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Liquid Chromatography: Still Striving for High Efficiency Drexel University Literature Seminar by Anna Caltabiano 17 May 2010
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Introduction
What is Chromatography? ,[object Object],Michael Semenovich Tswett  Sakodynskii,  Usp. Khromatogr. (1972), 9 (25), 9-25 ,[object Object],[object Object]
Why use Chromatography? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Chromatography today ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Liquid Chromatography (HPLC) instrument Solvent delivery Pump Autosampler Column Detector Injector
The Chromatographic Process –  Theoretical Considerations
Elution in Column Chromatography Mobile phase Stationary phase t 0 t 1 t 2 t 4 t 3
Intermolecular Interactions ,[object Object],[object Object],δ - δ + δ + δ - δ + δ - b) Polar  (dipole-dipole, dipole-induced dipole) δ - δ + e) Ionic H c) Hydrogen bonding NO 2 NO 2 d) Charge transfer
Chromatogram Column Time Chromatogram
Chromatogram (Cont) ,[object Object],a) b) 2 0 -2 W = 4 σ W 0.607  = 2 σ W 0.5  = 2.354 σ ,[object Object]
Retention Relationships t 0 t R t s t 0  – void volume; t R  –retention time, t s  – time spent in the mobile phase; L – column length; u 0  – superficial linear velocity of mobile phase; u – the average superficial linear velocity of analyte; V R  – retention volume; F – flow rate; R – the fraction of the molecules present in the mobile phase  at any time
Plate Number and HETP ,[object Object],W 0.1 B A and reduced  plate height: ,[object Object],[object Object]
Peak Capacity ,[object Object],[object Object],For isocratic separations: k max  is retention factor of the last peak; t g  is the gradient time; w is the base peak width that is assumed to be constant For gradient separations: ,[object Object],[object Object]
Variables that Affect Column Efficiency
Band Broadening and Mobile Phase Velocity ,[object Object],[object Object],[object Object],[object Object],d is contributions from longitudinal diffusion; sm – from stagnant mobile phase  mass-transfer, s – from stationary phase mass-transfer, e – from eddy diffusion; m – from mobile phase mass transfer.
Band Broadening and Mobile Phase Velocity (Cont) ,[object Object],a) Eddy Diffusion Mobile phase Particle b) Mobile Phase Mass-Transfer Particle ,[object Object]
Band Broadening and Mobile Phase Velocity (Cont) ,[object Object],c) Stagnant Mobile Phase Mass-Transfer d) Longitudinal diffusion ,[object Object],[object Object]
Band Broadening and Mobile Phase Velocity (Cont) ,[object Object],[object Object],Van Deemter plot  http://chromedia.org/chromedia
Column Length, Column Diameter and Particle Size η is the viscosity of the mobile phase ; K is the column permeability; dc is the column internal diameter. ,[object Object],[object Object],[object Object],[object Object],[object Object]
Temperature ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],kB is Boltzmann constant, d is the diameter of the solute
Existing approaches to improve LC efficiency
High Temperature Liquid Chromatography (HTLC) ,[object Object],[object Object],[object Object],[object Object],[object Object]
High Temperature Liquid Chromatography (HTLC) (Cont) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Monolithic Stationary Phase  ,[object Object],[object Object],[object Object],[object Object],[object Object],Tanaka et al.,  Analytical Bioanalytical Chemistry   (2003), 376, 298-301
Monolithic Stationary Phase (Cont) ,[object Object],[object Object],Wu et al.,  Analytica Chimica Acta  (2004), 523, 149–156
Fused-Core Stationary Phase Particles ,[object Object],[object Object],[object Object],[object Object],[object Object],http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/Product_Information_Sheet/t407044.Par.0001.File.tmp/t407044.pdf Fekete et al.,  Journal of Pharmaceutical and  Biomedical Analysis  (2009), 49, 64-71
Fused-Core Stationary Phase Particles (Cont) ,[object Object],Zheng et al.,  Journal of Pharmaceutical and Biomedical Analysis  (2009), 50, 815-822
Small-diameter (sub-2- μ m) totally porous stationary phase particles and UHPLC ,[object Object],[object Object],de Villiers et al.,  Journal of Chromatography A   (2006), 1127, 60-69
Small-diameter (sub-2- μ m) totally porous stationary phase particles and UHPLC (Cont) ,[object Object],[object Object],1.7um porous particle (Waters UPLC BEH)  Fekete et al.,  Journal of Pharmaceutical  and Biomedical Analysis  (2009), 49, 64-71 where  is the flow resistance factor related to the column packing structure   Φ  is the flow resistance factor related to the column packing structure
Small-diameter (sub-2- μ m) totally porous stationary phase particles and UHPLC (Cont) ,[object Object],[object Object],Gritti et al.,  Analytical Chemistry  (2009), 81, 3365-3384
Small-diameter (sub-2- μ m) totally porous stationary phase particles and UHPLC (Cont) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],q is the heat flux, Rc the column radius, and  λ  is the radial heat conductivity of the column packing
Conclusions
Conclusions ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
References ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
References (Cont) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Thank You! Tswett’s column Waters Acquity BEH (UPLC) column Agilent HPLC-Chip

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Liquid chromatography still striving for high efficiency2

Notes de l'éditeur

  1. Separations of complex mixtures on glass columns (tubes) packed with solid adsorbents, using liquids as eluents were introduced by a Russian botanist Michael Semenovich Tswett when he was carrying out experiments on chlorophyll extracts. His invented technique not only opened the door to understanding the mystery of the green leaf, but served as the basis to a new separation technique – chromatography. In his report “On a new category of adsorption phenomena” that was presented at the meeting of the Biological department of Warsaw Society of Natural Scientists on March 8 th , 1903, Tswett emphasized the development of a new separation method of substances dissolved in organic solvents: In his next paper published 3 years later, Tswett gives a name to this method and calls it chromatography: The term chromatography is coming from the Greek chroma (color) and graphein (to write). However, Tswett does not provide the origin of his term in his publications, and since tswett means color in Russian, it is possible that he named the method after himself, literally Tswett’s writing. Chromatography has evolved from Tswett’s time and now it’s comprised from a wide range of techniques that are based on Tswett’s simple separation process. Unified definition and nomenclature of chromatography was published in 1993 by IUPAC
  2. Chromatography is one of the most eminent discoveries of the 20 th century, and at the present it is one of the most important analytical techniques. Discoveries in biochemistry, biotechnology, medicine, agriculture, as well as in space science and industry would not have been possible without separation of complex mixtures. Separation of chemical components is essential in any type of chemical analysis as it allows purification as well as qualitative and quantitative measurements. Nowadays chromatographic separations possess such essential valuable characteristics as well-understood separation mechanisms, ease of use, sensitivity, selectivity, robustness, and they’re relatively fast. No other separation method is as powerful as a chromatographic method is, and only a few chemical analysis methods can be found specific to a particular compound.
  3. Chromatography significantly evolved since its discovery, and now more than sixty variants of the technique have been developed. Gas Chromatography ( GC ) is preferred to HPLC for analysis of gases, thermally stable low-boiling and higher boiling compounds due to GC being more efficient than HPLC. However, GC is applicable to samples volatile below 300ºC, and thus is not applicable to nonvolatile or very-high-boiling compounds. That is approximately 75% of all known compounds can’t be separated by GC. Supercritical Fluid Chromatography ( SFC ) utilizes columns and equipment similar to HPLC, however, the mobile phase is a supercritical fluid, usually a gas under critical pressure and temperature. In terms of separation efficiency, SFC takes place between HPLC and GC. Significant advantages of SFC over HPLC is that SFC can be used in separation of polymeric mixtures, while HPLC is unable to resolve polymeric species with high molecular weights, and that SFC is faster than HPLC since lower viscosity of the mobile phase allows operation at higher flow rates. Capillary Electrophoresis ( CE ) not being a form of chromatography, this separation technique is a rival to HPLC. The separation principle is based on the separation of charged compounds in the order of their mass-to-charge ratios (m/z) in a capillary, under the influence of an electric field. CE, as well as GC and SFC, is characterized by higher than HPLC separation efficiency. The drawback of CE is that its detection sensitivity is much lower than in HPLC. Regardless of HPLC being less efficient than some of the separation techniques, it is still a dominating technique routinely used in the industry due to its essential valuable characteristics comprised of well-understood separation mechanisms, ease of use, sensitivity, selectivity, precision and robustness. This technique has many applications including separation, identification, purification, and quantification of numerous biological and pharmaceutical mixtures of ever increasing complexity. Therefore, in the past decade, significant efforts have been undertaken to improve the efficiency of high-performance liquid chromatography, and successful results were achieved with the use of high temperature LC, monolithic and fused-core columns, and with the use of small-diameter (sub - 2 µm) stationary phase particles. Chromatographic process, the factors influencing chromatographic efficiency in HPLC and the means to improve efficiency are reviewed herein.
  4. Separation of the components of a mixture takes place in a narrow-bore tubing (column) packed with fine inert solid particles that hold stationary phase. The mobile phase surrounds particles as it percolates through the column. Sample mixture applied to the column as a discrete band (to in Figure 2) carried through the column by the flowing mobile phase. Sample components distribute themselves between the mobile phase and the stationary phase. Elution is promoted by continuous addition of fresh mobile phase into the column. The eluent moves down the column where further partitioning between the stationary and mobile phases occur (t1 in Figure 2) and a new equilibrium establishes. Components in the mobile phase adsorb onto the stationary phase, and components from the stationary phase migrate into the mobile phase (t2 in Figure 2). This process is repeated many times during the elution that eventually leads to separation (t3 in Figure 2) and detection (t4 in Figure 2) of components. Separation of components arises from differential retention of the solutes by the stationary phase. Components that prefer to reside in the stationary phase move down the column slower than those that prefer the mobile phase since solute movement can occur only in the mobile phase. This phase preference can be expressed by the distribution coefficient, K. The net retention of a solute is determined by molecular interactions solute-solute, solute-stationary phase, solute-mobile phase, and stationary-mobile phase interactions. Molecular interactions result from intermolecular forces which are all electrical. Gravitational and magnetic forces may be present, however, they are significantly weaker and therefore don’t affect solute retention. There are three types of intermolecular forces: 1) dispersion , 2) polar , and 3) ionic . Understanding of molecular interactions should allow prediction of retention depending on molecular structure.
  5. Dispersion forces, also called induced dipole-induced dipole forces or Van der Waals forces, result from charge fluctuations that originate from electron-nuclei vibrations. Iduced dipole induces a dipole in the adjacent atom. The original transient dipole favor electrostatic attraction with the induced dipole. Polar forces occur in a polar molecule that contains a dipole in the form of localized charges located on different parts of the molecule.These charges interact with opposite charges on other molecules. These interactions are always accompanied by the dispersive interactions. Polar forces include dipole-dipole (or orientation), dipole-induced (or induction) interactions, hydrogen bonding and charge transfer interaction. Hydrogen forces are specific to molecules containing oxygen, nitrogen or fluorine atom attached to hydrogen. Hydrogen Bonding arises from the attraction between the slightly positive charge on a hydrogen atom and can be described as a strong dipole-dipole attraction between an electronegative atom and a hydrogen atom bonded to another electronegative atom. Charge transfer or interactions result from the partial transfer of an electron between electron-rich and electron-poor molecules. Such interactions occur between aromatic or unsaturated compounds. Ionic interactions occur between charged ions (counter-ions). Ionic interactions are always accompanied by the dispersive interactions, and sometimes by polar interactions
  6. As sample components progress down the column, its molecules tend to spread out and occupy larger volumes within the column. The volume that is occupied by a compound’s molecules is called a band . When such band leaves the column (elutes), it is recorded by a detector as a peak in the chromatogram.
  7. Ideally, the eluted peak would have the same form as the graphical representation of one dimension probability function of the normal distribution of random errors (i.e. the normal error function or Gaussian function) [15]. The mathematical descriptions of the Gaussian function: Since the area and height are proportional to the amount of a compound injected, both these parameters can be employed in analytical determinations, however peak area is preferred for quantitation. Peak area is the true measure of analyte’s quantity if it is eluted intact and is detected linearly [17]. Although theoretically the peak area is equal to the zeroeth moment ( M0 ) of the time-dependence of the concentration, equation, practically the peak area can be calculated from a systematic summation of the detector signal that is (ideally) linearly proportional to concentration as depicted in equation.
  8. The time from the moment of injection to the baseline distortion is the average rate of mobile phase migration, void time . All analytes spend in the mobile phase. The time elapsed from the moment of injection to the center of the retained by the stationary phase peak is retention time, . The analyte is retained because it spends time in the stationary phase. This relationship can be described as The retention volume of the peak,, is related to the flow rate, F , and retention time by the expression   To compare the migration rates of solutes under different experimental conditions (column dimensions, flow rate), the retention factor k can be used:   Assuming that the fraction of molecules in the mobile phase is R (the fraction of the molecules present in the mobile phase at any time), then the fraction of molecules in the stationary phase is 1-R and Combining equations with R=u/uo:
  9. The width of chromatographic peaks, or a bands that are occupied by molecules of each compound, is finite. Depending on the width, , of such bands, the quality of their separation will vary, therefore it is important to understand the parameters influencing the band with. The band broadening process is measured by the column efficiency or the number of plate number N : W= 4sigma Equation demonstrates that peak width, increases in proportion to retention time. This signifies that peak width will increase as a compound progresses down the column. As previously mentioned (section 2.3), peaks are rarely Gaussian and very significant errors can result from using equations that assume Gaussian peak to calculate column efficiency. Prof. Foley & Dorsey urged the use of an exponentially-modified-Gaussian function based equation for plate count. where H is the height equivalent to one theoretical plate (HETP) and is a measure of column efficiency per unit length of column. The efficiency of chromatographic columns increases with the increase in plate number, while plate height becomes smaller. Since broadening of a Gaussian peak is described by the standard deviation and the variance , the variance per unit length of column can be used as a measure of efficiency: In order to compare columns over a range of particle sizes of the packing material and mobile phases, reduced plate height, h (a dimensionless parameter), is used.
  10. Also with decrease of dp.
  11. The column efficiency is a measure of band broadening. It is important to understand causes of band broadening in order to keep them to a minimum so that efficiency can be maximized. Peak width and retention time determine plate number for the column. Various processes that occur inside and outside the column contribute to the peak width. The total peak variance can be expressed as:
  12. Eddy Diffusion: This contribution to band broadening depends on the size and arrangement of particles packed into the column and is independent of flow rate. Mobile phase mass-transfer: The center of the stream moves faster with the flow rate increase, increasing band broadening
  13. Stagnant mobile phase and stationary phase mass-transfer: This contribution to band broadening increases with the flow rate [22]. Its contribution also depends on the size, shape and pore structure of the particles and the diffusion of the analyte. Longitudinal diffusion: This contribution to band broadening increases with time, and is present regardless of whether or not the mobile phase is flowing, and decreases with increasing the flow rate. Extra-column: . Often the extra-column contributions can be ignored, but it depends on the characteristics of the equipment and column size. The smaller the column diameter and the shorter the column, the smaller is the peak volume. The volumetric extra-column contribution can be decreased with increasing column length, column radius and retention factor, and the time-based contribution to extra-column band broadening can be minimized by choosing the correct detector settings, mostly the detector time constant. In addition, optimization of connecting tubing, injection volume, flow cell volume and geometry leads to maximum performance of a column.
  14. The magnitude of band broadening depends on the time of contact between the mobile phase and the stationary phase, and thus depends on the flow rate. A good measure of the band broadening is HETP. A plot of H (or h ) against a linear velocity is a curve with a minimum and a nearly linear increase of the H with linear velocity at high values of linear velocity. This relationship can be described by the van Deemter, the Giddings, the Knox, and several others equations.
  15. The best compromise between column efficiency, pressure drop and analysis time can be obtained with shorter columns (L=20-40mm) packed with small particles (1-2μm). But what would happen if column diameter was increased or decreased? In theory, efficiency should not depend on column diameter, but practically it has been observed that narrower-diameter columns are usually less efficient that larger-diameter ones. The peak volume (peak width in volumetric terms) decreases significantly as the column diameter is decreased. This translates into narrower peaks, and the narrower peaks are more susceptible to extra-column dispersion.
  16. For a given solute, and , where R is the gas constant, are temperature independent constants, therefore k usually decrease with increasing temperature. Thus, with temperature increase k decreases (equation 3.15), and linear velocity increases. This is a drawback since flow rates will have to be increased to avoid a loss in performance. It was shown that the elevation of column temperature while operating at constant flow rate below 0.4mL/min (below Van Deemter optimum) will result in the loss of efficiency. Higher flow rates should be used in order to maintain the same efficiency at the elevated temperatures. Increasing flow rates, in turn, will result in an increase in pressure. This suggests that increasing temperature will not reduce backpressure if one maintains constant column plate count. In any case, the advantage of the elevated temperature is the reduction in the analysis time with negligible loss in column efficiency
  17. High temperature liquid chromatography (HTLC), operation at temperatures 40 - 200º C, was first proposed by Antia et al. It was postulated that the use of higher temperatures would improve LC efficiency to values comparable to that of SFC, and shorten run time. As discussed in section, theoretically we may expect increase in efficiency with the increase in column temperature. However, practical results were thus far contradictory. In some instances, increase in column temperature led to improvement, had no effect, or even decreased column efficiency. A conclusion that can be drawn is that there are other factors that influence band broadening at high temperatures. Guiochon offered his view on a possible cause – radial temperature gradients across the column (this phenomenon is discussed further in section 4.4). He stressed that this temperature gradient should be kept to a minimum (not more than a few ºC) since even small gradients will cause large efficiency loss. He suggested that decreasing column diameter and insulating the column wall with a thick layer of plastic foam would alleviate the temperature gradient issue. Also, he emphasized that the temperature of incoming mobile phase should be controlled in order to keep it in the range of column wall temperature in order to prevent the formation of radial gradients. This issue, termed “thermal mismatch” occurs due to silica and polystyrene column packings being poor thermal conductors compared to stainless steel.
  18. There are other serious considerations that should be addressed before HTLC becomes a powerful tool. The major one is the stability of analytes. Another issue that still needs to be addressed is column stability. The stationary phase has to be stable for over an extended period of time. However, it’s not just the column that has to be stable at high temperatures, but the column hardware needs to be improved. Stationary phase and column hardware might be subject to differential expansion which would result in the formation of voids and channels in the packing material and led to additional band broadening. One more concern that is worth mentioning is that the mobile phase should be cooled down right after it elutes from the column and before it enters the detector, because the sensitivity of the detector is affected by the temperature of the mobile phase. This influence can be positive (the signal will be increased) or negative (the signal will be reduced). Teutenberg [41] sees the future of HTLC in miniaturization since it would allow alleviation of slow temperature equilibrium and would reduce peak broadening (increase efficiency) due to a better heat dissipation.
  19. This approach is based on reduction of the mass-transfer resistance by increasing the external porosity and the flow-through pore size of the packing. A monolithic column consists of a single piece of solid material that consists of interconnected skeletons and interconnected flow paths (Figure 10). An advantage that monolithic columns hold over particle packed columns is that it is possible to control the silica skeleton diameter and the macro- (~ 2 µm) and meso- (~ 10 nm) pore diameters during manufacture. The high porosity allows high permeability and thus low pressure drop. Compared to particles packed columns, monolithic columns can be operated at higher flow rates without loss in efficiency (Figure 11 [47]). In addition, small-sized skeletons of the material offer high efficiency. There are silica-based and polymer-based monoliths. Monolithic polymer-based columns were introduced in the late 1980s, early 1900s. These columns can be made by the copolymerization of monomers (e.g. styrene/divinylbenzene with monovinyl/divinyl methacrylate). A variety of monomers with different functionalities provides different selectivity. Polymer-based monoliths are widely used for the separation of large biomolecules, however the efficiency of polymer-based columns is lower than the efficiency of silica-based monoliths. Silica-based monoliths (Figure 10) were introduced later and became commercially available in 2000.They’re prepared as rods from a single piece of porous silica. The primary advantage of a silica-based monolith over a particle-packed column is high and variable external porosity. Monolithic columns can be made with 60% of external porosity, and 85% total porosity which is about 20% higher than what particle-packed column can offer. Due to higher porosity, the surface area and retention factors are smaller in monolithic columns than in particle-packed column. It is thought that heterogeneity of both the silica skeleton and the through-pores determines the efficiency of monolithic columns. Some drawbacks of silica-based monoliths include peak asymmetry (tailing), limited variety and dimensions of monolithic columns, limited stability at high pressures, temperatures and.
  20. The primary advantage of a silica-based monolith over a particle-packed column is high and variable external porosity.
  21. Fused-core particle technology was introduced by Kirkland in 1992 . The rationale behind this approach is based on reduction of the diffusion distance of analytes to minimize stagnant mobile phase mass-transfer (e.g. 0.25 - 0.5 µm – fused-core versus 1.8 – 3.5 µm – totally porous particle). Fused-core particles consist of a 1.7 µm solid core surrounded by a 0.5 µm porous silica shell (Figure 13). The shorter diffusion path reduces axial dispersion of solutes and minimizes peak broadening. Reduction in axial dispersion enables higher flow rates that can be used without sacrificing column performance. Fused-core particles are characterized by their narrow particle size distribution which allows packing of these columns with great ruggedness. The axial diffusion term (B-term) being 25% lower (due to the solid core in the particle) and the eddy diffusion term (A-term) being 20% lower (due to a narrow particle size distribution) for the solid-core particles than that for the porous particles in the HETP equation.
  22. The fused-core Halo column had the highest column efficiency and the smallest mass-transfer resistance for highly retained compounds
  23. It is evident that when the particle size is reduced, the minimum plate height is decreased and the optimum velocity (point on the curve at the minimum plate height) is increased. The flat part of the curve at higher linear velocities signifies that operating at the velocities above the optimal value results in an insignificant decrease in efficiency. Hence, columns with smaller particles can be used at higher linear velocity without sacrificing efficiency for analysis time.
  24. The improvement in peak efficiency, however, comes at cost of the high pressure required to flow mobile phase through a column when particle diameter decreases: For any particles, pressure drop can also be estimated by the following relationship… This table implies significant increase in efficiency, along with favorable reduction in analysis time at the expense of increasing pressure.
  25. Aside from the engineering challenges associated with the use of high pressures in liquid chromatography, at high flow rates, the friction of the mobile phase against the stationary phase generates heat. The rate of heat generation (power dissipation) is the result of pressure drop and flow rate: The heat, convectionally transported by the mobile phase along the column, conducts in the axial and radial directions , and evacuates into the air through the column walls and end-fittings . After asymptotically reaching a steady-state thermal equilibrium, the column temperature remains constant everywhere in the column and doesn't depend on time any more. At this point, the generated heat evacuates the column, producing radial and axial temperature gradients. In the axial direction, the temperature increases from the column inlet to the outlet, and in the radial direction, the temperature decreases from the center to the column wall. The amplitude of these gradients depends on the degree of the column's thermal insulation. The longitudinal temperature gradient is the greatest when the column temperature is radially uniform (the smaller the radial heat loss, the smaller the radial temperature gradient, and the larger the axial temperature gradient). This happens when there is no radial heat loss through the wall (the column is kept adiabatic), and the generated heat is evacuated through the column ends. The radial temperature gradient is the greatest when the column wall temperature is kept constant. This happens when there is no radial heat loss through the wall (the column is kept adiabatic), and the generated heat is evacuated through the column ends. The radial temperature gradient is the greatest when the column wall temperature is kept constant. Normally, columns are operated under intermediate conditions of thermal insulation where radial and axial temperature gradients co-exist. Temperature gradients have a negative impact on the column efficiency due to heterogeneous distribution of the mobile phase linear velocity, viscosity and density throughout the column, which affect the equilibrium constant of a compound distribution between the mobile and stationary phases (decreases with increasing temperature), and retention factors of the analytes. The difference between the temperatures at the inlet and the outlet of the column can be as much as 20K, and the difference between the temperatures of the center and the column wall can reach up to 6K if the column is operated under regular convection conditions. c
  26. Since the detector measures the average concentration in the effluent from the column, and the radial concentration profiles of the analytes form an arc in the column (efficiency lose due to radial temperature distribution that causes radial band velocity profile) the loss of efficiency is observed. When the temperature of the external column wall is kept constant, the efficiency loss is the largest [62] with the maximum amplitude of the radial temperature gradient (ΔT) between the column wall and its center, which can be expressed as: This equation suggests that the frictional heating effect can be reduced by decreasing the column diameter. It was shown that the efficiency losses can be kept to a minimum for particle diameters of 1.5–2.0 μm if column i.d.s of < 2mm are used