UMR ChemLabs Chromatography PCh 9-98

Gary L. Bertrand, Professor of Chemistry

Any system consisting of two or more equilibrium phases (liquid + vapor, solid + vapor, liquid + liquid, liquid + solid) has the ability to partition its components, depending on their relative affinities for the different phases. This relative affinity is defined as an equilibrium constant representing the ratio of the equilibrium activities of a component in two different phases. These activities are often approximated as concentrations (moles/volume, though in some cases the concentration may be raised to some mathematical power), and the equilibrium constant is called a distribution constant, or partitioning ratio. When one phase is a solid, a component is often adsorbed on the surface of the solid, and the activity is approximated as a surface concentration, moles/area. However, this activity may also be represented as moles/volume, since the total area of the adsorbing surface is proportional to the weight or the volume of the solid. That proportionality, however, may depend on the particle size and/or the porosity of the solid. Many methods for separating chemical mixtures are based on the different relative affinities the components may have for the two phases. Chromatography (Greek kromos = color, graphein = to write) methods utilize many repetitive partitionings to obtain separations between compounds (analytes) with very similar affinities. These methods involve a mobile phase (liquid or vapor) flowing over, around, or through a stationary phase (solid or immobilized liquid), carrying the analytes at different rates to a point where they may be detected and/or collected. Components with greater relative affinity for the mobile phase will move more rapidly with the mobile phase than components with lesser relative affinities for the mobile phase. With colored compounds, these movements may be seen very easily with column chromatography, paper chromatography, and thin-layer chromatography; all of which involve a liquid mobile phase and an adsorbing solid stationary phase. When a piece of adsorbent paper or cloth, such as paper towel or filter paper, is placed in contact with a liquid, such as water, the paper is wetted. The wetting is not constrained to the point of contact, however, but advances in all directions, including upward for 10 cm or more, by the capillary effect, or wicking. If spots of colored compounds have been placed on the paper slightly above the point of contact, some of these compounds will be carried upward with the liquid. If a spot contains a mixture of compounds, it may be separated into different colors climbing at different rates. This is the essence of paper chromatography. The distance traveled by a spot relative to that of the upper edge of the wetted area (the solvent front) is called the Retention Factor, Rf: Rf = (distance travelled by component)/(distance travelled by solvent). This retention factor is characteristic of the compound, the adsorbing medium (the paper), and the solvent. The relative affinity of the component (K) for the mobile phase relative to the stationary phase is related to the retention factor and the ratio of the volume of the mobile phase to the volume of stationary phase in the completely saturated paper (r): rK = Rf/(1 - Rf). [If the concentrations used in the equilibrium constant, K, are on a per weight basis, as in molality (moles/kg) or weight fraction, r is the ratio of the masses of the mobile and stationary phases.] The product Kr represents the equilibrium ratio of moles (or grams) of the component in the mobile and stationary phases in a small increment of the wetted paper. A component which moves with the solvent front has infinite affinity for the mobile phase relative to the stationary phase, and a component which does not move at all has zero relative affinity for the mobile phase. As spots of color move upwards on the paper, they become progressively more spread out and diffuse, in both vertical and horizontal directions. This band broadening reduces the ability of this method to separate materials by extending the length of the separation process (perhaps by placing the original spots at the top of the paper and allowing the solvent to carry them downward), and is thus inversely related to the efficiency of the method. Column chromatography involves an adsorbing solid which has been ground or powdered to a fairly uniform size, packed into a vertical column above a porous plug (perhaps a wad of cotton or glass wool), through which a liquid may slowly flow. Analytes are placed at the top of the column, and as solvent flows over them, they are carried as bands through the column at different rates, again depending on their relative affinities for the liquid and solid phases. The separated materials may be collected as they are eluted by the solvent. Instead of measuring the distance travelled, as in paper chromatography, one measures the amount of solvent required (Vmax) for the center of a band (the most concentrated part) to reach a designated point on the column, usually the end. The retention factor (Rf) is this volume divided into the dead volume (Vo), which may be approximated as the total volume of mobile phase in the column, or the volume of solvent required to elute a component which is known to have very little affinity for the solid phase. Rf = Vo/Vmax , and again, with r representing the volume of mobile phase to stationary phase in the packed column, the relative affinity (K) of the analyte for the mobile phase relative to the stationary phase is given by: rK = Rf/(Rf - 1) This experiment will quantitatively demonstrate these chromatographic principles by independently measuring the retention factor (Rf), the equilibrium constant (K), and the ratio (r) for the adsorption of dyes from aqueous solution onto chromatography paper. Retention factors will be measured for these and other known dyes, pure and mixed, and for several commercial inks and colorings. The weight ratio of stationary (solid) phase and mobile (water) phase is measured by weighing the dry paper, then thoroughly soaking it, blotting away any excess water, and re-weighing. The equilibrium constant is determined by spectrophotometric measurement of the relative concentrations of the dye in a measured volume of solution, before and after being adsorbed on a weighed sample of the paper.

Part A. Chromatography: 1. Draw pencil lines on two sheets of chromatography paper, parallel to the long sides, 0.5 to 1 cm from the edges. Along one of the lines (to be the bottom edge) mark off spaces about 1 cm apart on each of the sheets. Roll each paper into a cylinder, with the two lines circling the outside. Staple at the top and bottom to hold the cylinder in place, bridging the ends of the paper without overlapping. Place small spots of dyes at the spacing marks along the lower line, using toothpicks or any applicator to give a very small spot. Spot each sheet identically, and record where each dye or material is spotted. 2. Add a small amount of water to a petri dish, a few millimeters deep. and place one of the cylinders vertical in the petri dish. Watch the action of the advancing solvent front for a while, observing movements around the various spots. 3. Repeat step 2 with the second sheet, putting isopropanol instead of water into the petri dish. 4. It will take 5 - 10 minutes for the solvent fronts to reach the upper lines. 5. When each solvent front reaches the upper line, quickly remove the cylinder. Remove the staples and spread the paper on a clean dry paper towel. Measure the distance (D) from the lower line to the darkest point of each spot, and the distance (Do) between the lower line and the solvent front (the upper line). Enter these values on Data Sheets #1A and #1B, and calculate the Rf value for each spot on each sheet.

Part B. Direct Determination of K: 1. Obtain a piece of chromatographic paper (about15 cm x 4 cm). Weigh, and record weight (dry weight) to ± 0.001 g on Data Sheet #2. Accordion-fold the paper (1 cm pleats), and gently push it into a 6" test tube, trying not to rough up the paper in the process. With a graduated cylinder, add 10 ml of red dye #3 standard (about 15 ppm) to the test tube. Note the time. 2. Repeat Step 1 with a fresh piece of chromatography paper and 10 ml of blue dye #2 (about 20 ppm). Again note the time. 3. Cover both tubes with a small piece of Saran Wrap. Shake the tubes occasionally over the next hour, being careful not to contaminate the solutions, nor to spill any of the liquids. After each shaking, make sure that the paper is completely immersed in the solution. 4. After about 1 hour, prepare cuvettes for the Spectronic 20 as follows: A. distilled water (blank) B1. red dye #3 standard C1. red dye solution decanted from contact with paper from Step 1*. B2. blue dye #2 standard C2. blue dye solution decanted from contact with paper from Step 2*. 5. Gently withdraw the paper with a pair of tweezers, pat dry with paper towels, and record the weight (wet paper) to ± 0.001 gram. [r = (wgt of wet paper - wgt of dry paper)/wgt of dry paper] 6. Calibrate the instrument after it has warmed up for at least 15 minutes. 7. Set the absorbance of the blank to zero at wavelength of 520 nm, and record the absorbances of samples B1 and C1 above. 8. Set the absorbance of the blank to zero at wavelength of 620 nm, and record the absorbances of samples B2 and C2 above. * The solutions may need to be centrifuged to remove particulate matter.

Calculations: S = ppm of dye in standard ppm dye in solution @ equilibrium = S (AbsC/AbsB) ** ppm dye in paper = 10 S(1 - AbsC/AbsB)/(wgt of dry paper,g) K = ppm in solution/ppm in paper K = (wgt of dry paper, g)x(AbsC)/(AbsB - AbsC)(10 grams)

** C and B are solutions C1 and B1 or C2 and B2.