PAPER CHROMATOGRAPHY LAB REVIEW
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All 7 dyes have
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Conjugated systems
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Conjugated systems
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Long carbon structures with alternating carbon-carbon single and double bonds
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Lots of conjugated bonds means
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Color
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Chromatography
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Group of techniques used to separate colored mixtures into their component parts (often used for blood or drinking water analyses)
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Chromatography involves
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A stationary phase and a mobile phase
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Stationary phase can be
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Solid or liquid
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The mixture to be separated is placed
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On the stationary phase
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Mobile phase can be
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Liquid or gas
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Liquid chromatography
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The separation is based on preferential attraction of each component of the mixture to either stationary or mobile phase
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The mobile phase moves along the stationary phase,
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Carrying some or all of the mixture with it, resulting in the separation of the mixture components
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Attractions of components to mobile or stationary phase is due to
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Intermolecular interactions
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Paper chromatography
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Sample of mixture is put on chromatography paper, which acts as stationary phase; edge of paper placed in solvent, which acts as mobile phase
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Wicking occurs because
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Solvent is attracted to water molecules that are bound to cellulose fibers of paper
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In paper chromatography, the sample is
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Spotted on the origin line
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Once sample has been spotted,
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Bottom edge of paper is put in solvent and solvent moves up paper; when solvent front reaches sample, the sample components are attracted to either stationary or mobile phase
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Solvent front
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Leading edge of mobile phase
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Attraction depends on what property?
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Relative polarities of sample components and two phases
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The attraction is never
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All or nothing
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Equilibrium equation
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Component-mobile phase component-stationary phase
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As fresh solvent continually passes the sample,
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New equilibria are established
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Even as new equilibria are established,
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Any components that have dissolved in the mobile phase encounter fresh stationary phase and new equilibria are established
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Components of mixture move
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At different rates and separate, creating chromatogram
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Overall effect of these equilibria
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The movement of the components depends directly on their relative attractions for mobile and stationary phases
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Characterize movement of components in terms of a
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Retention factor (Rf)
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Retention factor equation
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Distance traveled by component/distance traveled by solvent front (cm)
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Highest possible Rf? Lowest?
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1.0, 0.0
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Need to cover the developing chromatogram because
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Solvent might evaporate and change its percent composition
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In this experiment you will
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Determine Rf value of 7 dyes in 3 solvents; identify the solvent system that most effectively separates these dyes; identify which of these dyes are present in various mixtures
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We want a solvent system for which sample components
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Have distinctly different Rf values
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We can conclude that a component is present if
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It has the same Rf value as known component
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Moblie phase
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The solvent
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Stationary phase
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The paper
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What happens to constitute tad of a spot that are completely soluble
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Move with the solvent
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Components that aren't soluble at all
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Stay in original spot
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Adsorb
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Molecules adhere to chromatography paper to different extents and those extents can tell us what a mixture is composed of
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Rf formula
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Distance spot traveled divided by distance solvent traveled
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Retention (rf) depends on
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The solvent used and the composition of filter paper
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Will rf change if if paper is left in solution for a longer time
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No bc it is a ratio that is constant under a certain set of conditions
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How is color a helpful in chromatography
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If 2 different metal ions have the same rf values they can be identified by color
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Chromogenic reagents
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Reagent that will impart color to a colorless spot
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Metals used in experiment
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Iron Cobalt Nickel Manganese Copper
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Solvent used
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Acetone-water-hydrochloride acid
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List chromogenic reagents used
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Ammonia DMG Hydrogen peroxide
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What did ammonia do
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Copper turns blue
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What did DMG do
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Made buckle appear bright red
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What did hydrogen peroxide do
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Manganese became visible
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What is paper chromatography used for?
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It is widely used for the separation and identification of compounds of biochemical interest.
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Why does the paper chromatography technique work?
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It works because of differences in the polarity and solubility of amino acids.
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What are paper and thin layer chromatography often used for and why?
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They are often used for the initial screening of urine samples for drug metabolites because they are inexpensive and quick.
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What are paper and thin layer chromatography based on?
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The differential partitioning of the samples (solutes) between a polar stationary phase and a nonpolar mobile phase.
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How does the solution in paper chromatography move up the paper?
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By capillary action.
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In paper chromatography, which parts of the solvent are in the stationary phase and which parts are in the mobile phase?
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The organic components of the solvent have a lower affinity for the paper and so they move up faster. They form the mobile phase. The water in the solvent is strongly absorbed to the polar -OH groups of the paper and so is the stationary phase.
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How do the solutions move up the paper in paper chromatography? Describe what the paper would look like...
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Solutes are applied near the bottom of the paper. The solvent moves up the paper and over the solutes. The individual solutes will be partitioned between the stationary and mobile phases according to their relative solubility in each phase. The more soluble the solute in the nonpolar, organic, mobile phase, the farther it will move.
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What determines the distance the solute travels?
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The distance the solute travels is relative to the distance the solvent moves is called the Rf value.
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What is the formula for calculating the Rf value?
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Rf= distance traveled by the solute from the origin/distance traveled by the solvent from the origin.
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How is the unknown identified in paper chromatography?
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The unknown is identified using the known samples. More specifically, identification of the unknowns is accomplished by a comparison of their Rf values and colors with those of the known reference amino acids.
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Are amino acids polar or non-polar? Explain. How are they separated?
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Amino acids are polar due to their carboxyl and amino groups attached to the alpha-carbon. They can be seperated based on the polarity of their R-groups. Some of these R-groups are more soluble in the mobile phase because of their nonpolar R-groups, while others are insoluble due to charged or highly polar R-groups.
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How are amino acids visualized in paper chromatography?
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Amino acids are visualized by spraying the completely chromatogram with ninhydrin reagent.
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How does ninhydrin reagent work?
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Ninhydrin reacts with primary amino acids to give a purple color and with proline, a secondary amino acid, to give a yellow color.
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Will polar or non-polar molecules travel farther in paper chromatography?
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Non-polar molecules will travel farther than polar molecules.
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What are some procedural errors that could have occurred in paper chromatography if no spots showed at all?
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(1) Forgot to spray the paper with ninhydrin. (2) Forgot to dry the chromatography paper (3) Did not spot the amino acids well enough.
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What is paper chromatography used for?
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It is widely used for the separation and identification of compounds of biochemical interest.e.g. forensic science samples
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Why does the paper chromatography technique work?
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It works because of differences in the polarity and solubility of amino acids.
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What are paper chromatography often used for and why?
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They are often used for the initial screening of urine samples for drug metabolites because they are inexpensive and quick.
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What are paper chromatography based on?
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The differential partitioning of the samples (solutes) between a polar stationary phase and a nonpolar mobile phase.
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How does the solution in paper chromatography move up the paper?
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By capillary action.
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In paper chromatography, which parts of the solvent are in the stationary phase and which parts are in the mobile phase?
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The organic components of the solvent have a lower affinity for the paper and so they move up faster. They form the mobile phase. The water in the solvent is strongly absorbed to the polar -OH groups of the paper and so is the stationary phase.
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How do the solutions move up the paper in paper chromatography? Describe what the paper would look like...
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Solutes are applied near the bottom of the paper. The solvent moves up the paper and over the solutes. The individual solutes will be partitioned between the stationary and mobile phases according to their relative solubility in each phase. The more soluble the solute in the nonpolar, organic, mobile phase, the farther it will move.
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What determines the distance the solute travels?
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The distance the solute travels is relative to the distance the solvent moves is called the Rf value.
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What is the formula for calculating the Rf value?
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Rf= distance traveled by the solute from the origin/distance traveled by the solvent from the origin.
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How is the unknown identified in paper chromatography?
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The unknown is identified using the known samples. More specifically, identification of the unknowns is accomplished by a comparison of their Rf values and colors with those of the known reference amino acids.
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What are some procedural errors that could have occurred in paper chromatography if no spots showed at all?
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(1) Forgot to spray the paper with ninhydrin. (2) Forgot to dry the chromatography paper (3) Did not spot the amino acids well enough.
