ADVERTISEMENTS:Apart from these there are certain modified forms of these chromatographic techniques involving different mechanisms and are hence categorized as modified or specialized chromatographic techniques. Column Chromatography:It is the preparative application of chromatography. It is used to obtain pure chemical compounds from a mixture of compounds on a scale from micrograms up to kilograms using large industrial columns. The classical preparative chromatography column is a glass tube with a diameter from 5 to 50 mm and a height of 50 cm to 1 m with a tap at the bottom.Slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
A solution of the organic material is pipetted on top of the stationary phase.This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent. Eluent is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column.The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. At the end of the column they elute one at a time. During the entire chromatography process the eluent is collected in a series of fractions.The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g., by analytical chromatography, UV absorption, or fluorescence. Coloured compounds (or fluorescent compounds with the aid of an UV lamp) can be seen through the glass wall as moving bands.The stationary phase or adsorbent in column chromatography is a solid.
The most common stationary phase for column chromatography is —C 18H 37, followed by alumina. Cellulose powder has often been used in the past. Also possible are ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are micro porous for an increased surface; though in EBA a fluidized bed is used.The mobile phase or eluent is either a pure solvent or a mixture of different solvents.
It is chosen so that the retention factor value of the compound of interest is roughly around 0.75 in order to minimize the time and the amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer chromatography (TLC) with the same stationary phase.A faster flow rate of the eluent minimizes the time required to run a column and thereby minimizes diffusion, resulting in a better separation. A simple laboratory column runs by gravity flow.
The flow rate of such a column can be increased by extending the fresh eluent filled column above the top of the stationary phase or decreased by the tap controls. Better flow rates can be achieved by using a pump or by using compressed gas (e.g., air, nitrogen, or argon) to push the solvent through the column (flash column chromatography).Automated flash chromatography systems attempt to minimize human involvement in the purification process. Automated systems may include components normally found on HPLC systems (gradient pump, sample injection apparatus, UV detector) and a fraction collector to collect the eluent. The software controlling an automated system will coordinate the components and help the user to find the resulting purified material within the fraction collector.
The software will also store results from the process for archival or later recall purposes. Paper Chromatography:It is an analytical technique for separating and identifying mixtures that are or can be coloured, especially pigments. This can also be used in secondary or primary schools in ink experiments. This method has been largely replaced by thin layer chromatography; however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.A small, ideally concentrated spot of solution that contains the sample is applied to a strip of chromatography paper about 1 cm from the base, usually using a capillary tube for maximum precision. This sample is absorbed onto the paper and may form interactions with it.
Any substance that reacts or bonds with the paper cannot be measured using -Solvent front technique. The paper is then dipped into a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container.The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper and to one another.
As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibers in the paper. Paper chromatography takes anywhere from several minutes to several hours. ADVERTISEMENTS:In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same values in a particular solvent. In these cases, two-way chromatography is used to separate the multiple-pigment spots. The chromatogram is turned by ninety degrees, and placed in a different solvent in the same way as before; some spots separate in the presence of more than one pigment.As before, the value is calculated, and the two pigments are identified.
The R f value (retention factor) is the distance travelled by a particular component from the origin (where the sample was originally spotted) as a ratio to the distance travelled by the solvent front from the origin. Rf values for each substance will be unique, and can be used to identify components. A particular component will have the same R f value if it is separated under identical conditions.After development, the spots corresponding to different compounds may be located by their colour, ultraviolet light, ninhydrin (Triketohydrindane hydrate) or by treatment with iodine vapours.
The final chromatogram can be compared with other known mixture chromatograms to identify sample mixture using the R n value. ADVERTISEMENTS:As in most other forms of chromatography, paper chromatography uses R n values to help identify compounds.
R f values are calculated by dividing the distance the pigment travels up the paper by the distance the solvent travels (the solvent front). Because R f values are standard for a given compound, known R n values can be used to aid in the identification of an unknown substance in an experiment. Thin Layer Chromatography:Thin-layer chromatography (TLC) is a chromatographic technique that is useful for separating organic compounds. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose immobilized onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated dissolved in an appropriate solvent is drawn through the plate via capillary action, separating the experimental solution.When the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir. The separated spots are visualized with ultraviolet light or by placing the plate in iodine vapour.
The different components in the mixture move up the plate at different rates due to differences in their portioning behavior between the mobile liquid phase and the stationary phase.It can be used to determine the pigments a plant contains, to detect pesticides or insecticides in food, in forensics to analyze the dye composition of fibers, or to identify compounds present in a given substance, among other uses. It is a quick, generic method for organic reaction monitoring. ADVERTISEMENTS:TLC plates are made by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulphate (gypsum) and water. This mixture is spread as thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic, and the resultant plate is dried and activated by heating in an oven for thirty minutes at 110°C. The thickness of the adsorbent layer is typically around 0.1-0.25 mm for analytical purposes and around 1-2 mm for preparative TLC.
Every type of chromatography contains a mobile phase and a stationary phase.The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.A small spot of solution containing the sample is applied to a plate, about one centimetre from the base. The plate is then dipped into a suitable solvent, such as ethanol or water, and placed in a sealed container.
The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent.Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the stationary phase. Results also vary depending on the solvent used. For example, if the solvent were a 90:10 mixture of hexane to ethyl acetate, then the solvent would be mostly non-polar. ADVERTISEMENTS:This means that when analyzing the TLC, the non-polar parts will have moved further up the plate. The polar compounds, in contrast, will not have moved as much. The reverse is true when using a solvent that is more polar than non-polar (10:90 hexane to ethyl acetate).
With these solvents, the polar compounds will move higher up the plate, while the non-polar compounds will not move as much.The appropriate solvent in context of thin layer chromatography will be one which differs from the stationary phase material in polarity. If polar solvent is used to dissolve the sample and spot is applied over polar stationary phase of TLC, the sample spot will grow radially due to capillary action, which is not advisable as one spot may mix with the other.Hence, to restrict the radial growth of sample-spot, the solvent used for dissolving samples in order to apply them on plates should be as non-polar or semi-polar as possible when the stationary phase is polar, and vice versa.As the chemicals being separated may be colourless, several methods exist to visualize the spots:1. Often a small amount of a fluorescent compound, usually Manganese-activated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a black-light(UV 254).
The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.2. Iodine vapours are a general unspecific colour reagent3.
Specific colour reagents exist into which the TLC plate is dipped or which are sprayed onto the plateOnce visible, the R f value of each spot can be determined by dividing the distance travelled by the product by the total distance travelled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate, and are not physical constants. Applications:In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a “co-spot” with both.
A small (3 by 7 cm) TLC plate takes a couple of minutes to run.The analysis is qualitative, and it will show if starting material has disappeared, product has appeared, and how many products are generated. Unfortunately, TLCs from low-temperature reactions may give misleading results, because the sample is warmed to room temperature in the capillary. One such reaction is DIBALH reduction of ester to aldehyde. Gas-Liquid Chromatography (GLC) or Simply Gas Chromatography (GC):It is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. The instrument used to perform gas chromatographic separations is called a gas chromatograph (also: aerograph, gas separator).A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample. A gas chromatograph uses a flow-through narrow tube known as the column, through which different chemical constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their various chemical and physical properties and their interaction with a specific column filling, called the stationary phase.As the chemicals exit the end of the column, they are detected and identified electronically.
The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). Other parameters that can be used to alter the order or time of retention are the carrier gas flow rate, and the temperature.In a GC analysis, a known volume of gaseous or liquid analyte is injected into the “entrance” (head) of the column, usually using a micro syringe (or, solid phase micro-extraction fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls or onto packing materials in the column.The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the analyte mixture are separated as they progress along the column and reach the end of the column at different times (retention time).A detector is used to monitor the outlet stream from the column; thus, the time at which each component reaches the outlet and the amount of that component can be determined. Generally, substances are identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention time of the analyte in the column. Auto Samplers:The auto sampler provides the means to introduce automatically a sample into the inlets. Manual insertion of the sample is possible but very rare nowadays.
Automatic insertion provides better reproducibility and time-optimization. Different kinds of auto samplers exist. Auto samplers can be classified in relation to sample capacity (auto-injectors VS. Auto samplers, where auto-injectors can work a small number of samples), to robotic technologies (XYZ robot VS rotating/SCARA-robot – the most common), or to analysis:i.
Static head-space by syringe technologyiii. Dynamic head-space by transfer-line technologyiv. Solid phase micro extraction (SPME). Inlets:The column inlet (or injector) provides the means to introduce a sample into a continuous flow of carrier gas. The inlet is a piece of hardware attached to the column head.Common inlet types are:1.
Port Injection Chromatography Degradation Of Cocaine To Ecgonidine
S/SL (Split/Split less) injector:A sample is introduced into a heated small chamber via a syringe through a septum; the heat facilitates volatilization of the sample and sample matrix. The carrier gas then either sweeps the entirety (split less mode) or a portion (split mode) of the sample into the column. In split mode, a part of the sample/carrier gas mixture in the injection chamber is exhausted through the split vent.2. On-column inlet:The sample is here introduced in its entirety without heat.3. PTV injector:Temperature-programmed sample introduction was first described by Vogt in 1979.
Originally Vogt developed the technique as a method for the introduction of large sample volumes (up to 250 µl) in capillary GC. Vogt introduced the sample into the liner at a controlled injection rate. The temperature of the liner was chosen slightly below the boiling point of the solvent.The low-boiling solvent was continuously evaporated and vented through the split line.
Based on this technique, Poy developed the Programmed Temperature Vaporizing Injector PTV. By introducing the sample at a low initial liner temperature many of the disadvantages of the classic hot injection techniques could be circumvented.4. Gas source inlet or gas switching valve:Gaseous samples in collection bottles are connected to what is most commonly a six-port switching valve. The carrier gas flow is not interrupted while a sample can be expanded into a previously evacuated sample loop. Upon switching, the contents of the sample loop are inserted into the carrier gas stream.5. P/T (Purge-and-Trap) system:An inert gas is bubbled through an aqueous sample causing insoluble volatile chemicals to be purged from the matrix. The volatiles are ‘trapped’ on an absorbent column (known as a trap or concentrator) at ambient temperature.
The trap is then heated and the volatiles are directed into the carrier gas stream. Samples requiring pre-concentration or purification can be introduced via such a system, usually hooked up to the S/SL port.6. SPME (solid phase micro extraction) offers a convenient, low-cost alternative to P/T systems with the versatility of a syringe and simple use of the S/SL port. Columns:Two types of columns are used in GC:1. Packed columns are 1.5-10 m in length and have an internal diameter of 2-4 mm. The tubing is usually made of stainless steel or glass and contains a packing of finely divided, inert, solid support material (e.g., diatomaceous earth) that is coated with a liquid or solid stationary phase.
The nature of the coating material determines what type of materials will be most strongly adsorbed. Thus numerous columns are available that are designed to separate specific types of compounds.2. Capillary columns have a very small internal diameter, on the order of a few tenths of millimeters, and lengths between 25-60 metres are common. The inner column walls are coated with the active materials (WCOT columns), some columns are quasi-solid filled with many parallel microspores (PLOT columns).
Most capillary columns are made of fused-silica with a polyimide outer coating. These columns are flexible, so a very long column can be wound into a small coil.3. New developments are sought where stationary phase incompatibilities lead to geometric solutions of parallel columns within one column.Among these new developments are:(i) Internally heated micro FAST columns, where two columns, an internal heating wire and a temperature sensor are combined within a common column sheath (micro FAST);(ii) Micro packed columns (1/16″ OD) are column-in-column packed columns where the outer column space has a packing different from the inner column space, thus providing the separation behaviour of two columns in one. They can be easily fit to inlets and detectors of a capillary column instrument.The temperature-dependence of molecular adsorption and of the rate of progression along the column necessitates a careful control of the column temperature to within a few tenths of a degree for precise work. Reducing the temperature produces the greatest level of separation, but can result in very long elution times. For some cases, temperature is ramped either continuously or in steps to provide the desired separation.
This is referred to as a temperature program. Electronic pressure control can also be used to modify flow rate during the analysis, aiding in faster run times while keeping acceptable levels of separation.The choice of carrier gas (mobile phase) is important, with hydrogen being the most efficient and providing the best separation. However, helium has a larger range of flow rates that are comparable to hydrogen in efficiency, with the added advantage that helium is non-flammable, and works with a greater number of detectors.
Therefore, helium is the most common carrier gas used. Detectors:A number of detectors are used in gas chromatography.
The most common are the flame ionization detector (FID) and the thermal conductivity detector (TCD). Both are sensitive to a wide range of components, and both work over a wide range of concentrations.While TCDs are essentially universal and can be used to detect any component other than the carrier gas (as long as their thermal conductivities are different than that of the carrier gas, at detector temperature), FIDs are sensitive primarily to hydrocarbons, and are more sensitive to them than TCD.However, an FID cannot detect water. Both detectors are also quite robust. Since TCD is non-destructive, it can be operated in series before an FID (destructive), thus providing complementary detection of the same eluents. Other detectors are sensitive only to specific types of substances, or work well only in narrower ranges of concentrations.They include:i. Discharge ionization detector (DID)ii.
Electron capture detector (ECD)iii. Flame photometric detector (FPD)iv.
Hall electrolytic conductivity detector (E1CD)v. Helium ionization detector (HID)vi. Nitrogen phosphorus detector (NPD)vii. Mass selective detector (MSD)viii.
Photo-ionization detector (PID)ix. Pulsed discharge ionization detector (PDD)Some gas chromatographs are connected to a mass spectrometer which acts as the detector. The combination is known as GC-MS. Some GC-MS are connected to an Nuclear Magnetic Resonance Spectrometer which acts as a back-up detector. This combination is known as GC-MS- NMR. Some GC-MS-NMR are connected to an infrared spectra which acts as a back-up detector. This combination is known as GC-MS-NMR-IR.
It must, however, be stressed that this is very rare as most analysis needed can be concluded via purely GC-MS. Methods:The method is the collection of conditions in which the GC operates for a given analysis. Method development is the process of determining what conditions are adequate and/or ideal for the analysis required. Conditions which can be varied to accommodate a required analysis include inlet temperature, detector temperature, column temperature and temperature program, carrier gas and carrier gas flow rates, the column’s stationary phase, diameter and length, inlet type and flow rates, sample size and injection technique.Depending on the detector(s) (see below) installed on the GC, there may be a number of detector conditions that can also be varied. Some GCs also include valves which can change the route of sample and carrier flow, and the timing of the turning of these valves can be important to method development. Carrier Gas Selection and Flow Rates:Typical carrier gases include helium, nitrogen, argon, hydrogen and air. Which gas to use is usually determined by the detector being used, for example, a DID requires helium as the carrier gas.
When analyzing gas samples, however, the carrier is sometimes selected based on the sample’s matrix, for example, when analyzing a mixture in argon, an argon carrier is preferred, because the argon in the sample does not show upon the chromatogram. Safety and availability can also influence carrier selection, for example, hydrogen is flammable, and high-purity helium can be difficult to obtain in some areas of the world.The purity of the carrier gas is also frequently determined by the detector, though the level of sensitivity needed can also play a significant role.
Typically, purities of 99.995% or higher are used. Trade names for typical purities include “Zero Grade”, “Ultra-High Purity (UHP) Grade”, “4.5 Grade” and “5.0 Grade”.The carrier gas flow rate affects the analysis in the same way that temperature does (see above). The higher the flow rates the faster the analysis, but the lower the separation between analytes. Selecting the flow rate is, therefore, the same compromise between the level of separation and length of analysis as selecting the column temperature.With GCs made before 1990s, carrier flow rate was controlled indirectly by controlling the carrier inlet pressure, or “column head pressure”.
The actual flow rate was measured at the outlet of the column or the detector with an electronic flow meter, or a bubble flow meter, and could be an involved, time consuming, and frustrating process. The pressure setting was not able to be varied during the run, and thus the flow was essentially constant during the analysis.Many modern GCs, however, electronically measure the flow rate, and electronically control the carrier gas pressure to set the flow rate. Consequently, carrier pressures and flow rates can be adjusted during the run, creating pressure/flow programs similar to temperature programs.
Inlet Types and Flow Rates:The choice of inlet type and injection technique depends on if the sample is in liquid, gas, adsorbed, or solid form, and on whether a solvent matrix is present that has to be vaporized. ADVERTISEMENTS:CCC can be operated in different modes depending on the type of separation required and sample to be separated.These different operation modes of CCC are as under:i. Head to tail:The denser phase is pumped through as the mobile phase.
Derived from terminology for Archimedean screw force.ii. Tail to head:The less dense phase is used as the mobile phase.iii. Dual Mode:The mobile and stationary phases are reversed part way through the run.iv. Gradient Mode:The concentration of one or more components in the mobile phase is varied throughout the run to achieve optimal resolution across a wider range of polarities. For example, a methanol-water gradient may be employed using pure heptane as the stationary phase. This is not possible with all binary systems due to excessive loss of stationary phase.v.
Elution Extrusion Mode (EECCC):The mobile phase is extruded after a certain point by switching the phase being pumped into the system. For example, during the elution portion of a separation using an EtOAc-water system running head to tail, the aqueous mobile phase is being pumped into the system. In order to switch to extrusion mode, organic phase is pumped into the system.This can be accomplished either with a valve on the inlet of single pump, or ideally with an integrated system of two or three pumps, each dedicated either to a single phase of a binary mixture, or to an intermediate wash solvent. This also allows for good resolution of compounds with high mobile-phase affinities. It requires only one column volume of solvent and leaves the column full of fresh stationary phase.vi.
PH Zone Refining:Acidic and basic solvents are used to elute analytes based on their pKa.
AbstractThe scientific literature of the detection and analysis of drugs of forensic interest, as published from 1992 through 2001, is reviewed. 1,377 references are included. IntroductionThis review presents a 10 year survey of the detection and analysis of drugs of forensic interest, aspublished in the mainstream scientific literature from 1992 through 2001. Analyses of drugs in post-ingestionbiological matrices are not included, except for select studies which provide structural, spectral, and/oranalytical data above and beyond routine toxicological 'screening' techniques.
In addition, due totheir inherently transitory nature, Internet references are not included. Finally, forensic associationnewsletters and 'underground' publications are not included.Articles are first organized by overall focus, and subcategorized (where applicable) by specific drug ordrug class, or instrumental technique.