1 Chromatography Pham Van Hung, PhD What on Earth did scientist do before Chromatography? - Extraction is based on the difference in solubility material is grounded, placed with a solvent which dissolves soluble compounds. A second extract solvent . The mixture is placed in a separatory funnel - Crystallization also based on the difference of solubility. The solubility is solved in a fixed volume of solvent. The purified compound crystallizes as solution cools, evaporates or diffuses - Distillilation separates components based on their volatility typically via vaporization-condensation method - Filtration separate components of a mixture based on their particle size. Used most often to separate a liquid from a solid What entices the scientists to Chromatography? Just like the previous techniques, chromatography is a way to separate two components based on a specific characteristic What makes chromatography so useful: The results are reproducible with better accuracy than the before mentioned separation techniques Chromatography can separate more complex mixtures than the previous techniques Chromatography is less time consuming and cheaper Brief History of Chromatography  1903 – Tswett, a Russian botanist coined the term chromatography. He passed plant tissue extracts through a chalk column to separate pigments by differential adsorption chromatography.  1915 R.M Willstatter, German Chemist win Nobel Prize for similar experiment.  1922 L.S Palmer, American scientist used Tswett’s techniques on various natural products.  1931 Richard Kuhn used chromatography to separate isomers oh polyene pigments; this is the first known acceptance of chromatographic methods. History of the Main techniques  1938 Thin Layer chromatography by Russian scientist N.A Izamailov and M.S Shraiber  1941 Liquid-Liquid partition chromatography developed by Archer John, Porter Martin and Richard Laurence Millington Synge  1944 Paper Chromatography one of the most important methods in the development of biotechnology  1945 Gas Chromatography 1 st analytical gas-solid (adsorption) chromatography developed by Fritz Prior  1950 Gas Liquid Chromatography by Martin and Anthony James; Martin won the Nobel Prize in 1952 British chemist Archer John Porter Martin, co- recipient, with Richard L. M. Synge, of the 1952 Nobel Prize in chemistry, "for their invention of partition chromatography." History of the Main Techniques  1966 HPLC named by Csaba Horvath, but didn’t become a popular method until 1970s  1950s Ion-Exchange chromatography declassified this technique  1970s Ion Chromatography was developed by Hamish Small and co-workers at the Dow Chemical company  1930s Affinity Chromatography was developed for the study of enzymes and other proteins 2 Chromatography A technique exploiting the interaction of the components of a mixture with a stationary phase and a mobile phase (solvent) in order to separate the components. Components are separated by different levels of adsorption to the stationary phase and solubility in the the mobile phase. Principles of Chromatography  Chromatography is used when there is a difference in the retention times of different components  Two types of phases 1) Stationary phase 2) mobile phases  Properties of Chromatographic Properties 1) immiscible stationary and mobile phases 2) an arrangement where a mixture is deposited at one end of the stationary phase 3) flow of the mobile phase towards the other end of the stationary phase 4) different rates of partitioning for each component 5) means for visualizing the separation of each component Column Chromatography Gas Liquid Chromatography (GLC) Gas Liquid Chromatography (GLC) High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) Paper Chromatography and Thin Layer Chromatography (TLC) Types of Chromatography Chromatography  There are two basic types of chromatography  Gas  Liquid  Liquid includes TLC and high performance liquid chromatography (HPLC) Thin-layer chromatography  TLC is a form of liquid chromatography consisting of:  A mobile phase (developing solvent) and  A stationary phase (a plate or strip coated with a form of silica gel)  Analysis is performed on a flat surface under atmospheric pressure and room temperature Principles of TLC  TLC is one of the simplest, fastest, easiest and least expensive of several chromatographic techniques used in qualitative and quantitative analysis to separate organic compounds  Michael Tswett is credited as being the father of liquid chromatography. Tswett developed his ideas in the early 1900’s. 3 TLC  The two most common classes of TLC are:  Normal phase  Reversed phase Normal Phase  Normal phase is the terminology used when the stationary phase is polar; for example silica gel, and the mobile phase is an organic solvent or a mixture of organic solvents which is less polar than the stationary phase. Reversed Phase  Reversed phase is the terminology used when the stationary phase is a silica bonded with an organic substrate such as a long chain aliphatic acid like C-18 and the mobile phase is a mixture of water and organic solvent which is more polar than the stationary phase. Adsorbents for TLC  Silica gel  Silica gel-F (Fluorescing indicator added)  Magnesium Silicate (Florisil)  Polyamides  Starch  Alumina Steps in TLC Analysis  The following are the important components of a typical TLC system:  Apparatus (developing chamber)  Stationary phase layer and mobile phase  Application of sample  Development of the plate  Detection of analyte Thin Layer (and Paper) Chromatography TLC plates are inert supports (glass, plastic, aluminium) with a thin veneer of chromatographic media (silica,etc…)  Apply a concentrated drop of sample (•) with a capillary or dropping tube to bottom of plate (origin pencil line) • • • • • Stand plate in a sealed vessel. • carefully add solvent (keep solvent level below sample). • Allow solvent to adsorb up the plate, drawing the sample with it. 4 Thin Layer (and Paper) Chromatography • • • • The ratio of distance travelled by the component (from origin) compared with the distance travelled by the solvent front (from origin) is called the R f value. Solvent front Solvent front x a b c R f of = a/x R f of = b/x R f of = c/x R f of = a/x R f of = b/x R f of = c/x Thin Layer and Paper Chromatography A solution of a mixture is applied as a spot/band at the bottom of the plate and allowed to travel with the solvent up the plate. A B C A+B+C standards Mixed standards Unknown + standards • • • • • • • • • • • • A+B+C ? ? standards Mixed standards Unknown + standards A B C A+B+CABC A+B+CA+B+CABC Column Chromatography  Similar to thin layer chromatography  Stationary phase = silica gel on support  Mobile phase = liquid solvent  In column chromatography, this stationary phase is packed into a vertical glass column.  Mobile phase moves down the column as a result of gravity. Column Chromatography  Blue compound = more polar  Adsorb more to the silica gel  Elutes slower  Yellow compound = less polar  Spends much of its time in the mobile phase  Elutes faster  Example of column chromatography separation: HPLC Introduction:  HPLC = improved form of column chromatography  Instead of the mobile phase moving through the column as a result of gravity, it is forced through the column under high pressure.  Typical operating pressures: 500-6000psi  To get improved separation – smaller sized packing material is required (<10µm).  Smaller packing = greater resistance to flow  Low flow rate = solute diffusion  Higher pressures needed to generate the needed solvent flow  Gravity is too slow- high pressure greatly speeds up the procedure. 1903: Russian botanist Mikhail Tswett  Separated plant pigments through column adsorption chromatography  Packed open glass column with particles  Calcium carbonate and alumina  Poured sample into column, along with pure solvent  As the sample moved down the vertical column, different colored bands could be seen.  Bands correlated to the sample components.  Coined the term chromatography from the Latin word meaning “color writing”. HPLC History 5 Early 1950s: First appearance of GC  Almost immediately became popular.  Work began on improving LC 1964: J. Calvin Giddings  Published a paper entitled “Comparison of the Theoretical Limit of Separating Ability in Gas and Liquid Chromatography” in the journal Analytical Chemistry.  Outlined ways to improve LC: smaller packing size, increased pressure  In theory, he demonstrated how LC could actually be more efficient than GC.  Increased number of theoretical plates HPLC History HPLC History 1966: Horváth  Built the first HPLC instrument and gave it its name  HPLC = High Pressure Liquid Chromatography. 1970’s: HPLC became popular with an increase in technology  Improved columns and detectors  Production of small silica packing material  By 1972 particle sizes less than 10µm were introduced  This allowed for more precise and rapid separations.  As new technology continued to develop, HPLC became more efficient.  HPLC = High Performance Liquid Chromatography Overview of the HPLC Process  Mobile phase pumped through column at high pressure.  Sample is injected into the system.  Separation occurs as the mobile phase and sample are pumped through the column.  Each sample component will elute from the column, one at a time, and will be detected by one of several possible detector types.  The response of the detector to each component eluted will be displayed on a chart or computer screen.  Known as a chromatogram.  Each compound eluted will show up as a peak on this chromatogram.  Data processing equipment are used to analyze the data generated. Diagram of HPLC Apparatus: 1. The end! Happy time now? . Millington Synge  1 944 Paper Chromatography one of the most important methods in the development of biotechnology  1 945 Gas Chromatography 1 st analytical. Liquid Chromatography (GLC) High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) Paper Chromatography and Thin Layer Chromatography