Performance comparison of three types of high-speed counter-current chromatographs for the separation of components of hydrophilic and hydrophobic color additives

The performance of three types of high-speed counter-current chromatography (HSCCC) instruments was assessed for their use in separating components in hydrophilic and hydrophobic dye mixtures. The HSCCC instruments compared were: (i) a J-type coil planet centrifuge (CPC) system with a conventional multilayer-coil column, (ii) a J-type CPC system with a spiral-tube assembly-coil column, and (iii) a cross-axis CPC system with a multilayer-coil column. The hydrophilic dye mixture consisted of a sample of FD&C Blue No. 2 that contained mainly two isomeric components, 5,5'- and 5,7'-disulfonated indigo, in the ratio of ～7:1. The hydrophobic dye mixture consisted of a sample of D&C Red No. 17 (mainly Sudan III) and Sudan II in the ratio of ～4:1. The two-phase solvent systems used for these separations were 1-butanol/1.3M HCl and hexane/acetonitrile. Each of the three instruments was used in two experiments for the hydrophilic dye mixture and two for the hydrophobic dye mixture, for a total of 12 experiments. In one set of experiments, the lower phase was used as the mobile phase, and in the second set of experiments, the upper phase was used as the mobile phase. The results suggest that: (a) use of a J-type instrument with either a multilayer-coil column or a spiral-tube assembly column, applying the lower phase as the mobile phase, is preferable for separating the hydrophilic components of FD&C Blue No. 2; and (b) use of a J-type instrument with multilayer-coil column, while applying either the upper phase or the lower phase as the mobile phase, is preferable for separating the hydrophobic dye mixture of D&C Red No. 17 and Sudan II.     

Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column

Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C₁₈ columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C₁₈ RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.     

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.     

Simultaneous separation of hydrophilic and hydrophobic compounds by using an online HILIC-RPLC system with two detectors

In the present study, we developed a novel online hydrophilic interaction chromatography (HILIC)-RPLC separation system for simultaneous separation of both hydrophilic and hydrophobic solutes in a complex sample with one injection. A HILIC Si column and a C18 column were hyphenated with an interface including two electronic 2-position valves, a solvent pump, and a solute transfer column. By using column-switching technique, the nonretained hydrophobic solutes were eluted out of the HILIC column and transferred into the C18 column to perform further separation. The hydrophilic solutes were separated on the HILIC column at the same time. One detector was equipped for each column to record the individual chromatograms. By separating a standard mixture and a traditional Chinese medicine (TCM) extract, the developed HILIC-RPLC system demonstrates its potential for "entire-components" separation of complex samples with improved peak capacity and throughput compared with the common single-column LC.     

Preparative isolation of flavonoid glycosides from Sphaerophysa salsula using hydrophilic interaction solid‐phase extraction coupled with two‐dimensional preparative liquid chromatography

An offline preparative two‐dimensional reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography coupled with hydrophilic interaction solid‐phase extraction method was developed for the preparative isolation of flavonoid glycosides from a crude sample of Sphaerophysa salsula . First, the non‐flavonoids were removed using an XAmide solid‐phase extraction cartridge. Based on the separation results of three different chromatographic stationary phases, the first‐dimensional preparation was performed on an XAqua C18 prep column, and 15 fractions were obtained from the 5.2 g target sample. Then, three representative fractions were selected for additional purification on an XAmide preparative column to further isolate the flavonoid glycosides. In all, eight flavonoid glycosides were isolated in purities over 97%. The results demonstrated that the two‐dimensional liquid chromatography method used in this study was effective for the preparative separation of flavonoid glycosides from Sphaerophysa salsula . Additionally, this method showed great potential for the separation of flavonoid glycosides from other plant materials.     

New Validated Methods for the Simultaneous Determination of Two Multicomponent Mixtures Containing Guaiphenesin in Syrup by HPLC and Chemometrics‐Assisted UV‐Spectroscopy

Abstract

High pressure liquid chromatographic (HPLC) and spectrophotometric methods are developed for the determination of two multicomponent mixtures containing guaiphenesin, dextromethorphane hydrobromide, and sodium benzoate together with either phenylephrine hydrochloride, chlorpheniramine maleate, and butylparaben (mixture 1) or ephedrine hydrochloride and diphenhydramine hydrochloride (mixture 2). The HPLC method depended on using an ODS column with mobile phase consisting of acetonitrile ?10 mM potassium dihydrogen phosphate, pH 2.7 (40∶60 v/v) containing 5 mM heptane sulfonic acid sodium salt (for mix 1) and a cyanopropyl column with mobile phase consisting of acetonitrile ?12 mM ammonium acetate, pH 5 (40∶60 v/v) (for mix 2) and UV detection at 214 nm. The cyanopropyl column is much less hydrophobic, less sterically restricted to the penetration of bulky solute molecules into the stationary phase, and has weaker hydrogen‐bond acidity than the ODS column. So the cyanopropyl column is more suitable for separation of components of mix 2. The chemometric‐assisted spectrophotometric method with, principal component regression (PCR) and partial least squares (PLS‐1) was used. For the chemometric method a calibration set of the mixture consisting of each compound in each mixture was prepared in distilled water. The absorbance data in the UV spectra were measured in the spectral region (210–240 or 210–224 nm for mix 1 and mix 2, respectively, as this range provided the greatest amount of information about the two mixture components). The spectrophotometric method does not require a separation step. The proposed methods were successfully applied for the analysis of the two multicomponents combinations in laboratory‐prepared mixtures and in commercial syrups, and the results were compared with each other.     

High Performance Liquid Chromatography of Enkephalin and Endorphin Peptide Analogs

Abstract

Reverse phase systems are presented which utilize a μ alkylphenyl column and ammonium acetate buffered aqueous acetonitrile mobile phases to separate mixtures of enkephalin and endorphin peptide analogs. High pressure liquid chromatographic separations of mixtures of enkephalin diastereomers as well as mixtures of other closely similar analogs have been developed.

Endorphin analogs were observed to be quite hydrophobic and required mobile phases containing 40% or more acetonitrile for reasonable elution times. The enkephalin analogs by comparison required 20% or more acetonitrile. Detection at both 254 and 280 nm was useful in recognizing the important peaks in the elution profile.     

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.     

Optimization of peptide separations in reversed-phase HPLC: Isocratic versus gradient elution

Summary We have determined the interaction behaviour of peptides with hydrophobic stationary phases on analytical columns using isnocratic or shallow gradient elution for the purpose of developing procedures for rapid optimization of conditions for preparative reversed-phase chromatography of peptides. From our investigation of the separation of two closely related decapeptides (differing by one methyl group), in a 1:1 molar ratio on an analytical C8 column, we have found that shallow gradients of 0.1% acetonitrile/min appeared to be the best compromise between resolution and a practical run time for preparative peptide separations. Up to 20mg of the two-peptide mixture was efficiently resolved on the analytical column, with >97% recovery of homogeneous peptides.     

Micropreparative separation of peptides derived from sodium dodecyl sulphate-solubilized proteins

A systematic investigation of the influence of the detergent sodium dodecyl sulphate (SDS) on micropreparative peptide separations on microbore reversed-phase high-performance liquid chromatographic columns is reported. A tryptic digest of bovine serum albumin and a mixture of synthetic peptides were used to monitor the separation behaviour of a 1.6 mm I.D. Nucleosil C18 column in the presence of various amounts of SDS. The data demonstrate that even traces of SDS in the sample reduce the separation efficiency and peptide recovery. An extraction method is presented which reduces the SDS content in peptide mixtures below the critical concentration without affecting significantly the recovery of individual peptides. After acidification of the sample, the detergent is extracted into heptane-isoamyl alcohol (4:1, v/v). In combination with chemical or enzymatic fragmentation techniques, this extraction method facilitates the sequence analysis of minute amounts of SDS-solubilized hydrophobic proteins. The applicability of the method is demonstrated on the example of the integral membrane protein bacteriorhodopsin.     

Use of Centrifugal Partition Chromatography and Proteins in The Preparative Separation of Amino Acid Enantiomers

Abstract

The growth of analytical methodologies for the separation of enantiomers has been impressive. Attention is now turning to the large scale separation of enantiomers. Often scaling-up sensitive analytical separations is ineffective and inefficient. Centrifugal partition chromatography (CPC) may be a viable alternative for the preparative separation of racemic mixtures in some cases. The use of proteins as chiral selectors in CPC is examined. Attention was focused on proteins that previously were used as bonded phases in analytical LC columns. The enzymatic properties of α-chymotrypsin allowed it to be used as a bioreactor in conjunction with CPC. When proteins are used as components of the stationary or mobile phase there can be problems with denaturation. However, when used in external incubation processes or as column bioreactors coupled with CPC, effective gram-scale separations can be performed. Tryptophan methyl ester was used as a model compound to evaluate this approach.     

Solvent-free MALDI-MS for the analysis of <Emphasis Type="Italic">β</Emphasis>-amyloid peptides via the mini-ball mill approach: Qualitative and quantitative advances

Manual and automated solvent-free mini-ball mill (MBM) matrix-assisted laser desorption/ionization (MALDI) analysis of mixtures of beta-amyloid peptides (1-11), (33-42), (1-42) and non-beta-amyloid component of Alzheimer's disease peptide yielded interpretable spectra for all of the peptides present regardless of their relative amounts in the samples. This was not the case for solvent-based MALDI analysis using traditional acidic aqueous/organic solvent conditions, which resulted in severe over-representation of hydrophilic peptide (1-11) and provided no spectra for insoluble amphiphilic peptide (1-42) even when present at 50% relative molar amount. Less accurate representation of components in mixtures by the traditional method appears to be a combination of poor dissolution of peptides in the solvent and preferential ionization of more hydrophilic peptides in the mixture. Consequently, only MBM provided a complete tryptic map of beta-amyloid (1-42) compared to 67% coverage by traditional MALDI. Acetonitrile (0.1% TFA) led to improved coverage only at a 50% molar ratio of peptide (1-42), but also to a side product of (1-42), Met oxidation (amino acid 35), a phenomenon not observed in MBM MALDI analysis. Traditional MALDI analysis resulted in over-representation of hydrophilic soluble beta-amyloid (1-11) in defined mixtures and autoproteolytic peptides of trypsin. In contrast, over-representation and under-representation were less pronounced in solvent-free MALDI in all of the investigated cases. Analysis of defined peptide and tryptic peptide mixtures showed that MBM MALDI yielded greater qualitative reliability, which also improved quantitative response relative to the solvent-based approach.     

基于聚倍半硅氧烷微球的亲水/反相混合模式色谱填料的制备与评价

亲水/反相混合模式色谱应用广泛,但pH使用范围有限,不利于碱性药物的分离。该工作利用巯基-烯基点击化学合成了单分散多孔的半胱氨酸改性乙烯基功能化聚甲基倍半硅氧烷(C-V-PMSQ)微球。元素分析表明半胱氨酸成功键合在微球表面。C-V-PMSQ微球为介孔结构,单分散性好且具有优良的化学稳定性。以几种常见的核苷和核酸碱基作为测试样品,考察其色谱保留行为,溶质的保留因子随流动相中水相含量的变化呈现典型的U型曲线,表明C-V-PMSQ固定相具有亲水/反相的双重保留特征。使用该固定相可以分离苯的同系物及一系列亲水性与疏水性化合物。另外在高碱性流动相条件下利用亲水和反相模式成功分离了中药苦参中的3种主要活性成分,表明它在分离碱性药物方面具有较大的优势。     

Exploratory Purification of Compounds from Aqueous Extracts of <Emphasis Type="Italic">Pinus massoniana</Emphasis> Lamb. by Two-Dimensional Preparative Liquid Chromatography

An off-line orthogonal two-dimensional preparative liquid chromatography method was developed for isolation of high-purity compounds from aqueous extracts of Pinus massoniana Lamb., which has efficient therapeutic properties in recipes of traditional Chinese medicines. A polar-enhanced reversed-phase (RP) column, XAqua C18 was selected for the first-dimensional separation. A HILIC column and a hydrophobic RP column were both explored in the second dimension. The preparation results indicated hydrophilic RP × HILIC mode would provide better separation efficiency for polar complexes, i.e., the second-dimensional separation was finished in less than 30 min and high-purity compounds were obtained. In this work, 16 fractions were collected efficiently in the first dimension with a recovery up to 96%, and six compounds with high purity and enough quantity were identified by NMR and high resolution mass spectrometry, including epicatechin, betuloside, taxifolin-3-O-xyloside, cedrusin-4-O-glucoside, massonianoside C and massonianoside D, and five are glycosides, including flavonoid glycosides, lignanoid glycosides and an arylbutanoid glycoside. Betuloside was first reported in the preparation of Pinus massoniana Lamb. This work demonstrated a flexible strategy of compromise in between time and resolution for selecting solvent and column to enhance preparative efficiency of polar complex systems.     

Separation and purification of intermediates for the preparation of naproxen from synthetic mixtures by countercurrent chromatography

Three key intermediates in the preparation of the nonsteroidal anti‐inflammatory drug naproxen were successfully separated and purified with high purity from synthetic mixtures by countercurrent chromatography with a selected biphasic solvent system. The biphasic solvent system composed of n‐hexane/ethyl acetate/methanol/water (9:1:9:1, v/v/v/v) was selected according to partition performance of the three components using thin‐layer chromatography. Fifty milligrams of the synthetic mixture after the three‐step reaction was injected into a preparative countercurrent chromatography separation column and yielded 3.5, 14.0, and 8.0 mg of three key intermediates with 95.0, 99.0, and 98.0% purity, and the recovery of each component was 65.2, 71.2, and 69.6%, respectively. The results indicated that countercurrent chromatography is an efficient alternative and economical method for the separation and purification of intermediate components from synthetic mixtures.     

Large-Scale Preparative Counter-Current Chromatography with a Coil Planet Centrifuge

Abstract

Development of the large-scale preparative countercurrent chromatographic schemes has been continued by increasing the diameter of the separation column. A 0.55 cm i.d. FEP tube was coaxially coiled around the holder (7.5 cm, 10 cm or 15 cm in diameter) of a horizontal flow-through coil planet centrifuge (15 cm revolutional radius). Performance of each column was evaluated on the separation of dinitrophenyl amino acid samples with a two-phase solvent system composed of chloroform, acetic acid, and 0.1N hydrochloric acid (2:2:1) by using both aqueous and nonaqueous phases as the mobile phase. Experiments with the short preliminary columns (114 ml capacity) revealed that the hydrodynamic distribution of the two solvent phases was sensitively affected by the helical diameter of the column. However, by choosing the proper elution mode of the mobile phase, satisfactory results were obtained with the helical diameters of 7.5 cm and 15 cm at a high flow rate of 500 ml/h under a moderate revolutional speed of 300 rpm. With the long coiled columns (750 ml capacity), the preparative capability of the present scheme was successfully demonstrated on separations of the 1g-quantity sample mixture under optimized operational conditions. Overall results indicated that the sample-loading capacity of the present scheme can be further increased by the use of longer and/or larger-diameter columns.     

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.     

Selective solvation effects in phase separation in aqueous mixtures

Selective solvation can be crucial in phase separation in polar binary mixtures (water–oil) with a small amount of hydrophilic ions or hydrophobic particles. They are preferentially attracted to one of the solvent components, leading to a number of intriguing effects coupled to phase separation. For example, if cations and anions interact differently with the two components, an electric double layer emerges at a liquid–liquid interface. The main aim of this paper is to show that a strongly hydrophilic (hydrophobic) solute induces precipitation of water-rich (oil-rich) domains above a critical solute density n_p outside the solvent coexistence curve.     

Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions

The application of a silica hydride modified stationary phase with low organic loading has been investigated as a new type of chromatographic material suitable for the separation and analysis of peptides with electrospray ionization mass spectrometric detection. Retention maps were established to delineate the chromatographic characteristics of a series of peptides with physical properties ranging from strongly hydrophobic to very hydrophilic and encompassing a broad range of pI values (pI 5.5-9.4). The effects of low concentrations of two additives (formic acid and acetic acid) in the mobile phase were also investigated with respect to their contribution to separation selectivity and retention under comparable conditions. Significantly, strong retention of both the hydrophobic and the hydrophilic peptides was observed when high-organic low-aqueous mobile phases were employed, thus providing a new avenue to achieve high resolution peptide separations. For example, simultaneous separation of hydrophobic and hydrophilic peptides was achieved under aqueous normal phase (ANP) chromatographic conditions with linear gradient elution procedures in a single run, whilst further gradient optimization enabled improved peak efficiencies of the more strongly retained hydrophobic and hydrophilic peptides.     

Efficient strategies for preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity > 95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.