1.1 HPLC

It is a technique which separates components in a mixture due to the differing time for each component to travel through a stationary phase when carried through it by a mobile phase.

Chromatography

An analytical technique used to separate, identify and quantify the individual components in any sample dissolved in liquid.

Liquid chromatography

This separation technique is suitable for non-volatile or thermally-unstable compounds.

Liquid Chromatography

These are the different types of chromatographic method.

• Paper

• Liquid

• Gas

• HPLC

It is is a chromatographic technique used to separate a mixture of compounds with the purpose of identifying, quantifying, or purifying the individual components of the mixture

HPLC

It is the is the moving phase in a chromatographic system, which can be liquid or gaseous.

Mobile phase (solvent)

It is the non-moving phase, consisting of a solid, liquid, or a solid/liquid mixture that is immobilized.

Stationary phase (column)

it is the component under study that moves with the mobile phase depending on its affinity to the solid phase

Solute

it refers to the solvent or mixture of solvents used in a chromatographic separation process.

Eluent

It is the resulting graph produced by the chromatograph instrument during a separation process

Chromatogram

An elution technique that maintains a constant eluent composition and is typically used for simple separations

Isocratic Mode

The principle is based on adsorption as well as partition chromatography depending on the nature of the stationary phase.

HPLC

In HPLC, if the stationary phase is solid, the principle is based on __.

adsorption chromatography

In HPLC, if the stationary phase is liquid, the principle is based on __.

partition chromatography

Examples of a moving phase.

• Acetonitrile

• Methanol

• Water

• Buffer

Examples of a stationary phase.

• C18

• C4

• Amino

In the quantitative analysis in chromatography, the quantification is based on __.

Peak area/height

What are the different separation modes?

• Normal-Phase

• Reversed-Phase

• Ion-Exchange

• Size Exclusion

It refers to the localization of electrons within a molecule, which can cause molecules to have negative and/or positive poles.

Polarity

True or false: If a sample has a carboxyl group (-COOH), its hydrophobicity becomes stronger.

False. If a sample has a carboxyl group, its hydrophobicity becomes weak.

True or false: If a sample has an aromatic group, its hydrophobicity becomes stronger (aka it repels water more strongly and is highly non-polar).

True

Among the following molecules, which exhibits the strongest hydrophobicity?

• Hexane (contains a 6-carbon −(CH2​)n​− chain)

• Acetic acid (contains a −COOH group)

• Ethanol (contains an −OH group)

• Glycine (contains −NH2​ and −COOH groups)

Hexane

If the sample contains these groups, its hydrophobicity becomes weak.

• carboxyl

• amino

• hydroxyl

It separates polar analytes by partitioning onto a polar, bonded stationary phase.

Normal Phase

It separates non-polar analytes by partitioning onto a non-polar, bonded stationary phase.

Reversed Phase

It is the most common mode of separation.

Reversed Phase Chromatography

It separates moderately polar analytes, placing this technique in between Normal and Reversed Phase, by using a pure stationary phase, such as aluminum/silica.

Adsorption

It separates organic and inorganic ions by partitioning them onto ionic stationary phases bonded to a solid support.

Ion Chromatography

It separates large molecules based on the paths they take through the stationary phase.

Size Exclusion Chromatography

What are the two types of elution modes?

• Isocratic Mode

• Gradient Mode

This elution mode uses varying eluent compositions and is used for complex separations.

Gradient Mode

True or false: Reversed-phase chromatography utilizes a polar stationary phase and a non-polar mobile phase.

False. Reversed-phase chromatography uses a non-polar stationary phase and a polar mobile phase.

True or false: The primary goal of separation in HPLC is to have the best resolution possible between components.

True

True or false: A C18 (ODS) column is a common type of non-polar stationary phase used in reversed-phase chromatography.

True.

What are the common stationary phases used in normal phase chromatography?

• Silica (SiO₂)

• Diol (-OH)

• Aminopropyl (-NH₂)

• Cyanopropyl (-CN-)

Compare the stationary phases of Normal Phase and Reversed Phase Chromatography.

• Normal Phase - Polar stationary phase

• Reversed Phase - Non-polar stationary phase

Compare the mobile phases used in Normal Phase and Reversed Phase Chromatography.

• Normal Phase - Non-polar mobile phase

• Reversed Phase - Polar mobile phase

What is the difference between adsorption chromatography and partition chromatography in relation to the stationary phase?

• Adsorption chromatography is the underlying principle when the stationary phase is a solid.

• Partition chromatography is the principle when the stationary phase is a liquid.

What is the formula of the Lambert-Beer’s Law?