FRS581 FORENSIC CHEMISTRY

Experiment 4:
TLC OF STEROIDS IN HERBAL MEDICINE

NAME: MUHAMAD NAZIM MUHAIMIN BIN AHMAD NASZERI

STUDENT ID: 2017345949

GROUP: AS2534B1

LECTURER’S NAME: SIR MOHAMED IZZHARIF BIN ABDUL HALIM

DAY & DATE OF EXPERIMENT: 26 MARCH 2019

DATE OF SUBMISSION: 2 APRIL 2019

GROUP MEMBER:

1. SITI AISHAH BT M USMAN (2017722963)

2. NADIA SHALINI BT MAZLAN (2017714037)
3. NURUL AZWANIE BT ROSLAN (20177027125)
4. NIK NURUL HANIS BT NIK MOHD KAMAL (2017941993)
EXPERIMENT 4: TLC of Steroids in Herbal Medicine

OBJECTIVE

To detect and visualize the presence of steroid in traditionally prepared herbal medicine.

INTRODUCTION

Steroids are organic compounds with a set chemical structure. Some steroids, such as
cholesterol, estrogen and testosterone, are quite common. There are also hundreds of others
found in animals, plants and even fungi, and each of these reacts a different way within the
organism, leading to a variety of effects. Human bodies naturally produce and use steroids to
aid in reproduction, regulate metabolism and enhance muscle and bone growth. As such, they
are very useful chemicals and, if used properly, can have numerous benefits. Within the media,
most negative publicity goes to steroid hormones, which can be used to trigger certain bodily
effects if taken. These substances can be classifies as sex steroids (gonadal steroids),
corticosteroids and anabolic steroids. Most cases of steroids abuse or misuse typically involve
anabolic steroids, although there have been some cases of where corticosteroids are the
substances of choice as well.

PROCEDURE

1. Mortar and pestle was used for powdered the sample.

2. All samples were protected from light and kept in desiccators at room temperature until
analysis.
3. Stock standard solution (0.5 mg/mL reference standard of steroids) was prepared in
dichloromethane.
4. 0.1 mg/Ml working solution was prepared by dilution of the standard solution in
dichloromethane.
5. Sample solution was prepared by extracted 1 g of each powdered sample with 5 mL of
dichloromethane.
6. Vortex was used for the mixture mixed thoroughly/
 7. Extraction solutions of samples and standard solutions of steroids was separated using
capillary tube onto 20x20 cm TLC plates containing a 250µm thick silica gel stationary
phase.
8. Mobile phase A that consisted of dichloromethane: methanol (9:1) and mobile phase B
that consisted of cyclohexane: ethyl acetate (1:3) were prepared.
9. The spotted TLC plate was placed in a closed chamber containing a layer of mobile
phase for 1 hour.
10. The plate was removed and dried at room temperature before the solvent front reached
the end of TLC plate.
11. The resulting TLC pattern was viewed under shortwave at 254 nm ultraviolet light.
12. The plate was sprayed with 10% sodium hydroxide solution and 1 % methanolic
tetrazolium blue solution.

RESULTS

In mobile phase A ; dichloromethane (9) : methanol (1)

Result Description
UV
-Examine under 312 nm
UV light
-no reaction for all sample
 Fluorescence
-5 sample were separated
for sample 2

-6 sample were separated

for sample 3

In mobile phase B ; cyclohexane (1) :ethyl acetate (1)

Result Description
UV
-Examine under 312 nm
UV light
-no reaction for all sample

Fluorescence
-5 sample were separated
for sample medicine 2

-4 sample were separated

for sample medicine 3
Spray with 10% sodium hydroxide solution and 1 % methanolic tetrazolium blue solution

Result Description
Mobile phase A
-Reference sample turns to
purple color
-Sample 1 turns to yellow
color
-No color change for
sample 2 and sample 3

Mobile phase B
-Sample 1, sample 2 and
sample 3 turns to yellow
color
-No color change for
reference sample
Reference value (Rf)

Sample Rf value
Mobile phase A Mobile phase B
Reference sample 0.5 -
Sample 1 0.86 0.67
Sample 2 - 0.93
Sample 3 - 0.93

DISCUSSION

In this experiment, there were 3 sample of herbal medicine used. Traditional diarrhea medicine
(sample 1), Phytonatal (sample 2) and Shaklee supplements (sample 3) were observed of their
steroids content with the reference sample. Two types of TLC mobile phase were used for the
further analysis. For mobile phase A it consist of dichloromethane : methanol (9:1 v/v) while
cyclohexane : ethyl acetate (1:3 v/v) for mobile phase B. The resulting TLC pattern was views
under shortwave at 312 nm ultraviolet light. All of the sample showed no reaction under UV
light. Furthermore, it also had been visualized under fluorescence. By using TLC using mobile
phase A, sample 2 and 3 are reacted under the fluorescence but not for reference sample and
sample 1. There were 5 sample separated in sample 2 while 6 sample separated in sample 3.
Meanwhile, in mobile phase B, both sample 2 and sample 3 also reacted under fluorescence.
TLC by using this mobile phase were managed to separate 5 compounds for sample 2 and 4
compounds for sample 3.

Both of the TLC plate with mobile phase A and mobile phase B were sprayed with 10% sodium
hydroxide solution and 1 % methanolic tetrazolium blue solution which to look up their
sensitivity, selectivity and specificity. In TLC plate with mobile phase A the reference sample
showed the purple color and yellow color for sample 1. Both sample 2 and 3 did not showed
any color changed. Meanwhile, in TLC with mobile phase B, only sample 1 showed the color
change which is yellow color and the other three samples did not showed any color change.

Thin layer chromatography (TLC) is a chromatographic technique used to separate the

components of a mixture using a thin stationary phase supported by an inert backing. It may be
performed on the analytical scale as a means of monitoring the progress of a reaction, or on the
preparative scale to purify small amounts of a compound. TLC is an analytical tool widely used
because of its simplicity, relative low cost, high sensitivity and speed of separation.

Similar to other chromatographic methods, thin layer chromatography was also based on the
principle of separation. The separation depends on the relative affinity of compounds towards
stationary and mobile phase. The compounds under influence of the mobile phase that driven
by capillary action, travel over the surface of the stationary phase. During this movement, the
compounds with higher affinity to stationary phase travel slowly while the others travel faster.
Thus separation of components in the mixture is achieved. Once operation occurs, the
individual components are visualizes as spots at a respective level of travel on the plate. Their
nature or characters are identified by means of suitable detection techniques.

The amount that each component of a mixture travels can be quantified using retention factors
(Rf). The retention factor of a particular material is the ratio of the distance the spot moved
above the origin to the distance of the solvent front moved above the origin. It can be calculated
using the formula:

distance spot travel

𝑅𝑓 =
distance solvent moved

Retention factors were useful in comparing the results of one chromatogram to the results of
another. The retention factor for a given material will remain constant with the conditions in
which chromatogram were ran unchanged (same mobile and stationary phase). This allows
unknowns to be compared to known materials. The unknown and known materials were not the
same compound if the retention factor does not match each other. Similar retention factors
suggest that two samples could be the same.

Some steroids can be incredibly harmful to people who addictively take them. People that abuse
anabolic steroids were related to improve physical performance and muscle growth. However,
in the purpose to improve in strength and performance, it also many unwanted short term effects
include acne, mood swing, fatigue, restlessness or agitation, decreased appetite, trouble
sleeping and decreased sperm count. Basically anabolic steroids which is typically liquids were
injected into areas of muscle that might cause the injection sites with infections or swelling. In
those taking doses up to 100 times the medically-appropriate levels, many side effect caused
by steroids will occur. Steroids’ side effect include shrinking of the testicles, excessive hair
growth in women, fertility issues, heart problems, elevated bloos pressure and stroke. In the
long term, anabolic steroid abuse can cause anger and aggression, paranoia, heart attack, stroke,
kidney failure and tumors in the liver.

Steroid also can be detected and analyzed by GC-MS apart from TLC. Many commercial GC-
MS instrument can provide the ultimate speed, accuracy and sensitivity. With the ability to
record and store in its memory several hundred mass spectra, such a system can detect and
identify substances present in only one-million-of-a-gram quantities. Furthermore, the
computer can be programmed to compare an unknown spectrum against a comprehensive
library of mass spectra stored in its memory. Therefore, the retention time of the steroids peak
and its mass spectrum may give an absolute identification of steroids.

Drug problems in Malaysia seem to be on the increase despite harsh penalties for those caught
supplying the drug. In Malaysia, opiates were the most commonly abused drug which is heroin
while the second most widely abused drug was methamphetamine. There was a growing market
for amphetamine type stimulants.

CONCLUSION

In conclusion, the entire samples (sample 1, sample 2 and sample 3) do not have any steroid
based on thin layer chromatography analysis.

QUESTIONS

1. Discuss the fundamental principle of thin layer chromatography.

Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a
solid (stationary phase) and a liquid (moving phase). Solids most commonly used in
chromatography are silica gel (SiO2 x H2O) and alumina (Al2O3 x H2O). Both of these
adsorbents are polar, but alumina is more so. Silica is also acidic. Alumina is available in
neutral, basic, or acidic forms. Thin Layer Chromatography (TLC) is a sensitive, fast, simple
and inexpensive analytical technique. It is a micro technique; as little as 10-9g of material can
be detected, although the sample size is from 1 to 100x10-6 g. TLC involves spotting the sample
to be analyzed near one end of a sheet of glass or plastic that is coated with a thin layer of an
adsorbent. The sheet, which can be the size of a microscope slide, is placed on end in a covered
jar containing a shallow layer of solvent. As the solvent rises by capillary action up through the
adsorbent, differential partitioning occurs between the components of the mixture dissolved in
the solvent the stationary adsorbent phase. The more strongly a given component of a mixture
is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the
more slowly it will migrate up the plate.

2. What it the justification for using Rf for comparison purpose?

Retention factors are useful in comparing the results of one chromatogram to the results of
another. If the conditions in which the chromatogram are run are unchanged (same mobile and
stationary phases), the retention factor for a given material should remain constant. This allows
unknowns to be compared to known materials. If the retention factor of an unknown does not
match that of a known material, they are not the same compound. Similar retention factors
suggest that the two samples could be the same, but is not proof. In reality, the retention factors
will vary slightly from sample to sample. Interactions of the individual components with each
other and the concentration of the component in the sample will both affect the Rf value.

REFERENCE

1. Saferstain.R (2015). Criminalistics-An Introduction to Forensic Science. Drug abuse

and drug evidenve.(11th ed., pp.278-311).

2. Clark.J (2007). Thin Layer Chromatography. Retrieved on November 15, 2016 from
http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html.