Characterization of High‐Pressure Liquid Chromatography Columns using Chromatographic Methods

Abstract

A great variety of columns for liquid chromatography (LC) are available in dimensions ranging from industrial scale to micro‐bore, nano‐bore, and capillary size, and on‐chip columns. The columns may be used in various liquid chromatography modes or in capillary electrochromatography, depending on the support materials and stationary phase chemistry. Every year many new column types are introduced on the market, with improved selectivity and efficiency, long lifetime, and mobile phase compatibility, intended for general use, for liquid chromatography/mass spectrometry (LC/MS) applications, proteomic research, or for the analysis of other specific sample types. Considerable improvement in pH, high‐temperature, and high‐pressure stability of new column types, together with advances in the instrumentation, enabled introduction of capillary, high‐temperature, and ultra‐high‐pressure HPLC into routine practice. Even though reversed‐phase mode is still by the most widely used in contemporary LC, applications of other separation modes (such as ion, normal‐phase, or high‐interaction liquid chromatography (HILC)) have become more frequent recently, because of unique separation selectivity for certain sample types.

Characterization of column quality is not a simple task, because a number of factors should be taken into account, that affect the selectivity, efficiency and resolution of sample separation and the reproducibility of chromatographic data. These include the type of the support, the arrangement and density of the stationary phase on the adsorbent surface, the homogeneity of the chromatographic bed, etc. Various physicochemical techniques are used for characterization of the properties of column packings however, most of them are suitable for bulk materials only and cannot be directly applied for commercial columns without damaging them. Not to destroy the columns, often precious and expensive, practicing chromatographers can apply chromatographic methods to characterize columns and evaluate their analytical suitability under real‐life conditions, where the intermolecular interactions between the analytes, the stationary phase, and the mobile phases affect the retention. The present review reports various chromatographic tests and strategies available for column evaluation.     

Gas chromatography of hydrocarbons using packed liquid chromatographic capillary columns

Capillary columns of 0.3-0.5 mm i.d. packed with 3- to 30-μm silica-based stationary phases for liquid chromatography were used for gas chromatographic separation of hydrocarbons. Column efficiencies were evaluated for various commercially available packing material. The best column efficiency was achieved with 5-μm octadecyl group bonded silica gel, the surface of which was coated with a poly (dimethylsiloxane) film. The 30-cm column produced 11,000 theoretical plates.     

Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 μm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 μm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 μm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 μm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 μm average particle size materials for clinical sample analysis.     

Effect of buffer on peak shape of peptides in reversed-phase high performance liquid chromatography

The effect of changing the buffer at constant low pH in the mobile phase is investigated with respect to the separation of a mixture of basic peptides. Considerably worse peak shapes, leading to poorer resolution of complex peptide mixtures, were obtained when using formic acid favoured in LC-MS applications compared with non volatile phosphate buffers or with trifluoroacetic acid (TFA). Poorer peak shapes were largely attributable to reduced column capacity for the peptides when using mobile phases of low ionic strength, due to the increased mutual repulsion of ions held on the hydrophobic column surface which is facilitated in these buffers. However, ion-pairing between the peptides and additives such as TFA or even phosphate may also lessen mutual repulsion effects, leading to greater column capacity. Overloading effects could be observed when sample masses around only 0.1 microg were injected on to standard size analytical columns in formic acid containing mobile phases; sample masses around only 1.5 microg may cause loss of half the system peak capacity in such mobile phases. Results were broadly comparable (after scaling sample size according to column diameter) on columns of both conventional (4.6 mm i.d.) and capillary (0.075 mm i.d.) dimensions. Ammonium formate may be a useful alternative buffer for some applications due to its higher ionic strength.     

二维液相色谱接口的改进及其在蛋白质组学研究中的应用

随着蛋白质组学、本草物质组学等组学概念的提出,所需分析的样品的成分越来越复杂,因此具有强大分离能力的多维液相色谱技术受到人们越来越多的关注。二维液相色谱中第二维的分离性能和速度是整个分离系统性能的关键。基于捕集柱模式,我们采用经特殊设计的流路系统,使得双捕集柱型接口具有预分离的功能。样品从第一维流出以后被富集在捕集柱1的柱头,经过脱盐后,正冲捕集柱,捕集柱1与第二维色谱柱联用对富集的样品进行分离,增加了第二维分离效率。当捕集柱上的样品全部被洗脱到第二维色谱柱上时,捕集柱2已经完成对第一维洗脱液中样品的捕集和脱盐,此时将阀进行切换,捕集柱2与第二维色谱柱直接相连进行洗脱。循环切换捕集柱1和捕集柱2,维持较高的阀切换频率,实现了第二维色谱柱的连续洗脱。因此保证了第二维分离具有较快速度,同时具有较高的分离效率。使用35 mm长捕集柱和十通阀为接口,以弱阴离子交换(WAX)色谱为第一维分离模式,以反相(RP)色谱为第二维分离模式,构建了WAX-RP二维液相色谱系统(2D-LC system)。以小鼠血清为样品对系统进行了初步评价。色谱流出曲线出现了明显的界面现象,这是由于捕集柱流动相中含有的较多盐分流出时的背景吸收造成的。同时,由于界面两侧的流动相黏度不同产生了黏性指进(VF)现象。当第二维色谱柱长度为50 mm时,理论上可将第二维分离效能提高70%。该接口可以应用于多种二维液相色谱模式,适用于蛋白质组学和本草物质组学研究中对于复杂样品的分离分析。     

Twenty years of glass capillary columns. An empirical model for their preparation and properties

Both the invention of the capillary drawing machine and the preparation and successful use of the first glass capillary column were achieved by Desty and his group in 1959. This paper attempts to describe the glass capillary column in a general way, including the spreading of liquid phases of different polarity and solid surfaces with different chemical and geometrical structures, and surface activity (as a source of essential column characteristjcs such as film stability, adsorptive and catalytic activity, acidity, and thermostability). The model is entirely based on experimental evidence. It is shown that today's apolar columns may closely approach ideal performance, such as is conceivable at the present time, while a severe gap still exists between the actual and the ideal characteristics of polar columns. The problem lies in the necessity of using considerably active support surfaces for polar coatings, yielding columns with reduced upper temperature limits, and of restricted suitability concerning the nature of sample. It is hoped that progress in the field of surface roughening may produce an inert support which is wettable by polar phases.     

Porous structure of the monolithic sorbent and separation properties of monolithic capillary columns in gas and liquid chromatography

Macroporous polymer based on polydivinylbenzene was used for the preparation of monolithic capillary columns with the diameter from 0.01 to 0.53 mm for separations by gas and liquid chromatography. The separation properties of the columns were studied by analysis of model systems of aromatic (in liquid chromatography) and light (in gas chromatography) hydrocarbons. The permeability was determined and the C parameter of the Van-Deemter equation was found for each column. The permeability of the majority of columns determined by gas chromatography is independent of the column diameter. The permeability of the same columns in liquid chromatography is also almost constant for the columns 0.53–0.1 mm in diameter; however, the permeability decreases sharply on going to columns of smaller diameter. In gas chromatography the value of the C parameter reflecting the effect of the mass transfer of the sorbate between the mobile and stationary phases on the smearing of a chromatographic peak in the column approximately the same for all columns. In liquid chromatography the value of the C coefficient in the Van-Deemter equation for the same capillary columns changes with a change in the column diameter and reaches a minimum for the columns 0.1 mm in diameter. The differences observed for the characteristics of the columns in gas and liquid chromatography are due to different structures of the macroporous monolith formed in columns of different diameter and to the effect of solvation of the monolith by the mobile phase under the conditions of liquid chromatography.     

Effect of high-temperature on high-performance liquid chromatography column stability and performance under temperature-programmed conditions

Six commercially available analytical (4.1 or 4.6 mm i.d.) columns were evaluated under temperature-programmed high-temperature liquid chromatography (HTLC) conditions to access their stability and performance at extreme temperatures. Seven components consisting of acidic, basic and neutral compounds were analyzed under temperature-programmed conditions and solvent gradient conditions using three different mobile phase compositions (acidic, basic and neutral). Each column was checked with a two-component test mix at various stages of the evaluation to look for signs of stationary phase collapse. Three zirconia based stationary phases studied exhibited column bleed under temperature-programmed conditions. The other three columns, a polydentate silica column, a polystyrene-divinylbenzene (PS-DVB) polymeric column, and a graphitic carbon column performed well with no evidence of stationary phase degradation. The R.S.D. for the retention times and efficiencies were less than 10% for most conditions, and not more than 15% during the course of the evaluation for each column. The polydentate silica stationary phase was temperature programmed to 100 degrees C, the PS-DVB stationary phase was temperature programmed up to 150 degrees C, and the graphitic carbon column was used with temperature programming up to 200 degrees C. Comparable peak capacities and similar retention behaviors were observed under solvent gradient and temperature-programmed conditions. Temperature programming with dynamic mobile phase preheating can replace solvent gradient analysis without a loss of peak capacity when used with 4.1 or 4.6 mm columns.     

外标归一化气相法测定天然气组成

 对SP 6000天然气分析仪的气路系统作了改进,实现了仪器自身标定。采用填充柱和毛细管柱分离,热导检测器和火焰电离检测器双检测器检测,外标 归一化法定量,简易、快速地测定了天然气组成。方法的准确度及精密度均符合气相法定量分析的要求。     

Packing and performance of microbore columns for adsorption and partition chromatography

Summary Microbore columns of 1 mm i.d. have turned out to be very suitable for the achievement of efficient columns.The packing procedure for stainless steel 1 mm i.d. columns from 5 to 100 cm in length was studied. Stationary phases used were: pure silica gel, octyl, octadecyl and amino bonded silicas. The main parameters (slurry composition, packing system, choice of materials) are discussed.Short columns packed with 3 or 5m particles allow high speed separations. A separation in 18 seconds is described.Very high plate numbers can be obtained with long columns. With 7–8m particles, a 1 m column can produce 50,000 plates (h=3). Columns can be joined without loss of efficiency. 270 000 theoretical plates were obtained on a 6 m adsorption column with a test mixture. In reversed-phase chromatography, bile acid sodium salts can be separated on a 1 m column. In adsorption chromatography, details are given of the separation of a polystyrene oligomer sample, as well as a light and a heavy petroleum distillate samples on a 2 m column with refractive index detection in the last-case.     

High-performance liquid chromatographic enantioseparations on monolithic silica columns containing a covalently attached 3,5-dimethylphenylcarbamate derivative of cellulose

Covalent immobilization of 3,5-dimethylphenylcarbamate derivative of cellulose was performed in situ onto native silica monoliths cladded in a 50 mm x 4.6 mm polyether ether ketone high-performance liquid chromatographic (HPLC) column. The covalent attachment of cellulose derivative in the range of 16-19% (w/w) was performed via an epoxide moiety. The column obtained by this technique combines the high enantiomer-resolving ability of the polysaccharide derivative with favourable dynamic properties of monolithic HPLC columns. The covalent attachment of the cellulose derivative enables this column to be used in combination with the mobile phases which are incompatible with coated-type polysaccharide columns due to solubility of chiral selector in some organic solvents.     

Polyimide polymer glass-free capillary columns for gas chromatography

Polymeric polyimide capillary tubing, both uncoated and coated with stationary phases of two polarities, is explored for use as capillary columns for gas chromatography (GC). These glass-free polyimide columns are flexible and their small winding diameter of less than a cm around a solid support makes them compatible for potential use in portable GC instruments. Polyimide columns with dimensions of 0.32 mm i.d. × 3 m are cleaned, annealed at 300°C, and coated using the static method with phenylmethylsilicone (PMS). Separations of volatile organics are investigated isothermally on duplicate sets of polyimide columns by GC with a flame ionization detector using split injection. Unlike the uncoated ones, the coated polyimide columns successfully separate Grob test mix classes of alkanes, amines, and fatty acid methyl esters. The relative standard deviations for retention time and peak area are 0.5 and 2.5 , respectively. With the 3 m PMS-coated column connected to a retention gap to permit operation at its optimum flow rate of 30 cm/s, a plate count of 3200 or plate height of 1 mm is possible. Lack of retention and tailing peaks are evident for the polyimide polymer capillary columns as compared to that of a 3 m commercial cross-linked PMS fused silica capillary. However, headspace analyses of an aromatic hydrocarbon mix and a Clearcoat automotive paint sample are viable applications on the PMS polyimide polymer column.     

Application of micropellicular poly-styrene/divinylbenzene stationary phases for high-performance reversed-phase liquid chromatography electrospray-mass spectrometry of proteins and peptides

Summary Short columns packed with highly crosslinked 2.3 μm poly-styrene/divinylbenzene (PS/DVB) particles were used for rapid and efficient separation of proteins and peptides by reversed-phase high-performance liquid chromatography at elevated temperatures. Enhancement of the diffusivities of the sample components at elevated temperatures together with the short diffusion pathlength with the micropellicular polymeric stationary phases were responsible for high efficiency, high speed of analysis, and short column regeneration times. Underivatized PS/DVB beads as well as PS/DVB microspheres which have been modified with polyvinylalcohol or octadecyl chains on the surface were synthesized, employed, and compared to HY-TACH-C18, a commercially available micropellicular octadecyl-silica stationary phase, for the separation of proteins, octapeptides and tryptic protein digests. Highest performance was obtained with the silica- and PS/DVB-based octadecyl stationary phases, which exhibited similar column efficiencies but different selectivities for proteins and peptides. The minimum detectability at 214 nm and the maximum loading capacity for ribonuclease A using analytical 30×4.6 mm I.D. columns were 10 ng (0.6 pmol) and 1 μg, respectively. Finally, reversed-phase HPLC with a 60×2 mm I.D. narrow-bore column packed with micropellicular octadecyl PS/DVB was coupled successfully to electrospray mass spectrometry at a flow-rate of 0.15 mL min⁻¹ and on-line full-scan mass spectra for molecular mass determination and identification of proteins in the lower picomol range were obtained.     

A comprehensive two-dimensional normal-phase × reversed-phase liquid chromatography based on the modification of mobile phases

A comprehensive orthogonal two-dimensional liquid chromatography (2D-LC) based on the modification of mobile phases was developed with a sample loop–valve interface. To improve the compatibility of mobile phases and analysis speed, some special solvents were chosen as the mobile phases, and the column temperature was elevated to decrease the viscosity of mobile phases of reversed-phase liquid chromatography (RPLC). Based on this principle, the first dimension was normal-phase liquid chromatography (NPLC) with a SiO₂ column, and the second dimension was reversed-phase liquid chromatography containing two tandem C18 columns. 1,4-Dioxane was used in the NPLC mobile phase, and isopropyl alcohol was employed in the RPLC mobile phase. Moreover, the elevated column temperature enabled the reduction of the backpressure and using tandem C18 columns to improve the resolving power in RPLC. The new comprehensive 2D-LC system and applied strategy offered a novel idea for construction of 2D-LC system. A traditional Chinese medicine, Zhengtian pill, was used as the test sample to evaluate the constructed 2D-LC system. 876 peaks were detected, and the peak capacity reached 1740.     

Rapid and high resolution capillary supercritical fluid chromatography (SFC) and SFC/MS of trichothecene mycotoxins

The potential application of capillary column supercritical fluid chromatography (SFC) and SFC/mass spectrometry (SFC/MS) for the separation and analysis of mycotoxins of the trichothecene group was examined. Trichothecenes present significant analytical problems for both gas and liquid chromatography with a major difficulty for the latter being the lack of sufficiently sensitive and selective detectors. Supercritical carbon dioxide mobile phases at temperatures up to 100 degrees C were used with deactivated fused silica columns coated with crosslinked stationary phases. Separations were obtained under pressure ramped conditions using long (15 m) 50-micron i.d. columns for several trichothecenes (diacetoxyscirpenol, deoxynivalenol, and T-2 toxin) and related higher molecular weight macrocyclic (roridin and verrucarin) trichothecenes. In addition, new rapid pressure programming techniques with short (less than 2m) 25- to 50-micron i.d. capillary columns were used to obtain fast separations in as little as 1 min. SFC/MS with ammonia chemical ionization provided high selectivity and sensitive detection (with approximately 1-pg detection limits) for trichothecene mixtures. The extension to complex sample matrices is discussed and the application of selective MS/MS detection is demonstrated.     

不锈钢宽口径填充毛细管液相色谱柱

 设计了一种零死体积的二通式柱尾结构和一种使匀浆填料均匀进入色谱柱管内的储料池 ,研究了制备内径在 0 5mm～ 1 0mm的不锈钢宽口径填充毛细管液相色谱柱的方法。详述了以不同牌号、规格的反相ODS类固定相制备的不同柱长的色谱柱的性能。通过折合板高 /折合流速关系和不对称因子对柱性能进行了评价 ,结果表明 ,该方法制备的色谱柱柱效达到理论值的 75 %以上、RSD为 6% ,稳定性也很好。将其应用于抗癫痫药物和氯苯类化合物的分析 ,结果令人满意。     

Amberlite XAD-4 as a Stationary Phase for Preparative Liquid Chromatography in a Radially Compressed Column

Abstract

Amberlite XAD-4, a nonpolar adsorbent with a particle size of 62 to 177 μm, was packed into 58 mm i.d. x 294 mm columns and radially compressed in the Waters Prep 500 preparative liquid chromatograph. Efficiency, loading capacity (multigram samples), resolution, recovery, and types of mobile phases were major parameters studied. Since XAD-4 is chemically inert mixed solvent and pH control from 1 to 13 can be successfully used in the mobile phase. Separations illustrating these advantages and the scope of the 58 mm i.d. column are described.     

Toward rapid preparation of capillary columns for electrochromatography use

CEC is a high performance electrodriving liquid phase separation technique. It does not need complex and sophisticated high pressure instrumentation for nanoflow driving. This is attractive for parallel multicolumn analysis. To this end, high throughput methods for column preparation are needed to support the use of multiple columns. In this study, we directly used CEC mobile phase solution as the packing solvent, and realized rapid preparation of capillary columns based on a single particle fritting technology. The method presented high preparation throughput compared with other reported methods based on various fritting technologies. The single particle fritting approach promoted column preparation throughput to 1 column/h, including all the fritting, packing and conditioning steps. The rapidly prepared columns showed consistently high efficiency of up to 150 000 plates per meter, and usefulness in reversed phase CEC of neutral, charged and biomolecules. With standard peptides as the sample, excellent long term reproducibility (better than 0.8%RSD, ten days, for retention times) was observed.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.     

台锥形柱尾的填充毛细管液相色谱柱

首次将台锥形柱尾用于填充毛细管液相色谱柱,并与环氧胶粘的柱尾连接方式进行比较,发现在柱内径≥０．３２ｍｍ时,前者的柱效比后者高出３０％～５０％,且明显改善柱效－流速特性。对比研究了不同内径柱（０．５３ｍｍ,０．３２ｍｍ,０．２５ｍｍ）采用两种尾接方式的塔板高度与流速的关系,对产生差异的原因进行了探讨。