Indirect detection in liquid chromatography. II. Response models for reversed-phase non-ionic systems

Summary Uncharged solutes devoid of inherent detectable properties can be monitored and quantified in reversed-phase chromatographic systems by including a non-ionic detectable component (probe) in the eluent. Response models have been developed, which are based on the fact that the distribution of the solute and the probe are connected to each other by common interaction effects, such as competition for the binding surface. The direction and magnitude of the indirect response can be predicted quantitatively by means of expressions which clearly show the influence of the retention of the solute relative to the probe as well as the effect of the solid phase coverage by the probe. The relatively low response obtained in non-ionic systems may be due to a low loading of the probe on the solid phase and to a simultaneous distribution of the solute in complexed and uncomplexed forms, the two distribution modes giving opposite response patterns.     

Indirect fluorometric detection in micro high-performance liquid chromatography

Summary Indirect fluorometric detection of non-electrolytes and inorganic anions were examined by using a commercially available fluorometer. The dynamic reserve achieved by the present system was (1.0–1.4)×10³, and it was about one-tenth of the value achieved by UV detection. The mass detection limit of 0.2 pmol was achieved for nitrate by using 3×10^–5 M sodium salicylate as the mobile phase. In the case of ion chromatography the mass detection limit was improved by using a low concentration mobile phase.     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for cations on polystyrene polymers

Summary The distribution equilibria of cationic compounds in reversed-phase chromatographic systems (ion-pair chromatography) have been studied on the basis of their effect on a detectable mobile phase component. The solid phase was a polystyrene-divinylbenzene copolymer and the detectable component, a quaternary ammonium ion, 1-methylpyridine. The solutes were mono- and divalent amines and quaternary ammonium ions. The cations can be retained by ion-pair adsorption and ion exchange. Expressions for the ion-pair retention of the solutes and the mobile phase cation (system peak) have been developed assuming Langmuir distribution of ion pairs to a solid phase with one kind of binding site. The validity of the expressions has been tested by evaluation of ion-pair distribution constants using non-linear curve fitting techniques. Good agreement for the constants of common ion pairs was obtained from different kinds of capacity ratio expressions. Ion exchange retention can appear beside ion-pair retention, and it has been observed in the pH range 1.6–6.1. The effect depends not only on cations in the mobile phase, but also on the nature of the buffering systems.     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for ionic compounds on μ Bondapak phenyl

Summary Retention models for ionic compounds in reversed-phase ion-pair chromatographic systems have been developed on the basis of the capacity ratios of solutes and ionic mobile phase components using indirect detection. The binding to the adsorbent, μBondapak Phenyl, has been expressed by ion-pair distribution constants and tested in the retention models by non-linear regression. All the ionic compounds followed adsorption models of the Langmuir type comprising two kinds of binding sites with widely different adsorption capacities and binding constants. The results indicate certain differences between the two sites regarding the binding of cationic and anionic components, respectively.     

Indirect conductimetric detection of cyclodextrins in liquid chromatography

Indirect conductimetric detection of cyclodextrins (CDs) was investigated in liquid chromatography (LC). The mobile phase contained an electrolytic substance which maintained high background, and CDs were indirectly detected owing to a depression of the background signal due to dilution of the mobile phase by the analyte molecules. The dynamic reserve, defined as the ratio of the background to its noise level, was (1–2) × 10⁵, and the detection limits at S/N = 3 were 23–40 ng for CDs when a microcolumn LC system was employed.     

Indirect conductimetric detection of nonelectrolytes in microcolumn liquid chromatography

Summary Indirect conductimetric detection of nonelectrolytes was investigated in microcolumn liquid chromatography. The mobile phase contained an electrolytic substance which maintained a high background and analytes were indirectly detected owing to a depression of the background signal. The dynamic reserve, defined as the ratio of the background to its noise level, was 9.3×10⁴, and the detection limits at S/N=3 were 10–32 ng for alcohols examined.     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for anionic analytes and mobile phase components on polystyrene polymers using an alkaline mobile phase

Summary Retention models for anionic analytes and mobile phase compounds in reversed-phase ion-pair chromatographic systems have been studied. The solid phase was a polystyrene-divinylbenzene copolymer. The analytes were mono- and divalent anions monitored by indirect detection technique. The mobile phase was highly alkaline and contained a detectable anion, sulfanilic acid.Expressions for the ion-pair retention of solutes and mobile phase components have been developed assuming Langmuir distribution of ion-pairs to a solid phase with one kind of binding site. The validity of the expressions and the origin of system peaks have been confirmed by determination of distribution constants using linear regression.     

Comparative study of some commercial chemically bonded phases in classical reversed-phase chromatography and in ion-pair reversed-phase liquid chromatography

Summary The behaviour of various octadecyl commercial bonded phases are compared in classical reversed-phase chromatography and in ion-pair reversed-phase chromatography. Great differences are exhibited by the packings studied according to the polarity of the solutes. Whereas hydrocarbonaceous bonded phases show very similar selectivity versus apolar or weakly polar solutes, great differences are observed when analyzing more polar solutes even when ion-pair reversed-phase chromatography is performed.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Optimization of reversed-phase ion-pair chromatography by over-pressurized thin-layer chromatography III. Over-pressurized thin-layer chromatography using 10-camphor sulfonic acid as ion-pair reagent

Summary The use of over-pressurized thin-layer chromatography in ion pair system using acidic type pairing reagent has been studied. The most important aspect when reversed phase ion-pair TLC system is used to apply a suitable procedure for pre-treatment of the sorbents. Because of the acidic type of ion pair reagents cannot be bounded to the surface of the layer by immersion or pre-development with the reagent solution, double coating technique was used for the pre-treatment, the plate was firstly immersed in an ethanolic solution of cetrimide, then the immersion has been repeated by an ethanolic solution of acidic ion pair reagent. The necessary coating of the sorbents can be achieved by this technique. To find the optimal conditions for reversed phase ion pair TLC separation of organic amines, 10-camphor sulfonic acid as reagent, different aliphatic, aromatic amines and diamines and heterocyclic nitrogen compounds, respectively as model compounds were selected. The dependence of the selectivity and efficiency of the separation on the sorbents, on the concentrations of reagents (cetrimide and camphor sulfonic acid) applied for both immersion and in the eluent were investigated in detail. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Detection by ion-pairing probes in reversed-phase liquid chromatography

Summary Compounds without own detector response can be detected and quantified by a UV-absorbing or fluorescent, ion-pairing probe in reversed-phase liquid-solid systems. Charged as well as uncharged samples give response by affecting the distribution of the probe between the phases. The probe is usually a hydrophobic organic ion. It is included in the mobile phase, which also contains a counter ion and an ion of the same charge as the probe, both fairly hydrophilic. The choice of properties and concentrations of the hydrophilic ions is essential for the response since too low and too high distribution of the mobile phase ions to the adsorbent will make the system insensitive. It is with UV-absorbing probes possible to get a response corresponding to a molar absorptivity of more than 3000 for ionic samples. Fluorescent probes have given 3–5 times higher sensitivity. Uncharged compounds give as a rule lower response. A combination of two UV-absorbing probes with different charge gives a considerable improvement of the response for samples with low retention. A simplified theoretical model for the detector response is suggested on the basis of detailed studies of the concentration changes in the eluted mobile phase.     

Column selectivity in reversed-phase liquid chromatography. VII. Cyanopropyl columns

Eleven cyanopropyl ("cyano") columns were characterized by means of a relationship developed originally for alkyl-silica columns. Compared to type-B alkyl-silica columns (i.e., made from pure silica), cyano columns are much less hydrophobic (smaller H), less sterically restricted (smaller S*), and have lower hydrogen-bond acidity (smaller A). Because sample retention is generally much weaker on cyano versus other columns (e.g., C8, C18), a change to a cyano column usually requires a significantly weaker mobile phase in order to maintain comparable values of k for both columns. For this reason, practical comparisons of selectivity between cyano and other columns (i.e., involving different mobile phases for each column) must take into account possible changes in separation due to the change in mobile phase, as well as change in the column.     

Retention models for isocratic and gradient elution in reversed-phase liquid chromatography

One- and multi-variable retention models proposed for isocratic and/or gradient elution in reversed-phase liquid chromatography are critically reviewed. The thermodynamic, exo-thermodynamic or empirical arguments adopted for their derivation are presented and discussed. Their connection to the retention mechanism is also indicated and the assumptions and approximations involved in their derivation are stressed. Special attention is devoted to the fitting performance of the various models and its impact on the final predicted error between experimental and calculated retention times. The possibility of using exo-thermodynamic retention models for prediction under gradient elution is considered from a practical point of view. Finally, the use of statistical weights in the fitting procedure of a retention model and its effect on the calculated elution times as well as the transferability of retention data among isocratic and gradient elution modes are also examined and discussed.     

Detection of albiflorin and paeoniflorin in Paeoniae Radix by reversed-phase high-performance liquid chromatography with pulsed amperometric detection

We have developed a reversed-phase high-performance liquid chromatography-pulsed amperometric detection (RP-HPLC-PAD) method for the detection of albiflorin and paeoniflorin in Paeoniae Radix and Wu-ji-san. Albiflorin and paeoniflorin were completely separated using 10% acetonitrile in 5 mM sodium phosphate buffer (pH 3.0) as an eluent and detected by PAD under alkaline conditions after using a post-column delivery system. The limit of detection (S/N = 3) and the limit of quantification (S/N = 10) were 0.10 and 0.35 ng for albiflorin, and 0.20 and 0.50 ng for paeoniflorin, respectively. The coefficients of linear regression were 0.9995 and 0.9999 for concentrations between 0.035 and 100 μg/mL. The intra- and inter-day precision (RSDs) was less than 3.56% in Paeoniae Radix and Wu-ji-san. The average recoveries from Paeoniae Radix and Wu-ji-san were 99.01–100.94% and 99.46–100.64%. This method shows higher selectivity than HPLC–UV method for analyzing albiflorin and paeoniflorin in Chinese medicinal preparation.     

Separation of inorganic anions by reversed-phase ion-pair chromatography with direct UV detection

In this paper, a separation method of the inorganic anions including I^–, NO ₂ ^– , NO ₃ ^– , IO ₃ ^– and SCN^– on the reversed-phase ion-pair chromatography with direct UV detection has been developed, and the limits of detection of these inorganic anions were determined. The effects of the organic modifier volume fraction and concentration of the ion-pair reagent on the retention of inorganic anions were discussed.     

Evaluation of the signal intensity of induced peaks in indirect photometric detection of nonelectrolytes in reversed-phase high-performance liquid chromatography

Summary We have experimentally evaluated the equation representing the peak area of induced peaks in indirect photometric detection of nonelectrolytes as a function of the amount and concentration of the analyte and the visualizing agent and their capacity factor, the phase ratio of the column and the absorptivity and the pathlength of detection. Aliphatic alcohols and hydrocarbons were used as the test analytes. Most of the analytes behaved according to the relationship expressed by the equation.     

Convergence of retention for small solutes in reversed-phase liquid chromatography

Summary It is shown theoretically that when the concentration of organic solvent in the mobile phase increases, or solute size decreases, log k values of small solutes in reversed-phase liquid chromatography (RPLC) will tend to have a minimum value called the convergence point. A theoretical model for evaluating the convergent coordinates of small solutes is presented by using a stoichiometric displacement model for retention (SMDR). The physical meaning of the coordinates of each kind of convergence are also elucidated. The convergence points have either two-dimensional coordinates with a common ordinate (the logarithm of the phase ratio of the column, log ) or threedimensional corrdinates with two common axes: — log and the logarithm of the molar concentration of the pure displacing agent in mobile phase, log a_D. The other axis relates to the nature of the solutes, such as carbon number of a homolog, van der Waal's surface area, hydrophobic fragment constant etc. for the latter and those and/or concentration axis for the former. The model was tested with published data and found to give a good fit.     

Statistical tests for the selection of the optimum parameters set in models describing response surfaces in reversed-phase liquid chromatography

Summary An appropriate procedure based on statistical criteria is suggested for the determination of the optimum set of model parameters for a given chromatographic system. The criteria employed are the t-ratio test, the rate of change in the sum of squares of residuals, the standard error of the fit, the F-test, and the C_P-test. The suggested procedure has been evaluated using two different models, one based on partition and the other on adsorption mechanisms, which describe the combined effect of pH and organic modifier content on the retention of ionogenic solutes in reversed-phase liquid chromatography. It is shown that all the criteria give almost converged results and therefore we may simply use the F-test, which seems to be the most sensitive and reliable criterion excluding any personal judgement. It is also found that the retention models tested show a different behavior towards their simplification. In particular, the use of a reduced equation of the partition model, selected on the basis of the suggested procedure, is necessary for the prediction of meaningful retention surfaces, whereas the decrease in the number of the adjustable parameters in the adsorption model offers only noise reduction and fitting simplicity, because no version of this model predicts abnormal retention surfaces.     

Refractive index detection in microbore column liquid chromatography

Summary An existing commercial refractive index detector was modified for use with microbore column LC systems. The detector utilizes the Fresnel method. The effect of band dispersion and dilution at the detector side is of extreme importance in connection with the miniaturized LC system. In the modified model the original heat-exchanger tube was removed and a stainless steel capillary was used for heat-exchanging. Gaskets having different cell volumes were also examined with respect to band broadening and sensitivity. The detection limit was 10ng for di-n-pentyl phthalate. The examples include the detection of phthalates, alcohols, n-paraffins, and kerosine.     

Indirect photometric detection of inorganic anions by microcolumn liquid chromatography using anthraquinone-disulfonate as visualization agent

Summary Disodium salts of several anthraquinone-disulfonic acids, which possess large molar absorptivity, were employed as the visualization agent for microcolumn ion chromatography with indirect photometric detection. Detection limits of inorganic anions were improved, e.g., concentration detection limits of chloride, nitrate and sulfate were 0.91–1.6 mol L^–1 at S/N=3, corresponding to mass detection limits of 18–32 fmol. The present system was applied to the determination of inorganic anions in tap water.     

Analysis of propantheline bromide and related quaternary ammonium drugs by reversed-phase,ion-pair liquid chromatography with two counterions in the eluent

Summary The addition of appropriate concentrations of an organic amine and an alkylsulphonate to the mobile phase in reversed-phase, ion-pair liquid chromatography can introduce unique selectivity in to the chromatographic system allowing separation of complex mixtures of basic, acidic and neutral compounds. As an example, the methodology for a specific stability-indicating determination of propantheline bromide, a quaternary ammonium anticholinergic agent, on several reversedphase stationary phases, was developed. The retention mechanism was studied and it was concluded that both ion-interaction processes and ion-exchange processes were involved in the separation technique developed with two counterions of opposite charge in the eluent.