Density gradients in packed columns: II. Effects of density gradients on efficiency in supercritical fluid separations

To investigate how fluid compressibility affects efficiency in supercritical fluid separations, band dispersion along a packed capillary column was measured from on-column elution rate profiles obtained under solvating gas chromatography (SGC) conditions; this allowed efficiency to be determined with respect to position along the column. Theoretical efficiency was also modeled. The model indicates that the primary cause of band broadening in SGC is high mobile phase velocity near the column outlet. However, the experimental results show that significant band broadening also occurs near the column inlet in a region that corresponds to high elution rates of the analyte. On-column detection also revealed spatial focusing of the analyte as it moves down the column density gradient.     

Changes in peak width and concentration at the inlet and outlet of a chromatographic column

The original plate model of chromatography is extended to the sorption process occurring at the column inlet and the desorption process at the column exit. At the column inlet it is shown that sufficiently wide feed bands undergo no change in concentration but a fall in band width, i.e., the volume of mobile phase occupied by the solute band is reduced. The reduction factor is (1 + k) where k is the mass distribution ratio (capacity factor). Narrower bands suffer partial reduction in both band width and concentration. On desorption at the outlet, however, the change is always in band width and not concentration. A perfect detector registers the true concentration-time profile of the band in the column if the solute mass fraction in the stationary phase is below 10^?3 at the column outlet. The risks of stripping the stationary phase at high solute concentrations in analytical and preparative or production gas chromatography are compared.     

锥型与圆柱型液相色谱制备柱的比较研究

用可视化柱上图形分析对入口内径大于出口内径的锥型液相色谱制备柱的样品谱带流型进行了研究。在一定的实验条件下，锥型柱与圆柱型柱的流动相流型曲线相反。对与锥型柱有相同长度、相同容积的圆柱型色谱柱的柱效、样品容量及峰高的比较研究表明：锥型柱优于圆柱型柱。锥型柱的样品容量约为圆柱型柱的2倍，柱效比圆柱型的高36％，色谱流出曲线峰值高于圆柱型柱12％。     

Tailoring Polymer Molecular Weight Distribution and Multimodality in RAFT Polymerization Using Tube Reactor with Recycle

Solution reversible addition fragmentation chain transfer (RAFT) polymerization of butyl acrylate in 50 wt% toluene, initiated with 2,2′‐azobisisobutyronitrile and mediated with 3‐benzyltrithiocarbonyl propionic acid, is carried out in a tube reactor of 1.65 mm inner diameter. The tube reactor is operated in three modes: batch tube reactor (inlet and outlet closed, recycle open), continuous tube reactor (inlet and outlet open, recycle closed), and loop tube reactor (inlet, outlet, and recycle all open). The effects of inlet and outlet flow rates, residence time, and recycle ratio on the polymerization rate and polymer molecular weight distribution (MWD) are systematically investigated. The dynamic and steady state kinetics of the three modes of operation are analyzed and compared. Polymer samples having multimodal MWD are generated using the loop reactor. It is found that the MWD and multimodality can be readily controlled by residence time (τ) and recycle ratio.     

Influence of the errors made in the measurement of the extra-column volume on the accuracies of estimates of the column efficiency and the mass transfer kinetics parameters

The influences of the errors made in the measurement of the extra-column volume of an instrument on the accuracies of the estimates made of the column efficiency and of the parameters of the mass transfer kinetics were investigated from an experimental point of view. A standard HP1090 apparatus (extra-column volume, approximately 50 micro L) was used to measure the efficiency of a Sunfire-C(18) RPLC column (column hold-up volume, approximately 1.50 mL). The first and second moments of the peaks of phenol (a retained compound) and of thiourea (a practically non-retained compound) were measured at six different temperatures between 22 and 78 degrees C, for flow rates between 0.10 and 4.70 mL/min (i.e., for linear velocities between 0.025 and 1.179 cm/s). Each series of measurements was successively made with the instrument being fitted with and without the column. The experimental HETP data must be corrected for the solute dispersion in the connected tubes in order properly to assess the true column efficiency. Even with a modern, high performance instrument, the dispersion of a non-retained compound is essentially due to the band broadening phenomena that take place in the extra-column volumes, the sum of all these extra-column band broadening contributions accounting for more than 80% of the total band broadening measured. The contribution of the sampling device is particularly deleterious since, for a 2 mu L injection, the maximum solute concentration in the peak that enters into the column is nearly ten-fold lower than that of the sample. Nevertheless, the impact of the extra-column volumes on the estimates of the kinetic parameters (e.g., molecular diffusion coefficient D(m) and effective particle diffusivity D(e)) remains negligible. Obviously, the relative error made on the column efficiency of a retained compound depends much on its retention factor. It decreases from 8 to 1% when the retention factor increases from 5 to 17.     

Broadening of peaks eluted before the solvent in capillary GC Part II: The role of phase soaking

Summary Phase soaking is a solvent effect which tends to reconcentrate peaks eluted after and to broaden peaks eluted before the solvent. The principles of the phase soaking effect on peaks eluted before the solvent are discussed. The broadening effect is distinguished from the broadening effect occurring in the flooded column inlet by partial solvent trapping. It was found that in most cases broadening by partial solvent trapping strongly predominated over broadening by phase soaking. Phase soaking was noticeable only in the neighbourhood of the solvent peak.Phase soaking does not broaden peaks eluted before the solvent as much as it re-concentrates those eluted after it. The two phase soaking effects on the front and the rear of the solvent band (that is, of the soaked zone) differ strongly from each other.Peak broadening by phase soaking is negligible for non-trapped components, because such solutes start their chromatography before a significant quantity of solvent enters the column. Phase soaking only broadens solute bands which were retained by the solvent in the column inlet, that is, bands already broadened by partial solvent trapping.     

Contribution of axial dispersion to band spreading in perfusion chromatography

A new interpretation of the band spreading data in perfusion chromatography is proposed by investigating the relative importance of axial dispersion in perfusive beds. Elution chromatography of proteins (bovine serum albumin and lysozyme) under non-retained conditions on two kinds of reversed-phase perfusive supports (POROS R1/H and POROS R2/H), which have different pore structures, were carried out to obtain the axial dispersion data. The Knox equation and some empirical correlations for dispersion coefficients in porous media were applied to correlate the experimental data. The influences of particle properties, solute molecular sizes and flow velocity on the dispersion coefficient were elucidated. Axial dispersion was recognised to be the main contributor to peak broadening in perfusion chromatography. The dependence of the height equivalent to a theoretical plate on flow-rate was found to be the result of the velocity dependence of the axial dispersion. The dispersion coefficient in a perfusive column can be well represented both by a power-law relationship and a correlation derived based on stochastic theory. Pursuant to these, it was found that pore size distribution of the perfusive particles and solute molecular size are important parameters, which influenced the dispersion results significantly.     

Mathematical model using non-uniform flow distribution for dynamic protein breakthrough with membrane adsorption media

A mathematical model has been investigated to predict protein breakthrough during membrane adsorption/chromatography operations. The new model incorporates a non-uniform boundary condition at the column inlet to help describe the deviation from plug flow within real membrane adsorption devices. The model provides estimated breakthrough profiles of a binding protein while explicitly accounting for non-uniform flow at the inlet of the separation operation by modeling the flow distribution by a polynomial. We have explored experimental breakthrough curves produced using commercial membrane adsorption devices, as well as novel adsorption media of nanolayered nanofiber membranes, and compare them to model predictions. Further, the impact of using various simplifying assumptions is considered, which can have a dramatic effect on the accuracy and predictive ability of the proposed models. The new model, using only simple batch equilibrium and kinetic uptake rate data, along with membrane properties, is able to accurately predict the non-uniform and unsymmetrical shape for protein breakthrough during operation of membrane adsorption/chromatography devices.     

Evaluation of packed capillary liquid chromatography columns and comparison with conventional-size columns

Apart from extracolumn effects peak dispersion in liquid chromatographic columns is caused by the column inlet, the packed bed, and the column outlet. A strategy applicable for independent evaluation of the individual sources of column band broadening was developed on the basis of the linear extrapolation method (LEM). This method was applied to compare the performance of packed capillary LC columns from various commercial suppliers with conventional-size columns. The columns differed widely in their performance with respect to peak shapes and widths for standard substances. The capillary columns were found well packed, but in some cases overall performance would benefit from improving the design of the area between the packed bed and the connecting capillaries, containing frits as well as dead volumes.     

Visualization of sample introduction in liquid chromatography columns. The effect of the frit diameter.

A previously developed on-column detection technique using 35 mm SLR cameras [J. Chromatogr. A 826 (1998) 1] was employed to visualize colored sample bands as they elute through frits of differing diameter. Head fittings containing a 4.0 mm frit and a 15.9 mm frit were mounted in a 17 mm I.D. glass column packed with C18 silica with an average particle size of 21 microns. A carbon tetrachloride mobile phase of matching refractive index to that of the silica provides clarity along the column diameter during band migration. The photographs of the migrating sample zones were scanned and analyzed with appropriate imaging software. The smallest diameter frit induced severely parabolic sample distributions at the column inlet compared to the larger frit. Local axial dispersion coefficient values, expressed as local reduced plate height, were calculated as well as local zone velocities at the column inlet. The results demonstrate clearly the need to match the diameter of the inlet frit to the I.D. of the column so as to avoid the initial onset of zone broadening due to the frit.     

Effect of depletion interactions on transport of colloidal particles in porous media

The influence of depletion interactions on the transport of micrometer-sized, negatively charged polystyrene latex particles through porous media was studied by analysis of particle breakthrough curves as a response to short-pulse particle injections to the inlet of a packed column of glass beads. The column outlet latex particle concentration profiles and the total amount of particles exiting the column were determined as a function of the concentration of small, silica nanoparticles in the solution and the bulk flow rate. Because of similar charges, the silica particles do not adsorb to either the latex particles or glass beads and thus induce an attractive depletion force between the latex particles and glass bead collectors. The total column outlet latex particle amount was calculated by integrating the measured breakthrough concentration curve and compared to the known amount of injected particles at the column inlet. It was found that the particle recovery was a decreasing function of the silica nanoparticle concentration and the carrier fluid residence time, and an increasing function of the velocity in the bed. In addition, removing the silica nanoparticles from the flowing solution caused a second outlet peak to appear, suggesting that some of the polystyrene particles were captured in secondary energy wells. The experimental data were interpreted using the predicted potential energy profile between a single particle and a glass bead, which was assumed to consist of electrostatic, van der Waals, and depletion components. The results indicate that secondary energy wells significantly affect particle transport behavior through porous media.     

Faster axial band dispersion in a monolithic silica column than in a particle-packed column

The contribution of molecular diffusion to peak broadening was studied in a reversed-phase HPLC system, consisting of a monolithic silica C18 column and methanol-water mobile phase. Study on the band broadening effect of holding a solute in a column or elution at very low linear velocity of mobile phase allowed facile determination of the contribution of the molecular diffusion term. Less obstruction against molecular diffusion, or the faster axial band dispersion in a monolithic silica column than in a particle-packed column, was found both in mobile phase and in stationary phase.     

The effect of column characteristics on the minimum analyte concentration and the minimum detectable amount in capillary gas chromatography

The need for faster and more efficient separations of complex mixtures of organic compounds by gas chromatography has led to the development of small inner diameter open tubular columns. Owing to their decreased plate height, extremely narrow peaks are obtained. When differently sized columns with equal plate numbers are compared, injection of a fixed amount of a solute will give the highest detector signals for the smallest bore columns. When P is defined as the ratio of the column inlet and outlet pressures, it can be seen from theory that under normalized chromatographic conditions the minimum detectable amount (Q_º) for a mass flow sensitive detector increases proportionally to the square of the column diameter for P = 1. In the situation of greater interest in the practice of open tubular gas chromatography where P is large, a linear relationship is derived between Q_º and the column diameter. It is a widespread misunderstanding, however, that narrow bore capillary columns should be used for this reason in trace analysis. If a fixed relative contribution of the injection band width to the overall peak variance is allowed, a decreased plate height drastically restricts the maximum sample volume to be injected. It is shown that the minimum analyte concentration in the injected sample (C_º) is inversely proportional to the column inner diameter when a mass flow sensitive detector is used. For actual concentrations less than C_º, sample preconcentration is required. The effect of peak resolution and selectivity of the stationary phase in relation to C_º and Q_º will be discussed as well. The validity of the given theory is experimentally investigated. Minimum analyte concentrations and minimum detectable amounts are compared using columns with different inner diameter.     

Fundamental chromatographic equations designed for columns packed with very fine particles and operated at very high pressures. Applications to the prediction of elution times and the column efficiencies

The wall temperatures of three Acquity-BEH-C18columns (2.1 mm x 50, 100, and 150 mm) and the temperature of the incoming eluent were maintained constant at 289 K, using a circulating water heat exchanger. The retention times and the band broadening of naphtho[2,3-a]pyrene were measured for each column as a function of the flow rate applied. Pure acetonitrile was used as the eluent. The flow rate dependence of neither elution volumes nor bandwidths can be accounted for by classical models of retention and HETP, respectively, since these models assume columns to be isothermal. Because the heat generated by friction of the eluent against the column bed increases with increasing flow rate, the column bed cannot remain isothermal at high flow rates. This heat is evacuated radially and/or longitudinally by convection, conduction, and radiation. Radial and axial temperature gradients are formed, which are maximum and minimum, respectively, when the temperature of the column wall is kept uniform and constant. The retention times that we measured match well with the values predicted based on the temperature distribution along and across the column, which we calculated and on the temperature dependence of the retention for the same column operated isothermally (i.e., at very low flow rate). The rate of band spreading varies along non-isothermal columns, so the HETP can only be defined locally. It is a function of the axial coordinate. A new contribution is needed to account for the radial thermal heterogeneity of the column, hence the radial distribution of the flow velocities, which warps the elution band. A new model, based on the general dispersion theory of Aris, allows a successful prediction of the unusually large bandwidths observed with columns packed with fine particles, operated at high flow rates, hence high inlet pressures.     

Experimental and modeling investigation of thorium biosorption by orange peel in a continuous fixed-bed column

In this research work process parameters and breakthrough modeling of thorium biosorption on orange peel in a fixed-bed column has been studied. Some experiments have been done with sorbent diameter, flow rate, bed height and feed inlet concentration. Breakthrough point decreased with decreasing the bed height, increasing feed inlet concentration and increasing flow rate. Meanwhile, sorption capacity increased with decreasing bed height and flow rate. The highest sorption capacity, 87.7 mg/g, occurred in a 0.4–0.8 mm sorbent diameter. The experimental results were fitted by models of Thomas, Yoon–Nelson and Modified Dose–Response (MDR). The MDR model showed better results.     

Effect of steric exclusion on the separation of proteins by hydrophilic size-exclusion chromatography

On the basis of studying the retention and band broadening of proteins on the TSK SW column, diffusion coefficients (Ds) of solute in stationary phase were obtained which elucidate the hydrodynamic process of chromatographic resolution of proteins by hydrophilic size-exclusion chromatography (SEC). After calculating the correlation between Ds and the molecular weight of the solute, the molecular dimensions of proteins in the process of chromatographic separation can be predicted. Deviations in diffusion coefficient of a protein from the calculated value reflect differences of measured molecular dimensions from molecular volumes predicted from the calibration curve of the SEC column. This study illustrates a convenient method for estimating the purity of proteins by SEC. Deviations from 2 lambda dp (where dp is the particle diameter) in the intercept of the theoretical plate height (H) versus flow-rate (U) curve from the band broadening equation H = CsU + 2 lambda dp + f(alpha M)T (where CsU represents mass transfer resistance caused by solute diffusion in the stationary phase and f(alpha M)T an added term for polydisperse solutes as proposed by Knox and McLennan [Chromatographia, 10 (1977) 75]) reflect impurities in the proteins.     

Visualization of sample introduction in liquid chromatographic columns. Contribution of a flow distributor on the sample band shape

The contributions to the radial distribution of the sample concentration across the column inlet and to the axial band dispersion resulting from a column header containing a distributor were evaluated using a band-visualization process entailing matching the refractive indices of the stationary and mobile phases in a glass column. This study illustrates graphically how a distributor fitted to the column can increase the axial dispersion of the sample band compared to an inlet containing only a frit. The distributor did not provide a uniform sample distribution across the column. In fact, for 17-mm inner diameter columns and high-porosity frits, the distribution was no better than with the frit having no distributor. However, when low-porosity frits were employed, improved peak shapes were obtained with a distributor. In addition, we observed that the inlet header configuration influenced dramatically the flow stream established along the column. The radial distribution of the efficiency of the columns was nearly homogeneous for those having only a frit but not for those having also a distributor. For the latter, the efficiency decreased from the column axis to its wall.     

Band broadening in size-exclusion chromatography of polydisperse samples

Understanding and controlling the band broadening is essential to obtain accurate molar-mass distributions by size-exclusion chromatography (SEC). In this paper, band broadening in SEC is reviewed from a contemporary perspective. The observed band broadening is due to dispersion inside and outside the chromatographic column (undesirable band broadening) and to the polydispersity of the sample (desirable SEC selectivity). The various contributors to band broadening are discussed. Integrity plots are introduced as a tool to evaluate the performance of specific SEC columns at given experimental conditions. For narrow polymer standards on single SEC columns the observed peak width is dominated by the chromatographic dispersion. MALDI-ToF-MS is demonstrated as an alternative to determine the PDI of narrowly distributed samples. The plate heights encountered at very high reduced velocities are found to be lower than expected. This is advantageous for fast separations by SEC.     

Using the liquid nature of the stationary phase in counter-current chromatography V. The back-extrusion method

The main feature of counter-current chromatography (CCC) is that the stationary phase is a liquid as well as the mobile phase. The retention volumes of solutes are directly proportional to their distribution coefficients K(D) in the biphasic liquid system used in the CCC column. Solutes with high K(D) coefficients are highly retained in the column. The back-extrusion method (BECCC) uses the fact that the liquid stationary phase, that contains the retained solutes, can be easily moved. Switching the column inlet and outlet ports without changing the liquid phase used as the mobile phase causes the rapid collapse of the two immiscible liquid phases inside the column, the previously stationary phase being gathered at the new column outlet. Then this previously stationary liquid phase is extruded outside the CCC column carrying the retained solutes. The back-extrusion method is tested with a standard mixture of five compounds and compared with the recently described elution-extrusion method. It is shown that the chromatographic resolution obtained during the back-extrusion step is good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. However, a major drawback of the BECCC method is that all solutes are split between the liquid phases according to their distribution ratios when the CCC column equilibrium is broken. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode, eluting solutes with small and medium distribution ratios. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column and produce a broad peak showing after the stationary phase extrusion.     

Converging flow chromatography in constant-pressure mode

Dispersion in chromatographic processes can be reduced to a minimum using converging columns and a curved frit at the outlet. Working at constant pressure at the inlet the internal packing is increasingly compressed by the accelerated flow. Thus the packed bed is stabilized. Under these conditions the observed flux at the outlet and the power input of the pump are inversely related to the viscosity of the eluting solute. Comparing converging flow chromatography (CFC) with classical axial flow chromatography (AFC) and radial flow chromatography (RFC) the stationary phase is used more efficiently in CFC.