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.     

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography has been investigated in the present study. Columns with inner diameters of 0.32, 0.53, 3.2 and 4.6 mm were compared with respect to retention and efficiency characteristics using temperature gradients from 30 to 90 degrees C with temperature ramps of 1, 5, 10 and 20 degrees C min(-1). The columns were all of 15 cm length and were packed with 3 microm Hypersil ODS particles. Alkylbenzenes served as model compounds, and the mobile phase consisted of acetonitrile-water (50:50, v/v). The study revealed that the column ID is not a critical limiting factor when performing temperature programming in LC, at least for columns narrower than 4.6 mm inner diameter in the temperature interval 30-90 degrees C. The retention times for all components on all columns were highly comparable, with similar peak profiles without any signs of peak splitting. The use of mobile phase pre-heating when using the larger bore columns was avoided by starting the temperature gradients close to ambient. However, the relative apparent efficiency was inversely proportional to column inner diameter, making the capillary columns generally more functional towards temperature gradients than the larger bore columns with respect to chromatographic efficiency. In addition, the capillary columns possessed higher robustness towards temperature programming than the conventional columns.     

Selectivity tuning in chiral dual column gas chromatography

The enantioselective tuning of two columns coupled in series is investigated in chiral high-resolution gas chromatography. Two columns with opposite enantioselectivities (Chirasil-L-Val and Chirasil-D-Val) are coupled in series via a T connector, and the relative retention of enantiomers chromatographed on the system is changed by varying the individual carrier gas flow rates in the coupled columns. The flow-rate ratio necessary for the required selectivity is calculated on the basis of the measured retention factors on the individual columns. The performance of this method for adjusting selectivity is studied by the separation of enantiomers of the N-TFA-O-methyl esters of six amino acids. It is demonstrated that the change of the coupling point carrier gas pressure, at the constant inlet and outlet pressures, may change the enantioselectivity of the given column series to such an extent that the enantiomer elution order may be reversed.     

Preparative isolation and dual column high-performance liquid chromatography of ginkgolic acids from Ginkgo biloba.

A chromatographic procedure for the preparative isolation of six different 6-alkylsalicylic acids (syn. ginkgolic acids) with as alkyl substituents C13:0, C15:0, C15:1, C17:1, C17:2 and, tentatively C17:3 from Ginkgo biloba leaves was developed. The procedure consisted of a combination of normal-phase, reversed-phase and argentation chromatography. The compounds were characterised by means of UV, ¹H-NMR and ¹³C-NMR spectroscopy, and mass spectrometry after silylation. A 15 cm C₁₈ RP-HPLC column connected in series with a 20 cm silver(I) loaded cation exchanger HPLC column in combination with the solvent methanol–water (93:7) acidified with 0.1% formic acid was capable of separating the ginkgolic acids C13:0, C15:1, C17:2, C15:0 and C17:1 within 21 min on an analytical scale. The separation is based on a combination of reversed-phase mechanisms and double bond complexation. Detection took place by UV at 311 nm. The separation is a good starting point for the development of a quantitative procedure for the five major ginkgolic acids in Ginkgo leaves and standardised extracts.     

Separation of polyesters by gradient reversed-phase high-performance liquid chromatography on a 1.5 microm non-porous column

Efficient separation of polyesters composed of a large number of individual oligomers was achieved on a 1.5 microm "non-porous" octadecylsilyl (ODS) silica support by gradient high-performance reversed-phase liquid chromatography (gRP-HPLC) with a mobile phase of acetonitrile, aqueous trifluoroacetic acid (0.2%) and tetrahydrofuran at ambient temperature and signal monitoring by UV absorption at 280 nm. Substantial signal splitting of oligomers in the low molecular weight (Mr) region is indicative that separation not only occurs with respect to molecular weight distribution (MWD) but also to chemical composition distribution (CCD) and functionality type distribution (FTD). Although separation according to CCD and FTD decreases with increasing number of oligomers, co-elution of species with identical number of repeat units but differing in either structure of repeat units or end-groups can be assumed from the relatively broad signals succeeding the aforementioned peaks showing at least partial resolution. Despite the observation that high Mr oligomers elute as sharp signals, the preceding observations suggest that each of these peaks presumably composes of more than one individual component. The polyester oligomers are eluted in the range of increasing Mr and therefore, either separation according to MWD or CCD/FTD was at least achieved for the low Mr sample constituents. Some principal mechanistic aspects of separation are discussed and adsorption seems to play the dominant role. The detection limit, defined as that sample amount yielding an unequivocal recognition on the base of its characteristic chromatographic fingerprint pattern was about 5,000 ppm for the pair Alftalat 3258 - Alftalat 3352 and 10,000 ppm for the pair Crylcoat 430 - Crylcoat 801.     

Development of an accelerated low-pH reversed-phase liquid chromatography column stability test

The important experimental design criteria for an accelerated low-pH RPLC column stability test are discussed. The influence of method variables on the amount and rate of retention-loss and the final optimized parameters for the accelerated low-pH RPLC stability test are presented. The retention-loss curves for selected C8 and C18 stationary phases are compared. These studies indicate that ligand chain length, functionality and bonding density play an important role in determining the low-pH stability of a stationary phase. Additionally, elemental analysis data are used to infer the mechanism responsible for the observed retention-loss under low-pH conditions.     

并联式双三元高效液相色谱系统快速检测土壤中6种蒽醌类化合物

建立了用于住宅建设污染土壤中1,8-二硝基蒽醌、1,5-二硝基蒽醌、1-氨基蒽醌、蒽醌、萘、1,4,5,8-四氯蒽醌等6种蒽醌类化合物含量的高效液相色谱分析方法。利用Ultimate双三元液相色谱仪的特点,设计了一套并联式双系统,采用2根相同的色谱柱(Dikma Diamonsil C18)交叉分析,节省了41.46%的分析时间。以50 mL N,N-二甲基甲酰胺-1,4-二氧六环-甲醇(5:20:25, v/v/v)为萃取剂,采用超声波辅助方法提取样品中的6种目标物。6种目标物的平均加标回收率为82.7%～101%,相对标准偏差为1.8%～3.2%,检出限(以信噪比为3计)为0.007～0.054 mg/kg,可满足北京地方标准DB11/T 811-2011的限量要求。该方法简便、准确,并能节省检测时间,有良好的重现性,可用于土壤中蒽醌类化合物的检测。     

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.     

Stationary-phase effects in gradient high-performance liquid chromatography

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample polarity range that can be covered using gradients of organic solvents in water. The polarity range available for gradient separations of samples containing compounds differing in the lipophilic parts of the molecules can be characterized by "gradient lipophilic capacity", Pl, based on the retention of standard compounds with a repeat lipophilic structural unit, such as a methylene group. The gradient lipophilic capacity is also suitable to characterize the separation possibilities of the columns in non-aqueous reversed-phase gradient elution of strongly non-polar compounds, such as triacylglycerols. In the same way, the suitability of various columns for reversed-phase gradient separations of oligomers can be characterized by "gradient oligomer capacity", as demonstrated in the example of oligo(ethylene glycols). To enable a comparison of the properties of stationary phases independent of column efficiency and dimensions, the gradient lipophilic capacity or the gradient oligomer capacity should be normalized for a "standard" column plate number, gradient range and volume (in column hold-up volume units). The gradient lipophilic capacity or the gradient oligomer capacity and the number of compounds that can be resolved during a gradient run decrease as the initial concentration of the strong solvent in the mobile phase increases and (or) the gradient time decreases. These quantities can be used to select a suitable column and to adjust the optimum gradient profile (the initial composition of the mobile phase and the gradient steepness) with respect to the time of analysis and the number of oligomers or other compounds with regular repeat structural groups that can be resolved during the gradient run.     

Control of column temperature in reversed-phase liquid chromatography

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.     

Determination of testosterone and its metabolites using liquid chromatography with elevated column temperature and flow-rate gradient

A rapid high performance liquid chromatography (HPLC) method for determination of testosterone and its metabolites in biological samples is described. The method combines a flow-rate gradient with elevated column temperature to obtain a complete separation of testosterone and its metabolites. The flow-rate gradient method dramatically reduces the analysis time (17 min) compared with isocratic elution (35 min). This method is simple and reproducible with relative standard deviations (R.S.D.) of <0.1% for retention time and 1-4% for peak area. It provides limits of detection (LOD) in the range of 0.05-0.1 μM for testosterone and its five major metabolites. We applied the method to analyze rat liver microsome samples incubated with testosterone, demonstrating a testosterone metabolic profile due to enzymatic activities in the microsome. Analysis of testosterone and its metabolites is important because metabolism of testosterone is a surrogate measure for cytochrome P-450 (CYP) enzymatic activity.     

Preparative isolation and dual column high-performance liquid chromatography of ginkgolic acids from Ginkgo biloba

A chromatographic procedure for the preparative isolation of six different 6-alkylsalicylic acids (syn. ginkgolic acids) with as alkyl substituents C13:0, C15:0, C15:1, C17:1, C17:2 and, tentatively C17:3 from Ginkgo biloba leaves was developed. The procedure consisted of a combination of normal-phase, reversed-phase and argentation chromatography. The compounds were characterised by means of UV, ¹H-NMR and ¹³C-NMR spectroscopy, and mass spectrometry after silylation. A 15 cm C₁₈ RP-HPLC column connected in series with a 20 cm silver(I) loaded cation exchanger HPLC column in combination with the solvent methanol–water (93:7) acidified with 0.1% formic acid was capable of separating the ginkgolic acids C13:0, C15:1, C17:2, C15:0 and C17:1 within 21 min on an analytical scale. The separation is based on a combination of reversed-phase mechanisms and double bond complexation. Detection took place by UV at 311 nm. The separation is a good starting point for the development of a quantitative procedure for the five major ginkgolic acids in Ginkgo leaves and standardised extracts.     

Optimization of two-dimensional gradient liquid chromatography separations

An almost orthogonal comprehensive two-dimensional liquid chromatography was developed for the separation of phenolic and flavone natural antioxidants by using combinations of a polyethylene glycol silica micro-column in the first dimension and a porous-shell fused-core C18 column in the second dimension, both in the reversed-phase mode. System orthogonality was improved using parallel gradients of acetonitrile in buffered mobile phase. A new approach was proposed to optimize matching segmented gradient profiles in the two dimensions. An algorithm was developed for automatic corrections of the shifts in retention in the second dimension induced by the parallel two-dimensional gradient operation technique. Using the porous-shell C18 column in the second dimension at elevated temperature (60 degrees C) and high pressure (480 bar) with optimized segmented profiles of the parallel gradients in the two dimensions, the overall separation time for comprehensive LC x LC was reduced to 30 min.     

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.     

Effect of column loading on the accuracy of analyzing pentalgin N tablets by gradient high-performance liquid chromatography

Two approaches for the high-performance liquid chromatographic analysis of multicomponent medical preparations were experimentally studied by an example of Pentalgin N tablets. One of them was the simultaneous determination of all components of the preparation; another was the independent analysis of concentrated and diluted solutions for determining the presentpresent in small and large amounts, respectively. The performance characteristics of the studied procedures were determined in the analysis of model solutions containing all active and adjuvant substances of the tablets. In the simultaneous determination of all components, the relative error was smaller because of the more sustained performance of the chromatographic column in the absence of its overload with the sample observed in the independent analysis.     

Overloaded gradient elution chromatography on heterogeneous adsorbents in reversed-phase liquid chromatography

Overloaded band profiles of phenol were measured on a C18-Kromasil column in gradient elution conditions. The mobile phase used was a mixture of methanol and water. The volume fraction of methanol was allowed to vary between 0 and 0.5. A general adsorption model, which expresses the amount of phenol adsorbed q* as a function of both its concentration C and the composition phi of the organic modifier (methanol) in the mobile phase, was empirically derived from previous independent adsorption experiments based on frontal analysis (FA) and frontal analysis by the characteristic point (FACP). Accordingly, the general model was an extension of the simplest heterogeneous model, the Bilangmuir model, to non-isocratic conditions. The low-energy sites followed the classical linear solvent strength model (LSSM), but not the high-energy sites whose saturation capacity linearly decreased with phi. The general model was validated by comparing the experimental and simulated band profiles in gradient elution conditions, in linear and non-linear conditions, as well. The band profiles were calculated by means of the equilibrium-dispersive model of chromatography with a finite difference algorithm. A very good agreement was observed using steps gradient (delta phi) from 0 to 50% methanol and gradient times t(g) of 20, 25, 30, 40, 60, 80 and 100 min. The agreement was still excellent for steps gradient from 5 to 45% (t(g) = 25 min), 5 to 35% (t(g) = 50 min), 5 to 25% (t(g) = 50 min) and 5 to 15% (t(g) = 50 min). Significative differences appeared between experience and simulation when the slope of the gradient (delta phi/t(g)) became too strong beyond 3.3% methanol per minute. This threshold value probably mirrored the kinetic of arrangement of the G18-bonded chains when the methanol content increased in the mobile phase. It suggested that the chromatographic system was not in a full thermodynamic equilibrium state when very steep mobile phase gradients were applied.     

High speed gradient elution reversed-phase liquid chromatography

A major disadvantage of gradient elution in terms of speed results from the need to adequately re-equilibrate the column. This work distinguishes two states of re-equilibration: (1) run-to-run repeatability and (2) full equilibration. We find that excellent repeatability (+/-0.002 min in retention time) is achieved with at most 2 column volumes of re-equilibration whereas full equilibration can require considerably more than 20 column volumes. We have investigated the effects of adding ancillary solvents (e.g. n-propanol, n-butanol) to the eluent and changing the particle pore size, initial eluent composition and type, column temperature and flow rate on the speed of full equilibration. Full equilibration seems to be more thermodynamically limited than kinetically controlled. Also, we show that the main limitation to reducing the full equilibration time is related to instrument design issues; a novel approach to overcome these instrumental issues is described.