Effect of first-dimension column film thickness on comprehensive two-dimensional gas chromatographic separation

In comprehensive two-dimensional gas chromatography (GC x GC), samples experience two-dimensional separation implemented by a modulator which helps preserve the first-dimension separation and facilitates the second-dimension separation by periodically collecting, focusing and launching the material from the primary column onto the secondary column with a different stationary phase. Column overloading in GC x GC is a considerable problem, aggravated by the fact that two columns are involved. Broad first-dimension peaks of an analyte help produce smaller fractions of the analyte in the second-dimension, reducing the chance of secondary column overloading. One of the means to generate broad peaks in the first-dimension is to use thick film primary columns. A series of primary columns of various film thickness were tested in the study, and the results indicate that when other conditions are kept constant, 1 microm film columns often provide better resolution in both first and second-dimension but at the expense of a much longer separation time; 0.1 microm is clearly inadequate for GC x GC separation; 0.5 and 0.25 microm film columns seem to be the best compromises.     

Overloading of the second-dimension column in comprehensive two-dimensional gas chromatography

Comprehensive two-dimensional gas chromatography (GC x GC) is based on a coupling of two GC columns of different characteristics by means of a device that allows portions of the effluent from the primary column to be injected onto the second dimension column for an additional separation. The time available for the separation in the second-dimension column is very short. Thus, this separation should be very efficient. The vast majority of GC x GC practitioners use very narrow bore columns for the second dimension. While this approach is justified in principle, if peaks in the second dimension overload this column, its peak capacity is severely reduced. A series of second-dimension columns of varying internal diameters, but similar phase ratios, were used to study these effects. The results indicate that 250 microm columns often provide comparable second dimension peak widths to 100 microm columns, while at the same time being less prone to overloading, indicating that they may often be a better choice than smaller diameter columns in the second dimension of GC x GC systems.     

利用超载洗脱技术在TSK Gel CM-5PW分析柱上进行溶菌酶百毫克级制备

本实验采用非线性色谱的展开方式之一-超载洗脱, 在普通的分析型离子交换柱TSK Gel-5PW (Φ7.5×75mm)上, 一次进样蛋白质混合样150mg, 成功地进行了溶菌酶的分离纯化, 回收率达90%。收集的馏份经透析和冰冻干燥后,通过高效毛细管电泳(HPCE)测定纯度, 得到了满意的结果, 活性回收率达88%。     

Preparation of a super‐long two column chromatography system and its application in separating glycosylated puerarin

Separation of Puerarin‐7‐O‐glucoside from its precursor, puerarin, using a common chromatography column packed with AB‐8 macroporous resin was unsuccessful. Therefore, in this study a 8 m super‐long flexible reinforced PVC column was externally added to the common column in order to improve the chromatography efficiency by increasing the number of theoretical plates. Both the PVC and common columns were separately packed with AB‐8 macroporous resin slurry. The packed PVC column was coiled after washing and stored until use. The microbial transformation mixture with puerarin‐7‐O‐glucoside and puerarin (250 mL) was loaded onto the common column, followed by washing with 2000 mL H₂O. After attaching the coiled external PVC column to the common column, a linear gradient of 10–30% ethanol was applied to elute the target compound. Two peaks appeared: peak I contained puerarin‐7‐O‐glucoside at 97.9% purity and 88.1% recovery rate, and peak II was puerarin at 98.7% purity and 87.0% recovery rate. The use of the coiled external flexible reinforced PVC column avoided spatial restriction for long columns, which made it much more convenient for column packing and chromatography operations. Furthermore, this method eliminated the resin blockage problem caused by stationary water pressure in a rigid vertical long column. Using an external super‐long column, the PVC tube was connected with the common column only during elution, which avoided delay in time period during sample loading and column washes associated with the use of long external columns. Copyright © 2009 John Wiley & Sons, Ltd.     

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using middle chromatogram isolated gel column coupled with positively charged reversed‐phase liquid chromatography

Positively charged reversed‐phase liquid chromatography was employed for the efficient preparative separation of isoquinoline alkaloids from Corydalis impatiens. Ten commercially available columns were compared for isoquinoline alkaloids analysis. While tailing, overloading, lower resolution, and buffer salts limited the application in purification of isoquinoline compounds of many of these columns, one positively charged reversed‐phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading isoquinoline compounds on a larger, preparative scale. The general separation process is as follows. First, isoquinoline alkaloids are enriched with Corydalis impatiens extract via a middle chromatogram isolated gel column. After column selection, separation is performed on an XCharge C18 analytical column, from which two evident chromatographic peaks are readily obtained. Finally, two isoquinoline alkaloids (protopine and corydamine) are selectively purified on the XCharge C18 preparative column. These results demonstrate that a middle chromatogram isolated gel column coupled with positively charged reversed‐phase liquid chromatography is effective for the preparative separation of isoquinoline alkaloids from Corydalis impatiens.     

色谱柱分类数据库用于指导天然药物化学对照品色谱纯度测定时色谱柱理性选择

由于中药化学对照品多数来源于动植物药材,很容易混有结构类似物,故有机杂质测定是可能影响其化学对照品赋值准确性的关键风险因素。中药化学对照品的有机杂质测定通常采用药典收载或文献报道的高效液相色谱法,这些方法通常仅规定“以十八烷基硅烷键合硅胶为填充剂”,无适宜色谱柱的品牌信息,或者实验室无文献所用的色谱柱品牌,而目前市场上已有800多种品牌的C₁₈柱,生产工艺的不同导致不同品牌C₁₈柱的选择性有差异,甚至差异显著。这很容易出现由于色谱柱选择不适宜而导致测定结果不准确的风险。该文采用国外色谱柱分类数据库指导对照品纯度考察时色谱柱的理性选择,尽可能减少色谱柱盲选可能导致的纯度结果不准确的风险。首先,用数据库挑选2根选择性差异显著的色谱柱（选择性因子F≥6）进行平行实验,以尽可能反映采用不同品牌色谱柱可能出现的分离效果差异。如果这2根色谱柱的分离效果及纯度测定结果无显著性差异,则可以交叉验证该对照品纯度测定的准确性。否则需要从数据库中选择另外1根与之前试验中分离效果更好、选择性相似的色谱柱进行纯度结果验证。在N-反式-p-对香豆酰基酪胺和表儿茶素没食子酸酯首批对照品的纯度考察中,使用了上述策略并验证了其有效性和科学性,计划推广应用至更多的中药化学对照品,特别当其可能含碱性或弱酸性化合物时,更应该尝试采用本文推荐的色谱柱选择策略交叉验证其纯度测定结果的准确性。     

Packings and stationary phases in preparative column liquid chromatography

Although the theoretical treatment of chromatographic processes on a preparative scale provides guidelines to the extent to which packing and stationary phase properties affect the target quantities such as sample input, throughput and resolution times sample input, a series of additional criteria were established to judge the quality of a packing in preparative column liquid chromatography. These include bed stability and flow resistance, chemical resistance and purity, solute accessibility, mass and biological recovery, fouling, regeneration and cost. Applying these criteria, the relative importance of physical and chemical structure parameters of packings and stationary phases was assessed. Commercial packings with mean particle diameters dp greater than 20 micron were listed for adsorption, size exclusion, ion-exchange and affinity chromatography. An analysis of the characteristic features of phase systems showed that adsorption media offer a high selectivity combined with adequate loadability, whereas ion exchangers and affinity media were best suited for biospecific solutes, particularly biopolymers, which can be attributed to their high selectivity and loadability.     

Preparative isolation of arylbutanoid‐type phenol [(‐)‐rhododendrin] with peak tailing on conventional C18 column using middle chromatogram isolated gel column coupled with reversed‐phase liquid chromatography

Reversed‐phase liquid chromatography coupled with middle chromatogram isolated gel column was employed for the efficient preparative separation of the arylbutanoid‐type phenol [(‐)‐rhododendrin] from Saxifraga tangutica. Universal C18 (XTerra C18) and XCharge C18 columns were compared for (‐)‐rhododendrin fraction analysis and preparation. Although tailing and overloading occurred on the XTerra C18 column, the positively charged reversed‐phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading at a on a preparative scale (3.69 mg per injection). The general separation process was as follows. First, 365.0 mg of crude (‐)‐rhododendrin was enriched from 165 g Saxifraga tangutica extract via a middle chromatogram isolated gel column. Second, separation was performed on an XTerra C18 preparative column, from which 73.8 mg of the target fraction was easily obtained. Finally, the 24.0 mg tailing peak of (‐)‐rhododendrin on XTerra C18 column was selectively purified on the XCharge C18 analytical column. These results demonstrate that the tailing nonalkaloid peaks can be effectively used for preparative isolation on XCharge C18 columns.     

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.     

Self-disproportionation of enantiomers of prazoles via achiral,gravity-driven silica gel column chromatography

(R)-Lansoprazole, (S)-pantoprazole, and (R)-rabeprazole are commonly used drugs for peptic ulcers. The self-disproportionation of the enantiomers of these prazoles in regular column chromatography is described herein. When non-racemic mixtures of the prazoles were eluted under achiral, gravity-driven silica gel column chromatography conditions, enantiomeric enrichment occurred in the first fractions, while enantiomeric depletion occurred in the last fractions. The pure enantiomers of all prazoles can be prepared from non-racemic starting materials using a simple procedure (achiral chromatography). Thus, a new method for obtaining a sample with very high enantiomeric purity was established. These results suggested that caution is required to avoid enantiomer fractionation during the purification of the asymmetric synthesis products by chromatography.     

High-performance liquid chromatography of carbapenem antibiotics in complex biological samples with column switching and simultaneous multichannel ultraviolet monitoring

During fermentative production of new carbapenem compounds of the OA-6129 type, many interfering by-products, exhibiting physicochemical properties very similar to the main components, are produced. The compounds of interest, especially OA-6129 A, B1, B2 and C, are difficult to quantify because of the complex biological matrix and the broad spectrum of by-products. High-performance liquid chromatography (h.p.l.c.) is used to quantify OA-6129 B2. In order to assign the chromatographic peaks clearly to the wanted substances, h.p.l.c., t.l.c, and a selective chemical degradation method proved to be necessary. This procedure was considerably simplified by combined use of a column-switching technique and a diode-array detector. The polar by-products and much of the interfering matrix were eliminated by column switching. Positive identification of OA-6129 B2 was feasible by monitoring chromatograms simultaneously at different wavelengths and by use of the diode-array detector. Additional information on uniformity and purity of the peaks was obtained by u.v.-absorbance ratio recording.     

Preparative anion-exchange chromatography of soybean trypsin inhibitor: the alternative of column-overload methods.

The objective of preparative separation is to purify the largest amount of material in the shortest time and at a minimum cost, i.e. to maximize throughout. One of the techniques for increasing throughput is to overload the column while maintaining purity and cycle time at the same level. This principle is applied in sample displacement mode chromatography, in which the column is overloaded with sample mixture until it is completely saturated. Soybean trypsin inhibitor was purified from a crude protein extract by this technique using an analytical anion-exchange column with small particle size (20 microns). The comparison of these results, using the criterion of throughput, with those derived from a conventional scale-up, using a 40-microns preparative column, led to the conclusion that the overloaded 20 microns column gave a higher throughput than the 40-microns column.     

Gas chromatographic determination of galactose in milk. Example of a switching valve used for the protection of the capillary column.

Galactose, a marker of heat treatment, has been analysed in milk as pentafluorobenzyloxime acetate by gas chromatography with flame ionization detection using a simple switching valve system. The procedure did not entail any prederivatization clean-up for lactose elimination from the sample. In a short pre-column, reagent and lactose derivative excess were separated and the galactose and internal standard derivatives were transferred to the analytical column by a four-port valve. Thus, the analytical column was protected from overloading, so avoiding rapid deterioration.     

IEX/RP二维液相色谱中弱保留组分收集模式的构建及系统评价

以十通阀和捕集柱接口形式,构建了弱阴离子交换/反相(WAX/RP)二维液相色谱分离系统.通过将第一维离子交换色谱分析中的前部集中洗脱出的弱保留组分收集后单独分析,剩余样品进一步采用二维液相色谱分析,可以有效避免第二维色谱柱对特殊样品局部集中流出导致的第二维分离超柱容量问题,提高了系统的整体分离能力.使用4种蛋白胰蛋白酶酶解后的多肽样品评价该系统,在不分流的系统中共检测到115个峰.对第一维分离的前15 min分流后得到的组分单独分析,共识别出58个峰,后续的二维分离中共得到78个峰.2种方法相比,第二种方法检测峰数增加了21个,一些低丰度的组分在弱保留组分收集后被识别.     

Semi-automated peak trapping recycle chromatography instrument for peak purity investigations

A peak trapping recycle chromatography system has been developed and optimized for peak purity assessment of active pharmaceutical ingredients analyzed by high performance liquid chromatography (HPLC). After being analyzed using a reversed phase analytical column, peaks of interest are trapped and are subsequently introduced to a recycle chromatography system. In addition to the increased effective length afforded the recycling system, the small selectivity difference between the analytical and recycling methods help separate potential impurities under the main peak. For more difficult to separate components, the increased efficiency of recycle chromatography provides the necessary resolution. Over 227,000 theoretical plates have been obtained in the recycle dimension for some compounds. The sensitivity of the system fell short of the target (0.1%), but it did show sensitivity (0.5%) comparable to other peak purity techniques commonly used in the pharmaceutical industry. The recovery and repeatability have also been shown to be adequate for peak purity assessment. The system has also been automated using a Visual Basic macro, simplifying the interface allowing it to be used as an open access instrument.     

Effects of large sample loads on column lifetime in preparative-scale liquid chromatography

In preparative-scale liquid chromatography of proteins, the use of high sample concentration and large sample mass may result in irreversible adsorption to the support surface. This can change the stationary phase characteristics, reduce the capacity, shorten the column lifetime and diminish the economic viability of a particular separation method. Column recycling and regeneration can influence the throughput (mass purified per time unit) and selectivity, and affect the reproducibility. The effects of large sample loads on column lifetime and performance were evaluated for three strong anion-exchange columns: (1) a silica support with a quaternized polyethyleneimine (PEI) coating, (2) a polymeric support with an adsorbed PEI coating which also was quaternized, and (3) a polymeric support with a proprietary quaternary amine stationary phase. The column capacity for proteins was measured by frontal chromatography and monitored as a function of cycle number. The column lifetime was determined by examining chromatographic properties subsequent to the frontal chromatography. The change in protein binding capacity was then compared to the change in nitrate binding capacity. The column performance was evaluated under analytical conditions by measuring the change in resolution of standard protein mixtures.     

Optimization of throughput and desorbent consumption in simulated moving-bed chromatography for paclitaxel purification.

In simulated moving-bed (SMB) applications, throughput and desorbent consumption are two key factors that control process cost. For a given adsorbent volume and product purity requirements, throughput and desorbent consumption depend on desorbent composition, column configuration, column length to diameter ratio, and adsorbent particle size. In this study, these design parameters are systematically examined for paclitaxel purification. The results show that if adsorbent particle size, column dimensions and column configuration are fixed, the higher the product purity required, the lower the throughput. If product purity and yield are fixed, the larger the solute migration speed ratio, the higher the throughput, and the lower the desorbent consumption. If total bed volume and product purities are fixed, the longer the separation zones, the higher the throughput, but the higher the desorbent flow-rate. An intermediate configuration gives the minimum desorbent consumption. If there are no limits on pressure drop or zone flow-rate, the larger the column length to diameter ratio, the smaller the adsorbent particle size, the higher the throughput, and the lower the desorbent consumption. If the maximum zone flow-rate is controlled by the pressure drop limit and not by the standing waves requirement, the longer the columns, the lower the zone flow-rates and the lower the throughput. For 150 microns adsorbent particles and a maximum zone flow-rate of 300 ml/min, a design with optimal throughput and desorbent consumption is found for paclitaxel purification.     

Semipreparative enantiomeric separation of omeprazole by supercritical fluid chromatography

Omeprazole, a widely used antiulcer drug, has been enantiomerically separated at semipreparative scale on a polysaccharide based chiral stationary phase by supercritical fluid chromatography (SFC). For this work, a modular supercritical fluid chromatograph was adapted to operate at semipreparative scale and a Chiralpak AD (250 mm x 10 mm) column was used. The effect of two organic modifiers (ethanol and 2-propanol) was studied, and different injection volumes and concentrations of the omeprazole racemic mixture were evaluated in order to obtain high enantiomeric purities and production rates. Better results were achieved using concentration overloading instead of volume overloading. The recoveries decreased when the requirements of enantiomeric purity or the load increased, but it was possible to recover 100% of both enantiomers at an enantiomeric purity higher than 99.9% under some loading conditions, like injecting 1 and 2 ml of a solution of 3g/l. As far as production rates are concerned, the best result for S-(-)-omeprazole at that purity (27.2mg/h) was achieved with sample concentrations of 10 g/l and the injection of 2 ml, while a volume of 4 ml was better in the case of R-(+)-omeprazole (20.5mg/h).     

Displacement chromatography of biomolecules

Displacement chromatography was used for the preparative-scale separation of peptides, antibiotics, and proteins. The feed components were both purified and concentrated during the separation processes. The components of a peptide mixture were separated on a reverse-phase analytical column using 2-(2-butoxyethoxy) ethanol as the displacer. The use of organic modifiers in the carrier along with an elevated column temperature of 45 degrees C enabled the efficient separation of relatively hydrophobic peptides by displacement chromatography. In addition, the throughput of the process was significantly increased by carrying out the separation at an elevated flow-rate with no adverse effect on product purity. The antibiotic cephalosporin C was isolated from impurities in a fermentation broth using 2-(2-butoxyethoxy)ethanol as the displacer along with a step change in column temperature. The proteins cytochrome c and lysozyme were purified on a weak cation-exchanger column using cationic polymers as the displacers. While polymers of 60 and 20 kilodaltons were both found to be good displacers for these proteins, only the lower molecular weight polymer was readily removed from the column by standard regeneration techniques.     

Purification of secoisolariciresinol diglucoside with column chromatography on a sephadex LH-20

A simplified and efficient method is developed for the large-scale purification of the secoisolariciresinol diglucoside (SDG) from defatted flaxseed after aqueous ethanol extraction. Extractant from defatted flaxseed with aqueous ethanol is hydrolyzed with basic solution, concentrated under vacuum, subjected to Sephadex LH-20 column chromatography, and eluted with aqueous ethanol of different concentrations. Elution is monitored by a UV detector at 280 nm, and fractions containing SDG are pooled, concentrated, and applied to a second column chromatography under the same conditions. Elution with water results in a better resolution of SDG [94.5% by high-performance liquid chromatography (HPLC)] than that with pure ethanol or 50% (v/v) aqueous ethanol. HPLC-photodiode array detection-mass spectrometry and NMR are applied to identify SDG and to determine the purity of the eluted fraction. This simplified purification scheme avoids toxic organic solvent used in the common silica gel separation process and, thus, increases the safety of the process.