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The complete report of Analytical Chemistry II Experiment with the title

Thin Layer Chromatography (TLC) was made by:
name : Helny Lydarisbo
id : 1513442002
group : IV
class : Chemistry Education of ICP
After checked and consulted by assistant and assistant coordinator. So, this report
was accepted.

Makassar, April 2017

Assistant Coordinator, Assistant,

Rahmania Tiara Lestari Paembonan

ID. 131344 ID. 131344

Known by,
Responsibility Lecturer

Drs. H. Muh. Yunus, M.Si

ID.
A. TITLE OF EXPERIMENT
Thin Layer Chromatography

B. OBJECTIVE OF EXPERIMENT
Separation of amino acids in a mixture by using thin layer chromatography
(TLC).

C. LITERATURE REVIEW
Chromatography is a technique in which a mobile phase (a gas or liquid)
carries the components of a mixture across a stationary phase (a solid or a liquid).
Separation of the mixture is based upon differences in migration rates between the
mixtures components due to their different absorptive tendencies for the mobile
or the stationary phases (Stanton,2010:29).
In his early papers Tswett (1905) stated that Chromatography is a method
in which the component which the mixture are separated on an adsorbent column
in a following system. Chromatography has progressed considerable from
Tswetts time and now includes a number of variations on the basic of the
separation process. Chromatography therefore encompasses a wide range of the
techniques. In order to bring a same order to the language of chromatography
IUPAC, the International of Union of Pure and Applied Chemistry, their updated
in nomenclature and definition for chromatography, United for Nomenclature
forChromatography (Braithwaite and Smith, 1999:2).
Historically, the word chromatography was used by Tswett in 1903 to
describe the separation of plant pigments by percolating a petroleum-ether extract
through a glass column packed with powdered calcium carbonate. Coloured zones
were produced by the various pigments migrating through the column at different
rates, the components being isolated by extrusion and sectioning of the calcium
carbonate packing. Modern chromatographic techniques are more complex and
are used for a wide variety of separations frequently involving colourless
substances, but the original term is retained. All the techniques depend upon the
same basic principle, i.e. variation in the rate at which different components of a
mixture migrate through a stationary phase under the influence of a mobile phase.
Rates of migration vary because of differences in distribution ratios.
Chromatography therefore resembles Craig counter-current distribution which has
been described in the previous section. In the Craig process, individual
equilibrations are performed in a series of separate vessels. If the walls of these
vessels are imagined to be non-existent so that the stationary phase is continuous,
and the mobile phase is allowed to move continuously rather than stepwise,
the situation would be closely analogous to that found in chromatographic
separations. In practice the liquid stationary phase is coated onto an inert,
granular or powdered solid support which is either packed into a column or spread
on a supporting sheet in the form of a thin layer. The solid stationary phases
used in some chromatographic techniques have no need of a support if packed
into a column but still require a supporting sheet for thin-layer
operation (Fifield and Kealay, 2000:80).
Thin-layer chromatography is a form of planar chromatography similar to
paper chromatography, but the stationary phase is a finely-divided sorbent spread
as a thin layer on a supporting flat plastic, aluminum or glass plate. Solutes
migrate through the stationary phase at rates determined by their distribution
ratios , those with the largest values moving the least, if at all, whilst those with
the smallest values moving with the advancing mobile phase, or solvent front.
Thin-layer chromatography is used primarily as a qualitative analytical technique
for the identification of organic and inorganic solutes by comparisons of samples
with standards chromatographed simultaneously (Kealey and Hanies, 2002:131).
Thin-layer chromatography (TLC) continues to be an important method
for qualitative analysis of plant products because of its inherent advantages many
samples can be analyzed simultaneously and quickly and multiple separation
techniques and detection procedures can be applied. The absence of a need for UV
activity (as in LC), paramagnetic properties (as in NMR), or volatility (as for GC)
makes TLC one of the most powerful and general analytical tools. It is clear from
the mobile phase systems comprising of harmful chemicals as one of the
component are not especially useful due to their strong toxic nature. It is now
highly recommended that avoid use of these toxic chemicals because these release
toxins in the environment. For the sustainable green environment
chromatographers are now devoted to develop new environmental friendly
chromatographic systems. The interest in TLC has increased with the
improvements in TLC instrumentation and methods and further in the last few
years with the development of methods for detection (Mohammad, 2010:43).
A major advantage of TLC is the speed of analysis on a per sample basis.
This is due to the short development distance (about 10 to 15 cm beyond the
origin) and the resultant short development time. Spotting samples along with
standards on the same plate allows them to be processed under identical
conditions in contrast to sequential analysis on a column. By far the greatest
number of chromatographic analyses are concerned with the measurement of only
one or two components of a sample mixture. Therefore, development conditions
are optimized for resolution of only the components of interest, while the
remainder of the sample material is left at the origin or is moved away from the
region of maximum resolution. Modern high-performance TLC (HPTLC) rivals
HPLC and GC in its ability to resolve complex mixtures and to provide analyte
quantification. The choice of mobile-phase components is not restricted by
concerns about deterioration of the coating material since layers are not reused or
by compatibility with a detector; thus solvents that are highly absorbing in the
ultraviolet region can beused. TLC provides for separations in the milligram to the
picrogram range (Patnaik, 2005:593).
Thin layer chromatography has the same principle with paper
chromatography except mobile phase is a thin layer of absorbent from the eluent
smooth over a plate of glass or aluminum. the most common substance is used as
absorbent is alumina, and silica powder silikagel. for organic compounds spoken
spraying the plate with sulfuric acid and then heated till compounds such as
charcoal and black stains (Tim Dosen Kimia Analitik II, 2016:13).
The mobile phase will carry the most soluble compounds the furthest up
the TLC plate. The compounds that are less soluble in the mobile phase and have
a higher affinity to the particles on the TLC plate will stay behind. R values: The
behavior of an individual compound in TLC is characterized by a quantity Known
as R and is expressed as a decimal fraction. The R is calculated by dividing the
distance the compound traveled from the original position by the distance the
solvent travelled from the original position (the solvent front). R = Distance of
centre of spot from starting point / Distance of solvent front from starting point.
The R value is a constant for each component only under identical experimental
condition (Bele and Khale, 2011:257-258).
Thin layer chromatography (TLC) is a routine analytical technique for the
separation and identification of drugs. Itssimplicity (require less sophisticated
apparatus), low cost, need for minimum sample clean up, allows this type
ofanalysis to be performed in remote areas. The TLC methods that have been
described for AMD (15, 16) were devoted to its qualitative identification, relied
on expensive densitometers, and/or do not indicate stability. Thepresent study
describes the development of a new alternative quantitative and not expensive
stabilityindicating TLC method for determination of AMD in bulk and capsule
forms (Askal, 2008: 156).
A chromatogram of standard phospholipids that were applied singly and as
the mixture.Three reference phospholipids of the highest purity,
phosphatidylinositol, phosphatidylcholineand lysophosphatidylcholine, were
used for recovery studies. Satisfactory recovery was accomplished for all three
phospholipids whether theywere applied individually or as a mixture. The
chromatographic separation of phospholipids from pooled rat liverTheindividual
spots were identified on the basis of the position of standard compounds and by
the detection methods listed above (Skipski, et al., 1964: 375).
During a chromatographic separation solute molecules are continually
moving back and forth betweenthe stationary and mobile phases. While they are
in the mobile phase, they are carried forward with it but remain virtually
stationary during the time they spend in the stationary phase. The rate of
migrationof each solute is therefore determined by the proportion of time it spends
in the mobile phase, or inother words by its distribution ratio.The original method
employed by Tswett involved surface adsorption wherethe relative polarities of
solute and solid stationary phase determine the rate of movement of that
solutethrough a column or across a surface. If a liquid is coated onto the surface of
an inert solid support, thesorption process is one of partition, and movement of the
solute is determined solely by its relativesolubility in the two phases or by its
volatility if the mobile phase is a gas. Both adsorption and partitionmay occur
simultaneously, and the contribution of each is determined by the system
parameters, i.e. the nature of the mobile and stationary phases, solid support and
solute (Fifield and Kealay, 2000: 80-81).

D. APPARATUS AND CHEMICALS

1 Apparatus
a Pencil (1 unit)
b Ruler (1 unit)
c Scissor (1 unit)
d Rough cloth (1 unit)
e Spray bottle (1 unit)
f Chamber (2 units)
g Drop pipette (2 units)
h Oven (1 unit)
i Clamp (1 unit)
2 Chemicals
a TLC plat (4 units)
b Capillary pipette (5 units)
c Alanine (H3C-CH-(NH2)-COOH)
d Glutamic Acid (HOOC-CH2-CH(NH2)-COOH)
e Histidine (C6H9N3O2)
f Tyrocine (HO-C6H6-CH(NH2)-COOH)
g X misture
h Eluent A solution (buthanol : acetic acid : water = 80 : 20 : 20)
i Eluent B solution (propanol : water = 70 : 30 v/v)
j Ninhydrin solution in buthanol contain 3% of acetic acid glacial (CH3COOH)
k Aquadest (H2O)

E. WORK PROCEDURES
1 The plate was cut to size.
2 Samples were spotted amino acid (alanine, glutamic acid, tyrosine and
histidine) and X mixture using capillary pipette just above the base line.
3 After that the plate was put in a chamber contain eluent A and B. Let a few
minutes, removed and drained. Sprayed with ninhydrine.
4 Let until some minutes
5 Heated in oven some minutes.
6 The value of Rf from amino acids and X mixture was calculated.

F. OBSERVATION RESULT
1 Eluent A
A
Spot Eluent
Component Color distance distance Rf
(cm) (cm)
Alanine Purple 0.7 6 0.116
Amino Histidine Purple 0.4 6 0.066
Standar Pink-
Glutamic Acid 2.7 6 0.450
d Purple
Tyrosine Purple 1.5 6 0.250
Mixture Purple 0.7 6 0.116
2 Eluent B
B
Spot Eluent
Component Color distance distance Rf
(cm) (cm)
Amino Alanine Purple 6.6 9.3 0.709
Histidine Purple 1.4 9.3 0.150
Standar
Glutamic Acid Purple 7.3 9.3 0.784
d Tyrosine Purple 7.7 9.3 0.827
Mixture Purple 6.4 9.3 0.688

G. ANALYSIS DATA
1 Eluent A
spot distance 0.7 cm
Rf Alanine = eluent distance = 6 cm = 0.116

spot distance 0.4 cm

Rf Histidine = eluent distance = 6 cm = 0.066

spot distance 2.7 cm

Rf Glutamic Acid = eluent distance = 6 cm = 0.450
 spot distance 1.5 cm
Rf Tyrosine = eluent distance = 6 cm = 0.250

spot distance 0.7 cm

Rf X Mixture = eluent distance = 6 cm = 0.116

2 Eluent B
spot distance 6.6 cm
Rf Alanine = eluent distance = 9.3 cm = 0.709

spot distance 1.4 cm

Rf Histidine = eluent distance = 9.3 cm = 0.150

spot distance 7.3 cm

Rf Glutamic Acid = eluent distance = 9.3 cm = 0.784

spot distance 7.7 cm

Rf Tyrosine = eluent distance = 9.3 cm = 0.827

spot distance 6.4 cm

Rf X Mixture = eluent distance = 9.3 cm = 0.688

H. DISCUSSION
Salah satu cara atau metode untuk memisahkan dan mengidentifikasi asam
amino yakni kromatografi lapis tipis. Pada dasarnnya kromatografi lapis tipis
(KLT) sangat mirip dengan kromatografi kertas, terutama pada cara
melakukannya namun perbedaannya hanya pada media pemisahannya, yakni pada
kromatografi lapis tipis digunakan lapis tipis adsorben halus yang tersangga
pada papan kaca, aluminium, atau plastik sebagai pengganti kertas. Lapisan
tipis adsorben ini pada proses pemisahan berlaku sebagai fasa diam,
sedangkan pada kromatografi kertas selulosa pada kertas sebagai fasa
diam (Tim Dosen Kimia Analitik).
Percobaan ini dilakukan dengan tujuan untuk memisahkan asam-asam
amino dalam suatu campuran dengan cara kromatografi lapis tipis. Prinsip dasar
dari kromatografi lapis tipis adalah pemisahan komponen kimia berdasarkan
prinsip partisi dan adsorpsi secara selektif karena adanya perbedaan daya serap
terhadap adsorben dan kelarutan komponen kimia terhadap cairan pengelusi.
Sedangkan, prinsip kerja KLT adalah penotolan, pengembangan, dan
pengidentifikasian, serta penentuan nilai Rf. Pada percobaan ini, yang merupakan
fase diam yaitu plat KLT yang mengandung silica dan fase geraknya adalah
larutan pengelusi yang terdiri dari pengelusi A dan B.
Asam amino yang digunakan pada percobaan ini adalah alanin, asam
glutamat, tirosin, dan histidin serta zat campuran sebagai pembanding dari asam
amino. Percobaan ini dimulai dengan pemotongan plat sesuai dengan ukuran
chamber dan digaris pada batas atas dan bawah dengan menggunakan pensil
diberi garis bawah sebagai tempat penotolan dan garis atas sebagai batas
perambatan eluen, digunakan pensil karena unsur penyusun dari pensil adalah
grafit (karbon) yang bersifat inert, sehingga tidak akan ikut melarut dan
mempengaruhi identifikasi noda, selain itu pensil juga tidak memiliki tinta seperti
pulpen sehingga walaupun terendam dalam larutan pengelusi, tidak akan
mengganggu perambatan noda sampel, kemudian plat KLT diaktivasi, fungsi dari
aktivasi yaitu agar plat steril atau bersih dari kotoran-kotoran yang dapat
mengganggu noda nantinya dan juga plat dapat terbebas dari molekul-molekul
H2O. Kemudian penotolan, setiap asam amino (alanin, tirosin, dan histidin dan
asam glutamat) dan sampel X, ditotolkan pada plat dengan menggunakan pipa
kapiler, karena diameternya yang sangat kecil sehingga penotolan tidak melebar
yang dimana apabila terlalu lebar atau besar noda yang ditotolkan maka akan
terjadi pelebaran noda saat dielusi sehingga sulit dalam penentuan nilai Rf
masing-masing noda.
Plat yang telah ditotolkan dan dikeringkan tadi, kemudian dimasukkan
kedalam chamber yang berisi larutan pengelusi A dan B. Tujuan digunakan dua
pengelusi yaitu untuk mengetahui pengelusi mana yang baik digunakan untuk
mengelusi asam-asam amino. Setelah itu, chamber tersebut ditutup dengan tujuan
untuk menjenuhkan udara dalam chamber dengan uap pelarut karena dengan
penjenuhan, pelarut tersebut akan berhenti menguap dan mempercepat
perambatan sampel sehingga dapat diperoleh noda yang baik. Selain itu proses
pengembangan yang dilakukan dalam chamber harus dalam keadaan jenuh karena
dalam kondisi ini, larutan akan menghasilkan uap yang akan membantu
perambatan larutan melalui fase diam. Ketika plat yang berisi noda dimasukkan
kedalam chamber, tidak boleh garis batas mencapai eluen karena totolan cuplikan
akan melarut dalam larutan pengembang dan noda tidak akan merambat keatas.
Setelah beberapa menit plat dikeluarkan dari chamber, setelah pelarut mencapai
puncak atau garis batas atas pada plat.
Kemudian plat tersebut disemprot dengan larutan ninhidrin untuk
memperjelas noda hasil elusi pada plat KLT. Larutan ninhidrin adalah oksidator
sehingga dapat memperjelas noda dengan mengikat asam amino bebas pada plat.
Setelah itu, plat diletakkan di dalam oven untuk mempercepat proses pengeringan
dan pembentukan noda. Proses pengeringan dan pemanasan asam amino akan
memutuskan ikatan rangkapnya sehingga akan memancarkan gelombang tertentu
yang dapat dilihat oleh visual (mata) berupa warna. Hasil pengamatan yang
diperoleh yaitu sampel asam amino dan sampel X masing-masing berwarna ungu
yang menandakan bahwa penyusun sampel X adalah asam amino. Hal ini sesuai
dengan teori yang menyatakan bahwa apabila asam amino direaksikan dengan
ninhidrin maka akan membentuk warna ungu.
Dengan munculnya warna dari hasil pengeringan, adapun nilai Rf yang
diperoleh pada pengelusi A yaitu, alanin = 0,116; histidin = 0,066; asam glutamat=
0,450; tirosin = 0,250 dan sampel X = 0,116. Sesuai hasil yang diperoleh dapat
disimpulkan bahwa sampel X merupakan larutan alanin selain karena warna noda
yang dihasilkan sama, nilai Rf-nya juga sama. Hasil yang diperoleh pada
pengelusi B yaitu, alanin = 0,709; histidin = 0,150; asam glutamat= 0,784; tirosin
= 0,827 dan sampel X = 0,688. Sesuai hasil yang diperoleh dapat disimpulkan
bahwa sampel X juga merupakan larutan alanin, selain karena warna noda yang
dihasilkan sama, nilai Rf-nya juga hampir sama dengan nilai Rf asam glutamat.
Jika dibandingkan dengan teori, nilai Rf asam amino standar yaitu,
alanin= 0,38; asam glutamat = 0,24; tirosin = 0,45; dan histidin = 0,11. Hasil yang
diperoleh berbeda dengan teori, karena nilai Rf tergantung pada eluen yang
digunakan. Pada teori tidak diketahui eluen apa yang digunakan, kemungkinan
eluen yang digunakan berbeda sehingga hasilnya juga berbeda. Faktor lain yang
mempengaruhi nilai Rf yaitu diameter noda, suhu, jenis pelarut, dan kepolaran,
kesetimbangan dan juga struktur kimia dari substrat yang akan dipisahkan.
Adapun persamaan reaksinya yaitu:
1. Alanin + ninhidrin

O O O
H3C
OH + OH
OH
N +
NH2 O O
O
(ungu)
alanine ninhidrin
O
CH3 + CO2 + 4H2O

2. Asam Glutamat + Ninhidrin

O O
O O
+2 OH N + CO2
HO OH OH
NH2 O O
O
asam glutamat (ungu)
ninhidrin
OH
O + CO2 + 4H2O
O

3. Histidin + Ninhidrin
O O
O
N OH +2 OH
N + CO2
OH
N NH2
O O O
H
histidin ninhidrin (ungu)

H
OH N
+ 4H2O +
O N

4. Tyrosin + Ninhidrin
 O O
O

OH
+2 OH
N + CO2
OH
NH2
HO O O O
tirosin ninhidrin (ungu)

+ 4H2O + O
OH

I. CONCLUSION AND SUGGESTION

1. Conclusion
a. The Rf value for eluen A such as alanin : 0.116, hystidin : 0.066, glutamic
acid : 0.450, tyrosin : 0.250, X : 0.116.
b. The Rf value for eluen B such as alanin : 0.709, hystidin : 0.150, glutamic
acid : 0.784, tyrosin : 0.827, X : 0.688.
c. The sample X is containing alanin.
2. Suggestion
Apperantice should be careful in spot because spot can affect the value of
Rf.

BIBLIOGRAPHY

Askal, Hassan F, Alaa S. Khedr, Ibrahim A. Darwish, et al. 2008. Quantitative

Thin-Layer Chromatographic Method for Determination of Amantadine
Hydrochloride. International Journal of Biomedical Science. Vol. 4. No. 2:
155-160.

Bele, Archana A And Anubha Khale.2011. An Overview On Thin Layer

Chromatography. Internasional Journal Of Pharmaceutical Science And
Research. Vol. 2(2): 256-267 Issn: 0975-8232

Braithwaite, A and F.J. Smith. 1999. Chromatographic Methods Fifth Edition. The
Netherlands: Kluwer Academic Publishers.
Fifield, F. W and D. Kealay. 2000. Principles and Practice of Analitical
Chemistry. United Kingdom: Blackwell Science, Ltd.

Kealey, D and P.J. Haines. 2003. Instant Notes Analytical Chemistry. United
States of America: Blos Scientific Publishers.

Mohammad, A. S.A. Bhawani And S. Sharma. 2010. Analysis Of Herbal Products

By Thin-Layer Chromatography: A Review. International Journal Of
Pharma And Bio Science. Vol 1(2):43

Patnaik, P. 2005. Deans Analytical Chemistry Handbook Second Edition. United

States of America: Mc-Graw Hill Handbooks

Skipski, V. P., et al. 1963. Quantitative Analysis of Phospholipids by Thin-Layer

Chromatography. Journal of Biochem. Vol. 90. No. 374.

Stanton, Bobby. Zhu, Lin and Charles H. Atwood. 2010. Experiments in General
Chemistry. USA : Cencage Learning.

Tim Dosen Kimia Analitik. 2017. Penuntun Praktikum Kimia Analitik II.
Makassar: Laboratorium Kimia FMIPA UNM.

ANSWER OF QUESTION
1. What happen if protein hydrolyzed by strong acid?
Answer :
If protein hydrolyzed by strong acid is it can be composer of amino acid
that discontinuance of peptide bond.

2. Explain the basic principle of TLC!

Answer :
The basic principle of thin layer chromatography is the difference of
substance solubility that used, the method can basically of velocity
differences of substance migration on plat.

3. Give the name of amino acid that identification based on the value of Rf!
Answer :
Amino acid that identification are glutamic acid.