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EXPERIMENT 4: TLC of Steroids in Herbal Medicine


To detect and visualize the presence of steroid in traditionally prepared

herbal medicine.


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.


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 250m 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.


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

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

-6 sample were
separated for sample

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

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

-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

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


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

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.

In conclusion, the entire samples (sample 1, sample 2 and sample 3) do

not have any steroid based on thin layer chromatography analysis.


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