Liquid Chromatography (Chrom-Ed Series)

This difference occurs despite the greater molecular weights and higher boiling points of heptane and heptene. On the stationary phase Carbopack, methanol, weakly dispersive, is eluted almost at the dead volume while the more dispersive solutes are extensively retained. An example of the use of induced dipoles to separate polarizable substances is afforded by the analysis of some aromatic and nitroaromatic hydrocarbons by LC using silica gel as the stationary phase.

The mobile phase was n-hexane at a flow-rate 50 ml per min. The two solutes are well separated and, as they have no permanent dipole, and as dispersive interactions with the silica gel are weak, they are selectively retained almost exclusively by induced dipole interactions. These interactions occur between the strong dipoles of the silanol groups on the silica gel surface and the induced dipoles on the aromatic nucleus resulting from their proximity to the silanol groups.

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To ensure that polar interactions dominate in the stationary phase the mobile phase consists of the dispersive solvent n-hexane. Other substances are present which also contain polar groups and thus, the sample lends itself to separation on the polar stationary phase, silica gel. The analysis was completed in less than 4 minutes using a short column 3. The column appears to be significantly overloaded, but the impurities are well still separated from the main component and a substance in the generic formulation that was not present in the This eBook is protected by Copyright law and International Treaties.

The mobile phase was Although, the ammoniacal methanol helped to decrease extreme polar activity from specially active adsorption sites on the silica surface, the overall interaction of the solutes with the stationary phase was predominately polar. In contrast solute interactions with the methylene dichloride in the mobile phase would be almost exclusively dispersive. Separations Based on Ionic Interactions Ionic materials are not volatile under the conditions normally employed in GC, so, ionic interactions cannot be exploited in GC stationary phases to control retention.

However, they are very important in LC, and ion exchange chromatography the name given to LC separations that employ ionic interactions to control retention is widely used to analyze ion mixtures. The use of ionic interactions to separate some alkali and alkaline earth cations is shown in figure The separation almost exclusively involved ionic interactions as any dispersive interactions between a metal ion and the stationary phase would be very small indeed. The Control of Chromatographically Available Stationary Phase Vs The volume of stationary phase that is made available to the solutes can be controlled in a number of ways.

Firstly, the stationary phase loading on the column can be varied to adjust the retention as required. A specific stationary phase loading may be selected, to either improve the resolution, or to reduce the analysis time, or in some instances, to increase the sample load. Sometimes, the stationary phase loading is reduced so the column is more amenable to specific compounds e.

Secondly, the stationary phase can contain molecules of a special shape that can only make c l o s e contact with molecules having a complementary shape. Other molecules can not interact so closely with the stationary phase and consequently, the stationary phase available to them will be restricted. This approach is exploited in chiral chromatography where the stationary phase is made to consist largely of a specific enantiomer that confers chiral selectivity to the distribution system Thirdly, the stationary phase can be attached to the surface of a porous support, and the pore size chosen to be commensurate with the size of the solute molecules to be separated.

Under such circumstances the molecules that are smaller than the pores will enter the matrix of the material and have more stationary phase available to them. Conversely, the larger molecules will be excluded from the pores and, consequently, come in contact with much less of the stationary phase. Size selectivity, achieved by the use of porous solids, is utilized in size exclusion chromatography SEC where solutes are separated almost exclusively This eBook is protected by Copyright law and International Treaties.

The separation of chiral compounds can be successfully utilized in both GC and LC; size exclusion chromatography, however, is not greatly used in GC and is almost exclusively confined to LC. The Effect of Stationary Phase Loading on the Performance of a Chromatographic System The stationary phase content of a column can affect a separation in two ways. The more stationary phase there is in a column, the more the solutes will be retained, the further they will be apart and the greater the separation. Any change in stationary phase, however, will change the retention of all solutes proportionally and thus the separation will only increase, if the peak widths remain unchanged.

Increasing the amount of stationary phase will usually increase the thickness of the stationary phase film, which, as is shown in Book 7 will increase peak dispersion. It follows that there will be a specific stationary phase loading that provides the best compromise between separation and band dispersion 6 and thus provides the maximum resolution. The loading can be quite critical for open tubular columns in GC. Thus, the stationary phase loading cannot be increased indefinitely to separate the peaks as, eventually, the peaks will start spreading to a greater extent than they are being separated.

Increasing the stationary phase load on a GC column packed or open tube will allow the sample placed on the column to be increased. A large sample is often necessary in trace analysis to provide sufficient material for detection. Under such circumstances the column may be overloaded giving a very broad asymmetric peak which may obscure the trace materials of interest.

This asymmetric dispersion is due to solute-solute interaction in the mobile and stationary phases causing a nonlinear adsorption isotherm. The subject of adsorption isotherms will not be discussed here and it is sufficient to say that the asymmetric dispersion can be reduced by increasing the stationary phase in the column.. A larger amount of stationary phase, will, even with a larger charge, reduce the sample concentration in the stationary phase and thus the deleterious high sample concentrations are never reached.

This is because, irrespective of the type of support material, the amount of stationary phase in an LC column is primarily determined by its surface area. In addition, the amount of available stationary phase on a bonded phase can be modified by adjusting the molecular size chain length of the bonded material. The chain length of the bonded material can be critical when separating proteins as dispersive interactions between the alkane chains and the dispersive hydrophobic groups of the protein can be strong enough to cause structural deconformation; i.

Reducing the chain length of the bonded material, the dispersive forces can be reduced significantly and the deconformation diminished. In practice, carbon chains only two or four carbon atoms long are among those most commonly used for separating labile proteins. Stationary Phase Limitation by Chiral Selectivity The extent to which an enantiomer can interact with the stationary phase depends on how close it can approach the molecules of the stationary phase. If the stationary phase is also chiral in nature, it is likely that one enantiomer in the sample will fit closely to the stationary phase surface whereas the other will be stearically excluded and thus have less stationary phase with which to interact.

The first chiral separations in GC were reported by Gil-Av et al. The use of chiral stationary phases in GC has been dogged by entantiomeric instability arising from the racemization of both the chiral stationary phase and the chiral solutes at elevated temperatures. In addition, at the elevated temperatures necessary to elute the solutes in a reasonable time, the chiral selectivity of the stationary phase can also be impaired.

A thermally stable chiral stationary phase was produced by Frank, Nicholson and Bayer 8 in by the co-polymerization of This eBook is protected by Copyright law and International Treaties. Figure 17 The Separation of the Enantiomers of a-Halocarboxylic Acid Esters on a b-Cyclodextrin-Based Stationary Phase This material was relatively stable up to oC with little racemization but, was not made commercially available until Presently, there are a number of effective GC chiral stationary phases available, some of the more effective being based on cyclodextrin,.

The separation of the enantiomers of an a-halocarboxylic acid ester on a fused silica open tubular column coated with a b-cyclodextrin product is shown in figure The column was 10 m long and operated at 60oC using nitrogen as the carrier gas. The use of LC for chiral separations is easier to carry out and generally more efficient. A number of racemic mixtures can be easily separated using a reverse-phase column and a mobile phase doped with a chiral reagent. In some cases, the reagent is adsorbed strongly on to the stationary phase, under which circumstances, the chiral selectivity resides in the stationary phase.

Conversely, if the reagent remains predominantly This eBook is protected by Copyright law and International Treaties. Camphor sulphonic acid and quinine are examples of mobile phase additives. The most common method used to achieve chiral selectivity is to bond chirally selective compounds to silica in a similar manner to a reverse phase e.

Figure 18 The Separation of Warfarin Isomers on a CYCLOBOND Column The cyclodextrins are produced by the partial degradation of starch followed by the enzymatic coupling of the glucose units into crystalline, homogeneous toroidal structures of different molecular sizes. Three of the most widely characterized are alpha, beta and gamma cyclodextrins which contain 6, 7 and 8 glucose units respectively. Cyclodextrins are chiral structures and the beta- cyclodextrin has 35 stereogenic centers.

The separation of the isomers of Warfarin is shown in figure The column was 25 cm long and 4. It is seen that an excellent separation has been achieved with the two isomers completely resolved. Stationary Phase Limitation by Exclusion Size Exclusion Chromatography might imply that solute retention was determined solely by the size of the molecule.

However, this can only be true if the magnitude of the interaction forces between the solute and each phase is the same. This situation can be closely approached by the appropriate choice of the mobile phase. Under such circumstances the larger molecules, being partially or wholly excluded, will elute first and the smaller molecules elute last. It is important to understand that, even when the dominant retention mechanism is controlled by molecular forces, if the stationary phase or supporting material contains pores of size commensurate with those of the solute molecules, exclusion will still partly control retention.

This is because the larger molecules will interact with less stationary phase and be eluted relatively faster than if they had interacted with the same amount of stationary phase as the smaller molecules. The two most common exclusion media used in LC are silica gel and macroporous polystyrene divinylbenzene resins. Figure 19 shows an exclusion chromatogram of a series of molecular weight standards obtained on silica gel. The column length was 50 cm and the mobile phase tetrahydrofuran THF. The THF would be strongly adsorbed on the silica surface and thus the solutes would be distributed between pure THF in the mobile phase and THF on the surface of the silica.

As a consequence, the interactions are virtually identical in the two phases and the retention was determined almost exclusively by stationary phase availability. Figure 19 The Separation of a Mixture by Exclusion Chromatography Until relatively recently, silica has been the most commonly used exclusion media for the separation of high molecular weight hydrocarbons and polymers. However, it was not so successful in the separation of polymeric materials of biological origin.

More recently the micro-reticular macroporous polystyrene gels have been introduced and found to be very useful for the separation of biopolymers by size exclusion. These materials have even replaced many of the traditional applications of silica gel. However, even if, by appropriate choice of the phase system, the solutes are separated, unless the peak dispersion is contained to allow the individual solutes to be eluted discretely, the mixture will not be resolved.

Peak Dispersion in a Chromatographic Column The first comprehensive approach to dispersion in chromatographic columns was taken by Van Deemter 8 who developed the dispersion This eBook is protected by Copyright law and International Treaties. Van Deemter's development did not take into account the compressibility of the mobile phase which was dealt with later by Katz, Ogan and Scott 9.

A simple form of this theory will be given that does not accommodate the compressibility of the mobile phase but a more detailed and comprehensive treatment is given in Books 6 and 7. Van Deemter et al. Van Deemter derived an expression for the variance contribution of each process to the overall variance per unit length of the column. Furthermore, as the individual dispersion processes can be assumed to be random and non- interacting, the total variance per unit length of the column can be obtained from a sum of the individual variance contributions.

The Multi-Path Effect The multi-path effect is diagramatically depicted in figure As a result, some will randomly travel shorter routes than the average and some longer. It follows, that those molecules taking shorter paths will move ahead of the mean and those that take the longer paths will lag behind the mean. This will result in a differential distance traveled dl as shown in figure 3. This differential flow of the solute molecules results in band dispersion. Longitudinal Diffusion Solutes when contained in a fluid naturally diffuse and spread driven by their concentration gradient.

Thus, in a chromatographic column a discrete solute band will diffuse in the gas or liquid mobile phase. It also follows, that because the diffusion process is random in nature, a concentration curve that is Gaussian in form will be produced. This diffusion effect occurs in the mobile phase of both GC and LC columns.

The diffusion process is depicted in figure Sample Mobile Phase Figure 21 Peak Dispersion by Longitudinal Diffusion The longer the solute band remains in the column, the greater will be the extent of diffusion. The time the solute remains in the column is inversely proportional to the mobile phase velocity, so, the dispersion will also be inversely proportional to the mobile phase velocity. The Resistance to Mass Transfer in the Mobile Phase During passage through a chromatographic column, the solute molecules are constantly and reversibly transferring from the mobile phase to the stationary phase.

This transfer is not instantaneous; time is required for the molecules to pass by diffusion through the mobile phase to reach the interface and enter the stationary phase. Those molecules close to the stationary phase enter it immediately, whereas those molecules some distance away will find their way to it some time later. Since the mobile phase is continually moving, during this time interval, those molecules that remain in the mobile phase will be swept along the column and dispersed away from those molecules that were close and entered the stationary phase immediately.

This process is depicted in figure The diagram shows 6 solute molecules in the mobile phase and the pair closest to the surface, 1 and 2 , enter the stationary phase immediately. While molecules 3 and 4 diffuse through the mobile phase to the interface, the mobile phase moves on. As a consequence, when molecules 3 and 4 reach the interface, they enter the stationary phase some distance ahead of the first two.

Finally, while molecules 5 and 6 diffuse to the interface, the mobile phase has moved even further down the column until molecules 5 and 6 enter the stationary phase ahead of molecules 3 and 4. Thus, the 6 molecules, originally relatively close together, are now spread out in the stationary phase. This explanation, although over-simplified, gives a correct description of the mechanism of mass transfer dispersion. Van Deemter derived the following expression for the variance contribution by the resistance to mass transfer in the mobile phase, s 2R M , This eBook is protected by Copyright law and International Treaties.

Solute molecules close to the interface will leave the stationary phase and enter the mobile phase before those that have diffused further into the stationary phase and have a longer distance to diffuse back. Thus, as those molecules that were close to the surface will be swept along in the moving phase, they will be dispersed from those molecules still diffusing to the surface.

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The dispersion resulting from the resistance to mass transfer in the stationary phase is depicted in figure Molecules 1 and 2 the two closest to the surface will enter the mobile phase and begin moving along the column. Their movement will continue while molecules 3 and 4 diffuse to the interface at which time they will enter the mobile phase and start following molecules 1 and 2 down the column.

By the time molecules 5 and 6 enter the mobile phase, the other four molecules will have been smeared along the column and the original 6 molecules will be dispersed. Van Deemter derived an expression for the variance from the resistance to mass transfer in the stationary phase, s 2R S , which is as follows: The relationship between H and sx is explained in Book 6. Hence the term "HETP equation" for equation This form of the Van Deemter equation is very nearly correct for LC but, due to the compressibility of the gaseous mobile phase in GC, neither the linear velocity nor the pressure is constant along the column.

Furthermore, as the diffusivity, Dm , is a function of pressure, the above form of the equation can only be approximate. However, equation 10 generally gives the correct form of the relationship between H and the linear velocity u. It also predicts that there will be an optimum velocity that gives a minimum value for H and thus, a maximum efficiency. Pressure corrections for retention volume and the height of the theoretical plate are derived in Books 6 and 7.

The Golay Equation for Open Tubular Columns The corresponding equation describing dispersion in an open tubular column was developed by Golay 10 for GC columns but is equally applicable to LC columns and to dispersion in connecting tubes. Consequently, the equation contains only three functions. One function describes dispersion from longitudinal diffusion and the other two describes dispersion from the resistance to mass transfer in the mobile and stationary phases, respectively.

The Golay equation takes the following form: Open tubular columns behave in exactly the same way as packed columns with respect to pressure. As the column is geometrically simple the respective functions of k' can also be explicitly developed. The Efficiency of a TLC Plate TLC plate efficiency is a measure of its capacity to restrain solute dispersion and maintain narrow spots as the solutes migrate along the plate. An explicit equation that describes the dispersion in TLC has not been rigorously developed, nevertheless, high efficiencies are realized in much the same way as they are in GC and LC.

Primarily, the particle size of the silica layer must be made as small as possible and the layer must be spread in a thin, homogenous film on the supporting plate. TLC plate efficiency is measured in a similar manner to column efficiency but slightly modified. It is very difficult, if not impossible, to identify the positions of the points of inflexion on a TLC spot, but if the visible edges of the spot are assumed to occur at four standard deviations of the spot distribution, then it is still possible to assess the efficiency.

If the diameter of the spot d , corresponds to four standard deviations, then applying the same rationale as with the packed column, This eBook is protected by Copyright law and International Treaties. Besides assuming that the visible limits of the spot correspond to four standard deviations, the basic assumption that the value of K is constant throughout the development, is also tacitly made and this is certainly not so. In fact, this procedure would give similar errors to those that would arise from calculating the efficiency of an LC column under conditions of gradient elution.

Nevertheless, the method does allow the relative performance of different plates to be accessed and in this way can be helpful. The Basic Column Chromatograph A chromatograph consists of five basic units and these units, although possibly designed differently for different systems, are essential for all types of chromatograph, including both gas and liquid chromatographs. The layout of all the five essential units is shown in figure They consists of a mobile phase supply, a sampling system, a column and column oven including a temperature controller and temperature programming system.

The temperature controller and programmer will probably have its own microprocessor which may also be under the control of the data acquisition and processing computer. The column eluent will pass to an appropriate detector and its associated electronics. There may be more than one detector and they may be employed in parallel or in series and individually controlled and monitored.

The final unit will be a computer and data acquisition system This eBook is protected by Copyright law and International Treaties. Mobile Phase Sampling System Supply Detector Detector Oven Column Detector Electronics Column Oven Oven Controller Potentiometric Recorder or Computer Data Acquisition and Processsing System Figure 24 The Basic Chromatograph The Mobile Phase Supply The first unit, the mobile phase supply, can range in complexity from a simple gas cylinder connected to a flow controlling valve for a gas chromatograph, to a complex multi-piston pump supplied by four or five solvent reservoirs and fitted with both flow programming and gradient elution facilities, each with its own controlling micro-processor.

For a gas chromatograph the gas supplies will vary depending on the type of detector employed and column that is used. If gases are being analyzed then a katharometer detector will probably be appropriate and thus helium will be used as the carrier gas to provide the best sensitivity. If a flame ionization detector FID is to be used then oxygen or air and hydrogen will be needed for the detector and helium or nitrogen must be available as the mobile phase.

If an argon ionization detector is to be used then an argon supply will be necessary. Flow controllers are usually supplied to each gas supply, often under microprocessor control and This eBook is protected by Copyright law and International Treaties. Such a system can provide flow programming facilities if so desired. The mobile phase supply for a liquid chromatograph usually has a capacity for at least four solvents which are normally housed in a flame proof environment and solvent vapors are removed by a suitable vapor scavenging device.

The solvents can be selected by a microprocessor or manually by suitable valves and these pass either directly to a dual piston pump for isocratic development or to a solvent programmer and thence to a pump for gadient elution. Care must be taken to minimize the volume existing between the solvent programmer exit and the sample valve otherwise the gradient will be distorted by the logarithmic decay function of the interstitial volume. The Sampling System Gas samples are generally placed on a GC column using an external loop sampling valve but liquid samples are usually injected onto the column by a syringe via a heated injector.

Sample are placed on an LC column directly with either an internal or external loop sample valve the valve being connected directly to the column. The external loop sample system, employing six ports, is depicted in figure The external loop sample valve has three slots cut in the rotor so that any adjacent pair of ports can be connected.

In the loading position, shown on the left, the mobile phase supply is connected by the rotor slot between port 4 and port 5 directly through to the column. In this position, the sample loop is connected across ports 3 and 6. Sample passes either from a syringe or other sample device into port 1 through the rotor slot to the sample loop at port 6 and the third slot in the rotor connects the exit of the sample loop to waste at port 2.

The sampling position is shown by the diagram on the right. On rotating the valve, the sample loop is interposed between the column and the mobile phase supply by connecting port 3 and 4 and ports 5 and 6 This eBook is protected by Copyright law and International Treaties. In the sampling position, the third rotor slot connects the syringe port to the waste port. Figure 25 The External Loop Valve After sampling, the rotor can be returned to the loading position, the system washed with solvent and the sample loop loaded in readiness for the next injection.

For analytical applications, the sample loop can have a volume ranging from 1 to 20 ml, but for preparative work, loops with sample volumes of 1 ml or more can be placed on a preparative column. Modern liquid chromatographs that are used for routine analysis usually include an automatic sampling device. This involves the use of some type of a transport mechanism that may take the form of a carousel or some form of belt conveyor system.

The transporter carries a series of vials that alternately contain sample and washing solvent. The sampling can involve a complex sequence of operations that are controlled by a microprocessor. The syringe plunger is operated pneumatically and the syringe is first washed with solvent, then rinsed with the sample, reloaded with the sample and the contents discharged into the column.

In routine analytical laboratories, which often have very sophisticated LC assemblies, there may also be a sample-preparation robot which will automatically carry out such procedures as extraction, concentration, derivatization, etc. The robot is usually programmable, so that a variety of separation procedures can be carried in a sequence that is unique for each sample.

In laboratories that have a high throughput of samples, an automatic sampling device is often essential for the economic operation of the laboratory. In contrast, LC column ovens cover a more limited range of temperatures viz. Temperature programming is an essential feature of all GC column ovens and is necessary to handle a sufficiently wide molecular and polarity range of samples. Linear programming is the most common although other functions of time are often available.

LC column ovens are rarely provided with temperature programming facilities as the technique appears to be far less effective compared with GC, gradient elution being a far more ffective alternative. The thermostatting medium used in GC ovens is almost exclusively 'forced air' as the heat capacity of the GC mobile phase i. Consequently, air has sufficient heat capacity to change the column temperature rapidly without significant cooling from the carrier gas.

Air ovens are also employed in LC column ovens but are far less effective as the mobile phase, a liquid, has a much higher heat capacity and thus a stronger cooling effect. This problem is partly alleviated by using mobile phase preheaters but these introduce a significant volume between the solvent supply system and the column which will distort the profile of any solvent gradient that is employed.

Nevertheless, a liquid thermostatting medium introduces difficulties when changing columns and with column detector connections and is thus, not commonly used. The temperature program can be controlled by a microprocessor incorporated in the programmer or can be controlled from a central computer that governs the operation of the whole instrument. The GC column can be a packed or open tubular and thus the oven must be capable of taking both. The open tubular column is by far the This eBook is protected by Copyright law and International Treaties.

Open tubular columns will always provide the highest efficiencies but, if correct operating procedures are adopted, in general, analyses carried out on packed columns, are likely to provide greater accuracy and better precision and repeatability. Packed GC columns are usually made of stainless steel or glass and open tubular column almost exclusively fused quartz.

Almost all LC columns are packed, although they can vary widely in length and diameter depending of the nature of the sample and the resolution required. They are usually manufactured of stainless steel or titanium reputed to provide greater stability for labile materials of biological origin and the connection to the sample valve and detector should be as short as possible and have a very small diameter to reduce extra column dispersion.

Detector and Detector Electronics There is a wide range of detectors available for both GC and LC each having their own particular areas of application. In general the more catholic the response, the less sensitive the detector and the most sensitive detectors are those that have a specific response. The performance of all detectors should be properly specified so that the user can determine which is most suitable for a specific application.

Such specifications are also essential to compare the performance of different detectors supplied by alternative instrument manufactures. Detector specifications should be presented in a standard form and in standard units, so that detectors can be compared that function on widely different principles.

The more important detector specifications are summarized in table 2. The Detector Output Most practical detectors must have a linear output, e. However, the output from some detector sensors may not be linearly related to the solute concentration and appropriate signal modifying circuits must be introduced into the detector electronics to provide a linear output e. It is best defined a function of the detector response and the noise level The detector response Rc can be defined as the voltage output for unit change in solute concentration or as the voltage output that would result from unit change in the physical property that the detector measures, e.

Detector noise is the term given to any perturbation on the detector output that is not related to an eluted solute. It is a fundamental property of the detecting system and determines the ultimate sensitivity or minimum detectable concentration that can be achieved. Detector noise has been arbitrarily divided into three types, 'short term noise', 'long term noise' and 'drift' all three of which are depicted in figure Short term detector noise can be easily removed by appropriate noise filters without significantly affecting the profiles of the peaks.

Its source is usually electronic, originating from either the detector sensor system or the amplifier. Long term noise consists of baseline perturbations that have a frequency that is similar to that of the eluted peak. This noise is the most significant as it is indiscernible from very small peaks in the chromatogram. Long term noise cannot be removed by electronic filtering without affecting the profiles of the eluted peaks. Long term noise usually arises from temperature, pressure or flow rate changes in the sensing cell.

Drift are baseline perturbations that have a frequency that is large to that of an eluted peak. Drift is almost always due to either changes in ambient temperature, changes in mobile flow rate, or column bleed in GC; in LC drift can be due to pressure changes, flow rate changes or variations in solvent composition. A combination of all three sources of noise is shown by the trace at the bottom of figure The detector noise is defined as the maximum amplitude of the combined short— and long-term noise measured over a period of 15 minutes.

The detector must be connected to a column and mobile phase passed through it during measurement. The detector noise ND is obtained by constructing parallel lines embracing the maximum excursions of the recorder trace over the defined time period as shown in figure Figure 27 Measurement of Detector Noise Detector sensitivity or minimum detectable concentration MDC is defined as the minimum concentration of solute passing through the detector that can be unambiguously discriminated from noise, conventionally taken when the signal to noise ratio is two and this criteria has been adopted for defining detector sensitivity.

Thus for a concentration sensitive detector, the detector sensitivity XD is given by This eBook is protected by Copyright law and International Treaties. The dynamic range DR extends from the minimum detectable concentration i. The dynamic range is not usually pertinent to general analytical work but is important in preparative chromatography.

The linear dynamic range or detector linearity is as important as sensitivity for any detector that is to be used for quantitative analysis. It is defined as the concentration range over which the detector response is linearly related to the concentration of solute passing through it. Because of the imperfections in mechanical and electrical devices practical detectors can only approach this ideal response.

A measure of linearity that is specified in numerical terms so that comparisons can be made between detectors can be obtained as follows. In addition if r is not unity but is known then appropriate corrections can be made to the response and improved accuracy can be achieved. The three remaining important specifications are pressure sensitivity which is particularly crucial if multidimensional chromatography is This eBook is protected by Copyright law and International Treaties.

In GC any connecting tube between the column and the detector must also be heated to a temperature above that of the oven to prevent condensation. Data Acquisition and Processing System A diagram of a chromatographic data acquisition and processing system is shown in figure Most systems have a means for real time monitoring the detector output either by using an ancillary recorder or by computer software, the chromatogram being drawn by the computer on the printer.

The data may be partially processed 'on the fly' or processed at the completion of the analysis. Thin Layer Chromatography Apparatus Thin layer chromatography appears to have been first developed and utilized by Schraiber in Schraiber working with Izmailov at the Khar'kov Chemistry and Pharmacy Research Institute employed the techniques for the analysis of pharmaceuticals. In her own words, " It occurred to us that a thin layer of the sorbent could be used in lieu of a strip of paper; also we felt that the flat bed could be considered as a cut-out of the adsorbent column.

We believed that in carrying out the separation process in such a layer, the process would be accelerated significantly. In our work, we deposited a drop of the solution being investigated on the flat adsorbent layer and observed the separation into concentric circular zones which could become visible because of their fluorescence in the light of a UV lamp. Unfortunately, Schraiber's work does not seem to have been heeded and the technique appears to have been rediscovered by Kirchner in Although thin layer chromatography TLC phase systems are basically the same as those used in LC, the equipment required is far simpler and very much less expensive.

Furthermore, as many separations can be carried out simultaneously by multiple spotting, analysis times are much shorter and there can be as many as 60 samples per plate which, in effect means that each analysis will only take about 5 seconds to complete. Resolution obtained from TLC is far less than that obtainable by LC but, as a result of the cost advantage, the technique is very widely used.

In fact, despite the many advances that have taken place in LC techniques over the past years, the use of TLC for routine analyses continues to grow. However, samples containing multiple components cannot be separated by TLC due to restricted plate capacity. Thin Layer Chromatography Chambers A diagram of two simple thin layer chromatography development chambers is shown in figure In the simplest case, the developing chamber can consist of a round or square glass jar fitted with a glass cap.

Sufficient solvent mixture is placed in the chamber to raise the level about a centimeter from the base. The plate is then placed in the chamber with the end where the samples have been placed dipping into the solvent and the cap replaced. The sample spots must not be at, or below, the surface of the solvent mixture or they will be washed from This eBook is protected by Copyright law and International Treaties. To improve the air space saturation with solvent, the walls, or part of the walls, of the chamber are sometimes covered with filter paper to act as a wick that soaks up the solvent and provides a greater surface area for evaporation.

The use of a paper wick is depicted on the right- hand side of figure The saturated solvent vapor in the chamber not only prevents solvent evaporating from the plate surface but partly controls the retention mechanism by surface deactivation. The solvents are selectively adsorbed on the surface of the stationary phase causing the solutes to interact, not with the native silica surface, but with the silica surface covered with the most strongly interacting solvent.

It should be emphasized, however, that the equilibrium between the solvent vapor and the plate will not be the same as the equilibrium between the solvent and the plate. One form of apparatus that can be used for the pre- equilibrium of a thin layer plate is shown in figure The apparatus is very similar to that used for normal development but a separate reservoir contains the developing solvent.

The plate is placed in the enclosure and allowed to come into equilibrium with the solvent vapor for a few minutes. The plate is then lifted and placed so that the end now dips into the developing solvent and the separation processed in the usual way. There are two main effects resulting from the pre- saturation of a TLC plate. These affects are depicted in figure Firstly the velocity of the solvent front is increased relative to that of the unsaturated plate. Secondly, as a significant amount of the solvent at the This eBook is protected by Copyright law and International Treaties.

In most cases, the effect of pre-saturation on the actual separation is small but can be important for special mixtures where subtle changes in retention can make the necessary difference. Continuous Plate Development The normal development of a thin layer plate is limited by its physical dimensions but a continuous development procedure has been used employing special equipment.

An apparatus used for the continuous development of a thin layer plate is shown in figure Plate Saturated by Solvent Unsaturated Plate Vapor Solvent Front Figure 31 Effect of Plate Saturation on Plate Development A wick transfers the solvent from the reservoir to the stationary phase coating which is sandwiched between the two glass plates.

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The solvent passes along the plate by surface tension forces in the usual way until it reaches the end of the plate. A small area at the end of the plate is exposed and heated electrically to evaporate the solvent as it arrives. In this way development can be continued and the system now resembles an LC column of lamina shape. The value of this technique is a little questionable as its intent is to simulate an LC column, in which case it would be preferable to use an LC column in the first place.

Sample volumes of 1 to 2 ml are common but when using the so called high performance TLC plates HPTLC plates coated with particles mm in diameter a maximum loading of about nl can only be tolerated. Ideally the sample area should be circular and not greater than 1 mm in diameter, on any HPTLC plate. As this requires the use of samples a few nanoliters in volume, most will need to be concentrated.

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Manually, the sample can be applied with a micro-pipette and the solvent is then allowed to evaporate, This procedure is repeated until sufficient sample is placed on the plate. Micro-syringes can also be employed to place a sample on to the plate. With care, and a little local heating, sample concentration can also be accomplished. The contents of the syringe are slowly but continuously discharged onto the plate and at the same time the solvent is progressively evaporated.

This procedure can be automated, using computer controlled syringes and, in this way, samples can be focused onto the plate and constrained to a very small area. There are various devices that are commercially available that will apply samples to a TLC plate either individually or as multiple groups. An example of a device for automatically concentrating a sample and then placing it on a plate is shown in figure A polymer film is placed over the plate surface and a vacuum applied to suck the film into the indentation.

A small quantity of sample solution is placed in the indentation and the solvent evaporated. This procedure is repeated until sufficient sample is present on the film for a satisfactory TLC separation. It is important that the sample is not evaporated to dryness as the transfer of solid materials to the thin layer plate can be very inefficient. When sufficient sample has been accumulated and the droplet is still liquid, the thin layer plate is placed over the film and a positive pressure now applied to the aperture at the center of the indentation.

The film extends to the surface of the TLC plate and the sample is transferred by contact, as a result of surface This eBook is protected by Copyright law and International Treaties. The plate is then conveyed to the solvent chamber and the separation developed in the usual way. If larger samples are required for semi preparative work, sample bands can be applied to the plate as opposed to sample spots.

Sample bands can be applied either by using TLC plates with concentrating zones or alternatively by using band applicators. A diagram of a TLC plate that includes a concentrating zone is shown in figure Concentrating Normal Retentive Zone Coating Glass Plate Figure 34 TLC Plate with Sample Concentrating Zone The concentrating zone is about 2 cm wide and consists of a coating made from relatively large particles of silica with a relatively low surface area and consequently low retentive capacity. The concentrating band is coated closely adjacent to the normal retentive coating which, consists of the usual particles mm in diameter but with a much higher surface area and, thus, much greater retentive capacity.

Several samples of a few microliters or more can be placed sequentially on to the concentrating zone and the solvent allowed to evaporate until there is an adequate quantity of sample on the plate. The sample is now spread along the concentration zone in a fairly broad band. When the plate is developed the solutes move rapidly through the concentration zone due to its low retentive character to the interface between the layers.

At the interface the solutes are slowed down by the more retentive layer and are thus focused as a sharp band at the front of the plate. As development proceeds, the solutes separate in the normal high retentive layer in the usual manner. This procedure has other advantages. If the sample is contaminated with salts or biological polymers, these will be trapped in the concentration zone and, thus, will not pass onto the separation region of the plate and effect the quality of the separation. The sample is atomized in a stream of air or nitrogen depending on the nature of the sample and its tendency to oxidation.

A diagram of the type of atomizer used in band application is shown in figure The range and the number of sweeps, are usually under computer control and the speed of movement is adjusted such that the solvent is able to evaporate from a given area of sample before it receives the subsequent dose.

After dosing, the plate is developed in the normal way. Some exceedingly novel and clever devices have been developed for TLC. These devices indeed improve the performance of the TLC analysis but are also expensive and in many cases tend to make the TLC system more like a liquid chromatograph. The great advantage of TLC is its low cost and its relatively high separating capability. If the required performance required is at the limit or beyond the capability of the technique, there is no point in trying to stretch it. The rational solution for the chemist or analyst would be to change to an alternative procedure such as liquid chromatography or to some other technique if more appropriate.

Chromatography Applications Gas chromatography has an entirely different field of applications to that of liquid chromatography. In general, gas chromatography is used for This eBook is protected by Copyright law and International Treaties. There are certain compounds, however, that can be separated with either techniques, and more importantly, many involatile substances such as amino acids, steroids and high molecular eight fatty acids can be derivatized to form volatile substances that can be separated by GC.

The derivatization must be highly reproducible and usually proceed to completion in order to maintain adequate accuracy. The capillary columns in GC can have much higher efficiencies than their LC counterpart and thus GC can more easily handle multicomponent mixtures such as essential oils.

On the other hand, only LC can separate the peptides, polypeptides, proteins and other large biopolymers that are important in biotechnology. Gas Chromatography Applications The most common hydrocarbon analysis carried out by GC is probably that of gasoline. The analysis of gasoline is typical of the type of sample for which GC is the ideal technique.

It is this type of multicomponent mixtures containing very similar compounds that need the high efficiencies available from GC for a successful analysis. The separation of a sample of gasoline carried out on a long open tubular column is shown in figure It is clear that the column had a very high efficiency which was claimed to be in excess of , theoretical plates. The column was m long and only mm I. Petrocol DH is specially designed stationary phase for the separation of hydrocarbons and consists of bonded dimethylsiloxane, a very dispersive type of stationary phase, retaining the solutes approximately in the order of their increasing boiling points.

Nonpolar or dispersive stationary phases are employed for the separation of hydrocarbons e. OV, which is also a polyalkyl- siloxane, is widely used in packed columns. Thus, due to the pressure correction the actual effective linear velocity would be much less than that see Book 7 Peak This eBook is protected by Copyright law and International Treaties. Helium was used as the carrier gas which was necessary to realize the high efficiencies with reasonable analysis times.

An excellent separation is obtained giving clearly separated peaks for the marker compounds which are of importance in fuel evaluation. Nevertheless, due to the complexity of the sample, exceedingly high efficiencies were necessary and so, the analysis time was about min. Long analysis times are directly related to the use of long columns The complete analysis was carried out using only 0.

High Temperature GC Stationary Phases The major limitation of gas chromatography is the stability of the stationary phase at high temperatures. The higher the polarity and the higher the molecular weight of the solutes, the higher the temperature necessary to provide adequate solute partial vapor pressure to allow a gas chromatographic separation to be realized. Similarly, the stability of the solutes at high temperature can also become a problem. The solute must be thermally stable so that the partial pressure is sufficiently high to allow elution in a reasonable time.

Nothing can be done with respect to the solute stability as this is determined by the nature of the sample. There are certain materials hat can be used as stationary phases at remarkably high temperatures. These materials are based on the polymerization of carborane substituted siloxanes.

The introduction of a phenyl group makes it slightly less thermally stable. An example of the use of Dexsil to separate some very high boiling waxes is shown in figure Stationary phases based on the carborane structure, can extend the temperature range of gas chromatography very significantly, However, having thermally stable stationary phases solves only half the problem, the solutes themselves must be equally stable.

Hydrocarbon Analysis Due to the perceived toxicity and carcinogenic character of the aromatic hydrocarbons, the presence of these materials is carefully monitored in all areas where they might enter the human food chain. The analysis of water for aromatic hydrocarbons, particularly surface water in those areas where contamination might take place, is a common assay made by the public analyst. It is essential to be able to measure concentrations in the ppb levels, and thus GC method employing a high sensitive detector is essential.

Nevertheless, even if a high sensitivity detector is employed, some sample concentration will be necessary to measure contaminants at such low levels. A diagram of the purge and trap system is shown in figure The purge was carried out at room temperature for 11 min.

The short length of packing with the higher surface area thus high adsorptive capacity was to used to ensure that none of the sample material was eluted through the adsorption bed and lost. A chromatogram of the separation that was obtained is shown in figure The open tubular column used was 60 m long, 0. This stationary phase is strongly polar and corresponds to a bonded polyethylene glycol. The strong fields from the hydroxyl groups polarize the aromatic nuclei of the aromatic hydrocarbons and thus retention was effected largely by polar interactions between the permanent and induced dipoles of the stationary phase and solute molecules respectively.

More than adequate separation is achieved and even the m and p xylenes are well resolved. This might indicate that a significantly shorter analysis was possible. The aromatic hydrocarbons were present This eBook is protected by Copyright law and International Treaties.

Figure 38 The Separation of 10 ppb Quantities of Aromatic Hydrocarbons from Water Essential Oils Without the use of gas chromatography the analysis of essential oils would be extremely difficult. Prior to the technique being developed, only the major components of the oils could be separated, achieved by distillation with high efficiency columns. The next free day is 30 th March Full details are available at: Watch out for the time zone on the download day!

LC Calculator from Agilent. I think this tool is great for checking out if a method is viable in terms of the back pressure when I want to change method parameters such as particle size, column length and internal diameter, and flow rate. This allows optimisation of the method without having to check these experimentally. Click here to access the tool, you can select the mobile app or web version. Agilent also provide an article describing the use of the calculator which may be helpful click here to view.

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