Unit 3- Chromatography.docx

Chromatography Introduction The number of analytes measured in clinical laboratories has increased dramatically in recent years This has been accompanied by an equally high demand for technologies that can accurately measure a large variety of compounds Liquid or gas chromatography (LC or GC) coupl...

Chromatography Introduction The number of analytes measured in clinical laboratories has increased dramatically in recent years This has been accompanied by an equally high demand for technologies that can accurately measure a large variety of compounds Liquid or gas chromatography (LC or GC) coupled to mass spectrometry is considered the gold standard for most testing due to its high sensitivity and specificity Basic Principles- separation technique based on charge on size how they are in different slovents Method in which components of a mixture are separated based on their differential interactions with two chemical or physical phases Mobile phase: travels through the system and carries the sample components once the sample has been applied or injected-sample Stationary phase: held within the system by a support- the system the mobile phase is moving through As a samples components pass through the stationary phase, the components that have the strongest interactions with it will be retained more and move through the system more slowly than components that have weaker interactions This leads to a difference in the rate of travel for these components and their separation Chromatography is often used to identify analytes and/or measure their concentrations Components Chromatograph- instrument used in chromatography that provides a response that is related to the amount of a compound that is exiting (or eluting) from a column as a function of the elution time or the volume of mobile phase that has passed through the system The resulting plot of the response versus time or volume is known as a chromatogram Terms The average time or volume that is required for a chemical to pass through the column is known as that chemical’s retention time (tR) or retention volume (VR) The elution time or volume for a compound that has no interaction with the stationary phase and is not retained is known as the void time (tM) or void volume (VM) Column Chromatography Type of chromatographic system that uses a column (or tube) to contain the stationary phase and support Planar Chromatography Support and stationary phase that are present on a plane, or open surface Further Classifications Chromatographic methods can be classified based on the mobile phase E.g., a method that uses a mobile phase (sample) that is a gas is called gas chromatography (GC); a liquid mobile phase is liquid chromatography (LC) It is also possible to divide methods according to the type of stationary phase that is present E.g., gas(mobile)-solid (stationary phase)chromatography (GSC) Planar Chromatography In this type of chromatography, the stationary phase is coated or placed onto a flat surface, or plane. The sample is added as a small spot or band on this surface This support is then placed into an enclosed container with one edge in contact with the mobile phase and the sample band located just beyond the point of contact The mobile phase is usually allowed to travel across the plane by means of capillary action After this movement has occurred for a given period of time, the support is removed from the mobile phase and dried prior to the analysis or measurement of the separated sample components The planar surface used may be a sheet of paper (paper chromatography) or other type of surface (thin-layer chromatography) With this method of chromatography, retention is described by the distance that compounds have traveled in a given amount of time, rather than the time or volume of mobile phase required for elution, as is used in column chromatography The distance travelled by a chemical from its point of application (Ds) in TLC is often compared with the distance that has been travelled by the mobile phase in the same amount of time (Df) This information can be used to calculate a measure of retention that is known as the retardation factor (Rf) Calculation: Retardation Factor = distance solute = Ds (Rf) distance solvent Df E.g., Distance of solute = 100 mm Distance from the origin to solvent front = 150 mm – know this for exam Rf = 100 = 0.67 150 Chemicals can be identified by comparing their Rf values with those for reference compounds that have been examined on the same stationary phase and support and mobile phase The separated components can be detected by their natural colour, by their response to UV light or through their fluorescence, or by their visualization with reagents that form coloured products This method tends to be used primarily for qualitative analysis One application is their use in the analysis of amniotic fluid to determine lecithin-to- sphingomyelin ratios Liquid Chromatography Type of chromatography in which the mobile phase is a liquid Based on the distribution of chemicals between a liquid mobile phase and a stationary phase LC is the dominant type of chromatography that is currently used for chemical analysis in clinical or biomedical laboratories Types of LC Adsorption- attaches to the surface of something almost like velcro Also known as liquid-solid chromatography Retention is based on the competition between the sample and the mobile phase for adsorptive sites on the solid stationary phase Three types of adsorbents are generally used: Polar acidic- adsorbs polar compounds and works well with basic substances- the acidic will attract the basic substances and vise versa Polar basic- adsorbs polar compounds and works well with acidic substances Nonpolar- include charcoal and polystyrene Not widely used in clinical laboratories because of technical problems with the preparation of the stationary phase Partition Is also referred to as liquid-liquid chromatography Separation of solute is based on relative solubility in a nonpolar solvent and aqueous solvent E.g., a polar liquid is used as the stationary phase, and a relatively nonpolar solvent or solvent mixture is used as the mobile phase The support in most types of partition chromatography is silica Ion- exchange Type of LC in which ions are separated by their adsorption onto a support that contains fixed charges at its surface Depending on the charge of the groups that make up the stationary phase, the types of ions that bind of the column may be either cations or anions Used to separate ions Size- Exclusion An LC technique that separates analytes based on size A porous support is used that has an inert surface with little or no interactions with the sample components The support should also have a range of pore sizes that are similar to the sizes of the compounds that are to be separated As a sample travels through the column, small sample components can enter all or most of the pores while larger components may enter only a few or none of the pores The result is a separation based on size, in which the larger components elute first from the column The beads have small openings large particles cant fit through the holes so they will pass quickly, the smaller ones will be trapped in the holes so it will take them longer to pass through Affinity- antigen antibody or enzyme and a substrate An LC method that makes use of biologically related interactions for the retention and separation of chemicals This method uses selective, reversible interactions that are found in many biological systems, such as the binding of an antibody with an antigen or the interactions of an enzyme with a substrate High-Performance Liquid Chromatography (HPLC) Uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution techniques- we usually have a computer detector Parts A pump forces the mobile phase through the column at a much greater velocity than that accomplished by gravity flow columns and includes pneumatic, syringe, reciprocating, or hydraulic amplifier pumps The stationary phase is packed into long stainless steel columns. Columns are often packed to provide less band broadening. The packing can be pellicular (inert core with a porous layer), inert and small particles, or macroporous particles. The most common material used for packing is silica gel. Sample injectors- a small syringe can be used to introduce the sample into the path of the mobile phase that carries it into the column. The best and most widely used method, however, is the loop injector. They have high reproducibility and are used at high pressures. Detectors- monitor the eluate as it leaves the column and, ideally, produce an electronic signal proportional to the concentration of each separated component. Spectrophotometers that detect absorbances in the visible and UV regions are most often used. Photodiode arrays and fluorescence detectors can also be utilized. Recorders- are used to record detector signal versus the time the mobile phase passed through the instrument, starting from the time of sample injection. The retention time is used to identify compounds when compared with standard retention times run under identical conditions. Peak area is proportional to concentration of the compounds that produced the peaks Gas Chromatography Type of chromatography in which gas is used as the mobile phase Can be used to separate and analyze compounds that either are naturally volatile or can be converted into a volatile form Mobile phase is typically an inert gas such as nitrogen or helium which have low densities and do not have any appreciable interaction with the sample but simply carry it through the column Separations in GC are based on differences in the vapour pressures of the injected compounds and in the interactions of these compounds with the stationary phase Types of Gas Chromatography Gas-Solid (GS) Type of GC in which the same material acts as both the stationary phase and the support In this method, chemicals are retained by their adsorption to the surface of the support (inorganic material like silica or alumina) Retention of an analyte on a GSC support will be affected by factors such as surface area of the support, size of the pores in the support, and the types of functional groups that are present on the support Gas-Liquid (GLC) The stationary phase is a liquid that is placed as a coating or a layer on the support One issue in GLC is that some of this liquid will eventually leave the column over time in a process known as “column bleed”- overtime the liquid will come out of the stationary phase, if we are interested in liquids its best to use hlc This results in a change in the ability of the GLC system to retain chemicals and may cause the signal of a detector to have high background or to be noisy as the stationary phase leaves the column This can be minimized by using a bonded phase instead of a liquid