PHBioChem (Lab) | Experiment 4: PAPER CHROMATOGRAPHY

Chromatography

method for separating mixtures into their components based on their physical and/or chemical properties

Chromatography

It is a method of separating mixtures into their components based on their physical or chemical properties, distributing its components between two phases.

Stationary Phase and Mobile Phase

CHROMATOGRAPHY: Distribute components into 2 phases, which are?

Thin Layer Chromatography

What chromatography was used in the experiment?

Stationary Phase

the medium or component that does not move; it is where separation occurs

Filter Paper

STATIONARY PHASE: This paper is for paper chromatography

Silica gel or alumina

STATIONARY PHASE: Thin plates coated with? (for Thin Layer chromatography)

Mobile Phase

the solvent = medium that accompanies the substance as it moves through the stationary phase (Can be both liquid or gas)

4:1:5

MOBILE PHASE: What was the ratio used in the experiment? (butanol:acetic acid:water)

Butanol, acetic acid and distilled water

MOBILE PHASE: In the experiment, a 4:1:5 ratio of ______, ______ and __________ was used.

Stationary Phase

Mixture placed on ________________ → Mobile Phase passes over the Stationary Phase → Mobile Phase dissolves the components of the mixture → Mobile Phase carries individual components at certain distance through the stationary phase, depending on their attraction to both phases

Mobile Phase

Mixture placed on Stationary Phase → __________ passes over the Stationary Phase → Mobile Phase dissolves the components of the mixture → Mobile Phase carries individual components at certain distance through the stationary phase, depending on their attraction to both phases

Mobile Phase

Mixture placed on Stationary Phase → Mobile Phase passes over the Stationary Phase → ____________ dissolves the components of the mixture → Mobile Phase carries individual components at certain distance through the stationary phase, depending on their attraction to both phases

Mobile Phase

Mixture placed on Stationary Phase → Mobile Phase passes over the Stationary Phase → Mobile Phase dissolves the components of the mixture → _______________ carries individual components at certain distance through the stationary phase, depending on their attraction to both phases

Sample

3 STEP PROCESS

1. Application of _________

2. Development of chromatogram by allowing the mobile phase to move up the paper

3. Calculation of Rf values and making conclusion

Chromatogram

3 STEP PROCESS

1. Application of sample

2. Development of ________ by allowing the mobile phase to move up the paper

3. Calculation of Rf values and making conclusion

Rf values

3 STEP PROCESS

1. Application of sample

2. Development of chromatogram by allowing the mobile phase to move up the paper

3. Calculation of ________ and making conclusion

Paper Chromatography

Separation of mixtures on the basis of the differences in solubility of the components between two immiscible solvents

Capillary Action and Solubility

PRINCIPLES INVOLVED IN PAPER CHROMATOGRAPHY

Capillary Action

Movement of liquid upward along narrow spaces (or porous material) w/out external forces like gravity

Solubility

The degree to which a material or solute dissolves into a solvent

Solubility

affinity of a substance for each phase, migration depends on polarity, MW, structure, shape, polarity

Rf value

The ratio of how the solute travels in the chromatograph versus how far the solvent front travels.

Rf = Distance traveled by solute / Distance traveled by solvent

Rf Value Formula

polar amino acids

Polar solvents will carry?

non-polar amino acid

Polar solvents will NOT carry?

High

Non-Polar amino acids = ____ Rf Value

Low

Polar amino acids = ____ Rf Value

Acidic amino acid

RF VALUE: This will migrate more than Basic amino acid

Ninhydrin

Amino acid mixtures: colorless

• thus, it is sprayed with this to make the color visible

Gas Chromatography, High-Performance Liquid Chromatography, Thin Layer Chromatography and Paper Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY

Inert Gas

DIFFERENT TYPES OF CHROMATOGRAPHY: What is the mobile phase of Gas Chromatography?

Column within the machine

DIFFERENT TYPES OF CHROMATOGRAPHY: What is the stationary phase of Gas Chromatography?

Gas Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: ideal in any scenario that needs to separate volatile mixtures.

Gas Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: Industries that commonly use this method of chromatography include: Pharmaceuticals, Medicine, Agriculture, Environmental, Industrial, Food and Beverage, Cosmetics and Cleaning

High-Performance Liquid Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: A favorite in many labs because of its efficiency using a liquid (or solvent) mobile phase, that pushes the sample through the machine using a high-pressure pump.

High-Performance Liquid Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: Since the machines do most of the work, this method requires the least amount of training.

High-Performance Liquid Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: Best for any scenarios that require separating non-volatile mixtures.

High-Performance Liquid Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: Industries that prefer this method include: Environmental, Biotechnology, Industrial, Pharmaceuticals, Forensics, Chemical Research, Beverage Research

Thin Layer Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: Lab technicians use a silica gel or alumina as its STATIONARY PHASE and coat it onto a thin glass or plastic plate.

Thin Layer Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: MOBILE PHASE is like high-performance liquid chromatography, in that it uses either a single solvent or a mixture of solvents.

Thin Layer Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: best suited for smaller experiments that require analyzing liquid or solvent compounds, the number of components, or the purity of compounds in a mixture.

Thin Layer Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: The most common industries that use this method include: Food Chemistry, Agriculture (mostly for pesticide analysis), Toxicology, Pharmaceuticals

Solvent or mixture of solvents

DIFFERENT TYPES OF CHROMATOGRAPHY: What is the mobile phase of Paper Chromatography?

Paper

DIFFERENT TYPES OF CHROMATOGRAPHY: What is the stationary phase of Paper Chromatography? (very absorbent piece that allows for liquid to pass through the pores.)

Paper Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: Most commonly used in forensics

• It helps forensic scientists identify DNA from fingerprints.

Paper Chromatography

DIFFERENT TYPES OF CHROMATOGRAPHY: Useful in the following industries and for the following uses as well: Food Coloring, Reaction, Monitoring, Plant Pigments, Scientific Studies (to identify organic and inorganic mixtures),