Chem Lab

Column and thin layer chromatography of plant pigments
CHEMISTRY
201L
EXPT 04
PAGE 01 - 12

Noel Angelo P. Kalacas*, Hanna Mae Laluces, Ina Bianca Lanuza
Department of Chemistry, College of Science

*Corresponding author; e-mail: [e-mail]
Abstract
Chromatography is a powerful technique for separating and/or identifying the components in a mixture. There are different types of chromatography and each has its own strengths and weaknesses. In this experiment, pigments of the chili pepper were extracted with the use of hexane, hexane-DCM, DCM, and DCM with methanol; then, the extract was introduced into the column and eluate were collected, this process is the column chromatography (CC). The purity of the components was determined by using the thin layer chromatography (TLC). Ultraviolet spectroscopy and iodine staining were used to visualize the developed TLC plate and the Retention Factor was measured.
Keywords: Chromatography, column chromatography, TLC and visualizing agent, elution, capsicum frutescens

Introduction
Chromatography can be defined as the separation of a mixture, in which the mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. Chromatography was first developed by the Russian botanist Mikhail Tswett in 1903 as he produced a colorful separation of plant pigments through a column of calcium carbonate. The principle behind chromatography is that different substances have different partition coefficient between mobile and stationary phases, and consequently each will move through a system at a different rate, resulting in complete separations.
There are various types of chromatography, depending on the physical states of the phases. Employing a gas, the mobile phase is termed as gas chromatography (GC) or vapor phase chromatography (VPC). Separations using gas chromatography involve vapor phase versus adsorption and/or equilibria. Liquid chromatography (LC) refers to any chromatographic process that employs a mobile liquid phase. Chromatographic separations can also be carried out using thin layer chromatography (TLC) and column chromatography (CC) which a variety of supports, including immobilized silica on glass plates.
Chromatography separates a substance into its component parts, which is very useful, as substances are often unique in their composition. It can identify a substance and show how it differs from others that may look alike on the surface. All types of chromatography are useful for analytical purposes. Under appropriate conditions, all types of chromatography can be used for preparative scale separations. In every type of chromatography there are three elements to be considered: the size of the sample (load), relative separation of components (resolution), and the speed.
It would be ideal if all three elements could be maximized so that the complete separation of samples of any desired size could be quickly achieved. In practice, generally two of these elements can be maximized at the expense of the third. For routine analytical work, resolution and speed are maximized at the expense of the load. In preparative scale separations, load and speed can be maximized but then separations are usually incomplete. Complete separations of large samples can be achieved but the overall operation is likely to be slow and tedious, and may involve the use of large quantities of solvent that must be distilled for reuse, or discarded.
In the experiment, Column Chromatography and Thin Chromatography were used.

Figure 1. Column Chromatography

Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. Not only it can be used to determine the number of components in a mixture, but it can also be used to separate and purify considerable quantities of those components for subsequent analysis. This is in contrast to paper chromatography, which is solely an analytical method. The disadvantage of a column chromatography is that it is time-consuming and tedious, especially for large samples. Analytical methods such as paper chromatography may be more suitable and easier to perform as an alternative to column chromatography.
Figure 2. Thin Layer Chromatography
Thin-layer Chromatography (TLC) is closely related to column chromatography. The adsorbent is coated on one side of a strip or plate of glass, plastic or aluminum. The solvent travels up by plate through capillary action. It has a number of advantages: It is simple, quick and inexpensive, and it requires only small amounts of sample. TLC is generally used as a qualitative analytic technique, such as checking the purity of a compound or determining the number of components in a mixture or column chromatographic function. In addition, TLC is useful for determining the best solvents for a column chromatographic separation. It can be used for an initial check on the identity of an unknown sample. Preparative plates can be carried out with special, thick-layered TLC plates.
Different solvent systems were used to elute through a column chromatography. These were hexane, hexane-dichloromethane, DCM, and DCM with methanol.
The solid phase (silica gel) is eluted with these solvent systems until fully solvated, the compound to be purified is then loaded onto the solvated solid phase, and the column is eluted with these solvent systems until the desired compound has come off the column.
The Retention or Retardation Factor (Rf value) is the ratio of the distance that the spot travelled relative to the distance moved by the solvent, which in this case is the DCM-hexane. The objective of the experiment is to separate and analyze the components of a plant pigment.
Results and discussion
The plant used in the experiment was a red chili pepper and the solvent systems used were hexane (C6H14), hexane-dichloromethane (C6H14-CH2Cl2), dichloromethane (CH2Cl2), and dichloromethane with methanol (CH2Cl2-CH4O) for the column chromatography, while the hexane-ethyl acetate (C6H14-C4H8O2) used in the TLC.
Column Chromatography
Four eluates were obtained from the extraction of the colored components of red chili pepper using Column Chromatography. Two clear, light yellow eluates, one lclear, light orange eluate, and one clear orange eluate were obtained respectively.
Table 1. Color of the eluates extracted using Colum Chromatography Test Tube # | Color of component | 1 | Clear, light yellow | 2 | Clear, light yellow | 3 | Clear, light orange | 4 | Clear orange |

Thin Layer Chromatography

Figure 3. TLC plate viewed under UV light
Figure 4. TLC plate subjected to Iodine crystals
With reference to Figure 3 (From top to bottom), the first spot, which was oval on the TLC plate is the second eluate, while the second spot is the third eluate collected from column chromatography. The first eluate travelled 4.8 cm from the origin, while the second eluate travelled 2.25 cm from the origin.
After the TLC plate was subjected to iodine crystals it was revealed that there was a third dark yellow oval spot in the fourth eluate of the chili pepper extract.

Table 2. Number of spots on the TLC plate Test Tube # | Number of spots | | UV | Iodine | 1 | 0 | 0 | 2 | 1 | 1 | 3 | 1 | 0 | 4 | 0 | 1 |

The spots in the developed TLC plate were not visible by the naked eye. It was placed in the UV light for viewing any compounds which are UV-active, particularly those with extended conjugation, aromatic rings, unsaturated carbonyls, etc. The TLC plate was then placed in a chamber of iodine crystals in order to visualize organic compounds that have an affinity to iodine, such as unsaturated and aromatic compounds.
Calculation of Rf (Retardation/Retention Factor)
After measuring the distance travelled for each spot, the Rf value was computed. The Rf is the ratio of time spent in the stationary phase relative to the time spent in the mobile phase.
The general formula for computing the Rf value is shown below:
Rf= distance of the spot from origindistance of the solvent from the origin
Since Rf value is a ratio, Rf doesn’t have any unit. Computation of the Rf value has been provided below:
Distance of solvent from origin: 5.7 cm
Rffirst spot= 4.85.7 =0.82
Rfsecond spot= 2.35.7=0.39
Rfthird spot= 4.75.7=0.82
The Rf value cannot exceed the value of 1. To avoid the decimal point, the Rf value is sometime multiplied by 100 and then described as the hRf value. The formula of hRf is obtained through the formula below: hRf=Rf ×100
To obtain the hRf for each spot, just multiply their Rf by 100.
Table 3. Retention factor of each spot Test Tube # | Distance of component from origin (cm) | UV | I2 | | | Rf | HRf | Rf | HRf | 1 | 0 | 0 | 0 | 0 | 0 | 2 | 4.8 | 0.84 | 84.2 | - | - | 3 | 2.25 | 0.39 | 39.5 | - | - | 4 | 4.7 | - | - | 0.82 | 82 |
The developed plate wasn’t able to show completely the separation of colors. The possible sources of error are from the spotting of the TLC plate. When the extracted pigments of chili pepper were spotted on the plate, it was not left completely dry before placing the succeeding spots, in addition to that, the spots weren’t small enough which have caused the color to disarray. Another source of error is not covering completely the developing chamber during the development of the TLC plate.
Experimental methodology
Pigments of the red chili pepper were extracted by pouring dichloromethane and eventually grinding it with sand to break the cells using mortar and pestle. The extracted pigments were set aside for a while.
A glass dropper was plugged with a small piece of cotton. The dropper was secured with a burette clamp. A slurry of silica gel with hexane was then prepared in a beaker. Using another clear dropper, a slurry of silica-hexane was pipetted into the glass dropper until it reaches the intended part, which was the curvy part near the top of the dropper, and the micro column was quickly packed, not allowing the silica to dry out.
Ten drops of the chili pepper extract was placed on top of the slurry using Pasteur pipette. The mixture was allowed to go down in a 50-mL beaker and get adsorbed. The pigment mixture was eluted using the first eluting solvent, hexane, and was allowed to drain down the column. The hexane was continuously added until a noticeable color band descended from the color. When the color was about to be eluted out of the column, the pigment was collected in a clean, dry, labeled test tube while the colorless eluate was discarded. The eluting solvent was changed to a hexane-dichloromethane after the color band using the first eluting solvent stopped descending. This time another color band descended, and was collected in another test tube. After no more color band descended, the eluting solvent was changed to dichloromethane and another color band was collected in another test tube. The final eluting solvent was dichloromethane with methanol, wherein another color band was collected in a different test tube. All pigment samples were covered to protect them from direct sunlight. After collecting the pigment samples, thin layer chromatography was performed.

Figure 5. Spotting of TLC plate
The colored eluates were applied on a TLC plate by equidistantly spotting each mark in the plate using a micro capillary tube. The spot was allowed to dry first before applying the succeeding spots. It was ensured that the spots made were small as possible so that when the plate develops, the colors would not be in disarray.
The developing chamber was prepared by placing the approximate amount of hexane with ethyl acetate in a beaker. The inner wall of the beaker was lined with filter paper in order for the TLC plate to stand. It was then covered with a watch glass and was allowed to equilibrate. The developing plate was carefully placed in the developing chamber. The solvent system was allowed to rise up until it reaches just 1 cm from the upper end. The developing plate was then removed carefully from the chamber and was allowed to dry.
The developed TLC was then placed inside a UV chromatography box. The components were then visualized and the spots were quickly traced using a pencil. The TLC plate was then put inside a chamber containing iodine crystals in order to view the colorless spots which the UV chromatography box wasn’t able to view. The Rf value for each spot was then calculated.
References
Fessenden, J., & Feist, P. (2001). Organic laboratory techniques. Australia: Brooks/Cole; pgs. 119-125, 133-138.
Robards, K. (1994). Principles and practice of modern chromatographic methods. San Diego, California: Academic Press, Inc.; pgs. 1-34,36-225.
Williams, T. (1946). An Introduction to chromatography. London: Blackie & Son Limited; pgs. 22-34, 47-50. http://curlyarrow.blogspot.com/2011/11/lets-talk-about-tlcs-part-5-iodine.html (retrieved on September 2, 2012) http://media.rsc.org/Modern%20chemical%20techniques/MCT5%20Chromatography.pdf (retrieved on September 2, 2012) http://www.umich.edu/~orgolab/Chroma/chromahis.html (retrieved on September 5, 2012)

References: Fessenden, J., & Feist, P. (2001). Organic laboratory techniques. Australia: Brooks/Cole; pgs. 119-125, 133-138. Robards, K. (1994). Principles and practice of modern chromatographic methods. San Diego, California: Academic Press, Inc.; pgs. 1-34,36-225. Williams, T. (1946). An Introduction to chromatography. London: Blackie & Son Limited; pgs. 22-34, 47-50. http://curlyarrow.blogspot.com/2011/11/lets-talk-about-tlcs-part-5-iodine.html (retrieved on September 2, 2012) http://media.rsc.org/Modern%20chemical%20techniques/MCT5%20Chromatography.pdf (retrieved on September 2, 2012) http://www.umich.edu/~orgolab/Chroma/chromahis.html (retrieved on September 5, 2012)