Organic Chemistry Post Lab #3 Chromatography - Analyzing Analgesics By Tlc And Isolation PDF

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Name: Danielle Curtis Partners: Gabbi Nguyen & Misturah Abdulkareem TA: Olapeju Oyesiku 2/8/2017 Experiment 3: Chromatography – Analyzing Analgesics by TLC and Isolation of Carotene by Column Chromatography

Introduction: Chromatography is an analytical technique that is often used in order to separate a mixture of two or more compounds, or ions, into their separate components1. Moreover, these components are separated so that their properties can be more thoroughly analyzed1. There are three different types of chromatography often used throughout organic chemistry, Thin Layer Chromatography (TLC), Gas Liquid Chromatography (GC) and Column Chromatography (CC)2. However, this experiment specifically focuses on the use of TLC and CC in order to separate a mixture. Thin Layer Chromatography (TLC) uses a plastic plate coated with a thin layer of silica to quickly and inexpensively analyze small samples3. A small dot of the mixture is applied to the baseline of the plate; the plate is then dipped in the appropriate solvent and is placed in a container2. The solvent moves up the plate via capillary action, meets the dot of the mixture on the baseline, the mixture is dissolved and is then carried up the plate by the solvent2. The components in the mixture can be identified because different compounds travel at different rates due to the differences in the solvent’s solubility and differences in the compounds attraction to the stationary phase3. TLC uses R-values to represent the distance traveled by the compound divided by the distance traveled by the solvent. It is these values that can then be compared to analyze the different compounds2. Column Chromatography (CC) is similar to TLC in terms of separating compounds. However, CC uses a column to separate and isolate compounds, rather than analyze different compounds2. In this experiment, CC uses a micro column packed with silica gel and sand to separate the pigments in spinach leaves into their respective

components2. Then, the approximate volume collected and the color of each component collected is recorded2. The separation that occurs during chromatography is caused by the respective components molecular interactions with two phases, a mobile phase and a stationary phase2. The mobile phase is a medium used in chromatography that has the ability to move through the stationary phase3. Moreover, in TLC and CC the mobile phase is most often an organic liquid2. Whereas, the stationary phase is a medium used in chromatography that does not move3. In this experiment, the stationary phase is the thin layer of silica that is on top of the plastic plate2. It is this ability to partition into separate components that allows for the mixture to be separated and analyzed2. Moreover, this ability to partition is based on the polarity and affinity between the two different phases2. The objective of this experiment was to analyze four analgesics: Acetaminophen, Aspirin, Caffeine and Ibuprofen; by Thin Layer Chromatography and identify an unknown analgesic by TLC comparison2. Furthermore, a secondary objective of this experiment was to isolate -carotene via liquid-solid extraction of spinach and purify this compound via column chromatography2. Experimental Procedure: TLC Procedure

#1 The solvent front lines and baselines wer drawn 1 cm from the top and bottom of the gel plate.

#2 The TLC was spotted with the known #4 analgesics The TLC (Acetaminophen, was Aspirin, Ibuprofen, developed Caffeine) under a UV in thetoTLC light ensure they chamber. were spaced evenly.

#3 9.5 mL of ethyl acetate was added to 0.5 mL of acetic acid, in a container, to make #5a solvent that was 95%the ethyl acetate Once solvent and 5%the acetic reached front acid. line on the silica plate, the TLC was removed from the chamber.

#7#6 The was was placed in an TheTLC solvent iodine chamber and left to allowed to sitevaporate for a fewand moments. It the was removed and placed TLC was checked under UV lamp. lamp and underthe a UV the dots of each component were circled lightly with a pencil.

#9 The previous steps were repeated. However, the second time around the 4 analgesics and the unknown were spotted onto the silica plate and viewed under the UV light. The unknown was determined using the data collected from knowns

#8 The distances from the baseline to where the dot had moved was measured and recorded.

CC Procedure #3 #2 #1 The spinach leaves were The micro column A mircro column #6 #5 up and the pigment ground was wet with was prepared with #8 #7 The ether was This was decanted was extracted with ethyl petroleum ether. ½ cm of sand and Petroleum ether was added thea column The mixture was added to dissolved with a 1:1 further into Vacuum acetate. to silica gel. and the column was pushed with a bulb the column, and the petroleum ether : ethyl flask and aspirated to the sand line while the yellow pigment column was pushed withacetate a mixture. over a steam bath. bulb to the sand line was collected in a test tube.

#9 -carotene was collected and the column was emptied by aspiration.

#4 The ethyl acetate was decanted into an Erlenmyer flask and dried with Na2SO4.

#10 The -carotene was checked for purity by using TLC and comparing it to the standard.

Chemicals Used: Name of Chemical IUPAC Name Formula Molar Mass Melting Point

Acetaminophen N-(4-Hydroxyphenyl)acetamide C8H9NO2 151.17 g/mol 170C

Aspirin 2-(Acetyloxy)benzoic acid C9H8O4 180.15 g/mol 139C

Properties

Skin/eye irritant Irritant if inhaled

Skin/eye irritant Irritant if inhaled

Chemical Structure

Name of Chemical IUPAC Name Formula Molar Mass Melting Point Properties

Caffeine

Ibuprofen

1,3,7-trimethyl-3,7-dihydro1H-purine C8H10N4O2 194.2 g/mol 238C Skin/eye irritant Irritant if inhaled

2-(4-isobutylphenyl)proponic acid C13H18O2 206.29 g/mol 75C Skin/eye irritant Irritant if inhaled

Chemical Structure

Name of Chemical IUPAC Name Formula Molar Mass Melting Point Boiling Point Properties

Chemical Structure

Silica Gel Silicon Dioxide SiO2 60.08 g/mol 1600C Skin/eye irritant Irritant if inhaled

Ethyl Acetate Ethyl Ethanoate C4H8O2 88.11 g/mol -83C 77C Skin/eye irritant Irritant if inhaled Flammable

Name of Chemical IUPAC Name Formula Molar Mass Melting Point Boiling Point Properties

Acetic Acid Ethanoic Acid C2H4O2 60.05 g/mol 16.6C 118.1C Skin/eye irritant Irritant if inhaled Flammable

Iodine Iodine I2 253.81 g/mol 113.7C 184.4C Skin/eye irritant Irritant if inhaled Corrosive

Sand Silicon Dioxide SiO2 60.08 g/mol 1600C

Sodium Sulfate Sodium Sulfate Na2SO4 142.04 g/mol 888C 1100C Skin/eye irritant

Chemical Structure

Name of Chemical IUPAC Name Formula Molar Mass Melting Point Boiling Point Properties

Name of Chemical IUPAC Name Formula Molar Mass Melting Point Boiling Point Properties

Skin/eye irritant Irritant if inhaled

Petroleum Ether Various Various Various 60C Skin/eye irritant Irritant if inhaled Flammable

Hexane Hexane C6H14 86.18 g/mol -95C 68C Skin/eye irritant Irritant if inhaled Flammable

Results: Table #1: Results for TLC Procedure Ibuprofen Caffeine Acetaminophen Aspiri

Unknown

Rf Values for Part 1 (w/o unknown) Rf Values for Part 2 (w/ Unknown)

0

0.217

0.7

n 0.883

0

0.185

0.677

0.892

unknown compound = Caffeine Table #2: Results for the CC Procedure

Rf Values Weight

Isolated -Carotene 0 1.12 g

Standard -Carotene 0 N/A

SAMPLE CALCULATIONS FOR Rf VALUES Rf = (distance traveled by compound)/ (distance traveled by solvent)

Rf (for Ibuprofen #1) = 0/6 = 0 Rf (for Ibuprofen #2) = 0/6.5 = 0

Rf (for Caffeine #1) = 1.3/6 = 0.217 Rf (for Caffeine #2) = 1.2/6.5 = 0.185

Rf (for Acetaminophen #1) = 4.2/6 = 0.7 Rf (for Acetaminophen #2) = 4.4/6.5 = 0.677

Rf (for Aspirin #1) = 5.5/6 = 0.883 Rf (for Acetaminophen #2) = 5.8/6.5 = 0.892

N/A 0.2

Rf (for unknown) = 1.3/6.5 = 0.2

Rf (for Isolated -Carotene) = 0/0 = 0 Rf (for Standard -Carotene) = 0/0 = 0 Discussion: During the TLC part of the experiment, two sets of Rf values were determined using the distance traveled by the compound divided by the distance traveled by the solvent. The first set of Rf values was for four different analgesics: ibuprofen, caffeine, acetaminophen and aspirin. It was found that the Rf value for ibuprofen was 0, the Rf value for caffeine was 0.217, the Rf value for acetaminophen was 0.7 and the Rf value for aspirin was 0.883. Based on the Rf values, it can be ascertained that the aspirin had the greatest affinity for the mobile phase. Where as, the ibuprofen did not move at all and therefore had a greater affinity for the stationary phase. Moreover, it can also be ascertained, from the Rf values, that aspirin moved the furthest up the silica gel due to capillary action. Where as, ibuprofen did not move up the silica gel at all. The second set of Rf values was similar to the first set, in the sense that they included all four analgesics used in the first set. However, the second set of Rf values also included a value for an unknown analgesic. The second set of Rf values was for four different analgesics: ibuprofen, caffeine, acetaminophen, aspirin; and an unknown analgesic. It was found that the Rf value for ibuprofen was 0, the Rf value for caffeine was 0.185, the Rf value for acetaminophen was 0.892, the Rf value for aspirin was 0.892 and the Rf value for the unknown was 0.2. It is clear that the second set of Rf values for the four analgesics was very similar to the first set of Rf values for the analgesics. By

comparing the Rf value for the unknown to the Rf value of the analgesics, the unknown can be easily identified. The Rf value for the unknown was 0.2, which most closely matches the Rf value for caffeine: 0.217 in the first set and 0.185 in the second set. Therefore, it can be ascertained that the unknown analgesic is most likely caffeine because their Rf values are the most similar. During the CC part of the experiment, spinach leaves were crushed up and underwent CC in order to isolate -Carotene from the other pigments in the spinach leaves. The -Carotene that needed to be collected was the first layer in the column, the yellow layer. This yellow layer then underwent TLC and was compared to the standard Carotene on a silica gel plate, under the UV light, to determine if the isolation of Carotene was successful or not. One mistake made throughout this part of the experiment was that the -carotene extracted from the spinach was aspirated out before it underwent the rest of the procedure. Therefore, the isolation of -carotene was unsuccessful for this experiment. However, -carotene was borrowed from another group so that it could be compared to the standard -carotene on a silica gel plate. It was found that neither the standard -carotene nor the isolated -carotene moved up the silica gel plate. However, under the UV light both -carotene dots appeared to be the same color. Nonetheless, based on the data collected from the CC procedure during the experiment, it can be ascertained that the isolation of -carotene was not successful. Conclusion: The objective of this experiment was to analyze four analgesics: acetaminophen, aspirin, caffeine and ibuprofen; by thin layer chromatography and identify an unknown analgesic by TLC comparison2. Moreover, another objective was to isolate -carotene by

liquid-solid extraction of spinach and purify the -carotene by column chromatography2. The data collected throughout this experiment reveals that the unknown analgesic was caffeine. The evidence that supports this are the Rf values for caffeine and the unknown. Both Rf values are 0.217 and 0.2 respectively. The fact that they are so similar gives way to the idea that the unknown could be caffeine. Moreover, the data collected throughout this experiment also reveals that the isolation of -carotene was unsuccessful due to mistakes made throughout out the experiment. For example, aspirating out the -carotene before completion of the procedure. The information and techniques used throughout this experiment are important because they have many real world applications. For example, TLC is often used for clinical, pharmaceutical and food testing4. In terms of clinical testing, TLC is used to test for the presence of drug abuse4. Where as for the pharmaceutical industry, TLC is used widely in the production and quality assurance applications4. Finally, in the food industry, TLC has many functions ranging from lipid separation to food dye analysis4. Overall, this lab accomplished what it set out to do. New skills and techniques were learned when TLC was used to identify an unknown analgesic and CC was used to isolate -carotene from spinach. Moreover, the data collected from the experiment illustrates how Rf values and TLC can be used to compare known compounds to unknown compounds, in order for them to be identified. These skills learned can then be applied to real life situation, such as in the industrial, pharmaceutical or food related fields. References:

[1] Khan Academny. Principles of Chromatography. Khan Academy. (accessed Feb 9, 2017) [2] Wildegirma, S. Experimental Organic Chemistry Lab Manual; University of South Florida: Tampa, FL, 2016; P. 15-19 [3] ChemGuide. Column Chromatography. ChemGuide. ChemGuide. (accessed Feb 9, 2017) [4] LCGC Editors. (2009, November 10) Thin Layer Chromatography. LCGC. LCGC . (accessed Feb 9, 2017)...