The effect of column characteristics on the minimum analyte concentration and the minimum detectable amount in capillary gas chromatography. Part II: The stationary phase film thickness

For a typical narrow bore (50 μm) and wide bore (320 μm) capillary column the effects of increased stationary phase film thickness (d_f) on the minimum detectable amount, Q_o, as well as on the minimum analyte concentration, C_o, are described. In treating the effect of an increased film thickness, two approaches can be followed; either the separation temperature is kept constant, resulting in larger values of the capacity ratio, k, or the column temperature is increased such as to keep k constant. For normalized chromatographic conditions the effects of both approaches on the minimum plate height, optimum carrier gas velocity, and required plate number are described, finally yielding expressions for Q_o and C_o for both mass flow and concentration sensitive detectors. At constant temperature, C_o always increases with the film thickness for mass flow sensitive detectors (e.g. FID). Wide bore thin film columns offer the lowest value of C_o attainable. For concentration sensitive detectors (e.g. TCD), C_o is affected neither by column diameter nor by film thickness. The Q_o–d_f plot for constant temperature shows a minimum, suggesting an optimum film thickness for mass flow sensitive as well as concentration sensitive detectors. The corresponding capacity ratio has a value between 0.5 and 1.5. At elevated temperatures (k constant) in combination with mass flow sensitive detectors, again an optimum film thickness exists, corresponding to a minimum value of C_o. For constant capacity ratio Q_o always increases with the film thickness for both types of detectors. As indicated above, in some situations the lowest values of C_o and Q_o are obtained at an increased film thickness, the effect being marginal. As an initial guideline, for the daily practice of capillary gas chromatography with respect to minimum values of C_o and Q_o, the use of thin film columns is to be preferred.     

Detector Systems in Capillary Gas Chromatography

Expressions for the minimum detectable amount Q_o and the minimum analyte concentration C_o as functions of the chromatographic parameters are derived for both mass and concentration sensitive detectors. The effects of pressure drop, column inner diameter, and film thickness are given. The minimum analyte concentration for mass flow sensitive detectors, C_o^m, can be reduced considerably by selecting the carrier gas velocity well above its optimum value (related to H_min), however, at the cost of long columns and long analysis times. For Q_o the improvements can be neglected, and so the analysis can best be performed at u_opt. When the flow rate in the detector, F_d, is equal to the column flow rate F_c, the maximum permissible detector volume of concentration sensitive detectors is proportional to d_c² up to d_c³, and so narrow bore columns require detectors of extremely small volume. Make-up gas has to be added when the actual volume is too large, thus worsening the detectability. Another approach, vacuum operation of the detector cell, appears to be very attractive. On the other hand, when wide bore columns are used in combination with small volume concentration sensitive detectors, very small values of Q_o^c and C_o^c are obtainable when the abundant carrier gas can be removed before entering the detector cell. Digital noise filtering can further reduce the obtainable Q_o and C_o values, especially for broad peaks and thus for wide bore columns.     

Hydrophilic interaction chromatography: A promising alternative to reversed‐phase liquid chromatography systems for the purification of small protonated bases

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed‐phase liquid chromatography mode (BEH‐C₁₈), an electrostatic repulsion reversed‐phase liquid chromatography C₁₈ column (BEH‐C₁₈+), a poly(styrene‐divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm³). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed‐phase liquid chromatography like conditions (with BEH‐C₁₈, BEH‐C₁₈+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed‐phase liquid chromatography columns.     

Influence of a water vapor admixture in the carrier gas on capacity coefficients of polar organic compounds

The influence of a water vapor admixture in helium, nitrogen, and carbon dioxide on capacity coefficients of C₃−C₅ alcohols and pyridine during chromatography process in capillary columns with polar (PEG-20M) and nonpolar (SE-30) stationary phases was studied. The introduction of a water admixture into the carrier gas, increases the capacity coefficient of polar organic compounds on the capillary column with PEG-20M and has almost no effect on this value in the case of SE-30. The change in retention of polar organic compounds on the capillary column with the PEG-20M polar phase occurs due to a change in the properties of the stationary phase when it adsorbs water from the mobile phase. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2258–2261, November, 1998.     

Chromatographic Properties of Monolithic Silica Capillary Columns for Polar and Nonpolar Compounds in Reversed-Phase HPLC

Retention properties of monolithic silica C₁₈ columns were examined for polar and nonpolar compounds. Monolithic silica columns were prepared from two kinds of starting materials, tetramethoxysilane (abbreviated as TMOS) or a mixture of TMOS and methyltrimethoxysilane (abbreviated as Hybrid), and modified with dimethyloctadecylchlorosilane (C₁₈). Retention factors of various compounds on Hybrid-C₁₈ are about twice as much as that on TMOS-C₁₈. The two types of columns showed very similar retention selectivity with few exceptions. TMOS-C₁₈ showed slight preference toward planar polynuclear aromatic hydrocarbons (PAHs) compared to bulky aromatic hydrocarbons, while Hybrid-C₁₈ showed opposite tendency. Some TMOS-C₁₈ columns showed peak tailing for PAHs, and also for basic compounds in an acidic mobile phase, while Hybrid-C₁₈ and majority of TMOS-C₁₈ columns resulted in good peak shape for these compounds.     

Comparison of High Performance Liquid Chromatography Methods with Different Detectors for Determination of Steroid Hormones in Aqueous Matrices

This paper presents the development and comparison of procedures for the qualitative and quantitative determination of five estrogenic compounds (17-α-ethynylestradiol, estrone, estradiol, estriol, and progesterone) in drinking water and wastewater samples. Five extraction columns and two disks were tested for their efficiency. The C₁₈ columns were superior on the basis of cost for solid phase extraction of drinking water or sewage. However, the best recoveries were achieved using extraction disks. High-performance liquid chromatography with diode array, fluorescence, and tandem mass spectrometry detectors were compared for selectivity, repeatability, and linearity of response. Solid phase extraction with high-performance liquid chromatography and tandem mass spectrometry was the most sensitive, efficient, and precise for the determination of hormones in drinking water and wastewater. This method satisfied analytical validation criteria and was characterized by a low limit of detection at the pg-ng/L level. The study also considered challenges that emerged for steroidal hormone determination in aqueous samples.     

Chromatographic characteristics of monolithic capillary columns with different polarities

Relative polarity values and retention indices for a mixture of normal paraffins C₅–C₁₀ and alkylbezenes C₆–C₉ are determined for four types of monolithic capillary columns based on divinylbenzene, ethylene glycol dimethacrylate, pentaerythritol tetraacrylate, and pentaerythritol triacrylate. It is established that the retention of polar sorbates increases with a rise in stationary phase polarity, while the retention of nonpolar compounds grows with a reduction in stationary phase polarity.     

Solvent Composition-Capacity Factor Dependencies of Iodoamino Acids

Abstract

The retention behaviour of several iodoamino acids on μBondapak C₁₈ columns has been investigated as the volume fraction, Ψ_s, of the organic solvent modifier was varied over the range 0.08<Ψ_s < 0.8 under low pH, low ionic strength conditions. Bimodal plots of the logarithmic capacity factor, log k′, versus Ψ_s were observed with selectivity reversals from a reversed phase to a polar phase elution mode occurring at Ψ_s ca 0.6 for acetonitrile-based eluents. The calculated slope parameters (s-values) of the iodoamino acids were similar to those found with other low molecular weight solutes but were considerably smaller than s-values observed with polypeptides or proteins of comparable capacity factor characteristics.     

Analysis of gases containing inorganic and organic compounds using a combination of a thick-film capillary column and a packed adsorption column

A gas chromatographic system consisting of one multiport valve, two (hot-wire and flame-ionization) detectors and two analytical columns (one thick-film capillary and one packed adsorption column) is used for the analysis of gas samples containing a number of inorganic compounds (hydrogen, argon, oxygen, nitrogen, carbon monoxide, and carbon dioxide) and organic compounds. Examples include samples containing hydrocarbons up to n-nonane and benzene and toluene. The system also permits the analysis of more complicated samples containing, for example, alcohols, in addition to hydrocarbons.     

Comparison of a C30 Bonded Silica Column and Columns with Shorter Bonded Ligands in Reversed-Phase LC

The Carotenoid S is a new C₃₀ bonded silica stationary phase, intended for reversed-phase chromatographic applications, which is more hydrophobic and consequently shows stronger retention in comparison to conventionally used C₁₈ stationary phases. We compared the non-polar selectivities of the columns for homologous alkylbenzenes in acetonitrile—water and methanol–water mobile phases and polar reversed-phase selectivities employing the interaction indices and the Linear Free Energy Relationship models. Further, we investigated possibilities of separations of structurally closely related compounds in the groups of phenolic acids, flavones, phthalic acids and related compounds and of acylglycerols on the new C₃₀ column and with different types of columns for reversed-phase chromatography, including shorter alkyl C₄, C₈, C₁₈ and phenyl bonded stationary phases. The C₃₀ column has in some aspects properties similar to the non-endcapped Nova-Pak column for separation of some acylglycerols with equal equivalent carbon numbers, but enables separations of longer chain triacylglycerols in a single gradient run.

     

Analysis of peptides and proteins using sub-2 μm fully porous and sub 3-μm shell particles

The objective of this study was to evaluate the potential of sub-2 μm totally porous particles and sub-3 μm shell particles for peptide and protein analysis. Specific analytical strategies must be developed for these biomolecules as their importance in the pharmaceutical industry increases and as their structural complexity involves some issues when classical LC conditions are employed. Attention was paid on comparing these different columns in various LC conditions (different temperatures, gradient times, and mobile phase flow rates). The comparison of the different supports was assessed considering columns characteristics (quality of packing, silanol activity, pore size, totally porous or shell particles). In this article, peptides were first analyzed with both column technologies. Similar results to those achieved with low molecular weight compounds were obtained (peak capacity >100 for t_grad around 3 min and columns dimensions of 2.1 mm id × 50 mm), but specific conditions were required (elevated temperature and the use of a volatile ion-pairing reagent, namely TFA). For peptide analysis following tryptic digestion, the goal was to improve peak capacity and resolution because of the large number of generated peptides. For this purpose, longer columns packed with porous sub-2 μm or shell sub-3 μm particles (i.e., 150 mm) and gradient times (i.e., up to 30 min) were tested. On the other hand, proteins in their intact forms have higher molecular weights (MW > 5000 Da) and a tertiary structure, thus requiring different conditions in terms of stationary phase hydrophobicity (C₄vs. C₁₈) and pore size (300 vs. 120 Å). In addition, there were issues with adsorption onto the LC system and/or the column itself. This study showed that proteins with MWs lower than 40,000 Da required chromatographic conditions close to those employed for peptide analysis. For larger proteins, a C₄ 300 Å stationary phase gave the best results, confirming theoretical predictions.     

Evaluation of efficiency and trapping capacity of restricted access media trap columns for the online trapping of small molecules

Restricted access media are generally composed from multi‐modal particles that combine a size excluding outer surface and an inner‐pore retention mechanism for small molecules. Such materials can be used for either online isolation and pre‐concentration of target small molecules or removal of small molecule interferences from large macromolecules, such as proteins in complex biological matrices. Thus, they are considered as enhanced online solid‐phase extraction materials. We evaluated the efficiency and trapping capacity of different semi‐permeable surface restricted access media columns (C₁₈, C₈, and C₄ inner pores) for four model small molecule compounds (dopamine hydrochloride, acetaminophen, 4‐hydroxybenzoic acid, and diethyl phthalate) having variable physicochemical properties. We further studied the effect of mobile phase flow rate (0.25, 0.5, 1, and 2 mL/min) and pH, using 98:2 0.5% acetic acid in water/ methanol (pH 2.88) and 5 mM ammonium acetate in 98:2 water/methanol (pH 6.61) as mobile phases. Breakthrough curves generated using frontal analysis were analyzed to determine important chromatographic parameters specific for each of the studied compounds. Experimental determination of these parameters allowed selection of the most efficient trap column and the best loading mobile phase conditions for maximal solute enrichment and pre‐concentration on restricted access media trap columns.     

Recent trends in ultra‐fast HPLC: New generation superficially porous silica columns

New generation columns, i.e. packed with superficially porous silica particles are available as trade names with following manufacturers: Halo, Ascentis Express, Proshell 120, Kinetex, Accucore, Sunshell, and Nucleoshell. These provide ultra‐fast HPLC separations for a variety of compounds with moderate sample loading capacity and low back pressure. Chemistries of these columns are C₈, C₁₈, RP‐Amide, hydrophilic interaction liquid chromatography, penta fluorophenyl (PFP), F5, and RP‐aqua. Normally, the silica gel particles are of 2.7 and 1.7 μm as total and inner solid core diameters, respectively, with 0.5‐μm‐thick of outer porous layer having 90 Å pore sizes and 150 m²/g surface area. This article describes these new generation columns with special emphasis on their textures and chemistries, separations, optimization, and comparison (inter and intra stationary phases). Besides, future perspectives have also been discussed.     

Modification of Selectivity in Reversed-Phase Liquid Chromatography of Polycyclic Aromatic Hydrocarbons Using Mixed Stationary Phases

Abstract

Monomeric and polymeric C₁₈ materials provide significantly different selectivities for polycyclic aromatic hydrocarbons (PAH) in reversed-phase liquid chromatography. Selectivity factors vary in a regular manner with respect to surface concentration of C₁₈ groups on different C₁₈ columns. In this study, we investigated the feasibility of “customizing” a C₁₈ column to provide an intermediate selectivity by mixing 5-μm polymeric C₁₈ material from two different lots with high and low C₁₈ surface concentrations. Polymeric C₁₈ materials from different production lots were mixed in ratios of 30/70, 50/50, and 70/30 (w/w). Selectivity factors for these columns were found to be similar to those predicted by the linear addition of the selectivities of the two individual phases. The PAH selectivities on these mixed columns were also found to be comparable to data obtained from coupled short columns of appropriate lengths each containing one of these different C₁₈ materials. These studies indicate that columns of specific selectivity can be prepared by either mixing two different C₁₈ materials or by coupling columns containing each of these different phases. The use of mixed phase columns is illustrated for the analysis of a fraction containing five condensed ring PAH isomers (molecular weight 278) isolated from an air particulate sample.     

Evaluation of an amide‐based stationary phase for supercritical fluid chromatography

A relatively new stationary phase containing a polar group embedded in a hydrophobic backbone (i.e., ACE ® C18‐amide) was evaluated for use in supercritical fluid chromatography. The amide‐based column was compared with columns packed with bare silica, C₁₈ silica, and a terminal‐amide silica phase. The system was held at supercritical pressure and temperature with a mobile phase composition of CO₂ and methanol as cosolvent. The linear solvation energy relationship model was used to evaluate the behavior of these stationary phases, relating the retention factor of selected probes to specific chromatographic interactions. A five‐component test mixture, consisting of a group of drug‐like molecules was separated isocratically. The results show that the C₁₈‐amide stationary phase provided a combination of interactions contributing to the retention of the probe compounds. The hydrophobic interactions are favorable; however, the electron donating ability of the embedded amide group shows a large positive interaction. Under the chromatographic conditions used, the C₁₈‐amide column was able to provide baseline resolution of all the drug‐like probe compounds in a text mixture, while the other columns tested did not.     

Properties and Applications of Mixed Packing Capillary Electrochromatography

Mixed packing capillary electrochromatography (MP CEC) with the stationary phase comprising a physical mixture of strong cation exchange (SCX) phase and octadecysilyl (ODS) phase was developed. With the existence of a sulfonic acid group on the surface of SCX, not only could the electroosmotic flow (EOF) remain high at low pH, but also the hydrophilicity of the stationary phase was increased greatly, leading to broad adaptable ranges of both pH and organic modifier concentration in the mobile phase. At the same time, with the coexistence of C₁₈ on the surface of ODS, both the retention and the resolution of samples were improved. Accordingly, MP CEC combined the advantages of both SCX and ODS columns. Effects of operation parameters on EOF and the capacity factors of solutes as well as the retention mechanism of such a column were studied systematically. In addition, MP CEC columns were used in the analysis of strong polar solutes as well as for the high speed separation of acidic, basic, and neutral compounds in a single run.     

Separation of dixanthogen and sulphur xanthates by H.P.L.C.: Disproportionation of Sulphur Dixanthates

High-performance liquid chromatography on C₁₃ reverse-phase or silica gel columns, with methanol—water (85:15) or n-hexane, respectively, as mobile phase, is used to separate dixanthogens and sulphur xanthates with different alkyl groups. Detection limits are as low as 1 ng (0.05 ppm) with u.v. detectors. When molecular emission cavity analysis is used for the detector, detection limits are reduced to ca. 0.25 ng (0.013 ppm). In aqueous methanol, alkyl sulphur dixanthates, (ROCS₂)₂S, disproportionate to yield mixtures of the corresponding dixanthogen, (ROCS₂)₂ , alkyl sulphur monoxanthates, (ROCS₂)₂S₂ , and dixanthates.     

Evaluation of Alkyl Bonded Silica and Solvent Phase Modifiers for the Efficient Elution of Basic Drugs on HPLC

Abstract

Nine representative drugs were used to evaluate the effects of alkyl bonded stationary phases containing type A and type B silica and the effects of an amine modifier on the efficiency of high performance liquid chromatographic elution of basic and acidic drugs. The theoretical plate count and asymmetry factor of the eluted peaks were compared to that of acetophenone as a reference to the maximal efficiency of each system evaluated. Zorbax^R C₈ was used as the stationary phase prepared from type A silica and Zorbax RX^R was used as the stationary phase prepared from the type B silica. The theoretical plate count and asymmetry factor of acetophenone was observed to be the same on both columns when analyzed in an acidic aqueous/acetonitrile mobile phase. An improvement in the efficiency and peak shape of the amine containing compounds was observed using the Zorbax RX^R stationary phase as compared to the efficiency and peak shape of these compounds on the Zorbax^R C₈ stationary phase. Interestingly, the acidic compounds salicylic acid and mefenamic acid showed better peak shapes on the Zorbax RX^R column than on the Zorbax C₈. For all drugs studied the theoretical plate count and asymmetry factor was better on both the Zorbax^R C₈ and the Zorbax RX^R stationary phases when the amine modifier triethylamine was used in the mobile phase. Except for salicylic acid, the theoretical plate count and asymmetry factor for each drug was similar on the Zorbax^R C₈ and the Zorbax RX^R columns when the amine modifier     

Evaluation of the Chromatographic Performance of Conventional, Polar-Endcapped and Calixarene-Bonded Stationary Phases for the Separation of Water-Soluble Vitamins

The performance of four different reversed-phase columns which included a conventional C₁₈ phase, a C₁₈ polar-endcapped phase, an ether-linked phenyl polar-endcapped phase and a calixarene-bonded phase has been systematically compared for the separation of mixture of some water-soluble vitamins containing basic, neutral and acidic compounds of different polarities, as well as different functional groups at three pH levels and different proportions of buffer/methanol. The characteristics of water-soluble vitamins make this combination of compounds very useful as a test mixture to check column performance with real samples. Due to the physical and chemical differences between these compounds, the type of chosen column has a significant influence on the chromatographic behavior. Results of this comparison show that the C₁₈ polar-endcapped phase was the most suitable for the separation of this group of vitamins. The presence of a polar group as an endcapping agent does not seem to influence the overall hydrophobic nature of the polar-endcapped stationary phases. At the same time, these phases displayed enhanced hydrogen bonding and silanol activity.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.