Thin Layer Chromatography and Gas Chromatography

Lab #6 – Thin Layer Chromatography and Gas Chromatography

Aim: Separating and identifying compounds by two types of chromatography, gas chromatography and thin layer chromatography.

Introduction: Chromatography is a method used for separating and identifying compounds in a mixture. The two types of chromatography used in this experiment were gas chromatography (GC) and thin-layer chromatography (TLC). Gas chromatography involves the equilibrium of the compounds between two phases, the stationary phase and the mobile phase. The stationary phase is a non-volatile liquid, usually a polymer with a high boiling point. For GC the mobile phase is an inert gas, the reason for an inert gas is so that it doesn’t interact with the compounds. The inert gas is called the carrier gas and It can be either helium or nitrogen. The compound that interacts strongly with the carrier gas gets carried by the gas over the liquid stationary phase. The compounds that interact more strongly with the stationary phase are retained longer in the GC giving us a retention time. Retention times can be affected by the structures of the compounds, the type of stationary phase, the column temperature, the length of the column, and the rate of flow of carrier gas. The separated compounds can be quantified. Some important components of the gas chromatograph are a rubber septum and heated injection port where the sample is added with a syringe. Another component is the carrier gas tank which is attached to a compartment on the machine where it will be mixing with the sample and converted to a vapor by a heater in that compartment. Then the sample and the carrier gas move into a coil within a column oven. The coil or the GC column is where the stationary phase is and the temperature of the heater in the oven can be controlled. The compounds exit at different times depending on their interactions and are detected then recorded.

In thin-layer chromatography (TLC), the equilibrium of compounds between two phases is also involved. This method requires silica gel or alumina as the adsorbent on a backing sheet of either plastic, glass, or aluminum that serves as the stationary phase. The mobile phase is called the eluent or the eluting solvent. The compounds placed on the plate are then carried by the eluting solvent over the solid stationary phase. The compounds can be separated by their differential interactions, the compounds that interact more strongly with the stationary phase are retained longer on the TLC plate. In order to carry out TLC you must first spot the TLC plate with the compound or compounds you are trying to identify and also spot it with the compound you are comparing it with in order to identify it. Then you must place the TLC plate in the solvent in a jar which is the mobile phase and let it rise up to the solvent front which is known as developing the TLC plate. Then you visualize the spots and calculate the Rf

Compound Name

2,4,4- trimethyl-1- pentene

2,4,4- trimethyl-2- pentene

Molecular Formula

C8H16

C8H16

Molecular Weight

112.21 g/mol

112.21 g/mol

Structure

Appearance

Vapor

Vapor

Melting Point

-101°C

-106°C

Boiling Point

101°C

104°C

Hazard

·         Very hazardous in case of skin and eye contact.

·         Seek medical attention if infected. Skin must be thoroughly washed

·         Highly flammable liquid and vapor

·         May be fatal if swallowed and enters airway

·         Can cause irritation in contact with skin or eyes

values of the components.

Table 1: Table of Physical Properties and Hazards – Pentenes

Compound Name

Silica

Ferrocene

Acetylferrocene 

Hexane 

Ethyl Acetate

Molecular Formula

SiO2

C10H10Fe

C12H12FeO

C6H14

C4H8O2

Molecular Weight

60.083 g/mol

186.035 g/mol

228.072 g/mol

86.178 g/mol

88.106 g/mol

Structure

Appearance

Fine white powder

Orange, crystalline solid with camphor-like odor

Orange to brown powder, crystals, or chunks 

Clear colorless liquids with a petroleum-like odor

Clear colorless liquid with a fruity odor

Melting Point

1710°C

173°C

81-83°C

-95°C

-83.8°C

Boiling Point

2230°C

249°C

160-163°C

69°C

77°C

Hazard

·         Causes skin irritation

·         Causes serious eye irritation

·         May cause respiratory irritation

·         Harmful if swallowed

·         Flammable solid

·         Toxic to aquatic life  

·          May be fatal if swallowed

·         May cause irritation of the digestive tract

·         May be fatal if swallowed and enters airways

·         Causes skin irritation

·         Causes serious eye irritation

·         May cause drowsiness or dizziness

·         Causes serious eye irritation

·         May cause drowsiness or dizziness 

Table 2: Table of Physical Properties and Hazards

Procedure: The first type of chromatography that was done was the TLC chromatography. This was done by first drawing a line 1cm from the bottom of the TLC plate with a pencil and drawing a line 1cm from the top of the TLC plate, the top line is known as the solvent front. Then we placed one spot for each ferrocene, acetylferrocene, and a mixture solution on the line 1cm from the bottom using a capillary tube. The TLC plate was then placed into a jar with hexane ethyl acetate, only a small amount of solvent was added in jar so that when the TLC plate was added it would be below the spotted line. If the solvent was above or where the spots were, they would be extracted into the solvent rather than running up the TLC plate. Once the solvent reached the solvent front the plate was removed. The Rf values were then recorded by measuring the distance that the compound traveled compared to how far the solvent traveled.

The second type of chromatography done was the GC chromatography. We first chose a mixture of 2.4.4-trimethyl-pent-1-ene, and 2,4,4-trimethyl-pent-2-ene, called GC7 and it was 1.7uL. We then took a micro syringe and washed it 3 times with acetone, then 1-2uL of the sample was loaded into the syringe making sure there are no bubbles in the syringe. The syringe is then inserted into the gas chromatograph and start was pressed on the GC machine as the same time the plunger was pressed down. A report was then displayed and printed. From this report the beaks could be used to calculate the peak areas and retention times.

Questions:

  1. Name the major components of the GC system.

The major components of the gas chromatograph are the rubber septum and heated injection port to add the sample with the syringe. Another component is the carrier gas tank which holds the carrier gas or the mobile phase.  There is also a compartment on the machine where the carrier gas mixes with the sample and they are converted to a vapor by a heater in that compartment. Another major component is the coil within a column oven. The coil or the GC column is where the stationary phase is and the temperature of the heater in the oven can be controlled. Another major component is the detector which interacts with the solutes that elute from the column and convert the interaction into an electronic signal for the recorder.

  1. What type of column did you use and what is the polarity of the column you used?

The column used was an open capillary wall coated tube. The polarity of the column used was an intermediate polarity given we used polymethylphenylsiloxane.

  1. Can you find the type of detector (thermal conductivity or flame ionization) you used by simply looking at the instrument layout?

By simply looking at the instrument layout we cannot tell what type of detector was used but the two differ in how they interact with the eluent. The thermal conductivity detector detects any species that differs from the carrier gas in thermal conductivity. The flame ionization detector uses voltage to accelerate reduced carbon ions towards plates for detection by an ammeter.

  1. What kinds of compounds can you separate by GC?

Volatile compounds or substances that can be vaporized without decomposition can be separated by GC.

Results:

TLC:   The Rf values were calculated for the acetylferrocene, ferrocene and the acetylferrocene/ferrocene mixture, using this formula.

Rf = Distance traveled by compound / Distance traveled by solvent

Distance traveled by solvent: 5.2cm

Distance traveled by Ferrocene (A): 3.6cm

Distance traveled by Acetylferrocene in mix (B): 0.7cm

Distance traveled by Ferrocene in mix(B) 3.6cm

Distance traveled by Acetylferrocene (C): 0.7cm

Rf Calculations:

Rf (Ferrocene): 3.6cm / 5.2cm = 0.692

Rf (Acetylferrocene): 0.7cm / 5.2cm = 0.135

Rf (Ferrocene in mix): 3.6cm / 5.2cm = 0.692

Rf (Acetylferrocene in mix): 0.7cm / 5.2cm = 0.135

Gas Chromatography: For this experiment composition of compounds in the mixture, the column efficiency, and the resolution of the compounds were calculated using these 3 formulas.

  1. The composition of a peak = (area of the peak / total area of all the peaks) x 100
  2. Column efficiency = N = 16 ((tR)/w)2

N = number of plates

tR = retention time

w = width of the plate

  1. Resolution = R = (tB – tA) / (wA + wB)/2)

tA = Retention time of compound A

tB = Retention time of compound B

wA = Width of compound A

wB = width of compound B

  1. Composition of a Peak

The composition of peak 1 = (20mm / 176mm) x 100 = 11.36%

The compostion of peak 2 = (75mm / 176mm) x 100 = 41.61%

The compostion of peak 3 = (81mm / 176mm) x 100 = 46.02%

  1. Column Efficiency

The column efficiency of peak 1 = 16(1.827min / 4mm)2 = 3.338 theoretical plates

The column efficiency of peak 2 = 16(2.815min / 4mm)2 = 7.924 theoretical plates

The column efficiency of peak 3 = 16(2.907 min / 4mm)2 = 8.451 theoretical plates

  1. Resolution

Resolution between 1 and 2 = 0.092 / 81875 = 0.0112

Resolution between 2 and 3 = 0.092 / 0.988 = 0.176

Resolution between 1 and 3 = 1.08 / 5.8945 = 0.183

Conclusions:

Based on my results from the TLC experiment, I did not have pure compounds. In position C of the attached photo two spots are present for acetylferrocene. This means that the acetylferrocene was not pure because it had a spot that appears to be ferrocene because it traveled up the same distance as the ferrocene spot in the mixture. We also had trouble with the spotting of the ferrocene, it seems that we did not spot enough onto the plate so as it moved up with the solvent, we were not able to see it. We inferred how far it traveled because it was moving up the plate the same way the mixture was and at the same time until we could no longer see it. The utilities for GC and TLC are alike in a few ways. They both deal with the use of stationary and mobile phases in which the mobile phase carries the compound over the stationary phase. They differ in that in GC the mobile phase is a known as a carrier gas and has to be an inert gas such as helium or hydrogen, in TLC the mobile phase is an eluting liquid solvent. The stationary phases used are also different, in GC the stationary phase is a non-volatile liquid and usually a polymer and in TLC the stationary phase is the silica gel on the plate. The results given by TLC are readily visible as you can see the spots move up the plate but there is more room for error. GC on the other hand can give more accurate results but the process inside the chromatographer is not visible. The utilities for TLC are much simpler since it is only the plate, the solvent, and the spots but they are similar to GC in the results we can obtain from them and what those results tell us.

References:

National Center for Biotechnology Information. PubChem Database. Silica, CID=24261, https://pubchem.ncbi.nlm.nih.gov/compound/24261 (accessed on Apr. 26, 2019)

National Center for Biotechnology Information. PubChem Database. Ferrocene, CID=7611, https://pubchem.ncbi.nlm.nih.gov/compound/7611 (accessed on Apr. 26, 2019)

National Center for Biotechnology Information. PubChem Database. Acetyl ferrocene, CID=11985924, https://pubchem.ncbi.nlm.nih.gov/compound/11985924 (accessed on Apr. 26, 2019)

National Center for Biotechnology Information. PubChem Database. Hexane, CID=8058, https://pubchem.ncbi.nlm.nih.gov/compound/8058 (accessed on Apr. 26, 2019)

National Center for Biotechnology Information. PubChem Database. Ethyl acetate, CID=8857, https://pubchem.ncbi.nlm.nih.gov/compound/8857 (accessed on Apr. 26, 2019)

National Center for Biotechnology Information. PubChem Database. 2,4,4-Trimethyl-1-pentene, CID=7868, https://pubchem.ncbi.nlm.nih.gov/compound/7868 (accessed on Apr. 26, 2019)

National Center for Biotechnology Information. PubChem Database. 2-Pentene, 2,4,4-trimethyl-, CID=7869, https://pubchem.ncbi.nlm.nih.gov/compound/7869 (accessed on Apr. 26, 2019)

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