HPLC: Principle and Application of High Performance Liquid Chromatography

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Questions and Answers

What is the primary function of an HPLC system?

To analyze pH levels

To measure temperature variations

To estimate molecular weights

To separate, quantify, qualify, or purify components in mixtures (correct)

In HPLC, why must the mobile phase be degassed?

To eliminate air bubbles (correct)

To decrease polarity

To increase viscosity

To enhance solubility

What is the main role of the stationary phase in liquid chromatography?

To pump the mobile phase through the column

To carry the sample through the column

To remain fixed in the column and facilitate separation (correct)

To introduce the sample into the column

Which of the following are commonly used as stationary phases in HPLC?

C18 and Silica Signup and view all the answers

Which component of HPLC is responsible for quantifying and identifying the sample components?

Detector Signup and view all the answers

Approximately how much sample volume is typically introduced into the column in HPLC?

5-20 µL Signup and view all the answers

During which decade did HPLC begin?

1960s Signup and view all the answers

What is the range of pump pressure normally used in HPLC?

400-600 bar Signup and view all the answers

Which improvement over time justifies the term 'High Performance' in HPLC?

Improvement in instrumentation and columns Signup and view all the answers

What aspect of HPLC distinguishes it from other forms of liquid chromatography?

Higher speed, efficiency, and sensitivity Signup and view all the answers

In liquid chromatography, what is the primary function of the mobile phase?

To carry the sample through the stationary phase Signup and view all the answers

What is one advantage of High-Performance Liquid Chromatography (HPLC) over Gas-Liquid Chromatography (GLC)?

HPLC has a wider choice of mobile and stationary phases. Signup and view all the answers

Which of the following is a use of HPLC in chemistry and biochemistry research?

Purifying chemical compounds. Signup and view all the answers

Which of the following describes an advantage of HPLC?

It allows for continuous monitoring of the column effluent. Signup and view all the answers

Why is HPLC considered to have a high degree of selectivity?

Availability of a very wide range of solvents and column packings. Signup and view all the answers

What characteristic of HPLC makes it suitable for analyzing complex mixtures?

High resolution power. Signup and view all the answers

Which of the following is a parameter in HPLC analysis?

Injection volume Signup and view all the answers

In what way is HPLC used in quality control?

Ensuring the purity of raw materials. Signup and view all the answers

What type of column packing is used in normal phase HPLC?

Polar (e.g., silica) Signup and view all the answers

Which mobile phase is typically used in reverse phase HPLC?

Water+miscible solvent Signup and view all the answers

For what type of compounds is ion exchange HPLC used?

Ions (anions and cations) Signup and view all the answers

What is one of the key advantages of using HPLC?

Fast and efficient separations Signup and view all the answers

HPLC can be used in which of these fields?

Both pharmaceutical and environmental analysis Signup and view all the answers

Which HPLC parameter is associated with controlling the temperature?

Oven temperature Signup and view all the answers

Which phase technique is best for water-sensitive compounds?

Normal phase Signup and view all the answers

Which of the following is NOT listed as a pharmaceutical application of HPLC?

Therapeutic drug monitoring Signup and view all the answers

HPLC can be used to quantify assays of which category?

Final products Signup and view all the answers

Which is an example of a bioscience application of HPLC?

Peptides Signup and view all the answers

What is an example of a consumer product application of HPLC?

Antioxidants Signup and view all the answers

Which of these is a clinical research application of HPLC?

Genomics Signup and view all the answers

HPLC helps to save lives in situations such as:

Accidents Signup and view all the answers

Which application is concerned with the detection of counterfeit drug products?

Pharmaceutical Signup and view all the answers

In which area does Therapeutic Drug Monitoring (TDM) primarily play a role?

Clinical research Signup and view all the answers

Which of the following applications helps uncover fraud in sports?

Support the quality of life Signup and view all the answers

What is one of the applications in genomics, proteomics, and metabolomics?

Clinical research Signup and view all the answers

Which of these is not a pharmaceutical application?

Genomics, proteomics, and metabolomics Signup and view all the answers

Study Notes

Liquid Chromatography High Performance Liquid Chromatography (HPLC)

HPLC system can quantitatively analyze substances in mixtures containing multiple ingredients by separating and detecting target substances.
It is used to separate, quantify, qualify, or purify single components from a sample containing various analytes in different matrices.

Principle of Work

Compounds are separated by injecting a sample mixture onto the column.
Different components in the mixture pass through the column at different rates due to differences in their partition behavior between the mobile phase and the stationary phase.
The mobile phase must be degassed to eliminate the formation of air bubbles.

History

Began in the 60's and was known as High Pressure Liquid Chromatography.
Instrumentation and columns improved over time, leading to high speed, efficiency, and high sensitivity.

Liquid Chromatography

A very small volume of sample is injected into a tube packed with a stationary phase (3-5 microns particles).
Separation is based on differential migration between the stationary and mobile phases.
Stationary Phase: the phase which remains fixed in the column, e.g. C18, Silica.
Mobile Phase: carries the sample through the stationary phase as it moves through the column.

Components of HPLC

Pump: pumps the mobile phase at a specific flow rate in mL/min, with a pressure of 400-600 bar.
Injector: introduces the sample into the column (about 5-20 µL).
Column: provides separation through high pressure created by the small particles.
Detector: quantifies and identifies the sample components and provides information to the computer.

HPLC Analysis Parameters

Mobile Phases: flow rate, composition.
Injection Volume.
Column.
Oven Temperature.
Wavelength.
Time Constant.

Separation Columns

Normal Phase: polar column packing, non-polar mobile phase, used for water-sensitive compounds, geometric isomers, cis-trans isomers, and chiral compounds.
Reverse Phase: non-polar column packing, polar mobile phase, used for polar, non-polar, ionizable, and ionic samples.
Ion Exchange: column packing contains ionic groups, mobile phase is buffer, used to separate anions and cations.
Size Exclusion: used for separating based on molecular size.

Advantages of HPLC

Separations are fast and efficient.
Continuous monitoring.
It can be applied to complex mixtures.
Accurate quantitative analysis.
Repetitive and reproducible results.
HPLC is more versatile due to the advantage of not being restricted by the choice of mobile phase and the choice of sample type.
Both aqueous and non-aqueous solvents are used.
A variety of solid supports are available.
It provides a method for very high degree of selectivity and sensitivity for single analysis.

Uses of HPLC

Analyzing complex mixtures.
Purifying chemical compounds.
Developing new separation techniques.
Isolating natural products.
Pharmaceutical and environmental analysis.
Ensure the purity of the product.

Applications of HPLC

Control and Improve Process Yields.
Quantify assays of final products.
Evaluate product stability and monitor degradation.
Analyze air and water pollutants.
Monitor materials that may jeopardize occupational safety or health.
Monitor pesticide levels in the environment.
Survey food and drug products.
Identify confiscated narcotics.
Check for adherence to label claims.
Chemical, pharmaceutical, bioscience, environmental, consumer product, and clinical applications.

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Description

Learn about the principle and application of High Performance Liquid Chromatography (HPLC) in separating, quantifying, and purifying substances in mixtures.