Chromatography Separation Methods

Separation Methods

• Separation is crucial in analysis of biological samples to avoid interference from other substances
• Two main approaches to separation: isolate the test substance or remove the interfering substance

Principles of Separation Techniques

• Separation techniques are based on differences in molecular characteristics such as polarity, ionic nature, size, and shape
• These differences affect the interaction between molecules and the stationary and mobile phases

Classification of Separation Techniques

• Based on molecular characteristics:
  • Polarity: Gas-liquid chromatography, Liquid-liquid chromatography, Liquid-solid chromatography
  • Ionic nature: Ion-exchange chromatography, Electrophoresis
  • Size (mass): Gel permeation chromatography, Dialysis, Ultracentrifugation
  • Shape: Affinity chromatography

Chromatography

• A technique used to separate and identify the components of a mixture
• Works by allowing molecules to distribute between a stationary and mobile medium
• Molecules that spend more time in the mobile phase are carried along faster

Thin Layer Chromatography (TLC)

• Uses a stationary phase (adsorbent) and a mobile phase (solvent)
• The sample is spotted on the plate and the solvent is allowed to move up the plate
• Components of the mixture are separated based on their interactions with the stationary and mobile phases
• Visualization techniques are used to identify the separated components, such as UV light, alkaloids, cardiac glycosides, sugars, and amino acids

Interpreting the Data

• Rf (retention factor) values are calculated for each spot and are characteristic for a given compound
• Rf values can be used to identify unknown substances by comparing them to known values
• Purity of a sample can be estimated from the chromatogram

Paper Chromatography

• A method of partition chromatography using filter paper as the carrier
• The factor governing separation is the partition between two immiscible phases
• General procedure involves choice of paper and solvent, desalting of sample, application of the sample, equilibration of paper, development, detection, and identification of substances

Multiple Chromatography

• Includes procedures where development is repeated after one development is completed
• A- multiple development: repeated development in the same direction with the same or different solvent systems
• B- two-dimensional chromatography: development in a direction perpendicular to the first, with a different solvent system

Column Chromatography (CC)

• A chromatographic method where the stationary phase is packed into a column and the mobile phase is a moving liquid or gas
• Stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or gravity

Open Column Chromatography

• Traditional column chromatography characterized by addition of mobile phase under atmospheric pressure
• Packing and operating the column involve packing, sample application, and elution techniques

Separation Methods

• The importance of separation in biological samples lies in the presence of substances that may interfere with the analysis or affect the quality of the results.
• Separation is necessary to either isolate the test substance or remove the interfering substances before analysis.

Principles of Separation Techniques

• Separation techniques are based on molecular characteristics, such as polarity, ionic nature, size, and shape.
• These characteristics are used to separate biomolecules based on their physical properties.

Classification of Separation Techniques

• Separation techniques can be classified based on molecular characteristics, including:
  • Polarity: volatility, solubility, adsorptivity
  • Ionic nature: charge
  • Size (mass): diffusion, sedimentation
  • Shape: ligand binding

Chromatography

• Chromatography is a technique used to separate and identify the components of a mixture.
• It works by distributing molecules between a stationary and a mobile phase.
• Molecules that spend most of their time in the mobile phase are carried along faster.
• Chromatography is a physical method of separation that uses a stationary phase and a mobile phase to separate components based on their distribution constants.

Thin Layer Chromatography (TLC)

• TLC is a method for identifying substances and testing the purity of compounds.
• It is a useful technique because it is relatively quick and requires small quantities of material.
• The stationary phase is a thin layer of adsorbent coated on a plate.
• The mobile phase is a developing liquid that travels up the stationary phase, carrying the samples with it.
• Components of the sample will separate on the stationary phase according to their adsorption and solubility properties.
• TLC is used to identify compounds by spotting known substances next to unknown substances on the same plate.
• The purity of a sample can be estimated from the chromatogram, with impure samples often developing as two or more spots, and pure samples showing only one spot.

Paper Chromatography

• Paper chromatography is a method of partition chromatography that uses filter paper strips as a carrier or inert support.
• The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases.
• One phase is usually water adsorbed on cellulose fibers in the paper (stationary phase), and the second is the organic solvent (mobile phase) that flows past the sample on the paper.

Multiple Chromatography

• Multiple chromatography includes all procedures in which the development is repeated after one development is completed.
• Examples include:
  • A- multiple development: repeated development in the same direction to resolve substances with close Rf values.
  • B- two-dimensional chromatography: development in a direction perpendicular to the first, with a solvent system different from that used initially.

Column Chromatography (CC)

• Column chromatography includes chromatographic methods in which the stationary phase is packed into a column.
• The mobile phase is a moving liquid or gas that is forced under pressure or gravity.
• Stationary phase is held in a narrow tube through which the mobile phase is forced.

Open Column Chromatography

• Traditional column chromatography is characterized by addition of mobile phase under atmospheric pressure.
• The stationary phase is packed in a glass column.

Packing and Operating the Column

• Packaging methods depend on the density of the solid, and techniques used include wet, dry, and slurry methods.
• Avoid inclusion of air bubbles.
• Sample application involves adding the sample evenly and in a concentrated solution to the top of the column, which is protected from disturbance.
• Elution techniques include:
  • Isocratic elution: addition of solvent mixture of fixed composition during the whole process.
  • Gradient elution: continuous or linear elution, in which there is a continuous change in the composition of the mobile phase over a period of time.
  • Step-wise or fractional elution: in which the change is not continuous, with a sudden change in the composition of the mobile phase followed by a period where the mobile phase is held constant.

Errors in Analytical Data

• Errors can be divided into two types: Random Error and Systematic Error
• Random Error:
  • Caused by unpredictable changes during an experiment
  • Affects measurements differently each time
  • Comes from unpredictable changes during an experiment
  • Examples: reading a volume from a different angle each time, variable volume delivered by auto pipettes
• Systematic Error:
  • Caused by instrumental factors or error of the methods
  • Affects measurements the same amount or by the same proportion
  • Examples: instrumental factors (measuring length with a metal ruler), error of the methods (not setting an instrument to zero)
• Both Random Error and Systematic Error affect the accuracy of results, but Systematic Error can be predicted and avoided

Quality of Data

• Variability refers to the spread out of a set of data
• Variability is due to two types of errors: Random Error and Systematic Error
• Importance of quality of data: to minimize errors

Assessment of Analytical Methods

• Analytical methods should be:
  • Precise
  • Accurate
  • Sensitive
  • Specific
• Assessment of analytical methods:
  • Accuracy: the closeness of the mean of a set of replicate analyses to the true value of the sample
  • Precision: the extent to which a number of replicate measurements of a sample agree with one another
  • Sensitivity: the ability to detect small amounts of the test substance

Quality Assurance in Analytical Biochemistry

• Quality control: implementing procedures to ensure quality of data
• Quality assessment: evaluating the quality of data
• Accreditation of laboratories: ensuring that laboratories meet certain standards

Objectives

• Importance of quality of data
• Type of errors
• Assessment of analytical methods