Biomedical subjects

David V McCalley

Publications and source records attributed to David V McCalley.

14 recordsLinked to original sources

PubMed2006

A study of retention and overloading of basic compounds with mixed-mode reversed-phase/cation-exchange columns in high performance liquid chromatography.

The retention and overload of bases were studied on two new mixed-mode, silica based phases possessing ionic carboxylate functionalities of different acidity embedded within a hydrophobic ligand (SiELC Primesep). At low pH, good peak shapes were obtained for small solute mass, suggesting that the mere presence of a mixed-mode hydrophobic/ionic retention mechanism is not responsible for the poor peak shape that can occur on conventional reversed-phases with ionised silanols. Somewhat inferior, but still acceptable peak shape for bases was obtained on a column containing a mixture of discrete ion exchange and reversed-phase particles (Hypersil Duet). In both types of column, the ionic sites favourably increased the capacity for ionised bases, reducing considerably the deterioration of peak shape with load observed with conventional RP columns. The combined ionic and reversed-phase interaction can give strong retention of bases under certain conditions, necessitating careful choice of stationary and mobile phase.

Chromatography, High Pressure Liquid↗

PubMed2006

Overload for ionized solutes in reversed-phase high-performance liquid chromatography.

Overloading occurs for submicrogram quantities of ionized solutes particularly when using low ionic strength mobile phases at low pH (e.g., formic acid), even with highly inert silica RP-HPLC columns of normal dimensions. Much higher loads can produce a sharp L-shaped peak with retention above the column void volume, in line with the hypothesis that a small number of high-energy sites fill first and are rapidly overloaded, followed by a much larger number of weaker sites. However, charged acids and bases show identical overloading behavior; overloading is reduced as the mobile-phase ionic strength is increased. These findings raise questions about the physical nature of the strong sites. The rapid overloading of silica and purely polymeric phases could be explained by mutual repulsion of ionic species or their inability to fully penetrate the hydrophobic structure of the phase. However, these alternative hypotheses cannot readily explain the high total saturation capacities obtained using frontal analysis. Ion pairing with trifluoroacetic acid may reduce overload, while the effect is less important for formate or phosphate buffers. A surface layer of acetonitrile is not a prerequisite for rapid overloading, as shown by studies using purely aqueous buffers.

Acetonitriles↗

PubMed2006

Study of overload for basic compounds in reversed-phase high performance liquid chromatography as a function of mobile phase pH.

The retention and overloading properties for eight basic solutes and two quaternary ammonium compounds were studied over the pH range 2.7-10.0 using phosphate and carbonate buffers. At low pH, a hybrid inorganic-organic silica-ODS phase (XTerra RP-18, 15 cm x 0.46 cm) showed substantial loss in efficiency when sample masses exceeded about 0.5 microg; these results were similar to those obtained previously on pure silica ODS and wholly polymeric phases, suggesting a common overloading mechanism. At pH 7-8.5, substantial improvements in loading capacity were obtained on XTerra due apparently to the unexpectedly strong influence of small decreases in solute ionisation. Data from the quaternary compounds suggested that silanol ionisation on this phase was still small even at intermediate pH. For many bases, loading capacity continued to improve as the pH was raised to 10, in line with the decrease in the proportion of ionised solute. However, for the highest pK(a) solutes, peak shape worsened at high pH, possibly due to the negative influence of increasing column silanol ionisation.

Amitriptyline↗

PubMed2005

Study of overloading of basic drugs and peptides in reversed-phase high-performance liquid chromatography using pH adjustment of weak acid mobile phases suitable for mass spectrometry.

Serious losses in column efficiency for ionised basic drugs and peptides occur due to overloading of C18 phases when weak acid mobile phases of low ionic strength suitable for mass spectrometric detection are used. Measurable changes in retention time and efficiency can be observed even for levels around 0.01 microg of basic drugs on 0.46 cm I.D. columns; the overloading process is a continuum, rather than an event which takes place only once a certain threshold value has been reached. Overloading can be reduced by increasing the mobile phase pH, e.g to the pKa of the weak acid, which increases the ionic strength, allowing a greater degree of ion pairing and perhaps also physical screening of the adsorbed solute ions by buffer ions. Buffer capacity is also optimum at its pKa. Silanol ionisation and kinetic tailing effects were mostly absent at aqueous pH up to 4.75 on the hybrid inorganic-organic C18 phase employed in this study, except when analysing the most highly charged peptides. The exact cause of overloading of these ionic species is unclear.

Buffers↗

PubMed2005

Comparison of an organic polymeric column and a silica-based reversed-phase for the analysis of basic peptides by high-performance liquid chromatography.

The performance of a purely polymeric and a Type B silica-based C18 reversed-phase column was compared for the analysis of the basic peptide bradykinin and some analogues in order to assess the contribution of silanol interactions to peak shape. Good peak shapes were obtained for small masses of these peptides (0.1 microg or less) using acidic mobile phases on both columns; however, both showed a similar and serious deterioration in peak shape with increasing sample mass. Loss of efficiency on both columns as sample mass increased was considerably more serious when using formic acid rather than trifluoroacetic acid (TFA) as a mobile phase additive. For example, the peak capacity for a 2.5 microg load of one bradykinin on the polymeric column was reduced to only 0.38 times its value for 0.1 microg when using 0.02 M formic acid, compared with 0.77 times its value when using the same concentration of TFA. This result can be attributed to the ion pair effect of TFA and its higher ionic strength, which reduce mutual repulsion of charged peptides when held on the hydrophobic surface of the phase. Addition of salt (KCl) to the formic acid mobile phase caused dramatic increases in retention on the polymeric column, which can also be attributed to ion-pairing effects between halide ions and peptides. The increase in retention with salt addition also confirms that there are no ionic retention sites on the polymeric phase at low pH. The general similarity in behaviour between the polymeric and silica column suggests that silanol groups have little involvement in the retention and overload behaviour of these peptides when using highly inert Type B silica phases.

Chromatography, High Pressure Liquid↗

PubMed2004

Rationalisation of unusual changes in efficiency and retention with temperature shown for bases in reversed-phase high-performance liquid chromatography at intermediate pH.

Despite the possibility of poorer peak shapes, analysis of pharmaceuticals and other bases using reversed-phase high-performance liquid chromatography (RP-HPLC) at intermediate pH gives useful increases in retention, selectivity and column loading capacity compared with low pH. Retention times of some bases showed anomalous increases with column temperature. Peak shapes for bases improved significantly at elevated temperature (up to 70 degrees C), with the weakest bases studied (pKa approximately 8) giving greater improvement than the strongest bases (pKa approximately 10). In contrast, quaternary ammonium compounds showed reduced retention with increasing column temperature (normal behaviour) and only modest improvements in peak shape. Considering these results, and pKa measurements of the bases made using capillary electrophoresis, it appears that increases in retention and improvement in efficiency may be influenced significantly by reduction in the pKa of bases with temperature, leading to reduction in protonation. It is less likely that efficiency improvements are due to the speeding up of the kinetics of silanol ion exchange, at least in the temperature range studied here.

Chromatography, High Pressure Liquid↗

PubMed2004

Effect of buffer on peak shape of peptides in reversed-phase high performance liquid chromatography.

The effect of changing the buffer at constant low pH in the mobile phase is investigated with respect to the separation of a mixture of basic peptides. Considerably worse peak shapes, leading to poorer resolution of complex peptide mixtures, were obtained when using formic acid favoured in LC-MS applications compared with non volatile phosphate buffers or with trifluoroacetic acid (TFA). Poorer peak shapes were largely attributable to reduced column capacity for the peptides when using mobile phases of low ionic strength, due to the increased mutual repulsion of ions held on the hydrophobic column surface which is facilitated in these buffers. However, ion-pairing between the peptides and additives such as TFA or even phosphate may also lessen mutual repulsion effects, leading to greater column capacity. Overloading effects could be observed when sample masses around only 0.1 microg were injected on to standard size analytical columns in formic acid containing mobile phases; sample masses around only 1.5 microg may cause loss of half the system peak capacity in such mobile phases. Results were broadly comparable (after scaling sample size according to column diameter) on columns of both conventional (4.6 mm i.d.) and capillary (0.075 mm i.d.) dimensions. Ammonium formate may be a useful alternative buffer for some applications due to its higher ionic strength.

Buffers↗

PubMed2004

Determination of ionisation constants of organic bases in aqueous methanol solutions using capillary electrophoresis.

The pKa of eight organic bases was determined in aqueous and aqueous methanol solutions of 0-70% (v/v) methanol using capillary electrophoresis. The bases investigated include compounds commonly used to test the activity of RP columns in HPLC. The variation of pKa with temperature in aqueous methanol solutions was also investigated and found to closely resemble temperature coefficients reported for bases in purely aqueous solutions. pKa values determined by CE were compared to those reported using NMR spectroscopy. The good agreement of the results is evidence that either technique is suitable to perform pKa measurements.

Acetonitriles↗

PubMed2003

Rationalization of retention and overloading behavior of basic compounds in reversed-phase HPLC using low ionic strength buffers suitable for mass spectrometric detection.

The retention and overloading behavior of some basic (and acidic) compounds has been studied on different RP-HPLC columns in buffers of varying ionic strength. Anomalous retention patterns of acids and bases were found on one phase in low-pH, volatile buffers such as formic acid, favored for mass spectrometric analysis. Unusual retention compared to that in higher ionic strength phosphate buffers is attributed to the presence of positively charged sites existing on this phase at low pH. Overloading of bases as well as acids is shown to be a function of mobile-phase ionic strength. This result is a logical consequence of previous suggestions that mutual repulsion of ions held on the hydrophobic surface of the stationary phase, rather than overload of silanols, is largely responsible for overloading on pure silica RP columns. Thus, overloading occurs much more readily in low ionic strength formic acid buffers. Appreciable loss of efficiency can occur in such buffers when only 50 ng of some bases is analyzed on a standard-sized column.

Journal Article↗

PubMed2003

Determination of pKa values of organic bases in aqueous acetonitrile solutions using capillary electrophoresis.

Capillary electrophoresis (CE) was used for the determination of ionisation constants (pKa) of a variety of organic bases in aqueous acetonitrile solutions over the range 0-60% (v/v) acetonitrile. These bases are used as test compounds in HPLC column evaluation, thus knowledge of their pKa in hydro-organic solutions is useful. The base pKa decreased with acetonitrile concentration and significant shifts from the aqueous pKa (up to -0.8) were found using 60% acetonitrile. The CE application was confirmed to be very suitable for fast and accurate pKa measurement in aqueous organic solutions.

Acetonitriles↗

PubMed2003

Comparison of peak shapes obtained with volatile (mass spectrometry-compatible) buffers and conventional buffers in reversed-phase high-performance liquid chromatography of bases on particulate and monolithic columns.

Retention factor, column efficiency and asymmetry factor were recorded for nine basic compounds on a number of RP-HPLC columns using phosphate and a variety of (MS-compatible) volatile mobile phase buffers of acid and neutral pH, in order to assess any effects of the buffer on performance. With formic or acetic acid, some phases gave partial or complete solute exclusion effects (reduced or negative k) compared with results using phosphate buffers at low pH. Despite its possible suppression of mass spectrometer sensitivity, trifluoroacetic acid was useful in enhancing retention times of relatively hydrophilic protonated bases, due to ion-pair effects. Peak shape was relatively poor on some pure silica-based ODS phases at pH 7 compared with results at acid pH. At low pH and at pH 7, ammonium and potassium phosphate gave very similar k, but the former may be preferable due to its volatile cation. Improved peak shapes, attributed to superior silanol masking effects, were obtained with ammonium phosphate at pH 7, but not at acid pH. Ammonium acetate gave acceptable peak shape at pH 7, but due to very limited buffer capacity, poor results were obtained for solutes having a pKa close to the mobile phase pH. Due to its instability, ammonium hydrogen carbonate is not a viable alternative buffer at pH 7.

Buffers↗

PubMed2002

Overloading study of bases using polymeric RP-HPLC columns as an aid to rationalization of overloading on silica-ODS phases.

The separation of ionized bases by reversed-phase liquid chromatography with alkyl silica columns often leads to severely tailed bands that are highly detrimental. Band shape and its dependence on sample mass are notably different when mobile-phase pH is changed, and this behavior has not been previously explained. Ionized silanols present in the stationary phase have been credited with a role in determining peak shape. In the present study, separations on two different polymer columns were compared with those previously obtained on alkyl silica phases. Because silanols are absent from polymer columns, this comparison enabled us to assess the role of silanols in separations on alkyl silica phases and to offer an explanation of why band shape changes with sample size and mobile-phase pH for both polymer and silica-based phases.

Journal Article↗

PubMed2002

Analysis of the Cinchona alkaloids by high-performance liquid chromatography and other separation techniques.

The Cinchona alkaloids, which include the pharmaceuticals quinine and quinidine, continue to have a wide variety of important uses. A number of different chromatographic procedures have been developed for the qualitative and quantitative analysis of these compounds in a variety of sample matrices. Reversed-phase HPLC using ODS columns in combination with acidic mobile phases, and UV detection, is the most widely used method. Nevertheless, precautions need to be taken due to the strong silanophilic interactions which can occur with these analytes and the column surface, which can lead to poor peak shape and resolution. Different selectivity may be achieved in HPLC separations by use of alternative stationary phases, or by varying mobile phase pH. The specificity of detection systems may be improved by use of photodiode array UV detectors, or especially mass spectrometers. Thin-layer chromatography (TLC) provides a cheap alternative analytical method, which is especially useful for qualitative analysis. High-performance TLC, gas chromatography, capillary electrophoresis and capillary electrochromatography are all methods which after some development, could prove useful for Cinchona alkaloid separations.

Chromatography, Gas↗

PubMed2002

Comparison of conventional microparticulate and a monolithic reversed-phase column for high-efficiency fast liquid chromatography of basic compounds.

The effect of mobile phase flow on column efficiency for a neutral compound together with weak and strong bases was compared for conventional microparticulate (3/3.5 microm and 5 microm) silica RP columns and a monolithic silica RP. For benzene, the minimum plate height (Hmin) at optimum flow-rate (mu(opt)) for weak bases was similar for the 5 microm and the monolith phases. However, the monolith generated much flatter Van Deemter curves, such that at high flow-rate (5 ml min(-1)) the plate height was nearly 3.5 times lower on the monolith. For weak bases analysed in unbuffered mobile phases, and stronger bases with acid phosphate buffer, increased tailing was obtained on the monolith compared with the conventional phases. Nevertheless, Van Deemter plots on the monolith still showed some advantages over particulate phases, even when asymmetry factor was included in the calculation of the plate height. However, at pH 7 considerable tailing of strong bases was found using the monolith; it is not clear whether this results from unique features of the monolith structure, or whether it is due merely to usual problems of silica activity. Van Deemter plots for conventional phases may be improved considerably by operating the column at elevated temperatures. At pH 3, these improvements are influenced to a considerable extent by increases in Dm, as shown by measurements of Dm using the Taylor-Aris procedure. However, at pH 7.0, improvements are much too substantial to be explainable wholly on this basis.

Buffers↗