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Chromatography Separation Of A Dye Mixture Biology Essay

Introduction: The purpose of this experiment is to exhibit the different techniques of chromatography to analyze the composition of complex mixtures. This lab uses Thin Layer Chromatography and Column Chromatography to separate the components of a mixture, which is a (1:1) solution of 0.2% fluorescein dye and 0.2% methylene blue in 95% ethanol. Both methods are based on the differential distribution of the parts of a mixture between two phases. The two phases include stationary phase and mobile phase.

Experimental:

The experimental procedure carried out for Thin Layer Chromatography was identical to the steps listed in the lab manual. Refer to Organic Chemistry Lab Manual Fall 2010 – Winter 2011 page 8 – 9.

The experimental procedure carried out for Column Chromatography was the nearly identical to the steps listed in the lab manual but with a slight modification. Instead of preparing a “slurry” of alumnia in ethanol and then adding it to the column, only approximately 6.0g of dry alumnia was added at first. Once the final layer of sand was added, 95% ethanol was used to wash down the whole column in order to create the required slurry in the middle of the column. For the rest of the steps, refer to Organic Chemistry Lab Manual Fall 2010 – Winter 2011 page 10.

Results:

Thin Layer Chromatography

Using (1:12:14) K2SO4:H2O:CH3CN as a solvent:

Spotting Solution (colour)

Distance Traveled from baseline (cm)

Solvent Front (cm)

Rf Value*

Methylene blue (blue)

4.1

5.3

0.77

Dye Mixture (blue)

4.0

5.3

0.75

Dye Mixture (yellow)

4.7

5.3

0.89

Flourescein dye (yellow)

4.65

5.3

0.88

*Refer to Appendix A for calculations of Rf value

Other observations:

The solvent moved up the plate rapidly after its introduction to the beaker.

Flourescein dye did not leave a large trail. It only has a dark yellow spot with light yellow tint around the edge.

Methylene blue left a large trail as it traveled upwards.

Dye mixture divided into two colours, that of blue and yellow. The yellow traveled further than the blue. Blue colour left a trail but the yellow did not.

Using 95% Ethanol as a solvent:

Spotting Solution (colour)

Distance Traveled from baseline (cm)

Solvent Front (cm)

Rf Value*

Methylene blue (blue)

2.4

4.2

0.57

Methylene blue (purple)

3.7

4.2

0.88

Dye Mixture (blue)

2.4

4.2

0.57

Dye Mixture (purple)

3.7

4.2

0.88

Dye Mixture (yellow)

0.1

4.2

0.02

Flourescein dye (yellow)

0.0

4.2

0.00

*Refer to Appendix A for calculations of Rf value

Other Observations:

There were purple impurities observed in Methylene blue and Dye Mixture. These moved further than any other colour from their respective spotting solution.

The Flourescein dye did not move up with the solvent. It remained on the baseline as a dark yellow spot with light yellow tint around the edge.

Aside from the purple impurity, the Dye mixture divided into yellow and blue colours. The blue colour traveled upwards but the yellow colour barely moved up from the baseline.

The blue colour from both, Methylene blue and Dye Mixture, left a trail while neither of the other two colours did.

Column Chromatography

Components of the Column Chromatography (initial stage):

Alumina slurry: thick and white coloured solution with a medicinal scent.

Sand: brown coloured

Cotton: white coloured

After eluting the column with 95% Ethanol (with the use of air pump):

The top layer sand quickly turned purple and stayed purple during the rest of the experiment.

A layer of blue coloured dye slowly traveled down through the alumina slurry. The slurry changed to blue colour while the dye was running through it but once the dye passed, the alumina slurry was cleared of most of the blue dye and returned to white colour.

Dye then reached the bottom layer sand and turned it purple. The sand stayed purple throughout the experiment.

The cotton was the next to change colours. It turned into a dark blue colour.

The beaker slowly filled up with blue coloured drops. The solution formed was blue in colour and translucent.

After eluting the column with 0.1M NaOH (Sodium Hydroxide):

The top layer sand stayed purple.

A yellow coloured dye (with a hint of blue) entered the alumina slurry. A layer of yellow coloured dye slowly traveled down through the alumina slurry. The slurry changed to yellow colour while the dye was running through it but once the dye passed, the alumina slurry was cleared of most of the yellow dye and returned to white colour.

When the dye reached the bottom layer of sand, the sand layer remained purple in colour.

The cotton turned from dark blue to a slightly less dark blue colour.

At first a few blue coloured drops went into the beaker. Though, yellow coloured drops followed and so the overall solution in the beaker had a greenish-yellow colour. The solution was translucent

Discussion:

Thin Layer Chromatography (TLC) is a type of chromatography technique that is usually used for analytical reasons. In this experiment, TLC was used to compare the Rf values and the distances between different types of dyes (Methylene blue and Flourescein dye) when 95% ethanol and (1:12:14) K2SO4:H2O:CH3CN are used as solvents. The solvents use capillary action to move up the thin layer, while carrying the components of mixture through polar attraction. This is because the solvent and the components of mixture are polar in nature. The mixture separates because different components have different degree of polar attraction to the solvent and so each component will travel at different speed for different distance. It was expected that the Methylene blue dye and dye mixture (blue) would travel the same distance and have identical Rf values (distance traveled by spot divided by distance traveled by solvent) since they are the same compound. While using (1:12:14) K2SO4:H2O:CH3CN as a solvent, it was recorded that Methylene blue dye and dye mixture (blue) traveled 4.1cm and 4.0cm above the baseline, respectively. Methylene blue dye had a Rf value of 0.77 while the dye mixture (blue) had a Rf value of 0.75. While using 95% ethanol as a solvent, it was recorded that Methylene blue dye and dye mixture (blue) both traveled 2.4 cm above the baseline. This meant that they both had a Rf value of 0.57. Hence, from the obtained results it could be clearly seen that Methylene blue dye and dye mixture (blue) had near identical figures, which agrees with the expected results. The human judgment error (in measuring distances with a ruler) most probably accounted for any differences between the distance traveled and Rf values.

Similarly, it was expected that the Flourescein dye and dye mixture (yellow) would travel the same distance above the baseline and so have identical Rf values as they are the same compound. Using the (1:12:14) K2SO4:H2O:CH3CN as a solvent, Flourescein dye and dye mixture (yellow) traveled 4.65cm and 4.7cm and has Rf values of 0.88 and 0.89, respectively. Using the 95% ethanol as a solvent, Flourescein dye and dye mixture (yellow) traveled 0.0cm and 0.1cm and had Rf values of 0.00 and 0.02, respectively. Hence, it can be seen that both of them behaved similarly to the solvent. The solvent barely displaced the two dyes. It can be seen that the obtained results agree with the expect results.

While performing TLC chromatography using 95% ethanol as a solvent, Methylene dye and dye mixture, both had purple impurities that traveled 3.7cm from the baseline. Since there was no impurity found from the Flourescein dye, it could be said that the purple impurity originated from to the Methylene dye. Additionally, since the purple impurity was not detected while using (1:12:14) K2SO4:H2O:CH3CN as a solvent, it could be stated that 95% ethanol solvent is more efficient to separate impurities.

Looking at the Methylene blue dye, dye mixture (blue), dye mixture (yellow), and Flourescein dye (ignoring purple impurity), it can be seen that all the different dyes had a greater Rf value when the solvent was (1:12:14) K2SO4:H2O:CH3CN compared to when the solvent was 95% ethanol. This also shows that dyes, Methylene blue dye and Flourescein dye, are more soluble in (1:12:14) K2SO4:H2O:CH3CN than 95% ethanol. This shows that (1:12:14) K2SO4:H2O:CH3CN has a relatively higher polarity than 95% ethanol as it moved all the dyes further (higher Rf values) than the ethanol solvent. Hence, since Flourescein dye required the relatively higher polarity solvent to move it, the dye must itself have a higher polarity than Methylene blue.

Flourescein dye had a greater Rf value than Methylene blue dye in the basic (1:12:14) K2SO4:H2O:CH3CN solvent. This shows that it had a longer mobile phase and shorter stationary phase when compared to Methylene blue dye and hence proves that Flourescein dye is weakly adsorbent (to alumina on the plate) and more attracted to the solvent than Methylene blue dye. Methylene blue dye had a lower Rf value as it is slightly basic in nature as shown by its structure (refer to Question 2) and so it was more attracted to the stationary alumina, causing it to ascend less with the solvent. The opposite can be said when using 95% ethanol as a solvent as Methylene blue dye had a Rf value while the Flourescein’s Rf value was non-existant.

Column Chromatography is used in order to separate mixtures composed of multiple components and make individual solutions. From the results obtained, it could be seen that Methylene blue dye component was separated from the dye mixture by 95% ethanol solvent as it eluted a blue coloured solution but Flourescein dye was not moved by this solvent at all as no traces of yellow were found. This is due to the fact that Methylene blue is a slightly basic compound as shown by its structure (refer to Question 2) and so it would be attracted to ethanol which is slightly acidic. (CITE) Additionally, 0.1M NaOH solvent was successful in eluting Flourescein dye as well as Methylene blue dye as it eluted the left over blue coloured dye and a stream of yellow coloured dye to form greenish-yellow compound. This proves that 95% ethanol has relatively lower polarity than NaOH as it could only elute one of the two dyes (using both chromatography methods) while NaOH was successful in eluting both dyes. Additionally, this means that Flourescein dye must have a high polarity as it required a relatively higher polarity solvent to help elute it from the column. Flourescein dye’s slightly acidic nature helped attract it to the basic NaOH solvent.

The advantage of TLC method was that it was fast and efficient in separating the dyes as it also showed impurities, but it can only be used to separate small amount of samples as the size of the plate is very small. Column Chromatography is useful for eluting large sample size and the eluents obtained could be purified through distillation.

Questions:

If the level of the solvent in the TLC chamber was higher than the spots at the bottom of the TLC plate then the spotting samples will merge and dissolve into the solvent instead of climbing up with the solvent through capillary action. The sample will dissolve because the amount of solvent would greatly overpower the minute sample size. Hence, the large polar attraction created by the sample will attract almost the entire spotting sample.

Structure of Methylene Blue dye:

Structure of Fluorenscein dye:

The circles on the structure indicate which group will interact with slightly acidic, surface Al-OH and Si-OH on alumina or silica in neutral solvent.

The solvent NaOH will mix with Alumina or Silica and create Al-O- and Si-O- groups. Due to the formation of these groups, there will be less available space for the dyes to form intermolecular bonds with alumina or silica. As a result, the dyes will be more freely available to move along with the solvent as there will be a greater attraction between the two. Hence, NaOH solvent would increase the mobility of the dyes.

Ion Exchange Chromatography is a type of chromatography that is based on highly-selective separation of compounds, cations, and anions based on charge. It is mainly used for separation of solution of proteins and other charged biological materials due to their difference in net charge. This chromatography is composed of cation exchange chromatography, where positive ions are attracted to negatively charged solid in column, and anion exchange chromatography, where negative ions are attracted to positively charged solid in column. The higher the charge on the ion, the greater the concentration that the elutant (usually NaOH) has to be in order to elute the desired molecule/ion. Hence, to elute a molecule/ion, a strong enough elutant (in terms of pH or ionic strength) must run down the column. More specifically, elution of separate components is obtained by steadily running down elutant while gradually increasing its pH or ionic strength to split the ionic attraction between the molecules/ions and the solid in the column.

Appendix A

Calculations for Rf value:

Rf = b/a= distance traveled by spot/distance traveled by solvent

Using (1:12:14) K2SO4:H2O:CH3CN as a solvent (Solvent Front = 5.3cm):

Methylene Blue: 4.1cm/5.3cm = 0.77

Dye Mixture (blue): 4.0cm/5.3cm = 0.75

Dye Mixture (yellow): 4.7cm/5.3cm = 0.89

Flourescein dye: 4.65cm/5.3cm = 0.88

Using 95% Ethanol as a solvent (Solvent Front = 4.2cm)

Methylene Blue (purple): 3.7cm/4.2cm = 0.88

Methylene Blue (blue): 2.4cm/4.2cm = 0.57

Dye Mixture (purple): 3.7cm/4.2cm = 0.88

Dye Mixture (blue): 2.4cm/4.2cm = 0.57

Dye Mixture (yellow): 0.1cm/4.2cm = 0.02

Flourescein dye: 0.0cm/4.2cm = 0.00

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