Peichang Lu

Effect of organic modifier concentration on retention in reversed-phase ion-pair liquid chromatography

Abstract The concentration of organic modifier in the eluent is one of the most important factors that affect the retention of ionized solutes in reversed-phase ion-pair liquid chromatography (RP-IPC). Linear regression analysis of ln vs. methanol concentration ( C b ) according to the equation ln was carried out; ln and c ip are constants for a given solute with a given column system, where ln is determined mainly by the electrostatic and non-electrostatic free-energy change of retention at C b  0, and c ip is mainly determined by the interaction behaviour between the ion-pair reagent, the ionized solute and the mobile phase. This equation has been confirmed experimentally. The absolute values of ln and c ip in RP-IPC are much larger than those in RP high-performance liquid chromatography (HPLC), which means that there is a much stronger effect of methanol concentration on retention in RP-IPC than in RP-HPLC. On the other hand, ionized compounds with the same kind and number of charges show almost the same value of electrostatic free-energy change, and ln and ln in RP-IPC can be well correlated with ln and ln in RP-HPLC.

The S index in the retention equation in reversed-phase high-performance liquid chromatography

The empirical retention equation log k′ = log k′w − Sϕ in reversed-phase high-performance liquid chromatography (RP-HPLC) was investigated to evaluate the properties of the parameter S. The S index, which is defined as the slope of log k′ versus volume fraction of the organic modifier (ϕ) was systematically examined as a function of bonded phase density, column type and temperature in RP-HPLC. The S index for a particular solute was observed to be nearly constant even when column systems with different C18 packing materials are used. The dependence of log k′ on eluent composition was found to be represented by parallel lines for a given solute for a variety of different stationary phases. The S index remains constant for a given solute despite the prolonged use of C18 column. The results showed that the S index is determined mainly by the interaction between the solute and the mobile phase. It was observed to decrease with the increasing column temperature for non-ionic solutes. Other factors influencing the measurement of the S index are discussed.

Effects of molecular structure on the S index in the retention equation in reversed-phase high-performance liquid chromatography

Abstract The S index in the retention equation log k′ = log k′w—Sϕ in reversed-phase high-performance liquid chromatography was systematically investigated as the function of molecular structure parameters. The S index, which has been observed to be nearly constant for a specific solute even when column systems with different C18 packing materials are used, was quantitatively correlated with the solvatochromic parameters of the solutes. The coefficients in the correlation of the S index with the solvatochromic parameters of the solutes were investigated and were found to be consistent with the results of using a solvatochromic comparison method. For non-polar compounds, a simplified linear relationship between S and the Van der Waals volume of the solute was observed. For homologues, a linear relationship between S and carbon number was found. Therefore, when other factors remain the same, increasing the size of the solute results in an increase in S whereas increasing the dipolarity or hydrogen bonding ability of the solute will result in a decrease in S.

Effects of molecular structure on parameters a, b and c in the fundamental retention equation

An important aspect of chromatography is the prediction of peak positions. For high-performance liquid chromatography (HPLC), a fundamental retention equation ln k′  a + b ln Cb + cCb has been derived by statistical thermodynamics. The effects of molecular structure on the parameters a, b and c in this equation were investigated theoretically and proved experimentally. The parameter b in reversed-phase (RP) HPLC approaches a low constant value, because there is very weak displacement caused by adsorption. However, in normal-phase (NP) HPLC, the displacement by adsorption is strong, and plays an important role in retention. The parameter b in NP-HPLC changes slightly when different batches of packing of the same size and with the same mobile phase are used, and the absolute value of this parameter increases slightly and approaches a constant value with increase in carbon number for homologous compounds. The parameter c in RP-HPLC is mainly determined by the difference between interactions in solute—strong solvent and solute—weak solvent systems and approaches a constant value even when column systems with the different packings and the same mobile phase are used. The parameter c in RP-HPLC can also be quantitatively correlated with structural parameters of the solute such as Van der Waals volume (VW), dipole moment (μA) and hydrogen bond energy (XAH). For non-polar or homologous compounds, a simplified linear relationship between the parameter c and VW can be established. The parameter c in NP-HPLC is affected not only by factors valid in RP-HPLC, but also by the displacement caused by adsorption of the solute on the surface. The parameter a in both RP- and NP-HPLC follows similar rules to parameter c. A linear relationship between k′ and amount of C18 in RP-HPLC was also found. A good linear relationship between the parameters a and c occurs only when two of the three structural parameters VW, μA and XAH for solutes are equal or close.

Separation of aqueous polythionates by reversed-phase ion-pair liquid chromatography with suppressor-conductivity detection

Abstract A method for the separation of sulphate, thiosulphate and four polythionates by reversed-phase ion-pair liquid chromatography with suppressor—conductivity detection is described. The limits of detection for these inorganic anions varied between 0.01 and 0.30 μg ml−1. The effects of the number of sulphur atoms in polythionates, the ion-pair reagent and the organic modifier concentration on retention were studied. It was observed that the logarithim of the capacity factors decreases linearly with the organic modifier concentration (Cb) and increases with the logarithm of the ion-pair reagent concentration (Cp) and the number of sulphur atoms in the polythionates. The absolute values of intercepts and slopes of the linear relationships of ln k′ vs. ln Cp and ln k′ vs. Cb increase linearly with increasing number of sulphur atoms in the polythionates. The method developed was applied to the determination of sulphate and polythionates in different gold extract solutions.

Measurement of partition coefficients by reversed-phase ion-pair liquid chromatography

Abstract Sixteen phenylamine- and naphthylaminesulphonic acids which are negatively charged and weakly retained or non-retained in reversed-phase high-performance liquid chromatography were used as model compounds to examine the quantitatively relationship between the calculated n -octanol-water partition coefficient (log P ) and the retention value (log k′ ) (or log k w ) and solute charges in reversed-phase ion-pair liquid chromatography (RP-IPC). It was observed that the correlation of log P vs . log k′ (or log k w ) for solutes with one negative charge has moderate regression coefficients of ca . 0.80, but the correlation of log P vs . log k′ (log k w ) and solute charges ( n e ) for solutes with different charges has a regression coefficient higher than 0.98. The log k′ (or log k w ) value in RP-IPC always make a positive contribution and the solute charges always makes a negative contribution to log P values. The proposed relationship between the log P value and tje log k′ value and solute charges makes it possible to predict the n -octanol-water log P values of ionic compounds from the retention values in RP-IPC, and is also useful in elucidating the retention mechanism in RP-IPC.

Advances in expert systems for high-performance liquid chromatography

Abstract The development of modules for the selection of the separation mode, stationary phase and mobile phase and for peak identification in expert systems for high-performance liquid chromatography is discussed. Both the rules and methods used in these modules and their theoretical basis are included. A program to select the separation mode and the stationary and mobile phases has been developed in which there are two modes of entry, the molecular structure of the sample and provision of the sample name. In the peak identification module, three methods for off-line peak identification, by transfer of retention values from one column to another column, by the relationship between retention values and molecular structure parameters and by the interaction index, and also a method for on-line peak identification by combination of the UV spectral parameters with the retention values, have been developed.

The retention equation in reversed-phase ion-pair chromatography

In this paper, by combination of the statistical thermodynamic method with the Freundlich isotherm, the retention equations to describe the effects of the mobile phase composition, concentration of the ion-pair reagent and the ionic strength on the amount of adsorbed ion-pair reagent and the retention value of the ionic solute have been reported by simultaneously considering the electrostatic and molecular interaction between solutes, ion-pair reagent and molecule or ion in each phase in the reversed-phase ion-pair chromatography. The validity of these retention equations has been confirmed by calculation of capacity factor of the different phenylamine and naphthylamine sulphonic acids during systematic change of concentration of the strong solvent, the ion-pair reagent and the ionic strength in the mobile phase.

Separation of phenylamine- and naphthylaminesulphonic acids by reversed-phase high-performance liquid chromatography

Abstract Mixtures of singly and multiply substituted phenylamine- and naphthylaminesulphonic acids, important intermediates in the synthesis of dyestuffs, were separated by reversed-phase liquid chromatography with UV detection. Linear regression analysis according to the retention equation ln k′ = ln k′w + cCb [where k′w is the extrapolated capacity factor at Cb = 0 and c is mainly determined by molecular interaction between the solute and the eluent; they are constants for a given solute at a given column system; Cb is the concentration of organic modifier (v/v)] with and without experimental data at Cb = 0 being taken into account was carried out. The regression coefficient in the former situation is much lower than that in the latter. A linear relationship between ln k′w (without taking the data at Cb = 0 into account) and the ln k′w measured at Cb = 0 was obtained. The effect of the eluent acidity on k′ is complex, indicating that the negatively charged, neutral and positively charged solutes simultaneously contribute to the retention. The retention value increases with increasing concentration of sodium chloride, but the linear regression analysis according to Horvaths equation ln k′ = A + Bm (where A and B are constants related to the physico-chemical behaviour of a given column system and m is the concentration of the inorganic salt) is not good, which may be caused by masking of the ionized silanol group and changing the eluent surface tension simultaneously.

Characterization of Immunochemical Reaction for Human Growth Hormone with its Monoclonal Antibody by Perfusion Protein G Affinity Chromatography and Capillary

An extremely rapid assay technique for antibody-antigen interaction using human growth hormone and its monoclonal antibody as an example has been developed by utilizing Protein G bound to perfusion chromatography matrices. The antibody and antigen were mixed and incubated at different molar ratios by keeping the concentration of antibody constant. The mixture of antibody and antigen solution was then injected onto the Protein G column. The complex of antibody and antigen formed in the sample solution was retained on the Protein G column as was the free antibody. The peak-area and height of the retained complex and antibody linearly increased with the molar concentration of the antigen when it was not in excess of the corresponding stoichiometric amount of antibody, however, those of the retained peak were constant when the concentration of antigen was in excess of that of the antibody. The validity of this method was confirmed by the results of capillary zone electrophoresis. The method developed cannot only be used to determine the biological activity between antibody and its antigen quickly, but also to determine the stoichiometry of immunological reactions between the antibody and antigen when a stable complex of them can be formed.