http://www.crawfordscientific.com/newsletter-2009-12-Peak-Tailing-HPLC.

html
Peak Tailing in HPLC Tailing peaks are the most common chromatographic peak shape distortion. A peak is classified as tailing if its asymmetry is greater than 1.2, although peaks with As values as high as 1.5 are acceptable for many assays. The peak asymmetry factor (As) is determined using the following equation; As = B / A Where; B = peak width after the peak centre at 10% peak height and A = peak width at baseline before the peak centre

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention. In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase. However, polar interactions with any ionised residual silanol groups residing on the silica support surface are also common. Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups, producing tailing peaks. This is illustrated in the figure shown below at a mobile phase pH >3.0.

Please have a look at our HPLC column selection guide.

Analyte secondary interactions with ionised silanols on the silica surface give rise to peak tailing. These interactions need to be minimized if good peak shape is to be achieved. How to Avoid Peak Tailing Operate at a lower pH. As Silanol groups are acidic, secondary interactions can be minimised by performing the chromatographic separation at a lower pH, thereby ensuring the full protonation of such ionisable residual silanol groups;

Of course this may lead to a decrease in retention time is ionisable basic analytes are present in the sample - this may need to be mitigated by a reduction in the organic modifier content of the mobile phase. Standard silica should not be used under pH 3 as this may induce silica dissolution.Agilent ZORBAX Stable Bond (SB) columns are designed to operate at low pH (<3). The five component mix of basic drug compounds in the chromatograms below consist of Phenylpropanolamine, Ephedrine, Amphetamine, Methamphetamine and Phenteramine (Peaks 1-5) respectively. The first chromatographam was obtained at a mobile phase pH of 7.0, and under these

separation conditions Peak 4, Methamphetamine, shows an Asymmetry of 2.35. Lowering the mobile phase pH reduces basic analyte interaction with residual silanol groups, thereby eliminating excessive peak tailing. The second chromatographic trace was acquired with a mobile phase pH of 3.0, and the Asymmetry of Methamphetamine under these separation conditions was reduced to 1.33.

Use a highly deactivated column. Alternatively an end capped column can be utilised. End-capping is a process whereby residual silanol groups are treated to convert them to substantially less polar surface functional groups. This has the effect of reducing the amount of secondary interaction they can potentially have with polar analyte molecules.

Chemical reagents typically used in the end-capping process include;

Trimethylchlorosilane (TMCS) Hexamethyldisilazane (HMDS)

End capping reduces the peak tailing seen for polar analyte compounds, by effectively blocking their interaction with potentially ionisable residual silanol groups. However, end-capping only reduces by approximately a further 50% the number of unreacted residual silanol groups. Due to steric hindrance factors such reactions are rarely very efficient in absolute terms, and consequently a fully end-capped column is not quite as it would seem! Agilent ZORBAX Eclipse Plus columns are highly endcapped and deactivated and are an excellent choice for method development or general laboratory use as they give rise to symmetrical peak shapes even with acidic, basic and highly polar analytes.

Consider the possibility of Mass Overload. If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded. Assess by simply diluting the sample x10 and re-assess the peak shapes in the resulting chromatogram. If necessary use a higher capacity stationary phase i.e. increased % carbon or pore size, use a column with an increased column diameter, or decrease the absolute sample amount or volume injected. Consider the possibility of Column Bed Deformation Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit. Substituting the column will quickly confirm the problem. If a void is suspected reverse the column, disconnect from the detector and wash in 100% strong solvent (at least 10 column volumes) which will remove any blocking contamination from the column inlet filter directly to waste. Check the manufacturers instructions as to whether the column s hould remain in the reversed flow orientation. Blockage of column frits can be avoided though regular replacement of solvents filers, use of in-line filters, and guard columns. More information on these parts can be found using the links below.

Solvent filters In-line filters Guard column

Work at High pH when analyzing basic compounds.

From a practical point of view it is generally usually only possible to use pH to suppress acid ionisation, as base ionisation requires pH values typically greater than 8, at which pH silica dissolution can occur. However, Agilent ZORBAX Extend HPLC Columns are able to operate extended pH, the silica surface being protected from dissolution by the use of bi- and tridentate ligands. These ligands are bidentate and are bridged using proprietary chemistry, prior to their application to the stationary phase. Their bridged structure affords steric protection against hydrolysis of the silica surface.

Bidentate Ligands of Agilent ZORBAX Extend

HPLC Columns

Work at High pH when analyzing basic compounds The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored. Confirm the presence of an interferent by changing the detection wavelength, or improve the resolution of the separation by using a column offering greater efficiency i.e. a longer column or a column packed with smaller sized particles. Agilent ZORBAX RRHT and RRHD columnsare designed to afford very high efficiency separations which may serve to separate the co-eluting peaks.

http://www.chromatographyonline.com/lcgc/article/articleDetail.jsp?id=572447&pageID=4
Fronting Peaks

Whereas tailing peaks are fairly ubiquitous in reversed-phase separations, fronting peaks are much more rare. Some workers will never see an instance of peak fronting. Figure 4 shows an instance of peak fronting that was encountered in my laboratory several years ago.1 This method used a highpH (pH 9.0) and highly aqueous (95% buffer) mobile phase with a silica-based C18 column. Column failure occurred with disturbing regularity after approximately 500 injections, with dramatic peak fronting. The source of peak tailing is described easily by secondary chemical interactions between the sample (often a basic compound) and the silica-based column-packing material. Peak fronting, however, is best described by a physical problem with the column. This can be explained by a column void, distorting the sample flow path as shown in Figure 3(c). In this instance, a small portion of the sample is able to move quickly down the column through a void or channel in the column, getting a head start on the bulk of the injected sample. This results in a fronting peak, as in Figure 4(b); when more than one peak is present, all the peaks will show fronting, as in Figure 3(c). In the present example, the method had two potential problem areas that converged to create the problem. First, the mobile phase pH of 9.0 was necessary to achieve the desired separation. The normal silica-based columns that are used for most LC applications are stable in the range of 2 < pH < 8. Below pH 2, the bonded phase is hydrolysed and lost; above pH 8, the silica particles tend to dissolve. Some columns, such as those using "hybrid" silica-based particles, are designed for operation at pH > 8. Another alternative is to use one of the polymer-particle columns that are not susceptible to base attack.

A second problem associated with the method of Figure 4 was the need to operate in a highly aqueous mobile phase (95% buffer) to get sufficient retention for the analyte of interest. One approach to improving operation in highly aqueous mobile phases is to use one of the embeddedpolar-phase or "AQ"-type columns that many manufacturers sell for use with 100% aqueous mobile phases. Another option for use of highly aqueous mobile phases that at least one manufacturer uses is to reduce the surface coverage of the C18 phase so that more of the polar silica surface is exposed. This was the type of column used for the separation of Figure 4. Unfortunately, two influences converged to make this column and mobile phase combination a problem high-pH mobile phase plus a low-coverage column. The column collapsed after approximately 500 injections, as indicated by a badly fronting peak [Figure 4(b)]. Fortunately, the peak area was not affected for our method, so failure during a sample batch allowed for accurate quantification of the samples, even with fronting peaks. The column, of course, was replaced before another sample batch was run. One can only speculate on how this deadly combination was missed during development and validation of the method, but by the time it was realized, correction of the problem would have required revalidation of the method. We found that the data were sufficiently accurate for the application and the method has performed well for more than 10000 samples. Yes, column replacement is expensive, but the column cost is only a small fraction of the overall analysis cost. A typical LCtandem mass spectrometry (MSMS) method such as this costs $50 $100/sample, so a $500 column replaced after 500 injections amounts to < 2% of the overall costs certainly not worth the expense of revalidating the method in the present instance.
Conclusions

Physical problems at the inlet of the column can cause split or doubled peaks or peak fronting. These problems often require replacement of the column. If the column life and the data quality are sufficient, it might not be worth changing the method to avoid problems. Two simple practices should minimize or eliminate the problems discussed this month. The use of a 0.5 m porosity in-line filter plus sample centrifugation or filtration should prevent problems associated with column blockage. Careful adherence to the recommended mobile-phase pH limits or selection of a column designed for high-pH work should have avoided the problems associated with fronting peaks.

http://www.restek.com/techtips/What-Causes-Fronting-Peaks

What Causes Fronting Peaks?

Fronting peaks occur when the sample capacity of the analytical column is exceeded. This overloading effect results from poor sample solubility in the stationary phase, injecting too much sample, or operating at a k value (capacity factor) that is too low. Not only is choosing the correct stationary phase important in achieving gaussian peak shapes, but choosing the correct film thickness and ID can aid in increasing column sample capacity. Using a k value between 3 and 5 will ensure good peak shape, as well as ensuring that the sample partitions in the stationary phase long enough to achieve separation.

http://pharmaresearchdevelopment.blogspot.com/2010/12/peak-tailing-peak-frontingrounded.html

Peak tailing, Peak Fronting, Rounded Peaks in HPLC chromatogram

Peak tailing

What you expect

What you Got

Disease
Active sites within the column Wrong pH Wrong column Void volume at inlet Wrong sample solvent

Medicine
Test with standard test mixture. If OK add competing base or acid modifier Correct Change

May need repacking

Dissolve sample in mobile phase

Peak fronting

What you Expect

What you Got

Disease
Column overload

Medicine
See this (Retention Times are not constant) Correct

Wrong pH Sample solvent incompatible with mobile phase Void volume at inlet

See this (Peak Tailing)

May need repacking Dissolve sample in mobile phas

Wrong sample solvent

http://medical-dictionary.thefreedictionary.com/gas-liquid+chromatography
gas-liquid chromatography (GLC) gas chromatography in which the sorbent is a nonvolatile liquid coated on a solid support. gas-solid chromatography (GSC) gas chromatography in which the sorbent is an inert porous solid.

http://www.4college.co.uk/a/Cd/Glc.php

Gas-Liquid Chromatography
o

Chromatography is an important analytical technique used by chemists to separate and identify the compounds in a mixture.

There are a number of different types of chromatography, in Whats in a Medicine, thin layer chromatography was covered.

All the different types of chromatography rely on the equilibrium established when a compound distributes itself between two phases; one mobile and the other stationary.

Different compounds distribute themselves to different extents and so move along with the mobile phase at different speeds.

In thin layer chromatography, the stationary phase is the solid support material (often the plastic chromatography plate). The mobile phase is the solvent that rises up the plate.

In gas-liquid chromatography, the principle is the same, but the mobile phase is an unreactive gas, such as nitrogen (the carrier gas), and the stationary phase comprises of a small amount of liquid held on a finely-divided inert solid support.

The solid support is in the form of a powder which is packed into a long, thin tube (the column).
o

The column is coiled inside an oven.

The Instrument The sample to be reacted is injected into the gas stream just before it enters the column.
o

The components of the mixture are then carried through the column in a stream of gas.

Each compound distributes itself between the phases to different extents and therefore emerges from the column at a different time.

Some of the compounds dissolve in the stationary solvents more readily than others; these travel through the column slower and so emerge last.

o o o

The most volatile compounds usually emerge first. A detector on the outlet tube monitors compounds emerging from the column. Signals from the detector are plotted out by a recorder as a chromatogram.

The chromatogram shows the recorder response against the time which has elapsed since the sample was injected into the column.

o o

Each component of the mixture gives rise to a peak on the chromatogram. The following shows a gas chromatogram of a premium grade petrol:

The time a compound is held on a column under given conditions is characteristic of each compound and is referred to as its retention time; this can be affected by many factors, such as:
o o o

The length and packing of the column. The nature and flow rate of the carrier gas. The temperature of the column.

o o

The retention time can be used to identify the different compounds. The instrument is calibrated with known compounds, so that the conditions are kept constant throughout the analysis.

o o

The area under each peak represents the amount of each compound present. Gas-liquid chromatography is very sensitive and can be used to detect small quantities of substances; it is often used in forensic tests.

In more sophisticated instruments, the outlet tube is connected to a mass spectrometer, so that each compound can be detected directly.

http://chemistry.mtu.edu/~djchesne/classes/ch5210/pdfs/GC_notes_1.pdf Gas-Liquid Chromatography (GLC) mobile phase - gaseous stationary phase - heavy viscous liquid coated on a porous solid support or the inner wall of a capillary tube mobile phase does not interact with solutes; serves only as a transport medium compare to distillation: both separations based on vapor-liquid equilibrium distillation: separation a function of the difference in vapor pressure of the pure compounds GLC: separation is a function of the difference in vapor pressure plus the difference in solubility of components in the stationary phase

thus, the potential for selectivity between two compounds is much better in GLC the efficiency of separation per column length unit: GLC >> distillation sample capacity: distillation >> GLC

http://www.dyerlabs.com/chemistry/gas.html
Gas Chromatography (GC); Gas-Liquid Chromatography (GLC); Gas-Solid Chromatography (GSC); Vapor-Phase Chromatography (VPC) Here the mobile phase is a gas, often nitrogen, but sometimes helium, hydrogen or occasionally another gas. It is called the "carrier gas". Gas-solid chromatography is relatively rare, but it is used to separate atmospheric gases. Common solids are charcoal, a synthetic zeolite called "molecular sieve", or a combination of the two. Solids 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 ("backflushing"), 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 sizes.) Gas-liquid chromatography begins with a more or less inert "support" with a high surface area. That is mixed 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 "packing", is "packed" into a column, such as a 6 mm inside diameter stainless steel tube about 200 mm long. No firm packing 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 packing. The ends of the column are plugged, often with glass wool, to hold the packing but allow gas flow. The column ends have fittings (often "Swagelok" brand) so that they can be connected to matching fittings in the "oven" of the gas chromatograph. The oven has three functions: 1. It keeps the column temperature constant. 2. It allows operation at elevated temperature (faster; perhaps necessary to vaporize the sample). 3. It allows "temperature programming", a controlled increase in column temperature during an analysis to make the slow-moving components move faster, reducing the time needed for the analysis, and reducing the diffusion which makes peaks broader. Because the mobile phase is gas, solubility in the mobile phase is not really a factor, but volatility (vapor pressure) of the components being analyzed is a close equivalent. Solubility 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 work has been done on "derivatizing" components to increase volatility. Much of the early work in gas chromatography involved separating hydrocarbon mixtures such as gasoline on columns in which the liquid phase was a silicone oil. The process acted somewhat like distillation with the 'pot' 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. When it is assumed that there is equilibrium between vapor and liquid at each plate, the plates are "theoretical plates". Increasing the number of plates gives better separation. Application of the theory to gas chromatography introduced the term "height of an equivalent theoretical plate", abbreviated HETP. This was (roughly) the distance along the column that gave the same separation as a (theoretical) plate in a distillation column. Great advances have been made in reducing that distance, increasing the number of "theoretical plates" in a length of column, and improving separation, or "resolution". Separation 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. One of the major theoretical advances was the "van Deemter equation" which related HETP 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 0.25 - 0.5 mm inside diameter (ID), with a thin coating (about 0.2 mm) of liquid phase on the wall, and from 10 meters to 100 meters long. This is a "wall coated open tubular" (WCOT) column. The coating on the wall can also be a combination of support and coating, giving a "support coated open tubular" (SCOT) column. The sample must be very small, and the detector must be very sensitive, but the number of theoretical plates is immense (e. g., 100,000) and very complex mixtures 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. If the column temperature is too high, the most volatile components will move practically as fast as the carrier gas, and will not be separated. But 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 vaporize into the carrier gas, resulting in column "bleed". Significant 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 zirconia particles, or to the inner surface of a capillary column, to make "bonded" phases. Very few analysts pack their own columns or make their own TLC plates now. They buy prepared columns or plates from suppliers. Wide varieties of both supports and coatings are available. For instance, Supelco is a major US supplier of ready-to-use columns and materials. (See http:www.sigmaaldrich.com, and select Supelco. A catalog may be even more helpful.) So far, I have ignored two major operations in gas chromatography: getting the sample onto the column, and recognizing the components as they elute with the carrier gas. Samples are applied to the column with an "injector". The early injectors were just 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 ("Hamilton" is a major syringe tradename). The syringe needle was pushed through the septum, and a quick slap of the syringe plunger injected a narrow pulse of sample onto the column. Similar but larger syringes could be used for gas samples. The rubber septa have been replaced by polymers with more heat resistance, and injectors now have short lengths of tubing ("sample loops") into which the sample is placed before the injector switches the loop into the gas flow. Automated injection systems can be loaded with numerous samples to be injected 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 "peak" on the recorder chart as each component is detected. Each peak appears at a characteristic "retention time". The degree of separation of the peaks is the "resolution". When separation is so complete that the recorder pen returns to its zero position between peaks, one has "baseline resolution". The peak height was used first as an easy measure of the quantity of that component in the sample. Peak area proved to be a better measure, so the peaks were "integrated".

Integration was originally done by cutting a piece of recorder paper below the peak and weighing it; by counting the squares on the recorder chart below the peak; or by measuring its area with a planimeter. Mechanical ("ball and disc") integrators followed, and electronic integrators appeared quickly. Because 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. Detectors do not have the same sensitivity for all molecules, so quantitative measurements require use of standards. Sometimes a fixed amount of an "internal standard" is applied to all samples and standards. The principal detectors are: Thermal conductivity (TC): 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 (approximately) constant in a steady flow of carrier gas. When the carrier gas contains molecules larger than those of the carrier gas, less heat is removed from the wire; its resistance increases; and the voltage across the wire increases. Sensitive to all components, but not very sensitive to any. Flame ionization detector (FID): The detector contains a small hydrogen-oxygen (or hydrogen-air) flame. Some substances, when they burn in the flame, produce ions which carry current, and the current is measured. Very sensitive, particularly for molecules having C-H bonds. Not at all sensitive to some other molecules (such as CCl4). Electron capture detector (ECD): A radioactive source produces ions, and an ion current is measured between positive and negative electrodes. Some components (especially those containing C-Cl bonds) capture ions, reducing the current. Very sensitive; complements FID. The preferred carrier gas is argon. Mass spectrometer (GC-MS): 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. (Remember that mass spectrometry is done in vacuum.) In the time-of flight (TOF) mass spectrometer, the sample is broken into ionized 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 second intervals. A computer displays and records the total ion current vs. time, the current of a specific ion (a specific mass/charge ratio) versus time, or the mass spectrum of a component. The computer also has a "library" of mass spectra. It compares the mass spectrum of each peak 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 50,000 or more components. An "ion-trap" detector is one type of mass spectrometric detector. Some of the less broadly useful detectors are the thermionic emission detector (TED), sensitive to nitrogen and phosphorus compounds (also called an NPD); flame photometric detector (FPD), sensitive to sulfur and phosphorus compounds; and the photoionization detector (PID). Except for the MS detector, chromatography only separates the sample components, but does not identify them. In routine analyses, the analyst knows what components are expected, and can separately run chromatograms of individual known standard compounds or mixtures, 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 rate; perhaps the inlet pressure; the column dimensions; the support and coating (together, the packing), the injection method and injection volume, the detector (perhaps with applied voltages), any integrator used, and attenuator settings. Retention times may appear in the procedure or be shown with a reproduction of the chromatogram.

http://goldbook.iupac.org/G02591.html
gas-solid chromatography
Comprises all gas chromatographic methods in which the stationary phase is an active solid (e.g. charcoal, molecular sieves). Separation is achieved by adsorption of the components of a sample. In gas chromatography the distinction between gas-liquid and gas-solid may be obscure because liquids are used to modify solid stationary phases, and because the solid supports for liquid stationary phases affect the chromatographic process. For classification by the phases used, the term relating to the predominant effect should be chosen.

http://www.chromatography-online.org/topics/gas/solid/chromatography.html

Gas-Solid Chromatography

Gas-solid chromatography is a chromatography separation technique in which the mobile phase is a gas (usually helium or nitrogen) and the stationary phase is a suitable adsorbent such as silica gel, alumina or carbon. The technique is mostly used for the separation of the permanent gases or the low molecular weight hydrocarbons. By employing special phase systems it has also been used for the separation of the halogens and other highly corrosive gases and vapors. Solute distribution occurs between the gaseous mobile phase (often called the carrier gas) and the surface of the adsorbent. The distribution isotherm of a solute between a gas and solid surface usually takes the form of the Langmuir Isotherm or the Freundlich Isotherm and, thus, only at very low concentrations of solute do the isotherms approach linearity. As the symmetrical shape of an elution curve is only realized if the adsorption isotherm is linear, or close to linear, very small samples must be placed on any column used in gas-solid chromatography. Heavy sample loading on gas-solid chromatography columns results is the formation of asymmetric peaks often with very long tails

http://en.wikipedia.org/wiki/Eddy_diffusion
Eddy diffusion, eddy dispersion, multipath, or turbulent diffusion is any diffusion process by which [1][2] substances are mixed in the atmosphere or in any fluid system due to eddy motion. In another [3] definition it is mixing that is caused by eddies that can vary in size from the small Kolmogorov microscales to subtropical gyres. Because the microscopic processes responsible for atmospheric mixing are too complex to model in detail, atmospheric modelers generally treat atmospheric mixing as a macroscopic "eddy" diffusion process. In this approach, the diffusion rate at each pressure level is parameterized by a quantity known as the eddy diffusion coefficient, K
[4]

(also sometimes called eddy diffusivity, with units of

).

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm

The Rate Theory of Chromatography A more realistic description of the processes at work inside a column takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate model, which assumes that equilibration is infinitely fast). The resulting band shape of a chromatographic peak is therefore affected by the rate of elution. It is also affected by the different paths available to solute molecules as they travel between particles of stationary phase. If we consider the various mechanisms which contribute to band broadening, we arrive at the Van Deemter equation for plate height; HETP = A + B / u + C u where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening. A - Eddy diffusion The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take 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 diffusion 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. If 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 - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If 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.

http://www.nature.com/nature/journal/v184/n4683/abs/184357a0.html

Eddy Diffusion in Chromatography

J. CALVIN GIDDINGS
Department of Chemistry, University of Utah, Salt Lake City, Utah.

AMONG the various diffusion and kinetic factors which are responsible for smearing chromatographic zones, the effect known as eddy diffusion has been subject to the most controversy. Its contribution to the height equivalent to a theoretical plate (H) is usually assumed to depend only upon the packing of a column and to be independent of the velocity of flow. This assumption may be questioned by virtue of recent experimental work in chromatography13. An equation will be derived here which predicts eddy diffusion to be dependent on velocity. This will be compared to the existing experimental evidence.

http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography

van Deemter Equation

In chromatography, it is important that the components in solution are adequately separated so that the separate components can be collected in their purest form. This becomes easier to do as the separation between the bands for each component have a greater separation between them. Also, it is ideal to have the bands of the individual components as narrow as possible. This is to say that it is best to have each component occupying as little space as possible within the column:

From this figure it can be seen that a better separation between narrow bands of components is ideal for easier collection of the individual samples. Band broadening is an especially important factor for this type of chromatography when separating colored compounds. When the bands of the components are narrow, most of the particles of that component are in close proximity with one another, which makes it easier to see the color of the bands. As the particles diffuse away from one another and broaden the component's band, the color of the band fades and can become more difficult to see, which may also make it harder to collect pure samples of the mixture's components. The extent of band broadening in chromatography is determined by the Van Deemter equation . This equation relates the efficiency of the chromatography procedure to three different factors. The Van Deemter equation is shown below: H = Au
1/3 3

+ B/u + Cu
4

Where H is the height equivalent of a theoretical plate (HETP) and u is the velocity (flow rate) of the mobile phase. The lower the resulting value of H is, the greater the efficiency of the procedure. So, ideally, a scientist will want to minimize all three terms in order to minimize H. The other three terms refer to factors that come into play while the chromatography is performed.

The A factor is determined by a phenomenon called Eddy Diffusion . This is also called the multi-path term, as molecular particles of a certain compound have a multitude of options when it comes to finding a pathway through a packed column. The following figure helps in visualizing Eddy diffusion:

Because there is an almost infinite number of different paths that a particle can travel by through a column, some paths will be longer than others. The particles that find the shortest path through the column will be eluted more quickly than those that travel a longer way. In the figure, particle B will be eluted before particle C, and both will be eluted before particle A. Since it is improbable for all particles of one compound to find the shortest path, there will be fractions of the component that will behave like particles A, B, and C. This leads to the broadening of the band. There is little a scientist can do to minimize the Eddy Diffusion factor, as it is influenced by the nature of column being used and by the particles' movement through that column. The A term is loosely affected by the flow rate of the mobile phase, and sometimes the affect of the flow rate is negligible. It is for this reason that sometimes the Van Deemter equation is written as such: H = A + B/u + Cu B/u is called the longitudinal diffusion term, and is caused by the components' natural migration from a place of high concentration (the center of the band) to a place of lower concentration (either side of the band) within the column. Diffusion occurs because molecules in a place of high concentration will tend to spread out to areas of
6

lower concentration to achieve equilibrium. Given enough time, diffusion will result in equilibrium of the diffusing fluid via random molecular motion. The figure below helps to visualize this phenomenon:

At time zero in the figure above, the particles of a compound are generally localized in a narrow band within the separating column. If the mobile phase flow rate is too small or if the system is left at rest, the particles begin to separate from one another. This causes a spread in the concentration distribution of that compound within the column, thus bringing about band broadening for the band of that particular compound. As the time that the system is left still approaches infinity, the compound reaches complete concentration equilibrium throughout the entire column. At this point, there is no definitive band for that component, as a single concentration of that compound is present throughout the entire column. Longitudinal diffusion is a chief cause of band broadening in Gas Chromatography, as the diffusion rates of gaseous species are much higher than those of liquids. It is for this reason that longitudinal diffusion is less of an issue in liquid chromatography. The magnitude of the term B/u can be minimized by increasing the flow rate of the mobile phase. Increasing the velocity of the mobile phase does not allow the components in the column to reach equilibrium, and so will hamper longitudinal diffusion. The flow rate of the mobile phase should not be increased in excess, however, as the term Cu is maximized when u is increased. Cu is referred to as the mass transfer term. Mass transfer refers to when particles are so strongly adhered to the stationary phase that the mobile phase passes over them without carrying them along. This results is particles of a

component being left behind. Since it is likely that more than a single particle of any given compound will undergo this occurrence, band broadening results. This results in a phenomenon called tailing, in which a fraction a component lags behind a more concentrated frontal band. Non-equilibrium effects can be caused by two phenomena: laminar flow and turbulent flow. Laminar flow occurs in tubular capillaries, and so is most prominent in Capillary Electrophoresis. Turbulent flow occurs as a result of particles becoming overwhelmed by the stationary phase and is more common in column chromatography. This occurrence can be visualized by observing the figure below:
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In the above figure, particles of the adsorbent solid become occupied by particles of the sample. If too many particles of the adsorbent are occupied, particle A will have nothing hindering it from flowing through the column. So, the particles of a single compound separate from one another. Also, as the mobile phase moves through the column, particles of the sample leave the stationary phase and migrate with the mobile phase. However, if the flow rate of the mobile phase is too high, many of the sample particles are unable to leave the stationary phase and so get left behind. These occurrences result in band broadening, as the individual particles of a single compound become less closely packed. The high flow rate of the mobile phase makes it more difficult for the components within the column to reach equilibrium between the stationary and mobile phase. It is for this reason that the Cu term is also called the non-equilibrium factor. Minimization of this factor can be achieved by decreasing the flow rate of the mobile

phase. Decreasing the flow rate of the mobile phase gives sample components more time to leave the stationary phase and move with the mobile phase, thus reaching equilibrium. By observing the Van Deemter equation, it can be deduced that an ideal mobile phase flow rate must be determined to yield the best (lowest) value of H. Decreasing the flow rate too much will result in an increase of the longitudinal diffusion factor B/u, while exceedingly increasing the flow rate will increase the significance of the mass transfer term Cu. So, H can be minimized to a finite limit depending on the various parameters involved in the chromatography being performed.