Injection profiles in liquid chromatography II: predicting accurate injection-profiles for computer-assisted preparative optimizations

In computer assisted optimization of liquid chromatography it has been known for some years that it is important to use experimental injection profiles, instead of rectangular ones, in order to calculate accurate elution bands. However, the incorrectly assumed rectangular profiles are still mostly used especially in numerical optimizations. The reason is that the acquisition of injection profiles, for each injection volume and each flow rate considered in a computer-assisted optimization requires a too large number of experiments. In this article a new function is proposed, which enables highly accurate predictions of the injection profiles and thus more accurate computer optimizations, with a minimum experimental effort. To model the injection profiles for any injection volume at a constant flow rate, as few as two experimental injection profiles are required. If it is desirable to also take the effect of flow rate on the injection profiles into account, then just two additional experiments are required. The overlap between fitted and experimental injection profiles at different flow rates and different injection volumes were excellent, more than 90%, using experimental injection profiles from just four different injection volumes at two different flow rates. Moreover, it was demonstrated that the flow rate has a minor influence on the injection profiles and that the injection volume is the main parameter that needs to be accounted for.     

Microbore columns in quantitative column liquid chromatography

Summary Some quantitative implications of the use of microbore columns in column liquid chromatography are investigated. Although for several pumps the flow rate stability at 50 l min^–1 is slightly worse than that of pumps operating at 1 ml min^–1, the quantitative performance with respect to repeatability and reproducibility of response factors equals that of conventional liquid chromatography. Thermostatting is strongly recommended for microbore column operation.     

Influence of injection parameters on column performance in nanoscale high-performance liquid chromatography

Summary The influence of the injection volume and the sample solvent on column efficiency has been evaluated in packed nano liquid chromatography using columns 150μ i.d. Evaluation of column performance was by means of reduced plate height (h) versus reduced velocity (v) for four polyaromatic hydrocarbon test compounds (PAHs). When compounds are dissolved in a weak solvent (such as MeCN: H₂O, 30∶70), and whatever the injection volume −60 or 200 nL-a gain in efficiency can be observed due to the well-known on-column focusing phenomenon, but keeping constant solute retention factors. Under optimized conditions (flow rate: 150 nLmin⁻¹, solvent sample MeCN: H₂O, 30∶70, injection volume 200 nL), a reduced plate height of 1.83 has been obtained for a 15 μm C₁₈ packing corresponding to 36000 plates m⁻¹, which illustrates the absence of any extracolumn band broadening under nano LC conditions.     

Prediction of retention times and peak widths in temperature-programmed gas chromatography using the finite element method

Optimization of separations in gas chromatography is often a time-consuming task. However, computer simulations of chromatographic experiments may greatly reduce the time required. In this study, the finite element method was used to predict the retention times and peak widths of three analytes eluting from each of four columns during chromatographic separations with two temperature programs. The data acquired were displayed in predicted chromatograms that were then compared to experimentally acquired chromatograms. The differences between the predicted and measured retention times were typically less than 0.1%, although the experimental peak widths were typically 10% larger than expected from the idealized calculations. Input data for the retention and peak dispersion calculations were obtained from isothermal experiments, and converted to thermodynamic parameters.     

Fundamental equation of the dual flow rate-solvent gradient elution in liquid chromatography

An alternative expression of the fundamental equation of multi-mode gradient elution involving simultaneous changes in mobile phase composition and flow rate is derived using simple kinetic arguments and graphic interpretation. The new expression consists of a system of two integral equations and provides an easy and direct way of predicting retention times under dual-mode gradient conditions.     

Column selectivity in reversed-phase liquid chromatography I. A general quantitative relationship

Retention factors k have been measured for 67 neutral, acidic and basic solutes of highly diverse molecular structure (size, shape, polarity, hydrogen bonding, pKa, etc.) on 10 different C18 columns (other conditions constant). These data have been combined with k values from a previous study (86 solutes, five different C8 and C18 columns) to develop a six-term equation for the correlation of retention as a function of solute and column. Values of k can be correlated with an accuracy of +/- 1-2% (1 standard deviation). This suggests that all significant contributions to column selectivity have been identified (and can be measured) for individual alkyl-silica columns which do not have an embedded polar group. That is, columns of the latter kind can be quantitatively characterized in terms of selectivity for use in the separation of any sample.     

Maximizing peak capacity and separation speed in liquid chromatography

The practical effects of gradient time and flow rate on the peak capacities of a range of analytes of differing molecular weights (MWs) and physico-chemical properties have been evaluated using ultra high pressure LC instrumentation with sub-2 mum and superficially porous particle phases. Optimum peak capacity, in RP gradient LC, for small molecules, including typical pharmaceutical drugs and peptides with MWs up to 1300, was demonstrated at a maximum flow rate for a given gradient time (i.e. up to 40 min). Flow rates significantly higher than the optimum in the van Deemter plots and also higher than those typically employed by the majority of the chromatographers today are recommended for gradient LC (i.e. up to 1.0 mL/min on 50-150x2.1 mm 1.7 mum columns). This recommendation is applicable for temperatures above 40 degrees C, i.e. temperatures typically utilized for separations employing sub-2 mum particles to reduce column back pressure. Van Deemter and pseudo van Deemter plots were determined and combined with chromatographic gradient elution theory to explain our unexpected observations. The derived models exhibited good agreement between experimental and predicted peak capacities (absolute average error 4%, max. error 12%).     

以离子液体作流动相添加剂的高效液相色谱法测定复方苦参注射液中4种生物碱

将离子液体1-丁基-3-甲基咪唑四氟硼酸盐(［BMIM］BF4)作为流动相添加剂建立了同时测定复方苦参注射液中4种主要生物碱的HPLC分析方法。以Agilent TC-C18柱(250 mm×4.6 mm, 5 μm)为分离柱,甲醇-0.1%磷酸水溶液(含2.2×10-4mol/L ［BMIM］BF4)(5:95, v/v)为流动相,流速1.0 mL/min,柱温30 ℃,进样量20 μL,在205 nm下检测。结果表明,苦参碱、槐定碱、氧化槐果碱以及氧化苦参碱的质量浓度分别在25.8～155.0 mg/L, 40.0～240.0 mg/L, 21.7～130.0 mg/L和37.5～225.0 mg/L范围内线性关系良好,相关系数均大于0.9990,平均回收率(n=9)在96.2%和98.9%之间。离子液体的加入能明显改善C18柱分离生物碱的色谱峰形并提高分离度。本法简便、快速、重复性好,可用于复方苦参注射液中生物碱的分离与测定。     

A simplex optimization of the experimental parameters in preparative liquid chromatography

Summary A study of the optimization of the experimental conditions for the purification or extraction of pure compounds by liquid chromatography is presented. Optimum values of the parameters of overloaded elution are derived for maximum production rate, using a Simplex algorithm and the procedure previously described for the simulation of the elution profiles of binary mixtures. The mobile phase flow velocity and the sample size have been optimized together in a first step, simulating the procedure followed in practice, when a column is available. In a second part, the influence of the column length and the average particle size of the packing material on the column performance as well as the trade-offs between the production rate and the yield are discussed.There are three major conclusions in this work. First, the optimum experimental conditions are often very different, depending whether one is primarily interested in the first or in the second eluted component of a mixture. Second, the column efficiency under analytical conditions is very important: it is traded-off for high flow rates, hence short cycle time and increased production rate. Third, the production rate depends strongly on the maximum pressure at which the equipment can be operated. Finally, the optimum production rate varies rather smoothly with the mobile phase velocity and the sample size, so a high yield (70% or more) can usually be obtained with a limited loss in production rate (30 to 60%).     

Repeatability and reproducibility of high concentration data in reversed-phase liquid chromatography. I. Overloaded band profiles on Kromasil-C18

Single-component adsorption-isotherm data were acquired by frontal analysis (FA) for six low-molecular-mass compounds (phenol, aniline, caffeine, theophylline, ethylbenzene and propranolol) on one Kromasil-C18 column, using water-methanol solutions (between 70:30 and 20:80, v/v) as the mobile phase. Propranolol data were also acquired using an acetate buffer (0.2 M) instead of water. The data were modeled for best agreement between calculated and experimental overloaded band profiles. The adsorption energy distribution was also derived and used for the selection of the best isotherm model. Widely different isotherm models were found to model best the data obtained for these compounds, convex upward (i.e. Langmuirian), convex downward (i.e. anti-Langmuirian), and S-shaped isotherms. Using the same sample size for all columns (loading factor, Lf approximately 10%), overloaded band profiles were recorded on four different columns packed with the same batch of Kromasil-C18 and five other columns packed with different batches of Kromasil-C18. These experimental band profiles were compared to the profile calculated from the isotherm measured by FA on the first column. The repeatability as well as the column-to-column and the batch-to-batch reproducibilities of the band profiles are better than 4%.     

Band splitting in overloaded isocratic elution chromatography I. The experimental evidence

A series of samples of increasing volume (from 0.001 to 4.0 cm3), containing the same constant concentration (40 g/l) of two simple compounds, ethylbenzoate and 4-tert.-butylphenol were injected on a Kromasil-C18 column with methanol-water (62:38. v/v) as the mobile phase. Complex band profiles were observed when the volume of the sample became large enough and strong band interference took place. The analysis of the fractions collected during the elution of the mixed band demonstrates that, for samples larger than 2 cm3, the band of 4-tert.-butylphenol is split into two separate bands, one eluted before and the other eluted after the band of ethylbenzoate. Such a phenomenon has never been observed yet in RPLC, under isocratic elution conditions.     

Method of analysis of a selected group of pyrethroids in soil samples using off-line flow-through extraction and on-column direct large-volume injection in reversed phase high performance liquid chromatography

An analytical method using flow-through extraction of a soil sample filled in a short HPLC column with methanol or methanol-water mixtures and large-volume injection in RP-HPLC has been developed for the simultaneous determination of residues of three pyrethroids--kadethrin, cypermethrin, and permethrin--from soil samples. The developed RP-HPLC method enables separation of four diastereoisomers of cypermethrin into three peaks and resolution of two diastereoisomers of permethrin. The UV photometric detection limits of direct on-column large-volume injection of 1.00 mL of extract were 30 ng/mL of kadethrin, 37 ng/mL of total content of cypermethrin, and 65 ng/mL of trans-permethrin, which corresponds to a pyrethroid soil content of around 0.3 mg/kg. Effects of extractant flow rate and optimal extractant volume on the percentage recovery of pyrethroids from Slovak soil samples were studied. Recovery studies were performed at 0.5- 5.0 microg/g fortification level of kadethrin and 1.0-2.5 microg/g fortification level of cypermethrin and permethrin in a soil sample. Recoveries ranged from 83 to 90% for kadethrin, from 87 to 94% for total cypermethrin, and from 85 to 98% for trans-permethrin. This work comprises a basic study aimed at elaboration of an RP-HPLC method of direct analysis of pyrethroids in a soil matrix at low concentration levels achieved by a "solid sample injection" in HPLC--on-line interfacing of analyte extraction, extract clean-up, and analysis.     

Indirect detection in liquid chromatography I. Response models for reversed-phase ion-pairing systems

Summary Ionic compounds without inherent detectable properties show good response in reversed phase ion-pairing systems containing a detectable ionic compound (probe) in the mobile phase. The basis for the indirect detection is the common interaction effect for solutes and probe caused by the limited binding capacity of the adsorbent. Quantitative models for solutes and probe with the same or opposite charges show a simple dependence of the response on the retention of the solute and the coverage of the binding surface by the probe. Applications of the models have shown good agreement between calculated and found response. The binding to sites of different kinds on the adsorbent and possibly ion-pair formation in the mobile phase have an influence on the indirect response. Uncharged solutes give low response in such systems owing to low binding to the competitive sites.     

A new method for the determination of mobile phase volume in normal and reversed-phase liquid chromatography

Summary A new method for the determination of the mobile phase volume (V _m) in liquid chromatography is presented based on the model regarding the retention of ionic solutes in the presence of eluent electrolytes. TheV _m value can be determined by measuring the retention volumes of two ions that have the same charge in two eluent electrolyte systems. Compared with the methods using isotopically labelled eluent components or inorganic salts asV _m markers, the method presented is proved to give more reasonableV _m values for both normal and reversed-phase liquid chromatography. As well as in binary mixed solvent systems, theV _m values in single solvent systems can be determined by this method.     

Role of the buffer in retention and adsorption mechanism of ionic species in reversed-phase liquid chromatography. I. Analytical and overloaded band profiles on Kromasil-C18

The influence of the pH, the concentration, and the nature of the buffer on the retention and overloading behavior of propranolol (pKa = 9.25) on Kromasil-C18 was studied at 2.75 < pH < 6.75, using four buffers (phosphate, acetate, phthalate, and succinate), at three concentrations, 6, 20, and 60 mM. The propranolol band profiles were recorded for three sample sizes, less than 1 microg and 375 microg (sample less concentrated than the buffer), and 7500 microg (band more concentrated than the buffer). Results showed that the buffer concentration, not its pH, controls the retention time of propranolol, in agreement with the chaotropic model. The retention factor depends also on the nature of the buffer, particularly the valence of the basic anion. At moderate loading, the band profiles are well accounted for by a simple bilangmuir model (no adsorbate-adsorbate interactions) with the monovalent anions H2PO4- (pH 2.75), HOOC-Ph-COO- (pH 2.75), HOOC-CH2-CH2-COO- (pH 4.16) and CH3COO- (pH 4.75), and by a bimoreau model (significant adsorbate-adsorbate interactions) with the bivalent anions OOC-Ph-COO- (pH 4.75), OOC-CH2-CH2-COO- (pH 5.61) and HPO4(2-) (pH 6.75). The isotherm were determined using the inverse method. The results show that both the saturation capacity and the equilibrium constant on the low-energy sites increase with increasing buffer concentration, a result similar to that observed with neutral salts. For bivalent anions, the adsorbate-adsorbate interactions are much stronger on the low than on the high energy sites. The density of high energy sites is lower and the equilibrium constant on the low energy sites are higher with bivalent than with univalent anions. These results are consistent with the formation of a propranolol-buffer (2:1) complex with bivalent anions.     

The varigrad and similar apparatus in gradient liquid chromatography: A systematic computational approach. Part 2: Applications

Part 2 deals with the application of the equations derived in Part 1 to the systematic calculations of the constant volume and hydrostatic Varigrads. Examples are given which illustrate the advantage of using the modified incremental method of gradient elution. The non-homogeneous Varigrad, where mixing chambers may vary in volume, is also treated.     

A systematic investigation of algorithm impact in preparative chromatography with experimental verifications

Computer-assisted optimization of chromatographic separations requires finding the numerical solution of the Equilibrium-Dispersive (ED) mass balance equation. Furthermore, the competitive adsorption isotherms needed for optimization are often estimated numerically using the inverse method that also solves the ED equations. This means that the accuracy of the estimated adsorption isotherm parameters explicitly depends on the numerical accuracy of the algorithm that is used to solve the ED equations. The fast and commonly used algorithm for this purpose, the Rouchon Finite Difference (RFD) algorithm, has often been reported not to be able to accurately solve the ED equations for all practical preparative experimental conditions, but its limitations has never been completely and systematically investigated. In this study, we thoroughly investigate three different algorithms used to solve the ED equations: the RFD algorithm, the Orthogonal Collocation on Finite Elements (OCFE) method and a Central Difference Method (CDM) algorithm, both for increased theoretical understanding and for real cases of industrial interest. We identified discrepancies between the conventional RFD algorithm and the more accurate OCFE and CDM algorithms for several conditions, such as low efficiency, increasing number of simulated components and components present at different concentrations. Given high enough efficiency, we experimentally demonstrate good prediction of experimental data of a quaternary separation problem using either algorithm, but better prediction using OCFE/CDM for a binary low efficiency separation problem or separations when the compounds have different efficiency. Our conclusion is to use the RFD algorithm with caution when such conditions are present and that the rule of thumb that the number of theoretical plates should be greater than 1000 for application of the RFD algorithm is underestimated in many cases.     

The Varigrad and similar apparatus in gradient liquid chromatography: A systematic computational approach. Part 1: Theoretical development

Different methods for producing a pre-assigned gradient in liquid chromatography are presented. A systematic approach to the calculations involved is given. The derived equations are mostly Poisson and Poisson summation distributions which are tabulated in the literature. The incremental method of gradient elution developed by Scott has been modified. In the modified apparatus two mixing chambers are used instead of one. This leads to an appreciable decrease in the number of reservoirs needed for the same precision in fitting a desired gradient. The application of the derived equations, together with other Varigrad modifications, will be given in Part 2 of this paper (to be published shortly in this Journal).     

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. I. Injection procedure and column stability

We report a method for reliable routine polymer sample introduction with minimal bias, a separation method of the first six linear and cyclic oligomers by liquid chromatography, quantification using group equivalents and long term method performance. Injecting a polymer sample in a mobile phase containing an aqueous non-solvent often results in blocked systems as the polymer precipitates in the connecting capillaries. In this first part we focus on a new injection technique, in which the dissolved polyamide is placed between two zones of formic acid, preventing the polymer to precipitate before it reaches the column. Development of this sandwich injection method makes direct injection of the polymer into an aqueous acetonitrile gradient feasible. The oligomeric polyamide recovery of this technique, extraction, dissolution/precipitation and direct injection on a hexafluoro-isopropanol (HFIP) gradient were compared. With the sandwich injection method the polymer remains on the column, slowly changing the stationary phase. The influence of this on resolution and retention was studied. Column stability allows sixty injections before cleaning or replacing the column is necessary.     

A systematic investigation of the effect of sample diluent on peak shape in hydrophilic interaction liquid chromatography

The aim of this study was to evaluate the importance of sample diluents to improve peak shapes in hydrophilic interaction liquid chromatography (HILIC), using low molecular weight (<1000 Da) analytes as well as peptides (with MW ranging between 1000 and 6000 Da) as model compounds. Various solvents were tested including water, acetonitrile, methanol, ethanol, propan-2-ol, dimethyl sulfoxide, and a number of combinations of them. For the analysis of small MW compounds, best peak shapes were obtained with sample dissolved in pure ACN but, IPA or a mixture of ACN/IPA (50:50, v/v) could represent a viable alternative in the case of solubility issues with pure ACN. For drug discovery applications, DMSO can be employed but in combination with at least 80% of ACN. For peptides analysis, acetonitrile, EtOH and IPA as sample diluents, provided similar chromatographic profiles, but pure EtOH or IPA were recommended to limit denaturation and samples solubility issues. Finally, whatever the nature of the compounds, it is recommended to add the lowest amount of water to the sample diluent, to maintain suitable peak shapes.