Comprehensive two-dimensional liquid chromatography

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LC×LC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LC×LC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LC×LC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.     

Development and validation of a liquid chromatography with mass spectrometry method to determine resveratrol and piceid isomers in beeswax

This paper represents the first report of a liquid chromatography coupled to electrospray ionization mass spectrometry method for simultaneously analyzing resveratrol and piceid isomers (cis and trans) in beeswax. An efficient extraction procedure has been proposed (average analyte recoveries were between 89 and 95%); this involved a solid–liquid extraction using a mixture of ethanol and water (80:20, v/v) and a concentration step in a rotary evaporator. The separation of all the compounds was achieved using a C₁₈ column and a mobile phase composed of ammonium formate 0.03 M in water and acetonitrile in gradient elution mode at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification ranged from 1.0 to 1.7 and 3.5 to 5.5 μg/kg, respectively. Finally, the proposed method was applied to analyze beeswax samples collected from experimental and organic apiaries.     

Isolation of the active constituents in natural materials by 'heart-cutting' isocratic reversed-phase two-dimensional liquid chromatography

A multidimensional heart-cutting reversed-phase HPLC separation approach, where two columns were operated independently via two six-port two-position switching valves, was employed in the isolation of a major bioactive found in the ethanol-water (80:20) crude extract of Clerodendrum floribundum. In this mode of operation, the specific productivity of the multidimensional approach under overload conditions was twice that of conventional gradient methods with the same loading factor. Isolated sample purities were greater than 99% with recoveries of 95%. The independent operation of each of the two columns employed in the multidimensional approach allowed the cycle time to be less than 7 min, compared to 23 min in the gradient elution single-dimension mode of operation.     

Analysis of triacylglycerol positional isomers by silver ion high performance liquid chromatography

Silver ion chromatography, utilizing two commercially available HPLC columns connected in series, was used to separate a variety of triacylglycerol positional isomers. The isomers had a fatty acid composition of ABA and AAB, where A and B were fatty acids containing zero to three double bonds. Isocratic solvent systems of 0.5% to 1.0% acetonitrile in hexane were used to separate the triacylglycerol isomers. This methodology can be used to rapidly determine the isomeric purity of synthesized or isolated triacylglycerols and to isolate specific positional isomers.     

Separation ofcis andtrans isomers of naturally occurring hydroxycinnamic acids by high-pressure liquid chromatography

Summary The separation of hydroxycinnamic acids has been investigated by high-pressure liquid chromatography using a micro C₁₈ bonded phase. Irradiation oftrans-ferulic and sinapic acids readily gave a mixture ofcis-trans isomers which can be also separated with the system described.     

Separation of xylidine isomers on heteroaromatic compounds by gas-liquid chromatography

Summary Nicotinic acid and 1H-benzotriazole appreciably resolved all the xylidine (dimethylaniline) isomers, while benzimidazole, nicotinamide and phthalimide can also separate all the isomers except 2,4- and 2,3-xylidines. The analyses were carried out on supports which were not treated with an alkali.     

Differentiating root and rhizome of panax notoginseng based on precursor ion scanning and multi heart-cutting two-dimensional liquid chromatography

Owing to increasing demand for Panax notoginseng-based medicines and health products, establishing a fast, simple, and reliable assay to analyze the chemical differences between its root and rhizome is important. Although previous studies showed that the chemical and biological differences between the root and rhizome of P. notoginseng seem to be small, efforts should be taken to investigate such differences to ensure the safety and efficacy of the products. This work describes a holistic approach that combines characteristic fingerprinting using ultra-high performance liquid chromatography-tandem mass spectrometry parent ion scanning with charged aerosol detection and targeted separation by online heart-cutting two-dimensional liquid chromatography, to identify and evaluate characteristic markers allowing differentiation of the root and rhizome. A total of five potential markers chikusetsusaponin L₅, ginsenoside Rb₂, stipuleanoside R_2, malonyl-ginsenoside Rb₁, and malonyl-ginsenoside Rd, were identified and confirmed by comparing chromatographic retention time, the accurate mass of molecular weight, and the fragments of secondary MS with the available reference materials. The results showed that all five markers were 2.8–7 times higher in content in the rhizome than in the root.     

Separation of nonionic surfactants according to functionality by hydrophilic interaction chromatography and comprehensive two-dimensional liquid chromatography

It is shown, that amphiphilic polymers--such as polysorbates and fatty esters of polyethylene glycol can be separated by comprehensive two-dimensional liquid chromatography using a reversed phase column (under critical conditions for the polyoxyethylene chain) and a HILIC column, which may arranged in different order. The mobile phases in both dimensions can be 93-97 wt% acetone water. As the retention of higher esters on the reversed phase column is very strong, this column should be used as the first dimension. On the HILIC column all fractions elute within a reasonably short time (at a flow rate of 2.5 ml/min within 2 min). With a flow rate of 0.1 ml/min in the first dimension, a full separation can be achieved in 90 min.     

Characterization of hydroxypropylmethylcellulose (HPMC) using comprehensive two-dimensional liquid chromatography

Various hydroxyl-propylmethylcellulose (HPMC) polymers were characterized according to size and compositional distributions (percentage of methoxyl and hydroxyl-propoxyl substitution) by means of comprehensive two-dimensional liquid chromatography (LC×LC) using reversed-phase (RP) liquid chromatography in the first dimension and aqueous size-exclusion chromatography (aq-SEC) in the second dimension. RP separation was carried out in gradient-elution mode applying 0.05% TFA in water and 1-propanol, while 0.05% TFA in water was used as mobile phase in aqueous SEC. A two-position ten-port switching valve equipped with two storage loops was used to realize LC×LC. Detection of HPMC was accomplished by charged-aerosol detection (CAD). Data processing to visualize chromatograms was carried out using Matlab software. The significant influence of the LC×LC temperature on (the retention of) HPMC was studied using a column oven which allowed accurate temperature control. Due to the phenomenon of thermal gelation, which is a result of methyl and hydroxypropyl substitution of anhydroglucose units from the cellulose backbone, we were able to obtain additional, specific information on compositional characteristics of various HPMC samples. As the retention behaviour of gelated and non-gelated polymer proved to be different, the fraction of the polymer that is gelated in the chromatographic column could be monitored at different temperatures. Moreover, the temperature at which half of the polymer is gelated could be correlated with the cloud-point temperature. As a result, differences in inherent cloud points of modified cellulose can be used as a further distinguishing property in "temperature-responsive" LC×LC.     

二维反相液相色谱制备五味子中的木脂素

应用自主研发的具有分离-富集模式的制备色谱工厂,建立了分离制备五味子木脂素有效部位及其单体化合物的方法。该方法首先以C₁₈（250 mm×4.6 mm,5 μm）为色谱柱,水和甲醇为流动相,分离度和保留时间为考察指标,采集4个不同梯度的色谱信息,通过XTool色谱专家系统软件模拟,确定五味子木脂素第一、二维色谱条件;然后采用线性放大的方法,以C₁₈（250 mm×30 mm,10 μm）为第一、二维分离柱,C₁₈（80 mm×30 mm,10 μm）为富集柱,水为富集稀释液,对五味子木脂素进行二维色谱分离纯化;最后第一维分离得到9个可重复组分,第二维分离得到20个高纯度化合物,其中有6个单体化合物。结果表明该法重现性良好,可以实现五味子木脂素的系统性分离,对五味子化学成分的研究具有重要意义。     

液相色谱测定土壤中4种硝基苯胺类化合物

建立了索氏提取-液相色谱-紫外检测器(SE-HPLC-UV vis)测定土壤中4种硝基苯胺同系物的方法。在以Hypersil BDS C18为色谱柱,55%甲醇为流动相,流速1 mL/min,检测波长230 nm时,4种物质可以在10 min内实现良好分离。对于索氏提取,本研究指出,采用二氯甲烷作为提取剂,能极大地消除HPLC-UV vis检测中的背景干扰。在单因素实验的基础上,得出最佳的提取条件如下:温度55℃,时间8h,提取剂用量60 mL。方法回收率>90%,RSD在1.9%～4.1%之间。     

Bias reduction in the quantitative analysis of a target analyte present in a limited quantity in human plasma using dual-mode heart-cutting two-dimensional liquid chromatography coupled with isotope dilution mass spectrometry

Dual-mode heart-cutting two-dimensional liquid chromatography (DMHC 2D-LC) was applied to isotope dilution mass spectrometry (IDMS) to reduce the bias in the quantitative analysis of a target analyte present in a limited quantity in human plasma. Based on a Waters I-Class LC system, the DMHC 2D-LC system was operated in one- and two-dimensional modes to facilitate the determination of heart-cutting time and the efficient trapping of the target LC eluate. Experiments to determine the feasibility of coupling with IDMS were performed with triple quadrupole mass spectrometry using folic acid standards and/or ¹³C₅-folic acid. To validate the performance of the DMHC 2D-LC/IDMS system on a complex sample, human plasma was analyzed for folic acid and the result was compared with that obtained using conventional single-column LC. The total run time of the DMHC 2D-LC system was 20 min, the same as that of the single-column LC system. The peak profile of the spiked ¹³C₅-folic acid obtained with single-column LC/MS was affected by matrix effects, but resolved with DMHC 2D-LC/MS, thus improving the accuracy of the analysis. The DMHC 2D-LC/IDMS system showed reliable performance in analyzing the target analyte in human plasma, eliminating matrix effects and saving analysis time.     

Retention behavior of dipeptide isomers in reversed-phase high-performance liquid chromatography

Summary The effects of eluent pH and organic modifier concentration on the capacity factor (k) and selectivity of dipeptide isomers were investigated. It has been observed that the variation in the logarithm of the capacity factor of the dipeptide isomers is linearly dependent on the organic modifier concentration (C_b), however, the selectivity is almost independent of it. Both capacity factor and selectivity were seriously affected by the pH of the eluent. Both the capacity factor and the intercept of the ln k vs. C_b plot increased with increasing van der Waals volume of the non-polar amino acid subunit of the dipeptides.     

Comprehensive two-dimensional gas chromatography using a high-temperature phosphonium ionic liquid column

A high-temperature ionic liquid, trihexyl(tetradecyl)phosphonium bis(trifluoromethane)sulfonamide, was used as the primary column stationary phase for comprehensive two-dimensional gas chromatography (GC × GC). The ionic liquid (IL) column was coupled to a 5% diphenyl/95% dimethyl polysiloxane (HP-5) secondary column. The retention characteristics of the IL column were compared to polyethylene glycol (DB-Wax) and 50% phenyl/50% methyl polysiloxane (HP-50+). A series of homologous compounds that included hydrocarbons, oxygenated organics, and halogenated alkanes were analyzed with each column combination. This comparison showed that the ionic liquid is less polar than DB-Wax but more polar than HP-50+. The most unique feature of the IL × HP-5 column combination is that alkanes, cyclic alkanes, and alkenes eluted in a narrow band in the GC × GC chromatogram; whereas, these compounds occupied a much larger portion of the DB-Wax × HP-5 and the HP-50+ × HP-5 chromatograms. Each column combination was used to analyze diesel fuel. The IL × HP-5 chromatogram displayed narrow bands for three major compound classes in diesel fuel: saturates, monoaromatics, and diaromatics. The IL column was used at temperatures as high as 290 °C for several months without any noticeable changes in column performance.     

在线单柱二维液相色谱法快速纯化牛胰腺中细胞色素C

用二维（弱阳,疏水）色谱柱首次完成了在线单柱二维液相色谱法快速纯化牛胰腺中的细胞色素C.在将牛胰腺粗提液进样到该二维色谱柱后,在弱阳离子交换模式下,以梯度洗脱方式进行一维色谱分离,并将分离得到的细胞色素C样品液收集到色谱仪的附加样品储液管内.然后将储液管中样品液全部排出,并二次进样到同一根二维色谱柱中,与此同时也完成了对该样品液的缓冲溶液交换,按疏水色谱（HIC）分离模式进行分离.最终对细胞色素C完成了第二维的HIC纯化.上述全部操作均为在线,在一具有正压的封闭体系中进行并可在52分钟内完成.细胞色素C的最终产品纯度高达94.7%（RSD=1.91%）,质量回收率为80.5%（RSD=2.20%）.预计此在线单柱二维液相色谱法也可能用于牛胰腺中其他功能蛋白的快速纯化,并可能将其放大到制备和生产规模.     

On-line parallel reversed phase two-dimensional liquid chromatography for high-throughput analysis of complex proteomic samples

A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is con- structed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by reversed phase chromatography (Hypersil BDS C18, 15 mm×4.6 mm I.D.) to separate the complex peptides from globin peptic hydrolysate. After the sample was loaded on the SCX column, the phosphate buffer (pH 4.0) was used to elute the peptides. Then, elutes flowed through the interface and the peptides focused on the head of the trapping columns (Hypersil BDS C18, 15 mm×4.6 mm I.D.) but salt passed into the waste. After the valve was switched, the samples were flushed with a backward flow into the RP analytical column. The peptides on the SCX were eluted with 12 discontinuous steps linearly increasing salt concentrations. The peptides enriched on the trapping column were desalted and separated by the RP columns. The resolution and the resolved peaks of the 2D SCX/RP system were greatly increased and the total peak capacity reached as high as 2280.     

Separation of isomeric and nonisomeric monoribonucleotides by isocratic ion pair high performance liquid chromatography

A simple, isocratic, high performance liquid chromatographic procedure is described for the first time for the separation of nine monoribonucleotides using the ion-pairing technique. An aqueous mobile phase containing 100 mM KH₂PO₄ and 12.5 mM tetramethylammonium hydroxide as the solvophobic ion, pH 3.9, was used with a reverse phase RP-18 column. The nine monoribonucleotides studied were separated and eluted in the following order: cytidine-5′ -phosphate, uridine-5′ -phosphate, cytidine-3′ -phosphate, guanosine-5′ -phosphate, uridine-3′ -phosphate, uridine-2′ -phosphate, adenosine-5′ -phosphate, guanosine-3′ -phosphate, and adenosine-3′ -phosphate. Generally the 5′ nucleotides eluted faster than the 3′ and the order of elution within each series was: cytidine, uridine, guanosine, and adenosine. The only nucleotide where three isomers were studied was uridine, and the 2′ eluted later than the 3′. Baseline separation was attained for a mixture containing four 3′ nucleotides and uridine-2′ -phosphate. When the four 5′ nucleotides were chromatographed, baseline separation was also obtained except between cytidine-5′ -phosphate and uridine-5′ -phosphate. The coefficient of variation of the retention characteristics, which reflected day-to-day variation, averaged 6.4%.