FLR 2 (1)
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Chemistry
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Jun 11, 2024
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Identification of an Unknown Drug By a Thin Layer of Chromatography
Experiment Performed: 1/23/2024
Introduction:
In the following experience, we were tasked to identify an unknown sample and or substance by
identifying the active ingredients present by using the Rf values determined by a thin layer of
chromatography (TLC). By comparing the Rf values of pure chemicals (Aspirin,
Acetaminophen, caffeine) to the Rf values to determine for the unknown sample of the identity
of the unknown and said drug can be determined.
Scheme/Equation
Equations and Calculations:
Rf: distance traveled by the spot/ distance traveled by the solvent.
Caffeine: 3.1 / 6 = 0.52
ASA: 4.1 / 6 = 0.68
AC: 3.5 / 6 = 0.58
U: 3.2 / 6 = 0.53
Sample
Acetaminophen
Caffeine
Acetylsalicylic Acid
Unknown
#___
Rf
0.58
0.52
0.68
0.53
Safety/Hazards:
Use gloves or forceps when working with silica gel on the TLC plates. When working with
UV light, DO NOT look directly at it as it damages your eyes. Always wear your goggles.
Procedure:
Obtain the 3 known compounds (Caffine, Aspirin, and Acetaminophen) and the 1 NSAID drug
sample (make sure to write the number of the unknown in the lab notebook). Find the Rf of the
known compounds and of the unknown sample by using the TLC analysis and through
observation of the spots. Prepare each sample, add a micro spatula of each sample into a vial of 2
ml of MeOH. Then gently draw on the base of the TLC plate with a pencil. Afterwards, spot the
dissolved solution on the plate by using a capillary tube. Place the TLC plate in the chamber w/
2-5 mL of ethylacetate. Make sure the TLC plate leaning on the side and not stuck on the side of
the beaker. Allow the plate to develop until the solvent line is near the top. Remove the TLC
plate with forecups, and draw in pencil a line along the solvent front so you can calculate the Rf,
irradiated with the UV lamp. Circle the spots with a pencil. Then sketch the developed TLC plate
to scale in your lab notebook for all 4 samples calculate the Rf values using the ruler and then
record them in the lab notebook. Once the Rf values have been calculated, run the unknown
compound with those present in the unknown sample, Finally, report the identity of the unknown
drug and provide rationale for the selection. For waste disposal place all the solvent waste in the
non-nal waste container.
Observations:
Some of the plates were lined up straight, but the instructor mentioned how it doesn’t have to
be 100% straight and long as the whole.
Discussion Questions:
1. Why must the baseline be above the solvent level in the TLC chamber?
The baseline must be above the solvent level in the TLC chamber because if the baseline is too
low then the samples will dissolve at the beginning of the experiment. Additionally, this will also
contaminate the solvent and it can yield inaccurate results.
2. Why are pencils used on TLC plates and pens never are?
Pens should never be used on TLC plates because the pigments in the ink are soluble in organic
solvents (ethyl acetate) and will also become part of the experiment's results. For pencils as long
as they have a soft edge pencil it won't tamper with the experiment or yield inaccurate results
because a soft pencil wouldn’t rip off the silica gel.
3. Why is it important to not let the solvent line travel past the end of the TLC plate?
It is important not to allow the solvent line to pass past the end of TLC plate because this would
produce a misleading result. Without a clear stopping point, some compounds will mix together
or run off to the edge of the plate. All cases dad to illegibility and incorrect results.
4. If a TLC was run in a solvent system that caused all the spots to show up at the baseline, how
could the system be adjusted so that useable Rf values could be determined?
If the TLC plate was run in a solvent system that caused all the spots to remain at the baseline,
increasing the polarity of the solvent would give useable Rf values with proper power the solvent
has. Ethyl acetate is one of the most common solvent systems alongside hexanes, is the most
polar in said systems. Should the spots remain at the baseline in a 20% EA% 80% hexane
solution, then the EA portion would head to be increased to 30% or 40% to accelerate the
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elution.
Discussion:
The examination of the thin layer chromatography (TLC) outcomes yields valuable insights into
the unidentified substance. The objective of the experiment was to pinpoint the unknown
substance by evaluating its Rf values against those of known compounds (Caffeine,
Acetaminophen, and Acetylsalicylic Acid). The Rf values derived for each compound played a
crucial role in the comparative analysis. Significantly, the Rf value for the unknown sample was
identified as 0.53, closely corresponding to Caffeine's Rf value of 0.52. The minute 0.01
difference and nearly identical travel distances strongly suggest that the unknown sample is
likely caffeine. Acknowledging potential sources of error and limitations in the experimental
procedure is essential. The alignment of the TLC plates was noted as less than perfect, with
clarification from the instructor that this imperfection did not markedly impact overall results.
Nonetheless, maintaining precise alignment could improve the accuracy of future
experiments.The selection of solvents in TLC holds importance for successful separation. In this
instance, ethyl acetate served as the solvent, with its polarity proving suitable for the separation
of the chosen compounds. The significance of the solvent system in achieving accurate Rf values
was underscored in the discussion questions, especially in addressing scenarios where all spots
emerge at the baseline. Adjusting the solvent system's polarity, such as increasing ethyl acetate
concentration, was recommended to enhance separation and obtain usable Rf values.The
discussion questions also prompted reflections on baseline placement, instrument choice (pencil
vs. pen), and the importance of preventing the solvent line from extending beyond the TLC
plate's end. These considerations are pivotal in ensuring result reliability and averting
experimental artifacts.
Conclusion:
The experiment effectively employed TLC to identify an unknown substance. The computed Rf
values, coupled with meticulous consideration of experimental conditions and potential sources
of error, confidently suggest that the unknown sample is likely caffeine. This analysis
underscores the significance of TLC in pharmaceutical analysis and emphasizes the importance
of methodological precision for accurate results. As demonstrated in the calculation the unknown
sample Rf was 0.53 which out of all the known samples was closest to caffeine, which was 0.52.
Since the Rf results have a 0.1 difference and have traveled a nearly identical distance it can be
concluded that the unknown sample is caffeine.
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My Answer: The HPLC analysis detects chromophores, compounds with multiple double bonds. Since water, ethyl acetate, and hexane all lack multiple pi bonds this will not be detected in the HPLC.
-Question for you, is this complete (and correct), and should I include anything about UV detection? Is UV detection used in HPLC? I thought it was only used on TLC to show compunds that arent visable.
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TABLE 1
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Total Volume
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