A study of the efficiency of monolithic silica gel capillary columns for gas chromatography

The efficiency and dynamic characteristics of seven silica-gel-based monolithic capillary columns were analyzed by separating on them a mixture of five light hydrocarbons. For helium carrier gas flowing at an optimum velocity, the height equivalent to a theoretical plate was found to be 0.15–0.20 mm, values comparable to those typical of packed capillary columns. An analysis of the Van Deemter curves for the columns under study demonstrated that the main contribution to the smearing of the chromatographic zone comes from the diffusional processes in the mobile phase while the mass transfer between the mobile and stationary phases plays only a minor role. At the same time, the parameter A in the Van Deemter equation, which characterizes the degree of column packing uniformity, was found to be negative. This result contradicts the classical theory of chromatography and calls for further studies of monolithic capillary columns.     

Divinylbenzene-based monolithic capillary columns in capillary liquid chromatography

The effect of the conditions of synthesis of divinylbenzene-based monolithic capillary columns on their chromatographic characteristics was studied. It was demonstrated that the porosity and permeability of the column change significantly even at small deviations from the optimum conditions of polymerization of the monolith in the column. By contrast, the minimum value of HETP proved to be only slightly sensitive to the conditions of synthesis, ranging within ～10–20 μm. The conditions of polymerization of the monolith were found to produce more pronounced effect on the slope of the right branch of the van Deemter curve (parameter C), with the flattest curve being observed for columns prepared under optimum conditions. The minimum value of HETP for polymer monolithic capillary columns was found to be similar to that for silica gel monolithic capillary columns, but the latter are characterized by C values approximately an order of magnitude lower.     

Effect of the pressure of the carrier gas on the parameters of the Van Deemter equation for monolithic silica gel gas chromatography columns

The chromatographic properties of monolithic capillary silica gel columns for gas chromatography were examined with the use of four different variants of the Van Deemter model. The corresponding experimental curves were measured for the elution of light hydrocarbons with the helium carrier gas in the isothermal mode at 60°C. Despite the models tested are based on different mechanisms of the smearing of chromatographic peaks, the values of the Van Deemter equation parameters proved to be very close to each other for three of the four models. All models yielded negative values of the parameter A. Physically reasonable values of the parameters of the Van Deemter equation were obtained only for the Giddings model, which takes into account the pressure drop across the column. At the same time, this model overestimated the contribution from diffusional smearing (parameter B). It was concluded that none of the models tested adequately described the chromatographic properties of monolithic capillary columns for gas chromatography.     

Significant viscosity dependent deviations from classical van Deemter theory in liquid chromatography with porous silica monolithic columns

Significant deviations from classical van Deemter behaviour, indicative of turbulent flow liquid chromatography, has been recorded for mobile phases of varying viscosity on porous silica monolithic columns at elevated mobile phase flow rates.     

LC with a Teicoplanin Aglycone Chiral Sorbent for the Separation of the Enantiomers of Non-Steroidal Anti-Inflammatory Drugs: An Evaluation of Chiral Capillary Columns

A capillary column with a teicoplanin aglycone (TAG) stationary phase (CSP) was used for enantioselective separation of selected profen non-steroidal anti-inflammatory drugs in capillary liquid chromatography (cLC). The effect of variations in the mobile phase composition on the retention and enantioselective separation was examined. The best resolution was attained in the mobile phase composed of 40/60 (v/v) methanol/1.0% triethylamine acetate buffer, pH 4.0 or 4.5. Under the optimized separation conditions, five of the set of eight analytes were enantioresolved with resolution values better than 0.9. Only fenoprofen was not enantioseparated in any system tested. The optimized separation conditions were used for evaluation of three chiral capillary columns (all prepared in the same way in our laboratory) in terms of the repeatability and reproducibility of the results. The run-to-run repeatability was expressed in terms of the relative standard deviation (RSD) values, obtained from ten independent measurements, for the following parameters: the retention factor for the first eluted enantiomer (k ₁), the selectivity (α), the enantioresolution (R), the theoretical plate count per meter for the first eluted enantiomer (N ₁) and the elution curve asymmetry for the first eluted enantiomer (As ₁). None of the RSD values exceeded 8%. The column-to-column reproducibility of these parameters ranged between 1 and 9%. The results obtained with TAG based CSPs in cLC (a laboratory packed capillary column) were compared with those obtained by classical high-performance liquid chromatography (HPLC) with a commercially available column. The cLC procedure provided a better enantioresolution and the elution curves had a better symmetry.     

Capillary columns with in situ formed porous monolithic packing for micro high-performance liquid chromatography and capillary electrochromatography.

Capillary columns with monolithic stationary phase were prepared from silanized fused-silica capillaries of 75 microns I.D. by in situ copolymerization of divinylbenzene either with styrene or vinylbenzyl chloride in the presence of a suitable porogen. The porous monolithic support in this study was used either directly or upon functionalization of the surface to obtain a stationary phase that was appropriate for the separation of peptides by capillary electrochromatography (CEC). The main advantages of monolithic columns are as follows. They do not need retaining frits, they do not have charged particles that can get dislodged in high electric field, and they have relatively high permeability and stability. Whereas such columns are designed especially for CEC, they find application in micro high-performance liquid chromatography (mu-HPLC) as well. Five different porogens were employed to prepare the monolithic columns that were examined for permeability and porosity. The flexibility of fused-silica capillaries was not adversely affected by the monolithic packing and the longevity of the columns was satisfactory. This may also be due to the polymerization technique, which resulted in a fluid-impervious outer layer of the monolith that precluded contact between the fused-silica surface and the liquid mobile phase. For the most promising columns, the conductivity ratios and the parameters of the simplified van Deemter equation, both in mu-HPLC and CEC, were evaluated. It was found that the efficiency of the monolithic columns in CEC was significantly higher than in mu-HPLC in the same way as observed with capillary columns having conventional particulate packing. This is attributed to the relaxation of band-broadening with electroosmotic flow (EOF) with respect to that with viscous flow. It follows then that the requirement of high packing uniformity to obtain high efficiency may also be relaxed in CEC. Angiotensin-type peptides were separated by CEC with columns packed with a monolithic stationary phase having fixed n-octyl chains and quaternary ammonium groups at the surface. Plate heights of about 8 microns were routinely obtained. The mechanism of the separation is based on the interplay between EOF, chromatographic retention and electrophoretic migration of the positively charged peptides. The results of the complex migration process, with highly nonlinear dependence of the migration times on the organic modifier and the salt concentration, cannot be interpreted within the framework of classical chromatography or electrophoresis.     

Porous structure of the monolithic sorbent and separation properties of monolithic capillary columns in gas and liquid chromatography

Macroporous polymer based on polydivinylbenzene was used for the preparation of monolithic capillary columns with the diameter from 0.01 to 0.53 mm for separations by gas and liquid chromatography. The separation properties of the columns were studied by analysis of model systems of aromatic (in liquid chromatography) and light (in gas chromatography) hydrocarbons. The permeability was determined and the C parameter of the Van-Deemter equation was found for each column. The permeability of the majority of columns determined by gas chromatography is independent of the column diameter. The permeability of the same columns in liquid chromatography is also almost constant for the columns 0.53–0.1 mm in diameter; however, the permeability decreases sharply on going to columns of smaller diameter. In gas chromatography the value of the C parameter reflecting the effect of the mass transfer of the sorbate between the mobile and stationary phases on the smearing of a chromatographic peak in the column approximately the same for all columns. In liquid chromatography the value of the C coefficient in the Van-Deemter equation for the same capillary columns changes with a change in the column diameter and reaches a minimum for the columns 0.1 mm in diameter. The differences observed for the characteristics of the columns in gas and liquid chromatography are due to different structures of the macroporous monolith formed in columns of different diameter and to the effect of solvation of the monolith by the mobile phase under the conditions of liquid chromatography.     

Influence of the diameter of monolithic capillary columns on their gas chromatography characteristics

Divinylbenzene polymer monolithic capillary columns were prepared on the basis of capillaries 0.01 to 0.53 mm in diameter. Separation properties of the columns were investigated with the use of a test mixture of light hydrocarbons. The permeability and C parameter in the Van Deemter equation were determined for all the columns. For the most part, the columns had similar characteristics: permeability was in the range (2.2 ± 0.2) × 10^?9 cm², with parameter C in the range (0.7 ± 0.2) × 10^?3 s (with n-butane as a sorbate). It was thus established that capillary diameter has only a slight effect on the efficiency of monolithic capillary columns (unlike packed capillary columns and microcolumns, whose properties, according to the literature data, depend strongly on the column diameter). The difference in properties between the narrowest monolithic column (capillary diameter 0.01 mm) and the others is explained by column overloading.     

Porous Monoliths: Stationary Phases of Choice for High Performance Liquid Chromatography in Various Formats

 Modern porous monoliths have been conceived as a new class of stationary phases for high performance liquid chromatography (HPLC) in classical columns in the early 1990s and later extended to the capillary format. These monolithic materials are prepared using simple processes carried out in an external mold (inorganic monoliths) or within the confines of the column (organic monoliths and all capillary columns). These methods afford macroporous materials with large through-pores that enable applications in a rapid flow-through mode. Since all the mobile phase must flow through the monolith, the convection considerably accelerates mass transport within the monolithic separation medium and improves the separations. As a result, the monolithic columns perform well even at very high flow rates. The applications of monolithic capillary columns are demonstrated on numerous separations in the HPLC mode.     

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.     

Capillary Electrochromatography on Methacrylate Based Monolithic Columns: Evaluation of Column Performance and Separation of Polyphenols

Fused-silica capillary columns (100 μm I.D.) englobing a porous monolithic stationary phase were prepared by in situ copolymerization of 2-ethylhexyl methacrylate, ethylene glycol dimethacrylate and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) in the presence of a porogenic mixture containing 1-propanol, 1,4 butanediol and water. The influence of the monomers ratio and the porogen solvent composition as well as the content of AMPS in the polymerization mixture on column total porosity and efficiency was investigated to attain minimum HETP values for the reversed-phase capillary electrochromatography separation of bioflavonoids. For the most promising column, the van Deemter plots, in both μ-HPLC and CEC, were also evaluated. In CEC the reduced plate height was found almost constant (1.6–2.0) within the range of linear mobile phase velocity between 0.2–2.0 mm s⁻¹. The chemical and mechanical stabilities of the monolithic column over a wide range of buffer pH (2-10) and time were satisfactory. Furthermore, the effects of mobile phase parameters, such as buffer concentration and organic modifier content, on the electroosmotic flow were studied systematically. CEC separations of standard mixtures of polyphenols, including flavonols, flavanones and flavanones-7-O-glycosides, were accomplished in less than 8 min. The CEC separation of the major flavanone glycoside constituents in the extract from a freshly squeezed grapefruit juice was also reported.     

Effects of the dynamic modification of stationary phases by sorbates in gas chromatography: The possibility of separating enantiomers in achiral systems

It is shown that the gas chromatographic separation of enantiomers on columns with achiral nonpolar stationary phases is principally possible as a result of the dynamic modification of stationary phases by sorbates under analysis. It is found that a number of key characteristic features is intrinsic to such separation: it can be only partial, it does not occur for all chromatographic columns, and it is observed only for some compounds and only within narrow ranges of quantities of sorbates that are close to the limits of mass overload of chromatographic systems. These characteristic features are illustrated by the examples of separating (1R,5R)-(+)- and (1S,5S)-(?)-α-pinenes on a WCOT column with an RTX-5 phase. The main characteristic feature of the separation of enantiomers as a result of the dynamic modification of stationary phases is the nonconformity of peaks in chromatograms with two individual enantiomers, compared to other ways and means for their separation; the first eluting peak belongs to the enantiomer that predominates in a mixture irrespective of its configuration, while the second peak corresponds to the racemic mixture of enantiomers; i.e., the ratio of peak areas in chromatograms does not correspond to the actual ratio of enantiomers in samples under analysis and is strongly distorted as a result of their incomplete separation. It is concluded that the separation of racemic mixtures in achiral systems is fundamentally impossible under any conditions, and this is one of the key criteria of the validity of the considered concept as a whole.     

Optimization of capillary parameters for gas chromatography

An HETP equation for the capillary column is developed that takes into account the dependence of gaseous diffusion on pressure, the compressibility of the mobile phase, together with the unique relationship between mobile phase velocity, and the resistance to mass transfer in the stationary phase. The equation is used to develop a procedure for column optimization and expressions are derived that allow the optimum column radius and optimum column length to be calculated for a given fixed inlet pressure. It is shown that fast, simple separations are optimally achieved using relatively short small diameter columns. Conversely, optimum performance for the separation of complex mixtures requiring higher efficiencies requires the use of long columns with relatively large diameters.     

Phase Ratio and Equilibrium Constant in RP-HPLC Obtained from Octanol/Water Partition Constant Through Solvophobic Theory

The experimentally known dependence in RP-HPLC of the retention factor k′ on octanol/water partition coefficient (K _ow) has been examined based on solvophobic theory. The result showed that the dependence provides a means for the evaluation of phase ratio (Φ) of RP-HPLC columns, and of the equilibrium constant for a given compound and mobile phase. Using this theory, the phase ratio was evaluated for a set of seven different C18 columns (five having fully porous particles and two core–shell particles), and the equilibrium constants were calculated for four homologous series of compounds in two mobile phase systems. One mobile phase was methanol/aqueous solution of 0.1% H₃PO₄, and the other was acetonitrile/aqueous solution of 0.1% H₃PO₄. Besides providing the values for Φ for the evaluated columns, the results of the study indicated that for a specific composition of the mobile phase and for a given compound displaying only hydrophobic interactions, the equilibrium constant K(X) for different C-18 columns is basically the same. The data were further used to provide guidance in the selection of a chromatographic column for a specific separation based on K _ow values and chemical structure of the analytes. The study indicated that the separation of compounds with identical polar groups (or no polar groups) and with very close values for the K _ow cannot be achieved based only on hydrophobic interactions that dominate the separation on RP-type columns. Only column that displays polar interactions may provide a solution to such separations. For hydrocarbons with close K _ow values, the separation cannot be achieved even on columns with some polarity. On the other hand, even compounds with equal K _ow values, but with different functionalities can be separated on RP-HPLC columns without involving polar interactions. The compounds with different K _ow values are expected to be easily separated on RP-HPLC columns.     

Evaluation of New Stationary Phases for the Separation of Fatty Acid Methyl Esters

In the analysis of fatty acids, one of the most commonly used tools is a GC separation of the fatty acid methyl esters (FAME). Many researchers perform this separation using a non-polar phase such the ubiquitous 5% phenyl / 95% methyl capillary columns found in most every chromatography laboratory. Numerous laboratories have also turned recently to polar phases such as 70% cyanopropyl columns, as this type of chemistry provides increased selectivity for unsaturated compounds, and thus improved separation of cis/trans and ω³/ω⁶ FAME isomers. Here, a series of columns nominally having 60, 70, 80, and 90% bis-cyanopropyl content have been tested for the separation of FAME isomers. Trends in retention and the influence of increasing phase polarity on effective and fractional chain lengths are highlighted to provide the FAME chromatographer with insight into which of these novel stationary phases might be best suited to their particular application. In addition, the elution temperatures (T_e) of the FAME and linear alkane standards are presented, as this information will be of value to comprehensive two-dimensional multidimensional GC (GC × GC) users who wish to use these columns in the primary dimension separation.     

High-resolution monolithic columns—a new tool for effective and quick separation

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a “classic” monolithic column, the first generation (Onyx? monolithic C₁₈, 100 mm?×?4.6 mm, Phenomenex); a “narrow” monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm?×?3 mm, Merck); and a recently introduced “high-resolution” monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm?×?4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile–phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.

Preparation of a Sulfoalkylbetaine-Based Zwitterionic Monolith with Enhanced Hydrophilicity for Capillary Electrochromatography Separation Applications

A novel monolithic stationary phase based on in situ copolymerization of zwitterionic monomer N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (DMMSA), pentaerythritol triacrylate (PETA), either methacrylatoethyl trimethyl ammonium chloride (META) or sodium 2-methylpropene-1-sulfonate (MPS) was designed as a multifunctional separation column for hydrophilic interaction capillary electrochromatography (HI-CEC). A significantly enhanced hydrophilicity was obtained on the poly(DMMSA-co-PETA-co-META or MPS) monolith, which was contributed by the high percentage of DMMSA in the polymerization mixture. A column efficiency of 200,000 plates/m was obtained and the monolithic column also displayed a satisfactory repeatability in terms of migration time with RSD values less than 1.1% (intra-day, n = 5) and 2.0% (inter-day, n = 3). Most importantly, the column was successfully applied to separation of a pool of neurotransmitters which are not well separated on commercial HILIC packing materials. A baseline separation of the 12 model components was obtained with good selectivity, symmetrical peak shape and high column efficiency with BGE consisting of 20 mM ammonium formate (pH 3.0) in ACN/H₂O (80/20, v/v).     

A new method for assessing the effect of residual silanol groups on total retention in reversed-phase HPLC

A new approach is proposed to assess the effect of residual silanol groups (hydrophilicity) on the retention of polar substances in reversed-phase high-performance liquid chromatography (HPLC). It is shown that the numerical characteristic of the relative position of trend lines in the coordinates logk(B) vs. logk(A) of the relative retention parameters (at different mobile phase compositions) for any stationary phase relative to the stationary phase selected as a standard is preferable to the separation selectivity of the same pair of substances. Full information can be obtained by determining the retention of corresponding adsorbates with two different mobile phase compositions for each column under study. It was shown that the pair of adsorbates p-toluidine-p-cresol exhibits higher sensitivity to a change in the hydrophilicity of stationary phases as compared to the pair aniline-phenol. However, the use of a nonpolar substance as a reference compound is more informative.     

Modeling chromatographic dispersion: a comparison of popular equations

An analyte that is introduced onto a column as a finite band broadens as it moves along the column. This band-broadening is generally attributed to three independent processes, including flow path inequalities, molecular diffusion, and resistance to mass transfer. Many equations have been derived in attempts to mathematically model the process. Some of the more popular of these include the equations of van Deemter, Giddings, Horvath and Lin, and Knox. Although the basis of each equation is theoretically different, the differences among them are minor, and most of the equations can be used to adequately fit plate height data. Chromatographers often collect efficiency data to monitor the performance of their columns, and then use one of the above equations to fit the data. The choice of which equation to use can be daunting, since the theories are conflicting. Using an extensive collection of data, we have compared these equations on the basis of the resulting fit. This study was performed using analytes covering a wide range of retention values and mobile phases of differing strengths. The Foley-Dorsey equation was used to calculate the number of theoretical plates for the efficiency study and special precautions were taken to ensure that the observed broadening was due to only processes occurring in the column and that the peaks were adequately sampled. The variance from extra-column sources was measured and subtracted from the system variance. Although more complex equations gave very slightly better fits, 50 years after its introduction the van Deemter equation remains an incredibly accurate representation of band-broadening processes.     

Dynamics of capillary electrochromatography. II. Comparison of column efficiency parameters in microscale high-performance liquid chromatography and capillary electrochromatography.

In capillary electrochromatography (CEC) the flow of the mobile phase is generated by electrosmotic means in high electric field. This work compares band spreading measured experimentally in several packed capillaries with electrosmotic flow (EOF) and viscous flow under otherwise identical conditions. The data were fitted to the simplified van Deemter equation for the theoretical plate height, H = A + B/u + Cu, in order to evaluate parameters A and C in each mode of flow in the different columns. The ratio of these two parameters obtained with the same column in microscale HPLC (mu-HPLC) and CEC was used to quantify the attenuation of their contribution to band spreading upon changing from viscous flow (in mu-HPLC) to electrosmotic flow (in CEC). The capillary columns used in this study were packed with stationary phases of different pore sizes as well as retentive properties and measurements were carried out under different mobile phase conditions to examine the effects of the retention factor and buffer concentration. In the CEC mode, the value of both column parameters A and C was invariably by a factor of two to four lower than in the mu-HPLC mode. This effect may be attributed to the peculiarities of the EOF flow profile in the interstitial space and to the generation of intraparticle EOF inside the porous particles of the column packing. Thus, band spreading due to flow maldistribution and mass transfer resistances is significantly lower when the mobile phase flow is driven by voltage as in CEC, rather than by pressure as in mu-HPLC.