Theoretical Plates The concept of the theoretical plate arose from distillation column theory and was borrowed

by A J. P. Martin to develop the first elution curve theory for the chromatography column Neither in a distillation column, nor in a chromatographic column, does equilibrium e ist in any part of the distribution system. This non!equilibrium condition arises from the dynamic nature of the distribution systems as the mobile phase is continually flowing past the stationary phase. The solute does not spend sufficient time at any point in the column for equilibrium to be achieved. To avoid this difficulty in the theoretical treatment of retention, the column is considered to be divided into a number of theoretical plates or cells and each is allotted a finite height "or length# that will allow the solute sufficient theoretical dwell!time for equilibrium to ta$e place. %t is clear that the faster the equilibrium, the smaller the theoretical plate, and the more theoretical plates there will be in the column. Thus, the more efficient column has more theoretical plates. The plate theory was critici&ed when first developed, as it was claimed that equilibrium was not achieved in the column and, thus, the theoretical approach was invalid. 'owever, the theoretical plate concept was introduced specifically to attend to this non! equilibrium problem The theory gave rise to the elution equation, the equation for resolution, and the equation used for calculating the column efficiency, all of which, have been e haustively validated e perimentally over many years. Gas Chromatography (GC); Gas-Liquid Chromatography (GLC);

Gas-Solid Chromatography (GSC); Vapor-Phase Chromatography (VPC) 'ere the mobile phase is a gas, often nitrogen, but sometimes helium, hydrogen or occasionally another gas. %t is called the (carrier gas(. )as!solid chromatography is relatively rare, but it is used to separate atmospheric gases. *ommon solids are charcoal, a synthetic &eolite called (molecular sieve(, or a combination of the two. +olids typically adsorb so strongly that some adsorbed components do not pass through the column "in a reasonable time# and are

removed by reversing the flow of mobile phase through the column "(bac$flushing(#, usually at a high temperature. "The term (molecular sieve( is often used generically, but was originally a tradename for certain specific types with specific pore si&es.# )as!liquid chromatography begins with a more or less inert (support( with a high surface area. That is mi ed with a solution of the liquid phase in a volatile solvent. Then the solvent is evaporated in a rotary evaporator, leaving the support with a (coating( of the liquid phase. The coated support, now called a (pac$ing(, is (pac$ed( into a column, such as a , mm inside diameter stainless steel tube about -.. mm long. No firm pac$ing is done, but the column is often vibrated to be sure that the particles settle without leaving voids. The column is usually coiled, before or after pac$ing. The ends of the column are plugged, often with glass wool, to hold the pac$ing but allow gas flow. The column ends have fittings "often (+wagelo$( brand# so that they can be connected to matching fittings in the (oven( of the gas chromatograph. The oven has three functions/ 0. -. 2. %t $eeps the column temperature constant. %t allows operation at elevated temperature "faster1 perhaps necessary to %t allows (temperature programming(, a controlled increase in column

vapori&e the sample#. temperature during an analysis to ma$e the slow!moving components move faster, reducing the time needed for the analysis, and reducing the diffusion which ma$es pea$s broader. 3. 4ecause the mobile phase is gas, solubility in the mobile phase is not really a factor, but volatility "vapor pressure# of the components being analy&ed is a close equivalent. +olubility in the stationary "liquid# phase is also important, and some stationary phases interact chemically with certain types of sample components. The components to be separated must have a reasonable vapor pressure at the column temperature, or they will not move at all. Much wor$ has been done on (derivati&ing( components to increase volatility. Much of the early wor$ in gas chromatography involved separating hydrocarbon mi tures such as gasoline on columns in which the liquid phase was a silicone oil. The

process acted somewhat li$e distillation with the 5pot5 temperature gradually increasing, and some of the theoretical treatments were derived from distillation theory. An industrial distillation is done in a vertical column with a series of perforated plates inside. 6hen it is assumed that there is equilibrium between vapor and liquid at each plate, the plates are (theoretical plates(. %ncreasing the number of plates gives better separation. Application of the theory to gas chromatography introduced the term (height of an equivalent theoretical plate(, abbreviated '7TP. This was "roughly# the distance along the column that gave the same separation as a "theoretical# plate in a distillation column. )reat advances have been made in reducing that distance, increasing the number of (theoretical plates( in a length of column, and improving separation, or (resolution(. +eparation could also be improved by increasing the column length, but that requires longer analysis times, during which the components can diffuse, perhaps actually reducing resolution and reducing the ability to detect them. 8ne of the ma9or theoretical advances was the (van :eemter equation( which related '7TP to carrier gas flow rate. The number of (plates( is still important for chromatograph columns. Molecules equilibrate more rapidly between liquid and gas phases if both phases are very thin. That led to the (capillary( column, which is typically a fused silica capillary of about ..-; ! ..; mm inside diameter "%:#, with a thin coating "about ..- mm# of liquid phase on the wall, and from 0. meters to 0.. meters long. This is a (wall coated open tubular( "6*8T# column. The coating on the wall can also be a combination of support and coating, giving a (support coated open tubular( "+*8T# column. The sample must be very small, and the detector must be very sensitive, but the number of theoretical plates is immense "e. g., 0..,...# and very comple mi tures can be separated. The capillary usually has a coating of polymer or aluminum on the outside for protection. Many real samples have components with a very wide range of volatility. %f the column temperature is too high, the most volatile components will move practically as fast as the carrier gas, and will not be separated. 4ut with a lower column temperature, the less volatile components will hardly move at all. That situation can be handled by (temperature programming(/ increasing the column temperature at a controlled rate.

Another problem with high temperature is that the (liquid( may vapori&e into the carrier gas, resulting in column (bleed(. +ignificant bleeding interferes with analysis, and can destroy the column by removing the liquid phase. The liquid "coating# is now very often a polymer that resists bleeding and decomposition, and which may improve selectivity for certain types of molecules. More recently, it has been possible to bond molecules of the liquid phase chemically "covalently# to silica or &irconia particles, or to the inner surface of a capillary column, to ma$e (bonded( phases. <ery few analysts pac$ their own columns or ma$e their own T=* plates now. They buy prepared columns or plates from suppliers. 6ide varieties of both supports and coatings are available. >or instance, +upelco is a ma9or ?+ supplier of ready!to!use columns and materials. "+ee http/www.sigmaaldrich.com, and select +upelco. A catalog may be even more helpful.# +o far, % have ignored two ma9or operations in gas chromatography/ getting the sample onto the column, and recogni&ing the components as they elute with the carrier gas. +amples are applied to the column with an (in9ector(. The early in9ectors were 9ust that/ they provided access to the end of the column, which was sealed by a rubber (septum(. A liquid sample was drawn into a small syringe calibrated in microliters "('amilton( is a ma9or syringe tradename#. The syringe needle was pushed through the septum, and a quic$ slap of the syringe plunger in9ected a narrow pulse of sample onto the column. +imilar but larger syringes could be used for gas samples. The rubber septa have been replaced by polymers with more heat resistance, and in9ectors now have short lengths of tubing "(sample loops(# into which the sample is placed before the in9ector switches the loop into the gas flow. Automated in9ection systems can be loaded with numerous samples to be in9ected automatically "overnight, for instance#. As the carrier gas leaves the column, it flows immediately into a (detector(. The output of the detector is recorded, giving a (pea$( on the recorder chart as each component is detected. 7ach pea$ appears at a characteristic (retention time(. The degree of separation of the pea$s is the (resolution(. 6hen separation is so complete that the recorder pen returns to its &ero position between pea$s, one has (baseline

resolution(. The pea$ height was used first as an easy measure of the quantity of that component in the sample. Pea$ area proved to be a better measure, so the pea$s were (integrated(. %ntegration was originally done by cutting a piece of recorder paper below the pea$ and weighing it1 by counting the squares on the recorder chart below the pea$1 or by measuring its area with a planimeter. Mechanical "(ball and disc(# integrators followed, and electronic integrators appeared quic$ly. 4ecause the amounts of the sample components vary greatly, it may be necessary to (attenuate( strong signals so that the recorder does not overshoot the chart paper. :etectors do not have the same sensitivity for all molecules, so quantitative measurements require use of standards. +ometimes a fi ed amount of an (internal standard( is applied to all samples and standards. The principal detectors are/ Thermal conductivity "T*#/ A wire in the gas flow is heated by a constant electrical current. The electrical resistance of a hot wire depends on its temperature, which is "appro imately# constant in a steady flow of carrier gas. 6hen the carrier gas contains molecules larger than those of the carrier gas, less heat is removed from the wire1 its resistance increases1 and the voltage across the wire increases. +ensitive to all components, but not very sensitive to any. >lame ioni&ation detector ">%:#/ The detector contains a small hydrogen!o ygen "or hydrogen!air# flame. +ome substances, when they burn in the flame, produce ions which carry current, and the current is measured. <ery sensitive, particularly for molecules having *!' bonds. Not at all sensitive to some other molecules "such as **l3#. 7lectron capture detector "7*:#/ A radioactive source produces ions, and an ion current is measured between positive and negative electrodes. +ome components "especially those containing *!*l bonds# capture ions, reducing the current. <ery sensitive1 complements >%:. The preferred carrier gas is argon. Mass spectrometer ")*!M+#/ The gas leaving the column "usually a capillary# first goes through a separator which passes the sample molecules to the mass spectrometer while removing most of the carrier gas molecules. "@emember that mass spectrometry is done in vacuum.# %n the time!of flight "T8># mass spectrometer, the

sample is bro$en into ioni&ed fragments, and the ions are accelerated into a (drift tube( by an electrical pulse. The light ions are accelerated more than the heavier ones, and arrive sooner at the other end of the tube, where the ion current is measured versus time to give a mass spectrum. The heaviest ion is quite often the (molecular ion(, the whole molecule plus or minus a hydrogen ion. The whole process is repeated at about A second intervals. A computer displays and records the total ion current vs. time, the current of a specific ion "a specific massBcharge ratio# versus time, or the mass spectrum of a component. The computer also has a (library( of mass spectra. %t compares the mass spectrum of each pea$ with the spectra in its library to identify the individual components. This does assume that the component is in the library, but such libraries include ;.,... or more components. An (ion!trap( detector is one type of mass spectrometric detector. +ome of the less broadly useful detectors are the thermionic emission detector "T7:#, sensitive to nitrogen and phosphorus compounds "also called an NP:#1 flame photometric detector ">P:#, sensitive to sulfur and phosphorus compounds1 and the photoioni&ation detector "P%:#. 7 cept for the M+ detector, chromatography only separates the sample components, but does not identify them. %n routine analyses, the analyst $nows what components are e pected, and can separately run chromatograms of individual $nown standard compounds or mi tures, so that the substance which elutes at a certain (retention time( is assumed to be a certain compound. A report or procedure will probably state the instrument manufacturer and model, the carrier gas and its flow rate1 perhaps the inlet pressure1 the column dimensions1 the support and coating "together, the pac$ing#, the in9ection method and in9ection volume, the detector "perhaps with applied voltages#, any integrator used, and attenuator settings. @etention times may appear in the procedure or be shown with a reproduction of the chromatogram. Introduction >ew methods of chemical analysis are truly specific to a particular analyte. %t is often found that the analyte of interest must be separated from the myriad of individual

compounds that may be present in a sample. As well as providing the analytical scientist with methods of separation, chromatographic techniques can also provide methods of analysis. *hromatography involves a sample "or sample e tract# being dissolved in a mobile phase "which may be a gas, a liquid or a supercritical fluid#. The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will ta$e longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase. Techniques such as '.P.=.*. "'igh Performance =iquid *hromatography# and ).*. ")as *hromatography# use columns ! narrow tubes pac$ed with stationary phase, through which the mobile phase is forced. The sample is transported through the column by continuous addition of mobile phase. This process is called elution. The average rate at which an analyte moves through the column is determined by the time it spends in the mobile phase. istri!ution o" analytes !et#een phases The distribution of analytes between phases can often be described quite simply. An analyte is in equilibrium between the two phases1 Amobile Astationary The equilibrium constant, K, is termed the partition coefficient1 defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase. The time between sample in9ection and an analyte pea$ reaching a detector at the end of the column is termed the retention time "t@ #. 7ach analyte in a sample will have a different retention time. The time ta$en for the mobile phase to pass through the column is called tM.

A term called the retention factor, k', is often used to describe the migration rate of an analyte on a column. Cou may also find it called the capacity factor. The retention factor for analyte A is defined as1 k'A D t @ ! tM B tM t
@

and tM are easily obtained from a chromatogram. 6hen an analytes retention

factor is less than one, elution is so fast that accurate determination of the retention time is very difficult. 'igh retention factors "greater than -.# mean that elution ta$es a very long time. %deally, the retention factor for an analyte is between one and five. 6e define a quantity called the selectivity factor, a , which describes the separation of two species "A and 4# on the column1 a D k '4 B k 'A 6hen calculating the selectivity factor, species $ elutes "aster than species %& The selecti'ity "actor is al#ays greater than one& %and !roadening and column e""iciency To obtain optimal separations, sharp, symmetrical chromatographic pea$s must be obtained. This means that band broadening must be limited. %t is also beneficial to measure the efficiency of the column. The Theoretical Plate (odel o" Chromatography The plate model supposes that the chromatographic column is contains a large number of separate layers, called theoretical plates. +eparate equilibrations of the sample between the stationary and mobile phase occur in these (plates(. The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the ne t.

It is important to remem!er that the plates do not really e)ist 1 they are a figment of the imagination that helps us understand the processes at wor$ in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N "the more plates the better#, or by stating the plate height1 the Height Equivalent to a Theoretical Plate "the smaller the better#. %f the length of the column is , then the '7TP is '7TP D !N The number of theoretical plates that a real column possesses can be found by e amining a chromatographic pea$ after elution1

where "0B- is the pea$ width at half!height. As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mi ture. The *ate Theory o" Chromatography A more realistic description of the processes at wor$ inside a column ta$es account of the time ta$en for the solute to equilibrate between the stationary and mobile phase "unli$e the plate model, which assumes that equilibration is infinitely fast#. The resulting band shape of a chromatographic pea$ is therefore affected by the rate of elution. %t is also affected by the different paths available to solute molecules as they travel between particles of stationary phase. %f we consider the various mechanisms which contribute to band broadening, we arrive at the <an :eemter equation for plate height1 '7TP D A # $ ! u # % u where u is the average velocity of the mobile phase. A, $, and % are factors which contribute to band broadening.

+ddy

di""usion

The mobile phase moves through the column which is pac$ed with stationary phase. +olute molecules will ta$e different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths. B Longitudinal di""usion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. %f the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion. C *esistance to mass trans"er The analyte ta$es a certain amount of time to equilibrate between the stationary and mobile phase. %f the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes. Van eemter plots A plot of plate height vs. average linear velocity of mobile phase.

+uch plots are of considerable use in determining the optimum mobile phase flow rate. *esolution Although the selectivity factor, a, describes the separation of band centres, it does not ta$e into account pea$ widths. Another measure of how well species have

been separated is provided by measurement of the resolution. The resolution of two species, A and 4, is defined as

4aseline resolution is achieved when & D 0.; %t is useful to relate the resolution to the number of plates in the column, the selectivity factor and the retention factors of the two solutes1

To obtain high resolution, the three terms must be ma imised. An increase in N, the number of theoretical plates, by lengthening the column leads to an increase in retention time and increased band broadening ! which may not be desirable. %nstead, to increase the number of plates, the height equivalent to a theoretical plate can be reduced by reducing the si&e of the stationary phase particles. %t is often found that by controlling the capacity factor, k', separations can be greatly improved. This can be achieved by changing the temperature "in )as *hromatography# or the composition of the mobile phase "in =iquid *hromatography#. The selectivity factor, a, can also be manipulated to improve separations. 6hen a is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. %n these cases, k' is optimised first, and then a is increased by one of the following procedures/ 0. -. 2. 3. *hanging mobile phase composition *hanging column temperature *hanging composition of stationary phase ?sing special chemical effects "such as incorporating a species which

comple es with one of the solutes into the stationary phase# *e'ie# your learning

Cou should now be familiar with the terms used in chromatography, how species become separated from one another, and how various conditions can be manipulated to obtain well!resolved chromatograms with a minimum elution time.