K.-G. Wahlund

Peak tailing and retention behaviour of tricyclic antidepressant amines and related hydrophobic ammonium compounds in reversed-phase ion-pair liquid chromatography on alkyl-bonded phases

Abstract Peak tailing of tricyclic antidepressant amines was studied on a range of alkyl-bonded silicas in reversed-phase ion-pair chromatographic systems. Mobile phases were mixtures of methanol and buffers containing phosphate or bromide as counter-ions. Alkylammonium ions added to the eluent eliminated peak tailing and decreased the retention of the amines. A retention model for ion-pair adsorption is used for evaluation of the results. It is found that the stationary phase contains adsorption sites with different ability to retain ammonium compounds and uncharged compounds.

Application of an asymmetrical flow field-flow fractionation channel to the separation and characterization of proteins, plasmids, plasmid fragments, polysaccharides and unicellular algae

The asymmetrical flow field-flow fractionation channel has been improved by introduction of a different sample loading method, downstream central injection, which reduces sample relaxation and focusing time considerably and allows the concentrations of material in the channel, thereby enabling the loading of very large sample volumes. The performance of the channel was demonstrated by the separation of a protein from its dimer, retention of nucleic acids, i.e., plasmids and enzymatically cleaved plasmids, hyaluronate and unicellular algae.

Ion-pair chromatography of acidic drug metabolites and endogenic compounds.

Liquid-liquid chromatographic systems based on ion-pair partition with silica microparticles as the support for the stationary phase have been used for the separation of anionic compounds of biochemical and pharmacological interest. A high separating efficiency can be obtained with both aqueous and organic mobile phases and the retention is easily regulated by the nature and the concentration of the quaternary ammonium counter ion, present in the aqueous phase. The influence of the composition of the liquid phases on the selectivity and separating efficiency has been studied, as well as equilibration methods and the stability of the systems. Examples are given of separations of sulphonamides, barbiturates, glucuronic and sulphuric acid conjugates of steroidal compounds and phenols glycine conjugates of carboxylic acids (hippuric, nicotinuric and salicyluric acid) and anionic metabolites of biogenic amines (indoleacetic, benzoic, mandelic and phenylacetic acid derivatives).

Reversed-phase ion-pair partition chromatography of carboxylates and sulphonates

Abstract A reversed-phase partition chromatographic system for separation of organic anions as ion pairs with quaternary ammonium ions has been developed. Commercial, hydrophobized silica supports are used with 1-pentanol as stationary phase and aqueous solutions of tetrabutylammonium (TBA) as mobile phase. The separation of aromatic sulphonates and benzoic acid derivatives is demonstrated. The use of the TBA concentration of the mobile phase to regulate the capacity factor of the anions, as a means of improving separation by gradient elution and direct injection of large sample volumes, is demonstrated. The isolation of nicotinic acid from human serum samples is shown.

Improved separation speed and efficiency for proteins, nucleic acids and viruses in asymmetrical flow field flow fractionation

The performance of the asymmetrical flow field-flow fractionation channel has been improved by the use of a much thinner (0.12-mm) channel than before and by flow programming (stepwise gradient elution). The thinner channel contributes to a decreased zone broadening which enables complete resolution in a shorter time. Three protein peaks, representing molecular weights from 12,000 to 136,000, wer completely resolved within 3 min. Flow programming speeds up the elution of the high-molecular-weight materials which occur late in a fractogram. This enabled separation of a small plasmid fragment (700 base pairs) from the large amounts of a big fragment (4600 base pairs) in 30 min and improved detection of a presumed trimer of albumin. Two viruses (1.8 10(6) and 50 10(6) daltons) were eluted as narrow peaks within 5 min.

Reversed-phase ion-pair chromatography of drugs and related organic compounds

Summary Procedures are given for high-performance ion-pair chromatography of organi ammonium compounds (e.g., noradrenaline, dopamine, synephrine, ephedrine, zimelidine, imipramine, desipramine, amitriptyline and nortriptyline) with dihydrogen phosphate, bromide, cyclohexylsulphamate, dicyclohexylsulphamate or octylsulphate as counter ions and 1-pentanol as the liquid stationary phase on LiChrosorb-RP. With methylene chloride as the stationary phase, the highly hydrophilic noradrenaline and adrenaline can also be separated. A long-chain alkylammonium compound must be added to the system in the chromatography of hydrophobic amines and all types of quaternary ammonium compounds to prevent peak deformation. Both the liquid stationary phase and the support affect the selectivity.

Reversed-phase ion-pair partition chromatography of phenylacetic, mandelic and pyridinecarboxylic acid derivatives

Phenylacetic, mandelic and pyridinecarboxylic acids of physiological interest have been separated by ion-pair partition chromatography with 1-pentanol as stationary phase and tetrabutylammonium as counter ion in the aqueous mobile phase. The stability of the chromatographic system is very high and the support is spontaneously re-coated with stationary phase. Untreated plasma samples can be injected in relatively large volumes without serious loss of separating efficiency. A venting system was found efficiently to reduce disturbing early peaks on injection of biological samples.

Procedures for direct injections of untreated blood plasma into liquid chromatographic columns with emphasis on a pre-column venting technique.

The stability of reversed-phase liquid chromatographic systems when untreated blood plasma samples are applied was studied with respect to retention, peak efficiency and column back-pressure. The influence of the content of organic solvents in the eluent, the flow-rate and the particle diameter of the support was investigated. Many of the column-deteriorating effects seem to be due to denaturation of the plasma proteins and the kinetics of this process. The stability is increased by decreasing the content of organic modifier in the eluent. The flow-rate should be moderate, as both high and low flow-rates give decreased stability. The stability increases with increasing particle size of the solid phase. A small pre-column is used (1) as a guard column and (2) to effect a pre-separation of the solute from plasma matrix components by a pre-column venting technique. The pre-column venting technique considerably increases the lifetime of the system and especially the separation column. Under optimal conditions hundreds of injections (10 microliters of plasma) can be performed without loss of stability.

Stationary phase effects in reversed-phase liquid chromatography of acids and ion pairs

Summary Reversed-phase partition chromatography has been performed with 1-pentanol as the stationary phase, applied on to the hydrophobic support by adsorption from an aqueous mobile phase containing a low concentration of 1-pentanol (1.9–2.5%). By ion-pair chromatography of car☐ylates with tetrapropylammonium and tetrabutylammonium as counter ions, ion pairs are retained by partition to the adsorbed layer of liquid stationary phase. Also for hydrophilic car☐ylic acids, which are retained in uncharged form as the acids, the dominant retention mechanism is partition to the liquid stationary phase, while hydrophobic car☐ylic acids are retained by an interaction with the hydrophobic support. The interaction of the hydrophobic acids with the support can be regulated by varying the amount of 1-pentanol stationary phase, which is effected by changing the concentration of 1-pentanol in the mobile aqueous phase.

Elimination of peak deformation in the liquid chromatographic separation of a strongly protein-bound drug from directly injected blood plasma samples.

Direct injection of blood plasma samples into reversed-phase columns resulted in skewed chromatographic peaks for the drug naproxen. The skew is shown to be due to strong binding of naproxen to albumin present in the blood plasma. Methods to eliminate the peak skew have been investigated. They include changes of the composition of the eluent and of the sample solution in order to decrease the degree of binding of the drug to albumin. The methods studied were dilution, addition of displacers, change of pH and change of methanol concentration. Calculations based on known binding constants indicate that the degree of peak skew was directly influenced by the degree of protein binding of the drug in the sample solution.