A.

Experiment title : Determining Amino Acid Type In A Sample

B. Experiment Date : Wednesday, October 10th 2018 (09.30-12.00)
C. Experiment Purpose :
 Determining the vitamin C level of Papaya
D. Basic Theory
E. Amino acids are basic unit of protein. Amino acids contain an amino group and a
carboxylic group. Amino acids play major role in regulating multiple processes
related to gene expression, including modulation of the function of the proteins
that mediate messenger RNA (mRNA) translation (Scot et al., 2006). Amino acids
are utilized in formation of protein. If amino acids are deficient, then protein
synthesis does not occur. As a result protein deficiency disease may occur. It is
necessary to take balanced diet containing all essential amino acids. Specific
amino acids are known to acutely and chronically regulate insulin secretion from
pancreatic β-cells in vivo and in vitro (Lorraine et al., 2006). Amino acids are
categorized as acidic, basic and neutral amino acids. Some amino acids are not
synthesized in the body and it is necessary to take them in diet. Such types of
amino acids are called essential amino acids. Some amino acids are synthesized
in the body and there is no needs to take them in diet, such type of amino acids
are called non essential amino acids. Some amino acids are synthesized in the
body but their production is insufficient such type of amino acids are called
semi-essential amino acids (Hellwinkel, 2001; IUPAC-IUB, 1968). Amino acids
help in tissue protein formation. Some amino acids are involved in enzyme
formation. Hormones like insulin, growth hormone and glucagon are made up of
amino acids. Adrenaline, nor-adrenaline and thyroxin are made up of single
amino acid. Glutathione, a physiologically active peptide is also made up of
amino acids. Amino acids are involved in synthesis of melanin. It has been
studied that amino-acid balance in cancer patients often differs from that in
healthy individuals, because of metabolic changes (Jun et al., 2010). In liver
cirrhosis functions of dendritic cells (DCs) are impaired and cirrhotic patients
may show decreased levels of plasma branched-chain amino acids (Eiji et al.,
2007).
Glutamic acid It is an acidic amino acid. It helps in synthesis of glutathione. It is
converted to alpha ketoglutaric acid in transamination reactions.
GABA It is called gamma amino butyric acid. It is formed from glutamic acid. It is
a neurotransmitter.
Tyrosine Tyrosine is usually used in synthesis of thyroid hormones. It is also
utilized to form Dihydroxyphenylalanine, noradrenaline and adrenaline. Cysteine
It is a sulpher containing amino acid. It is obtained from methionine. Dopa Dopa
is a neurotransmitter. It is converted to dopamine. Dopamine deficiency results
in parkinsonism. Tryptophan Tryptophan is produced from Niacin. Niacin
deficiency results in pellagra. Proline Proline is an amino acid that is
hydroxylated to hydroxyproline in presence of vitamin c. If vitamin c deficiency
occurs. It leads to a disease condition called Scurvy. Scurvy is characterized by
swollen gums and bleeding upon pressing the gums.
Glycine Glycine is helpful in formation of bile acids. It combines with colic acid to
form glycocholate. It converts benzoic acid to hippuric acid in the liver. It is also
a component of glutathione. It is utilized in biosynthesis of creatine, heme and
purines. Citrulline It is a necessary component of urea cycle where it is formed
from ornithine and carbamoyl phosphate in presence of an enzyme called
ornithine transferase. Citrulline combines with aspartate and form
arginosuccinate in urea cycle. Ornithine It is also a component of urea cycle. It is
formed from arginine by action of arginase. During this reaction urea synthesis
occurs and carbon dioxide is produced.
Essential amino acids Non essential amino Special amino acids
acids
Lysine Cysteine GABA

Methionine Tyrosine DOPA

Valine Serine Citrulline

Tryptophan Alanine Ornithine

Isoleucine Asparagines Taurine

Histidine Aspartic acid

Phenylalanine Glutamic acid

Threonine Glycine

Leucine Hydroxylysine

Arginine Proline

AMINO ACIDS IN PLANTS Amino acids are present in plant and form protein.
Plants synthesize amino acids from the carbon and oxygen that is obtained from
air and hydrogen from water in the soil. Amino acids play important role to
increase yield and overall quality of crops. Amino acids are absorbed through
stomas in plants. It has been observed that amino acids influence the
physiological activities of the plant. Plant mutants for amino acid transporter
genes are now being used to study the physiological functions of many of the
cloned genes (Wolf et al., 1998).
Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture for
qualitative and quantitative analysis. Proteins can be purified based on
characteristics such as size and shape, total charge, hydrophobic groups present
 on the surface, and binding capacity with the stationary phase. Four separation
techniques based on molecular characteristics and interaction type use
mechanisms of ion exchange, surface adsorption, partition, and size exclusion.
Other chromatography techniques are based on the stationary bed, including
column, thin layer, and paper chromatography. Column chromatography is one
of the most common methods of protein purification.

Chromatography is based on the principle where molecules in mixture applied

onto the surface or into the solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid of a mobile phase. The
factors effective on this separation process include molecular characteristics
related to adsorption (liquid-solid), partition (liquid-solid), and affinity or
differences among their molecular weights [1, 2]. Because of these differences,
some components of the mixture stay longer in the stationary phase, and they
move slowly in the chromatography system, while others pass rapidly into mobile
phase, and leave the system faster [3].
Based on this approach three components form the basis of the chromatography
technique.

 Stationary phase: This phase is always composed of a “solid” phase or “a layer of

a liquid adsorbed on the surface a solid support”.
 Mobile phase: This phase is always composed of “liquid” or a “gaseous
component.”
 Separated molecules

The type of interaction between stationary phase, mobile phase, and substances
contained in the mixture is the basic component effective on separation of
molecules from each other. Chromatography methods based on partition are very
effective on separation, and identification of small molecules as amino acids,
carbohydrates, and fatty acids. However, affinity chromatographies (ie. ion-
exchange chromatography) are more effective in the separation of
macromolecules as nucleic acids, and proteins. Paper chromatography is used in
the separation of proteins, and in studies related to protein synthesis; gas-liquid
chromatography is utilized in the separation of alcohol, esther, lipid, and amino
groups, and observation of enzymatic interactions, while molecular-sieve
chromatography is employed especially for the determination of molecular
weights of proteins. Agarose-gel chromatography is used for the purification of
RNA, DNA particles, and viruses [4].
Stationary phase in chromatography, is a solid phase or a liquid phase coated on
the surface of a solid phase. Mobile phase flowing over the stationary phase is a
gaseous or liquid phase. If mobile phase is liquid it is termed as liquid
chromatography (LC), and if it is gas then it is called gas chromatography (GC).
Gas chromatography is applied for gases, and mixtures of volatile liquids, and
solid material. Liquid chromatography is used especially for thermal unstable, and
non-volatile samples [5].
The purpose of applying chromatography which is used as a method of
quantitative analysis apart from its separation, is to achive a satisfactory
separation within a suitable timeinterval. Various chromatography methods have
been developed to that end. Some of them include column chromatography, thin-
layer chromatography (TLC), paper chromatography, gas chromatography, ion
exchange chromatography, gel permeation chromatography, high-pressure liquid
chromatography, and affinity chromatography [6].

Types of chromatography

 Column chromatography
 Ion-exchange chromatography
 Gel-permeation (molecular sieve) chromatography
 Affinity chromatography
 Paper chromatography
 Thin-layer chromatography
 Gas chromatography
 Dye-ligand chromatography
 Hydrophobic interaction chromatography
 Pseudoaffinity chromatography
 High-pressure liquid chromatography (HPLC)

Column chromatography
Since proteins have difference characteristic features as size, shape, net charge,
stationary phase used, and binding capacity, each one of these characteristic
components can be purified using chromatographic methods. Among these
methods, most frequently column chromatography is applied. This technique is
used for the purification of biomolecules. On a column (stationary phase) firstly
the sample to be separated, then wash buffer (mobile phase) are applied (Figure
1). Their flow through inside column material placed on a fiberglass support is
ensured. The samples are accumulated at the bottom of the device in a tme-, and
volume-dependent manner [7].

Ion- exchange chromatography

Ion- exchange chromatography is based on electrostatic interactions between
charged protein groups, and solid support material (matrix). Matrix has an ion
load opposite to that of the protein to be separated, and the affinity of the protein
to the column is achieved with ionic ties. Proteins are separated from the column
either by changing pH, concentration of ion salts or ionic strength of the buffer
solution [8]. Positively charged ion- exchange matrices are called anion-exchange
matrices, and adsorb negatively charged proteins. While matrices bound with
negatively charged groups are known as cation-exchange matrices, and adsorb
positively charged proteins (Figure 2) [9].

Gel- permeation (molecular sieve) chromatography

The basic principle of this method is to use dextran containing materials to
separate macromolecules based on their differences in molecular sizes. This
procedure is basically used to determine molecular weights of proteins, and to
decrease salt concentrations of protein solutions [10]. In a gel- permeation column
stationary phase consists of inert molecules with small pores. The solution
containing molecules of different dimensions are passed continuously with a
constant flow rate through the column. Molecules larger than pores can not
permeate into gel particles, and they are retained between particles within a
restricted area. Larger molecules pass through spaces between porous particles,
and move rapidly through inside the column. Molecules smaller than the pores are
diffused into pores, and as molecules get smaller, they leave the column with
proportionally longer retention times (Figure 3) [11]. Sephadeks G type is the
most frequently used column material. Besides, dextran, agorose, polyacrylamide
are also used as column materials [12].

Affinity chromatography
This chromatography technique is used for the purification of enzymes, hormones,
antibodies, nucleic acids, and specific proteins [13]. A ligand which can make a
complex with specific protein (dextran, polyacrylamide, cellulose etc) binds the
filling material of the column. The specific protein which makes a complex with
the ligand is attached to the solid support (matrix), and retained in the column,
while free proteins leave the column. Then the bound protein leaves the column
by means of changing its ionic strength through alteration of pH or addition of a
salt solution (Figure 4) [14].

Paper chromatography
In paper chromatography support material consists of a layer of cellulose highly
saturated with water. In this method a thick filter paper comprised the support,
and water drops settled in its pores made up the stationary “liquid phase.” Mobile
phase consists of an appropriate fluid placed in a developing tank. Paper
chromatography is a “liquid-liquid” chromatography [15].

Thin-layer chromatography
Thin-layer chromatography is a “solid-liquid adsorption” chromatography. In this
method stationary phase is a solid adsorbent substance coated on glass plates. As
adsorbent material all solid substances used. in column chromatography (alumina,
silica gel, cellulose) can be utilized. In this method, the mobile phase travels
upward through the stationary phase The solvent travels up the thin plate soaked
with the solvent by means of capillary action. During this procedure, it also drives
the mixture priorly dropped on the lower parts of the plate with a pipette upwards
with different flow rates. Thus the separation of analytes is achieved. This upward
travelling rate depends on the polarity of the material, solid phase, and of the
solvent [16].
In cases where molecules of the sample are colorless, florescence, radioactivity or
a specific chemical substance can be used to produce a visible coloured reactive
product so as to identify their positions on the chromatogram. Formation of a
visible colour can be observed under room light or UV light. The position of each
molecule in the mixture can be measured by calculating the ratio between the the
distances travelled by the molecule and the solvent. This measurement value is
called relative mobility, and expressed with a symbol Rf. Rf. value is used for
qualitative description of the molecules [17].

Gas chromatography
In this method stationary phase is a column which is placed in the device, and
contains a liquid stationary phase which is adsorbed onto the surface of an inert
solid. Gas chromatography is a “gas-liquid” chromatography. Its carrier phase
consists of gases as He or N2. Mobile phase which is an inert gas is passed through
a column under high pressure. The sample to be analyzed is vaporized, and enters
into a gaseous mobile phase phase. The components contained in the sample are
dispersed between mobile phase, and stationary phase on the solid support. Gas
chromatography is a simple, multifaceted, highly sensitive, and rapidly applied
technique for the extremely excellent separation of very minute molecules. It is
used in the separation of very little amounts of analytes [18].

Dye- ligand chromatography

Development of this technique was based on the demonstration of the ability of
many enzymes to bind purine nucleotides for Cibacron Blue F3GA dye [19]. The
planar ring structure with negatively charged groups is analogous to the structure
of NAD. This analogy has been evidenced by demonstration of the binding of
Cibacron Blue F3GA dye to adenine, ribose binding sites of NAD. The dye
behaves as an analogue of ADP-ribose. The binding capacity of this type
adsorbents is 10–20-fold stronger rhat that of the affinity of other adsorbents.
Under appropriate pH conditions, elution with high-ionic strength solutions, and
using ion-exchange property of adsorbent, the adsorbed proteins are separated
from the column [20, 21].

Hydrophobic interaction chromatography (HIC)

In this method the adsorbents prepared as column material for the ligand binding
in affinity chromatography are used. HIC technique is based on hydrophobic
interactions between side chains bound to chromatography matrix [22, 23].

Pseudoaffinity chromatography
Some compounds as anthraquinone dyes, and azo-dyes can be used as ligands
because of their affinity especially for dehydrogenases, kinases, transferases, and
reductases The mostly known type of this kind of chromatography is immobilized
metal affinity chromatography (IMAC) [24].

High-prssure liquid chromatography (HPLC)

Using this chromatography technique it is possible to perform structural, and
functional analysis, and purification of many molecules within a short time, This
technique yields perfect results in the separation, and identification of amino
acids, carbohydrates, lipids, nucleic acids, proteins, steroids, and other
biologically active molecules, In HPLC, mobile phase passes throuıgh columns
under 10–400 atmospheric pressure, and with a high (0.1–5 cm//sec) flow rate. In
this technique, use of small particles, and application of high presure on the rate of
solvent flow increases separation power, of HPLC and the analysis is completed
within a short time.
Essential components of a HPLC device are solvent depot, high- pressure pump,
commercially prepared column, detector, and recorder. Duration of separation is
controlled with the aid of a computerized system, and material is accrued [25].

Application areas of chromatography in medicine

Chromatography technique is a valuable tool for biochemists, besides it can be
applied easily during studies performed in clinical laboratories For instance, paper
chromatography is used to determine some types of sugar, and amino acids in
bodily fluids which are associated with hereditary metabolic disorders. Gas
chromatography is used in laboratories to measure steroids, barbiturates, and
lipids. Chromatographic technique is also used in the separation of vitamins, and
proteins.

F. TOOLS AND MATERIALS

Tools :
1. Mortar 1 piece
2. Volumetric Flask 1 piece
3. Erlenmeyer 3 pieces
4. Buret 1 set

Materials :

1. I2 Solutions Sufficiently
2. Aquades Sufficiently
3. Amylum Solution Sufficiently
4. Papaya 10 grams

G. LANES WORK
1. Blanco Titration

20 mL Aquadest

- Poured into Erlenmeyer

- Added with 3 drops of 1% starch

- Titrated with standard iodine 0,01 N solution

Volume of I2
 2. Reactions with Alkali

10 grams of
Papaya
- Peeled and weighed as much as 10 grams

- Crushed with mortar until get slurry

- Put into 100 mL volumetric flask

- Added aquadest to the boundary max

- Waited 15 minutes while sometimes being

shaken slowly

- Filtered it

Filtrate
Residue
- Taken filtrate as much as 10 mL

- Put into Erlenmeyer

- Added 3 drops 1% starch

- Added 20 mL aquadest

- Titrated with standard iodine 0,01 N solution

Volume of I2
 H. Result Of The Experiment

Exp. Experiment Procedure Observation Result Hypothesis / Reaction Conclusion

No
Before After

1. Making emulsifying solution - N- butanol : - N-butanol + Based on experiment

colorless glacia acetic the amino acid pf the
(aq)
25 mL n-butanol - Glacial acetic acid : sample is cysteine
acid : colorless colorless ℎ𝑥
- Added 6 ml of glacial acetic acid a. Rf sample = ℎ𝑜
(glacial acetic acid)
- Added 25 ml of aquadest - Aquadest : - (+) aquadest 1,2 𝑐𝑚
- Mixed with shaken = 8,5 𝑐𝑚 = 0,141
colorless : colorless (aq)
- Placed in chromatography thank ℎ𝑥
b. Rf C = =
- ℎ𝑜
Eluent (N-butanol )
1,1 𝑐𝑚
= 0,129
8,5 𝑐𝑚
ℎ𝑥
c. Rf A = ℎ𝑜 =
1,9 𝑐𝑚
= 0,225
8,5 𝑐𝑚

(ester)

+ H2O
2. Determinig of Amino Acid Sample - Chromatograph - Dripped The value of sample is

Crhomatograph paper 4x10 cm paper : white solution 0,141, which is the

paper A,B,C,D : type of amino acid in
- Dropped 4 kind of solution ABCD - Eluent : stain is not sample is cycteine
side by side with distance 1 cm cololress visible
from each abd 0,5 from the of (Nynhidrin)
solution - (+) put into
solution
- Each droplet to be dried before - Histidine : eluen, until
next drop placed on it colorless eluen wet
- The large of mark dont untl 0,4 cm solution all parts of
- Hung up the paper in the
chromatpgraphy cabinet for some - Glysin : TLC : stain
hour colorless is not
(Amino acids)
- Observed solution visible
- Taken out form chromatography
- Cysteine : - (+) put into
cabinet after eluen each upper limit
- Dried the paper at 105-110℃ for 5 colorless oven : stain
mins solution not visible
- Dried the paper for 1 min in oven
- Sample : turbid - sprayed
solution with (ruheman blue)
Amino acid stain
ninhydrin
- calculated Rf of each stain and
writen the color of stain
the Rf Order of polarity level :
Rf of each amino acids
Value :
a. Rf A : Aquadest > n-butanol>
0,226 glacal acetic acids
b. Rf C :
 Rf amino acids based on
0,130
theories:
c. Rf D
Cysteine = 0,4
(sample)
Histidine = 0,11
: 0,142
Glysin = 0,26
I. Analysis And Explanation
1. Making of emulsifying media
Eluen of emulsifying solution consists of 3 types of solution. They are
water, n-buthanol, and acetate acid. Those 3 solutions having different level of
polarity. Where Water>n-buthanol>acetate acid order of polarity characteristic.
The reason why using 3 kinds of solutions is used to solute the amino acid based
on its solvent. Because every amino acid has different solubility of solution. There
is sample of amino acid that can solute in water, but also there is another sample
that can dilute in unipolar or semi-polar solvent. So the eluen as moving phase of
chromatography. Every amino acid has its different solvent type. So, when the 3
samples of amino acid is tested it will move upward based on its solvent type.
Characteristic of emulsifying media is folatil. It means it’s easy to
evaporate. This characteristic giving positive impact to the chromatography
process, because it will accelerate chromatography process. Because the solution
will easy to be absorbed by paper of plate. The emulsifying solution consist of n-
buthanol-water-acetate acid. Between n-buthanol and water is polar solution. While
acetate acid is non-polar solution. So, when mixed the solution become semi-polar
solution.

2. Determining of Amino Acid Sample

Chromatography is the process of identification of sample based on polarity
level. So, when the sample is aimed to identify it used to chromatography based on
polarity level differences. When 4 samples of experiment they are: histidine,
sisteine, glisine, and sample of amino acid. While thin layer chromatography is the
process of adsorption based on coefficient distribution differences. The emulsifying
sample based on amino acid distribution of sample. This experiment included into
qualitatively experiment, because it used to know the type of amino acid in sample
by calculating the Rf.
The plate of chromatography made from cellulose, alumina, and kelisgar.
Before chromatography process, firstly the plate is heated up until 1040 C that used
to reduce the water inside plate. So, when chromatography process is conducted it
 will not influence the result of amino acid identification. Because if, there is water
inside plate it will influence the absorption of emulsifying solution. Emulsifying
solution will not move upward easily and the amino acid sample will be spread.
Because, water is polar solution that will dilute the amino acid. So, if the plate
contain water it will dilute the amino acid. The heating process of Chromatography
plate should not be done with too high temperature. It means it not more than 1030
C. if it is too high temperature it will dehydrates the plate. The plate should not too
dehydrates, because it will influence the chromatography process. If it is too
dehydrates it will not attrack the amino acid to patch the plate. The plate also will
be broken if heated up too high temperature. The plate consists of cellulose, so if it
is heated too high it will denaturation the cellulose. And the last reason why it
should not be heated up too high temperature is it will destruct the polarity of plate,
so the structure will be broke and the water will bounded to.
After the plate is tested with emulsifying media it will heated up with oven
again. To evaporate and testing the amino acid with ninhydrin to check the presence
of amino acid. If the heating process is too high temperature it is okay. Because the
amino acid will not denaturated because of heat.
The result of experiments are for Rf1 (histidine) = 0.089 cm, Rf2 (Glysine)
= 0.9 cm, Rf3 (Sistein) = 0.04 cm, and Rfs = 0.01 cm. While, theoretically result for
Histidine Rf is 0.11 cm, Glisine Rf is 0.26, and sistein is 0.4 cm.
J. Conclusion
From the experiment result, there is no calculation result of Rf that same
with theoretically with experimentally. Maybe there are several factors affecting
this. The factors will be explained in discussion part.
K. Discussion
Some factors that affecting spreading of sample
are firstly the heating process of plate is too short. So that
the water inside plate cannot evaporate perfectly. When
the sample is dropped into plate the sample will spread.
Because the polarity of water that will influence the amino
acid. The second factor is dropping process of sample is
 wrong. Because, when dropping samples, the tools is too short. So, its cannot
perfectly drop the sample into plate.

L. Refferences
Eiji K, Noriatsu K, Yoshiyuki U, Tooru S (2007). Extracellular BranchedChain Amino Acids,
Especially Valine, Regulate Maturation and Function of Monocyte-Derived Dendritic Cells, J.
Immun., 79: 7137- 7146.
Hellwinkel D (2001). Systematic Nomenclature of Organic Chemistry, Springer-Verlag, Berlin
und Heidelberg, Germany, pp. 209-210.
Lorraine B, Katrin B (2006). Amino Acid Metabolism, β-Cell Function, and Diabetes. Diabetes,
(55)2: 39-47
Scot R, Leonard S, (2006). New functions for amino acids: effects on gene transcription and
translation. Am. J. Clin. Nut., 83(2): 500-507.
Wilchek M, Chaiken I. An overview of affinity chromatography in affinity chromatography–
Methods and protocols. Humana Press. 2000:1–6.

M. Attachment
Questions and Answer
Questions
1. Calculate the level of vitamin C in the papaya!
2. Draw the vitamin C structure!
3. Mention the disease or symptom that appear, caused by deficiency of vitamin C!
4. Mention the food that contain vitamin C!
5. Mention the function of vitamin C for body!
Answers
1. known: V blanco: 0,6 mL
V I2: 1,1 mL
V I2: 1 mL
V I2: 0,9 mL
1,1 +1+0,9
V average = ( ) = 1 mL
3
V solution= v sample- v blanco = (1-0,6) ml = 0,4 mL
V sample= 10 mL
V dilution= 100 mL
Mass sample= 10 g = 1000 mg
Ask:…..?
Answer:
a. Level(mg) =
𝑉𝐼2 .𝑁𝐼2 0,4 𝑚 𝑙.0,01𝑁
a= 0,01 = 0,88 = x 0,88 mg = 0,352 mg
0,01
𝑉 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 100 𝑚𝐿
level (mg)= a x = 0,352 x = 3,52 mg
𝑉 𝑠𝑎𝑚𝑝𝑙𝑒 10 𝑚𝐿
100 𝑔
level (mg/100g) = level (mg) x 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔)
100 𝑔
= 3,52 mg x 10.000 𝑚𝑔
= 0,0352 mg/100g
 𝑉 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑎
Level (%) = x x 100 %
𝑉 𝑠𝑎𝑚𝑝𝑙𝑒 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
100 𝑚𝐿 3,52 𝑚𝑔/𝑚𝐿
= x x 100%
10 𝑚𝐿 10.000 𝑚𝑔
= 0,352%.
b. Evaluate calculation vitamin C in Papaya (Experiment 1)
Ask: levels vitamin C in papaya..?
Answer=
1. (Experiment 1)
VI2 = VI2 sample - VI2 blanco = (1,1-0,6) = 0,5 mL
 Level (mg)
VI2 .𝑁I2 0,5 𝑚𝐿 .0,01 𝑁
A= 0,01 x 0,88 = x 0,88 (mg) = 0,44 mg
0,01
𝑉 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 100 𝑚𝐿
Level Vit C (mg) = a x = 0,44 mg x = 4,4 mg
𝑉 𝑠𝑎𝑚𝑝𝑙𝑒 10 𝑚𝐿
100 𝑔
Level Vit C (mg/100g) = level vit C (mg) x 𝑚𝑎𝑠𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔)
100 𝑔
= 4,4 mg x 10.000 𝑚𝑔
= 0,044 mg/100g
𝑉 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑎
Level vit C (%) = 𝑉 𝑠𝑎𝑚𝑝𝑙𝑒 x 𝑚𝑎𝑠𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔) x 100%
100 𝑚𝐿 0,44 𝑚𝑔
= x 10.000 𝑚𝑔 x 100%
10 𝑚𝐿
= 0,044%
2. (Experiment 2)
VI2 = VI2 sample - VI2 blanco = (1-0,6) = 0,4 mL
 Level (mg)
VI2 .𝑁I2 0,4 𝑚𝐿 .0,01 𝑁
A= 0,01 x 0,88 = x 0,88 (mg) = 0,352 mg
0,01
𝑉 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 100 𝑚𝐿
Level Vit C (mg) = a x = 0,44 mg x = 3,52 mg
𝑉 𝑠𝑎𝑚𝑝𝑙𝑒 10 𝑚𝐿
100 𝑔
Level Vit C (mg/100g) = level vit C (mg) x 𝑚𝑎𝑠𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔)
100 𝑔
= 3,52 mg x 10.000 𝑚𝑔
= 0,0352 mg/100g
𝑉 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑎
Level vit C (%) = 𝑉 𝑠𝑎𝑚𝑝𝑙𝑒 x 𝑚𝑎𝑠𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔) x 100%
100 𝑚𝐿 0,352 𝑚𝑔
= x 10.000 𝑚𝑔 x 100%
10 𝑚𝐿
= 0,0352%
3. (Experiment 3)
4. (Experiment 1)
VI2 = VI2 sample - VI2 blanco = (0,9-0,6) = 0,3 mL
 Level (mg)
VI2 .𝑁I2 0,3 𝑚𝐿 .0,01 𝑁
A= 0,01 x 0,88 = x 0,88 (mg) = 0,264 mg
0,01
𝑉 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 100 𝑚𝐿
Level Vit C (mg) = a x = 0,264 mg x = 2,64 mg
𝑉 𝑠𝑎𝑚𝑝𝑙𝑒 10 𝑚𝐿
100 𝑔
Level Vit C (mg/100g) = level vit C (mg) x 𝑚𝑎𝑠𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔)
 100 𝑔
= 2,64 mg x 10.000 𝑚𝑔
= 0,0264 mg/100g
𝑉 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑎
Level vit C (%) = 𝑉 𝑠𝑎𝑚𝑝𝑙𝑒 x 𝑚𝑎𝑠𝑠 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔) x 100%
100 𝑚𝐿 0,264 𝑚𝑔
= x 10.000 𝑚𝑔 x 100%
10 𝑚𝐿
= 0,0264%
(0,044+0,0352+0,0264)
Average of level vitamin C (mg/100g) = mg/100g
3
0,1050
= mg/100g = 0,0352 mg/100g
3
(0,044+0,0352+0,0264)
Average of level vitamin C (%) = %
3
0,1050
= % = 0,0352 %
3

2.

3. bleeding and swollen gums, frequent nose bleeds, dry split hair, slow wound
healing, iron deficiency, dry and wrinkled skin.

4. Orange, tomato, guava, papaya, mango.

5. to prevent the disease and cancer, decreasing the risk of heart attack, increasing
body immunity, increasing mood, reducing runny nose.

N. Documentary

Figure 1. Papaya cutting

process Figure 2. Papaya pounding Figure 3. Papaya weighing
process process
 Figure 6. Amyllum Solution

Figure 4 Making Papaya

Figure 5 Filling the Buret with
Solution
Iodine

Figure 7. Filtrating the

papaya solution
Figure 8. Titrating the blanco Figure 9. The result of
solution with iodine titration
 Figure 11. Sample 1

Figure 12. Sample 2

Figure 10. Sample 3