Column selectivity in reversed-phase liquid chromatography. VIII. Phenylalkyl and fluoro-substituted columns

As reported previously, five solute-column interactions (hydrophobicity, steric resistance, hydrogen-bond acidity and basicity, ionic interaction) quantitatively describe column selectivity for 163 alkyl-silica, polar-group and cyano columns. In the present study, solute retention and column selectivity for 11 phenyl and 5 fluoro-substituted columns were compared with alkyl-silica columns of similar ligand length. It is concluded that two additional solute-column interactions may be significant in affecting retention and selectivity for the latter columns: (a) dispersion interactions of varying strength as a result of significant differences in bonded-phase polarizability or refractive index and (b) pi-pi interactions in the case of phenyl columns and aromatic solutes. These 16 phenyl and fluoro columns were also characterized in terms of hydrophobicity, steric resistance, hydrogen-bond acidity and basicity, and ionic interaction.     

Column selectivity in reversed-phase liquid chromatography. V. Higher metal content (type-A) alkyl-silica columns

Retention measurements involving 16 test solutes have been carried out for 38 type-A alkyl-silica columns and three bonded-zirconia columns. These measurements have been analyzed in terms of a model previously developed for type-B columns, so as to yield values of five column selectivity parameters (H, S*, A, B, C) for each type-A column. Overall differences in selectivity between type-A and -B columns can be related to the average values of H, S*, etc. for each column type. Compared to type-B columns, type-A columns provide generally stronger retention for carboxylic acids, while solutes that are more hydrophobic or less bulky are more retained on type-B columns. Hydrogen-bond acceptors (e.g. aliphatic amides) and cations (e.g. protonated bases) are strongly retained on type-A versus type-B columns. Compared to type-B columns, bonded-zirconia columns show much increased retention of cations and reduced retention of hydrogen-bond acceptors. Because of relatively large differences in the selectivity of bonded-zirconia, type-A, and type-B columns, it will prove difficult to find columns of different type (e.g. a type-A and a type-B column) which have equivalent selectivity. Type-A columns also tend to be more different from each other (in terms of selectivity) than is the case for type-B columns. As a result, the replacement of a given type-A column by an "equivalent" type-A column also appears unlikely, except for samples that do not contain ionized compounds.     

The hydrophilic interaction like properties of some reversed phase high performance liquid chromatography columns in the analysis of basic compounds

The hydrophilic interaction chromatography (HILIC) like properties of an ACE cyano (CN) HPLC column was studied for the separation of some basic compounds. Good separation of a test mix of basic compounds was obtained with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate. The retention times of the basic compounds decreased with increased ionic strength or with increased water content in the mobile phase. When Trishydroxymethyl aminomethane (Tris) (pK(a) 8.0), which is a weaker amine than ammonia (pK(a) 9.3), was used as an additive in the mobile phase retention of the basic compounds was increased. The ACE CN column gave excellent peak shapes for all the basic compounds. The utility of the column for impurity profiling of two basic drugs was tested and some impurities in oxprenolol were characterised by interfacing with Fourier transform mass spectrometry. It was also observed that ACE butyl and ACE phenyl columns retained basic compounds when the columns were eluted with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate.     

Column selectivity in reversed-phase liquid chromatography. IV. Type-B alkyl-silica columns

Columns for reversed-phase HPLC (RP-LC) can be characterized by five, retention-related parameters: H (hydrophobicity), S (steric selectivity), A (hydrogen-bond acidity), B (hydrogen-bond basicity), and C (cation-exchange behavior). In the present study, values of the latter parameters have been measured for 92 type-B (low metals content) alkyl-silica columns and compared to column properties such as ligand length, ligand concentration, pore diameter, and the presence or absence of end-capping. With the exception of five columns of unusual design, retention factors, k, for 16 representative test compounds were correlated with values of H, S, etc., within an average ±1.2% (1 standard deviation, SD), suggesting that all significant solute–column interactions are recognized by these five column parameters. A single-valued function F_s is proposed to measure differences in selectivity for any two RP-LC columns whose values of H, S, etc., are known. This allows the easy selection of columns whose selectivity is desired to be either similar to or different from a starting column, for application in either routine analysis or method development.     

Temperature gradients in HPLC columns due to viscous heat dissipation

Summary Temperature effects in HPLC columns due to viscous heat dissipation are examined. For the case when the thermostatted column wall and mobile phase at the column inlet are at the same temperature an explicit solution of the heat transport equation is given. The predicted temperature profile is parabolic at large distances from the column entrance; the magnitude of the effect is proportional to the square of the mobile phase velocity, and is of the order of a few degrees centigrade. At the upper end of the column a relaxation occurs over a length of a few centimers. Experimental results confirm the validity of the predictions made and indicate that the various assumptions and approximations are justified. Plate height curves obtained with two mobile phases with differing viscosities show a much smaller efficiency for the less viscous mobile phase. The curves show an upward curvature at high reduced velocities. Both phenomena can be related to thermal effects. It is concluded that viscous heat dissipation constitutes an obstacle to obtaining higher speed and efficiency in HPLC by the use of smaller particles. Possible remedies, such as the use of smaller bore columns or special thermostatting devices, look troublesome from the experimental point of view.     

Peptide separation in normal-phase liquid chromatography. Study of selectivity and mobile phase effects on various columns.

An experimental procedure for peptide separation by normal-phase liquid chromatography (NPLC) was proposed in previous papers. In the present study, the chromatographic behavior of amino, cyano, amide, diol and silica columns, which have been used in non-aqueous NPLC, is investigated anew. The amino column was not appropriate for peptide separation because of poor recovery. The cyano column could not be used due to lack of retention. The amide, diol and silica columns were useful for peptide separation. The chromatograms on amide, diol and silica columns were a little different when the mobile phase composition was changed. The recovery of peptides was good: diol > amide > silica. Repeatability and reproducibility using amide, diol and silica columns was satisfactory.     

Column selectivity in reversed-phase liquid chromatography I. A general quantitative relationship

Retention factors k have been measured for 67 neutral, acidic and basic solutes of highly diverse molecular structure (size, shape, polarity, hydrogen bonding, pKa, etc.) on 10 different C18 columns (other conditions constant). These data have been combined with k values from a previous study (86 solutes, five different C8 and C18 columns) to develop a six-term equation for the correlation of retention as a function of solute and column. Values of k can be correlated with an accuracy of +/- 1-2% (1 standard deviation). This suggests that all significant contributions to column selectivity have been identified (and can be measured) for individual alkyl-silica columns which do not have an embedded polar group. That is, columns of the latter kind can be quantitatively characterized in terms of selectivity for use in the separation of any sample.     

Comparison of different HPLC systems for analysis of galantamine and lycorine in various species of Amaryllidaceae family

Retention parameters of galantamine and lycorine standards were determined on different columns, i.e., octadecyl silica, SM C18, and strong cation-exchange (SCX) columns with different aqueous mobile phases. Retention of alkaloids was investigated on C18, SM C18 columns with mobile phase containing 5% MeCN, 20% acetate buffer at pH 3.5, and 0.025 ML^?1 diethylamine (DEA), and on SCX column with mobile phase containing 8% MeCN and phosphate buffer at pH 2.5. Better results were also obtained in ion-exchange chromatographic system. On the basis of results obtained in different chromatographic systems, simple, rapid, and sensitive high-performance liquid chromatography methods were developed for determining lycorine and galantamine in plant extracts from various species belonging to Amaryllidaceae family. Extracts were prepared from various parts of plants collected at different times of the growing season.     

Effect of high-temperature on high-performance liquid chromatography column stability and performance under temperature-programmed conditions

Six commercially available analytical (4.1 or 4.6 mm i.d.) columns were evaluated under temperature-programmed high-temperature liquid chromatography (HTLC) conditions to access their stability and performance at extreme temperatures. Seven components consisting of acidic, basic and neutral compounds were analyzed under temperature-programmed conditions and solvent gradient conditions using three different mobile phase compositions (acidic, basic and neutral). Each column was checked with a two-component test mix at various stages of the evaluation to look for signs of stationary phase collapse. Three zirconia based stationary phases studied exhibited column bleed under temperature-programmed conditions. The other three columns, a polydentate silica column, a polystyrene-divinylbenzene (PS-DVB) polymeric column, and a graphitic carbon column performed well with no evidence of stationary phase degradation. The R.S.D. for the retention times and efficiencies were less than 10% for most conditions, and not more than 15% during the course of the evaluation for each column. The polydentate silica stationary phase was temperature programmed to 100 degrees C, the PS-DVB stationary phase was temperature programmed up to 150 degrees C, and the graphitic carbon column was used with temperature programming up to 200 degrees C. Comparable peak capacities and similar retention behaviors were observed under solvent gradient and temperature-programmed conditions. Temperature programming with dynamic mobile phase preheating can replace solvent gradient analysis without a loss of peak capacity when used with 4.1 or 4.6 mm columns.     

Study on conical columns with different conical angles for semi-preparative liquid chromatography

The dynamic flow profiles and separation performances in conically shaped preparative liquid chromatographic columns (inlet i.d. larger than outlet i.d.) with three different angles (7, 10 and 15) were studied and compared with cylindrical column of the same length and internal volume. The shapes of dynamic flow profiles were studied by on-column visualization method. The transparent chromatographic columns made of polymethyl methacrylate (PMMA), packed with C18 bonded silica, were immerged into a cubic pool filled with glycerol to eliminate the cylindrical and conical lens effect. The flow profiles of colored iodine solution in the columns were observed clearly using cyclohexane as mobile phase since the refractive indices of C18, column wall and the mobile phase are very close. In the conical column of 15 degrees (20-7 mm i.d.) the mobile phase in the central region migrated slower than in wall region as it moved toward the column outlet, while in the conical column of 7 degrees (17-11 mm i.d.) the mobile phase in the central region migrated faster than in wall region just like in cylindrical column. We found that a plug-like flow profile was generated in the conical column of 10 degrees (18-9 mm i.d.) during the whole migration process. A carmine and brilliant blue mixture was used as a probe to test the separation ability of the columns. The resolutions of the two compounds on the conical column of 7, 10, 15 and on the cylindrical column were 0.6, 1.57, 1.29 and 0.8, respectively.     

Two-dimensional and serial column reversed-phase separation of phenolic antioxidants on octadecyl-, polyethyleneglycol-, and pentafluorophenylpropyl-silica columns

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.     

A strategy to develop fast RP-HPLC methods using monolithic silica columns

Since the appearance of monolithic silica, much work has been done describing the properties of monolithic silica columns. Meanwhile the transferability of analytical methods from conventional to monolithic silica columns has been intensively investigated [1-5]. RP HPLC method development strategies for conventional columns should be updated or scaled to meet the higher performing monolithic column technology. Because of the high permeability of monolithic silica columns it should be possible to decrease the time for method development by applying high isocratic flow rates. Here we suggest a clear strategy for method development using monolithic columns. The strategy will be applicable for various sample compositions, e. g., acidic, basic, or neutral. The applicability of monolithic columns for especially complex separations of basic mixtures without the need of using a highly basic mobile phase that harms the column will be pointed out in this work. This work will describe in detail the actual method development process. For better understanding of our strategy, the influence of flow rate, column length, mobile phase composition, pH, and temperature will be discussed. Details about the application of a flow program will be mentioned.     

Prospects for monolithic nano-LC columns in shotgun proteomics

We report a premier side-by-side comparison of two leading types of monolithic nano-LC column (silica-C(18), polystyrene) in shotgun proteomics experiments. Besides comparing the columns in terms of the number of peptides from a real-life sample (Arabidopsis thaliana chloroplast) that they identified, we compared the monoliths in terms of peak capacity and retention behavior for standard peptides. For proteomics applications where the mobile phase composition is constrained by electrospray ionization considerations (i.e., there is a restricted choice of ion-pairing modifiers), the polystyrene nano-LC column exhibited reduced identification power. The silica monolith column was superior in all measured values and compared very favorably with traditional packed columns. Finally, we investigated the performances of the monoliths at high flow rates in an attempt to demonstrate their advantages for high-throughput identification.     

Relevance of pi-pi and dipole-dipole interactions for retention on cyano and phenyl columns in reversed-phase liquid chromatography

Previous work suggests that pi-pi interactions between certain solutes and both phenyl and cyano columns can contribute to sample retention and the selectivity of these two column types versus alkylsilica columns. Recent studies also suggest that dipole-dipole interactions are generally unimportant for retention on cyano columns. The present study presents data for 44 solutes, three columns and two different mobile phases that were selected to further test these conclusions. We find that pi-pi interactions can contribute to retention on both cyano and phenyl columns, while dipole-dipole interactions are likely to be significant for the retention of polar aliphatic solutes on cyano columns. When acetonitrile/water mobile phases are used, both pi-pi and dipole-dipole interactions are suppressed, compared to the use of methanol/water.     

Column selectivity in reversed-phase liquid chromatography II. Effect of a change in conditions

The isocratic retention of 67 widely-different solutes in reversed-phase liquid chromatography (RP-LC) has been investigated as a function of temperature and mobile phase composition (% B) for three different C18 columns. Similar studies were also carried out in a gradient mode, where temperature, gradient time and solvent type were varied. These results show that changes in retention with these conditions are similar for each of these three columns. This suggests that relative column selectivity as defined by experiments for one set of experimental conditions will be approximately applicable for other conditions, with the exception of changes in mobile phase pH-which can affect values of the column parameter C (a measure of silanol ionization). Column selectivity as a function of pH was explored for several columns.     

The hydrophobic-subtraction model of reversed-phase column selectivity

A recently developed treatment of reversed-phase column selectivity (the hydrophobic-subtraction model) is reviewed and extended, including its characterization of the selectivity of different column types (e.g., C1-C30, cyano, phenyl, etc.). The application of this model to retention data for various solutes and columns has provided new insights into the nature of different solute-column interactions and their relative importance in affecting sample retention and separation. Reversed-phase columns can be characterized by five selectivity parameters (H, S*, A, B and C), values of which are summarized here for more than 300 different columns. The selection of columns of either equivalent or different selectivity is readily achievable on the basis of their values of H, S*, etc. The development of the hydrophobic-subtraction model, its use in characterizing the selectivity of different reversed-phase liquid chromatography (RP-LC) columns, and its application to various practical problems as described here began in 1998. The original inspiration for this project owes much to Jack Kirkland, who also contributed actively to the initial studies that laid the foundation of this model; he has since provided other important support to this project. Jack and one of the authors (LRS) have enjoyed a strong professional relationship and personal friendship for the past 35 years, and it is the privilege of the authors to dedicate this paper and the work that it represents to Jack. His contributions to HPLC column technology have extended from the mid-1960s into the present century, and it is impossible to conceive of present day HPLC practice without Jack's contributions over the years. In this and other ways, his position as a pioneer and key implementer of HPLC is widely recognized. We wish Jack well in the years to come.     

Development of dual gradient column in liquid chromatography

The dual gradient column, in which both the chemical property of the stationary phase and the flow velocity in the mobile phase are heterogeneous longitudinally along the column, is developed to obtain the mobile phase gradient-like elution in an isocratic condition. Here, the step-wise dual gradient columns were prepared by connecting an inlet column (I.D. 50 microm, packed with ODS) serially to an outlet column (I.D. 100-200 microm, packed with the mixture of ODS and C1 [9:1]). The retention behavior of alkylbenzenes was able to be controlled in the dual gradient column depending on the variation in the flow velocity. Moreover, the change in retention behavior induced by the flow velocity variation for the dual gradient columns was quite different from that by the variation in organic modifier content of the mobile phase in isocratic elution for a single gradient column and can induce the similar effect with an ordinary gradient elution in a mobile phase composition.     

Reversed phase liquid chromatography with UV absorbance and flame ionization detection using a water mobile phase and a cyano propyl stationary phase Analysis of alcohols and chlorinated hydrocarbons

The development of liquid chromatography with a commercially available cyano propyl stationary phase and a 100% water mobile phase is reported. Separations were performed at ambient temperature, simplifying instrumental requirements. Excellent separation efficiency using a water mobile phase was achieved, for example N=18 800, or 75 200 m(-1), was obtained for resorcinol, at a retention factor of k'=4.88 (retention time of 9.55 min at 1 ml min(-1) for a 25 cmx4.6 mm i.d. column, packed with 5 mum diameter particles with the cyano propyl stationary phase). A separation via reversed phase liquid chromatography (RP-LC) with a 100% water mobile phase of six phenols and related compounds was compared to a separation of the same compounds by traditional RP-LC, using octadecylsilane (ODS), i.e. C18, bound to silica and an aqueous mobile phase modified with acetonitrile. Nearly identical analysis time was achieved for the separation of six phenols and related compounds using the cyano propyl stationary phase with a 100% water mobile phase, as compared to traditional RP-LC requiring a relatively large fraction of organic solvent modifier in the mobile phase (25% acetonitrile:75% water). Additional understanding of the retention mechanism with the 100% water mobile phase was obtained by relating measured retention factors of aliphatic alcohols, phenols and related compounds, and chlorinated hydrocarbons to their octanol:water partition coefficients. The retention mechanism is found to be consistent with a RP-LC mechanism coupled with an additional retention effect due to residual hydroxyl groups on the cyano propyl stationary phase. Advantages due to a 100% water mobile phase for the chemical analysis of alcohol mixtures and chlorinated hydrocarbons are reported. By placing an absorbance detector in-series and preceding a novel drop interface to a flame ionization detector (FID), selective detection of a separated mixture of phenols and related compounds and aliphatic alcohols is achieved. The compound class of aliphatic alcohols is selectively and sensitively detected by the drop interface/FID, and the phenols and related compounds are selectively and sensitively detected by absorbance detection at 200 nm. The separation and detection of chlorinated hydrocarbons in a water sample matrix further illustrated the advantages of this methodology. The sensitivity and selectivity of the FID signal for the chlorinated hydrocarbons are significantly better than absorbance detection, even at 200 nm. This methodology is well suited to continuous and automated monitoring of water samples. The applicability of samples initially in an organic solvent matrix is explored, since an organic sample matrix may effect retention and efficiency. Separations in acetonitrile and isopropyl alcohol sample matrices compared well to separations with a water sample matrix.     

Effect of Column Dimensions on HPLC Separations Using Constant Volume Columns

Abstract

The effect of column dimension on resolution, sample capacity, retention time, efficiency and mobile phase composition were studied, using both constant flow rate and constant linear velocity. The four columns selected (A = 238 × 3.2 mm, B = 153 × 4.0 mm, C = 116 × 4.6 mm and D = 50 × 7 mm) had the same volume. K¹ values were found to be constant, within experimental error, for all columns. At constant linear velocity, the retention time was found to be a linear function of column length, while at constant flow rate retention time was constant for all columns. The longest column (A) generated the largest N values while columns 3 and C gave the lowest H values, for dilute solutions, while they decreased with decreasing column length. On the other hand, it was observed that as the sample size increased, N generated by column A decreased more rapidly and eventually fell below the values generated by columns B and C. These two columns (B & C) can tolerate a larger sample size with less reduction in N value than the longest column. It is important to note that although there were minor differences in performance between columns B and C, there were significant differences between them (B and C) and the other two columns (A and D). Column A offered the highest sensitivity (narrower peaks) for dilute solutions, while columns B and C offered higher loadability. The volume of organic modifier in the mobile phase affected the retention equally in the four columns. It was also found that equal separation (a) was obtained for each column at constant flow rate and constant linear velocity, except with the latter the retention times were longer.     

Chromatographic studies of unusual on-column degradations of aniline compounds on XBridge Shield RP18 column in high pH aqueous mobile phase

This paper reports unusual on-column degradations of aniline compounds on Waters XBridge Shield RP18 column when ammonium hydroxide in water and acetonitrile were used as mobile phases in liquid chromatography. The change of the level of on-column degradation of a model compound (Compound 1) with time was observed in the first fifteen injections when started at 60 °C. During a subsequent cooling program from 60 °C to 10 °C with a 10 °C interval, the levels of the degradation products of Compound 1 changed with the change of temperature and reached a maximum at 40 °C. The on-column degradation of Compound 1 was observed when started at 10 °C in the first injection, however, the magnitude of the change of the level of on-column degradation of Compound 1 with time in the first fifteen injections was much smaller than that at 60 °C. During a subsequent heating program from 10 to 60 °C with a 10 °C interval, the levels of the degradation products of Compound 1 increased with the increase in temperature but without a maximum. The change of the degradation product levels of this model compound in the heating process is not super-imposable with that in the cooling process, which demonstrates the degree of the degradation also depends on the heating or cooling process. Column history studies demonstrated that the on-column degradation of Compound 1 changed dramatically on the used columns at both starting temperatures while the dependency of heating and cooling processes on on-column degradation still existed. The unusual on-column degradation of Compound 1 on the used columns can be regenerated in a very similar fashion with an acetic acid column-wash procedure, but is not identical to that on the new column. Similar degradations of other commercially available aniline compounds were also observed with this high pH aqueous mobile phase system.