Lab 6 Report Identification of compounds from unknown mixture PDF

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Lab 6 Report Identification of compounds from unknown mixture ONLINE...

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10/2/20 Lab 6: Identifying Compounds from Unknown Mixture using TLC Introduction: Thin Layer chromatography is utilized in this experiment to separate compounds based on their polarity. Thin Layer Chromatography (TLC) is a type of chromatography that is used for small-scale separations of mixtures and for identification of compounds in an unknown mixture. To conduct a thin layer chromatography a TLC plate is required that is covered in silica gel that is more polar and will act as the stationary phase. A mobile phase is also required which is a solvent that is usually less polar so it can interact with the less polar compounds spotted on the plate and bring them up. The more polar compounds have a greater affinity to interact with the silica gel and are absorbed near the bottom of the plate. In this experiment, a more polar solvent of 95:5 ethyl acetate/acetic acid will be utilized as the mobile phase. This is because the compounds of caffeine, acetylsalicylic acid (aspirin), and phenacetin involved in this experiment are mostly polar. Utilizing a more polar mobile phase will yield a better separation on the even more polar silica gel covered TLC plate. If less polar solvent was used for the mobile phase, then all of the compounds would be absorbed at the bottom of the polar stationary phase because none of the polar compounds would want to interact with the less polar mobile phase to be brought up and separated. To identify the two compounds in the mixture, the mixture itself will be spotted on the plate, along with the pure three possible compounds. Once the mixture is eluted on the TLC plate and observed under UV light it yields two different spots because both compounds in it have varying polarities. Those two spots are matched to the individual spots of the three pure compounds spotted and compounds in mixture are identified. Rf values are calculated to confirm. Separating and identifying the unknown compounds are important in order to take the necessary protocol for the acid/base extractions that will be performed in the next experiment. Figure 1: TLC plate F Results and Observations: distance travelled by compound distance travelled by solvent TLC Results - Rf of Spots MIXTURE UNKNOWN F

Retention factor (Rf)=

Mixture Spot Aspirin 1: X/Y = 0.88 X/Y = 3.8cm/4.3cm = Mixture Spot 0.88 2: X/Y =0.28

Phenacetin X/Y= 3.3cm/4.3cm = 0.77

Distance travelled by solvent: 4.3 cm

Discussion and Conclusion:

Caffeine X/Y = 1.2cm/4.3cm= 0.28

To identify the compounds in the unknown mixture, a small amount of each compound (aspirin, caffeine, and phenacetin was dissolved in about 1mL dichloromethane in a culture tube. A small amount of unknown mixture was also dissolved in 1mL dichloromethane. All culture tubes were labelled and the same corresponding labelled were written on the TLC plate with a pencil and a baseline was drawn on the TLC plate. With different capillary tubes the TLC plate was spotted with the dissolved mixture and each of the 3 dissolved pure compounds. A solvent chamber was created by cutting filter paper and placing it into the side of the beaker. A small amount of solvent 95:5 ethyl acetate/acetic acid was added into the beaker just enough to cover the base. Solvent was swirled in the beaker to cover it in solvent fumes. The spotted TLC plate was placed into the solvent chamber leaning against filter paper and left in there until solvent line has reached the top. TLC plate was removed and left to dry, it was then placed under UV light to observed distance the spots travelled. Spots were circled and Rf values were calculated. According to the Rf calculations and the TLC plate under UV light, the unknown mixture was successfully separated into 2 compounds on the TLC plate with 95:5 ethyl acetate/acetic acid. The mixture spot 2 yielded and Rf value of 0.28 and Caffeine also yielded and Rf value of 0.28. This confirmed that one of the compounds in the mixture was in fact Caffeine. Caffeine was lower on the plate because its structure was the most polar structure out of the three. The amine and amide functional groups create large dipoles giving the structure more polar characteristics. The caffeine molecule was quickly absorbed by the more polar stationary phase because it was able to dissolve more quickly with the polar silica gel and had a greater affinity towards it. The mixture spot 1 yielded a Rf value of 0.88 and the acetylsalicylic acid (aspirin) spot also yielded a Rf value 0.88. This confirmed that the second compound in the mixture was acetylsalicylic acid (aspirin). Aspirin had a higher Rf value and travelled further up the plate because its structure was slightly less polar than phenacetin and a lot less polar than caffeine. Aspirin’s structure has a benzene ring with a ester functional group and a carboxylic acid group attached on the same side. Aspirin had a greater affinity towards the slightly less polar mobile phase because its benzene ring makes it slightly less polar and was able to dissolve in the mobile phase quickly allowing it to travel so high. Overall, this unknown mixture was identified as caffeine and acetylsalicylic acid mixture. Phenacetin did not have a spot that matched in the mixture column of TLC plate, so phenacetin was not one of the compounds in the mixture. An error that could have occurred was using too much sample when spotting the plate. This could have caused the spot to travel in a very long elongated streak making it hard to calculate Rf value and compare it to the spots defined on the mixture. This can make the identification process of the compounds in the mixture very hard. Another error is using too little sample on the plate. If not, enough sample was used, it can be very hard to observe where the spot travelled under UV light because the spot would not be pigmented enough, again making it hard to identify compound in mixture. Also, if too polar of a solvent was used then it would be very hard to separate the compounds, all of the compounds would not be accurately separated based on their polarities and all end up going to the top of plate. The compounds would react more with the solvent than the silica gel. The stationary phase is already very polar, using a too polar solvent defeats the purpose of thin layer chromatography. Another error could be trying to elute a spot of crystalline material which is insoluble in the eluent. This will not yield any results on the TLC because there is no way for it to interact with the TLC plate correctly and the reaction would not occur. A soluble solvent is necessary.

Flow chart of Acid Base extraction 1) Weigh 3. 00g of Aspirin/caffeine mixture. Caffeine:

Aspirin (acetlysalicylic acid)

2) dissolve mixture in about 30mL dichloromethane in Erlenmeyer flask and transfer dissolved solution into a separatory funnel. The stopcock of the funnel should be closed. Add about 5mL of the Erlenmeyer flask, rinse it, and put the rest into funnel to ensure all of the sample has left the flask. 3) Add sodium bicarbonate to separatory funnel, add ground glass stopper, pick up separatory funnel and shake it vigorously for about 30 seconds. Vent funnel frequently to prevent buildup of pressure. Place funnel back on stand and let it rest until water layer and organic layer are clearly separated 4) place a beaker under the separatory funnel and open stopcock to allow bottom organic layer to drain into the flask. Drain up until the upper liquid reaches stopcock. Label flask as organic layer (caffeine) 5) place another beaker labelled water layer (aspirin) and drain the rest of the fluid in separatory funnel.

Extracting from Water layer: once aspirin sodium salt is present in top water layer, it has been separated from caffeine, and need to get it back to its neutral form. Add HCl to water layer in baker

Aspirin precipitates out of solution because It’s not soluble in water due to acid being added, able to separate it out of aqueous layer now.

Extracting from Organic Layer: Add drying agent Na2SO4 into flask with dichloromethane layer.

Filter out the sodium sulfate through gravity filtration with flute filter paper, funnel and round bottom flask

Evaporate the dichloromethane from the mixture with caffeine using rotary evaporation. use vacuum filtration and Buchner funnel apparatus Caffeine solid

to collect all of the solid aspirin precipitate and heat it up to ensure all of the water has evaporated from the precipitate.

Aspirin solid...