Gas chromatography–mass spectrometry
Gas chromatography–mass spectrometry is an analytical method that combines the features of gas-chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation, identification of unknown samples, including that of material samples obtained from planet Mars during probe missions as early as the 1970s. GC-MS can be used in airport security to detect substances in luggage or on human beings. Additionally, it can identify trace elements in materials that were thought to have disintegrated beyond identification. Like liquid chromatography–mass spectrometry, it allows analysis and detection of tiny amounts of a substance. GC-MS has been regarded as a "gold standard" for forensic substance identification because it is used to perform a 100% specific test, which positively identifies the presence of a particular substance. A nonspecific test indicates that any of several in a category of substances is present.
Although a nonspecific test could statistically suggest the identity of the substance, this could lead to false positive identification. The first on-line coupling of gas chromatography to a mass spectrometer was reported in 1959; the development of affordable and miniaturized computers has helped in the simplification of the use of this instrument, as well as allowed great improvements in the amount of time it takes to analyze a sample. In 1964, Electronic Associates, Inc. a leading U. S. supplier of analog computers, began development of a computer controlled quadrupole mass spectrometer under the direction of Robert E. Finnigan. By 1966 Finnigan and collaborator Mike Uthe's EAI division had sold over 500 quadrupole residual gas-analyzer instruments. In 1967, Finnigan left EAI to form the Finnigan Instrument Corporation along with Roger Sant, T. Z. Chou, Michael Story, William Fies. In early 1968, they delivered the first prototype quadrupole GC/MS instruments to Stanford and Purdue University.
When Finnigan Instrument Corporation was acquired by Thermo Instrument Systems in 1990, it was considered "the world's leading manufacturer of mass spectrometers". The GC-MS is composed of two major building blocks: the mass spectrometer; the gas chromatograph utilizes a capillary column which depends on the column's dimensions as well as the phase properties. The difference in the chemical properties between different molecules in a mixture and their relative affinity for the stationary phase of the column will promote separation of the molecules as the sample travels the length of the column; the molecules are retained by the column and elute from the column at different times, this allows the mass spectrometer downstream to capture, accelerate and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass-to-charge ratio; these two components, used together, allow a much finer degree of substance identification than either unit used separately.
It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process requires a pure sample while gas chromatography using a traditional detector cannot differentiate between multiple molecules that happen to take the same amount of time to travel through the column, which results in two or more molecules that co-elute. Sometimes two different molecules can have a similar pattern of ionized fragments in a mass spectrometer. Combining the two processes reduces the possibility of error, as it is unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore, when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it increases certainty that the analyte of interest is in the sample. For the analysis of volatile compounds, a purge and trap concentrator system may be used to introduce samples; the target analytes are extracted by mixing the sample with water and purge with inert gas into an airtight chamber, this is known as purging or sparging.
The volatile compounds move into the headspace above the water and are drawn along a pressure gradient out of the chamber. The volatile compounds are drawn along a heated line onto a'trap'; the trap is a column of adsorbent material at ambient temperature that holds the compounds by returning them to the liquid phase. The trap is heated and the sample compounds are introduced to the GC-MS column via a volatiles interface, a split inlet system. P&T GC-MS is suited to volatile organic compounds and BTEX compounds. A faster alternative is the "purge-closed loop" system. In this system the inert gas is bubbled through the water until the concentrations of organic compounds in the vapor phase are at equilibrium with concentrations in the aqueous phase; the gas phase is analysed directly. The most common type of mass spectrometer associated with a gas chromatograph is the quadrupole mass spectrometer, sometimes referred to by the Hewlett-Packard trade name "Mass Selective Detector". Another common detector is the ion trap mass spectrometer.
Additionally one may find a magnetic sector mass spectrometer, however these
Capillary electrochromatography is a chromatographic technique in which the mobile phase is driven through the chromatographic bed by electroosmosis. Capillary electrochromatography is a combination of two analytical techniques, high-performance liquid chromatography and capillary electrophoresis. Capillary electrophoresis aims to separate analytes on the basis of their mass-to-charge ratio by passing a high voltage across ends of a capillary tube, filled with the analyte. High-performance liquid chromatography separates analytes by passing them, under high pressure, through a column filled with stationary phase; the interactions between the analytes and the stationary phase and mobile phase lead to the separation of the analytes. In capillary electrochromatography capillaries, packed with HPLC stationary phase, are subjected to a high voltage. Separation is achieved by electrophoretic migration of solutes and differential partitioning. Capillary electrochromatography combines the principles used in HPLC and CE.
The mobile phase is driven across the chromatographic bed using electroosmosis instead of pressure. Electroosmosis is the motion of liquid induced by an applied potential across a porous material, capillary tube, membrane or any other fluid conduit. Electroosmotic flow is caused by the Coulomb force induced by an electric field on net mobile electric charge in a solution. Under alkaline conditions, the surface silanol groups of the fused silica will become ionised leading to a negatively charged surface; this surface will have a layer of positively charged ions in close proximity which are immobilised. This layer of ions is called the Stern layer; the thickness of the double layer is given by the formula: δ = ϵ r ϵ 0 R T 2 c F 2 where εr is the relative permittivity of the medium, εo is the permittivity of vacuum, R is the universal gas constant, T is the absolute temperature, c is the molar concentration, F is the Faraday constant When an electric field is applied to the fluid, the net charge in the electrical double layer is induced to move by the resulting Coulomb force.
The resulting flow is termed electroosmotic flow. In CEC positive ions of the electrolyte added along with the analyte accumulate in the electrical double layer of the particles of the column packing on application of an electric field they move towards the cathode and drag the liquid mobile phase with them; the relationship between the linear velocity u of the liquid in the capillary and the applied electric field is given by the Smoluchowski equation as u = ϵ r ϵ 0 ζ E η where ζ is the potential across the Stern layer, E is the electric field strength, η is the viscosity of the solvent. Separation of components in CEC is based on interactions between the stationary phase and differential electrophoretic migration of solutes; the components of a capillary electrochromatograph are a sample vial and destination vials, a packed capillary, electrodes, a high voltage power supply, a detector, a data output and handling device. The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution.
The capillary is packed with stationary phase. To introduce the sample, the capillary inlet is placed into a vial containing the sample and returned to the source vial; the migration of the analytes is initiated by an electric field, applied between the source and destination vials and is supplied to the electrodes by the high-voltage power supply. The analytes separate as they migrate due to their electrophoretic mobility, are detected near the outlet end of the capillary; the output of the detector is sent to a data output and handling device such as an integrator or computer. The data is displayed as an electropherogram, which reports detector response as a function of time. Separated chemical compounds appear as peaks with different migration times in an electropherogram. Avoiding the use of pressure to introduce the mobile phase into the column, results in a number of important advantages. Firstly, the pressure driven flow rate across a column depends directly on the square of the particle diameter and inversely on the length of the column.
This restricts the length of the column and size of the particle, particle size is less than 3 micrometer and the length of the column is restricted to 25 cm. Electrically driven flow rate is independent of length of size. A second advantage of using electroosmosis to pass the mobile phase into the column is the plug-like flow velocity profile of EOF, which reduces the solute dispersion in the column, increasing column efficiency. Chromatography Electrophoresis High-performance liquid chromatography Capillary electrophoresis Electrochromatography Smith, N. "Capillary ElectroChromatography" Available at:https://www.beckmancoulter.com/wsrportal/bibliography?docname=AP8508ACECPrimer.pdf Bartle, K. D. Capillary ElectroChromatography Published by The Royal Society of Cambridge. ISBN 0-85404-530-9
Diffusion is the net movement of molecules or atoms from a region of higher concentration to a region of lower concentration. Diffusion is driven by a gradient in chemical potential of the diffusing species. A gradient is the change in the value of a quantity e.g. concentration, pressure, or temperature with the change in another variable distance. A change in concentration over a distance is called a concentration gradient, a change in pressure over a distance is called a pressure gradient, a change in temperature over a distance is called a temperature gradient; the word diffusion derives from the Latin word, which means "to spread way out.” A distinguishing feature of diffusion is that it depends on particle random walk, results in mixing or mass transport without requiring directed bulk motion. Bulk motion, or bulk flow, is the characteristic of advection; the term convection is used to describe the combination of both transport phenomena. An example of a situation in which bulk motion and diffusion can be differentiated is the mechanism by which oxygen enters the body during external respiration known as breathing.
The lungs are located in the thoracic cavity, which expands as the first step in external respiration. This expansion leads to an increase in volume of the alveoli in the lungs, which causes a decrease in pressure in the alveoli; this creates a pressure gradient between the air outside the body at high pressure and the alveoli at low pressure. The air moves down the pressure gradient through the airways of the lungs and into the alveoli until the pressure of the air and that in the alveoli are equal i.e. the movement of air by bulk flow stops once there is no longer a pressure gradient. The air arriving in the alveoli has a higher concentration of oxygen than the “stale” air in the alveoli; the increase in oxygen concentration creates a concentration gradient for oxygen between the air in the alveoli and the blood in the capillaries that surround the alveoli. Oxygen moves by diffusion, down the concentration gradient, into the blood; the other consequence of the air arriving in alveoli is that the concentration of carbon dioxide in the alveoli decreases.
This creates a concentration gradient for carbon dioxide to diffuse from the blood into the alveoli, as fresh air has a low concentration of carbon dioxide compared to the blood in the body. The pumping action of the heart transports the blood around the body; as the left ventricle of the heart contracts, the volume decreases, which increases the pressure in the ventricle. This creates a pressure gradient between the heart and the capillaries, blood moves through blood vessels by bulk flow down the pressure gradient; as the thoracic cavity contracts during expiration, the volume of the alveoli decreases and creates a pressure gradient between the alveoli and the air outside the body, air moves by bulk flow down the pressure gradient. The concept of diffusion is used in: physics, biology, sociology and finance. However, in each case, the object, undergoing diffusion is “spreading out” from a point or location at which there is a higher concentration of that object. There are two ways to introduce the notion of diffusion: either a phenomenological approach starting with Fick's laws of diffusion and their mathematical consequences, or a physical and atomistic one, by considering the random walk of the diffusing particles.
In the phenomenological approach, diffusion is the movement of a substance from a region of high concentration to a region of low concentration without bulk motion. According to Fick's laws, the diffusion flux is proportional to the negative gradient of concentrations, it goes from regions of higher concentration to regions of lower concentration. Sometime various generalizations of Fick's laws were developed in the frame of thermodynamics and non-equilibrium thermodynamics. From the atomistic point of view, diffusion is considered as a result of the random walk of the diffusing particles. In molecular diffusion, the moving molecules are self-propelled by thermal energy. Random walk of small particles in suspension in a fluid was discovered in 1827 by Robert Brown; the theory of the Brownian motion and the atomistic backgrounds of diffusion were developed by Albert Einstein. The concept of diffusion is applied to any subject matter involving random walks in ensembles of individuals. Biologists use the terms "net movement" or "net diffusion" to describe the movement of ions or molecules by diffusion.
For example, oxygen can diffuse through cell membranes so long as there is a higher concentration of oxygen outside the cell. However, because the movement of molecules is random oxygen molecules move out of the cell; because there are more oxygen molecules outside the cell, the probability that oxygen molecules will enter the cell is higher than the probability that oxygen molecules will leave the cell. Therefore, the "net" movement of oxygen molecules is into the cell. In other words, there is a net movement of oxygen molecules down the concentration gradient. In the scope of time, diffusion in solids was used. For example, Pliny the Elder had described the cementation process, which produces steel from the element iron through carbon diffusion. Another example is well known for many centuries, the diffusion of colors of stained glass or earthenware and Chinese ceramics. In modern science, the first systematic experimental study of di
A cylinder has traditionally been a three-dimensional solid, one of the most basic of curvilinear geometric shapes. It is the idealized version of a solid physical tin can having lids on bottom; this traditional view is still used in elementary treatments of geometry, but the advanced mathematical viewpoint has shifted to the infinite curvilinear surface and this is how a cylinder is now defined in various modern branches of geometry and topology. The shift in the basic meaning has created some ambiguity with terminology, it is hoped that context makes the meaning clear. In this article both points of view are presented and distinguished by referring to solid cylinders and cylindrical surfaces, but keep in mind that in the literature the unadorned term cylinder could refer to either of these or to an more specialized object, the right circular cylinder; the definitions and results in this section are taken from the 1913 text and Solid Geometry by George Wentworth and David Eugene Smith. A cylindrical surface is a surface consisting of all the points on all the lines which are parallel to a given line and which pass through a fixed plane curve in a plane not parallel to the given line.
Any line in this family of parallel lines is called an element of the cylindrical surface. From a kinematics point of view, given a plane curve, called the directrix, a cylindrical surface is that surface traced out by a line, called the generatrix, not in the plane of the directrix, moving parallel to itself and always passing through the directrix. Any particular position of the generatrix is an element of the cylindrical surface. A solid bounded by a cylindrical surface and two parallel planes is called a cylinder; the line segments determined by an element of the cylindrical surface between the two parallel planes is called an element of the cylinder. All the elements of a cylinder have equal lengths; the region bounded by the cylindrical surface in either of the parallel planes is called a base of the cylinder. The two bases of a cylinder are congruent figures. If the elements of the cylinder are perpendicular to the planes containing the bases, the cylinder is a right cylinder, otherwise it is called an oblique cylinder.
If the bases are disks the cylinder is called a circular cylinder. In some elementary treatments, a cylinder always means a circular cylinder; the height of a cylinder is the perpendicular distance between its bases. The cylinder obtained by rotating a line segment about a fixed line that it is parallel to is a cylinder of revolution. A cylinder of revolution is a right circular cylinder; the height of a cylinder of revolution is the length of the generating line segment. The line that the segment is revolved about is called the axis of the cylinder and it passes through the centers of the two bases; the bare term cylinder refers to a solid cylinder with circular ends perpendicular to the axis, that is, a right circular cylinder, as shown in the figure. The cylindrical surface without the ends is called an open cylinder; the formulae for the surface area and the volume of a right circular cylinder have been known from early antiquity. A right circular cylinder can be thought of as the solid of revolution generated by rotating a rectangle about one of its sides.
These cylinders are used in an integration technique for obtaining volumes of solids of revolution. A cylindric section is the intersection of a cylinder's surface with a plane, they are, in general and are special types of plane sections. The cylindric section by a plane that contains two elements of a cylinder is a parallelogram; such a cylindric section of a right cylinder is a rectangle. A cylindric section in which the intersecting plane intersects and is perpendicular to all the elements of the cylinder is called a right section. If a right section of a cylinder is a circle the cylinder is a circular cylinder. In more generality, if a right section of a cylinder is a conic section the solid cylinder is said to be parabolic, elliptic or hyperbolic respectively. For a right circular cylinder, there are several ways. First, consider planes that intersect a base in at most one point. A plane is tangent to the cylinder; the right sections are circles and all other planes intersect the cylindrical surface in an ellipse.
If a plane intersects a base of the cylinder in two points the line segment joining these points is part of the cylindric section. If such a plane contains two elements, it has a rectangle as a cylindric section, otherwise the sides of the cylindric section are portions of an ellipse. If a plane contains more than two points of a base, it contains the entire base and the cylindric section is a circle. In the case of a right circular cylinder with a cylindric section, an ellipse, the eccentricity e of the cylindric section and semi-major axis a of the cylindric section depend on the radius of the cylinder r and the angle α between the secant plane and cylinder axis, in the following way: e = cos α, a = r sin α. If the base of a circular cylinder has a radius r and the cylinder has height h its volume is given by V = πr2h; this formula holds. This formula may be established by using Cavalieri's principle. In more generality, by the same principle, the volume of an
High-performance liquid chromatography
High-performance liquid chromatography is a technique in analytical chemistry used to separate and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material; each component in the sample interacts differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column. HPLC has been used for manufacturing, legal and medical purposes. Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components; the active component of the column, the adsorbent, is a granular material made of solid particles, 2–50 μm in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles.
The pressurized liquid is a mixture of solvents and is referred to as a "mobile phase". Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent; these interactions are physical in nature, such as hydrophobic, dipole–dipole and ionic, most a combination. HPLC is distinguished from traditional liquid chromatography because operational pressures are higher, while ordinary liquid chromatography relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, 30–250 mm length. HPLC columns are made with smaller adsorbent particles; this gives HPLC superior resolving power when separating mixtures, which makes it a popular chromatographic technique. The schematic of a HPLC instrument includes a degasser, pumps, a detector; the sampler brings the sample mixture into the mobile phase stream.
The pumps deliver composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software provide data analysis; some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as photodiode array or based on mass spectrometry. Most HPLC instruments have a column oven that allows for adjusting the temperature at which the separation is performed; the sample mixture to be separated and analyzed is introduced, in a discrete small volume, into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are a function of specific physical interactions with the adsorbent; the velocity of each component depends on its chemical nature, on the nature of the stationary phase and on the composition of the mobile phase.
The time at which a specific analyte elutes is called its retention time. The retention time measured under particular conditions is an identifying characteristic of a given analyte. Many different types of columns are available, filled with adsorbents varying in particle size, in the nature of their surface; the use of smaller particle size packing materials requires the use of higher operational pressure and improves chromatographic resolution. Sorbent particles may be polar in nature. Common mobile phases used include any miscible combination of water with various organic solvents; some HPLC techniques use water-free mobile phases. The aqueous component of the mobile phase may contain acids or salts to assist in the separation of the sample components; the composition of the mobile phase may be kept constant or varied during the chromatographic analysis. Isocratic elution is effective in the separation of sample components that are different in their affinity for the stationary phase. In gradient elution the composition of the mobile phase is varied from low to high eluting strength.
The eluting strength of the mobile phase is reflected by analyte retention times with high eluting strength producing fast elution. A typical gradient profile in reversed phase chromatography might start at 5% acetonitrile and progress linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition may be part of
Size-exclusion chromatography known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, in some cases molecular weight. It is applied to large molecules or macromolecular complexes such as proteins and industrial polymers; when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are composed of dextran polymers, agarose, or polyacrylamide; the pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a used polymer characterization method because of its ability to provide good molar mass distribution results for polymers; the main application of gel-filtration chromatography is the fractionation of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic-soluble polymers.
Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges. The amount of time a solute remains within a pore is dependent on the size of the pore. Larger solutes will have access to vice versa. Therefore, a smaller solute will remain within the pore for a longer period of time compared to a larger solute. Another use of size exclusion chromatography is to examine the stability and characteristics of natural organic matter in water. In this method, Margit B. Muller, Daniel Schmitt, Fritz H. Frimmel tested water sources from different places in the world to determine how stable the natural organic matter is over a period of time. Though, size exclusion chromatography is utilized to study natural organic material, there are limitations. One of these limitations include. If precise molecular weight is required, other methods should be used; the advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate, that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to separate.
The technique is combined with others that further separate molecules by other characteristics, such as acidity, basicity and affinity for certain compounds. With size exclusion chromatography, there are short and well-defined separation times and narrow bands, which lead to good sensitivity. There is no sample loss because solutes do not interact with the stationary phase; the other advantage to this experimental method is that in certain cases, it is feasible to determine the approximate molecular weight of a compound. The shape and size of the compound determine. To determine approximate molecular weight, the elution volumes of compounds with their corresponding molecular weights are obtained and a plot of “Kav” vs “log” is made, where K a v = / and Mw is the molecular mass; this plot acts as a calibration curve, used to approximate the desired compound's molecular weight. The Ve component represents the volume at which the intermediate molecules elute such as molecules that have partial access to the beads of the column.
In addition, Vt is the sum of the volume within the beads. The Vo component represents the volume at which the larger molecules elute, which elute in the beginning. Disadvantages are, for example, that only a limited number of bands can be accommodated because the time scale of the chromatogram is short, and, in general, there must be a 10% difference in molecular mass to have a good resolution; the technique was invented by Grant Henry Lathe and Colin R Ruthven, working at Queen Charlotte's Hospital, London. They received the John Scott Award for this invention. While Lathe and Ruthven used starch gels as the matrix, Jerker Porath and Per Flodin introduced dextran gels. A short review of these developments has appeared. There were attempts to fractionate synthetic high polymers, it was recognized immediately that with proper calibration, GPC was capable to provide molar mass and molar mass distribution information for synthetic polymers. Because the latter information was difficult to obtain by other methods, GPC came into extensive use.
SEC is used for the analysis of large molecules such as proteins or polymers. SEC works by trapping smaller molecules in the pores of the adsorbent; this process is performed within a column, which consists of a hollow tube packed with micron-scale polymer beads containing pores of different sizes. These pores may be depressions on channels through the bead; as the solution trav
Thin-layer chromatography is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, coated with a thin layer of adsorbent material silica gel, aluminium oxide, or cellulose; this layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture is drawn up the plate via capillary action; because different analytes ascend the TLC plate at different rates, separation is achieved. The mobile phase has different properties from the stationary phase. For example, with silica gel, a polar substance, non-polar mobile phases such as heptane are used; the mobile phase may be a mixture, allowing chemists to fine-tune the bulk properties of the mobile phase. After the experiment, the spots are visualized; this can be done by projecting ultraviolet light onto the sheet. Chemical processes can be used to visualize spots. To quantify the results, the distance traveled by the substance being considered is divided by the total distance traveled by the mobile phase.
This ratio is called the retardation factor. In general, a substance whose structure resembles the stationary phase will have low Rf, while one that has a similar structure to the mobile phase will have high retardation factor. Retardation factors are characteristic, but will change depending on the exact condition of the mobile and stationary phase. For this reason, chemists apply a sample of a known compound to the sheet before running the experiment. Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitative analysis.
This method is referred to as HPTLC, or "high-performance TLC". HPTLC uses thinner layers of stationary phase and smaller sample volumes, thus reducing the loss of resolution due to diffusion. TLC plates are commercially available, with standard particle size ranges to improve reproducibility, they are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate and water. This mixture is spread as a thick slurry on an unreactive carrier sheet glass, thick aluminum foil, or plastic; the resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the absorbent layer is around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC. The process is similar to paper chromatography with the advantage of faster runs, better separations, the choice between different stationary phases; because of its simplicity and speed TLC is used for monitoring chemical reactions and for the qualitative analysis of reaction products.
Plates can be labeled before or after the chromatography process using a pencil or other implement that will not interfere or react with the process. To run a thin layer chromatography plate, the following procedure is carried out: Using a capillary, a small spot of solution containing the sample is applied to a plate, about 1.5 centimeters from the bottom edge. The solvent is allowed to evaporate off to prevent it from interfering with sample's interactions with the mobile phase in the next step. If a non-volatile solvent was used to apply the sample, the plate needs to be dried in a vacuum chamber; this step is repeated to ensure there is enough analyte at the starting spot on the plate to obtain a visible result. Different samples can be placed in a row of spots the same distance from the bottom edge, each of which will move in its own adjacent lane from its own starting point. A small amount of an appropriate solvent is poured into a glass beaker or any other suitable transparent container to a depth of less than 1 centimeter.
A strip of filter paper is put into the chamber so that its bottom touches the solvent and the paper lies on the chamber wall and reaches to the top of the container. The container is closed with a cover glass or any other lid and is left for a few minutes to let the solvent vapors ascend the filter paper and saturate the air in the chamber.. The TLC plate is placed in the chamber so that the spot of the sample do not touch the surface of the eluent in the chamber, the lid is closed; the solvent moves up the plate by capillary action, meets the sample mixture and carries it up the plate. The plate should be removed from the chamber before the solvent front reaches the top of the stationary phase and dried. Without delay, the solvent front, the furthest extent of solvent up the plate, is marked; the plate is visualized. As some plates are pre-coated w