I.

INTRODUCTION

Chromatography is a group of separation methods for individual compounds or compound-type

mixtures that involves distribution of the mixture components between stationary and mobile
phases; all chromatographic systems rely on the fact that a substance placed in contact with
these two phases, will equilibrate between them (Kebbekus 2005). There are different types of
chromatography that may be used depending on the physical states of the phases involved in the
mixture.

Adsorption chromatography uses surface adsorption in order to separate a solid stationary

phase and a liquid mobile phase; conversely, partition chromatography involves solubility
principles, wherein a liquid stationary phase supported by an insoluble solid and liquid or
gaseous mobile phase.

In adsorption chromatography, the stationary phase, over which the liquid mobile phase is
permitted to flow, adsorbs the mixture to be separated. Transferrance of the adsorbed
compound between the stationary and mobile phase is an equilibrium process, as shown by
Equation 4.1 below.

[EQN 4.1.]

The polarity of the molecule ultimately decides the extent of adsorption, as well as the activity
of the adsorbent and the polarity of the liquid mobile phase; the more polar the functional
group, the greater the chance of adsorption on the surface of the stationary phase. The actual
separation of components in a mixture, on the other hand, is dependent on each component's
relative values of the adsorption-desorption equilibrium constants (K); individually, the
components will move with the mobile phase, though with varying rates which results to their
separation into different regions (as basis or spots) in the stationary phase.

Thin-layer chromatography (TLC), a form of solid-liquid and adsorption chromatography, wherein

the stationary phase (often alumina or silica gel) is applied as a thin coating on a plastic sheet or
glass slide, called a TLC plate (Cortes 2007). This form of chromatography is most useful in
monitoring the progress of reactions, detecting intermediates in reactions, analyzing crude
products or an unknown mixture, determining component count, and for ensuring the efficiency
of a purification process.

Paper chromatography, similar to TLC, is used in rapid analysis of reaction mixture components
and tentatively, as a means for identification; it is an example of liquid-liquid partition
chromatography. The stationary phase of this form of chromatography is paper, specifically filter
paper that is made of highly purified cellulose which, being a polyhydroxy compound, absorbs
and strongly retains water molecules; the mobile phase is the chosen solvent. The mixture to be
separated is placed in small spots near the bottom of the filter paper and the solvent is allowed
to travel upward through capillary action. Separation occurs due to the mixture component's
differing affinities in the polar stationary and mobile phase which is a relatively nonpolar solvent
or solvent system; between water and the solvent, there is a continuous, repetitive exchange of
solutes, though those that are more soluble in the mobile phase spend more time in the solvent
a rise up through the paper much quicker.

A compound will move up the TLC plate or paper strip at a rate relative to that of the solvent
front, (see Figure 4.1.). The relative mobility, or the Rf value, of the compound is defined by
Equation 4.1. below, where the origin is the midpoint of the original spot.

[EQN 4.1.]

By measuring the distance from the origin to the point of greatest density corresponding to the
compound, the distance traveled by the compound may be obtained. The Rf value is dependent
on a certain set of defined conditions, which include absorbent, solvent, temperature, and
humidity, used to determine the identity of the compound.

Spotting is an important technique in chromatography, done with a capillary tube and applied to
a single area repeatedly (waiting for the sample to dry each time so that it may be adsorbed) to
produce solid support known as the "spot"; the spot becomes more concentrated the more
times a solution of the sample is added on top of another (Cortes 2007). There are several
precautions that must be followed when spotting a solution onto chromatographic paper in
order to ensure the return of a proper result with little to no errors, such as: (1) the sample spot
must be placed high enough on the paper so that when placed in the developing chamber, it will
not dissolve due to it being in contact with the solvent, (2) sample spots should be kept small to
minimize overlapping or trailing that might leave the paper, as this may affect the Rf values, (3)
in TLC, it is important to keep the developing chamber tightly covered while the developing
chromatogram is being developed, as this will prevent the stationary phase from drying out
before the process completes (Libal 2017), and finally (4), the solvent fron must not be allowed
to reach the top of the paper, as this may cause erroneous Rf values and cause nearby spots to
coalesce (Cortes 2007).

VI. DISCUSSION

Paper chromatography, consisting of a solid stationary phase and liquid mobile phase, was used
to separate the pigments of the Moringa oleifera or malunggay plant, which is popularly called
the "miracle tree" and is a monogeneric plant in the family Moringaceae (Nweze and Nwafor
2014). A "pigment" refers to a molecule that absorbs and reflects light, and different pigments
appear in different colorsbecause they have differing light absorption and reflection capacities
concerning varying colors of light (Carrington and Long 2006). Plants come in many different
colors and therefore pigments, with most of said pigments playing a vital role in permitting the
plant to realize photosynthesis, such as chlorophyll and carotenoids (Mbaiguinam, Tarkodjiel and
Ngakou 2014).

In the exercise, three different solvent systems were used as the mobile phase in three different
trials in order to compare the efficiency and completeness of the separation: petroleum ether,
9:1 petroleum ether:acetone, and 9:1:1 petroleum ether:diethyl ether:acetone.

The resulting chromatograms, as seen in Figure 4.6., showed that a faint yellow pigment had
been separated from the original green pigment as the solvent front passed through the spot.
The green color of the leaves is due to the presence of chlorophyll (A and B), while the faint
yellow color that followed is due to the presence of carotenoids (such as B-carotene and
xanthophyll), which are antioxidants known to be present in Moringa oleifera (Mbaiguinam et al.
2014); an antioxidant is a substance that has the ability to neutralize free radicals which cause
cellular damage by removing electrons from surrounding molecules (Carrington and Long 2006).
While other pigments may also be present, not all were shown on the filter paper; this may be
due to the type of solvent system used or the degree of difficulty in separation of these other
components.

Based on the results, 9:1 petroleum ether:acetone is the solvent of choice due to its quick and
clear separation of the pigments. The reason for this is because the pigments present in
Moringa oleifera, chlorophyll and carotenoids, have differing polarities. Chlorophyll is nearly
identical in its two forms except for the boxed portion seen in Figure 4.9., however both
compounds have oxygen atoms present in their structures which makes them somewhat polar.
Carotenoids on the other hand, such as B-carotene and xanthophyll which are also nearly
identical except for the boxed portion seen in Figure 4.10., are essentially nonpolar.

[Figure 4.9.] Skeletal structures of chlorophyll a & b.

[Figure 4.10.] Skeletal structures of B-carotene & xanthophyll.

Petroleum ether is a a mixture of nonpolar compounds including pentane, hexane, and 2-

methylhexane, and due to the present symmetry each carbon of the molecules, the only
significant intermolecular forces are dispersion forces; conversely, acetone has a structure in
which the central carbon of the parent chain creates a definite molecular dipole due to the
presence of oxygen (Marsden 2016). These pure solvents, creating a solvent system together, are
able to distinguish between the polar and nonpolar molecules. Petroleum ether should dissolve
the nonpolar compounds and move them along the paper, while acetone does similarly with the
polar compounds (Marsden 2016).

Besides separation of plant pigments, paper chromatography was also used in the identification
of an unknown amino acid. Portions of phenylalanine, tyrosine, and aspartic acid were spotted
onto a single filter paper in the order seen in Figure 4.3. The filter paper was then curled into a
cylinder, stapled, and then placed in a developing chamber with the developing solvent. Initially,
upon removing and drying the filter paper, no spots of separation are seen. However, after
coating the filter paper in ninhydrin solution and drying it, the different colored spots begin to
show (see Figure 4.7.). Ninhydrin, originally yellow in color, reacts with a free alpha-amino
group (NH2-C-COOH) which is present in all amino acids, yielding the purple color seen in the
chromatogram (Amrita Vishwa Vidyapeetham Virtual Lab 2011).
As seen from the data in Table 4.4., the Rf values showed increasing values, in both
measurements on the same chromatogram, from aspartic acid (0.21 and 0.19) to tyrosine (0.43
and 0.38) to phenylalanine (0.59 and 0.57). The unknown amino acid had the Rf values of 0.18
and 0.20, which were closest to that of aspartic acid, and the chromatogram itself also showed
that aspartic acid and the unknown acid travelled the same distance and also exhibited similar
shades of violet (see Figure 4.7.), thus the unknown acid was determined to be aspartic acid.

It can be said that the more polar a certain substance, the higher its Rf value, thus nonpolar
compounds move more rapidly up the stationary phase, or filter paper, than polar compounds
do; the eluting power of solvents also increase with polarity. In relation to the structures of
phenylalanine, tyrosine and aspartic acid in Figure 4.11., the presence of an aromatic ring in
phenylalanine and tyrosine make them less soluble and less polar than aspartic acid, which is
why aspartic acid has the smallest distance from the origin and the smallest Rf value; between
phenylalanine and tyrosine there is a difference of one extra alcohol group in the structure of
tyrosine that makes it more soluble than phenylalanine, thus tyrosine does not travel as far (see
Figure 4.7.).

[Figure 4.11.] Skeletal structures of phenylalanine, tyrosine, and aspartic acid.

Thin layer chromatography or TLC was then used in the purity analysis of benzoic acid, alongside
pure naphthalene for comparison. Spotted onto a single TLC plate with a silica slurry, benzoic
acid dissolved in acetone was placed beside naphthalene dissolved in m-nitroaniline, then left in
a developing chamber with the developing solvent. The results were difficult to see before
placement an iodine chamber, as both solutions were clear, but the iodine sublimes and will
absorb to organic molecules in the vapor phase, causing the organic spots on the plate will turn
brown and become identifiable (Penn State University 2006). Then viewing the plate under
ultraviolet (UV) light showed the solvent fronts, as seen in Figure 4.8. Oftentimes both the use of
an iodine chamber and UV lamp is needed, though some compounds are not "UV active", or
do not absorb light at the wavelength of 254 nm, and therefore cannot be seen under UV light
(Penn State University 2006).

On the TLC plate, the purity of naphthalene can be seen in the lack of spots between the starting
point and the final spot, showing that however in comparison, some spots were seen between
that of the original benzoic acid and the final spot, which means that the benzoic acid used is not
completely pure due to the presence of other separable components. However, in terms of
further analysis, test of purity can be done by isolating certain portions of the paper or TLC plate
and dissolving in an appropriate solvent, and their absorption can be characterized at specific
wavelengths using spectrophotometric methods (Bhanot 2014).

Many chemical substances are colorless, similar to the solutions of benzoic acid and
naphthalene, and difficult to identify in chromatograms. As such, use of the iodine chamber and
viewing under UV light are examples of methods for visual detection. Some methods are general
and can expose all the components in the mixture, similar to the addition of ninhydrin solution
and subsequent heating of the filter paper in the identification of amino acids, while other
methods are very specific and can only react with certain chemical types (Scott 1996). Other
examples include using reagent spray or an atomizer filled with the substance needed for visual
detection, such as a sulfuric acid spray or chromic-sulfuric acid spray (though both methods are
destructive and solutes are unrecoverable after detection) wherein the solutes are partially
oxidized and leave behind easily discernible black carbon deposits (this method helps detect
most involatile organic compounds) (Scott 1996).

Similarly, as it is difficult to visualize colorless solutions, it is far more difficult to visualize

chemical reaction, as they cannot all be observed visually. However, as many chemical reactions
occur in homogenous solution, TLC or paper chromatography is one of many methods that can
be used to separate reactants from the products for visual identifiction, which can be used to
monitor reaction progress. In TLC chromatography, aliquots of the substances undergoing the
reaction can be taken and placed onto the same TLC plate (one spot for starting material, one
spot for the reaction mixture, and one spot for the product, if possible); as the reaction
approaches completion, the spot of the starting material will be used up and unseen in the
reaction mixture, thus presenting the finished product with possible by-products (Chemical
Forums 2008). This type of monitoring also works similarly with paper chromatography, as taking
several samples over the course of the reaction should show the decrease of reactants and
increase of products.

Chromatography can be used to separate a large variation of substances, as was exhibited in this
exercise, however certain typesof chromatography have their limitations. While paper
chromatography can assist in the determination of amino acids, it cannot be used to separate
and identify very volatile compounds. Its solid phase is a filter paper, which is largely composed
of cellulose fibres, and as the solvent rises through the paper, very volatile substances may
vaporize immediately upon contact with the mobile phase and cause it to be unidentifiable.
Similarly, TLC chromatography should also not be used with very volatile compounds, as when a
TLC plate is removed from a developing chamber, the developing solvent should be evaporated,
a process which can sometimes be achieved through heating the plate; a very volatile compound
would also vaporize along with the developing solvent, thus making it unable to identify. Usually
gas-liquid chromatography is used with voaltile liquids that do not decompose at temperatures
around their boiling points.

VII. SUMMARY AND CONCLUSIONS

In this exercise, paper chromatography and thin-layer chromatography were employed as

methods of separation, identification and analysis.

The objectives were met and the procedure was completed with slight modifications.

IX. Recommendations
Instead of using piperine for the TLC analysis, recrystallized benzoic acid from exercise 2 was
used and compared to pure naphthalene.

VIII. REFERENCES

A. Books

SCOTT R.P.W. 1996. Chromatographic Detectors: Design: Function, and Operation. Boca Raton,
Florida: CRC Press, LLC. 363-340.

B. Journals

MBAIGUINAM M., TARKODJIEL M., NGAKOU A. 2014. Proximal and Elemental Composition of
Moringa oleifera (Lam) Leaves from Three Regions of Chad. Journal of Food Resource Science
3(1): 12-20.

NWEZE N.O., NWAFOR F.I. 2014. Phytochemical, Proximate and Mineral Composition of Leaf
Extracts of Moringa oleifera Lam. from Nsukka, South-Eastern Nigeria. Journal of Pharmacy and
Biological Sciences 9(1): 99-103.

C. Web Sources

BHANOT D. 2014. Applications of Paper Chromatography. Retrieved September 30, 2017, from:
http://lab-training.com/2014/11/28/applications-paper-chromatography/

CARRINGTON C., LONG, T. 2006. Paper Chromatography Separates Plant Pigments. Retrieved
September 30, 2017, from: https://msu.edu/course/plb/106/paperchromatography_s06.doc

CHEMICAL FORUMS. 2008. TLC - How can it be used to monitor the extent of reaction? Retrieved
September 30, 2017, from: http://www.chemicalforums.com/index.php?topic=28041.0

CORTES, S. 2007. Recitation Notes for Experiment #5 A&B: Thin Layer Chromatography. Retreived
on September 21, 2017, from:
https://www.utdallas.edu/~scortes/ochem/OChem_Lab1/recit_notes/recit_exp5_tlc.pdf

KEBBEKUS, B.B. 2005. Chromatography. Retrieved on September 21, 2017, from:

https://web.njit.edu/~kebbekus/analysis/4CHROMAT.htm

LIBAL, A. 2017. What Is the Purpose of the Filter Paper in the Thin-Layer Chromatography (TLC)
Process? Retrieved September 21, 2017, from: http://sciencing.com/purpose-filter-paper-
thinlayer-chromatography-tlc-process-16302.html

MARSDEN S. 2016. Separation of Plant Pigments by Paper Chromatography. Retrieved

September 30, 2017, from: http://www.chemtopics.com/unit06/pchrom.pdf

PENN STATE UNIVERSITY. 2006. Thin-Layer Chromatography. Retrieved September 30, 2017,
from: http://courses.chem.psu.edu/chem36/Experiments/PDF%27s_for_techniques/TLC.pdf
VISHWA VIDYAPEETHAM VIRTUAL LAB. 2011. Quantitative Estimation of Amino Acids by
Ninhydrin. Retrieved on September 30, 2017, from: vlab.amrita.edu/?
sub=3&brch=63&sim=156&cnt=1