Effect of the organic modifier concentration on the retention in reversed-phase liquid chromatography I. General semi-thermodynamic treatment for adsorption and partition mechanisms

A semi-thermodynamic treatment is adopted to account for adsorption or partition of solute molecules from aqueous mobile phases on/in reversed-phase liquid chromatography stationary phases. The theoretical expressions of ln k' versus organic modifier content are tested against 10 data sets covering a variety of solute molecules. It is shown that the mean field approximation, adopted widely in ptevious studies, is marginally valid in aqueous mobile phases, especially in the presence of solute molecules, and the lattice model approximation, which is also used in relevant studies, is a poor approximation. Clear conclusions about the validity of either the adsorption or the partition model for the retention mechanism could not be drawn. The equations of the adsorption model describe all data sets absolutely satisfactorily and yield a physically reasonable picture about the behavior of modifier and solvent at the adsorbed layer. However, the high applicability of the adsorption model may not safely entail the validity of the adsorption mechanism at a molecular level, especially in the case of solutes with small and non-polar molecules, where our analysis gives strong indications about the validity of the partition mechanism. The next steps needed for the final elucidation of the retention mechanism in reversed-phase chromatographic columns are indicated.     

反相液相色谱中溶剂强度规律的研究

 以溶质计量置换保留模型 (SDM R)为依据 ,通过研究 3种正链醇同系物溶剂置换剂对 14种正链醇同系物溶质色谱保留行为的影响 ,发现计量置换参数Z(对应 1mol溶质被吸附时从溶质与固定相接触处释放出的溶剂的总摩尔数 )、lgI(与 1mol溶质对固定相亲和势有关的常数 )和 j(与 1mol溶剂对固定相亲和势有关的常数 )均随着同系物置换剂相对分子质量的增大而减小 ,并呈现出线性变化 ,表明溶剂强度与溶剂分子的大小有关 ,分子愈大 ,溶剂洗脱能力愈强 ,并遵循同系物规律。     

Comparison of capillary electrochromatography with high-performance liquid chromatography for the analysis of pirimicarb and related compounds.

The applicability of capillary electrochromatography (CEC) to the analysis of pirimicarb and structurally related pyrimidines has been investigated. Methods were developed to improve the separation of closely related compounds. Resolution was achieved both by the use of running buffers containing a mixture of two organic modifiers to increase selectivity and reduce retention times. Solvent composition step gradients were used to separate compounds of widely differing retention factors. A comparison has been made between HPLC and CEC using identical separation parameters and the same stationary phase, from which two important conclusions are drawn. First, it has been shown that values of k' for the compounds analyzed were the same in both techniques. Secondly, although it is evident that CEC produces higher efficiencies than HPLC when running buffers with high organic solvent content are used, as the aqueous content of the running buffer is increased the efficiencies achieved in CEC and HPLC converge until they become equivalent. This is contrary to the theoretical model which predicts efficiencies are inherently higher using electrically rather than pressure driven flow. Disadvantages of the limited control of flow-rate in CEC in comparison with HPLC, are shown.     

Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography

The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.     

Errors involved in the existing B-term expressions for the longitudinal diffusion in fully porous chromatographic media Part I: computational data in ordered pillar arrays and effective medium theory

Numerically solving the effective diffusion in a simplified representation of a chromatographic bed, it was found that the B-term expressions that have up to now been used in the literature, and which can all be reduced to either Deff=(gamma mDm+k'gamma sDs)/(1+k') or Deff=(gamma meDm+k'Dpart)/(1+k'), can no longer be considered to be unconditionally valid. This could be demonstrated by showing that the simulated diffusion data are in agreement with the mathematically sound effective medium theory (EMT), whereas the B-term expressions used up to now in literature are in conflict with the EMT, a theory that is widely accepted in all other fields of science. It is also shown that the use of the existing B-term expressions can lead to very large measurement errors (up to a 100% and more) for the determination of the stationary phase diffusion coefficient gamma sDs from peak parking experiments. The representation of the B-term diffusion should in the future hence be based on the Deff expressions that can be derived from the EMT. These are physically sound and are also more accurate than the classical B-term expressions used up to now.     

毛细管电色谱中组分保留因子表达式的讨论

在组分的电泳因素基本不影响其色谱行为的前提下,推导出组分保留因子表达式k*CEC=k′-μepμeo+μep(Ⅰ)及k**CEC=k′-μep-μ0epμeo+μep(Ⅱ),两者可以互相补充。同时对毛细管电色谱的相关文献中组分保留因子的两种表达式kCEC=k′+k′ μepμeo+μepμeo(Ⅲ)及kCEC=k′-μep/μeo1+μep/μeo(Ⅳ)进行了讨论,指出了表达式(Ⅲ)推导中引用组分电泳迁移距离的错误。但表达式(Ⅰ)和(Ⅳ)在某些条件下也不能有效反映组分的电泳淌度μep及k′对kCEC的影响     

Use of the surfactant 3-(3-cholamidopropyl)-dimethyl-ammoniopropane sulfonate in hydrophobic interaction chromatography of proteins

Isocratic hydrophobic interaction chromatography of five proteins has been carried out using mobile phases containing the surfactant 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS). Linear relationships were found between log k' and ammonium sulfate concentrations for all the proteins with CHAPS in the submicellar concentration range. The slope of such a plot decreases monotonically as CHAPS concentration is increased. To a first approximation, the effect of CHAPS on protein retention can be explained in terms of a competitive binding model. However, CHAPS does show differential effects on the elution of proteins, substantially altering selectivity. The use of a normalized capacity factor, k'/k'o, proves useful for comparing retention times of different proteins as a function of CHAPS concentration. The magnitudes of k'/k'o were found to be inversely correlated with the slopes of plots of log k' vs. ammonium sulfate concentration in the absence of CHAPS. Adsorption isotherms for CHAPS were determined over the working range of ammonium sulfate. The binding of CHAPS to the SynChropak Propyl stationary phase and its effects on retention were found to be readily reversible. For each protein, plots of k'/k'o vs. surface concentration of CHAPS were superposable for data obtained at different salt concentrations. These findings support a competitive binding model. A simple geometric argument for stationary phase occupancy provides a qualitative explanation for the observed surfactant selectivity.     

非等时空距灰色模型在液相色谱保留值研究中的应用

本文运用灰色理论中的非等时空距ＧＭ（１，１）模型，对液相色谱中的流动相组成及容量因子的关系进行了研究。所建灰色模型经过检验其精度为Ｉ级。实验结果表明，不仅等时空距灰色模型，而且非等时空距灰色模型，同样适用于色谱保留值的研究。     

Retention prediction in ternary solvent reversed-phase liquid chromatography systems based on the variation of retention with binary mobile phase composition

An extension of the treatment adopted in a recent paper [P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 946 (2002) 33] was used to derive expressions describing the variation of solute retention k with composition in ternary reversed phase liquid chromatography, RP-LC, solvent systems. The equation of the partition model obtained in this way for a ternary mobile phase was identical to that previously derived using the solubility parameter concept. This equation as well as two new expressions of In k versus organic modifiers content were tested in a variety of ternary solvent systems in order to examine the possibility of predicting retention behavior of solutes under ternary solvent mixture elution conditions from known retention characteristics in binary mobile phases. It was demonstrated the superiority of both new equations derived in this paper to that previously proposed and applied to date in ternary solvent mixtures.     

Retention time and peak width in the combined pH/organic modifier gradient high performance liquid chromatography

The purpose of this work was to test the applicability of the current theory to predict the peak retention time and the peak width in the combined pH/organic modifier gradient reversed phase high performance liquid chromatography (RP HPLC). A series of 38 isocratic measurements have been conducted for a wide range of pH and methanol contents for ketoprofen (weak acid) and papaverine (weak base). It served to find the model describing dependence of retention factor and the height equivalent of a theoretical plate (HETP) on pH and organic modifier content. The information gathered in the isocratic mode was used to simulate retention times and peak widths for 30 various methanol gradients, 25 pH gradients, and 3 combined pH/methanol gradients. The simulations were compared with the experimental data. We also proposed a simplified version of this model that was parameterized based on 12 initial organic modifier gradients carried out for different pHs and for the 20 min and 60 min gradient development times. The full and the simplified model described the experimental data very well. In conclusion, the proposed modeling approach allowed predicting analyte retention times and peak width for various pH and organic modifier changes. Its simplified version required only 12 initial experiments and seems to be very promising in the optimization RP HPLC separations for complex samples and for conditions providing peak compression.     

Insights into the retention mechanism on an octadecylsiloxane-bonded silica stationary phase (HyPURITY C18) in reversed-phase liquid chromatography

Plots of the retention factor against mobile phase composition were used to organize a varied group of solutes into three categories according to their retention mechanism on an octadecylsiloxane-bonded silica stationary phase HyPURITY C18 with methanol-water and acetonitrile-water mobile phase compositions containing 10-70% (v/v) organic solvent. The solutes in category 1 could be fit to a general retention model, Eq. (2), and exhibited normal retention behavior for the full composition range. The solutes in category 2 exhibited normal retention behavior at high organic solvent composition with a discontinuity at low organic solvent compositions. The solutes in category 3 exhibited a pronounced step or plateau in the middle region of the retention plots with a retention mechanism similar to category 1 solutes at mobile phase compositions after the discontinuity and a different retention mechanism before the discontinuity. Selecting solutes and appropriate composition ranges from the three categories where a single retention mechanism was operative allowed modeling of the experimental retention factors using the solvation parameter model. These models were then used to predict retention factors for solutes not included in the models. The overwhelming number of residual values [log k (experimental) - log k (model predicted)] were negative and could be explained by contributions from steric repulsion, defined as the inability of the solute to insert itself fully into the stationary phase because of its bulkiness (i.e., volume and/or shape). Steric repulsion is shown to strongly depend on the mobile phase composition and was more significant for mobile phases with a low volume fraction of organic solvent in general and for mobile phases containing methanol rather than acetonitrile. For mobile phases containing less than about 20 % (v/v) organic solvent the mobile phase was unable to completely wet the stationary phase resulting in a significant change in the phase ratio and for acetonitrile (but less so methanol) changes in the solvation environment indicated by a discontinuity in the system maps.     

反相离子对色谱中有机溶剂浓度和离子对试剂浓度对保留值的影响

我们测定了氨基苯磺酸和氨基萘磺酸在反相离子对色谱中不同乙腈/水, 甲醇/水配比和离子对试剂浓度下的保留值,并把两种冲洗剂下的保留值和关系式lnk'=a+C~c~b中的参数a,C作线性关联,得到很好的相关性,这表明有机溶剂乙腈和甲醇对选择性并无显著的影响,但乙腈有更大的冲洗强度.证明关系式lnk'=A+Blnc~p+C~c~ b能较好地描述有机溶剂和离子对试剂浓度对保留值的影响, 但当离子对试剂浓度较高时该关系式不成立.同时提出了有机溶剂浓度和离子对试剂浓度"等同效应" 的概念     

Modeling the combined effect of temperature and organic modifier content on reversed-phase chromatographic retention. Effectiveness of derived models in isocratic and isothermal mode retention prediction

Models considering simultaneously mobile phase organic content and column temperature were developed in this study by an extension of different equations describing the influence of temperature on solute retention. This extension was achieved by two methods: a semi-thermodynamic and a direct combination of equations expressed separately the dependence of the retention upon each of these factors. The above approaches gave a great number of expressions for the logarithm of the solute retention factor in terms of both temperature and organic content in the mobile phase, ln k(T,phi), determined from the dependence of the standard enthalpy of the retention process on T. From the final expressions of ln k(T,phi) we tested only those with the minimum number of adjustable parameters, i.e. those that correspond to a constant standard enthalpy of the retention process. For this test we examined the retention behaviour of a sample of alkylbenzenes in aqueous acetonitrile eluents. These compounds exhibit ln k versus 1/T plots with a very small curvature. We found that a new equation for ln k(T,phi) based on the adsorption model for retention performs better than all the others. The average percentage prediction error ranges from 0.7 to 1.4%.     

应用基团贡献法关联抗生素的高效液相色谱的保留值

应用ＵＮＩＦＡＣ基团贡献模型计算了４种头孢菌素在ＯＤＳ柱上及不同配比甲醇－水体系中的活度系数，采用多元线性回归法关联了头孢菌素－甲醇－水体系的反相高效液相色谱的保留值，可预测不同配比流动相下的容量因子，经验证实验值与计算值的对误差小于１０％。     

Chromatographic and electrophoretic migration parameters in capillary electrochromatography

Chromatographic retention factor, k', as defined in high-performance liquid chromatography (HPLC) in terms of the migration times of the separand and the inert tracer, has limited applicability to capillary electrochromatography (CEC) when both chromatographic and electrophoretic processes determine the magnitude of the overall migration rates of the separands. This situation is unlike that in HPLC, where k' serves as a useful peak locator for the various sample components, as well as, provides thermodynamic insights into the interactions between the components and the stationary phase. Most publications have borrowed the definition of k' from HPLC and applied it on CEC. However, due to the dual separation mechanisms that are in action in CEC, the system is significantly complicated in comparison to that of HPLC. This paper discusses the impossibility of defining with a k' which would have all the attributes that it has in regular chromatography. The interplay of the two separation mechanisms in determining the overall migration process in CEC is discussed and the various definitions of the electrochromatographic retention factor are presented.     

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.     

Characterization of retentivity of reversed phase liquid chromatography columns

There are dozens of commercially available reversed phase columns, most marketed as C-8 or C-18 materials, but with no useful way of classifying their retentivity. A useful way of ranking these columns in terms of column "strength" or retentivity is presented. The method utilizes a value for ln k'(w), the estimated retention of a solute from a mobile phase of 100% water, and the slope of the plot of ln k' vsE(T)(30), the solvent polarity. The method is validated with 26 solutes varying in ln k'(w) from about 2 to over 20, on 14 different reversed phase columns. In agreement with previous work, it is found that the phase volume ratio of the column is the most important parameter in determining retentivity. It is strongly suggested that manufacturers adopt a uniform method of calculating this value and that it be made available in advertising, rather than the uninterpretable "% carbon".     

Comparison of ion-pairing and ion-suppressing liquid chromatographic methods for the determination of pyrimethamine and ormetoprim in chicken feed

A high-performance liquid chromatographic (HPLC) method is developed to simultaneously determine pyrimethamine (PYR) and ormetoprim (OMT) in chicken feed. In the ion-pairing HPLC determination of PYR and OMT, the relation between the retention factor (k') and the concentration of the organic phase (acetonitrile) shows a characteristic curve. The k' value first decreases and then increases slowly with increasing concentrations of acetonitrile, but then increases rapidly when the acetonitrile concentration increases to 90%. Resolutions (Rs) of PYR and OMT decrease gradually when the concentration of organic phase increases. Increasing the concentration of the pairing ion sodium 1-octanesulfonate (PIC B-8) can decrease the k' and Rs values. Optimum values of k' and Rs are obtained using 82% acetonitrile in 0.005 M PIC B-8. In ion-suppressing HPLC, varying the concentration of Na2HPO4 has little effect on either the k' or Rs values of PYR or OMT at pH 7.5. However, at pH 4.0, k' and Rs decline when the concentration of Na2HPO4 increases. In general, ion-pairing HPLC generates more satisfactory results than ion-suppressing HPLC. Using 82% acetonitrile in water containing 0.001M PIC B-8 as the mobile phase, linear calibration curves are obtained in the range from 1 to 5 mg/L of PYR and OMT. Sulfamonomethoxine, sulfadimethoxine, sulfaquinoxaline, trimethoprim, amprolium, clopidol, and nicarbazin do not interfere with the detection of PYR or OMT. The recoveries of PYR from spiked feed at 1 and 5 mg/Kg are 73.0% and 72.0%, respectively, and those of OMT from spiked feed at 3 and 7 mg/Kg are 50.3% and 53.6%, respectively.     

Martin's rule revisited. Its molecular sense and limitations

The linear relation ln k' = Bn + ln A between the retention factor k' in liquid adsorption chromatography (LAC) and the number of repeat units n within a homologous series of oligomers is called Martin's rule. This empirical relation was supported by the retention behavior of the homologous series of different classes of oligomers but had no theoretical justification. In this paper, it is demonstrated that Martin's rule is a consequence of the general theory of liquid chromatography and the molecular sense of coefficients B and A is clarified: B is the Gibbs energy of the repeat unit of the long polymer chain adsorbed at the wall surface, and A is a combination different parameters which characterize the column and the adsorption correlation length H. The theory predicts the deviations from the linear dependence under conditions of weak adsorption between repeat units and stationary phase when H is close to radius of gyration Rg. Experimental data for retention volumes and selectivity of poly(ethylene glycol)s are given for normal and reversed-phase LAC on different columns in acetone-water and methanol-water as mobile phases. These data show excellent agreement between the theory and experiments. It is shown that Martin's rule holds under special conditions, which are theoretically defined by the relation H > Rg/1.5.