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