Encyclopedia of chromatography by jack cazes 2

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Optical Activity Detectors Hassan Y Aboul-Enein Ibrahim A Al-Duraibi Pharmaceutical Analysis Laboratory, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia Introduction Optical activity detectors are capable of specifically detecting chiral compounds, taking advantage of their unique interactions with polarized light Much of the work on the development of prisms and other devices for the production of polarized light was done in the early part of the nineteenth century However, the measurement of optical activity is often used for enantiomeric purity determination of chiral compounds, which by definition have either a center or plane of asymmetry Enantiomers rotate the plane of polarized light in opposite directions, although in equal amounts The isomer that rotates the plane to the left (counterclockwise) is called the levo isomer and is designated (Ϫ), whereas the one that rotates the plane to the right (clockwise) is called the dextro isomer and is designated (ϩ) Questions of optical activity are of extreme importance in the field of asymmetric chemical synthesis and in the pharmaceutical industry Detection Principle Figure shows the basic optimal system of the optical rotation detector, which is based on the nonmodulated polarized beam-splitting method The light radiated from the light source is straightened by the plane polarizer, then to the lens for beam formation and concentration, and then to the flow cell The plane-polarized light which goes through the flow cell is rotated by optically active substances (chiral compounds) according to their specific optical rotations and concentrations The light then enters the polarized beam splitter and is divided into two beams according to the polarized beam directions These beams are detected by two photodiodes as shown The angle of the plane polarizer is adjusted so that the two photodiodes may receive the same beam intensity when no optically active substance is present in the flow cell When optically active substances are present in the flow cell, the difference between the beam intensities received by the two photodiodes is not zero Therefore, the difference has a linear relation Fig Optical rotation detector with specific optical rotation and concentration of the optically active substance and can be expressed by V0 ϭ K3a 4C where V0 is the difference of beam intensities received by the two photodiodes (i.e., output of signal level), K is a constant determined by cell structure and light intensity of the light source, [a] is the specific optical rotation of the chiral compound, and C is the concentration of the chiral compound Polarimetry Theory Most forms of optical spectroscopy are usually concerned with the measurement of the absorption or emission of electromagnetic radiation Ordinary, natural, unreflected light behaves as though it consists of a large number of electromagnetic waves vibrating in all possible orientations around the direction of propagation If, by some means, we sort out from the natural conglomeration only those rays vibrating in one particular plane, we say that we have plane-polarized light Of course, because a light wave consists of an electric and a magnetic component vibrating at right angles to each other, the term “plane” may not be quite descriptive, but the ray can be considered planar if we restrict ourselves to noting the direction of the electrical component Circu- Encyclopedia of Chromatography DOI: 10.1081/E-Echr 120005246 Copyright © 2002 by Marcel Dekker, Inc All rights reserved lar polarized light represents a wave in which the electrical component (and, therefore, the magnetic component also) spirals around the direction of propagation of the ray, either clockwise (“righthanded” or dextrorotatory) or counterclockwise (“left-handed” or levorotatory) If, following the passage of the plane-polarized ray through some material, one of the circularly polarized components, say the left circularly polarized ray, has been slowed down, then the resultant would be a planepolarized ray rotated somewhat to the right from its original position In addition, lasers have been incorporated into two optical rotation methods to date: polarimetry and circular dichroism Optical Rotation and Optical Rotatory Dispersion A polarimeter measures the direction of rotation of plane-polarized light caused by an optically active substance The specific optical activity of an asymmetrical molecule varies with the wavelength of the light used for its determination This variation is called optical rotatory dispersion (ORD) In ORD, rotations are measured over a range of wavelengths rather than at a single wavelength, usually covering the ultraviolet (UV) as well as the visible region Circular Dichroism In this technique, the molecular extinction coefficients of a compound are measured with both left and right circularly polarized light, and the difference between these values is plotted against the wavelength of the light used The phase angle between the projections of the two circularly polarized components is altered by passage through the chiral medium, but their amplitudes will be modified by the degree of absorption experienced by each component This differential absorption of left- and right-circularly polarized light is termed circular dichroism (CD) So, circular dichroism measurements provide both absorbance and optical rotation information simultaneously Circularly Polarized Luminescence Spectroscopy Circularly polarized luminescence spectroscopy (CPLS) is a measure of the chirality of a luminescent excited state The excitation source can be either a laser or an arc lamp, but it is important that the source of excitation be unpolarized to avoid possible photoselection artifacts The CPLS experiment produces two Optical Activity Detectors measurable quantities, which are obtained in arbitrary units and related to the circular polarization condition of the luminescence It is appropriate to consider CPLS spectroscopy as a technique that combines the selectivity of CD with the sensitivity of luminescence The major limitation associated with CPLS spectroscopy is that it is confined to emissive molecules only Vibrational Optical Activity The optical activity of vibrational transitions has been conducted The infrared (IR) bands of a small molecule can easily be assigned with the performance of a normal coordinate analysis, and these can usually be well resolved One of the problems associated with vibrational optical activity is the weakness of the effect Instrumental limitations of infrared sources and detectors create additional experimental constraints on the signal-to-noise ratios Two methods suitable for the study of vibrational optical activity have been developed: Vibrational Circular Dichroism: Vibrational circular dichroism (VCD) could be measured at good signal-to-noise levels Vibrational optical activity is observed in the classic method of Grosjean and Legrand Raman Optical Activity: The Raman optical activity (ROA) effect is the differential scattering of left- or right-circularly polarized light by a chiral substrate where chirality is studied through Raman spectroscopy Fluorescence-Detected Circular Dichroism Fluorescence-detected circular dichroism (FDCD) is a chiroptical technique in which the spectrum is obtained by measuring the difference in total luminescence obtained after the sample is excited by left- and right-circularly polarized light For the FDCD spectrum of a given molecular species to match its CD spectrum, the luminescence excitation spectrum must be identical to the absorption spectrum Factors Affecting the Measurement of Optical Rotation The rotation exhibited by an optically active substance depends on the thickness of the layer traversed by the light, the wavelength of the light used for the measurement, and the temperature of the system In addition, if the substance being measured is a solution, then the Optical Activity Detectors concentration of the optically active material is also involved and the nature of the solvent may also be important There are certain substances that change their rotation with time Some are substances that change from one structure to another with a different rotatory power and are said to show mutarotation Mutarotation is common among the sugars Other substances, owing to enolization within the molecules, may rotate so as to become symmetrical and, thus, lose their rotatory power These substances are said to show racemization Mutarotation and racemization are influenced not only by time, but also by pH, temperature, and other factors Of course, rotations that determined for the same compound under the same conditions are identical Therefore, in expressing the results of any polarimetric measurement, it is, therefore, very important to include all experimental conditions Temperature Temperature changes have several effects on the rotation of a solution or liquid An increase in temperature increases the length of the tube; it also decreases the density, thus reducing the number of molecules involved in the measurement It causes changes in the rotatory power of the molecules themselves, due to association or dissociation and increased mobility of the atoms, and affects other properties In addition, temperature changes cause expansion and contraction of the liquid and a consequent change in the number of active molecules in the path of the light The unique ability of the optical rotation detector to respond to the sign of rotation allows precise enantiomeric purity determination even if the enantiomers are only partially resolved The sign of rotation is also useful in establishing enantiomer elution order Because the optical rotation detectors only respond to optically active compounds, enantiomeric purity determination to precisions of better than 0.5% can be achieved and is possible in even the complex mixtures The detection can also be used as part of a flow injection analysis system to determine amount and enantiomeric purity of a drug in dosage form The applications using optical rotation detectors include the following: 3 Qualitative analysis of chiral compounds, including drugs, pesticides, carbohydrates, amino acids, liquid crystals, and other biochemicals Determination of enantiomeric purity of chiral compounds Monitoring an enzymatic reaction Qualitative analysis of proteins Use as a conventional polarimeter However, the disadvantages of optical rotation detectors may be limited by shot or flicker noise, which are dependent on the optical and mechanical properties of the system or by noise in the detector electronics Generally, the usefulness of this technique has been limited by the lack of sensitivity of commercially available instruments Suggested Further Reading Allenmark, S., Techniques used for studies of optically active compounds, in Chromatographic Enantioseparation: Methods and Application, 2nd ed., Ellis Horwood Ltd., London, 1991 Beesley, T E and R P W Scott, An introduction to chiral chromatography, in Chiral Chromatography, John Wiley & Sons, Inc., New York, 1998, pp 1–11 Dodziuk, H., Physical methods as a source of information on the spatial structure of organic molecules, in Modern Conformational Analysis, Elucidating Novel Exciting Molecular Structures, VCH, New York, 1995, pp 48 –54 Edkins, T J and D C Shelly, Measurement concepts and laser-based detection in high-performance micro separation, in HPLC Detection: Newer Methods (G Patonay, ed.), VCH, New York, 1992, pp 1–15 Goodall, D M and D K Lloyd, A note on an optical rotation detector for high-performance liquid chromatography, in Chiral Separations (D Stevenson and D Wilson, eds.), Plenum Press, New York, 1988, pp 131–133 Sheldon, R A., Introduction to optical isomersion, in Chirotechnology: Industrial Synthesis of Optically Active Compounds, Marcel Dekker, Inc., New York, 1993, pp 25 –27 Weston, A and P R Brown, HPLC and CE Principles and Practice, Academic Press, San Diego, CA, 1997 Yeung, E S., Polarimetric detectors, in Detectors for Liquid Chromatography (E S Yeung, ed.), John Wiley & Sons, New York, 1986, pp 204 –228 Optical Quantification (Densitometry) in TLC Joseph Sherma Lafayette College, Easton, Pennsylvania, U.S.A Introduction Quantitative evaluation of thin-layer chromatograms can be performed by direct, in situ visual, and indirect elution techniques Visual evaluation involves comparison of the sizes and intensities of color or fluorescence between sample and standard zones spotted, developed, and detected on the same layer The series of standards is chosen to have concentrations or weights that bracket those of the sample zones After matching a sample with its closest standard, accuracy and precision are improved by respotting a more restricted series of bracketing standards with a separate sample spot between each of two standard zones Accuracy no greater than –10% is possible for trained personnel using visual evaluation The determination of mycotoxins in food samples is an example of a practical application of visual comparison of fluorescent zones The elution method involves scraping off the separated zones of samples and standards and elution of the substances from the layer material with a strong, volatile solvent The eluates are concentrated and analyzed by use of a sensitive spectrometric method, gas or liquid column chromatography, or electroanalysis Scraping and elution must be performed manually because the only commercial automatic micropreparative elution instrument has been discontinued by its manufacturer The elution method is tedious and timeconsuming and prone to errors caused by the incorrect choice of the sizes of the areas to scrape, incomplete collection of sorbent, and incomplete or inconsistent elution recovery of the analyte from the sorbent However, the elution method is being rather widely used (e.g., some assay methods for pharmaceuticals and drugs in the USP Pharmacopoeia) Introduction to Densitometry In order to achieve the optimum accuracy, precision, and sensitivity, most quantitative analyses are performed by using high-performance thin-layer chromatography (TLC) plates and direct quantification by means of a modern optical densitometic scanner with a fixed sample light beam in the form of a rectangular slit that is variable in height (e.g., 0.4 –10 mm) and width (20 mm to mm) Densitometers measure the difference in absorbance or fluorescence signal between a TLC zone and the empty plate background and relate the measured signals from a series of standards to those of unknown samples through a calibration curve Modern computer-controlled densitometers can produce linear or polynomial calibration curves relating absorbance or fluorescence versus weight or concentration of the standards and determine bracketed unknowns by automatic interpolation from the curve Samples and standards are best applied using an automated instrument such as the one shown in Fig Use of manual spotting and less efficient TLC plates results in greater errors and poorer reproducibility in quantitative results Instrumental Design and Scanning Modes A commercial densitometer and a schematic diagram of the light-path arrangement used in scanning are shown in Fig The plate is mounted on a moveable stage controlled by a stepping motor drive that allows each chromatogram track to be scanned in or against the direction of development A tungsten or halogen lamp is used as the source for scanning colored zones in the 400 – 800-nm range (visible absorption) and a deuterium lamp for scanning ultraviolet (UV)-absorbing zones directly or as quenched zones on phosphorcontaining layers (F-layers) in the 190 – 450-nm range The monochromator used with these continuouswavelength sources can be a quartz prism or, more often, a grating The detector is a photomultiplier or photodiode placed above the layer to measure reflected radiation [Some scanners (e.g., Fig 2) make use of a reference photomultiplier in addition to the measuring photomultiplier in the single-beam mode; the reference photomultiplier puts out a constant signal that is compared to the signal from the measuring photomultiplier to produce a difference signal that is more accurate than a direct signal from a single measuring photomultiplier would be.] Encyclopedia of Chromatography DOI: 10.1081/E-Echr 120005247 Copyright © 2002 by Marcel Dekker, Inc All rights reserved Optical Quantification (Densitometry) in TLC Fig Photograph of the DESAGA Densitometer CD 60 with a superimposed schematic diagram of the light path including (right to left) the source lamp, two mirrors, grating monochromator, mirror, beam splitter, plate with chromatograms to be scanned, reference and measuring detectors (reflection) above the plate and detector (transmission) below the plate (Courtesy of DESAGA GmbH, Weisloch, Germany.) Fig Automatic TLC sampler (ATS 3) used for computercontrolled application of precisely controlled volumes of samples and standards between 10 nL and 50 mL from a rack of vials as spots or bands to preselected origins on a plate (Courtesy of Camag Scientific Inc., Wilmington, NC.) For normal fluorescence scanning, a high-intensity xenon continuum source or a mercury vapor line source is used, and a cutoff filter is placed between the plate and detector to block the exciting UV radiation and transmit the visible emitted fluorescence For fluorescence measurement in the reversed-beam mode, a monochromatic filter is placed between the source and plate and the monochromator between the plate and detector In this mode, the monochromator selects the emission wavelength, rather than the excitation wavelength as in the normal mode Simultaneous measurement of reflection and transmission, or transmission alone, can be carried out by means of a detector positioned on the opposite side of the plate (Fig 2) Ratio-recording double-beam densitometers, which can correct for background disturbances and drift caused by fluctuations in the source and detec- tor, were designed earlier with two photomultiplier detectors simultaneously recording the two beams (double beam in space), but, today, such densiotmeters are equipped with a chopper and one detector (double beam in time) For dual-wavelength, single-beam scanning, which will correct for scattering of the absorbed light by subtracting out the (presumably equal) scattering at a nonabsorbed wavelength, a light beam is selected by a mirror and passes through two separate monochromators to isolate the two different wavelengths The two beams are alternated by a chopper and recombined into a single beam representing a difference signal at the detector Zigzag or meandering scanning with a small point or spot of light is possible with densitometers having two independent stepping motors to move the plate in the X and Y axes Computer algorithms integrate the maximum absorbance measurements from each swing to produce a distribution profile of zones having any shape The potential advantages of scanning with a moving light spot are offset by problems with lower spatial resolution and errors in data processing, and the method is not as widely used as conventional scanning of chromatographic tracks with a fixed slit Some densitometers have the ability to rotate the plate while scanning for measurement of circular and anticircular chromatograms Single-wavelength, single-beam, fixed-slit scanning is most often used and can produce excellent results Optical Quantification (Densitometry) in TLC when high-quality plates and analytical techniques are employed Spectral Measurement Many modern scanners have a computer-controlled motor-driven monochromator that allows automatic recording of in situ absorption and fluorescence excitation spectra These spectra can aid compound identification by comparison with stored standard spectra, test for identity by superimposition of spectra from different zones on a plate, and check zone purity by superimposition of spectra from different areas of a single zone The spectral maximum determined from the in situ spectrum is usually the optimal wavelength for scanning standard and sample areas for quantitative analysis Data Handling The densitometer is connected to a recorder, integrator, or computer A personal computer with software designed specifically for TLC is most common for data handling and automation of the scanning process in modern instruments With a fully automated system, the computer can carry out the following functions: data acquisition by scanning a complete plate following a preselected geometric pattern with control of all scanning parameters; automated peak searching and optimization of scanning for each fraction located; multiple-wavelength scanning to find, if possible, a common wavelength for all substances to be quantified, to optically resolve fractions incompletely separated by TLC, and to identify fractions by comparison of spectra with standards cochromatographed on the same plate or stored in a spectrum library through pattern recognition techniques; baseline location and correction; computation of peak areas and/or heights of samples and codeveloped standards and processing of the analog raw data to quantitative digital results, including calculation of calibration curves by linear or polynomial regression, interpolation of sample concentrations, statistical analysis of reproducibility, and presentation of a complete analysis report; and storage of raw data on disk for later reintegration, calibration, and evaluation with different parameters Calibration Curves Densitometric calibration curves relating absorption signal and concentration or weight of standards on the layer are usually nonlinear, especially for higher amounts of standards, and not pass through the origin Fluorescence calibration curves are generally linear and pass through the origin, and analyses based on fluorescence are more specific and 10 –1000 times more sensitive The advantages of fluorescence measurement may be realized for nonfluorescent compounds by prechromatographic or postchromatographic derivatization reactions with suitable fluorogenic reagents Because the incident monochromatic light is absorbed, reflected, and scattered by the opaque layer material, the theoretical relationship between amount of absorption and amount of substance does not follow the simple Beer–Lambert law that is valid for solutions The Kubelka–Munk equation is the most accepted theoretical relationship for TLC, but its use is not necessary because of the ability of densitometer software to handle empirical nonlinear regression functions Image Analysis (Videodensitometry) Video camera systems are available from several manufacturers for documentation and densitometric quantification of TLC plates As an example, the Camag VideoScan instrument consists of a lighting module with short- and long-wave UV and visible sources upon which the layer is placed, a chargecoupled device (CCD) camera with zoom and longtime integration capability, and a PC under MSWindows control with frame grabber, monitor, and printer The available software for quantitative evaluation allows the display of the tracks of the chromatogram image acquired with the video camera as analog curves and calculation of their peak properties (Rf , height, area, height percent, and area percent) For quantification, the computer creates a standard curve from the areas or heights of the standards and interpolates unknown values from the curve Video scanners have potential advantages, including rapid data collection, simple design with virtually no moving parts, and ability to quantify two-dimensional chromatograms, but they have not yet been shown to have the required capabilities, such as sufficient spectral discrimination or the ability to illuminate the plate uniformly with monochromatic light of selected wavelength, to replace slit-scanning densitometers Current video scanners can measure spots in the visible range in transmittance, reflectance, or fluorescence modes, but they cannot perform spectral analysis Optical Quantification (Densitometry) in TLC Applications and Practical Aspects of Densitometry Densitometric quantification has been applied to virtually every type of analyte and sample For example, the greatest number of applications is for the analysis of drug and pharmaceutical compounds, most of which have structures including chromophores that cause strong UV absorption These compounds are readily quantified in the fluorescence quenching mode on Flayers or in the direct UV absorption mode on unimpregnated layers Lipids are compounds that are not easily analyzed by gas chromatography (GC) or highperformance liquid chromatography (HPLC) because they lack volatility and the presence of a chromophore leading to UV absorption The most successful way to quantify lipids is by densitometry after separation and detection on the layer with a chromogenic reagent, most notably phosphomolybdic acid The quantification of amino acids after detection with ninhydrin is another example of densitometry in the visible absorption mode Fluorescence densitometry has been applied to the determination of naturally fluorescent compounds (e.g., quinine in tonic water) or compounds derivatized with a fluorogenic reagent pre-TLC or post-TLC (e.g., amino acids reacted with fluorescamine, or carbamate pesticides with dansyl chloride after hydrolysis) The steps in a typical densitometric quantitative analysis, regardless of analyte type, are the following: Prepare a standard reference solution Prepare a sample solution in which the analyte is completely dissolved and impurities have been reduced to a level at which they not interfere with scanning of the analyte Choose a layer and mobile-phase combination that will separate the analyte as a compact zone with an Rf value in the range 0.2 – 0.8 Apply the standard and sample aliquots to the layer using an instrument (Fig 1) or manually with a micropipette, onto preadsorbent, laned plates Generally, three or four standard zones are applied in constant volumes from a series of standard solutions with increasing concentrations, or in a series of increasing volumes from a single standard solution The sample volume applied must provide an amount of analyte zone with a weight or concentration that is bracketed by the standard amounts Develop the plate in an appropriate chamber and dry the mobile phase under in a fume hood or oven Apply a detection reagent, if necessary, by spraying or dipping The reagent should produce a stable colored, UV-absorbing, or fluorescent zone having high contrast with the layer background Scan the natural or induced absorption or fluorescence of the standard and sample zones on the plate using a densitometer with optimized parameters Generate a calibration curve by linear or polynomial regression of the scan areas and weights of the standards and interpolate the weights in the sample zones from the curve Calculate the concentration of analyte in the sample from the original weight of the sample, the original total volume of the sample test solution, the aliquot volume of the test solution that is spotted, the interpolated analyte weight in that spotted volume from the calibration curve, and any numerical factor required because of dilution or concentration steps needed for the test solution to produce a bracketed scan area for the analyte zone in the sample chromatogram 10 Validate the precision of the TLC analysis by replicated determination of the sample and accuracy by comparison of the results to those obtained from analysis of the same sample by an established independent method or calculation of recovery from analysis of a spiked preanalyzed sample or spiked blank sample The following are some advantages of TLC densitometry compared to HPLC: The simultaneous analysis of multiple samples on a single plate leads to higher sample throughput (lower analysis time) and less cost per sample Up to 36 tracks are available for samples and standards on a 10-cm ϫ 20-cm high-performance TLC plate The ability to generate a unique calibration curve using standards developed under the same conditions as samples on each plate (insystem calibration) leads to statistical improvement in data handling and better analytical precision and accuracy and eliminates the need for an internal standard for most analyses Detection is versatile and flexible because the mobile phase is removed prior to detection Because the detection process is static (the zones are stored on the layer), multiple, complementary detection methods can be used Optical Quantification (Densitometry) in TLC Storage of the chromatogram also allows scanning to be repeated with various parameters without time constraints and assures that the entire sample is available for detection and scanning Less sample cleanup is often required because plates are not reused Every sample is analyzed on a fresh layer without sample carryover or cross-contamination Solvent use is very low for TLC, both on an absolute and per-sample basis, leading to reduced purchase and disposal costs and safety concerns Suggested Further Reading Fried, B and J Sherma, Thin Layer Chromatography— Techniques and Applications, 4th ed., Marcel Dekker, Inc., New York, 1999, pp 197–222 Jaenchen, D E., Instrumental thin layer chromatography, in Handbook of Thin Layer Chromatography, 2nd ed (J Sherma and B Fried, eds.), Marcel Dekker, Inc., New York, 1996, pp 129 –148 Petrovic, M., M Kastelan-Macan, K Lazaric, and S Babic, Validation of thin layer chromatography quantitation with CCD camera and slit-scanning densitometer, J AOAC Int 82: 25 –39 (1999) Pollak, V A., Theoretical foundations of optical quantitation, in Handbook of Thin Layer Chromatography (J Sherma and B Fried, eds.), Marcel Dekker, Inc., New York, 1991, pp 249 –281 Poole, C F and S K Poole, Chromatography Today, Elsevier, New York, 1991, pp 649 –734 Prosek, M and M Pukl, Basic principles of optical quantitation in TLC, in Handbook of Thin Layer Chromatography, 2nd ed (J Sherma and B Fried, eds.), Marcel Dekker, Inc., New York, 1996, pp 273–306 Robards, K., P R Haddad, and P E Jackson, Principles and Practice of Modern Chromatographic Methods, Academic Press, San Diego, CA, 1994, pp 180 –226 Optimization of Thin-Layer Chromatography Wojciech Prus Technical University of jód´z, Bieisko-Biaia, Poland Teresa Kowalska Institute of Chemistry, Silesian University, Katowice, Poland Introduction The principal task of chromatography is the separation of mixtures of substances By “optimization” of the chromatographic process, we mean enhancement of the quality of the separation by changing one or more parameters of the chromatographic system An ability to foresee, correctly, the direction and scope of these changes is the most important goal of each optimization procedure Use of chemometrics to devise procedures suitable for the most crucial stage of optimization, optimization of selectivity, is generally performed in three steps: Selection of the experimental method which best suits the analytical problem considered At this stage, a chromatographic technique is chosen that ensures that the best possible range of retention parameters is obtained for each individual component of the separated mixture Establishing the experimental conditions that enable quantification of the influence of the optimized parameters of a chromatographic system on solute retention Fixing the experimental conditions at values that provide the optimum separation selectivity Chemometric optimization of the chromatographic system consists, in fact, in predicting local maxima in multiparametric space and, then, in further deciding which of these parameters is global with regard to the overall efficiency of a given chromatographic system Quality of Chromatographic Separations Elementary Criteria ference between their respective retention parameters; that is, the difference between their RF values, ¢RF ϭ RF2 Ϫ RF1 (1) or between their RM values, ¢RM ϭ RM1 Ϫ RM2 ϭ log k1 ϭ log a k2 (2) where k1 and k2 are the capacity (retention) factors of the chromatographic bands and a is the separation factor The terms most frequently used to characterize the separation of two chromatographic bands are the separation factor, a, aϭ k1 k2 (3) where k1 Ͼ k2 , and the resolution, RS [1], RS ϭ 21z2 Ϫ z1 ϭ w1 ϩ w2 2l¢RF w1 ϩ w2 (4) where z1 and z2 are the distances of the geometric centers of two chromatographic bands, and 2, from the origin, l is the distance from the origin to the mobile phase front, and w1 and w2 are the diameters of the two chromatographic bands, measured in the direction of eluent flow Other elementary criteria include the separation factor, S [2], Sϭ k2 Ϫ k1 k1 ϩ k2 ϩ (5) the peak-to-valley ratio of the bands, P [3], Pϭ f g (6) (where f and g are, respectively, the average peak height and valley depth, characteristic of a given pair of neighboring solutes on a chromatogram), the fractional peak overlap, FO [4], The simplest way of quantifying the separation of two chromatographic bands, and 2, is to calculate the dif- Encyclopedia of Chromatography DOI: 10.1081/E-Echr 120005248 Copyright © 2002 by Marcel Dekker, Inc All rights reserved FO ϭ An Ϫ An, nϪ1 Ϫ An, nϩ1 An (7) Optimization of Thin-Layer Chromatography (where An is the surface area of the part of the band originating from the pure single compound, An, nϪ1 is the surface area of the fractional overlap of the nth and (n Ϫ 1)th bands, and An, nϩ1 is the surface area of the fractional overlap of the nth and (n ϩ 1)th bands), and the selectivity parameter, RR [5], RR ϭ RF1 RF2 (8) where RF1 Ͼ RF2 nϪ1 R nϪ1 S Si, iϩ1 i, iϩ1 rϭ q ϭ q RS S iϭ1 iϭ1 (10) where n is the number of the chromatographic bands, Criteria for the Quality of Chromatograms RS ϭ One method which can be used to establish the optimum conditions for the separation of a complex mixture (i.e., not only a pair) of compounds consists in searching for the maximum of a function denoted the chromatogram quality criterion The evaluation of separation selectivity can be conducted with the aid of different criteria of chromatogram quality such as the sum of resolution, ͚ RS [6], the sum of separation factors, ͚ S [2], and other sums and products of elementary criteria, selected examples of which are the resolution product, ß RS [7], q RS ϭ exp a a ln RS b , the product of the separation factors, ß S [8], the product of the fractional peak overlap, ß FO [9], and the product of the peak-to-valley ratio of the bands, ß P [10] There are also other, more complex criteria, including the normalized resolution product, r [11], (9) nϪ1 RSi, iϩ1 nϪ1 a iϭ1 and Sϭ nϪ1 Si, iϩ1 nϪ1 a iϭ1 and the minimum RS [8], RS, Ն x or max RS, (11) The minimum of a is used as a criterion of the quality of chromatograms in liquid chromatography [12] Other criteria are the total peak overlap, w [13], w ϭ a exp1Ϫ2RS 2, (12) the informing power, Pinf [14], n Pinf ϭ a log2 Si (13) iϭ1 and the chromatographic response function, CRF [10], n CRF ϭ a ln Pi (14) iϭ1 (where Pi is the peak-to-valley ratio for the ith pair of chromatographic bands) Performance of the Chromatographic System One measure of the performance of a given chromatographic system is the number of the theoretical plates per chromatographic band (N) In its simplest form, this can be defined as Nϭ Fig Graphical interpretation of the selected elementary criteria: (a) resolution, Rs ; (b) the peak-to-valley ratio, P; (c) the fractional peak overlap, FO l H (15) where l is the distance from the origin to the eluent front and H is the height equivalent to one theoretical plate (H is sometimes also denoted HETP) The average height equivalent to one theoretical plate (H ) can be calculated from the relationship [15] Zeta-Potential In the example in Fig 1, a mixture of anions is analyzed in a Tris-acetate buffer of pH A 2% change in zeta-potential, from Ϫ50 to Ϫ51 mV, results in a dramatic shift in migration time of peaks with longer migration times Suggested Further Reading migration time [min] Fig Effect of capillary zeta-potential on the analysis of a mixture and anions in an uncoated fused-silica capillary in a 10 mM Tris-acetate buffer of pH Normal zeta-potentials are around 50 mV at this pH Beckers, J L., Isotachophoresis, some fundamental aspects, Thesis, Eindhoven University of Technology, 1973 Boˇcek, P., M Deml, P Gebauer, and V Dolník, Analytical Isotachophoresis, VCH, Weinheim, 1988 Everaerts, F M., J L Beckers, and Th P E M Verheggen, Isotachophoresis: Theory, Instrumentation and Applications, Elsevier, Amsterdam, 1976 Hjertén, S Chromatogr Rev 9: 122 (1967) Jorgenson, J W and K D Lucaks, Science 222: 266 (1983) Kenndler, E., J Capillary Electrophoresis 3(4): 191 (1996) Li, S F Y., Capillary Electrophoresis—Principles, Practice and Applications, Elsevier, Amsterdam, 1992 VanOrman, B B., G G Liversidge, G L McIntire, T M Olefirowicz, and A G Ewing, J Microcol Separ 2: 176 (1990) MARCEL DEKKER, INC • 270 MADISON AVENUE • NEW YORK, NY 10016 ©2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc Zirconia–Silica Stationary Phases for HPLC: Overview and Applications R Andrew Shalliker Sindy Kayillo University of Western Sydney, Sydney, New South Wales, Australia INTRODUCTION This review details the development of zirconia –silica stationary phases in liquid chromatography Zirconia – silica stationary phases are just one member of a group of mixed oxide materials that have recently been studied in liquid chromatography Other members of this group include alumina – silica, magnesia – silica, and titania – silica.[1,2] Each of these mixed oxide composites contains silica, which is the most widely employed stationary phase material in modern high-performance liquid chromatography (HPLC) The dominance of silica in the chromatographic industry reflects the many desirable properties that silica affords the chromatographer Silica can be prepared in a wide variety of pore size and particle size materials, and can be easily modified to yield stationary phases with a wide range of differing selectivities For instance, the C18 column displays vastly different retention behavior to that of native silica and is the most widely employed reversed phase chromatography column Other types of modified silica stationary phases include, but are not limited to, the C8 column for intermediate retention of nonpolar compounds in reversed phase LC, the C4 column for protein work, and the phenyl, nitrile, and amino columns Silica also has a high mechanical strength, making it suitable for the preparation of columns under high pressure; when operated under suitable conditions, these columns have a long life span Collectively, each of these attributes has made silica the material of choice as a stationary phase or stationary phase support In many respects, the high efficiency of today’s chromatography column owes much to the properties of silica However, as a chromatographic stationary phase, silica has some limitations The siloxane linkage is the Achilles heel in any silica-based material, being easily hydrolyzed at low pH Furthermore, silica has a high solubility in an alkaline environment, especially at higher temperatures, leading to a loss in column bed integrity This, in general, limits the use of silica and modified silica to pH regions between and Another limitation of silica is the strong and often irreversible adsorption of basic species, occurring as a consequence of Encyclopedia of Chromatography DOI: 10.1081/E-ECHR 120013344 Copyright D 2002 by Marcel Dekker, Inc All rights reserved the hydrogen bonding between the solute and the surface silanol groups This belies one of the most desirable features of a stationary phase; that being good adsorption and good desorption characteristics Many modified silica-based stationary phases are consequently subjected to rigorous procedures that attempt to protect the solute from the residual hydroxyl groups through processes such as end capping Resolution (Rs) in a chromatographic system is a function of three essentially independent variables: the capacity factor (k’) (defined generally by the solvent strength), the number of theoretical plates (N), and the selectivity (a) according to the well-known equation given below:   pffiffiffiffi k Rs ¼ N a 1ị ỵ k0 Of these three variables, changes in the selectivity very often yield the most substantial gain in resolution, albeit with results that are often unpredictable One of the driving forces in the development of new stationary phases is the change in selectivity to yield improvements in separation A second driving force is developing materials that overcome the limitations of silica as noted above One of the more successful stationary phase materials that have been developed is that of zirconia and zirconiabased stationary phases, which are now commercially available as a high-grade chromatographic product As a stationary phase, zirconia has several important attributes Zirconia is a highly rigid support able to withstand the rigors of conventional column-packing processes Zirconia is temperature-stable and is insoluble in alkaline and acidic solvents, allowing mobile phases with pHs between and 14 to be employed as eluents This is particularly useful for protein work, allowing ‘‘dirty’’ columns to be cleaned with strong base such as NaOH The surface of zirconia is, however, heterogeneous, containing Lewis acid, Bronsted acid, and Bronsted base sites, which leads to a multitude of retention mechanisms that may complicate the elution process.[3] However, because zirconia is highly resistant to solvent attack, virtually any MARCEL DEKKER, INC • 270 MADISON AVENUE • NEW YORK, NY 10016 ©2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc mobile phase modifying agent can be employed to control any unwanted interactions Consequently, the highly functionalized zirconia surface can be fully utilized to maximize selectivity Zirconia can also be synthesized in a variety of particle size and pore size materials, in much the same manner as silica Furthermore, zirconia can be modified to yield a variety of stationary phases with different selectivities Nawrocki et al.[3] published an excellent review on zirconia for chromatographic use, and hence a detailed discussion on zirconia is not warranted in this text Recent studies in stationary phase development have lead chromatographers to explore the concept of mixed oxide chromatographic supports and materials for use in liquid chromatography These include alumina –silica, magnesia – silica, titania – silica, and zirconia –silica.[1,2] The driving force behind the development of these mixed oxide supports comes from the possibility of attaining a surface with properties that exploit the best attributes of both components in the mixed oxide For example, the band tailing of basic species on magnesia –silica columns is substantially reduced compared to silica columns due to the reduced concentration of acid sites on the surface of the magnesia – silica composite.[1] In the current text, we present an overview on the development of zirconia – silica composites as stationary phase supports and then detail some applications for which this support has been employed PREPARATION OF ZIRCONIA –SILICA MIXED OXIDE SUPPORTS The demand for mixed oxide chromatographic-quality stationary phase materials is small and only a few methods that detail their preparation are available in the literature There are, however, numerous examples of the preparation of zircon powders and Ref [4] is an example of such a process In general, these types of powders are unsuitable for chromatographic stationary phases because their particle sizes are in the submicron range with very broad distributions The synthesis of mixed oxide supports for chromatographic applications can essentially be divided into two types: coprecipitation methods and coating methods Coprecipitation Methods Kaneko et al.[1,2] employed a coprecipitation method, which involved a base-catalyzed procedure that precipitated irregular particles of zirconia –silica mixed oxides Specifically, a zirconium salt (zirconyl chloride octahydrate) was added to an acidified solution of sodium metasilicate ennehydrate The solution was made alkaline Zirconia – Silica Stationary Phases for HPLC: Overview and Applications (pH 7– 9) to allow the precipitation of the mixed oxide gel The gel was aged for 10 hr, collected by filtration and washed to remove residual chloride ions The gel was dried at 110°C and then ground to an appropriate mesh size The resulting precipitate was regarded as an intimate mixture of silica and metal oxide gel (in this case, zirconia) The silica framework was partly occupied with a widely spread metal oxide, hence decreasing the surface area of the silica gel itself.[1,2] In this procedure, the gel was not subjected to thermal treatment above 110°C Zhang, Feng, and Da[5] improved on the method of Kaneko et al.[1,2] by employing an oil emulsion sol – gel procedure, resulting in the formation of mixed oxide spherical particles (spherical particles being a desirable property of a modern LC packing) In their method, sodium metasilicate pentahydrate was hydrolyzed in an acidic aqueous solution and zirconyl chloride octahydrate was added with stirring The resulting mixture was suspended in a surfactant-stabilized oil and homogenized for 10 at a speed calibrated to yield 5-mm spherical particles Precipitation of the gel was initiated by the slow addition of base The resulting hydrogel was collected and washed to remove residual solvent and dried at 120°C for hr, after which the material was calcined at 600°C for hr Coating Processes Coating processes have been used by a number of researchers Chicz, Shi, and Regnier[6] coated commercially available 15 – 20 mm Vydac silica particles with zirconia The surface coating was obtained by reacting a combination of 2,4-pentanedione, zirconium isopropoxide, and toluene (1:3:3) (v/w/v) with silica for hr The zirconylclad silica was then vacuum-dried overnight and calcined at 500°C for hr, after which the material was washed with 2-propanol and then vacuum-dried The zirconylclad silica was further modified with polyethyleneimine (PEI) to produce an ion-exchange surface that was tested for retention of proteins The pore diameter of the resulting zirconyl-clad silica (22.6 nm) was approximately 75% of the original Vydac silica (300 nm), while the surface area decreased marginally ($10%) Peixoto, Gushiken, and Baccan[7] employed a similar coating process to prepare zirconized silica Silica particles were added to a solution of ZrCl4 in anhydrous ethanol The mixture was refluxed for hr under a nitrogen atmosphere The zirconized silica was then collected and dried at approximately 125°C, before being hydrolyzed and washed in water to remove the residual chloride ions The final product was dried at 120°C for hr The resulting zirconized silica had a 3.25% ZrO2 surface coverage Melo et al.[8,9] modified the method of Peixoto et al and coated 10-mm silica particles with MARCEL DEKKER, INC • 270 MADISON AVENUE • NEW YORK, NY 10016 ©2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc Zirconia – Silica Stationary Phases for HPLC: Overview and Applications zirconia by reacting the silica with zirconium(IV) butoxide in toluene according to the following reaction process:[8] n SiOH ỵ ZrOBuị4 ! SiOịn ZrOBuị4n ỵ nBuOH SiOịn ZrOBuị4n ỵ nịH2 O ! SiOịn ZrOHị4n ỵ nÞBuOH where SiOH stands for silanol groups on the silica surface Following hydrolysis, the zirconized silica contained 15.3% Zr on the surface More recently, a similar procedure for the preparation of zirconia-coated silica microspheres was employed by Tsurita and Nogami,[10] who coated 3-mm silica spheres with zirconia derived from zirconyl chloride octahydrate The resulting zirconia –silica spheres were subjected to thermal treatment in order to bring about crystallization and the subsequent development of the porous structure within the material A coating process was also employed by Shalliker et al.[11] where zirconia –silica mixed oxide supports were prepared by coating preformed zirconia microspheres with silica, the reverse process to the methods of Chicz et al.,[6] Peixoto et al.,[7] Melo et al.,[8,9] and Tsurita and Nogami.[10] In this method,[11] zirconia microspheres were first formed using a sol –gel method The resulting spherical zirconia hydrogels were suspended in water and a solution of hydrolyzed sodium metasilicate pentahydrate was added The quantity of sodium metasilicate pentahydrate varied depending upon the desired Zr/Si ratio in the final composite The solution ($200 mL) containing the zirconia microspheres and the hydrolyzed sodium metasilicate pentahydrate was heated to 60°C, allowing the slow evaporation of water to a final volume of 15% of the original amount The microspheres were collected by filtration and then washed Calcination of the material followed, which influenced the crystallization and resulting surface properties of the support.[11–15] Extended calcination at 1300°C resulted in the complete homogenization of the support, yielding zircon.[11] et al.[3] It is perhaps important to note that zirconia is amorphous until subjected to temperatures that exceed the phase transition temperature required to form the tetragonal phase (usually between 440 and 470°C depending on the procedure used to prepare the zirconia) Once thermal treatment exceeds this phase transition temperature, a metastable tetragonal phase predominates—where, after cooling, zirconia slowly transforms to the stable monoclinic form As thermal treatment of the zirconia is required to obtain a suitable pore-structured stationary phase, most zirconias prepared for chromatographic purposes are predominantly monoclinic, or perhaps more correctly polymorphous.[3] Complete conversion from the amorphous phase to the monoclinic phase requires a substantial degree of thermal treatment and then time to allow the tetragonal phase to form the monoclinic phase In contrast, the most common form of silica as a stationary phase material is usually the amorphous form,[16] but, on occasion, Cristobalite has been reported.[10] Silica doping of the zirconia, however, has been shown to have a marked effect on the crystallization of the zirconia phase.[4,5,11 – 15] As the concentration of silica increased, the phase transition temperature from the amorphous phase to the metastable tetragonal phase increased.[12] This was independent of whether the material was prepared through a coprecipitation process [5] or by a coating process.[11] For example, the relationship between the phase transition temperature and the percentage weight of silica in the zirconia– silica composite is shown in Fig As the concentration of silica doping increased, the phase transition temperature increased monotonically and was linear until 700°C.[11] Furthermore, the addition of the silica stabilized the metastable PROPERTIES OF ZIRCONIA– SILICA MIXED OXIDE SUPPORTS Crystallization The crystalline nature of zirconia microspheres prepared for chromatographic purposes is dependent upon the thermal history of the zirconia A detailed discussion on the porous nature of zirconia and silica is not warranted in this text but is covered in detail in a review by Nawrocki Fig Plot showing the relationship between the phase transition temperature from the amorphous to the tetragonal state and the composition of silica in the composite material (From Ref [12].) MARCEL DEKKER, INC • 270 MADISON AVENUE NEW YORK, NY 10016 â2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc Zirconia – Silica Stationary Phases for HPLC: Overview and Applications tetragonal phase, which delayed the transformation to the monoclinic phase.[11] In general, increased silica content decreased the apparent crystallinity Studies by Tsurita and Nogami[10] on zirconia-coated silica microspheres support the findings described above In their work, as the concentration of Zr doping increased (or, in other words, the relative concentration of Si decreased), there was a corresponding increase in the concentration of tetragonal zirconia Furthermore, increasing concentrations of ZrO2 suppressed the crystallization of silica gel Pore Structures Many of the most important properties of a chromatographic stationary phase relate to the pore structure, which provides a high surface area and pore volume for solute interaction for the chromatographic retention process to occur As such, the pores should be of a suitable size to allow the solutes of interest to have unimpeded entry and exit to and from the pores Microporous materials are of little interest in the chromatographic industry They provide a high surface area, but one that is inaccessible to the vast majority of solutes Mesoporous materials are the most sought after, with pore sizes between and 50 nm, although macroporous materials find specialized use in the separation of macromolecules, e.g., Ref [16], but at the cost of a reduced surface area Ideally, the pore shape should represent that of a cylinder and should be described by a type IV nitrogen sorption isotherm[17] as depicted in Fig 2(a) These types of isotherms are common in many industrial adsorbents with mesoporous structures The hysteresis loop in the type IV isotherm is usually associated with capillary condensation in a mesoporous structure These hysteresis loops can form several shapes; however, from a chromatographic aspect, the most important type of hysteresis loop is the type H1 (Fig 2(b)), which represents pores that are uniform in shape and with a narrow size distribution Many chromatographic surfaces display type H2 or intermediate H1 – H2 hysteresis loops, which in a simplified form represents surfaces that have pore structures with wide bellies and narrow necks (‘‘ink bottle’’ pores).[17] In general, the pore size and subsequent surface area of zirconia-containing supports is a function of the thermal history, i.e., the crystalline nature Calcination of the material at high temperatures leads to an increase in the pore size and a reduction in the surface area, as a consequence of particle sintering and crystallization.[3] Kaneko et al.[1] measured the physical properties (pore size distributions, specific surface areas, and pore volumes) of their stationary phases using nitrogen adsorption isotherms The surface area and pore diameter of their stationary phase material reflected the absence of calcination at temperatures high enough to induce crystallization Their stationary phase material was predominantly microporous, with a high surface area (122 m2/g) No mention was made with regard to the pore shape In comparison, the stationary phases prepared using the coprecipitation process of Zhang, Feng, and Da[5] with Fig (a) Types of physisorption isotherms (b) Types of hysteresis loops (From Ref [17].) MARCEL DEKKER, INC • 270 MADISON AVENUE • NEW YORK, NY 10016 ©2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc Zirconia – Silica Stationary Phases for HPLC: Overview and Applications thermal treatment at 600°C were mesoporous The mean pore diameters were 8.6 nm for a composite containing 3.4% Si and 5.9 nm for a composite containing 15.8% Si These stationary phase materials had surface areas of 29 and 76 m2/g, respectively It is of interest to note that the surface area for the composite containing the higher silica content was greater than the surface area for the composite with the lower silica content The average pore diameter of the material with the low silica content was greater than the average pore diameter of the composite with higher silica content This reflects the decrease in crystallinity associated with an increase in the concentration of silica doping,[5] and the change in density due to the different zirconia concentrations The composite materials prepared by Zhang, Feng, and Da[5] were reported as being type IV isotherms with H1 hysteresis loops, making them ideally suited as chromatographic adsorbents The pore structures of zirconia – silica composites prepared by Shalliker et al.[11–15] via a coating procedure were substantially different to those reported by Zhang, Feng, and Da.[5] In studies by Shalliker et al.,[13–15] the pore characteristics were determined using inverse size exclusion chromatography and nitrogen sorption The inverse size exclusion technique was employed to measure the chromatographically available pore surface, while the nitrogen sorption measurements were carried out for comparative purposes with information already available in the literature A range of composites were prepared with either or 10 mol% silicon.[14,15] These composites were calcined for hr at either 700 or 810°C Calcination of these materials at 810 or 700°C yielded composites that were either predominantly amorphous or tetragonal depending on the concentration of the silicon doping The increase in the phase transition temperature compared to that of native zirconia was consistent with the degree of silicon doping For a composite material containing 10 mol% Si and calcined for hr at 810°C, a type IV nitrogen sorption isotherm with an H3 hysteresis loop was obtained.[15] The type H3 hysteresis loop extended from the high relative pressure region to P/Po approximately 0.42, indicating that the surface had a broad pore size distribution The close proximity of the adsorption and desorption branches indicated that the entrances of the pores offered little restriction to the pore body This type of hysteresis loop was consistent with a surface containing slit-like pores The BET-specific surface area of the composite was 2.7 m2/g and the pore size distribution was very broad and skewed significantly to the macroporous region, with a maximum at 3.4 nm.[15] The specific pore volume of this material was very low (0.0056 mL/g), consistent with a macroporous material In comparison, the specific pore volume of the stationary phases prepared by Zhang, Feng, and Da[5] were 0.062 and 0.112 mL/g for the 3.4% Si and 15.8% Si composites, respectively The specific pore volume of the stationary phase prepared by Kaneko et al.[1] was 0.17 mL/g The size exclusion curve for polystyrenes that eluted from a column prepared from the 10 mol% silicon composite of Shalliker et al.[14] also indicated that the material had a broad pore size distribution that extended well into the macroporous region No exclusion limit was determined despite the elution of polymers with molecular weights in excess of 1.8 million Da The mean pore diameter determined from the inverse size exclusion method was 56.2 nm (essentially a macroporous support) and the specific surface area agreed remarkably well with the BET-specific surface area (2.75 compared to 2.7 m2/g) Reducing the calcination temperature from 810 to 700°C, which was below the phase crystallization temperature for the same 10 mol% silicon phase, yielded an amorphous material The mean pore diameter was 8.9 nm and there was a significant microporous contribution Because of the microporous nature of this amorphous material, the surface area was almost 20-fold greater with a pore volume almost threefold higher than the predominantly tetragonal phase material obtained following calcination at 810°C.[14] Reducing the concentration of silicon to mol% and calcining at 700°C yielded a very similar material to the one containing 10 mol% silicon, which was also calcined at 700°C However, there was a significant concentration of the tetragonal phase in the mol% silicon material, which was not the case for the 10 mol% silicon phase calcined at 700°C The phase transition temperature of mol% silicon support was approximately 700°C, hence the presence of the tetragonal phase Fig illustrates the cumulative pore volumes of these three supports This figure depicts the change in porosity as a function of the silica doping Fig Cumulative pore frequency plots of composite zirconia – silica stationary phases (a) ZS5-700, (b) ZS10-700, and (c) ZS10-810 (From Ref [14].) MARCEL DEKKER, INC • 270 MADISON AVENUE NEW YORK, NY 10016 â2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc concentration and the thermal treatment of the support For instance, for both phases calcined at 700°C, approximately 50% of the pores were less than nm in diameter, whereas there were essentially no pores of this diameter for the material calcined at 810°C The authors concluded in their study that because of the unfavorable pore shape, such supports would be unsuitable for chromatographic purposes Several researchers have investigated the inclusion of high melting point salts during the calcination processes in the synthesis of silica,[10,18] zirconia,[19] and zirconia – silica composites.[10,13–15] The function of the salt is to improve the pore structure of the material The pores are homogeneously filled with a salt solution (typically sodium chloride), which is allowed to evaporate to dryness leaving behind salt deposited in the pores Upon sufficient heating, both the salt and the stationary phase material partially liquefy and phase separation occurs.[18] The size and shape of the pores depend on the nature of the salt and on the calcination conditions The stationary phase and salt solidify when cooled, and residual salt can be removed by washing, leaving behind widened pores In the case of silica, this procedure has been used to prepare wide pore (>1000 nm) materials designed for bioseparations.[18] The surface structure of a LiChrosphere Si100 stationary phase that has been subjected to pore widening using a salt impregnation process is illustrated in Fig Salt impregnation during calcination Zirconia – Silica Stationary Phases for HPLC: Overview and Applications transformed mesoporous zirconia with surfaces that had type IV nitrogen isotherms with H1 –H2 hysteresis loops to surfaces with type H1 hysteresis loops No substantial increase in the overall pore diameter was observed although the salt transformed ‘‘ink bottle’’-shaped pores to cylindrical-shaped pores.[19] Salt impregnation of zirconia– silica composites resulted in materials that had type H3 hysteresis loops being transformed to materials with intermediate type H1 – H2 hysteresis loops.[15] The addition of salt resulted in a more than 15-fold increase in the pore volume and a greater than sixfold increase in the surface area (measured by the BET method).[15] The pore size distribution changed from being a very broad distribution for a zirconia– silica composite calcined without salt to one being mesoporous when calcined with salt Even with the aid of salt impregnation, evaluation of the nitrogen sorption isotherms showed evidence of pores with hindered access The mean pore radius was 21.0 nm when measured from the adsorption branch and 10.0-nm mean pore radius when measured from the desorption branch of a nitrogen sorption isotherm The difference between the two mean pore radii determined from the adsorption and desorption branches indicated that the pores contain wider bellies with some degree of restriction near the pore entrance Hence, the authors’ concluded that these surfaces contained a significant degree of type H2 hysteresis and the hindered pore access limited their chromatographic suitability.[15] Surface Acidity Fig Scanning electron micrograph of LiChrosphere Si100 soaked in NaCl solution and dried, heated to 800°C for hr, washed out with water, and dried (From Ref [18].) The surface of zirconia is known to be basic, with a pKa of between 10.5 and 12.8 The acidity of silica is dependent upon the state of hydrolysis, typically imparting a slight acidic nature to the surface.[3] Very little information has been published with respect to the acid/base properties of zirconia– silica composites and this is not surprising given the limited experience chromatographers have with these types of stationary phases Zhang, Feng, and Da[5] determined that the acidity of their zirconia– silica composite increased as the concentration of silica increased This finding was consistent with an increase in the concentration of acidic hydroxyl groups associated with the increase in silica content In contrast, Kaneko et al measured the acid/base properties of zirconia– silica mixed oxide supports via an amine titration method.[1] They found that in a comparison between the acidity of the four mixed oxides (alumina –silica, titania –silica, magnesia – silica, and zirconia –silica), only the zirconia– silica composite contained strong acid sites—despite the fact that both alumina and silica are more acidic than zirconia.[3] All four mixed oxides exhibited stronger acidity than native silica, which is somewhat surprising given the findings of Zhang, Feng, and Da.[5] This may indicate that routine preparation of these mixed oxide supports is sen- MARCEL DEKKER, INC • 270 MADISON AVENUE NEW YORK, NY 10016 â2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc Zirconia – Silica Stationary Phases for HPLC: Overview and Applications sitive to the process employed including thermal treatment, dehydroxylation, and rehydroxylation Surface Modification Two excellent reviews that detail procedures for the preparation of bonded phase supports have recently been published by Leonard[16] and Buchmeiser.[20] One of the most popular methods of surface chemical modification involves the use of organosilanes These organosilanes react with the surface metal hydroxyl groups and form a surface, which may be represented as M– O – R, where R represents an alkyl chain and M represents the metal (i.e., silica, zirconia, titania, etc.) One important factor that must be stated, however, is that the order of stability of M –O –R bonds increases in the order of M=Si > Zr > Ti > Al.[16] Improvements in the hydrolytic stability can be achieved by shielding the siloxane bond with, for instance, polymeric phases or an intensive multivalent attachment of the bonded layer.[16] As zirconia – silica composites are relatively new surfaces in chromatography, few studies have detailed methods that modify the surface One research group, however, has reported the preparation of reversed phase zirconia– silica stationary phases.[8,9] In studies by Melo et al.,[8,9] a C8 stationary phase material was prepared by grafting poly(methyloctasiloxane) (PMOS) to the zirconia – silica composite followed by a gamma irradiation immobilization procedure Surface modification was achieved by drying the zirconia– silica particles in air at 120°C for 24 hr PMOS was dissolved in dichloromethane at a concentration to yield g PMOS + support per 12 mL dichloromethane, resulting in a packing material with 50% w/w PMOS The mixture (PMOS + zirconia –silica particles + dichloromethane) was stirred for hr at room temperature after which the solvent was allowed to evaporate The PMOS-sorbed zirconia – silica microspheres were then subjected to gamma irradiation at either 80 or 120 kGy using a Co-60 source Columns prepared from the grafted and gamma-immobilized PMOS were tested for performance and compared to PMOS-sorbed silica and PMOS-sorbed zirconia –silica stationary phases that were not subjected to gamma irradiation The authors concluded that the gamma irradiation of the PMOS-sorbed zirconia– silica composite improved the chromatographic performance for neutral and basic species Furthermore, the gamma-irradiated PMOS-sorbed zirconia– silica stationary phase was shown to have superior chromatographic performance following alkaline stability trials when compared to PMOS-sorbed silica or zirconia –silica stationary phases that were not subjected to gamma irradiation This was despite there being an apparent loss in sorbed PMOS (as indicated by a reduction in the capacity factor) following washing in very basic (pH 10) solvents No retention information was presented on the performance of gamma-irradiated PMOS-sorbed silica in comparison to gamma-irradiated PMOS-sorbed zirconia –silica An inorganic anion-exchange resin based on a zirconia– silica support was prepared by Chicz, Shi, and Regnier.[6] The Lewis acid sites were employed as adsorption sites that permanently retained a PEI coating which then acted as an anion-exchange ligand In this study, a continuous film of cross-linked PEI was achieved These surfaces were then tested for the analysis of ovabumin and bovine serum albumin Through chance discovery, Shalliker et al.[13,21] prepared a surface-modified normal phase zirconia –silica composite In studies designed to optimize the pore structure of zirconia– silica composites, sodium iodide was used as a salt for pore widening experiments However, following calcination and subsequent washing to remove residual salt, the stationary phase was left with a permanent brown-yellow discoloration, indicating the presence of iodide remaining on the surface Elemental analysis confirmed this to be so Consequently, the authors considered that the presence of iodide may protect the solute from the Lewis acid sites on the surface of the support.[21] Normal phase retention studies showed a reduction in the degree of retention of basic solutes together with an improvement in the peak shape of basic compounds such as pyridine and aniline Alkaline Stability One of the advantages of zirconia –silica composite stationary phases is supposedly a reduction in the solubility of the surface in alkaline solvents However, only a limited amount of information detailing the alkaline stability of the surface is currently available Chicz, Shi, and Regnier [6] demonstrated that a zirconia – silica composite that was coated with a PEI coating was stable in boiling alkaline solvent (1 M KOH) for 24 hr with no visual change in the stationary phase material In contrast, a silica phase coated with PEI dissolved within 30 in boiling M KOH As mentioned above, Melo et al.[8,9] also observed stability in their reversed phase modified support following passage of 5000 column volumes of mobile phase (NH4 + /NH3 buffer) at pH 10 A reduction in retention was observed, but the column efficiency and resolution remained essentially constant APPLICATIONS Commercially, zirconia– silica composites have, at present, found limited use in liquid chromatography due to the popularity and dominance of silica and silica-based supports The highly competitive market and the drive to satisfy consumer demand has ensured manufacturers of silica-based supports to investigate methods that overcome or limit the shortfalls of the surface, particularly MARCEL DEKKER, INC • 270 MADISON AVENUE • NEW YORK, NY 10016 ©2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc with respect to the solubility of silica in aqueous and basic solvents The synthesis of the new generation hybrid silica (XTerra) is an example of these developments.[20] Surface protection of the silica surface, with appropriate care being taken during the dehydroxylation and rehydroxylation of the surface also improves the behavior of basic solutes There is, at present, very limited commercial interest in developing zirconia– silica composites and, consequently, applications on these surfaces are limited to researchers who prepare their own packing materials Nevertheless, studies that have been published so far detail differences in selectivities between silica or zirconia and zirconia –silica composites The employment of basic test substances such as pyridine and aniline in normal phase chromatography serves as a useful indicator of selectivity and secondary adsorption effects This is particularly true for surfaces such as zirconia as these basic compounds display strong affinity toward Lewis acid sites and are also sensitive to the presence of hydroxyl groups With that in mind, Kaneko et al [1] used a mixture of benzene, dimethyl phthlate, and pyridine to observe the retention characteristics of four mixed oxide supports in a hexane (99%)/methanol (1%) mobile phase Pyridine was not observed to elute from either the column packed with silica or from the column packed with the zirconia– silica composite The column prepared from a silica– magnesia stationary phase gave the best separation, while extreme band tailing of the pyridine was observed on columns prepared from silica –alumina and silica– titania Fig illustrates the results that were Fig Chromatograms obtained on four mixed oxide packing materials Solute: = benzene; = dimethyl phthalate; = pyridine Column packing material: (a) silica – zirconia; (b) silica – alumina; (c) silica – titania; (d) silica – magnesia; (e) silica gel Mobile phase, n-hexane containing 1% methanol: flow rate, mL/min; column temperature, ambient; detection, UV 254 nm (From Ref [1].) Zirconia – Silica Stationary Phases for HPLC: Overview and Applications Fig Relationship between the kSi’ – Zr /kZr ’ and the pKb of basic compounds (From Ref [14].) obtained The authors concluded that because their zirconia– silica composite contained the highest concentration of strong acid sites, pyridine could not be eluted, whereas a broad tailing pyridine band was observed for the silica – titania and silica– alumina columns, both of which had a slightly lower concentration of strong acid sites than the zirconia – silica stationary phase The silica – magnesia stationary phase with only weak acid sites gave the best separation and a symmetrical peak for pyridine In contrast, pyridine and other basic test solutes in normal phase conditions were observed to elute from zirconia– silica composites prepared by Zhang, Feng, and Da.[5] The retention of the basic solutes was greater on the zirconia –silica composite stationary phase than on a native zirconia stationary phase The ratio kSi ’ – Zr /kZr ’ , where kSi ’ – Zr and kZr ’ are measures of the capacity factor on the zirconia –silica stationary phase and the zirconia phase, respectively, decreased with an increase in the pKb of the test solute, as illustrated in Fig Retention was also greater on the stationary phase containing the greater concentration of zirconia However, the elution order of the aniline and p-nitroaniline was reversed when the surface concentration of zirconia increased from 3.4% to 15.3% and this was attributed to the stronger acid properties of the 15.8% zirconia – silica composite Neutral compounds (PAHs) were also shown to be retained on the surface of the zirconia– silica composites and zirconia, and this was attributed to interactions between p electrons and Lewis acid sites.[5] The retention of acidic compounds was also studied by Zhang, Feng, and Da[5] and they MARCEL DEKKER, INC • 270 MADISON AVENUE NEW YORK, NY 10016 â2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc Zirconia – Silica Stationary Phases for HPLC: Overview and Applications Table Capacity factor and asymmetry factors for pyridine in a normal phase separation on various silica and zirconia stationary phases Stationary phase Capacity factor Asymmetry factor Silica Zirconia Zirconia – silica NaI impregnated zirconia – silica 9.50 0.99 0.86 0.24 2.450 1.840 1.795 1.330 The ion-exchange properties of zirconia– silica surfaces were also evaluated by Chicz, Shi, and Regnier;[6] however, their study was conducted on surfaces modified with a layer of PEI A number of different stationary phase base support materials were employed in their study including zirconia –silica They found that retention was dependent on the amount of PEI bound to the surface, leading them to suggest that base materials with similar (From Ref [21].) suggested that the crystal structure of the support may be a factor in determining the extent of retention In their study, retention of acidic compounds increased in the order of zirconia– silica (3.4% Zr) < zirconia < zirconia – silica (15.8%) The addition of silica to the zirconia improved the peak asymmetry Shalliker et al.[21] used pyridine and aniline as test solutes in normal phase chromatography Strong retention of pyridine was observed on a native silica column (k’ = 9.5), while the sample was only weakly retained on a native zirconia column (k’$1) Even less retention was observed for pyridine on a zirconia – silica column (k’ = 0.86) All pyridine bands were severely tailed with asymmetry factors as high as 2.4 for pyridine eluting from the silica column However, elution of pyridine from a sodium iodide impregnated zirconia– silica column occurred with a capacity factor of 0.26, but most importantly, the peak tailing decreased substantially, as shown in Table Similar results were observed for the elution of aniline However, selectivity differences resulted in the elution order of pyridine and aniline being reversed between the sodium iodide impregnated zirconia – silica column and the unmodified zirconia –silica column In general, iodide impregnation decreased retention, and improved peak shape The ion-exchange properties of zirconia– silica surfaces were utilized in a study by Peixoto, Gushiken, and Baccan.[7] In this study, zirconica – silica microcolumns were employed for the selective extraction and enrichment of chromium(VI) in a flow injection system The hydrated zirconia – silica surface adsorbed cations or anions depending on the pH At low pH, the surface behaved as anion exchanger by a mechanism in which OH À was displaced from the surface according to:[7] ỵ ZrOH þ Hþ þ Cr2 O2À ! Zr Cr2 O7 þ H2 O They showed that the method was suitable for the analysis of Cr(VI) in natural waters with little interference from common ions normally found in these water samples Fig Chromatograms of a mixture of acetone (1), benzonitrile (2), benzene (3), toluene (4), and naphthalene (5), obtained from a zirconized silica column with a radiation-immobilized PMOS coating (a) before initiating the stability test and (b) after completing the stability test with 5000 column volumes each of neutral and alkaline (pH 10) mobile phases (From Ref [8].) MARCEL DEKKER, INC • 270 MADISON AVENUE • NEW YORK, NY 10016 ©2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc 10 charge density, surface area, and pore diameter (in other words, materials with the same capacity to adsorb the PEI coating) should behave in much the same manner The advantage of the zirconia –silica base support over silica was that the zirconia– silica phase was stable in alkaline solvents whereas the silica phase rapidly dissolved Only one research group has illustrated the reversed phase retention behavior of solutes on zirconia –silica modified surfaces In the study by Melo et al.[8,9] on PMOS gamma-irradiated modified surfaces, the reversed phase retention behavior of a test mixture containing acetone, benzonitrile, benzene, toluene, and naphthalene was evaluated The authors illustrated good resolution of the five-component text mixture as shown in Fig Retention, however, decreased after the stationary phase was washed with 5000 column volumes of base (pH 10) Despite the base washing, uniform peak shape was maintained, with only a slight reduction in resolution as discussed in ‘‘Alkaline Stability’’ above CONCLUDING REMARKS In searching for new separation processes, the chromatographer develops new types of stationary phase materials in order to gain improvements in selectivity The development of zirconia– silica composites is an example which—although, at present, applications on these surfaces are limited to their development—typifies the ongoing search Whether the separations achieved on the zirconia– silica composites are better or worse than those achieved on either zirconia or silica is immaterial to the fundamental process of discovery The important factor is that the separation is different and, therefore, an alternative separation strategy may be employed In addition to the differences in selectivity that may be found when new surfaces are developed, beneficial surface properties may also be realized These may be related to aspects such as the pore structure, the acidic or basic properties, or the solubility of the support In the specific case of zirconia –silica composites, the stationary phase could be prepared through relatively simple processes using either coprecipitation or coating methods The addition of silica to the zirconia matrix increases the phase transition temperature from the amorphous phase to the tetragonal phase, which in turn stabilizes the tetragonal phase The pore structure can be controlled through processes similar to those employed in the preparation of zirconia However, the type of pore structure obtained appears to be dependent on the method of preparation Calcination in the presence of salts improves the pore shapes Zirconia– silica phases can also be surface-modified and C8 and ion-exchange media have been prepared Composite zirconia– silica stationary Zirconia – Silica Stationary Phases for HPLC: Overview and Applications phases coated with PEI were shown to have a lower solubility in alkaline solvents than silica modified in the same manner The ion-exchange properties of zirconia – silica stationary phases were utilized in the analysis of natural waters, where the concentration of chromium(VI) was enriched in a flow injection system that incorporated a microcolumn packed with zirconia– silica stationary phase Several studies have shown the elution behavior of basic solutes on these composites, and one study illustrated that modifying the surface with a Lewis base could minimize peak tailing Future work in stationary phase design will probably continue to include studies on mixed oxide composites although these will continue to constitute only a small fraction of the stationary phase materials made for general purpose use Many of the mixed oxide supports will be custom-made for specific applications, and at least in the short term, these types of stationary phases will be made by individual research groups REFERENCES Kaneko, S.; Mitsuzawa, T.; Ohmori, S.; Nakamura, M.; Nobuhara, K.; Masatani, M Separation behaviour of silicacontaining mixed oxides as column packing materials for liquid chromatography J Chromatogr., A 1994, 669, – Kaneko, S.; Mitsuzawa, T.; Ohmori, S.; Nakamura, M.; Nobuhara, K.; Masatani, M Silica-containing mixed oxides gels prepared by a coprecipitation method as novel packing materials for liquid chromatography Chem Lett 1993, 1275 – 1278, and references cited therein Nawrocki, J.; Rigney, M.P.; McCormick, A.; Carr, P.W Chemistry of zirconia and its use in chromatography J Chromatogr., A 1993, 657, 229 – 282 Kanno, Y Thermodynamic and crystallographic discussion of the formation and dissociation of zircon J Mater Sci 1989, 24, 2415 – 2420 Zhang, Q.-H.; Feng, Y.-Q.; Da, S.-L Preparation and characterization of silica – zirconia supports for normal phase liquid chromatography J Liq Chromatogr Relat Technol 2000, 23 (10), 1461 – 1475 Chicz, R.M.; Shi, Z.; Regnier, F.E Preparation and evaluation of inorganic anion-exchange sorbents not based on silica J Chromatogr 1986, 359, 121 – 130 Peixoto, C.R.M.; Gushiken, Y.; Baccan, N Selective spectrophotometric determination of trace amounts of chromium(VI) using a flow injection system with a microcolumn of zirconium(IV) oxide modified silica gel Analyst 1992, 117, 1029 – 1032 Melo, L.F.C.; Collins, C.H.; Collins, K.E.; Jardin, I.C.S.F Stability of high performance liquid chromatography columns packed with poly(methyloctylsiloxane) sorbed and radiation-immobilized onto porous silica and zirconized silica J Chromatogr., A 2000, 869, 129 – 135 Melo, L.F.C.; Jardin, I.C.S.F Development of C8 station- MARCEL DEKKER, INC • 270 MADISON AVENUE • NEW YORK, NY 10016 ©2002 Marcel Dekker, Inc All rights reserved This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc Zirconia – Silica Stationary Phases for HPLC: Overview and Applications 10 11 12 13 14 15 ary phases immobilized by g-radiation on zirconized silica for high-performance liquid chromatographic applications J Chromatogr., A 2000, 845, 423 – 431 Tsurita, Y.; Nogami, M Preparation of porous supports in the SiO2 – ZrO2 – Na2O system from microspherical silica gels J Mater Sci 2001, 36, 4365 – 4375 Shalliker, R.A.; Rintoul, L.; Douglas, G.K.; Russell, S.C A sol – gel preparation of silica coated zirconia microspheres as chromatographic support materials J Mater Sci 1997, 32, 2949 – 2955 Shalliker, R.A.; Douglas, G.K.; Rintoul, L.; Russell, S.C The analysis of zirconia – silica composites using differential thermal analysis, Fourier transform – Raman spectroscopy and X-ray scattering/scanning electron microscopy Powder Technol 1998, 98, 109 – 112 Shalliker, R.A.; Douglas, G.K.; Rintoul, L.; Comino, P.R.; Kavanagh, P.E The measurement of pore size distributions, surface areas, and pore volumes of zirconia and zirconia – silica mixed oxide stationary phases using size exclusion chromatography J Liq Chromatogr Relat Technol 1997, 20 (10), 1471 – 1488 Shalliker, R.A.; Douglas, G.K The development of stationary phase supports for liquid chromatography: I Examination of the pore structure of zirconia – silica composites using size exclusion chromatography J Liq Chromatogr Relat Technol 1998, 21 (12), 1749 – 1765 Shalliker, R.A.; Douglas, G.K.; Elms, F.M The devel- 11 16 17 18 19 20 21 opment of stationary phase supports for liquid chromatography: II Examination of the pore structure of zirconia – silica composites using nitrogen sorption J Liq Chromatogr Relat Technol 1998, 21 (12), 1767 – 1781 Leonard, M New packing materials for protein chromatography J Chromatogr., B, Biomed Sci Appl 1997, 669, – 27, and references cited therein Sing, K.S.W.; Everett, D.H.; Haul, R.A.W.; Moscou, L.; Pierotti, R.A.; Rouquerol, J.; Siemieniewska, T Reporting physisorption data for gas/solid systems with special reference to the determination of surface area and porosity Pure Appl Chem 1985, 57, 603 – 619 Novak, I.; Berek, D Structural inhomogeneities in widepore silica gels J Chromatogr., A 1994, 665, 33 – 36, and references cited therein Shalliker, R.A.; Douglas, G.K.; Comino, P.R.; Kavanagh, P.E Examination of various size zirconias for potential chromatographic applications Powder Technol 1997, 91, 17 – 23 Buchmeiser, M.R New synthetic ways for the preparation of high performance liquid chromatography supports J Chromatogr., A 2001, 918, 233 – 266 Shalliker, R.A.; Rizk, M.; Stocksiek, C.; Sweeney, A.P Retention behaviour of basic solutes on zirconia – silica composite stationary phase supports in normal phase liquid chromatography J Liq Chromatogr Relat Technol 2002, 25 (4), 561 – 572 Zone Dispersion in Field-Flow Fractionation Josef Jancˇa Université de La Rochelle, La Rochelle, France Introduction All separation processes are inherently accompanied by zone broadening, which is due to the dynamic spreading processes dispersing the concentration distribution achieved by the separation [1] As long as the relative contributions of these dispersive processes decrease, the efficiency of the separation increases A conventional empirical parameter describing, quantitatively, the efficiency of any separation system is the number of theoretical plates per separation unit, N, or the height equivalent to a theoretical plate (the theoretical plate height) H defined by Nϭ a VR b , sV Hϭ L N (1) where VR is the retention volume, sV is the standard deviation of the elution curve (fractogram) of a uniform retained sample, expressed at the same retention volume units, and L is the length of the separation unit; in the case of the field-flow fractionation (FFF), it is the length of the fractionation channel Figure 1a shows a model fractogram and the method of graphical determination of the efficiency of the involved separation system A more accurate procedure for the determination of the efficiency of the separation system from the experimental fractogram consists in the numerical calculation of the statistical moments of the fractogram (viz of the first statistical moment, which corresponds to the maximum of the fractogram and, thus, to the retention volume): VR ϭ g Vihi g hi (b) (c) (2) where hi are the heights of the fractogram at the corresponding retention volumes Vi The square root of the second central moment corresponds to the standard deviation of the fractogram and, thus, to its width: sV ϭ a (a) g 1Vi Ϫ VR 2hi g hi b 1/2 (3) Fig Zone broadening and its correction (a) Schematic demonstration of the evaluation of the efficiency of the separation system of FFF; (b) dependence of the theoretical plate height on the linear flow velocity; (c) schematic demonstration of the application of the correction for the zone broadening on a model fractogram Encyclopedia of Chromatography DOI: 10.1081/E-Echr 120005356 Copyright © 2002 by Marcel Dekker, Inc All rights reserved Zone Dispersion in Field-Flow Fractionation It must be stressed that the terms band or zone broadening, spreading, and dispersion used here are equivalent from the viewpoint of their physical meanings The dimensionless parameter x is a complex function of the retention parameter l which, itself, can be approximated by the relationship lim R ϭ 6l (6) lS0 Another approximate relationship holds between x and l: Processes Contributing to Zone Broadening Several dispersive processes contribute to zone broadening: longitudinal diffusion, nonequilibrium and relaxation processes, spreading due to the external parts of the whole separation system, such as the injector, detector, connecting capillaries, and so forth It has theoretically been found [2] that the resulting efficiency of the FFF, characterized by the height equivalent to a theoretical plate, can very accurately be described by Hϭ xw2ͳv1x2ʹ 2D ϩ ϩ a Hext Rͳv1x2 ʹ D (4) where D is the diffusion coefficient of the retained species, R is the retention ratio, ͳv1x 2ʹ is the average linear velocity of the carrier liquid, w is the thickness of the fractionation channel, x is the dimensionless parameter defined later, and ͚ Hext is the sum of all contributions to the zone broadening from the external parts of the separation channel (injector, detector cell, and connecting capillaries) The retention ratio R is defined as Rϭ V0 VR (5) where V0 and VR are the retention volumes of the unretained and retained species, respectively Graphical representation of the particular spreading processes, as a function of the average linear velocity of the carrier liquid and of their sum, which results in a curve exhibiting a minimum, are shown in Fig 1b At very low linear velocities of the carrier liquid, the longitudinal diffusion, represented by the first term on the righthand side of the Eq (4), plays a dominant role of all zone-spreading processes The relaxation and nonequilibrium processes, represented by the second term on the right-hand side of the Eq (4), become more important when increasing the velocity The result is that H passes through the above-mentioned minimum As the diffusion coefficients of the macromolecules and particles are relatively low, the contribution of the longitudinal diffusion is almost negligible and the optimal efficiency (the minimum on the resulting curve) appears at a very low flow velocity The zone spreading due to the external elements of the fractionation channel can be minimized by reducing their volume lim x ϭ 24l3 (7) lS0 Equation (7) is a very important relationship because, with respect to Eq (4), it indicates that in the most practical range of the linear flow velocities, above the optimal flow, the efficiency of the separation in FFF increases very rapidly with the retention ratio This is rather an exceptional case among separation methods and the importance of this behavior has to be regarded with respect to the fact that the FFF methods and techniques are especially convenient for the fractionation of large and polydisperse species, such as macromolecules and particles As the retention usually increases with the molar mass or particle size in polarization FFF, the efficiency is higher in the high molar mass or large particle size domain This is one of the reasons why the FFF methods are particularly competitive in this field of application Nevertheless, the difficulty to separate the large polydisperse species enough, independently of the method or technique used, impose the necessity to introduce, and apply the powerful numerical methods, which are able to evaluate the amplitude of the zonebroadening contribution to the apparent molar mass distribution (MMD) or particle size distribution (PSD), calculated by a simple data treatment of the experimental fractograms, and to correct, casually, the MMD and PSD data for zone broadening Whenever the zone spreading attains a level not acceptable from the point of view of the analytical results obtained by a simple data treatment of the raw fractograms, a correction for the zone broadening must be applied Relaxation Relaxation represents an important contribution to the zone broadening and merits mention in particular, especially because it can be reduced by an appropriate experimental procedure [3] The concentration distribution of the fractionated sample across the channel established immediately after the injection is far from the steady state, which is formed progressively during the elution This leads to additional zone broadening However, if the flow is stopped after the injection for a Zone Dispersion in Field-Flow Fractionation time necessary to achieve a steady state, the shift of the retention volume and the zone spreading can substantially be reduced So-called secondary relaxation broadening can occur when the intensity of the field varies rapidly during the elution (e.g., by programming) Correction for the Zone Broadening The fractogram of a polydisperse sample is a superposition of the separation and of the zone broadening This is shown in Fig 1c, where the spread zones of the discrete species are overlapped and the fractogram is, in fact, a convolution of all individual zones Whenever the zone spreading is important, the accurate MMD or PSD can be calculated from the experimental fractograms only by using a correction procedure An efficient correction method applicable in FFF [4] was derived from well-known correction procedures used in size-exclusion chromatography [5] A raw fractogram h(V) is a convolution of the fractogram corrected for the zone broadening g(Y) and the spreading function G(V, Y) which is a detector response to a uniform species having the elution volume Y: h1V2 ϭ Ύ q PSD only On the other hand, whenever a nonuniform spreading function with the sS dependent on the retention has to be used, the correction to be applied is h1V2 ϭ 1/2 1V Ϫ Y2 G1V, Y ϭ a b exp a Ϫ b 2ps2S 2s2S (9) where the standard deviation sS should be independent of the elution volume This independence is valid in FFF only within a not very wide range of the elution volumes Equation (9) can correctly be applied to the fractograms of the samples with a narrow MMD or 1/2 1V Ϫ Y 2 R exp¢Ϫ ≤ dY 32p1sS 1Y2 4 321sSY2 (10) References (8) The spreading function can be approximated by g 1Y2B which is convenient for samples exhibiting wide MMD or PSD Equation (10) can be numerically solved The graphical representation of a model result of the application of the described correction procedure is shown in Fig 1c Practical utility of the described correction procedure of the experimental fractograms was demonstrated first by applying it to the real fractograms of a polymer latex obtained from sedimentation FFF [4] and was subsequently confirmed [6 – 8] g 1Y 2G1V Ϫ Y2 dY Ύ q J C Giddings, Dynamics of Chromatography, Marcel Dekker, Inc., New York, 1965 M E Hovingh, G H Thompson, and J C Giddings, Anal Chem 42:195 (1970) J Jancˇ a, Field-Flow Fractionation: Analysis of Macromolecules and Particles, Marcel Dekker, Inc., New York, 1988 V Jáhnová, F Matulík, and J Jancˇ a, Anal Chem 59: 1039 (1987) A E Hamielec, in Steric Exclusion Chromatography of Polymers (J Jancˇ a, ed.), Marcel Dekker, Inc., New York, 1984 M R Schure, B N Barman, and J C Giddings, Anal Chem 61: 2735 (1989) Y Mori, K Kimura, and M Tanigaki, Spec Publ Roy Soc Chem 102: 290 (1992) J.-C Vauthier and P S Williams, J Chromatogr 805: 149 (1998) ... analysis of organic acids by thin layer chromatography, such as chloroform, ethyl acetate, methanol, benzene, etc Encyclopedia of Chromatography DOI: 10.1081/E-ECHR- 120 041 125 Copyright D 20 04 by Marcel... film 120 °C for min, to 22 5°C at 4°C/min mL (splitless), T = 21 0°C FID 350° 20 0 – 28 0°C at 8°C/min HP1, 50  0 .25 mm J&W DB-5, 30 m  0. 32 mm, 0 .25 -mm film Split injection T = 25 0°C FID 28 0°C... chloroform/MEOH 25 4 A = water/ACN (85:15), B = ACN/water (85:15)b 20 mL, mL/min 20 mL, 1.5 mL/min 28 0 ACN/tetrahydrofuran (THF) (95:5) 25 4 22 5 MEOH/phosphate buffer, pH 5.5 (1:1) 20 mL, 0.8 or 0.17 mL/min 20
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