Column Chromatography and Thin-Layer Chromatography Lab Report

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Abstract

Chromatography is a versatile technique used for the separation and quantitative determination of components in mixtures. This experiment focuses on two chromatography methods: column chromatography and thin-layer chromatography (TLC) to separate ortho and para-nitrophenol products generated from the reaction between nitric acid and phenol. The experiment also includes the analysis of product melting points to assess their purity.

Introduction

Chromatography is a powerful method for separating and quantifying components within a mixture based on their differing polarities. It involves a stationary phase, such as a TLC plate or column, and a mobile phase, typically a solvent, to facilitate the separation.

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In this experiment, the reaction between nitric acid and phenol produces a mixture of ortho and para-nitrophenol. Column chromatography is employed to separate these two products based on their polarity, and thin-layer chromatography (TLC) is used to analyze the composition of the separated products. Additionally, the melting points of the products are determined after separation.

Procedure

The experiment was divided into three main parts:

Preparation of Reaction Mixture:
- 9.1 ml of 30% nitric acid was placed in a three-neck round-bottom flask.
- A thermometer, stirring bar, and addition funnel were added.
- 2.3641 g of phenol was dissolved in 10 ml of distilled water and added to the addition funnel.
- The nitric acid was cooled to 0˚C using an ice bath before adding the phenol mixture to prevent premature reaction.
- Stirring was maintained at 5-10˚C while adding the phenol mixture.
- After complete addition, the ice bath was removed, and stirring continued for 30 minutes at room temperature.
- 40 ml of distilled water was added to quench the reaction, and the mixture was cooled again to 0˚C.
- The top aqueous layer was decanted, leaving the viscous oil at the bottom, which was washed twice with 40 ml of water.
- The remaining product was dissolved in 100 ml of diethyl ether and transferred to a separatory funnel.
- 100 ml of water was added, shaken thoroughly to mix, and left to separate before draining the aqueous and organic layers.
- Sodium sulfate was added to the organic layer until some crystals swirled freely, then it was filtered off and washed with diethyl ether.
- The filtrate was evaporated under reduced pressure using a rotary evaporator.
Isolation and Separation of Ortho and Para-Nitrophenol Products:
- 0.7492 g of crude product was dissolved in 2 ml of ethyl acetate for column chromatography.
- A column was prepared with glass wool, sand, and silica gel, moistened with a 7:3 hexane/ethyl acetate solution.
- The product mixture was added to the column and pushed through using an inflation bulb.
- The eluting solvent, 7:3 hexane/ethyl acetate solution, was added to facilitate product separation.
- Fractions containing the separated products were collected into test tubes for TLC analysis.
Identification of Product Components:
- A diluted sample (2 mg of crude product in 2 ml of ethyl acetate) was prepared for thin-layer chromatography.
- A silica-coated TLC plate was labeled with a starting line 0.6 cm from the bottom and an end line 0.4 cm from the top.
- Compounds from each fraction were spotted onto the TLC plate using a capillary tube.
- The TLC plate was developed in a chamber containing a 7:3 hexane/ethyl acetate solution.
- Potassium permanganate solution was used to visualize the spots on the TLC plate.
- Rf-values for each fraction were calculated and are presented in Appendix 4.

Results & Discussion

The polarity of ortho and para-nitrophenol products influences their behavior during chromatography.

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Ortho-nitrophenol, with close proximity between the hydroxyl and nitro groups, tends to form stronger intermolecular bonds. In contrast, para-nitrophenol, with the hydroxyl and nitro groups on opposite sides, is less likely to form such bonds. This results in para-nitrophenol having a smaller Rf-value compared to ortho-nitrophenol, indicating slower movement from the starting line during TLC.

The experiment yielded a percentage yield of 51.26%, which suggests some loss, possibly due to the formation of by-products. The rapid temperature increase during the addition of phenol may have contributed to by-product formation. Stirring speed control and observation were challenging due to condensation on the flask's surface. Inadequate mixing or excessive stirring speed could also lead to by-products.

The selectivity percentages for ortho and para-nitrophenol were 44.69% and 55.31%, respectively, deviating from the expected 2:1 ratio. Inconsistencies in fraction volume collection and the use of the same solvent throughout the experiment may have resulted in mixed products, affecting the selectivity percentages.

The melting points of ortho and para-nitrophenol were found to be 43.5˚C and 114.2˚C, respectively, slightly different from the literature values of 45˚C and 115˚C, indicating potential impurities or contamination.

Conclusion

Chromatography is a valuable technique for isolating, separating, and identifying components within a sample based on their polarity differences. In this experiment, the use of different polarity solvents could have improved the separation efficiency. Despite some challenges and deviations from expected results, chromatography remains a useful tool for future analytical and separation purposes.

Appendix 1 – Result Data with Calculations

Test-tube	Compounds
1 - 4	Phenol
5 - 9	Ortho-nitrophenol
10 - 16	Mixture of Ortho and Para-nitrophenol
17 - 24	Para-nitrophenol
25 – 30	Solvent

Number of moles of phenol: 0.0487 mol

Number of moles of 30% nitric acid: 50 mmol

Number of moles of overall crude product: 0.02512 mol

Theoretical mass of overall crude product: 3.29 g

Percentage Yield of the Reaction: 51.26%

Number of moles of ortho-nitrophenol: 0.0112 mol

Number of moles of para-nitrophenol: 0.0138 mol

Selectivity of ortho-nitrophenol: 44.69%

Selectivity of para-nitrophenol: 55.31%

Retention factor (Ortho-nitrophenol): 0.73125

Retention factor (Para-nitrophenol): 0.38125

Appendix 2 – Rf-value Data

Test-tube	Rf values (Point 1)	Rf values (Point 2)	Compound Average Rf values
1	0.825	NA	Phenol: 0.825
2	0.825	NA
3	0.825	NA
4	0.825	NA
5	0.825	NA	Ortho-nitrophenol: 0.73125
6	0.825	0.725
7	0.825	0.725
8	0.825	0.725
9	0.75	NA
10	0.725	0.5	Mixture of Ortho and Para-nitrophenol
11	0.725	0.5
12	0.725	0.5
13	0.725	0.525
14	0.725	0.525
15	0.725	0.5
16	0.5	NA
17	0.4	NA	Para-nitrophenol: 0.38125
18	0.4	NA
19	0.4	NA
20	0.375	NA
21	0.375	NA
22	0.375	NA
23	0.35	NA
24	0.35	NA
25	0.475	NA	Solvent
26	0.45	NA
27	0.45	NA
28	0.45	NA
29	0.45	NA
30	0.4	NA

Solvent

Test-tube	Rf values
25	0.475
26	0.45
27	0.45
28	0.45
29	0.45
30	0.4

Column Chromatography and Thin-Layer Chromatography Lab Report. (2024, Jan 02). Retrieved from https://studymoose.com/document/column-chromatography-and-thin-layer-chromatography-lab-report