EXPERIMENT 1 CHROMATOGRAPHY Chromatography is the ability to separate a mixture using partitioning characteristics of the components to remain in a stationary phase

e (a fixed medium) versus a mobile (or moving) phase. Once a component is separated from the mixture, it can be isolated and quantified. The identification of the components requires a detector(s), as chromatography is just the process of separation, not identification. Some separation methods rely on physical differences between the components of a mixture. Chromatography exploits differences in solubility, adsorption, ionic charge, and affinities. Adsorption of solute on surface of stationary phase (usually for polar non-ionic compounds) Ion Exchange attraction of ions of opposite charges (usually for ionic compounds anions/cations) Partition - uses relative solubility of analyte in mobile and stationary phases Size Exclusion (gel filtration, gel permeation) separates molecules by size Affinity specific interactions (e.g. antibody to protein)

Several types of chromatography are common: paper chromatography, thin-layer chromatography (TLC), liquid-liquid chromatography, gas chromatography (GC), supercritical fluid chromatography (SFC), and high performance liquid chromatography (HPLC). Chromatography allows a mixture of different chemicals to be distributed or partitioned between a stationary phase and a mobile phase (eluent or solvent). The mobile phase may be a liquid or a gas; the stationary phase is typically a solid. As the mobile phase flows over the stationary phase, the components in the mixture are carried along. The more soluble a component is in the mobile phase the faster it will be transported along the stationary phase. The more strongly a component is adsorbed to the stationary phase, the slower it will be transported by the mobile phase. As the mixture moves over the stationary phase, the components in the mixture move further and further apart into discrete zones. Paper chromatography uses ordinary filter paper (primarily cellulose) as the stationary phase. Thin-layer chromatography (abbreviated TLC) uses a thin glass plate coated with either aluminum oxide (alumina) or silica gel as the solid phase. The mobile phase in both is a solvent chosen according to the properties of the components in the mixture. Relevant to our experiment, in paper chromatography, a stain/drop of the mixture of substances is spotted along a line near one end of a rectangular piece of filter paper. The paper is the stationary phase and the line is called the origin. The lower edge of the paper is placed in a developing solvent as the mobile phase. Capillary action causes the solvent to flow up the paper at a uniform rate creating a "wet" line across the paper. This line is called the solvent front. When the solvent front reaches a spot, the components of the spot will begin to migrate upward with the mobile phase. Each component will have a characteristic chemical affinity for the paper and a characteristic chemical affinity for the solvent. These affinities are competitive: The component's affinity for the paper tends to hold the component in one place, but its affinity for the solvent tends to make the component follow the solvent as it moves upward. A component with a strong affinity for the paper and a weak affinity for the solvent will move more slowly than a component with a weaker affinity for the paper and a stronger affinity for the solvent. Chromatography may be preparative or analytical and the two are not mutually exclusive. The purpose of preparative chromatography is to separate the components of a mixture for more advanced use and is also a form of purification. Page 1 of 8

Analytical chromatography is done normally with smaller amounts of material and is used for measuring the relative proportions of analytes in a mixture. Some types of preparative chromatography are Elution (liquid) Chromatography, Displacement Chromatography, Reverse Phased Chromatography (RPC), and Preparative HPLC. Preparative HPLC is used for the isolation and purification of valuable products in the chemical and pharmaceutical industry as well as in biotechnology and biochemistry. Elution chromatography can be carried out under constant or continuous change mobile phase composition, or step elution conditions. A feed mixture is injected into the column inlet, through which the components then migrate. The feed components migrate through the column at different speeds, which are a function of the mobile phase velocity and the distribution of the compounds between the mobile and stationary phases. Preparative elution chromatography is generally carried out under overloaded (mass or volume) conditions to increase the product throughput. In volume overloading, the sample concentration is maintained in the linear region of the isotherm and volume is increased until the throughput is optimized. A fundamental problem with this technique is the under-utilization of the column and the corresponding low throughputs. In mass overloading, the sample concentration is increased beyond the linear adsorption region, resulting in asymmetric band profiles. A combination of volume and mass overloading is commonly used to maximize throughput. Displacement chromatography is a preparative chromatographic technique for protein separations. In this a displacer compound is used as opposed to step elution chromatography. The displacer is selected such that it has a higher affinity for the stationary phase than any of the feed components. A large volume of the feed mixture is loaded onto the column, followed by a constant front of the displacer solution. The displacement process is based on the competition of solutes for adsorption sites on the stationary phase according to their relative binding affinities and mobile phase concentrations. The action of the displacer causes the feed components to migrate through the column at velocities greater than that dictated solely by their individual adsorption isotherms. The product components exit from the column as higher concentrated pure material, in the order of increasing affinity of adsorption. The separation mechanism in reversed phase chromatography depends on the hydrophobic binding interaction between the solute molecule in the mobile phase and the immobilized hydrophobic ligand, i.e. the stationary phase. RPC is an adsorptive process, which relies on a partitioning mechanism to effect separation. The solute molecules partition, establishing an equilibrium, between the mobile phase and the stationary phase. The distribution of the solute between the two phases depends on the binding properties of the medium, the hydrophobicity of the solute and the composition of the mobile phase. Initial experimental conditions favor adsorption of the solute from the mobile phase to the stationary phase. The mobile phase composition is then modified to favor desorption of the solute from the stationary phase back into the mobile phase. The rate theory of chromatography describes the shapes and breadths of elution peaks in quantitative terms based on a random-walk mechanism for the migration of molecules through a column. Quantitative chromatography is based on a comparison of either the height or the area of an analyte peak with that of one or more standards. If conditions are controlled properly, both of these parameters vary linearly with concentration. Peak height: The height of a chromatographic peak is obtained by connecting the base lines on the two sides of the peak by a straight line and measuring the perpendicular distance from this line to the peak. This measurement can ordinarily be made with reasonably high precision and yields accurate results, provided variations in column conditions do not

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alter peak width during the period required to obtain chromatograms for sample and standards. The variables that must be controlled closely are column temperature, eluent flow rate, and rate of sample injection. Peak area: Peak area is independent of broadening effects caused by the variables mentioned in the previous paragraph. From this standpoint, therefore, area is a more satisfactory analytical parameter than peak height. On the other hand, peak heights are more easily measured and, for narrow peaks, more accurately determined. A simple method that works well for symmetric peaks of reasonable widths is to multiply peak height by the width at one-half peak height. Calibration with standards: The most straightforward method for quantitative chromatographic analyses involves the preparation of a series of standard solutions that approximate the composition of the unknown. Chromatograms for the standards are then obtained, and peak heights or areas are plotted as a function of concentration. A plot of the data should yield a straight line passing through the origin; analyses are based on this plot. Frequent standardization is necessary for highest accuracy.

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Results of the Experiment 1. Photos please see below. The pen marks are Sharpie: 1=S, Crayola: 2=C, Vis--Vis: 3=V 2. Rf values please see the attached lab notebook 3. a. i. Ink from Vis--Vis separated well in 25% ethanol. Ink from Crayola was beginning to separate towards the top, but the separation didnt complete, and the edges were not well defined. Ink from Sharpie spread out into a dark purple shade, without clear boundaries. These data points indicate Crayola is the most polar, followed by Vis--Vis, with Sharpie not very polar. ii. In 95% ethanol, Sharpie was beginning to demonstrate separation into components the best amongst the three and had travelled the farthest. Vis-a-Vis was beginning to separate out, but had not travelled at all. Similarly, Crayola did not travel far and separation into components did not occur (although signs of blue had begun to appear towards the top).These data points indicate Sharipe is the most non polar, with Crayola being the most polar. b. We had used the green Crayola marker which separated into yellow right away when placed in 25% ethanol, with almost no signs of blue. When placed in 95% ethanol, separation only began to occur towards the end, where signs of blue were visible, but no indication of yellow. This would suggest that yellow is less polar due to its visibility and lack of travel in 25% ethanol, and that blue is highly polar as it travelled the farthest in both the solutions

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Post-Lab Questions 1. If left for too long in the beaker, the paper may have undistinguishable streaks or be blank. The sample inks would travel through the length of the paper due to capillary action, and determining distance of the solvent front would be difficult, making the Rf calculations unreliable, if at all possible. 2. Increasing the length of the paper would result in better chances for the components to parse out. This would be at the expense of time. 3. The farther the pigment from the starting line, the more non polar it is. Stationary phase is polar, and any pigments that stop travelling indicate their polar nature. Mobile phase tends to be more nonpolar than water. 4. It will be interesting to see how three different (markers) filter papers in the same beaker behave. These filter papers would be longer than the ones used in this experiment. This could lead to less smearing and possible better indication of pigments. Also, a mechanism (clamp?) to hold the filter paper(s) vertical will allow the pigments to reach the solvent front uniformly.

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