PARTITION CHROMATOGRAPHY By: Claudia V. Payumo Lordelle D. Ragas Introduction Chromatography is a technique for separating or analyzing a mixture of gases, liquids, or dissolved substances. This is brought about by means of two immiscible substances, one of which (the mobile phase) transports the sample mixture through the other (the stationary phase). The mobile phase may be a gas or a liquid; the stationary phase may be a liquid or a solid, and may be in a column, on paper, or in a thin layer on a glass or plastic support. Introduction The components of the mixture are adsorbed or impeded by the stationary phase to different extents and therefore become separated. The technique is used for both qualitative and quantitive analyses in biology and chemistry. Column chromatography In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column and the mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture. Typical Column Chromotography Column chromatography on a large scale in the 1950s. The chemist uses a ladder to refill eluent. He operates not one but 11 columns. Barely visible are Erlenmeyer receptacles on the floor. Paper Chromatography Paper chromatography is an analytical technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary schools in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool Thin Layer Chromatography (TLC) Thin layer chromatography (TLC) is a technique used to separate mixtures. It involves a stationary phase consisting of a thin layer of adsorbent material, immobilized onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated is then dissolved in an appropriate solvent and is drawn up the plate via capillary action, separating the experimental solution based on the polarity of the components of the compound in question. Partition Chromatography Separation of similar substances by repeated extraction by two miscible liquids. Partition chromatography is referred to as liquidliquid chromatography (LLC). Separation is based upon Nernst’s Law: Nernt’s Law When the ratio of concentrations expresses a distribution value for a single chemical species, the constant is designated a partition coefficient or distribution coefficient K and may be expressed mathematically as: K = Cl --Cu Where Cl represent concentration in the lower phase; while Cu represent concentration in the upper phase. In order to explain the mechanics involved in transferring solute molecules through a column during a chromatographic process, the plate theory is illustrated as follows: Plate Theory In the plate theory the entire length of the chromatographic column is considered to be composed of many small, identical cells called theoretical plates. Each cell contains a pair of immiscible solvents (ether & water). The lower phase is called the stationary phase, and the upper phase, the mobile phase. A mixture of solutes is introduced in the the first cell (0). Equilibrium is established, and the upper phase of each cell is then transferred to the next cell down to the next cell down the column. Each volume of upper phase eluting from the column is called a plate volume. The process of equilibration between transfers and the transfer of the upper phase of each cell to the next cell is continued until negligible amounts of solute remain in the stationary phase. Container # 0 1 2 3 4 ether Row A water Row B 50 50 Row C Row D Row E Row F 25 25 25 25 12.5 25 12.5 12.5 25 12.5 6.25 18.75 18.75 6.25 6.25 18.75 18.75 6.25 3.125 12.5 18.75 12.5 3.125 3.125 12.5 18.75 12.5 3.125 Detector Exit-Outlet Theoretical Distribution of a single solute after varrying numbers of transfers. Distribution of Solute in a Five-Plate Partition Chromatography System Plate Volume No. Fraction of Solute 0 0.00 1 0.00 2 0.00 3 0.00 4 0.00 5 0.031 6 0.078 7 0.117 8 0.137 9 0.137 10 0.123 11 0.103 12 0.081 13 0.061 14 0.044 15 0.031 16 0.020 17 0.014 18 0.009 Distribution of solute in partition-elution chromatography 0.16 Fraction of Solute 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Plate Volume No. Thank you!