Various Mixture Separation Processes Prepared by Mr. D. Scott, Celina High School (The material in this outline came from various internet resources including, but not limited to Wikipedia.)
Disclaimer: This outline is meant to be an overview or introduction and is not intended to be thoroughly descriptive or instructional with regard to an particular separation process contained herein.
Adsorption is a process that occurs when a gas or liquid solute accumulates on the surface of a solid or a liquid (adsorbent), forming a molecular or atomic film (the adsorbate). It is different from absorption, in which a substance diffuses into a liquid or solid to form a solution. The term sorption encompasses both processes, while desorption is the reverse process. Activated carbon adsorption explained: The best example of activated carbon adsorption explained as a process is in a wastewater treatment plant where millions of gallons of polluted, unfiltered water enters into a system and must be treated before being transported to nearby water systems. Activated carbon has an affinity for various organics and is use for organic contaminant removal from water supplies. There are several forms of activated carbon: powdered activated carbon (PAC) is ground loose carbon. Adsorption isotherm tests are run to determine the effectiveness on the substance and its concentration in the surrounding solution at equilibrium. Granular Activated Carbon (GAC) is typically used in drinking water systems where liquid flows through stationary beds through the adsorpbent. In generic terms, the carbon is heavier in weight than water and traps contaminants into its material. Adsorption is indicative in most natural physical, biological, and chemical systems, and is widely used in industrial applications such as activated charcoal, synthetic resins and water purification. Adsorption, ion exchange and chromatography are sorption processes in which certain adsorptives are selectively transferred from the fluid phase to the surface of insoluble, rigid particles suspended in a vessel or packed in a column.
Centrifugation is a process that involves the use of the centripetal force for the separation of mixtures, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. In chemistry and biology, increasing the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate ("pellet") to gather on the bottom of the tube. The remaining solution is properly called the "supernate" or "supernatant liquid". Since "supernatant" is an adjective, its usage alone is technically incorrect, although many examples can be found in scientific literature. The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette. Common Centrifugation Vocabulary and Formulas: • • • • •
Pellet: hard-packed concentration of particles in a tube or rotor after centrifugation. Supernatant: The clarified liquid above the pellet. Adapter: A device used to fit smaller tubes or centrifugal devices in the rotor cavities. RPM: Revolutions Per Minute (Speed). Rmax: Maximum radius from the axis of rotation in centimeters.
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Rmin: Minimum radius from the axis of rotation in centimeters. RCF: Relative centrifugal Force. RCF = 11.17 x Rmax (RPM/1000)2 K-factor: Pelleting efficiency of a rotor. Smaller the K-factor, better the pelleting efficiency.
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S-value: the sedimentation coefficient is a number that gives information about the molecular weight and shape of the particle. S-value is expressed in Svedberg units. The larger the Svalue, the faster the particle separates. Pelleting time: time taken to pellet a given particle. T = K/S where T= pellet time in hours. K = K-factor of the rotor, and S = sedimentation coefficient.
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These diagrams show change over time as the mixture is spun. Spinning in a centrifuge causes the effective g-force (centripetal force) to act like an increase in gravity that both enables and accelerates the settling process. Mixtures that won’t settle under normal earth gravity can settle under greater gravitation-like forces.
Chromatography is the collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture of different dissolved substances in a "mobile phase" through another material called a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated. Chromatography may be preparative or analytical. Preparative chromatography seeks to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography normally operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture. The two are not mutually exclusive.
Chromatography 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.
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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.
Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated. • •
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A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation. Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. The effluent is the mobile phase leaving the column. An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing. The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). 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 solvent consist of a carbonate/bicarbonate solution and the sample is the anions being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated. 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 sample is the matter analysed 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. 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 stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography.
Types of Chromatography Planar Chromatography
Original location spot of the mixture.
Separated portions of the mixture appear as “bands” or “spots” on the surface of the chromatography substrate. The easiest part of the mixture for the solvent to carry is located the farthest from the original spot.
Thin layer chromatography is used as shown above to separate components of chlorophyll. Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography). Paper Chromatography Paper chromatography is a technique that involves placing a small dot of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent. Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance. Thin layer chromatography Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.
Column chromatography As the mobile phase (solventlike liquid in this case) passes downward through the column, the different parts of the mixture travel through at different rates. The different rates of travel result in a separation of the mixture.
Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample. In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash). The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.
Gas chromatography Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" . Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.
Liquid chromatography Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC). In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more.
Affinity chromatography Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labelled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.
Supercritical fluid chromatography Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.
Ion exchange chromatography Ion exchange chromatography utilizes ion exchange mechanism to separate analytes. It is usually performed in columns but the mechanism can be benefited also in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins using FPLC.
Size exclusion chromatography Size exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able to enter the pores of the media and; therefore, take longer to elute, whereas larger molecules are excluded from the pores and elute faster. It is generally a low resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the structure of purified proteins.
Reversed-phase chromatography Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.
Two-dimensional chromatography In some cases, the chemistry within a given column can be insufficient to separate some analytes. It is possible to direct a series of unresolved peaks onto a second column with different physicochemical (Chemical classification) properties. Since the mechanism of retention on this new solid support is different from the first dimensional separation, it can be possible to separate compounds that are indistinguishable by one-dimensional chromatography.
Fast protein liquid chromatography Fast protein liquid chromatography (FPLC) is a term applied to several chromatography techniques which are used to purify proteins. Many of these techniques are identical to those carried out under high performance liquid chromatography.
Countercurrent chromatography Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids. It involves mixing a solution of liquids, allowing them to settle into layers and then separating the layers.
Chiral chromatography Chiral Chromatography involves the separation of stereoisomers. In the case of enantiomers, these have no chemical or physical differences apart from being three dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes. Chiral chromatography HPLC columns (with a chiral stationary phase) in both normal and reversed phase are commercially available.
Crystallization is the (natural or artificial) process of formation of solid crystals from a uniform solution. Crystallization is also a chemical solid-liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs. The crystallization process consists of two major events, nucleation and crystal growth. Nucleation is the step where the solute molecules dispersed in the solvent start to gather into clusters, on the nanometer scale (elevating solute concentration in a small region), that becomes stable under the current operating conditions. These stable clusters constitute the nuclei. However when the clusters are not stable, they redissolve. Therefore, the clusters need to reach a critical size in order to become stable nuclei. Such critical size is dictated by the operating conditions (temperature, supersaturation, etc.). It is at the stage of nucleation that the atoms arrange in a defined and periodic manner that defines the crystal structure — note that "crystal structure" is a special term that refers to the relative arrangement of the atoms, not the macroscopic properties of the crystal (size and shape), although those are a result of the internal crystal structure.
The crystal growth is the subsequent growth of the nuclei that succeed in achieving the critical cluster size. Nucleation and growth continue to occur simultaneously while the supersaturation exists. Supersaturation is the driving force of the crystallization, hence the rate of nucleation and growth is driven by the existing supersaturation in the solution. Depending upon the conditions, either nucleation or growth may be predominant over the other, and as a result, crystals with different sizes and shapes are obtained (control of crystal size and shape constitutes one of the main challenges in industrial manufacturing, such as for pharmaceuticals). Once the supersaturation is exhausted, the solid-liquid system reaches equilibrium and the crystallization is complete, unless the operating conditions are modified from equilibrium so as to supersaturate the solution again.
Decantation is a process for the separation of mixtures, carefully pouring a solution from a container, leaving the precipitate (sediments) in the bottom of the container. Usually a small amount of solution must be left in the container, and care must be taken to prevent a small amount of precipitate from flowing with the solution out of the container. It is generally used to separate a liquid from an insoluble solid, e.g. in red wine, where the wine is decanted from the potassium bitartrate crystals. Another example is when separating the oil from water after getting them as a mixed liquid out of olives in order to extract olive oil.
Drying is a mass transfer process resulting in the removal of water moisture or moisture from another solvent, by evaporation from a solid, semi-solid or liquid (hereafter product) to end in a solid state. To achieve this, there must be a source of heat, and a sink of the vapor thus produced. In the most common case, a gas stream, e.g., air, applies the heat by convection and carries away the vapor as humidity. Other possibilities are vacuum drying, where heat is supplied by contact conduction or radiation (or microwaves) while the produced vapor is removed by the vacuum system. Another indirect technique is drum drying, where a heated surface is used to provide the energy and aspirators draw the vapor outside the room. Freeze drying or lyophilization is a drying method where the solvent is frozen prior to drying and is then sublimed, i.e., passed to the gas phase directly from the solid phase, below the melting point of the solvent. Freeze drying is often carried out under high vacuum to allow drying to proceed at a reasonable rate. This process avoids collapse of the solid structure, leading to a low density, highly porous product, able to regain the solvent quickly. In biological materials or foods, freeze drying is regarded as one of the best if not the best method to retain the initial properties. It was first used industrially to produce dehydrated vaccines, and to bring dehydrated blood to assist war casualties. Now freeze drying is increasingly used to preserve some foods, especially for backpackers going to remote areas. The method may keep protein quality intact, the same as the activity of vitamins and bioactive compounds. In turn, the mechanical extraction of the solvent, e.g., water, by centrifugation, is not considered "drying". The ubiquitous term dehydration may mean drying of water-containing products as foods, but its meaning is more vague, as it is also applied for water removal by osmotic drive from a salt or sugar solution. In medicine, dehydration is the situation by which a person loses water by respiration, sweating and evaporation and does not incorporate, for whatever reason, the "makeup" water required to keep the normal physiological behavior of the body. Drying may be either a natural or an intentional process. The process of extreme drying is called desiccation.
Distillation is a technique used to separate mixtures of liquids. It is a method of separating chemical substances based on differences in their volatilities in a boiling liquid mixture. Distillation usually forms part of a larger chemical process, and is thus referred to as a unit operation. Commercially, distillation has a number of uses. It is used to separate crude oil into more fractions for specific uses such as transport, power generation and heating. Water is distilled to remove impurities, such as salt from sea water. Air is distilled to separate its components - notably oxygen, nitrogen and argon - for industrial use. Distillation of fermented solutions has been used since ancient times to produce distilled beverages with a higher alcohol content.
Laboratory distillation set-up: 1: Heat source 2: Still pot 3: Still head 4: Thermometer/Boiling point temperature 5: Condenser 6: Cooling water in 7: Cooling water out 8: Distillate/receiving flask 9: Vacuum/gas inlet 10: Still receiver 11: Heat control 12: Stirrer speed control 13: Stirrer/heat plate 14: Heating (Oil/sand) bath 15: Stirrer bar/anti-bumping granules 16: Cooling bath.
Electrophoresis is a separation technique used with organic molecules, such as protein. The proteins are placed in a gel. A voltage is applied and the molecules move through the gel because they are charged. The gel restricts the motion so that different proteins will make different amounts of progress in any given time.
Evaporation this separation technique removes a volatile liquid solvent from its dissolved or suspended components.
Extraction Processes Leaching is the process of extracting a substance from a solid by dissolving it in a liquid. Brewing of tea and coffee can similarly be considered as leaching process.
Solvent extraction, also known as liquid-liquid extraction and partitioning, is a method to separate compounds based on their relative solubilities in two different immiscible liquids, usually water and an organic solvent. It is an extraction of a substance from one liquid phase into another liquid phase. Liquid-liquid extraction is a basic technique in chemical laboratories, where it is performed using a separatory funnel. This type of process is commonly performed after a chemical reaction as part of the work-up.
Solid-phase extraction (SPE) is a separation process that is used to extract compounds (called analyte) from a mixture of impurities. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water samples, beverages, soil, animal tissue, and consumer products. The separation ability of solid phase extraction is based on the preferential affinity of desired or undesired solutes in a liquid, mobile phase for a solid, stationary phase through which the sample is passed. Impurities in the sample are either washed away while the analyte of interest is retained on the stationary phase, or vice-versa. Analytes that are retained on the stationary phase can then be eluted from the solid phase extraction cartridge with the appropriate solvent.
Flocculation is a process where a solute comes out of solution in the form of floc or "flakes." The term is also used in colloid chemistry to refer to the process by which fine particulates are caused to clump together into floc. The floc may then float to the top of the liquid, settle to the bottom of the liquid, or can be readily filtered from the liquid. Flocculation and sedimentation are widely employed in the purification of drinking water as well as sewage treatment, stormwater treatment and treatment of other industrial wastewater streams.
Filtration • •
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Mesh, bag and paper filters are used to remove large particulates suspended in fluids, eg. fly ash, membrane processes including microfiltration, ultrafiltration, nanofiltration, reverse osmosis, dialysis (biochemistry) utilising synthetic membranes can separate micrometre-sized or smaller species. Oil-water separation - gravimetric separator used to remove suspended oil droplets from wastewaters in oil refineries, petrochemical and chemical plants, natural gas processing plants and similar industries.
Some common every-day filters
Technique for folding filter paper:
Precipitation is the formation of a solid in a solution during a chemical reaction. When the reaction occurs, the solid formed is called the precipitate, and the liquid remaining above the
solid is called the supernate.
Recrystallization is a procedure for purifying compounds. Typically the mixture of "compound A" and "impurity B" are dissolved in the minimum amount of solvent to fully dissolve the mixture i.e. a saturated solution. The solution is then allowed to cool. As the solution cools the solubility of compounds in solution drops. This results in the desired compound dropping (recrystallizing) from solution. The slower the rate of cooling, the bigger the crystals formed . The crystallization process requires an initiation step. Once a small crystal has formed, more crystals can grow from that crystal. Since "Compound A" is in excess this will usually result in these crystals forming first and thus leaves a greater ratio of impurity in solution. Thus the resulting solid is more pure than the original mixture.
Sedimentation describes the motion of molecules in solutions or particles in suspensions in response to an external force such as gravity, centrifugal force or electric force.
Sieving In general, a sieve separates wanted/desired elements from unwanted material using a tool such as a mesh, net or other filtration or distillation methods. The word "sift" derives from this term. A strainer is a type of sieve typically associated with separating liquids from solids.
Sublimation is a technique used by chemists to purify compounds. Typically a solid is placed in a vessel which is then heated under vacuum. Under this reduced pressure the solid volatilizes and condenses as a purified compound on a cooled surface, leaving the non-volatile residue impurities behind. This cooled surface often takes the form of a cold finger. Once heating ceases and the vacuum is released, the purified compound can be collected from the cold surface. Usually this is done using a sublimation apparatus.
Winnowing Wind winnowing is an agricultural method developed by ancient cultures for separating grain from chaff. It is also used to remove weevils or other pests from stored grain. In its simplest form it involves throwing the mixture into the air so that the wind blows away the lighter chaff, while the heavier grains fall back down for recovery. Techniques included using a winnowing fan (a shaped basket shaken to raise the chaff) or using a tool (a winnowing fork or shovel) on a pile of harvested grain.
