Chromatography Part 1 PDF

# Chromatography Part 1 - Demi Marie S. Aguilar, RMT ## Objectives At the end of the discussion, students should be able to: - Define words related to Chromatography - Identify the different components of chromatography - Understand the different modes of separation (concepts and properties) and...

# Chromatography Part 1 - Demi Marie S. Aguilar, RMT ## Objectives At the end of the discussion, students should be able to: - Define words related to Chromatography - Identify the different components of chromatography - Understand the different modes of separation (concepts and properties) and their corresponding chromatographic types. ## Definition of Terms | Term | Definition | |---|---| | Mobile Phase | Liquid or gas that flows through a chromatography system | | Stationary Phase | Solid or Liquid phase of a chromatography system on which the materials to be separated or selectively absorbed | | Adsorbent | Solid material that takes up the particles of the substance passing through it | | Support | Solid base which supports the liquid film in partition chromatography | | Development | The process where mobile phase is allowed to flow over | | Eluate| The analyte material that emerges from the chromograph | | Elution | The process of extracting one material from another by washing with a solvent | | Resolution| Quantitative measure of how well two elution peaks can be differentiated in a chromatographic separation | | Chromatography| Refers to the group of techniques used to separate complex mixtures on the basis of different physical interactions between the individual compounds and the stationary phase of the system. | ## Basic Component of a Chromatographic Technique | Component | Description | |---|---| | Mobile Phase/Eluent | - Carries the sample - Gas/Liquid form | | Stationary Phase | - Solid/Liquid - It is through which the mobile phase flows | | Column | - Holds Stationary phase | | Eluate | - The separated components | ## Chromatography - This image describes chromatography as a process where a leaf extract is run through a column containing calcium carbonate and ether/ethanol is used as the mobile phase. It depicts the separation of chlorophyll pigments. ## Modes of Separation | Adsorption | Partition | Steric Exclusion | Ion-Exchange Chromatography | |---|---|---|---| ### Adsorption Chromatography - Liquid-Solid Chromatography - Oldest form of Chromatography - Not widely used in the laboratory - Competition between the sample and the mobile phase for absorptive sites on the stationary phase - Mostly preferred as a preparative method used for separating large quantities into separate mixtures of solutes with widely varying polarity and structure - MOST SOLUBLE= MOVES FASTEST - This image depicts adsorption chromatography as a process where a mixture is separated based on the adsorption/desorption steps of its components. It shows how a more soluble component, Y, will move faster down the column than component X. ### Common Adsorbents Used in Adsorption Chromatography | Adsorbent | |---|---| | Alumina | | Magnesia Carbonate | | Magnesium Silicate | | Calcium Carbonate | | Starch | | Silica Gel | | Carbon | | Magnesia | | Aluminum Silicate | | Sugar and Calcium Phosphate | ### Properties of a Good Adsorbent 1. Insoluble in the solvents used 2. Small particle size (powder should be small to give a large surface area for greater adsorption) 3. Colorless (Colored substances are visualized) 4. Does not react with or catalyze the composition of substances to be separated ### Adsorbent Strength - Measured by the rate in which a zone travels in an elution experiment - THE GREATER THE RATE, THE WEAKER THE ADSORBENT - The adsorbent strength/power of the adsorbent would facilitate the separation of the mixture in its components. Influencing factors are: - Solvent used/employed - Surface area * The greater the surface area, the greater the amount of solute capable of being adsorbed. ### Strong Adsorbents - Alumina - Fuller's Earth - Charcoal - Silica Gel ### Intermediate Adsorbents - Magnesia - Calcium Carbonate - Calcium Phosphate ### Weak Adsorbents - Sucrose - Starch - Talc - Inulin - This image depicts the relationship between polarity and solvent strength. The less polar the solvent, the stronger the absorption of the analyte. ### Mixture separation based on Polar Function Group | Stationary Phase | Mobile Phase | |---|---| | Acid Polar (Ex. Silica Gel) | Single Solvent | | Basic Polar (Ex. Alumina) | Mixture of 2 or more solvents (depending on the analytes to be desorbed)| | Non-polar (Ex. Charcoal) | Mixture of 2 or more solvents (depending on the analytes to be desorbed)| ### Partition Chromatography - Liquid-liquid chromatography - Separation of solute is based on relative solubility in the organic/non-polar solvent and an aqueous/polar solvent. - The solid adsorbent is replaced by the stationary liquid that is only partially miscible with the flowing liquid as the mobile phase. - Stationary phase may be coated with an inert chromatographic support - In its simplest form, partition/extraction is performed in a separatory funnel - This image shows how solutes dissolved in a liquid phase coated on a surface of solid support are separated during partition chromatography. ### Process of Partition Chromatography 1. Molecules containing polar and nonpolar groups in an aqueous solution are added to an immiscible organic solvent. 2. After vigorous shaking, two phases are allowed to separate and are visualized. 3. Polar molecules remain in the aqueous solvent. Nonpolar molecules are extracted in the organic solvent. Since the two liquids are immiscible the solute molecules are portioned in two phases however this is not an absolute separation because it is based on relative polarity. 4. Results in the partitioning of the solute molecules into two separate phases. - This image compares the separation of solutes in a solvent extraction method versus chromatography. It points out the differences and similarities between the methods and how the distribution of compounds between the two phases depends on the surface area between two liquids. ### Partition Coefficient - The ratio of the concentration of the solute in the two liquids - Determines the rate at which a solute moves between the two phases - Compounds most soluble in the mobile phase move through the column faster and are easily eluted. - Compounds with high partition coefficients are retained in the stationary phase Expressed as: $$K = \frac{solute \ in \ stationary \ phase}{solute \ in \ mobile \ phase}$$ ### Modern Partition Chromatography - Uses PSEUDO-LIQUID STATIONARY PHASES that are chemically bonded to the support of HMW polymers that are insoluble in the mobile phase. #### Partition Systems | System | Description | |---|---| | **Normal Phase** | - Mobile solvent is less polar than the stationary phase | | **Reverse Phase** | - Mobile solvent is more polar than the stationary phase - More useful in the separation of drugs and other nonpolar substances | | **Support** | - Coated to the stationary phase - Solid material that acts as support for the liquid | ### Properties of a Good Support 1. **INERT**: the solid support should be as inert as possible to the substances to be separated 2. Adsorptive properties 3. Retain stationary phase and expose a large surface area to the mobile phase 4. Mechanically stable and should impede solvent flow ### Most Commonly Used Solid Supports | Support | Description | |---|---| | **Silica gel** | - Strong absorptive properties - 28-200 mesh silica gel is usually used - Almost always used with water or buffered aqueous solution as stationary phase | | **Cellulose powder** | - Used in columns as an alternative to the use of cellulose in the form of sheets as in paper chromatography - Only ashless cellulose powder are used: this permits separation of large quantities of materials | | **Diatomaceous earth** | - Commercially available as kieselguhr or celite | ### Steric Exclusion - Variation of liquid-solid chromatography - Separate solute molecules based on their size and shape #### Partition Systems | Phase| Description | |---|---| | **Mobile phase** | Aqueous solvent | | **Stationary phase **| Chromatographic column packed with gels that are in beads containing a network of pores in which small molecules may pass | - This image depicts how size-exclusion chromatography separates molecules according to their size. ### Early Methods of Steric Exclusion - **Gel filtration** - Uses hydrophilic beads of: - Cross-linked dextran or Sephadex - Biogel-P or Polyacrylamide gel - Agarose or Cephalus gel - **Gel permeation chromatography** - Uses hydrophobic gel beads of polystyrene with a non-aqueous mobile phase ### Ion-Exchange Chromatography - Separated by magnitude and change of ionic species - Separation is based on how the analytes are charged and bind to the ion exchange stationary phase - A process wherein ions of the sample and mobile phase exchange with the ions of the stationary phase - Ions with a greater charger are held most strongly and this can be controlled by changing the pH and/or the ion exchange media - Reversible exchange of ions between solid and liquid phases without radical change in the structure of the solid phase - This image shows how ion-exchange chromatography separates molecules according to their charge and affinity for the resin particles. #### Partition Systems | Phase | Description | |---|---| | **Stationary phase** | - With covalently bound ions and are insoluble in water - Consisting of large polymers of substituted benzene, silicates, or cellulose derivatives - With charged functional groups that are immobilized as side chains on resin beads | #### Cation/Anion exchange resins - Solutes that are eluted with a solution with low to medium salt concentration containing competing counter ions - Change in pH and ionic concentration allows separation of organic and inorganic ions #### Mobile Phase - **Cation Exchange resin** - Cations are free to react with hydrogen (H+) ions - Sodium is used for cation exchange - Positively charged molecules are attracted to a negatively charged solid support - **Anion Exchange Resin** - Anions are free to react with hydroxyl (OH-) ions - Chloride is used for anion exchange - Negatively charged molecules are attracted to a positively charged solid support. - This image shows how ion-exchange chromatography is used to separate molecules based on their charge. ### Uses of Ion-Exchange Chromatography 1. Remove interfering substances from a solution. - Ex: Water filtration, cations and anion resins are mixed together and used to deionize water. 2. Used for amino acid and hemoglobin analysis 3. Used to separate mixtures of small organic and inorganic ions and charged molecules 4. Concentrate dilute ion solutions ## Sources - Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2013). Clinical chemistry: principles, techniques, and correlations. 8th ed. - Burtis, Carl. et al. (2013) Tietz Textbook of Clinical Chemistry and Molecular Diagnostics (5th edition). - Anderson, Shauna C., PhD & Cockyane, Susan, PhD. (2003) Clinical Chemistry, Concepts and Application - Calbreath, Donald F. (1992), Clinical Chemistry: A Fundamental Textbook - Coiner, Diane. Analytical Techniques & Instrumentation in Clinical Chemistry. # Thank You! # End of Chromatography Part 1