Analysis of Analgesic Drug by Thin Layer

Chromatography (TLC)

A Project Report Submitted

In Partial Fulfillment of the Requirements
For The Degree Of

Bachelor of Pharmacy (B. Pharm) Course

By

Atal Baghel
335032
University Roll No.:
Reg No -20-MSDP-02

Under the Supervision of

Mr. Yashpal Singh
Associate Professor, Department of Pharmacy

Submitted to

Mahender Singh College of Pharmacy, Palwal

(Affiliated to Pt. B. D. SHARMA UNIVERSITY OF HEALTH SCIENCES, Rohtak)
Deeghot, Palwal
Palwal (121102) Haryana India

JULY, 2024
 BONAFIDE CERTIFICATE

Certified that Atal Baghel (University Roll No.: 335032) has carried out the project work

presented in this report entitled “Analysis of Analgesic Drug by Thin Layer Chromatography

( TLC) for the award of Bachelor of Pharmacy (B. Pharmacy) Course from Pt. B. D. Sharma

University of Health Sciences, Rohtak under our/ my supervision.

The report embodies the result of review work, and studies are carried out by the student himself.

Mr. Yashpal Singh Dr. Vivek Gupta

Assistant Professor Principal

(MSDP) (MSDP)

(Supervisor)

Forwarded By :
Atal Baghel
 ACKNOWLEDGEMENT

Acknowledgement & celebration are very essential to fuel passion, making people feel valid & valuable,
and giving the team a real sense of progress that makes it all worthwhile.

With this real feeling, I consider myself fortunate enough to acknowledge my all inspirers and well-wishers.
Since, 4 years journey of graduation, I came in contact with many kind-hearted people who ensured my
success for project submission. This is high time to express my forehanded gratitude for them. I can hardly
find any word enough to pay homage to the Almighty God and my parents whose tremendous support,
encouragement, love and blessing were a real source of inspiration and will remain so forever

I am very grateful to Dr. Vivek Gupta Principal, MAHENDER SINGH COLLEGE OF PHARMACY,
for providing the necessary infrastructure and resources to accomplish my research work and for sharing
his invaluable scientific knowledge and experience during my study.

At this moment of accomplishment, I would like to express my deepest sense of appreciation and
profound gratitude to my revered supervisor,Mr Yashpal Singh, Associate Professor, MAHENDER
SINGH COLLEGE OF PHARMACY, for his excellent guidance

I am very grateful to Ms. Kanika Mangla, Course Coordinators (8th semester) for providing the
necessary support, knowledge and resources to accomplish my graduation and project work and for
sharing their invaluable scientific knowledge and experience during my Project.

Finally, I would like to thank everybody important to the successful realization of the project.

(Atal Baghel)
 TABLE OF CONTENTS

S.NO. CONTENTS PAGE NO.

1. Chapter 1- Introduction 6

2. Chapter 2 – Aims & 0bjectives 7-12

3. Chapter 3 - Literature Review 13-14

4. Chapter 4 – Methodology 15

5. Chapter 5 – Result & Discussion 16-19

6. Chapter 6 – Conclusion 20

7. Chapter 7 – References 21-22

 LIST OF FIGURES

FIGURE TOPIC PAGE NO.

NO.

2.1 Wide Mouth Bottal 8

2.2 TLC Plates 9

 LIST OF TABLES

S. NO. T PAGE NO.

O
P
I
C

5.1 Observation Table 16-19

Conclusion Table
 ABSTRACT

Objective:
Identification of compounds Thin layer chromatography can be employed in
purification, isolation and identification of natural products like volatile oil or
essential oil Fixed oil, waxes, alkaloids, glycosides, steroids etc.
Examination of reaction Reaction mixture can be examined by thin layer
chromatography to access whether the reaction is completed or not. This method
is also used in checking other separation process and purification process like
distillation molecular.

METHOD :

Dissolve a few milligrams of material in a volatile solvent creating a dilute

solution creating a dilute solution. Choose a volatile solvent that completely
dissolve the sample. However, if it is partially soluble, since such only low
concentration are needed, normally you will be able to observe the compound.

Once the sample is prepared, a spotting capillary must be used to add the sample
to the plate.

RESULT:

Analgesic drug sample were taken. The sample solution was prepared in ethanol.
The analgesic drug was analysed and separating by thin layer chromatography
technique.. Separation of mixture of analgesic drugs (unknown drugs) were done
by TLC Method.
 INTRODUCTION

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.
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.

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 chromatography's (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, eater, 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.

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 lor thermal unstate,
annonvoa 1 c samples.

The purpose of applying chromatography which is used as a method () quantitative analysis

apart from its separation, is to achieve a satisfactory separation within a suitable time
interval. 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.

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

• Pseudo affinity chromatography

• High-pressure liquid chromatography (HPLC)

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. The separation of analytes is achieved.
This upward traveling rate depends on the polarity of the material, solid phase, and of
the solvent. In cases where molecules of the sample are colorless, florescence,
radioactivity or a specific chemical substance can be used to produce a visible color
reactive product so as to identify their positions on the chromatogram. Formation of a
visible color 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 distances
travelled by the molecule and the solvent. This measurement value is called relative.
Aim To analyze the analgesic drugs by using thin layer chromatography techniques.

Objective

1. To learn the analytical technique of thin layer chromatography.

2. To identify the compounds based on the Re value.
3. To investigate the effect of solvent system polarity on TLC Re value retention times.
4. To check purity of given sample.
5. Identification of compounds like acids, alcohol, protein, antibiotic, steroids etc.
6. To Evaluate the Reaction process by using purification process.
7. To keep a check on the performance of other separation processes.
8. Purity of any sample purity of sample carried out with TLC. Comparison is done
between the sample and the standard or authentic sample; if any impurity is
detected, then it shows extra spots and this can be detected easily.
9. Identification of compounds Thin layer chromatography can be employed in
purification, isolation and identification of natural products like volatile oil or
essential oil Fixed oil, waxes, alkaloids, glycosides, steroids etc.
10. Examination of reaction Reaction mixture can be examined by thin layer
chromatography to access whether the reaction is completed or not. This method
is also used in checking other separation process and purification process like
distillation molecular.
11. Biochemical analysis TLC is extremely useful in isolation of separation of
biochemical metabolites or constituents from its body fluids, blood plasma,
serum, urine etc.
12. In TLC methodology is increasingly used in chemistry for separation and
identification of compounds which are closely related to each other. It is
inorganic chemistry.
13. In pharmaceutical industry Various pharmacopoeias have adopted TLC technique
for detection of impurity in a pharma chemical.
14. Various medicines like hypnotics, sedative, anti-convulsant, tranquillizer,
analgesics, local anesthesia, steroids have been tested qualitatively by TLC
method.
15. In food and Cosmetics industry, TLC method is used for separation and
identification of color preservative and sweetening agents, various cosmetics
products.
 REVIEW OF LITERTAURE

1. M algorzateDolowy and Zagrodzka et al gives that mesterolone is a synthetic

androgenic steroid indicating a weak anabolic activity. A new, sample in use, and
economical TLC densitometric method in normal phase system (NP-TLC) has
been developed and validated for the identification and quantitative
determination in bulk drug and tablet formulation (NP-TLC) analysis was
performed on plates pre-coated with silica gel 60254as the stationary phase
using chloroform- acetone (4010, V/V) as mobile phase. Densitometric analysis
was carried out at 745nm after staining with phosphomolybdic acid. oulieucta.

2. S. Kumar et al concluded that In this study, an attempt was made to review the
basic principles and the importance of TLC in research in general and in
phytochemistry in particular Thin layer chromatography is a simple, cost
effective, and easy to operate planer chromatographic technique which has been
used in general chemistry laboratories for several decades to routinely separate
chemical and biochemical compound. Traditionally, chemical and optical method
are employed to visualize to analyte spots on the TLC plate. Also it has a wide
application in identified impurities in a compounds. Study highlights the review
on TLC and its application of quantitative estimation of bioactive compounds
from medicinal plants

3. Julian Heep et al conclude that desorption/ ionization included by neutral

clusters (DINeC) was employed for mass spectroscopy of oligopeptides and lipide
after separation by means of thin layer chromatography, Clear and
fragmentation free spectra were obtained from the TLC plates without any
sample treatment. Mass resolvedchromatograms were deduced when scanning
the TLC plates with the cluster beam along the direction of solvent movement.
Using vancomycin and non-covalently bound complexes, the soft nature of
DINEC movement was demonstrated also when used in combination with TLC. As
a test application, TLC and DINeC-MS were employed to separate and detect
different phospholipids obtained from egg yolk.
4. Arya Aman Karim et al conclude that Diabetes mellitus (DM) is a group of
metabolic disorders that are characterized by hyperglycemia which results from
defects in insulin release or its efficient use by the human body. Although
significant progress has been made to manage DM and related complications, it
remains a major global health problem. To this end, the search for new
antidiabetic drugs from traditionally claimed medicinal plants is important. Aloe
megalacantha Baker is an endemic plant used traditionally to treat diabetes in
Ethiopia. This study aimed to investigate antidiabetic activity of isolates from the
leaf of A. megalacantha Baker in streptozotocin-induced diabetic mice.

5. Ammar Altemimi et al concluded that the use of medicinal plants has been
reported throughout human history. In the fight against illnesses, medicinal
plants represent the primary health care system for 60% of the world's
population. Flavonoids are polyphenolic compounds with active anti- microbial
properties; they are produced in plants as pigments. Quercetin, myricetin, and
rutine are among the most well-known and prevalent flavonoids in plants, with
an antioxidant activity capable of decreasing the oxidation of low-density
lipoproteins [LDLs). To date, this research is the first of its kind to employ a
coupled thin-layer chromatography (TLC) and a densitometric quantification
method with a Box-Behnken design (BBD) response surface methodology (RSM)
for optimization of ultrasonic-assisted extraction and determination of rutin and
quercetin from peach and ellagic acid and myricetin from pumpkin fruits.

6. Colin F Poole et al., concluded that the purpose of this article is to identify core
technologies with the potential to influence the development of thin-layer
chromatography over the next decade or so. Core technologies are identified as
(i) methods to provide a constant and optimum mobile phase velocity (forced
flow and electro osmotically driven flow). (ii) video densitometry for recording
multidimensional chromatograms, (iii) in situ scanning mass spectrometry, and
(iv) bioactivity monitoring for selective detection. In combination with two-
dimensional, multiple development and coupled column- layer separation
techniques these core technologies could dramatically increase the use of thin-
 layer chromatography for the characterization of complex mixtures. It is also
demonstrated that thin-layer chromatography has strong potential as a
surrogate chromatographic model for estimating biopartitioning properties. To
convert these opportunities into practice the current state of the art of the core
technologies is described and the principle obstacles to progress identify

7. llowarth et al currently there is paucity of evidence in the literature in relation

to normative values for diffusing capacity of carbon monoxide (DLCO) and total
lung capacity (TLC) among Indigenous Australians Hence, in this study we
assessed the DECO and TLC parameters among Indigenous Austrations in
comparison to Australian Caucasian counterparts.

8. Mahmoud A. Tantawy et al described sensitive, accurate and precise

spectrophotometric, TEC densitometric and High Performance liquid
chromatographic (HPLC) methods for simultaneous determination of olanzapine
and fluoxetine HCL Two spectrophotometric methods were developed namely
first derivative (D1) and derivative ratio (DDT) methods. The TLC method
employed aluminum TLC plates precoated with silica gel GF254 as the stationary
phase and methanol toluene ammonia (7 301 by volume) as the mobile phase,
where the chromatogram was scanned at 235 mm The developed HPLC method
used a reversed phase C18 column with sociaticcution. The mobile phase
composed of phosphate buffer pH 4.0 acetonitrile triethylamine 153 47.0.03 by
volume) at flow rate of 1.0 ml min! Quantitation was achieved with UN detection
at 235 mm. The methods were validated according to the International
Conference on Harmonization (ICH) guidelines. The selectivity of the proposed
methods was tested using laboratory - prepared mixtures. The developed
methods were successfully applied for the determination of olanzapine and
thoxetine HCI in bulk powder and combined capsule dosage form.

9. Karideste Streptomisin Sülfat et al developed thin layer chromatography

(TLC)-bioautography for identification and quantification of streptomycin sulfate
in shrimp. Materials and Methods TLC of streptomycin sulfate standard solation
was carried out using silica gel F254 and 7.5% of KH2PO4 solution as stationary
and mobile phase, respectively. Result The retardation factor of the streptomycin
 sulfate standard was 0.51 and the selectivity of streptomycin sulfate was 4.1 with
the presence of kanamycin sulfate in the shrimp.
10. Emily Kerr, Caroline West, et al Digital image analysis of the separation results
of colorless analytes on thin layer chromatography (TIC) plates usually involves
using tail software either a UV scanner or UV lamp station with a digital camera
or a densitometer. Here, a low cost alternatives setup for quantitative TLC digital
image analysis is demonstrated using a universal staining reagent (iodine vapor),
an office scanner and a commonly available software (Microsoft Paint) for
analysis of red, green and blue colors (RGB values) Urinary creatinine is used as a
model analyte to represent a sample in complicated biological matrices.
Separation was carried out on a silica gel plate using a butanol - NH4OH - H20
(401050, v/v) mobile phase with a 6cm solvent front.
11. Waldemar Vollmer et al; Peptidoglycan encases the bacterial cytoplasmic
membrane to protect the cell from lysis due to the turgor. The final steps of
peptidoglycan synthesis require a membrane an chored substrate called lipid II,
in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via
a pyrophosphate moiety. Lipid II is the target of antibiotics and several
antimicrobial peptides, and is degraded by attacking enzymes involved in
bacterial competition to induce lysis. Here we describe two protocols using thin
layer chromatography (TLC) and high pressure liquid chromatography (HPLC),
respectively, to assay the digestion of lipid II by phosphatases such as Calcium M
or the LXG toxin protein from Streptococcus intermedius. The TLC method can
also monitor the digestion of undecaprenyl (pyrophosphate, whereas the HPLC
method allows to separate the di-, mono- or unphosphorylated disaccharide
pentapeptide products of lipid II.
12. Isabelle A Kagan et al described the common screen for plant antimicrobial
compounds consists of separating plant extracts by paper or thin layer
chromatography (PC or TLC), exposing the chromatograms to microbial suspensions
(eg fungi or bacteria is in broth or agar), allowing time for the microbes to grow in a
humid environment, and visualizing zones with no microbial growth. The
effectiveness of this screening method,known as bioautography, depends on both
the quality of the chromatographic separation and the care taken with microbial
culture conditions. This paper describes standard protocols for TLC and contact
biography with a novel application to amino acid fermenting bacteria. The extract is
separated on flexible (aluminum backed) silica TLC plates, and bands
 are visualized under ultraviolet (UV) light. Zones are cut out and incubated face
down onto agar inoculated with the test microorganism. Inhibitory bands are
visualized by staining the agar plates with tetrazolium red. The method is applied
to the separation of red clover phenolic compounds and their screening for
activity against Clostridium hyper ammonia producing bacterium (HAB) that is
native to the bovine rumen.
13. S.J. Thakkar et al Khanda, an important ploy Herbal mineral Ayurvedic
formulation was analyzed by employing various parameters which included
physicochemical parameters, quantitative estimation of sugar and TLC profile
The evolved parameters will be useful for quality control.
14. Emily Kerr, Caroline West, and Supaporn Kradtap Hartwell et al Digital
image analysis of the separation results of colorless analytes on thin layer
chromatography (TLC) plates usually involves using special tail software image
generated from either a UV scanner or UV lamp station with a digital camera or a
densitometer Here, à low cost alternatives setup for quantitative TLC digital image
analysis is demonstrated using a universal staining reagent (iodine vapor), an office
scanner and a commonly available software (Microsoft Paint) for analysis of red,
green and blue colors (RGB values). Urinary creatinine is used as a model analyte to
represent a sample in complicated biological matrices. Separation was carried out
on a silica gel plate using a butanol NH4OH H20 (401050, v/v) mobile phase with a
6cm solvent front. It is important that the TLC plate be stained evenly and with
sufficient staining time. Staining the TLCplateina23.4-188-68 on chamber containing
about 70g iodine crystals yielded comparable.
15. Catherine E. Costello et al Gangliosides and sulfatides (STS) are acidic
glycosphingolipids (GSE) that have one or more stack acids or sulfate substituents, in
addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long-
chain base. TLC is a widely employed and convenient technique for separation and
characterization of GSLs. When TLC is directly coupled to MS, il provides both the
molecular max and structural information without further purification. Here, after
development of the TLC planes, the structural analyses of acidic GSLA, including
gangliosides and STs, were investigated using the liquid extraction surface analysis
(LESA) and CAMAG TEC-MS interfaces coupled to an ESI OSTAR Pulsar quadrupole
orthogonal TOP mass spectrometer Coupling TLC with ESI - MS allowed the
acquisition of High mass spectra of the cific GSLs with high sensitivity and in mass
accuracy, without the loss of sialic acid residues that
 frequently occurs during low-pressure MALDI MS. These systems were then
applied to the analysis of total lipid extracts from bovine brain. This allowed
profile of many different lipid classes, not only glucoside and STs, but also SMS,
neutral GSLs, and phospholipids.
16. Daniel W. Vomhof et al thin layer chromatography (TIC) a new analytical tool,
has found many applications in the separation and qualitative identification of small
quantities of organic compound The simple sugars are one clinics of compounds for
which TLC as yet has not been satisfactory The present work investigates possible
procedures to improve the usefulness of cellulose thin layers in separating sugars
Several solvent systems previously used in the paper chromatographic separation of
sugars were evaluated with this medium Two colorimetric procedures previously
used for the quantitative analysis of sugars separated by paper chromatography
have been employed in this study is an effort to make thin layer chromatography
even more effective for the analysis of signs The applicability of these methods to
the field of plan physiology is demonstrated using Gossypium var 4-42 as the test
plant. The sugars present in the free state in various tissues of this plant were
separated and qualitatively identified. The quantities of fructose, glucose, and
sucrose were then determined for each tissue.
17. R. Aliso - Fernandez et al we describe the history and current implementation
of an (TL) method, vertical sandwich - type layer chromatography upitnous
evaporative TLC with fixed mobile phase volume, that is convenient for detecting
and identifying reaching sugars of clinical relevance in the paper - borne blood
and urine samples collected in tall screening This method facilitates screening by
providing a considerable degree of standardization of chromatographic results.
Among some 555,000 newborns to which it has been applied has detected 10
cases of classical agacskase deficiency, 2 cases of glucosuria and 3 cases of
transitory neonatal diabetes mellitus the only false negatives we are aware of
two cases of galactose 4 epimerase deficiency detected by in term mass
spectrometry Screening for sugars in urine has allowed the detection when the
accompanying blood sample was invalid because of transfusion or parenteral
feeding. The conclusion is that this inexpensive procedure is very useful for the
detection of relevant information in circumstances where others fail.
18. Bernhard SURHOLT and Eric A. et al The rate of substrate cycling between
glucose and glucose 6-phosphate was measured lists of the hawk moth. 2 The insect
was injected with 12-3H.2-4Ciglucose, and after periods of time at rest or flying the
 animal was freeze clamped. Separation of glucose and hexose monophosphate
from the tissues was performed by paper chromatography and TLC and the 31
and 14C radio activities in these compounds were measured. On the basis of the
3H/4C ratios in these compounds and the measured rate of glycolysis, the rate of
cycling was calculated. The rates of cycling were 0.03, 0.10, 0.06 and
3.9umol/min per g for fat body at rest and during flight and for flight muscle at
rest and during flight respectively. 4 The marked increase in the cycling rate
between glucose and glucose 6-phosphate upon flight contrasts with the finding
of Clark, Bloxham, Holland & Lardy [(1973) Bio chem. J. 134, 589-5971 in the
bumble bee, in which this condition inhibited cycling. It is suggested that the
increased rate of cycling increases the sensitivity of glucose phosphorylation to
changes in the concentrations of effectors of hexokinase should it be necessary
to increase the rate of glycolysis in muscle, for example, to increase power
output of the flight muscle for increased speed of flight.
19. Waldemar Vollmer et al encases the bacterial cytoplasmic membrane to protect
the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis
require a membrane anchored substrate called lipid II, in which the peptidoglycan
subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid
II is the target of antibiotics and several antimicrobial peptides, and is degraded by
attacking enzymes involved in bacterial competition to induce lysis. Here we
describe two protocols using thin layer chromatography (TLC).
20. Lisova and Jin Rios et al gives radio thin layer chromatography (radio - TLC) is
commonly used to analyze purity of radiopharmaceuticals or to determine the
reaction conversion when optimizing radiosynthesis processes. In applications
where there are few radioactive species, radio TLC is preferred over radio high
performance liquid chromatography due to its simplicity and relatively quick
analysis time. However, with current radio-TLC methods, it remains cumbersome
to analyze a large number of samples during reaction optimization. In a couple of
studies, Cerenkov luminescence imaging (CLI) has been used for reading radio
TLC plates spotted with a variety of isotopes. We show that this approach can be
extended to develop a high-throughput approach for radio - TLC analysis of many
samples.
21. Paul E. Kendra et al developed a method that combined thin layer chromatography
(TLC) of tea tree essential oil (TTO) with short range bioassays to isolate attractive
kairomones for male C After development, the TLC chromatogram indicated that
 TTO separated into five major spots, designated as zones 1 to 5. When the TLC
plate was exposed to flies, zones I and 3 were strongly attractive to male C.
capitate to confirm activity, the developed TLC plate was cut into five zones
which were then tested in short - range bioassays. Again, flies were observed to
aggregate around zones 1 and 3, which corresponded with Rf values of 0.93 and
0.59. In addition, zones 1 to 5 were separated u sing preparative - TLC, and
olfactory responses to volatile emissions from the five fractions were quantified
by electroantennography (EAG). Highest amplitude EAG responses were
recorded with fractions 1 and 3, further supporting the bioactivity of these
samples. In conclusion, a TLC based bioassay system can provide an effective,
rapid screening protocol for initial isolation of insect kairomones from complex
mixtures such as essential oils or plant extracts. Further analysis of TTO fractions
1 and 3 is needed to identify the specific constituents attractive to male C.
22. Jesus Perez et al TLC has traditionally been used to analyze lipids isolated from
membrane complexes Here, we describe a method based on the combination of
TLC and SDS-PAGE to qualitatively analyze the protein / lipid profile of
membrane complexes such as those of lung surfactant. For this purpose, native
lung surfactant was applied onto a silica TLC plate in the form of an aqueous
suspension, preserving not only hydrophilic proteins associated with lipids but
also native protein lipid interactions. Using native membrane complexes in TLC
allows the differential migration of lipids and their separation from the protein
components. As a result, (partly) delipidated protein - enriched bands can be
visualized and analyzed by SDS PAGE to identify proteins originally associated
with lipids. Interestingly, the hydrophobic surfactant protein C, which interacts
tightly with lipids in native membrane complexes, migrates through the TLC
plate, configuring specific bunds that differ from those corresponding to lipids or
proteins. This method therefore allows the detection and analysis of strong
native like protein - lipid interactions Lopez Rodriguez, E., N. Roldan, B. Garcia -
Alvarez, and J. Pérez - Gil, Protein and lipid fingerprinting of native like
membrane complexes by combining TLC and protein electrophoresis.
 METHODOLOGY

REQUIREMENT

• Apparatus used: Beaker, Glass rod, TLC plate, Development chamber, capillary
tubes, TLC spreader, Iodine chamber, Hot air oven, Measuring Scale, Pipette,
Measuring cylinder etc.

• Chemical used: Silica gel G Powder, Distilled water, Aspirin, Paracetamol,

Acetone, Acetic Acid, Chloroform, Caffeine Powder, Ibuprofen Powder

PROCEDURE:

A. Preparation of TLC Plate: Thin layer chromatographic plates were prepared by

silica gel G slurry and prepared by pouring method. Then TLC plates were air
dried for 15-20 minutes and then activated by keeping in Hot air oven for 1 hour.
B. Spotting the TLC plate
One advantage TLC has over other separation methods, is that it is truly a
microscale technique.

Only a few micrograms of material in solution is necessary to observe the solute

on a TLC plate.

Dissolve a few milligrams of material in a volatile solvent creating a dilute

solution creating a dilute solution. Choose a volatile solvent that completely
dissolve the sample. However, if it is partially soluble, since such only low
concentration are needed, normally you will be able to observe the compound.

Once the sample is prepared, a spotting capillary must be used to add the sample
to the plate.
 The spotting capillaries must be extremely small. In fact, the opening at the end
of a regular Pasteur pipet is too big for spotting a TLC plate. CG capillary columns
denoted by restek, a chromatography company located in Bellefonte, are used to
spot the plates. This column are very small bore and can be cut into three inch
sections to provide very good TLC spotting tubes. The solution can be drawn up
the tube by capillary action (hence the name) and spotted on the plate at the
hash mark labeled in pencil. This is known as the origin and is shown below. Since
a TLC can run three, if not four mixtures at one time, it is very important to
properly label the plate. Notice that pencil is always used to mark a TLC plate
since the graphite carbon is inert.

If organic link is used to mark the plate, it will chromatograph just as any other
organic compound and give correct results.

To spot the plate, simply touch the end of capillary tube to the coated side of the plate.

The solvent should evaporate quickly leaving your mixture behind on the plate. you may
have to spot the plate a couple of times to ensure the material present, but do not spot
too much sample.
If too much solute is added to the plate, a poor separation will result.

Smearing, smudging and spots that overlap will result making identification of separated
component difficulties.

C. DEVELOPMENT- Once the dilute solution of the mixture has been spoted on the
plate, the next step is the development, just like paper chromatography the
solvent must be in contact with the stationary phase below figure shows a wide
mouth bottle commonly used to develop TLC plates

The bottle is filled with a small amount of mobile phase and capped with a cork. In
addition a piece filter paper is put in the bottle to help create an atmosphere saturated
with solvent. use your tweezers to place the plate in the development chamber oils
from your fingers, can sometimes smear or ruin a tie plate. Also make sure the origin
spot are not below the solvent level in the chamber. If the spot are submerged in the
solvent, they are washed off the plate and lost, once the solvent has run with in a
centimeter of the top of the plate, remove it with tweezers. Using a pencil, immediately
draw a line across the plate where the solvent front can be seen the proper location of
this solvent front line will be important for later calculation.

D. VISUALIZATION-
Visulization of spot were done by keeping them in the iodine chamber (saturated
with iodine vapour). The spot were visualized as dark spot on the TLC Plates.
Another techniques for visualization are such as Some organic compound are
colored. If you are fortunate enough to be separating organic molecules that are
colored such as dyes, inks or indicator, then visualizing the separated spot is easy.
However, since most organic compound are colorless, the first method does not
always work.

In most cases observing the seprated spot by uv light works well.tic plate
normally contain a fluorescent indicator which make the tic plate glow green
under uv light of wavelength 254 nm.

Compound that absorb uv light will quench the green fluorescence yielding the
dark purple or bluish spot on the plate, simply put the plate under a uv lamp, and
the compound become visible to the nacked eyes lightly circle the spot, so that
you will have a permanent record of their calculation.

Another useful visualizing technique is an iodine chamber, iodine sublimes and

will absorb to organic molecule in the vapor phase. The organic spot on the plate
will turn brown and can be easily identify. Also circle these absorb spots, since
the color stain will eventually fade from the plate. sometimes a combination of
both UV lamp and iodine is needed to observe all the spots. some compound are
not UV active that is, they do not absorb light at the wavelength of 254mm. Using
both method will ensure correct identification of all the spot on the TLC plate.
 E. Observations & Calculations:
The Rf value is the "reatardation factor" or the "ratio-to-front" values expressed
in the decimal fraction,

Observation analysis of analgesic drug by using thin layer chromatography

Analysis of Paracetamol
Rf value Distance travel by solute/ Distance travel by solvent
Rf value= 7.26

= 0.66
11

Analysis of Aspirin

Rf value Distance travel by solute/Distance travel by solvent

Rf value=711.04 = 0.64

Analysis of lbuprofen

Rf value Distance travel by solute/Distance travel by solvent

Rf value=311.85 = 0.35

Analysis of Caffeine

Rf value Distance travel by solute Distance travel by solvent

Rf value=1011.12 = 0.92

Observation table

s.no. Name of Drugs Observed or calculated Reference Rf value

Rf value (Literature Values)

1. Paracetamol 0.66 0.64

2. Aspirin 0.64 0.60

3. Ibuprofen 035. 0.40

4. Caffeine 0.92 0.89

 RESULT AND DISCUSSION

Analgesic drug sample were taken. The sample solution was prepared in ethanol. The
analgesic drug was analysed and separating by thin layer chromatography technique..
Separation of mixture of analgesic drugs (unknown drugs) were done by TLC Method.
Firstly we have determine which components is to be separated. We have searched the
literature with standards of each of the components in order to compare against your
unknown. Once you have run a TLC of our mixture of unknown drugs and all of the
standards, We have visualized the spots by using iodine chamber and under the
Ultraviolet cabinet/(UV) light. The dark spot were appeared which indicates one
component of mixture or unknown drugs. Compounds that are UV active will appear
when the TLC plate is exposed to UV light. To compare the spots, we have measured the
Rf (retention factor) of each separated component Then Rf values of separated
component is compared with value to each of the standards. Rf values of separated
drugs were found to be

Paracetamol-0.66

Aspirin -0.64

Ibuprofen-0.35

Caffeine-0.92
 Conclusion
Analgesic drug sample were taken. The sample solution was prepared in ethanol. The
analgesic drug was analysed and separating by thin layer chromatography technique. The Rf
value of standard drug and mixture of two drugs (unknown sample) was calculated

s.no. Name of Drugs Observed or calculated Reference Rf value

Rf value (Literature Values)

1. Paracetamol 0.66 0.64

2. Aspirin 0.64 0.60

3. Ibuprofen 035. 0.40

4. Caffeine 0.92 0.89

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