RESULTS AND DISCUSSION

In this experiment, two types of chromatography

based on solid-liquid chromatographic methods

were used for the purpose of separation and

purity determination. Column chromatography,

which runs a solvent that moves down slowly

through a glass column with packed adsorbent,

was used to separate the colored components of

malunggay leaves. On the other hand, thin layer

chromatography (TLC) which utilizes a stationary

phase composed of a thin layer of adsorbent

material on a glass plate or plastic sheet, was

used to separate the components of each

collected eluate to overall determine their purity

based on the retention values (Rf values)

obtained.

Starting with column chromatography for

separating the colored components of the

malunggay leaves, hexane-acetone was

determined to be the suitable solvent system to

be used as it run through the glass column with

packed adsorbent without dissolving the solid.

This reagent was continuously added up on the

column to prevent it from running dry, as the

pure extract of malunggay leaves was prepared

with the same reagent, to be added next to

perform the chromatography process.

As the pure extract slowly moved down the

column, 3.0 mL each of the solvents, specifically

hexane-acetone, acetone, and acetone-methanol

respectively, were added continously at the same

time while collecting different colors of eluates in

separate test tubes. For this, four different-

colored eluates were collected, of which two were

mixed to provide three distinct-colored eluates,

particularly, pale yellow, green, and yellow green.

The following table presents the data collected

under column chromatography.

Table 1. Characteristic Color and Volume of

Collected Eluate (Column Chromatography)

Color of ComponentVolume of Eluate

(Drops)

1 Pale yellow

2 Green

3 Yellow Green

These eluates were then used as individual

samples for spotting the TLC plates which were

then put inside the developing chamber for

completing the thin layer chromatography. The

movement of these eluates along the TLC plate

coated with silica-gel were possible through the

principle of capillary action.[1] Retention factors

were then determined by measuring the distance

of each component from the origin and by

dividing this to the solvent front, represented by

the formula:

Rf=Distanceof sample¿origin ¿Distanceof solvent ¿

origin¿

Table 2. Characteristic Color and Distance of

Component from Origin (X) with Determined

Rf Value

Color of

Component

Distance of

component

from origin (X)

in cm

Rf Value

1 Pale Yellow 4.89 cm 0.75

2 Green 4.79 cm 0.74

3 Yellow Green 0.42 cm 0.42

After the Rf values were calculated, several

deducations were made. The difference in the Rf

values of the components are based from their

solubility and the strength of their adsorption to

the adsorbent, in the case the TLC plate coated

with silica gel. The lower the Rf value of the

component, the more soluble it is to the solvent

system used, which was hexane-acetone. This is

based on the solubility principle ‘like dissolves

like’. The more soluble the component in the

solvent system is adsorbed more firmly to the

TLC plate.[1] With this principle, it was deduced

that the third component is non-polar, like the

polarity of the solvent sytem hexane-acetone.

From the Rf values calculated, it is also possible

to determine the identity the component by

comparing the experimental Rf values with the Rf

values of known components. The spots on the

TLC plate is also an indicator of purity although it

is not an absolute indicator of it. A pure

compound would appear as a single spot on the

plate and several spots are indications of

impurities.[2] It is also deduced that how close the

Rf values of a component to a known compound

is an indication of the purity of the component.

These explanations is now correlated with the

results obtained from the chromatogram shown

below. The third component has a polarity alike

with the solvent system, which was non-polar,

since it had the lowest Rf value out of the three

components. The several spots on the

Chromatogram, specifically in the second

component is also an indication that the

components we have separated still contain some

impurites. These impurites can be from the

different apparatus used and even from the

different solvents used in Column

Chromatography.

Figure 1.

Chromatogram under UV light

Figure 2. Dried Chromatogram with Markings

REFERENCEFrom the Internet[1] University of Massachusetts. (n.d.). Thin layer Chromatography [PDF]. Retrieved from http://www.chem.umass.edu/~samal/269/tlc.pdf

[2] Interpretation of experimental data [PDF]. (2011, August). Retrieved from http://chemistry.syr.edu/totah/che276/support/6a1.handouts/4.DataInterp.pdf