Paper Chromatography Science Background


All matter can be placed into two categories - pure substances and mixtures. Pure substances are either elements or compounds, and mixtures can be classed as suspensions, solutions, or colloids. The substances in a mixture are not chemically combined, so therefore they can be separated through some physical process. A heterogeneous mixture is when the mixture is made up of parts that are dissimilar (sand is a heterogeneous mixture). Homogeneous mixtures (also called solutions) are uniform in structure (milk is a homogeneous mixture). A sugar cube floating in water is a heterogeneous mixture, whereas sugar dissolved in water is a homogeneous mixture.

A suspension is is a heterogenous fluid containing solid particles that are sufficiently large for sedimentation. Unlike colloids, suspensions will eventually settle. An example of a suspension would be sand in water.

A solution is a homogeneous mixture composed of two or more substances. In such a mixture, a solute is dissolved in another substance, known as a solvent. A common example is a solid, such as salt or sugar, dissolved in water, a liquid.

A colloid is where relatively large molecules of one substance remain mixed and stable due to electric charge repulsions. The repulsion prevents coagulation and promotes the even dispersion of such particles through a mixtures.. This repulsion occurs because colloidal particles contain an equal number of positive and negative ions (charged atoms), but the negative ions form a layer surrounding the particle. Thus, the particles are electrically neutral but still tend to repel one another to spread out evenly through the dispersing medium. Milk, gelatin, clay, and smoke are all colloids that combine solids, liquids, and gases in different ways. Emulsions are a subset of colloids where the two components are both liquids.

Many mixtures, such as inks and food colorings, consist of two or more dyes. To separate the dyes, a small portion of the mixture is placed onto an absorbent material, such as filter paper. A liquid called a solvent is absorbed onto one end of the filter paper. The solvent soaks the filter paper, dissolving the ink. If a dye in the ink dissolves well, it will move along the paper at the same rate as the solvent. If another dye in the ink doesn't dissolve as well, it will not move as far.

Chromatography history

Chromatography was developed in the early 1900's when a Russian scientist, Mikhail Tsvett, was searching for a way to separate the hidden red and yellow pigments from green leaves. Like many students, Tsvett knew that the colored pigments were present in green leaves, but remained hidden until the chlorophyll broke down in the fall, allowing the leaves' true colors to appear. He crushed green leaves into a thick solution, and discovered that when this solution was mixed with a certain powder, different colors appeared in specific areas of the powder. The hidden colors in the leaves separated to different areas, depending on how easily they were absorbed by the powder.

Years later, two British researchers, Martin and Synge, improved upon Tsvett's procedure and developed a process called paper chromatography. They were able to use this technique to separate the amino acids in a protein, and were awarded the Nobel prize in chemistry in 1952 for their work in paper chromatography.


A very approximate, but useful, description of white light such as the sunlight reaching the earth, is that the light is a mixture of light of several colors. Specifically, we can construct white light by overlapping three beams of light, one each of red, green, and blue. These are the additive primaries. Where all three overlap we see white light. Subtractive color systems start with white light. Colored inks, paints or films placed between the viewer and the light source or reflective surface (such as white paper)subtract wavelengths from this white, and make a color.The subtractive primaries are cyan (blue + yellow (red and green)), magenta (red and blue), and green). Dyes of these colors are frequently used to make colored photographs and inks such as are found in markers. If a piece of yellow cellophane, for example, is placed in front of a white light source, such as a flashlight or slide projector, a beam of yellow light is formed. Since white light can be described as a mix of red, green and blue and since yellow is a mixture of red and green, but no blue, we can say that the yellow cellophane has allowed red and green light to pass through, but has subtracted or absorbed the blue light.

Mixtures of subtractive primary dyes can be used to make inks of different colors. For example, it is common to prepare green ink by mixing yellow and cyan subtractive primary dyes. The origin of this color can be visualized by considering the effect of placing cyan and yellow cellophane filters in front of a white light source.

When dyes are mixed in the ink on a paper surface, light reflecting from the paper after passing through the dyes is now colored. In this example the cyan dye absorbs the red light and the yellow dye absorbs the blue light. Only the green light is not absorbed and so the ink is seen as green.

Paper Chromatography Chemistry

Molecules with similar arrangements of their atoms or molecular structures are attracted to each other. Water (H 2O) molecules have two hydrogen atoms at a 104° angle with the oxygen at the vertex. Because of this structure the oxygen end of the molecule has a small negative electrical charge and the hydrogen end has a small positive charge. Liquid water is held together by the attraction between the charges on adjacent water molecules. A molecule with these charged regions is called a polar molecule.

Cellulose is the molecule which is the basic component of paper. It is a very long molecule (a polymer) in which thousands of rings of six atoms each are linked together like beads. Cellulose has polar -OH regions, like water, and these are attracted to the -OH groups on adjacent cellulose chains helping to hold the fibers together in paper. Water molecules are also attracted to these regions and when paper is wet it loses strength because the water molecules get between the cellulose chains and weaken the attraction between them. When the end of a piece of paper is dipped into water the water molecules keep finding new places (polar regions) to stick to and so the water molecules climb up the paper being replaced by new water molecules below. Other molecules which might be dissolved in the water will also be carried up the paper. This is the basis of paper chromatography. As the water moves up, it will carry with it the dye molecules. The more the dye is attracted to the paper, the slower it will travel up the paper, possibly not at all. If two or more dyes have been mixed to form an ink, then they may move at different rates as the water moves up the paper. If this happens, they will separate and we can identify them by their colors.

A very good way to compare dyes on different chromatograms is to measure the distance each dye moves relative to the solvent. This is called the R f value of the dye and it provides a way to judge whether two different dyes are the same. If the R f values are close and the dyes have the same color, than they are probably the same. The R f value is calculated by dividing the distance that the leading edge of the dye spot moved by the distance that the solvent has moved. Since the dye cannot move any farther than the solvent moves, the maximum value for the R f is 1.0. This would be found if the dye did not stick to the cellulose molecules at all. If it was strongly attracted to the cellulose then the R f would be very small because the dye would move very little compared to the solvent.

Related separation techniques

Thin-layer Chromatography. The stationary phase is a thin layer of silica gel or starch dried on a glass or metal plate. The mixture to be separated is placed at the bottom of the plate and the plate is placed in a closed chamber of solvent, which is the mobile phase. The solvent moves across the gel or starch the same way that solvent moves through paper.

Gel Chromatography. A porous gel packed into a column of glass is the stationary phase and the mixture to be separated is poured into the top of the column. Various samples of liquids exiting from the column at the bottom (eluents) are collected and further analyzed. The time it takes for a certain molecule to travel through the column is unique for that type of molecule. This technique is often used to separate different sizes of large biological molecules because the size of the pores in the gel can be controlled easily.

Gas Chromatography. In this technique, the mixture to be analyzed is in gaseous form. The gases in the mixture react with a coating on the inside of a glass tube (stationary phase) and are carried by inert gases such as helium (mobile phase). Gas components exit the tube at different times and are often further analyzed using a spectrophotometer. This technique can be used to separate complex mixtures of aromas and flavors, or to help analyze pollutants in the air.


Lecithin can be purified out of egg yolks (in Greek lekithos—λεκιθος) or soy beans and stored for a long time without refrigeration. Lecithin is a key building block of cell membranes—it is found in every cell in your body. It is made primarily of phosphotidylcholine. The oldest known emulsifier is beeswax, which was used in skin lotion by the Greek physician Galen (131–201 AD). But it was not until the early 19th century that egg yolk became the first emulsifier used in food applications. Since the shelf life of products based on egg yolk is rather short, in the 1920s lecithin derived from soybean was introduced as a food emulsifier. However, it was probably the invention of margarine by the French chemist Hippolyte Mège-Mouriès in 1869 that contributed most to the use of food emulsifiers on an industrial scale.