Introduction

Chromatography operates on the principle that the components of a mixture will distribute

unequally between two immiscible liquids (which is also the basis for separations by extraction).

There are two phases in chromatography: the mobile and stationary phase. The most frequently

used normal stationary phase for liquid chromatography is adsorbent that serves as a stationary

phase. Adsorbent in TLC (thin-layer chromatography) is usually alumina (Al2O3) or silica gel

(silicic acid SiO2 x H 2O), both of which are polar. Silica gel is slightly acidic, and alumina is the

most polar between the two.

Substances that have carboxyl groups and other polar functional groups are more strongly

adsorbed than those that contain less polar moieties-such as those present in alkenes and alkyl

halides. When sample compounds having highly polar functionalities, more polar eluants may be

required to effect a TLC analysis.

A solvent should not be too polar because it may bind strongly to the adsorbent and force the

solute to remain in the mobile phase. If this happens, then the components will move rapidly up

the TLC plate, offering little opportunity to establish the solid-liquid equilibria required for

separation. The eluting solvent must be less polar than various components of the mixture to

obtain an effective separation.

The polarity of a compound will influence its run through the TLC plate depending on the

eluting power of the solvents. For example, if there are two compounds that are being separated

that differ in only one -OH group (-OH is a very polar group) and the eluting solvent is

petroleum ether , the compound that lacks the –OH group will be placed on top of the compound

that has the –OH group. This will happen because the polarity of the compound can either

increase or decrease the eluting power as it runs through the TLC plate. Water (polar compound)

will have a higher eluting power with the polar stationary phase than petroleum ether (non-polar

compound), but petroleum ether will have a higher eluting power with a non-polar stationary

phase than water. Polarity affects the R f-value because it depends on the distance traveled by the

substances on the TLC plate.

The R f-value (retention factor) is calculated by Equation 1.1. The R f-value for a compound is a

physical constant that is given for a set of chromatographic conditions. The adsorbent and eluting

solvent should be recorded along with the experimentally determined R f-values. For example say

the solvent traveled 18 cm, and the first compound can be found at 1.3 cm. To find the R f-value,

one would simply plug in the numbers in Equation 1.1. After plugging in these values, one

would find the R f-value to be .072 cm.

R f=distance traveled by substancedistance traveled by solvent

Equation 1.1

Thin Layer Chromatography (TLC) is a form of solid-liquid adsorption chromatography. It’s a

rapid analysis of small quantities of samples. Both column and TLC cannot be used on volatile

compounds having boiling points below 150ᵒC (760 torr). Column chromatography depends on

the same fundamental principles as TLC, but it allows one to separate multigram amounts of

mixtures; however, column chromatography takes a considerable longer time than TLC.

Data and Observations

(From Left to Right: TLC Plate 1, TLC Plate 2, and TLC Plate 3)

-TLC Plate 1 Mobile Phase

-60% Petroleum Ether

-40% Acetone

-TLC Plate 2 Mobile Phase

-70% Petroleum Ether

-30% Acetone

-TLC Plate 3 Mobile Phase

-80% Petroleum Ether

-20% Acetone

Results and Discussion

The eluent that was most efficient for the separation of pigments was the solution of 60%

petroleum ether and 40% acetone. The range of R f-values for this eluent were good because all

the compounds on this plate were separated more than those on the other two plates. The highest

R f-value (R f=0.92) on this plate was higher than those on the other plates (R f=0.51 and R f

=0.53). Since the eluent solvent was the most effective for separation, then that must mean that

this eluent is less polar than the various components of the mixture. In this experiment, acetone

has a high eluting power with polar stationary phases, and petroleum ether has a high eluting

power with nonpolar stationary phases.

Beta carotene (Figure 1) is a non-polar compound because it lacks any polar groups, and lutein

(Figure 2) is polar because it has two oxygen-hydrogen (-OH) groups. On the TLC plates lutein

is always the first one to separate, and beta carotene is always at the top of the plate (nearest to

the solvent line). Since the eluent is mostly petroleum ether (high power with non-polar

stationary phases), then that would mean that the compound that traveled further on the TLC

plate- beta carotene- is non-polar. Chlorophyll b (Figure 3) is more polar than chlorophyll a

because it has an extra –OH group-which makes it more polar. On the TLC plate chlorophyll a is

above chlorophyll b because the latter is more polar. The less polar compound will be above the

more polar compound.

Figure 1

Figure 2

Figure 3

Conclusion

TLC is a good technique for rapid analysis of small quantities of samples. Its power depends on

choosing an appropriate adsorbent-in this experiment silica gel was used to separate the spinach

pigments. An appropriate eluant must also be chosen to achieve a good separation. The eluant

can separate both polar and non-polar compounds, but it must also be significantly less polar

than the various components of the mixture.

Works Cited

Experimental Organic Chemistry 5th Edition

http://www.webexhibits.org/causesofcolor/images/content/chphyll.gif

http://upload.wikimedia.org/wikipedia/commons/4/44/Beta-carotene-2D-skeletal.png

http://upload.wikimedia.org/wikipedia/commons/1/1d/Lutein.png