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Chromatographic separation methods Chem 313 Chapter I What is Chromatography Chromatography is described as the processes which allow the resolution of mixtures by affecting separation of some zones or in phases different from those in which they are originally present, irrespective of the...

Chromatographic separation methods Chem 313 Chapter I What is Chromatography Chromatography is described as the processes which allow the resolution of mixtures by affecting separation of some zones or in phases different from those in which they are originally present, irrespective of the nature of the force or forces causing the substances to move from one phase to another. Chromatography as an analytical tool, is now one of the most widely applicable separating techniques involving the resolution of mixtures, the comparison of substances, the determination of identity and purity of the compounds. There are different types of chromatography: such as paper, thin layer, or column chromatography (amongst others), each with its own strengths and weaknesses. Chromatography systems have a stationary phase (which can be solid or liquid) and a mobile phase (usually liquid or gas). Chromatographic Terms: The analyte: is the substance that is to be separated during chromatography. Analytical chromatography: is used to determine the existence and possibly also the concentration of analyte(s) in a sample. A bonded phase: is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing. A chromatogram: is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture  A better definition: The mobile phase consists of the sample being separated/ analyzed and the solvent that moves the sample through the column. In one case of HPLC The mobile phase: is the phase which the solvent consists of a moves in a definite direction. It may carbonate /bicarbonate solution be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical- fluid chromatography, SFC). The stationary phase: is the substance which is fixed in place for the chromatography procedure. The sample: is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste. Preparative chromatography: is used to purify sufficient quantities of a substance for further use, rather than analysis. The retention time: is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. The solute: refers to the sample components in partition chromatography. The solvent: refers to any substance capable of solubilizing other substance, and especially the liquid mobile phase in LC. The two mutually phases ‫الحالتين المتبادلتين‬in chromatography are: 1. Stationary phase: Which are the constant phase on which the components of the mixture will move on it through Stationary phase mobile phase (non volatile liquid or active solid). 2. Mobile phase: Which are the moving phase carriers the components over stationary phase (liquid or gas). Mobile phase  Chromatography class (based on the type of mobile phase physical state) Liquid chromatography Gas Chromatography Partition chromatography Adsorption Chromatography The separation process depend on the distribution of the components of the mixture Column Paper Thin Layer between the mobile phase and Chromatography chromatography Chromatography the stationary phase, (CC) (PC) (TLC) proportionally according to the partition coefficient Chromatographic Methods: 1. Partition chromatography: (Solution-solution distribution) in which the mobile phase is liquid or gas and stationary phase are liquid thus the separation process depends on the distribution of the components of the mixture between the mobile and stationary phase, proportionally according to the partition coefficient (Paper and column chromatography). 2. Adsorption chromatography: In which the mobile phase is liquid or gas and stationary phase are solid adsorbent thus the separation process depends on the distribution of the components of the mixture between the mobile phase and the surface of the solid adsorbent (TLC, adsorption column chromatography). Chapter II  Paper Chromatography (PC) Theory of Paper Chromatography: 1. Surface adsorption: Substance adsorbed to filter paper by passing a solvent through it, which would elute substance in a mixture. 2. Ion exchange: In the resolution of mixtures of ions, some exchange must occur with polar constituents of cellulose and with impurities present in the paper. 3. Partition between solvents: Partition between two immiscible phases is the most important factor. After equilibration of the paper with vapor of a solvent saturated with water, solvent development separations. The movement of a solute zone was explained as follows: 1. The cellulose fibers have a strong affinity to water present in solvent phase, but very little for organic liquid. 2. The paper acts as a stationary aqueous phase contain the solute. 3. Partition of the compounds occurs between the mobile organic phase and the stationary water phase. 4. Some of the solute leaves the paper and enters the organic phase, so when the liquid reach as section of the paper contain no solute. 5. Partition occurs again, but this time the solute transferred from the organic phase to paper phase. The effect of this partition is transfer of the solute from point to another point, which take some distance along the paper in the direction of solvent flow. Paper is made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose Experimental Technique: 1. Choice of filter paper: a. Degree and celerity of separation. b. Diffuseness of spots. c. Degree of formation of stains or streaks presumably caused by impurities in the paper. 2. Modified filter paper: By modifying filter paper chemically, it may be possible for the paper to become less hydrophilic, behave like an ion exchange resin. 3. Preparation of sample: a. Desalting of samples: If the sugars or amino acids have concentrated inorganic salts, we must desalt the sample before the chromatographic process by ion exchange treatment. b. Ultra filtration: For the analysis of small organic molecules in blood plasma, it is essential to remove the proteins by centrifugation. 4. Application of sample (Spotting): Probably the most important single factor for successful paper chromatography is the proper application of the sample onto the filter paper. Spot must be small and we can do that by using clean capillary tubes. All spots must be in the same size. 5. Solvent development: a. Choice of solvents: The choice of suitable developing solvents may be governed by: i. Slower moving solvents produce rounder and less diffuse spots. ii. The rate of movement of solvent is governed by its viscosity, surface tension, and density. iii. It is also observed that the Rf increase by raising the water content of a miscible pair of solvents. b. Effect of temperature and saturation: It is preferable to run a chromatographic separation at constant temperature. A change in temperature from one run to another will change the Rf value as the partition coefficient in the system is change. c. Effect of chamber size: i. Above the critical solvent volume the Rf values are at a minimum, even when the solvent volume increased. ii. Below the critical solvent volume the Rf values are at a maximum. 6. Separation process (Chromatography): The used eluent should ideally have the following properties: i. The individual components of the eluent should be obtainable easily and at fairly low cost, but should be of sufficient purity of direct use. ii. The eluent should be stable in air and when mixed with small quantities of acids. iii. The eluent must be simple at mixing. iv. The components should be relatively nonvolatile. v. It should be easy removal from the sheet after the run. vi. It should remain homogenous throughout the range of temperature. vii. It should not react with any of the substances to be separated. viii. The Rf values should vary from 5-95% of the sheet. 7. Detection, Identification, and Quantitation: Detect the spot zones either by U.V. light or by a chemical locating reagent and cut each zone alone. The following characteristics are desirable in the locating reagents: a. It should be cheap and pure. b. It should be stable both alone and in the solution. c. It should react rapidly with the compound in the cold. d. It should be inert towards residual solvent on the paper. e. It should not be toxic, if heating is required. f. It should not be a health hazard. 8. Extraction: Extract each zone by boiling with suitable organic solvent. 9. Evaporation: Evaporate the solvent. 10. Quantitation: Then you can make identification and quantitation for the separated compounds. The type of development depends upon the class of compounds to be investigated; the following are the types of development: 1. Ascending development: This simplest of all the methods. It consists of dipping the lower end of the paper into the solvent and then allowing it to rise up the paper by capillary action. Proper care is taken to ensure that the lower end of the paper containing spots (base line) is so placed so that it is above the solvent depth. As the solvent ascends, it does so against the gravitational force. The advantage of Ascending Paper Chromatography Technique: a. The paper is easily dried. b. A large number of separation can be done. c. This technique is very suitable for routine and control work and it is valuable and quick. 2. Descending development: In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper. The liquid moves down by capillary action as well as by the pull of the gravity. In this case the flow is more rapid as compared to ascending method. In one way this method is found to be most suitable and preferable over ascending method. Compounds with low Rf values are not completely separated by ascending method but this can be done by following descending method. The advantage of Descending Paper Chromatography Technique: a. The solution can be used again. b. It help for good separation. c. It is very easy technique. Factors Influencing (Rf) Values: 1. Temperature: As the temperature rises, the viscosity of the solvents (eluent) will decrease, then Rf will increase. 2. Grade of paper: Solvents travel faster along thick than thin papers, faster along open weave than close weave papers. 3. Existence of polyionic forms: Certain compounds, particularly the basic amino acids can exist in a number of ionic forms each having its own Rf value. This can be overcome by using a solvent of sufficiently high or low pH such that one of the ionic species. 4. Labile compounds: Sulfur amino acids decompose in many solvents during the actual flow. This can be overcome by conversion it into more stable form prior to chromatography. 5. Rf values: Rf values vary depending on the direction of solvent flow. (Ascending of Descending chromatography). 6. Length of solvent flow: Rf values determine after short runs will be different from those obtained after long runs. Chapter III  Thin Layer Chromatography (TLC) 1. The technique of thin layer chromatography (TLC) can be divided into two discrete techniques, namely the preparation of suitable TLC plates and chromatography on such plates. Once the plates are prepared, the technique of chromatography is identical with that used for paper. 2. Paper chromatography on a sheet (or layer) of cellulose, was highly successful in the field of ionic and polar molecules. It was highly unsuccessful in the field of non-polar or lipid molecules. TLC on silica gel brought the chromatography of lipids into use as a simple routine technique comparable with the paper technique for amino acids. Paper TLC plate 3. Not only were the separation as simple to perform but they occurred in minutes instead of hours, the technique required smaller quantities of materials as the spots were smaller reagents appeared more sensitive but, also, more corrosive location reagents could be used on the inert silica gel layer. 4. The thin separating layers of granular material or stationary phase is placed on a support plate of glass, metal, or plastic. The mixture to be separated in the form of a solution is then applied in spots or bands at the origin. 5. After the plate has been placed in a tightly closed chamber (Jar) containing a suitable single or mixed solvent, mobile phase, separation takes place during capillary migration which is termed development. Factors influencing separation: a. Nature of the adsorbent or stationary phase. b. The solvent mixture or mobile phase. c. Chamber atmosphere, which should be saturated with vapor. Stationary phase: 1. The stationary phase is prepared from one of the adsorbents, which are manufactured specially for TLC. 2. The dried layers must have a uniform appearance and form a good bond with support. 3. Normally, the plates have dimensions of 20 x 20 cm. 4. For analytical purpose a thickness of o.25 µm and for preparative purposes a thickness of 1.0 to 1.8 mm is used. Types of adsorbents: a. Silica gel and kieselguhr. b. b. Alumina c. Organic adsorbents as starch, normal cellulose powder, cellulose and ion exchange powder. adsorbents Acidic or basic Components to be separated Silica gel Acidic Acidic and neutral substances Alumina Bascic Basic and neutral Cellulous powder Neutral Soluble compounds Preparation of silica gel plate material: 1. Silica gel G or Merck for TLC (30 g) is placed in a dry, clean wide mouth flask, and 60 ml of distilled water is added. 2. Shake gently until a uniform mixture is formed, then vigorously until the mixture is uniform without air bubbles being formed. 3. The fluid suspension is then immediately poured into an applicator and is spread out uniformly in one smooth movement. 4. This quantity will coat 20 x 20 cm glass plates in 0.25 cm thick. Drying the plates: The plates are allowed to stand overnight (8-12 hours) in a dry room, then dried for 15 minute with a fan and activated for 30 minute at 110 oC in a special oven where they are placed in a vertical position in a drying rack. Storage: The dried plates should be stored in absence of moisture and laboratory vapors. Mobile phase (Solvents or eluent): 1. The mobile phase is the transport medium and consists of one or more (several) solvents. 2. It migrates in the stationary phase, i.e. the porous layer by capillary forces. 3. The elution effect increases with the polarity of the solvent, e.g. non polar hexane has a weak elution effect, chloroform has a medium ‒ strong effect and methanol has a strong elution effect. How are compounds separated on TLC? 1. The adsorption of unsaturated hydrocarbons increases with the number of the double bonds and with the number of these which are conjugated. 2. If functional groups are introduced into a hydrocarbon, the adsorption affinity increases in the following sequence: Me < OMe < C=O < OH < COOH Development of separation: 1. Linear ascending is the most popular technique and also the simplest one. The chromatogram is developed by allowing the eluent to ascend the plate by capillary action (10-15 cm). 2. A number of modifications of linear development techniques have been developed to meet particular difficulties when simple development will not provide adequate separation: a. Stepwise development: Some samples containing substances, which differ widely in polarity are not easily separated by one solvent system. Here one can develop again with a second, more polar solvent system. The process can be continued until all components are separated. The technique can be used in reverse, starting with a polar solvent system and moving onto solvents, which are less polar. b. Continuous development: The standard plate, which is 20 cm in length occasionally is too short for adequate separation of a mixture of compounds. Therefore, a continuous development technique is used in which only one mobile phase is allowed to ascend in the normal way. Provision is made at the top of the layer for evaporation, which a “cut off” in the lid of the chromatography tank. As the developing solvent evaporates from the top of the sorbet layer a continuous flow of the mobile phase is maintained. c. Multiple development: It is applied to improve separation by increase elution. After drying the plate, developing again with the same solvent system was carried out. This method can be repeated till good separation takes place. d. Two-Dimensional development: It is used with a complex mixture of substances, such as extracts from plant materials. This can be done by spotting the sample at one corner of a plate and running it. So that the mixture is partly resolved along one edge of the plate. The plate is then dried, turned through 90o, and run in a different solvent system. The line of the partially resolved mixture forms the starting line for this second development. This technique has proved to great value for separating amino acids. Detection: a. The colored compounds may be easily located. b. U.V. detection of the unsaturated compounds (Violet absorption). c. Chemical detection: i. Iodine vapor: The developed chromatogram is placed in a tank with a few crystals of iodine (I2) on the bottom and left for a few minutes. The iodine tends to accumulate at points where the compounds are giving dark brown spots on a pale yellowish background. ii. Spray reagent: Such as H2SO4 in MeOH and this for carbohydrates (sugar moiety), which burn after heating. Uses of the Thin-layer Chromatography: 1. To determine the number of the components in a mixture: TLC affords a quick and easy method for analyzing such things as a crude reaction mixture, or an extract from some plant substance. 2. To determine the identity of two substances: If the two substances spotted on the same TLC plate give spots in the identical locations, they may be identical. If the spot positions are not the same the substances cannot be the same. It is possible for two closely related compounds that are not identical to have the same positions ona TLC plate. 3. To monitor the progress of a reaction: By sampling a reaction from time to time it is possible to watch the reactants disappear and the products appear using TLC. Thus, the optimum time to halt the reaction can be determined, and the effect of changing such variables as temperature, concentration, and solvents can be followed without having to isolate the product. 4.To determine the effectiveness of a purification: The effectiveness of the distillation, crystallization, extraction, and other separation and purification methods can be monitored using TLC. 5. To determine the appropriate conditions for a column chromatography: Thin-layer chromatography (TLC) is generally unsatisfactory for purifying and isolating macroscopic quantities of material: however, the adsorbents most commonly used for TLC ‒ silica gel and alumina ‒ are used for column chromatography, discussed in the next chapter. Column chromatography is used to separate and purify up to about a gram of a solid mixture. The correct adsorbent and solvent used to carry out the column chromatography can be determined rapidly by TLC. 6. To monitor column chromatography: As column chromatography is carried out, the solvent is collected in a number of small flasks. Unless the desired compound is colored the various fractions must be analyzed in some way to determine which ones have the desired components of the mixture. TLC is a fast and effective method for doing this.