Chromatography :Chromatography


Chromatography :Chromatography Definition: Chromatography is a separation method in which a mixture is applied as a narrow initial zone to a stationary, porous sorbent and the components are caused to undergo differential migration by the flow of the mobile phase, a liquid or a gas.


History :History Michael Tswett (1872-1919), in 1906, published a paper describing the separation and isolation of green and yellow chloroplast pigments by column adsorption chromatography and stated that “Chromatography is a method in which the components of a mixture are separated on an adsorbent column in a flowing system”. “Chroma” is Greek for “color.” “Graphein” is Greek for “to write.”


History :History


History :History 1903-1906 Tswett invented chromatography with use of pure solvent to develop the chromatogram; devised nomenclature; used mild adsorbents to resolve chloroplast pigments. 1930-1932 Karrer, Kuhn, and Strain used activated lime, alumina and magnesia absorbents. 1935 Holmes and Adams synthesized synthetic organic ion exchange resins. 1938 Reichstein introduced the liquid or flowing chromatogram, thus extending application of chromatography to colorless substances.


History :History 1938 Izmailov and Schraiber discussed the use of a thin layer of unbound alumina spread on a glass plate. 1939 Brown had the first use of circular paper chromatography. 1940-1943 Tiselius devised frontal analysis and method of displacement development. 1941 Martin and Synge introduced column partition chromatography. 1944 Consden, Gordon, and Martin first described paper partition chromatography.


History :History 1947-1950 Boyd, Tompkins, Spedding, Rieman, and others applied ion-exchange chromatography to various analytical problems. 1948 Lederer and Linstead applied paper chromatography to inorganic compounds. 1951Kirchner introduced thin-layer chromatography as it is practiced today. 1952 James and Martin developed gas chromatography.


History :History 1956 Sober and Peterson prepared first ion-exchange celluloses. 1956 Lathe and Ruthvan used natural and modified starch molecular sieves for molecular weight estimation. 1959 Porath and Flodin introduced cross-linked dextran for molecular sieving. 1964 J. C. Moore developed Gel permeation chromatography as a practical method.


Theoretical Concept:Distribution Ratio :Theoretical Concept:Distribution Ratio Chromatography is a separation technique where component molecules (solutes) in a sample mixture are transported by a mobile phase over a stationary phase. Attraction of the solute for the stationary phase results in retardation of its movement through the chromatography system.


Theoretical Concept:Distribution Ratio :Theoretical Concept:Distribution Ratio Each component or solute is distributed between the two phases with an equilibrium established defined by the distribution ratio Thus for component S [SS] [SM] where [SS] is the concentration of S in a unit volume of the stationary phase, and [SM] is the concentration of S in a unit volume of the mobile phase.


Theoretical Concept:Distribution Ratio :Theoretical Concept:Distribution Ratio The distribution ratio, KS (also called as partition ratio or partition coefficient), for A, is therefore KS = [SS] / [SM]


Theoretical Concept:Distribution Ratio :Theoretical Concept:Distribution Ratio Each component separated will have a different value for K, reflecting their relative affinities for the stationary phase; the generalized form of the distribution equation for each component is K = CS / CM


Chromatographic Technique Classifications :Chromatographic Technique Classifications Adsorption Chromatography: The stationary phase is a solid on which the sample components are adsorbed. The mobile phase may be a liquid (liquid-solid chromatography) or a gas (gas-solid chromatography). The components distribute between the two phases through a combination of sorption and desorption processes.


Chromatographic Technique Classifications :Chromatographic Technique Classifications Partition Chromatography The stationary phase of partition chromatography is a liquid supported on an inert solid. The mobile phase may be a liquid (liquid-liquid partition chromatography) or a gas (gas-liquid chromatography, GLC).


Chromatographic Technique Classifications :Chromatographic Technique Classifications Ion Exchange and Size Exclusion Chromatography Ion exchange chromatography uses an ion exchange resin as the stationary phase. The mechanism of separation is based on ion exchange equilibria. In size exclusion chromatography, solvated molecules are separated according to their size by their ability to penetrate a sieve like structure (the stationary phase).


Chromatographic Technique Classifications :Chromatographic Technique Classifications Affinity Chromatography Affinity chromatography uses highly specific interactions between one kind of solute molecule and a second molecule covalently attached (immobilized) to the stationary phase.


Gas Chromatography :Gas Chromatography History Metal packed columns Glass packed columns Metal capillary columns Glass capillary columns Chemically bonded fused-silica capillary columns


Gas Chromatography :Gas Chromatography Requires the analyte to be thermally stable, reasonably volatile, and have a molecular weight of less than ~ 500 amu. The mobile phase is an inert gas such as He, H2 or N2. The stationary phase is a liquid that is immobilized on the surface of a solid support by adsorption or by chemical bonding. Gas chromatographic separation occurs because of differences in the adsorption equilibria between the gaseous components of the sample and the stationary phases.


Gas Chromatography :Gas Chromatography Basic components of a GC system


Sample Injection :Sample Injection Samples are introduced into the injector port via a glass syringe with a capacity of 1 – 10 mL. Gas-tight syringes are available for injecting gases and vapors with Teflon-tipped plungers for improved sealing of the plunger with the syringe barrel against the backpressure created by the inlet pressure of the injector.


Sample Injection :Sample Injection


Sample Injection Systems :Sample Injection Systems Features of Injection System Rapid clean switching or injection of the sample into the mobile phase with no tailing or dispersion of sample Correct inlet temperatures high enough to vaporize instantaneously all components in a sample without decomposition and condensation Minimum dead volumes to avoid diffusion of the sample in the mobile phase Design of the overall inlet system for good precision (better than 1%) No contamination of samples or catalytic effects No loss of retention of sample in the inlet system No septum bleed or leak


Sample Injection Systems :Sample Injection Systems Split Injectors Split Injectors are used for more concentrated samples since only a small fraction of the injected sample is introduced into the column.


Sample Injection Systems :Sample Injection Systems Split Injectors Split Vent: A small volume of the carrier gas flows into the column (1-4 ml/min) while a much higher volume (10-100 ml/min) flows out of the split line (split vent).


Sample Injection Systems :Sample Injection Systems Split Injectors Split Ratio: The split ratio is the ratio of the carrier gas flow in the column and out of the split vent. Typical split ratios range from 1:100 to 1:1000. Lower split ratios introduce more sample into the column. Using a 1:50 split ratio introduces approximately 1/50 (or 2%) of the sample into the column while a 1:100 split ratio introduces about 1/100 (or 1%) of the sample into the column.


Sample Injection Systems :Sample Injection Systems Splitless Injectors Splitless injection is suitable for trace level determinations in trace analysis where the analytes may be in ppm (µg/ml) concentration.


Sample Injection Systems :Sample Injection Systems Splitless Injectors Upon sample vaporization, the vapors are mixed with the carrier gas. At 15-60 seconds after the injection, the injector automatically enters the "purge on" mode.


Sample Injection Systems :Sample Injection Systems Cold Trapping (Solvent Effect) One requirement of splitless injections is that the initial column temperature be at least 10oC below the boiling point of the sample solvent. Since the column temperature is below the solvent boiling point, the sample solvent condenses at the front of the column.


Sample Injection Systems :Sample Injection Systems On-column Injector On-column injectors deposit the sample directly into the column without utilizing sample vaporization. The biggest drawback to on-column injections is column contamination.


GC Column Ovens :GC Column Ovens Column temperature is an important variable that must be controlled to a few tenths of a degrees for precise work. The column is ordinarily housed in a thermostatically controlled oven. Desirable characteristics of the chromatograph oven are: Rapid temperature response to follow accurately the temperature program profile Low thermal mass for fast cool-down at the conclusion of the analysis.


GC Column Ovens :GC Column Ovens Oven temperature programming An isothermal GC run does not yield a satisfactorily separated mixture of analytes. If the column temperature is high enough to give satisfactory peaks for the less volatile compounds, the low-boiling constituents will be less well-resolved. The solution is to raise the column temperature during a chromatographic run, so that for a homologous series peaks emerge at regular intervals.


GC Column Ovens :GC Column Ovens


Chromatographic Columns :Chromatographic Columns Packed Columns: Stationary phase is a liquid that is coated onto a solid support. The column may be made of glass or metal and typically 2 - 6 mm i.d. and 1 - 3 m in length. Advantages: more concentrated samples may be analyzed; more solvent may be injected; can be used in preparative applications. Disadvantage: analyte separation is less favorable; unsuitable for trace analysis.


Chromatographic Columns :Chromatographic Columns Capillary Columns: stationary phase is a liquid that is bond to the inner surface of the column. The column may be 0.2 - 0.7 mm i.d. and 10 - 100 m long Advantages: high resolution chromatography; multitude of liquid phases; inert surface upon which to coat the liquid phase. Disadvantages: small sample size; long chromatographic runs; “quirky” behavior depending upon chromatographic conditions.


Capillary Columns :Capillary Columns Fused silica, glass and stainless steel are the primary tubing materials. Fused silica tubing has recently become the preferred type because it produces flexible inert columns. Metal columns are generally avoided since catalyzed reactions with the analytes may occur. Capillary columns are constructed of three parts - fused silica tubing, polyimide coating and stationary phase


Glass Capillary Columns :Glass Capillary Columns Wall coated open tubular (WCOT) capillary columns are the most commonly used GC columns. Current column technology uses the surface properties of pure silica tubing to immobilize the stationary phase producing extremely stable inert columns.


Glass Capillary Columns :Glass Capillary Columns Fused Silica Tubing The fused silica used to manufacture capillary columns is synthetic quartz typically containing less than 1 ppm metallic impurities. Silylation Process Silanol groups (Si-OH) on the tubing surface are reacted with a silane type of reagent. Typically, a methyl or phenyl-methyl silyl surface is created for most columns


Glass Capillary Columns :Glass Capillary Columns Polyimide Coating Immediately after the drawing process, the outer surface of the tubing is coated with polyimide. This fills any flaws in the tubing. It also provides a strong, waterproof barrier.


Glass Capillary Columns :Glass Capillary Columns Stationary Phases The suitability of a stationary phase for a particular application depends on the selectivity and the degree to which polar compounds are retarded relative to what their retardation would be on a completely non-polar stationary phase. A method to select the appropriate stationary phase for analysis of a sample mixture is to consider the polar characteristics of the analytes and select a stationary phase of similar polarity.


Glass Capillary ColumnsLiquid Phases :Glass Capillary ColumnsLiquid Phases Cross-linked stationary phase Commercial equivalent Dimethylsiloxane BP1, DB1, HP1, SE30,OV1, CPSil 55% Diphenyidimethylsiloxane BP5, DB5, HP2, SE54 14% Cyanopropylphenyidi- BP10, DB1701, OV1701 methylsiloxane 50% Trifluoropropylmethyl- OV210, DB-210, QF-1 siloxane 50% Cyanopropylphenyidi- BP225, OV225 methylsilaxane Polyethylene glycol BP20, DB-WAX, W20M Cyanopropylsilarylene BPX70 Dimethylsiloxane-carborane HT5 copolymer Dimethylsilarylene BPX5


Glass Capillary Columns :Glass Capillary Columns Stationary Phases Polysiloxanes Polysiloxanes are the most common stationary phases. They are available in the greatest variety and are the most stable, robust and versatile.


Glass Capillary Columns :Glass Capillary Columns Stationary Phases Polysiloxanes The most basic polysiloxane is the 100 % methyl substituted such as DB-1 or HP-1. When other groups are present, the amount is indicated as the percent of the total number of groups.


Glass Capillary Columns :Glass Capillary Columns Stationary Phases Polysiloxanes A low-bleed phase is available which incorporates phenyl or phenyl type groups into the backbone of the siloxane polymer. The phenyl group strengthens and stiffens the polymer backbone which inhibits stationary phase degradation at higher temperatures.


Glass Capillary Columns :Glass Capillary Columns Stationary Phases Polyethylene Glycols Stationary phases with “wax" or “FFAP" in their name are some type of polyethylene glycol. They are less stable, less robust and have lower temperature limits than most polysiloxanes. With typical use, they exhibit shorter lifetimes and are more susceptible to damage upon over heating or exposure to oxygen.


Glass Capillary Columns :Glass Capillary Columns Stationary Phases Gas-solid Stationary Phase Gas-solid stationary phases are comprised of a thin layer (usually <10 mm) of small particles adhered to the surface of the tubing. They are called porous layer open tubular (PLOT) columns. Various derivatives of styrene, aluminum oxides and molecular sieves are the most common PLOT column stationary phases.


Glass Capillary Columns :Glass Capillary Columns Stationary Phases Gas-solid Stationary Phase PLOT columns are very retentive. Hydrocarbon and sulfur gases, noble and permanent gases, and low boiling point solvents are some of the more common compounds separated with PLOT columns.


Chromatographic Columns :Chromatographic Columns Cross-section of GC columns: (a) 1/8 in. packed column (b) thin film WCOT column (c) thick film WCOT column (d) 1/16 in. micropacked column (e) PLOT column (f) SCOT column)


Capillary Column Dimensions :Capillary Column Dimensions Column Length Resolution is a function of the square root of column length. Shorter column lengths are intended for samples containing a relatively small number of compounds especially if they are not very similar in structure, polarity or volatility. Most analyses are performed with intermediate column lengths (20 - 30 meters). Increased retention will be obtained with longer columns.


Capillary Column Dimensions :Capillary Column Dimensions Column Diameter The internal diameter will have a direct impact upon the efficiency, retention characteristics and sample capacity of a column. Smaller diameter columns are more efficient than larger diameter columns. As column diameter decreases, the retention of a given solute will increase providing no other changes to the chromatographic system have been made. Larger diameter columns have greater sample capacities. Typical column diameters range from 0.18 mm to 0.53 mm.


Column Diameter :Column Diameter Effect of column diameter on retentionA: DB-5, 30 m x 0.25 mm I.D., 0.25 µmB: DB-5, 30 m x 0.32 mm I.D., 0.25 µm


Capillary Column Dimensions :Capillary Column Dimensions Film Thickness Increasing film thickness will cause a substantial increase in the retention of a solute. Thin film columns are useful for the analysis of low volatility or high boiling samples. Film thickness runs from 0.10 mm to 5.00 mm. Effect of film thickness on retention  A: DB-5, 30 m x 0.32 mm I.D., 0.25 µm B: DB-5, 30 m x 0.32 mm I.D., 1.0 µm


Properties of the Capillary Column :Properties of the Capillary Column Bonded and Cross-linked Stationary Phases Cross-linked stationary phases have the individual polymer chains linked via covalent bonds. Bonded stationary phases are covalently bonded to the surface of the tubing. Columns with bonded and cross-linked stationary phases can be solvent rinsed to remove contaminants. Most polysiloxanes and polyethylene glycol stationary phases are bonded and cross-linked.


Properties of the Capillary Column :Properties of the Capillary Column Column Bleed Column bleed is the continuous elution of the compounds produced from normal degradation of the stationary phase and increases with higher temperatures. On average, polar stationary phases have higher column bleed, and significant bleed occurs at lower temperatures. Column bleed increases as a column is used. Exposing the column to oxygen (air) and/or consistently using the column at or near its upper temperature limit for prolonged periods accelerates the onset of higher column bleed.


Detectors :Detectors


Detectors :Detectors Thermal Conductivity Detector Measures the ability of a substance to transport heat from a hot region to a cold region. Are simple and universal. Are not sensitive enough for capillary columns.


Detectors :Detectors Flame Ionization Universal organic detector Forms ions when compounds are burned


Detectors :Detectors Mass Selective Detector (Mass Spectrometer)


GC/MS :GC/MS


Selective Ion Monitoring :Selective Ion Monitoring