Unit 4

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APPROVAL SHEET

Complete report of Analytical Chemistry II with the title “Thin Layer


Chromatography (TLC)” made by:

Name : Alif Gautama

Reg.Number : 1413440010

Class : ICP-B Chemistry

Group : V (five)

have been checked by Assistant and Assistant Coordinator. So, this report is
accepted.

Makassar, Juneth 2016


Assistant Coordinator Assistant,

Heril Hidayat Niah Mukramah


ID:1313041002
Known by,
Responsibility Lecturer

(Maryono, S.Si, Apt., M.M, M.Si)


ID 19760304 200501 2 002
A. TITLE OF EXPERIMENT
Thin Layer Chromatography
B. OBJECTIVE
Separation amino acid in a mixture with thin layer chromatography.
C. LITERATURE REVIEW
Although published work in which paper chromatography (PC) is used
continous to the appear , the use of the method has long since passed its peak.
The developments in thin layer, gas and high performance column
chromatography, with the increased speed and degree of instrumentation that they
offer, have diverted attention from the cheaper, less the sophisticated and the
poorer performing inferior the resolution and the log analysis times (several
hours) paper methods. Nonetheless, PC is useful as a low cost approach to
teaching the fundamentals ad practice of chromatography. The following is
intended as no more than an overview and the intereseted reader is directed to the
references cited herein and to the numerous reviews and bibliographies containing
detailed information on the techniques of paper chromatography; a current listing
of papers published appears in the bibliography section of the journal of
chromatography. Various types of sample separations on paper have been
described as forerunners of paper chromatography, among them the method of
Runge in 1850 for separating inorganic mixtures, and the process called 'capilarry
analysis' (Smith, 1999: 81-82).
The capillary analysis, the chromatographic technique used, was
developed by FriedliebRunge in the mid-1850s and was later refined by Friedrich
Goppelsroeder. The principle of the analysis was that substances were absorbed
on filter paper directly from the solutions in which they were dissolved; they then
migrated to different points on the filter paper. The capillary analysis differed
from paper chromatography in that no developing solvent was used. We find that,
from these humble beginnings 150 years ago, the direct descendant of this
technique, paper chromatography, is still widely used in evaluating
radiopharmaceuticals (Adamovics, 1997 : 2).
The Paper chromatography (PC) is simple and cheap but lacks the
separating power and versatility of thin-layer chromatography (TLC) which has
largely replaced it. Both require only inexpensive equipment and reagents, and,
unlike the various forms of column chromatography, comparisons can be made
between a number of samples and standards chromatographed simultaneously.
Gas (GC) and high performance liquid chromatography (HPLC) are
complementary techniques best suited to the separation of volatile and nonvolatile
mixtures, respectively. Both these techniques are instrumentally-based and
computer-controlled, with sophisticated software packages and the ability to
separate very complex mixtures of up to 100 or more components. HPLC is
particularly versatile, having several alternative modes suited to different types of
solute. For example, ion-exchange (IEC) and ion chromatography (IC) are modes
that enable mixtures of either anionic or cationic solutes to be separated.Size-
exclusion (SEC) and chiral chromatography (CC) are two additional modes used
for separating mixtures of high relative molecular mass solutes and enantiomers,
respectively (Kealey, 2002 : 120-121)
Paper chromatography has been successfully applied for the separation of
various degradation products of pectic substances. These degradation products are
obtained by acid or enzymatic hydrolysis and more recently by heating with the
cation exchange resin, Permutit Q, in a sealed tube. The last method of hydrolysis
yields a solution of galacturonic acid, oligogalacturonides, galactose, arabinose,
and rhamnose as detected by chromatography. A general chromatographic method
for the detection and separation of pectic substances has not been developed till
now. The aim of this paper is to present a method for chromatographic and
electrophoretic separation on paper of different pectin preparations. The method to
be described was based on the following properties of pectic substances:
1. The relative solubility of pectinic acid (pectin) in water and its insolubility in
ethanol suggested that either water (buffer) or a mixture of ethanol-water could be
used as solvent for paper chromatography of pectin.
2. Pectin is thought to consist mainly of polygalacturonic acid esters. These
should give similar reactions as carboxylic acid esters. The use of hydroxylamine
in alkaline solution to convert polygalacturonic acid esters into hydroxamates
followed by ferric chloride as color reagent appeared to be promising for the
detection of esterified polygalacturonic acid units.
3. Pectic substances show a relatively high mobility in electrophoresis. This
suggested that after the migration in an electric field the pectic substances could
be detected by spraying the paper with hydroxylamine-ferric-ehloride reagent.
(Ujejski, 1957).
Chromatography is the collection term for a set of laboratory techniques
for the separation of mixtures. The mixture is dissolved in a fluid called the
“mobile phase”, which carries it through a structure holding another material
called the “stationary phase”. The various constituents of the mixture travel at
different speeds, causing them to separate. The separation is based on differential
partitioning between the mobile and stationary phases. Subtle differences in a
compound’s partition coefficient result in differential retention on the stationary
phase and thus changing the separation. Paper chromatography is a technique that
involves placing a small dot or line of sample solution onto a strip of
chromatography paper. The paper is placed in a jar containing a shallow layer of
solvent and sealed. As the solvent rises through the paper, it meets the sample
mixture which starts to travel up the paper with the solvent. This Paper is made of
cellulose, a polar substance, and the compounds within the mixture travel farther
if they are non–polar. More polar substance, bond with the cellulose paper more
quickly and therefore do not travel as far. In Paper chromatography, paper is
marked with the plant extract and is placed in a developing chamber with a
specified solvent. The solvent carries the dissolved pigments as it moves up the
paper. This shows the variation of the pigments from one fertilizer effected plant
to another and finally compared with the control plant pigments. The distance of
the pigment travels is unique for the pigment in set conditions and is used to
identify the pigment. The ratio is the retention factor Rf value.
The distance travelled by the pigment (cm )
RF =
The distance travelled by the solvent (cm)
(Kumari, 2012)
D. APPARATUS AND CHEMICALS
1. Apparatus
a. Chamber 1 piece
b. Tweezers 2 pieces
c. Pencil 1 piece
d. Ruler 1 piece
e. Cutter 1 piece
f. Capillary pipe 5 pieces
g. Beaker 600 mL 1 piece
h. Erlenmeyer 1 piece
i. Measuring cylinder 1 piece
j. Spraying bottle 1 piece
k. Stopwatch 1 piece
2. Chemiclas
a. Ninhydrine 0,3%
b. Eluent A (butanol : Acetic acid : water = 80 : 20 :20)
c. Eluent B (propanol : water = 70 : 30)
d. Standard amino acid solution (Alanine, Glutamic Acid, Histidin, Tyrosin)
e. Mixture X solution
f. Aquadest
E. WORK PROCEDURES
1. Cutted the TLC plate as two plates, each measuring 7cm long and 3cm wide.
2. Prepared a solution of standard amino acids and the mixed solution x, then do
spotted one drop of solution of amino acids in the two (2) plate (TLC plate)
are different
3. Layed each plate on top of elution solution A and B and let elution creeping
up to the mark.
4. Lifted and drying the plate, then spray with ninhydrin solution.
5. Dried the plates above the water bath
6. Calculated Rfof amino acids and the mixed solution x analyzed.
F. OBSERVATION RESULT
Rf value
Component Name of amino acid
Eluent A (cm) Eluent B (cm)
0,69
Rf = =
3
0,3 0,23
Rf = = 0,1
Alanine 3 2,19
Rf = =
I 0,9 3
Glutamate acid Rf = = 0,3
II 3 0,73
standard
III Tyrosin - 2,49
Rf =
0,69 3
IV Rf = = 0,23
Histidine 3 =0,83
2,01
Rf = =
3
0,67
0,648
Rf = = 2,4
Mixture Sample x1 3 Rf = = 0,8
3
0,216

G. DATA ANALYSIS
a. Eluent A
1. Histidin
Known :distance stains = 0,3 cm
distance eluent = 3 cm
Asked :Rf ?
distance stains 0,3 cm
Answer:Rf = = = 0,1
distance eluent 3 cm

2. Glutamate acid
Known :distance stains = 0,9 cm
distance eluent = 3 cm
Asked :Rf ?
Answer:
distance stains 0,9 cm
Rf = = = 0,3
distance eluent 3 cm
3. Tyrosin
Known :distance stains = 0cm
distance eluent = 3 cm
Asked :Rf ?
Answer:
distance stains
Rf = =0
distance eluent

4. Alanin
Known :distance stains = 0,69
distance eluent = 3 cm
Asked :Rf ?
Answer:
distance stains 0,69 cm
Rf = = = 0,23
distance eluent 3 cm

5. sample x1 solution
Known :distance stains = 0,648 cm
distance eluent = 3 cm
Asked :Rf ?
Answer:
distance stains 0,648 cm
Rf = = = 0,216
distance eluent 3 cm

b. Eluent B
1. Histidin
Known :distance stains = 0,69 cm
distance eluent = 3 cm
Asked :Rf ?
distance stains 0,69 cm
Answer:Rf = = = 0,23
distance eluent 3 cm
2. Glutamate acid
Known :distance stains = 2,19 cm
distance eluent = 3 cm
Asked :Rf ?
Answer:
distance stains 2,19 cm
Rf = = = 0,73
distance eluent 3 cm
3. Tyrosin
Known :distance stains = 2,49 cm
distance eluent = 3 cm
Asked :Rf ?
Answer:
distance stains 2,49 cm
Rf = = = 0,83
distance eluent 3 cm

4. Alanin
Known :distance stains = 0,69 cm
distance eluent = 3 cm
Asked :Rf ?
Answer:
distance stains 2,01cm
Rf = = = 0,67
distance eluent 3 cm

5. sample x1 solution
Known :distance stains = 2,4 cm
distance eluent = 3 cm
Asked :Rf ?
Answer:
distance stains 0,24 cm
Rf = = = 0,8
distance eluent 3 cm

H. DISCUSSION
Kromatografi lapis tipis merupakan suatu metode pemisahan campuran
menjadi komponen – komponennya berdasarkan perbedaan cepat rambatnya.
Percobaan ini bertujuan untuk memisahkan asam amino dalam suatu campuran
dengan cara kromatografi lapis tipis (KLT). Pada percobaan ini plat KLT dari
alumina adalah fase diamnya dan larutan pengelusi A dan pengelusi B adalah fase
geraknya. Larutan pengelusi A terdiri atas butanol, asam asetat dan air dengan
perbandingan 80 : 20 : 20. Sedangkan larutan pengelusi B terdiri atas 2 – Propanol
dan air dengan perbandingan volume 70 : 30. Penggunaan dua macam larutan
pengelusi yang berbeda bertujuan untuk membandingkan komposisi pengelusi
yang baik untuk digunakan pada pemisahan KLT., yaitu pengelusi yang
kepolarannya sesuai dengan komposisi kepolaran pada permukaan plat KLT.
Dimana semakin panjang rantai karbon pada alkohol, maka kepolarannya semakin
kecil.

Pada tahap penotolan alanin, asam glutamat, tyrosin, histidin dan sampel
X ditotolkan pada KLT dengan menggunakan pipa kapiler, yang berfungsi untuk
menotolkan asam glutamat, tyrosin, alanin, histidin dan sampel X. Pipa kapiler
digunakan agar noda tidak menyebar pada saat penotolan sehingga akan
memudahkan dalam pengamatan sebab pipa kapiler memiliki diameter yang kecil
sehingga pada saat ditotolkan maka besar noda tidak akan lebar. Penotolan
diusakan tidak terlalu banyak karena akan mempengaruhi besar noda. Penotolan
yang terlalu besar tidak baik untuk penampakan noda karena nodanya dapat
melebar kesamping atau kebawah.

Setelah ditotoli asam-asam amino standar dan sampel X, kemudian plat


dikeringkan dengan tujuan untuk menguapkan HCl yang terdapat pada larutan.
Setelah kering, plat KLT dielusi dalam chamber sampai batas rambat kertas harus
dijaga agar tidak terendam dalam eluen, karena apabila kertas terendam maka
noda tidak akan merembes tetapi akan bercampur dengan eluen. Ketika eluen
mencapai batas rambat, kemudian plat dikeringkan. Selanjutnya plat disemprotkan
dengan menggunakan ninhidrin agar noda-noda hasil pemisahahn dapat diamati,
karena sebelum penyemprotan dengan ninhidrin noda-noda warna belum nampak,
kemudian plat dipanaskan dalam penangas air hingga warnanya timbul.
Pemanasan ini berfungsi untuk menghilangkan pengelusi yang masih terikat pada
lapisan adsorban, akibat hilangnya pengelusi tersebut akan memudahkan terjadiny
areaksi antara ninhidrin dengan asam-asam amino serta sampel X.

Dari hasil percobaan yang dilakukan, dapat diketahui nilai Rf untuk


masing-masing asam amino, pada pengelusi A : Histidin = 0,1 ; asam glutamat =
0,3 ; tyrosin = - ; Alanin = 0,23 ; dan senyawa X = 0,216. Harga Rf pada
pengelusi B : Histidin = 0,23 ; asam glutamat = 0,73 ; tirosin = 0,0,83 ; Alanin=
0,67; dan senyawa X = 0,8. Terdapat asam amino yang tidak memiliki noda
dikarenakan penotolan yang terlalu kecil dan pengeringan yang tidak maksimal.
Nilai Rf untuk pengelusi A lebih kecil dari pada pengelusi B, hal ini terjadi karena
pengelusi A terdiri dari dari pelarut yang kurang polar yaitu butanol, meskipun
terdapat air yang bersifat polar serta asam asetat pemberi suasan aasam, akan
tetapi konsentrasi butanol dalam pengelusi A jauh lebih besar dari air dan asam
asetat. Pengelusi B mempunyai kepolaran yang sama dengan asam-asam amino
karena terdiri dari propanol dan air sehingga noda yang tampak pada plat sangat
jelas, dilihat dari rembesannya. Identifikasi senyawa X berdasarka nnilai Rf yang
dihasilkan, dapat diketahui bahwa senyawa X tersebut merupakan tirosin ,karena
mempunyai nilai Rf yang hamper sama.
A. CONCLUSION AND SUGGESTION
1. Conclusion
Based on the trial can be concluded that the amino acids in a mixture can be
separated by thin layer chromatography, the basic principle is based on the speed
difference between the distribution of a component pase stationary and mobile
phases. For identification can be seen by staining and Rf value and concluded that
the mixture (X) is analine and tyrosine with Rf each is 0,26 and 0,96..
2. Suggestion
It is expected that the practitioner to be more careful - careful when penotolan
and pengelusian, because it affects the price of Rf
BIBLIOGRAPHY

F.J. Smith. A. Braithwaite. 1999. Cromatographic Methods. Netherlands. Kluwer


Academic Publisher.
Ujejski, Ludwik. 1957. The Detection and Separation of PecticSubstances by
Paper Chromatography andPaper Electrophoresis. Journal of Science.
Vol. 57. No. 1
D. Kealey. P.J. Haines. 2002. Analytical Chemistry. U.K.: BIOS Scientific
Publishers Ltd.
John A. Adamovics. 1997. Chromatographic Analysis of Pharmaceuticals. New
York : Marcel Dekker, Inc.
kumari, S.P Kiran. Y. Satya vani. V Sridevi.M.V.V. Chandana Lakshmi. 2012.
Separation And Observation Of Plant Pigments In Fertilizers Effected
Medicinal Plants Using Paper Chromatography. International Journal Of
Engineering Science & Advanced Technology. Vol. 2. No.2

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