reaction of ninhydrine with
stains
tyrosine
 Stain of A:
red
 Stain of B:
yellow
 Stain of C:
purplish red
 Stain of D:
yellow
I. ANALYSIS AND EXPLANATION
The purpose of this experiment is to determine the amino acid content
in the sample using TLC (Thin Layer Chromatography). Chromatography is
based on the principle in which molecules in a mixture are applied to a
surface or into a solid, and the stationary liquid phase (stable phase) separates
from each other while moving with the help of the mobile phase. Factors that
are effective in this separation process include molecular characteristics
related to adsorption (liquid-solid), partition (liquid-solid), and affinity or
differences in molecular weight. Because of this difference, some components
of the mixture stay longer in the stationary phase, and they move slowly in
the chromatographic system, while others enter the mobile phase more
quickly, and leave the system more quickly [ CITATION Ozl16 \l 1057 ].
1. Preparation of Eluent
The purpose of the first experiment is to make eluen for TLC.
The first step, 25 mL of n-butanol which is colorless solution is taken
into a beaker glass. Then, into the beaker glass added 6 mL of glacial
acetic acid which is colorless and has strong odor. Glacial acetic acid
is acetic acid which does not contain water or pure acetic acid liquid
or acetic acid anhydrous. Then, added 25 mL of aquades and shaked.
At this step an esterification reaction occurs between n-butanol and
acetic acid which will produce n-butyl acetate or butyl ethanoate as
the eluent. When carboxylic acid reacted with alcohol, they produce
an ester. The reaction is:
CH3COOH (aq) + CH3CH2CH2CH2OH (aq) 
CH3COOCH2CH2CH2CH3 (aq) + H2O (l)
The selection of n-butanol as the materials of making eluent is
based on the differences of the polarity. There are three type of
polarity that are aquades is a polar compound, n-butanol is a semi
polar compound, and glacial acetic acid is a non polar compound. The
selection of the three differences of polarity because amino acids also
has a different characteristic of the polarity. So that, the amino acids
can go up when spotted in the TLC plate.
 After that, the mixture is put inti the chamber and waited for an
hour an a half. The purpose of this step is to make the esterification
reaction perfectly complete because the esterification reaction is took
time. When the esterification reaction is finished, it can be identified
by the appearance of a distinctive pungent odor (banana scent). If the
odor can't smell, it can be ascertained that the esterification reaction
has not been completed perfectly.
2. Determination of Amino Acids Component
The purpose of the second experiment is to determine the amino
acids in the sample. The amino acid samples using in this experiment
is alanine, glycine, and tyrosine. The Rf standard of alanine is 0,277.
Then the Rf standard of glycine is 0,246 and tyrosine is 0,539
[ CITATION Tag16 \l 1057 ].
The first step is to draw the upper border about 0,5 cm from the
upper side of the plate, the the bottom border about 1 cm from the
bottom side of the plate and about 0,5 cm from left and right side f the
plate using pencil. The function of using a pencil is because the pencil
does not interfere with the movement of the sample. It's different if we
use a pen. Pen ink may interfere with sample movement. If the sample
movement is interrupted, the Rf value is inaccurate, so the sample
content cannot be accurately determined.
Then the next step is give a sample point using pencil with a
distance between each sample is about 1 cm. Then oven the plate at
105-110oC for 1 minutes. This step has purpose to remove water
molecules so that the absorption process is not disturbed. In addition,
it is also used to open the pores of the TLC plate made of silica so that
when spotting the amino acid samples, it can can enter the pores.
Next step is dripped 4 types of solution which is solution A is
alanine, solution B is glycine, solution C is tyrosine, and solution D is
a unknown sample solution with capillary pipe and the maximum
stain diameter is 0,4 cm. Wait until the sample is dried and added the
sample again. Then, put the plate into the chamber until the plate
touches the bottom of the plate using tweezers. Next, close the
chamber and wait until the mobile phase touches the top of the plate.
Then, take out the plate and put into the oven at 105-110 oC for 5
minutes. This step has purpose is to remove the remaining eluent on
the TLC plate. The eluent on the TLC plate must be removed because
when it sprayed with ninhydrin, no reaction will occur. Then put into
oven again at 100-105oC for 3 minutes. This third oven is intended
because the reaction of ninhydrin and amino acids will occur at hot
temperatures. ninhydrin acts as an oxidizing agent. and amino acids
will be oxidized so that the ninhydrin will bind to the amine group to
form a complex compound and this complex compound will give the
color spots on the TLC plate.
Reaction between ninhydrin and amino acid:
O

COOH
OH
+ H2N C H

OH R
O

N + RCHO + CO2 + 3H2O

Reaction between ninhydrin and alanine:

O

OH
O
H
C (aq) + +
H3 N C C O- (aq)
OH
CH3
O
 O O

N (aq) +

O O

H3 C CHO (aq) + CO2 (g) + 3H2O (l) + H+ (aq)

Reaction between ninhydrin and glycine:

O

OH
O
H2
C (aq) + NH2 C C OH (aq)

OH

O O

N (aq) +

O O

HCHO (aq) + CO2 (g) + 3H2O (l)

Reaction between ninhydrin and tyrosine:
O

OH
O
H
C (aq) + NH3+ C C O- (aq)
OH OH2

OH

O O

N (aq) +

O O

H2
HO C CHO (aq) + CO2 (g) + 3H2O (l) + H+ (aq)

After that, we can calculate the Rf value based on the stain that
appear in the plat. Use the following formula:
the distance takenby the sample
Rf =
the distance takenby eluent
From the calculation, the Rf value of sample A (alanine), sample B
(glycine), sample C (tyrosine), and sample D are:
1 cm
Rf Alanine = = 0,29
3,5 cm
 0,9 cm
Rf Glycine = = 0,26
3,5 cm
1,7 cm
Rf Tyrosine = = 0,48
3,5 cm
0,9 cm
Rf Sample D = = 0,26
3,5 cm

J. CONCLUSION
The sample is the amino acid glycine, because based on the calculation,
the Rf value of sample is 0.26. This means that it is close to the R f value of
glycine. The Rf value of sample B is the same as sample D so it can be
concluded that sample D is the amino acid glycine.

K. REFERENCES
Carey, F. A. (2000). Organic Chemistry. USA: Mc. Graw Hill.

Coskun, O. D. (2016). Separation Techniques: Chromatography. North Clin

Istanbul, 156-160.

Friedman, M., & Sigel, C. W. (1966). A Kinetic Study of the Ninhydrin

Reaction. Journal of Biochemistry, 478-485.

Gandjar, I. G., & Rohman, A. (2012). Analisis Obat Secara Spektrofotometri

dan Kromatografi. Yogyakarta: Pustaka Pelajar.

Hudaib, T., Brown, S., Wilson, D., & Eady, P. E. (2016). Identification of
Free Amino Acids in Several Crude Extracts of Two Legumes Using
Thin-Layer Chromatography. Journal of Planar Chromatography, 145-
146.

Kumar, D., & Rub, M. A. (2019). Study of the Reaction of Ninhydrin with
Tyrosine in Gemini Micellar Media. Journal RSC Advances, 22129-
22136.

Lehninger, A. L. (1982). Dasar Dasar Biokimia. Jakarta: Erlangga.

Mukhriani. (2014). Ekstraksi, Pemisahan Senyawa dan Identifikasi Senyawa

Aktif. Jurnal Kesehatan, 361-367.
Petraco, N. D., Proni, G., & Jackiw, J. (2006). Amino Acid Alanine
Reactivity with the Fingerprint Reagent Ninhydrin. Journal Forensic
Science, 1267-1275.

Poedjiadi, A. (1994). Dasar Dasar Biokimia. Jakarta: Universitas Indonesia.

Sudarmadji, S. (1989). Analisis Bahan Makanan dan Pertanian. Yogyakarta:

Universitas Gadjah Mada.

Suganda. (1997). Kromatografi Lapis Tipis. Temu Ilmiah Nasional Bidang

Farmasi. Bandung: ITB.

Wardhani, L. K., & Nanik, S. (2012). Uji Aktivitas Antibakteri Ekstrak Etil
Asetat Daun Binahong (Anredera scandens (L.) Moq.) terhadap
Shigella flexneri Beserta Profil Kromatografi Lapis Tipis. Jurnal
Ilmiah Kefarmasian, 1-16.
L. ANSWER OF THE QUESTION
1. What are the advantages and disadvantages in the paper chromatography
separation method?
Answer:
Advantage:
 In paper chromatography the equipment used does not need
complicated tools. Good results can be obtained with very simple
equipment and materials.
 Separate compounds can be detected on paper and can be
immediately identified directly.
 The cost required is relatively cheaper when compared with other
separation techniques.
 Preparative paper chromatography requires a larger paper than for
analysis. The advantage is that the load arm of the Rf number
becomes large so that the measurement of R f is a valuable
parameter in exposing new compounds.
Disadvantage:
 Cannot conduct quantitative analysis on sample components, only
limited to qualitative analysis.
 The time is longer than other adsorbents, but shorter than TLC.
 Cannot use H2SO4 reagent because cellulose will decompose.
2. Can chromatography methods be used for quantitative analysis?
Answer :
The paper chromatography method can be used for both quantitative and
qualitative analysis. Quantitative analysis was carried out based on the
comparison of the Rf of the sample substance with the Rf value of the
standard substance. In order for quantitative analysis to run well, the
following points must be considered:
 The trial conditions must be the same, because the value of Rf
depends on these conditions.
 Must try with different solvents.
  The presence of a stain on the chromatogram does not mean the
presence of a single substance in the sample.
3. What are the factors that affect the Rf value?
Answer :
 Solvent. This is due to the importance of the partition coefficient,
so that very small changes in solvent composition can cause
changes in the Rf value
 The presence of other ions, for example the presence of chloride in
the separation carried out with a nitrate solution
 Mixed properties. Various compounds are partitioned between
equal volumes of the fixed phase and the mobile phase. The nature
of the mixture almost always affects the solubility characteristics of
one another and also affects the value of Rf
 Paper. The main effect of paper on the Rf value arises from changes
in ions and absorption, which are different for different types of
paper. Paper affects the flow rate and affects the balance of the
partition
 Temperature. Changes in temperature change the partition
coefficient and flow velocity
 Size of the vessel. The volume of the vessel affects the
homogeneity of the atmosphere, thereby affecting the evaporation
rate of the solvent component of the paper. If a large vessel is used,
there is a tendency to propagate longer, as the solvent composition
changes along the paper, then the partition coefficient will change
as well. Two factors, namely evaporation and composition affect
the Rf value.
 Adsorbent quality
 Layer thickness. The thicker the Rf layer, the smaller
 Room chromatography saturation (chamber chromatography).
M. DOCUMENTATION ATTACHMENT
Documentation Description
Preparation of tools and materials

Making a scavengers; 25 ml n-
butanol

Making a scavengers ; 25 ml n-
butanol + 6 ml acetic acid

Making a scavengers; 25 ml n-
butanol + 6 ml acetic acid + 25 ml
aquades

Placed in a chamber
TLC plates that have been
bordered

Sample’s spotty process

The process of oven at a

temperature of 105-110 °C

Hanged in a chamber to be
saturated

Oven it returns with a temperature

of 105-110 °C
Sprayed with ninhydrin

Oven it returns with a temperature

of 105-110 °C

Sample color stain results

N. CALCULATION ATTACHMENT
Sample A (Alanine)
1 cm
Rf Alanine = = 0,29
3,5 cm
Sample B (Glycine)
0,9 cm
Rf Glycine = = 0,26
3,5 cm
Sample C (Tyrosine)
1,7 cm
Rf Tyrosine = = 0,48
3,5 cm
Sample D
0,9 cm
Rf Sample D = = 0,26
3,5 cm
O. HANDSWRITTING ATTACHMENT
Procedure
Interim Report