Chromatography- Analyzing Analgesics by TLC and Isolation of B-Carotene by Column Chromatography
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Chromatography- Analyzing Analgesics by TLC and Isolation of B-Carotene by Column
Chromatography
TA: Maria Ballivan
CHM 2210L Section 030
Introduction
Chromatography is simply separating components of a mixture (ThermoFisher,2019). In organic chemistry, this separation is based on the difference in attractions of the compounds in the mixture, to mobile phase and stationary phase. There are three types of chromatography that is used widely in organic chemistry which includes Thin Layer Chromatography, Gas Liquid Chromatography, and Column Chromatography (Weldegrima, 2023). The two methods utilized in this experiment are Thin Layer Chromatography and Column Chromatography. Thin Layer Chromatography (TLC) is a simple and fast way to analyze and identify compounds. TLC utilizes polarity differences, mobile and stationary phases to identify compounds. If the sample placed on a TLC plate has a greater attraction towards the mobile phase it will travel more faster and if the sample has greater attraction towards the stationary phase, it will travel slower (Weldegrima, 2023). The opposing solvent will be different from the samples in polarity, and on which phase it has more attraction to on the TLC plate. This allows for proper identification and analyzing of the sample compounds. The second type of chromatography used in this experiment is column chromatography. This type of chromatography is more useful in purification, separation, and isolation of compounds (Weldegrima, 2023). This technique also uses the differences in polarity and differences in the attraction towards stationary and mobile phases to separate compounds. In column chromatography, the stationary phase is achieved via silica gel or similar compound (Chem. Libre, 2020). The solvent flows through the silica gel in a column and is essential in the purification component of column chromatography. Experimental Section Thin Layer Chromatography -
Obtain silica gel with fluorescent indicator
-
Draw line (about 1 cm from edge of plate)
-
Spot TLC with the four compounds -
Check the spots under the UV light -
Add 95% ethyl acetate and 5% acetic acid in 10ml chromatography chamber
-
Put TLC in chamber -
Remove plate when solvent gets ½ cm from the end -
Remove and mark when solvent stopped -
Let it dry -
Check under UV light again -
Record Rf values -
Obtain another TLC -
Mark starting point -
Spot with unknown -
Check under UV light -
Place TLC in iodine chamber for 2 mins -
Check under UV light again and record Rf values and identify the unknown
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Column Chromatography -
Fill microcolumn with halfway with silica gel -
Add ½ cm of sand on top of the silica gel and clamp the column -
Weigh out 2g of defrosted spinach -
Add spinach and 15ml of ethyl acetate into an Erlenmeyer flask -
Crush spinach with spatula for 15 mins -
-
Decant the solution into another Erlenmeyer flask -
Add anhydrous sodium sulfate -
Remove the remaining solvent with an aspirator trap, over a steam bath -
Dissolve the solid in 5-6 drops of 1:1 solution of ethyl acetate and petroleum ether solution -
Save a couple drops in vial for TLC in a closed vial -
-
Obtain 6 small test tubes -
Wet the column with petroleum ether and let it drop down, use air to push the solvent down. -
Add spinach and ether to the column -
Keep the column running until the first colored substance
-
Collect the colored liquid in test tubes and record values
-
Transfer colored fractions to a 50 ml Erlenmeyer flask and evaporate solvent with aspirator
-
-
Obtain TLC plate
-
-
-
-
Spot b-carotene, crude and purified sample -
Place TLC plate in chromatography chamber -
Identify and record Rf values. Table of Chemicals Chemical Name
Physical Properties Chemical Properties Chemdraw
Acetaminophen
MM: 151.1 g/mol
BP:420 °C
MP: 169 °C
Crystalline, white Skin and eye irritation may cause respiratory infection
Aspirin MM: 180.158 g/mol
BP: 140°C
MP: 136 °C
Irritation to mucus membrane May cause eye
Crystalline, white
irritation
Caffeine
MM: 194.19 g/mol
BP: 178 °C
MP: 236.5 °C
Clear liquid
Harmful if swallowed Ibuprofen MM: 206.29 g/mol
BP: 314.6°C
MP: 76 °C
Clear liquid
Harmful if swallowed May cause eye and respiratory irritation
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B-Carotene
MM: 536.873 g/mol
BP: 633°C
MP: 180°C
Orange pigment
No hazards are currently
in place
Acetic Acid
MM: 60.052 g/mol
BP: 118° C
MP:16.6 °C
Crystalline, white
Eye damage and upper respiratory damage Silicon Dioxide
MM: 60.08 g/mol
BP: 2950 °C
MP: 1600°C
Colorless crystals Irritation to upper respiratory tract if inhaled
Hexane
MM: 86.18 g/mol
BP: 69 °C
MP: -95°C
Colorless liquid May cause irritation to skin, nose, lungs and throat Ethyl Acetate MM: 88.11 g/mol
BP: 77.1 °C
MP: -83.6°C
Colorless liquid Flammable, and may cause eye irritation Petroleum Ether
MM: 86.178 g/mol
BP: 60- 95°C
MP: -40 °C
Clear, colorless liquid Flammable, may be fatal
if swallowed Results TLC 1
Compound Distance Rf value Acetaminophen 3.55
(3.55/5.00)= 0.71
Aspirin 3.70
(3.70/5.00)= 0.74
Ibuprofen 4.15
(4.15/5.00)= 0.83
Solvent
5
(5.00/5.00) = 1
TLC Plate 2
Compound Distance Rf value Caffeine
2.25
(2.55/5.00)= 0.45
Aspirin 3.70
(3.70/5.00)= 0.74
Unknown*
3.55
(3.55/5.00)= 0.71
Solvent
5.00
(5.00/5.00) = 1
*The unknown was identified to be acetaminophen. TLC Plate 3 Compound
Distance
Rf value Crude B-Carotene 3.8
(3.81/6.1) = 0.623
Purified B- Carotene (#1)
2.7
(2.71/6.1)= 0.443
Standard B-Carotene 2.5
(2.5/6.1)= 0.409
Solvent 6.1
(6.1/6.1)=1
Discussion In thin layer chromatography, Rf is defined as the simple ratio of the distance travelled by a compound divided by the distance travelled by the solvent. In this experiment, the first TLC plate
the Rf value for acetaminophen was 0.71. This means the acetaminophen moved more than halfway from the solvent front. The recorded Rf value for aspirin was 0.74. This number also indicates the aspirin moved more than halfway from the solvent front and went to around 74% as
the solvent front. The Rf value for 0.83. This is indicating that the aspirin moved 83% as far as the solvent front. In the second TLC plate the Rf value of caffeine is 0.45, this indicates that caffeine moved only 43% as the solvent front. The Rf value of aspirin was 0.74, this means it
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moved over 50% as the solvent front. The unknown had a distance traveled value of 3.55 and a Rf value of 0.71. This is the same as acetaminophen and it can be concluded that the identity of the unknown is acetaminophen
. To determine if B-carotene was correctly isolated the Rf values must match up. This essentially means both compounds, the purified B-carotene and standard B-
carotene travelled the same distance. In this experiment the Rf, value for the purified b-carotene was 0.443. The Rf value for the standard was 0.409. The values are not the same, which then means B-carotene was not correctly isolated. Conclusion
The theoretical background and results are connected. This is because the use of TLC plates was successful in producing different Rf values. Also, in this experiment we were able to identify the unknown in the second TLC plate. Though we did not correctly isolated B-carotene with a hundred percent accuracy, the technique was still effective and correct. The experimental data reveals that chromatography is an efficient technique in isolating and identifying unknown compounds. This technique can apply to multiple different situations, including forensic and drug testing. Chromatography can be used to test for unknown compounds in blood, like drugs, poison, or alcohol. This can then aid in an investigation lead by the forensic and law enforcement
(Today, 2021). In all, this experiment accomplished what it was set out to do and showed the effectiveness of chromatography.
Works Cited
ChemLibre. "The Retention Factor." Chemistry LibreTexts
, 15 Nov. 2021, chem.libretexts.org/. Accessed 10 Feb. 2023.
ThermoFisher. "What is Chromatography and How Does It Work?" Ask a Scientist
, ThermoFisher Scientific, 17 Oct. 2019, www.thermofisher.com/blog/ask-a-
scientist/what-is-chromatography/. Accessed 10 Feb. 2023.
Today, Chromatography. "5 Uses of Chromatography in Everyday Life." Chromatography Today
, 2021, www.chromatographytoday.com/news/industrial-news/39/breaking-
news/5-uses-of-chromatography-in-everyday-life/32639. Accessed 10 Feb. 2023.
Weldegirma, Solomon. 2008. Experimental Organic Chemistry, Laboratory Manual,. 11th ed.
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