Paper chromatography is a simple, analytical technique used to separate the components of a mixture. It is an inexpensive method requiring only small quantities of material, making it accessible for various applications. The separation occurs because of the differential movement of the mixture’s components through a stationary phase and a mobile phase.
This technique is based on differential solubility, where components distribute themselves between the stationary phase (typically filter paper) and the moving liquid solvent (mobile phase). The paper’s cellulose structure holds water molecules, which serve as the true stationary phase, while the solvent moves through the paper by capillary action.
Preparing the Experiment: Materials and Setup
The foundation of a successful experiment begins with careful preparation of the materials. The stationary phase is usually high-quality filter paper. Handle the paper only along the edges to prevent skin oils from contaminating the surface and interfering with the solvent flow.
A starting line, known as the baseline, is drawn horizontally across the paper using a pencil, typically 1.5 to 2 centimeters from one end. Pencil is used because the graphite will not dissolve in the mobile phase, unlike ink. Samples are applied directly onto this baseline, often using a fine capillary tube to ensure the spot remains small and concentrated.
The mobile phase, or solvent, is selected based on the components being separated; common choices include water, rubbing alcohol, or a mixture. The solvent’s polarity influences the separation. The developing chamber (a glass jar or beaker) is prepared by pouring a small amount of solvent into the bottom, ensuring the solvent depth is less than the distance between the paper’s edge and the pencil baseline.
The Chromatography Procedure: Step-by-Step
Once the paper and solvent are prepared, the separation process, known as developing the chromatogram, can begin. The sample mixture is applied to the baseline as a small, concentrated spot and allowed to dry completely before the paper is placed in the chamber. Multiple spots can be applied along the baseline, provided they are spaced far enough apart to prevent components from bleeding into one another.
The paper strip is carefully suspended inside the developing chamber, typically by taping it to a pencil resting across the top. Ensure the bottom edge is submerged in the solvent. The sample spots on the baseline must remain entirely above the surface of the solvent. If the spots touch the solvent directly, the sample will dissolve into the pool, and no separation will occur.
As the experiment runs, the solvent moves up the paper against gravity due to capillary action, carrying the dissolved components with it. Components travel at varying speeds, depending on their relative affinity for the stationary paper versus their solubility in the mobile solvent. The solvent front, the highest point the solvent reaches, is monitored until it is close to the top edge of the paper.
When the solvent front is near the top, the paper is immediately removed from the chamber. The exact position of the solvent front must be marked immediately with a pencil before the paper dries, as this line is necessary for later calculations. The paper, now called a chromatogram, is then allowed to air-dry completely.
Interpreting the Results and Calculating Rf
The dried chromatogram displays a series of separated spots, with each distinct spot generally representing a single component. Components highly soluble in the mobile phase and with less attraction to the stationary paper travel the furthest. Those with a stronger affinity for the paper remain closer to the baseline.
For components that are not naturally colored, such as amino acids, a visualization step is necessary to make the spots visible. This often involves spraying the dried paper with a specific detecting agent, like ninhydrin, which reacts to produce distinct purple or yellow-colored spots. When using chemical sprays, take safety precautions, such as working in a fume hood and wearing gloves, as the chemicals can be irritants.
To quantitatively analyze the results, the Retention Factor (\(R_f\)) value is calculated for each separated spot. The \(R_f\) value is a ratio that quantifies how far a particular component has traveled relative to the solvent front. The calculation requires two measurements from the original pencil baseline: the distance traveled by the center of the component spot and the total distance traveled by the solvent front.
The formula is \(R_f = \frac{\text{Distance traveled by component}}{\text{Distance traveled by solvent front}}\). Since the component cannot travel further than the solvent, the \(R_f\) value will always be a number between zero and one. Comparing the calculated \(R_f\) value of an unknown substance to known \(R_f\) values under identical conditions helps in compound identification.
Practical Uses of Paper Chromatography
Paper chromatography is utilized across several scientific disciplines due to its effectiveness and simplicity.
Biochemistry
The technique is routinely employed for the separation and identification of complex biological mixtures, including the analysis of amino acids and the separation of various carbohydrates and sugars.
Environmental Monitoring
The technique is valuable in environmental monitoring, where it can be used to test water and soil samples for the presence of pollutants or heavy metals.
Educational and Forensic Applications
In educational settings, it is a common exercise to separate colored pigments found in plant extracts, such as chlorophyll, or to analyze dyes present in ink. The method’s ability to analyze very small quantities of material also makes it a powerful tool in forensic science for identifying trace evidence.