Preparative reversed-phase liquid chromatography of peptides. Isocratic two-step elution system for high loads on analytical columns

We have developed further our novel sample displacement chromatography (SDC) methodology to carry out preparative separations on analytical equipment and 15-cm analytical columns for sample loads < or = 200 mg. Thus, a two-step isocratic SDC protocol was developed and applied to the purification of important biologically active peptides, i.e. bradykinin antagonists of 10 and 11 residues. Following sample loading in 100% aqueous solvent at a concentration of approximately 7-10 mg/ml (with sample loads varying from 67 to 200 mg) onto a small C18 column (150 x 4.6 mm I.D., made up of three 50-mm columns attached in series), we applied isocratic elution with aqueous acetonitrile at two concentrations, the first (lower concentration) to displace hydrophilic impurities off the column and the second (higher concentration) to displace pure product from the column; hydrophobic impurities remain trapped on the column. This modified SDC approach promises to allow great flexibility in purifying peptides, at high yield of pure product (> 99% purity), and encompassing a range of sample hydrophobicities as well as sample loads (< or = 200 mg) varying by as much as a factor of three.     

酪蛋白多肽的制备和色谱分离方法

为了得到低成本的多肽,本文利用胰蛋白酶对酪蛋白进行了充分的酶解。采用分析级反相高效液相色谱-电喷雾质谱联用技术(RP-HPLC/ESI-MS)分析了酶解产物各组分的组成,并通过改变流动相的梯度洗脱程序,优化了分析级色谱条件以充分分离相对含量较高的多肽组分;将优化的分析级色谱条件直接放大到制备级RP-HPLC中,在程序控制下通过紫外吸收信号结合ESI-MS信号共同引导实现了多肽的全自动化分离和收集。整个过程方便快捷,经过这样一个单一的分离步骤,得到了多个纯度较高的多肽。除此之外,本文还考察了流动相的酸碱性、柱上样量等因素对该体系制备级分离的影响,并对一次分离中分辨率不好的亲水性多肽混合物进行了二次分离,得到了多个新的多肽。本文建立的多肽制备方法为多肽和多肽材料的广泛应用提供了一种选择。     

A Novel Approach to Reversed-Phase Preparative High-Performance Liquid Chromatography of Peptides

Abstract

In this study, we describe a novel approach to preparative liquid chromatography which takes advantage of the different relative hydrophobicities of components of a sample mixture, so that when a column is optimally loaded with an aqueous solution of the sample mixture, there is competition among the sample components for the adsorption sites on the hydrophobic stationary phase. The more hydrophobic components compete more successfully for these sites than more hydrophilic components, which are displaced and immediately eluted from the column. Thus, the major separation takes place in water. Subsequent treatment with an aqueous organic modifier is only required to wash retained components off the column and takes no part in the major separation process. This approach was applied to the preparative purification of mixtures of closely-related peptides, representing the crude peptide mixtures typically obtained from solid-phase peptide synthesis. The excellent separation profiles and high yields of pure peptide products on analytical columns reported in this study demonstrate that this methodology has great potential for preparative separation of a major component from hydrophilic and/or hydrophobic impurities.     

Peptide separations using fluorescence detection.

The formation of fluorophores by the action of o-phthalaldehyde with amino acids and peptides has provided a highly sensitive assay for these compounds. A relatively simple system for the analysis and separation of peptides, in the range 5 nmole to 10 micromole, normally derived from enzymic digestion of proteins, is described. The system comprises a gradient-generating device feeding volatile pyridine buffers via a pump to a column of cation-exchange resin. Eluate from the column is fed through a proportioning pump to a fluorocolorimeter, output from which is displayed on a recorder. For analytical runs the eluate is mixed with o-phthalaldehyde in borate buffer containing Brij 35 and 2-mercaptoethanol prior to its passage into the detector. For preparative work the eluate stream is split, one reacting with 0-phthalaldehyde, the other for collection. Results on the analysis and preparation of tryptic peptides derived from cytochrome c and Salmonella histidinol dehydrogenase are discussed.     

High-Performance Hydrophobic Interaction Chromatography of Proteins on TSKgel Phenyl-5PW Preparative Column

Abstract

TSKgel Phenyl-5PW preparative column of 200 × 55 mm I.D. was evaluated with respect to resolution, sample loading capacity and applications to the purification of enzymes. The preparative column provided similar separations as analytical column (75 × 7.5 mm I.D.) and 150 × 21.5 mm I.D. preparative column. The sample loading capacity was 200 – 1000 mg depending on the sample. If the slight decrease in resolution is acceptable, much more samples could be applied. Lipoxidase, phosphoglucose isomerase and lactate dehydrogenase could be purified to a great extent with high recovery of activity (more than 80%).     

Appropriate column configurations for the rapid analysis and semipreparative purification of the radiolabeled drug flutamide by high-performance liquid chromatography

Flutamide, marketed as Eulexin, is used for treatment of metastic prostatic carcinoma. Purity of a radiolabeled batch for metabolism studies was first determined by reversed-phase HPLC on a 5 microm, 150x4.6 mm analytical column. The separation was then scaled up to give a semipreparative column (5 microm, 250x10 mm) purification procedure. Fraction analysis was done on a short rapid analysis (5 microm, 50x3.0 mm) column. Analysis of the final product was performed on the analytical column. All columns were YMC-Pack ODS-AQ. The analytical work involved large mass injections in order to have the required amounts of radioactivity needed for accurate impurity profile determinations, and the preparative work involved masses much larger than the calculated scale-up values. Ultraviolet and radiochromatograms of the drug on the various column configurations are compared. A 95.7% recovery of product was obtained, with radiochemical purity increased from 95.0 to 99.8%.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.     

Application of preparative liquid chromatography to the isolation of enantiomers of a benzodiazepinone derivative.

The isolation of milligram amounts of the enantiomers of a benzodiazepinone derivative was performed on an analytical cellulose tribenzoate-based column by multiple repetitive injections. An enantiomeric purity greater than 98% was required. First, an analytical method was developed to maximize the resolution by adjusting the mobile phase composition, flow-rate and most importantly the column temperature. Then the preparative separation was optimized by adjusting the sample size and detecting the sample where its UV absorbance was low. The locations of the cut points were determined by use of detector response levels. The method development, preparative separations and analytical assays of the fractions obtained were all performed on analytical columns.     

Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1mm to 9.4mm I.D.)

The present study examines the effect of reversed-phase high-performance liquid chromatography (RP-HPLC) column diameter (1mm to 9.4mm I.D.) on the one-step slow gradient preparative purification of a 26-residue synthetic antimicrobial peptide. When taken together, the semi-preparative column (9.4mm I.D.) provided the highest yields of purified product (an average of 90.7% recovery from hydrophilic and hydrophobic impurities) over a wide range of sample load (0.75-200mg). Columns with smaller diameters, such as narrowbore columns (150x2.1mm I.D.) and microbore columns (150x1.0mm I.D.), can be employed to purify peptides with reasonable recovery of purified product but the range of the crude peptide that can be applied to the column is limited. In addition, the smaller diameter columns require more extensive fraction analysis to locate the fractions of pure product than the larger diameter column with the same load. Our results show the excellent potential of the one-step slow gradient preparative protocol as a universal method for purification of synthetic peptides.     

Purification of Solid-Phase Synthesized Peptides on the Coil Planet Centrifuge

Abstract

The analytical flow-through coil planet centrifuge, an instrument for countercurrent chromatography, performs the preparative purification of synthetic peptides. Various two-phase solvent systems have been tried with either phase mobile to purify many synthesized peptides. A series of N-terminal fragment peptides of cholecystokinin octapeptide (CCK 26–33) were synthesized by solid-phase techniques and purified on the coil planet centrifuge. The peptides were sulfated and chromatographed again. For hydrophobic peptides, purification is effected in solvent systems with a mobile aqueous phase. The n-butanol, acetic acid and water system (4:1:5 by volume) with the lower phase mobile was utilized. For sulfated peptides, the neutral system, 0.2 M ammonium acetate and n-butanol was generally applied.     

Rapid prediction and optimization of concentration conditions for preparative fractions by solid-phase extraction

SPE is an effective tool for concentrating preparative fractions isolated from a complex sample. To guarantee high efficiency and recovery of concentration, the concentration conditions could be optimized by predicting the breakthrough volume (V(B)). In this study, a method of predicting V(B )of unknown compounds in preparative fractions at any isocratic mobile phase composition with the analytical retention parameters a and c is described. The a and c values and the relationship between half peak width (W(1/2)) and retention time of a model analyte were measured using the analytical elution mode on an SPE column, and the V(B )and retention volume (V(R)) predicted with the a and c values were validated with breakthrough experiments. However, it is impossible to measure the a and c values of multiple compounds in a complex system directly on an SPE column with a low number of theoretical plates. The correlation of the a and c values between the SPE and analytical columns was developed so that the analytical data could be transferred to the SPE column. With the calculated a and c values, we could optimize the concentration conditions on the basis of the predicted V(B )and the volume of the preparative fraction.     

Capillary scale monolithic trap column for desalting and preconcentration of peptides and proteins in one- and two-dimensional separations

Monolithic columns based on poly-(styrene-divinylbenzene) (PS-DVB) were utilized both for preconcentration (in 10 mm x 0.20 mm I.D. format) and analytical separation (in 60 mm x 0.20 and 0.10 mm I.D. format) of peptides and proteins in column switching micro-scale high-performance liquid chromatography. A special holder for short monolithic preconcentration columns was designed and pressure durability tests approved long-term stability up to 400 bar. An 11-20% decrease in the average peak widths of nine peptides was obtained upon combining a preconcentration column with an analytical column as compared with a setup using an analytical column only. Trapping efficiency, especially for small and hydrophilic peptides, was optimized by using 0.10% heptafluorobutyric acid instead of 0.050% trifluoroacetic acid as solvent additive during sample loading. Using a 10 mm x 0.20 mm I.D. preconcentration column, loadabilities between 0.5 and 1.6 microg were determined by frontal analysis of proteins and bioactive peptides, respectively. A 100-fold concentration followed by direct on-line intact mass determination is demonstrated for diluted (3 micromolL(-1)) protein solutions. The applicability of the monolithic preconcentration column for multidimensional chromatography was tested by off-line two-dimensional separation, combining strong cation-exchange chromatography and ion-pair reversed-phase chromatography. Peptide identification data from digested protein mixtures demonstrated reproducibilities of 46-75% in triplicate analyses, and confident peptide identifications of low abundant peptides even in the presence of a 650-fold molar excess of high abundant peptides.     

The use of ammonium hydroxide as an additive in supercritical fluid chromatography for achiral and chiral separations and purifications of small, basic medicinal molecules

This study describes using 0.1% of a 28-30% ammonium hydroxide solution as an additive to alcohol modifiers in SFC to improve chromatographic peak shapes for basic molecules. Ammonium hydroxide's high volatility leaves no residual additive in the purified sample unlike classical additives in preparative chromatography such as diethylamine and triethylamine. We demonstrate that the silica support is stable despite having ammonium hydroxide in the modifier by running a durability study for over 350 h (105 L of solvent, 105,000 column volumes) on an analytical Chiralcel OJ column and a second study for 30 h (7.2 L, 14,400 column volumes) on an analytical Lux Cellulose-1 column. The peak shape of small, basic molecules is greatly improved with the use of ammonium hydroxide and this improvement is very similar to those having 0.1% diethylamine as a mobile phase additive. Electrospray ionization is also enhanced with the presence of ammonium hydroxide compared with that of diethylamine. We have found that the age of the 28-30% bottle of ammonium hydroxide solution can have significant effects on the chromatography and we describe how this can be overcome. Finally, we analyzed 23 racemic and basic compounds on six different chiral stationary phases and found there to be very little chiral selectivity difference between ammonium hydroxide and diethylamine, triethylamine, ethanolamine and isopropylamine.     

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.     

Application of a thermal desorption cold trap injector to multidimensional GC and GC-MS

The Thermal desorption Cold Trap injector (TCT) was used as a part of modified multidimensional GC (MDGC) or MDGC mass spectroscopy (MS) systems. These systems were based on a preparative GC (GC1), an analytical GC (GC2), or GC-MS and the TCT. The TCT was mounted on the GC2 or GC-MS. Analysis was carried out as follows: first, the volatile compounds heart-cut after separation on the GC1 column were adsorbed onto the Porapak Q column out of the GC1 oven. This Porapak Q column was then coupled to the TCT, and the volatile compounds adsorbed on the Porapak Q were thermally desorbed, cold trapped, and injected onto an analytical column in the GC2 or GC-MS. Repeatability of the retention time (RT) and area % of model samples consisting of citronellol, decanol, and geranyl acetate was examined. Also, the volatile compounds present at very low concentrations in ethanol solution were concentrated on the Porapak Q column. These were injected onto the analytical column by the same method as described above, and the repeatability of the RT and area % on the chromatogram was examined. In the two experiments, the standard deviation of the RT and area % for each compound was about 0.02 and less than 2.85, respectively. A commercial geranium oil was successfully analyzed by this technique. The results indicate that this modified MDGC and MDGC-MS system are very useful for detection and determination of compounds in complex mixtures.     

A Simple Liquid Chromatographic Method for Analysis and Isolation of the Unsaturated Components of Anacardic Acid

Abstract

Good analytical and preparative separation of the saturated, monoene, diene and triene components of anacardic acid have been achieved by reverse phase HPLC on a C₁₈ column by isocratic elution with methanol-aqueous acetic acid.     

Development of a custom high-throughput preparative liquid chromatography/mass spectrometer platform for the preparative purification and analytical analysis of compound libraries

Solution-phase parallel synthesis has had a profound impact on the speed of compound synthesis delivering relatively pure compounds (>80%) in short order. However, to develop structure activity relationships (SAR) for a compound series, each library member should preferably be >95% pure. Historically, achieving and quantifying such high-purity criteria for each library member proved to be the slow step for most lead discovery groups. To address this issue, significant modifications have been made to a commercial Agilent preparative LC/MS system to allow for the general mass-guided purification of diverse compound libraries. The custom modifications include (1) the "DMSO slug" approach for the purification of samples with poor solubility; (2) an active splitter to reduce system back-pressure, reduce the delay volume, and allow for a variable split ratio; (3) a sample loading pump for the quick purification of large, dilute samples; (4) a preparative column-selection valve to quickly change column selectivity or sample loading; and (5) an analytical injector with a separate flow path for crude reaction or fraction analyses.     

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.     

反相液相色谱法制备纯化柠檬苦素类似物配糖体

 利用反相制备液相色谱结合吸附树脂柱色谱和离子交换色谱方法 ,从甜橙种子的提取物中纯化制备了一种柠檬苦素类似物配糖体 ,经 NMR测定为奥巴叩酮配糖体。     

Präparative Hochdruck-Säulenflüssigkeits-Chromatographie

Several aspects of the apparatus and column technology of modern preparative column liquid chromatography are described together with a review of the present state of the art. We can state: one can today so construct and fill preparative high-speed columns that the separation and isolation of individual components from mixtures in quantities sufficient for tentative identification (spectroscopic and CHN analysis c. 1 mg), structural elucidation, or mechanical, physical, electrical and last but not least, biological tests (ca. 100 mg) is possible. Additionally, by optimal use of “scale up” columns one can provide an additional tool, which may be used for research and development on the gram scale and may be described as a modern time- and money-saving technique. Unfortunately, up until now, only few systematic studies and applications which make full use of the preparative possibilities of modern column chromatography have been published. Only a few of the instrument manufacturers and accessory suppliers are active in this area. For this reason, compared to the rate of development in analytical aspects, the development of commercial preparative equipment is less well advanced. There remain, therefore, several questions open regarding apparatus and operation of such preparative columns, e.g. batch versus continuous operation, or maximum attainable diameter of columns.