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