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DATE: June 12, 2014
PRACT. REPORT ON ANALYSIS OF QUININE PREPARATION BY TLC:
AIM: To analyses the Quinine preparations by TLC-based MINLAB.
INTRODUCTION AND THEORY:
Thin-layer chromatography (TLC) is a chromatography technique used to
separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet
of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent
is known as the stationary phase.
Figure.1.Separation of black ink on a TLC plate.
Thin layer chromatography is the main screening method being used today in order
to determine if a drug product meets label specifications and is legal. TLC can be
employed for the identification of drug substances, to estimate drug substance
content and to detect related substances that may be regarded as impurities. This
technique allows the active ingredient to be recognized by comparison with a
known drug standard. The approach using TLC chromatography is cheap, specific
and sensitive. The TLC procedures are therefore preferred, as they are capable of
giving semi-quantitative information on the active ingredient and also on any
related substances in the dosage forms.
Principle: The TLC technique consists of placing a spot of drug sample on a thin
layer of silica attached to a plate of glass, aluminum or plastic. The plate is then

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inserted into a vessel containing a solvent mixture. By capillary action, the solvent
creeps up the adsorbent material, silica, alumina, or cellulose, and dissolves the
sample. The drug sample consists of a mixture of drug and inactive ingredients.
These compounds will have various affinities to the adsorbent matrix and will
migrate with the solvent at various speeds. This characteristic effectively separates
out a mixture of compounds.
After migration of the solvent is complete, individual components can be
visualized by chemical treatment or ultraviolet (UV) absorbance. The distance that
the components migrate is characteristic for each compound; therefore the active
ingredient can be recognized by comparison with a known drug standard. The
solvent can be modified to increase resolution between various components.
This method is relatively inexpensive, specific and sensitive. It is commonly used
to assess drug quality. For example in our experiment we analyses the Quinine
preparations.
Quinine is a natural white crystalline alkaloid having antipyretic (fever-reducing),
antimalarial, analgesic (painkilling), and anti-inflammatory properties and a bitter
taste. It is a stereoisomer of quinidine, which, unlike quinine, is an antiarrhythmic.
Quinine contains two major fused-ring systems: the aromatic quinoline and the
bicyclic quinuclidine. Check structure below;
Figure 2.Structure of Quinine

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APPARATUS AND MATERIAL USED:
1) APPARATUS USED:
Two heavy-duty suitcases-Minilab kits contain the essential components which
are:
a) Aluminium chromatographic plates,
b) 50 ml glass bottle
c) 10ml vials
d) Stop watch
e) Developing and detection chambers
f) 25ml , 5ml and Micro Pipette
g) 25ml Measuring cylinders
h) UV lamps of 254nm wavelength.
i) Pens and pencils and ruler
j) Safety pipette filler
k) Beakers
l) Mortar and pestle.
m) Filter paper.
n) Round bottom flasks
Figure 3. Two heavy-duty suitcases contain the essential components.

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2) MATERIAL USED:
The materials we used in our experiment were;
a) 300mg reference Quinine tablet.
b) 300mg sample Quinine tablet.
c) Aqueous methanol
d) Distilled water.
e) Conc. Ammonia solution.
PROCEDURES:
A. Preparation of stock standard solution:
 300mg reference tablet was grinded by using mortar and pestle and wash
down the powder completely with 3ml of water into a 50ml glass bottle.
 Then the bottle was closed and shakes it for 1minute.
 After that 27ml of methanol was added and close again the bottle. Then
shaken again for 3minutes and was left to stand for 5minutes until all
insoluble material settles down.
 After above time the glass bottle was label as Quinine stock standard
solution.
B. Preparation of working standard solution:
QUININE WORKING STANDARD
SOLUTION 100%=1.25mg/ml
QUININE WORKING STANDARD
SOLUTION 80%=1.0mg/ml.
10ml was pipette into vial
 1ml of stock standard solution.
 Then 7ml of methanol was
added
 Finally closed, shaken and
labeled as Quinine working
standard solution 100%
10ml was pipette into vial
 1ml of hazy stock standard
solution
 Then we added 9ml of
methanol
 Then Finally closed, shaken
and labeled as Quinine
working standard solution
80%

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C. Preparation of Quinine stock sample solution (10mg/ml):
 300mg tablet was grinded by using mortar and pestle and wash down the
powder completely with 3ml of water into a 50ml glass bottle, then shaken
for 1minute and
 There after 27 ml of methanol was added.
 The bottle was closed and shaken for another 3minutes. Then left to stand
for 5minute until all insoluble material settles down
 After above time and ensuring that all insoluble material settles down, then
the bottle was labeled as Quinine stock sample solution.
D. Preparation of working sample solution:
 1ml of hazy stock sample solution was pipetted into 10ml vial
 Then 7ml of methanol was added, closed, shake and labeled as Quinine
working sample solution.
E. Preparation of developing chamber:
 20ml of methanol was pipette into the developing chamber jar then 0.5ml of
conc. Ammonia solution was added.
 Then the jar was closed and mixes thoroughly. Then the chamber’s wall was
line with filter paper.
 After wait for about 15minutes for chamber to saturating. Normally this time
was used for spotting as mention below.
F. Spotting (To Loading the TLC plate with sample solution):
 Firstly an origin line was mark about 1.5cm from the bottom edge with
pencil.
 Then 2micro litre of each working standard solution (both 100% as no. 1
and 80% as no. 4) was applied by using micro pipette.
 Then 2micro litre of each working sample solution (both as no. 2 and no. 3
for sample solution) also as above step was apply by using micro pipette.
 Then spots were waited till all dried off.
 After that the uniformity of all spots was check with UV light of 254nm.as
shown in figure.4 below

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Figure.4 Observation of uniformity of spots by UV LIGHT LAMP of 254nm.
G. Development:
 After 15minutes ,carefully the loaded plate above was placed into the
developing chamber, then the jar was close we wait until the solvent front
has moved three quarter of the length of the plate (approximately
developing time about 20 minutes)
 Then the plate was removed and marked the solvent front
 There after the plate was dried simply by air although there is another way
of dying by use of hot plate but were not applying that.
H. Detection:
he chromatoplate was observe with UV light by using UV light lamp 254nm.
I. The final chromatoplate observed at 254nm:
Then the spots observed above were marked as:
 Run no.1=2.0µl of Quinine 100% standard solution
 Run no.2=2.0µl of sample solution
 Run no.3=2.0µl of sample solution
 Run no.4=2.0µl of Quinine 80% standard solution
RESULTS AND CALCULATIONS:
By using ruler we measured the distance travelled by components (let’s say be X)
and we recorded as below:

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 Run no.1(X1)=3.9cm
 Run no.2(X2)=3.8cm
 Run no.3(X3)=3.8cm
 Run no.4(X4)=3.8cm
And distance travelled by solvents/distance of solvent front (Let’s say be Y)
=7.7
Then we calculated Rf values (reference values) as follows:
From the formula below;
Figure 5. Below is the chromatoplate we obtained after developing and marks
the spots under UV light

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Then we calculated the Rf values (reference values) as summarized in Table: 1
below.
Table: 1
Run no. 1 2 3 4
X(cm) 3.9 3.8 3.8 3.8
Y(cm) 7.7 7.7 7.7 7.7
Rf= Rf100= Rfs1= Rfs2= Rf80=
ANSWER 0.5065 0.4935 0.4935 0.4935
DISCUSSION:
By utilize standard TLC technology we are able to analyze some commonly
adulterated counterfeit drug compounds. We able to identify the active ingredient
by comparison of distance of travel (RF value) between the sample spot and an
authentic standard spotted on the same plate.
We obtained these points;
 As we done in above experiment it appeared that Rf values for our sample
solutions are completely same/corresponding to that for Quinine 80%
standard solution which equal to 0.4935. But it’s slightly below to Quinine
100% standard solution which equal to 0.5065 may due to error since by in
approximately they correlated.
 Also the spots` color, size and intensity produced by sample solutions, are
nearly equal to that of Quinine 80% standard solution, but slightly less to
that of Quinine 100% standard solution.
Hence: we conclude that the sample solutions from tablets which claims to
contain QUININE as active ingredients they REAL contain quinine since the
reference value correspond to those of standard exactly same to Quinine 80%

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standard solution but near same to Quinine 100% standard solution and color
under UV-light, size and intensity of spots are within range of Quinine 80%
and 100% standard solution. Therefore the drug is of good and required
quality.
SOURCES OF ERROR:
1) Human error which are the parallax errors and the calculation errors.
2) Environmental contamination.
3) Human error due to contamination.
4) Error in spotting and loading sample.
5) Error in indicating solvent front and distance travelled by components.
6) Lack of visual equity.
CONCLUSSION:
Global Pharma Health Fund e.V. Mini-labs (GPHF-Minilab) is being used in 70
countries to help identify counterfeit and substandard drugs. These minilabs, which
contain simple materials (as described above) and that utilize standard TLC
technology are able to analyze some 40 commonly adulterated counterfeit drug
compounds. The Minilab supplies a collection of authentic secondary standard
tablets and capsules in sealed plastic tubes. The Minilab analysis identifies the
active ingredient by comparison of distance of travel (RF value) reference value
between the sample spot and an authentic standard spotted on the same plate, and
semi-quantitative proof of content is made visually comparing the color, size, and
intensity between the sample spot and reference spots for each method of
detection.
As above we analyzed Quinine preparations by comparing the Rf values of sample
solution and that of standard we obtained it’s corresponding to Quinine 80%
standard solution and approximately to Quinine100% standard solution. Also color
under UV-light, size and intensity of spots are within range of Quinine 80% and
100% standard solution. Hence is of a good or required quality.
Advantages of TLC: It analytical tool for

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 Identification and Quantification of substances
 Detection of impurities and degradation products of drugs, and
 It is cheap and simple apparatus
 Several samples can be analyzed simultaneously.
 Great flexibility in terms of stationary and mobile phases.
 No detection problems in the case of non-elution, thermal instability and
masking by solvent, since the applied substance remains on the plate.
ACKNOWNLEDGEMENT:
1) TO MR. EDSON
2) TO. Dr. KAALE
3) TO. MY FELLOW STUDENTS.
4) TO MY MOTHER.
REFERENCES:
1) Experiment protocol prepared by MEDCHEM. Department-MUHAS
2) USP-NF 2007
3) Harry W. Lewis and Christopher J. Moody (13 Jun 1989). Experimental
Organic Chemistry: Principles and Practice (Illustrated ed.). Wiley
Blackwell. pp. 159–173. ISBN 978-0-632-02017-1.
4) Thin Layer Chromatography: http://www.reachdevices.com/TLC.html
5) TLC plates as a convenient platform for solvent-free reactions Jonathan M.
Stoddard, Lien Nguyen, Hector Mata-Chavez and Kelly Nguyen Chem.
Commun., 2007, 1240 - 1241, doi:10.1039/b616311d
6) Joseph Sherma, Bernard Fried (1991): Handbook of Thin-Layer
Chromatography (= Chromatographic Science. Bd. 55). Marcel Dekker,
New York NY, ISBN 0-8247-8335-2.