Preparative Reversed-phase Liquid Chromatography Research Articles

Urea complexation combined with rapid preparative reversed-phase liquid chromatography to separate α-linolenic acid from perilla seed oil: Purity, yield, and oxidation stability

Perilla seed oil (PSO) is a major source of α-linolenic acid (ALA). In order to better separate ALA from PSO and to improve its purity and yield, a purification method for ALA that combines urea complexation (UC) with rapid preparative reversed-phase liquid chromatography (RP-RPLC) has been studied. Within the selected ranges of conditions, four one-factor experiments were found to have significant effects on the purity and yield of ALA from PSO. A response surface methodology (RSM) has been applied to optimize UC for separating and purifying ALA from PSO, which yielded optimal parameters of 95% ethanol/urea (w/w) = 5:1, urea/esterified fatty acids (E-FAs) (w/w) = 2:1, complexation time 16.3 h, and complexation temperature 17 °C. The optimal purity and optimal yield of the one-time purification ALA (OTP-ALA) obtained by UC thereby reached 86.977 ± 0.082% and 34.696 ± 0.948 %, respectively. Two-time purification conditions of OTP-ALA by RP-RPLC were also determined, and the purity of the two-time purification ALA (TTP-ALA) was increased to 99.200 ± 0.061%, corresponding to a yield of 53.888 ± 0.904 %. The effect of different added amounts of α-tocopherol on the oxidation stability of TTP-ALA has also been studied. Accelerated oxidation tests showed that the addition of α-tocopherol effectively inhibited the oxidation of TTP-ALA by reducing the peroxide value (PV). of ALA separation and purification in PSO (triglycerides refers to the collective term for all fatty acid glycerides in PSO). • An improved method for purification of ALA from perilla seed oil was developed. • RSM has been used to optimize the separation of ALA from perilla seed oil. • The combined use of the two methods results in ALA purity exceeding 99.2 %. • Add an appropriate amount of α-tocopherol improved the oxidation stability of ALA.

Systematic activity-oriented separation and structure-activity relationship of tobacco cembranoids

Cembranoids are a class of glandular trichome exudates with a variety of structural variations and biological activities, and are found in high quantities in tobacco (Nicotiana tabacum L.). However, few studies have conducted preparative separation and examined the activities of cembranoids other than (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (α-CBT-diol, 1) and (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (β-CBT-diol, 2); thus, the structure-activity relationship (SAR) of cembranoids remains unclear. Here, to deduce the SAR of cembranoids, raw cembranoid extract was systematically separated through an activity-oriented approach with preparative reversed-phase liquid chromatography. Each fraction was identified, and their antifungal, insecticidal, and cytotoxic bioactivities were evaluated. Composition analysis and activity detection of primary fractions (Fr.1–Fr.10) revealed that cembranoid activity might be influenced by the number of hydroxyl groups and degree of unsaturation (macrocycle and double bonds). Sub-fraction analysis showed that the antifungal activity of cembranoids was related to the number of hydroxyl groups and double bonds, and that their cytotoxic activity was affected by the type and position of substituents. Furthermore, all four factors could influence insecticidal activity. By conducting in-depth studies of single cembranoids, their SARs were summarized. The existence of Δ11,12/Δ7,8 and hydroxylation at C-6/C-4 contributed to the antifungal activity of cembranoids. Variation in the type and position of substituents also significantly affected antifungal activity, of which Δ11,12 was the most influential factor. Additionally, the change in substituent configuration at C-4 and C-12 had no significant effect on antifungal activity. The insecticidal activity of cembranoids benefited from the existence of 4-hydroxyl, which greatly enhanced their binding affinity; however, functional groups substituted at other positions decreased insecticidal activity. Cytotoxic activity was positively correlated with α-configuration at C-4 and hydroxylation at C-6 but inversely correlated with 4-methoxy substitution. Among them, 6-hydroxyl formed the core; the α-/β-configuration at C-12 and 4-/12-substitution of cembranoids did not affect their insecticidal activity. Insecticidal activity might play a key role by restraining acetylcholinesterase activity through hydrophobic interactions between the cembranoid carbon skeleton and residues located at the acetylcholinesterase binding pocket. These findings may provide a foundation for the development of novel cembranoid-based pesticides and medicines.

Preparative isolation of apple flavan-3-ols monomers and oligomers using pH-zone-refining centrifugal partition chromatography combined with reversed-phase liquid chromatography

Flavan-3-ols (catechin monomers and procyanidins) are the main class of polyphenols in apples and are found in high concentrations in cider apple varieties. They are known to be involved in bitterness and astringency in apple-based beverages, and also contribute to polyphenol nutritional intake.Therefore, highly purified flavan-3-ol fractions isolated from raw materials are needed to study their various properties. For this purpose, a gentle strategy combining pH-zone-refining centrifugal partition chromatography (pH-ZRCPC) and preparative reversed-phase liquid chromatography (Prep-RPLC) was developed to recover one hundred milligrams of a high purity apple flavan-3-ol fraction.First, pH-ZRCPC fractionation in descending mode was optimized to remove hydroxycinnamic acid derivatives using a biphasic mixture composed of ethyl acetate/n-butanol/water (3/2/5, v/v). Trifluoroacetic acid and sodium hydroxide were used as retainer and eluter, in the upper and lower phases, respectively. Secondly, Prep-RPLC separation was carried out in isocratic mode at 20% ACN to remove dihydrochalcones. Finally, from one gram of a crude polyphenol extract, four hundred and nine milligrams of a highly purified fraction of flavan-3-ols with an average degree of polymerization close to 3.1 was obtained with 73% recovery.

Production and Characterization of Medium-Sized and Short Antioxidant Peptides from Soy Flour-Simulated Gastrointestinal Hydrolysate.

Soybeans (Glycine max) are an excellent source of dietary proteins and peptides with potential biological activities, such as antihypertensive, anti-cholesterol, and antioxidant activity; moreover, they could prevent cancer. Also, soy contains all the essential amino acids for nutrition; therefore, it represents an alternative to animal proteins. The goal of this paper was the comprehensive characterization of medium-sized and short peptides (two to four amino acids) obtained from simulated gastrointestinal digestion. Two different analytical approaches were employed for peptide characterization, namely a common peptidomic analysis for medium-sized peptides and a suspect screening analysis for short peptides, employing an inclusion list of exact m/z values of all possible amino acid combinations. Moreover, fractionation by preparative reversed-phase liquid chromatography was employed to simplify the starting protein hydrolysate. Six fractions were collected and tested for antioxidative activity by an innovative antioxidant assay on human gastric adenocarcinoma AGS cell lines. The two most active fractions (2 and 3) were then characterized by a peptidomic approach and database search, as well as by a suspect screening approach, in order to identify potential antioxidant amino acid sequences. Some of the peptides identified in these two fractions have been already reported in the literature for their antioxidant activity.

A selective comprehensive reversed-phase×reversed-phase 2D-liquid chromatography approach with multiple complementary detectors as advanced generic method for the quality control of synthetic and therapeutic peptides.

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3mm ID column is used in the second dimension. Recording data with a UV detector at 215nm after 1D separation provides the common generic 1D chromatogram. However, after the 2D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.

Separation of Piper kadsura Using Preparative Supercritical Fluid Chromatography Combined with Preparative Reversed-Phase Liquid Chromatography

We have developed a separation strategy that employed preparative supercritical fluid chromatography (prep-SFC) combined with preparative reversed-phase liquid chromatography (prep-RPLC) to purify compounds from Piper kadsura. The complexity of the sample was effectively reduced by two steps of prep-SFC. The key experimental parameters of prep-SFC were systematically optimized, including an “average-pressure rule” scaling-up method for adjustment of the backpressure of the system. A mixed modifier consisting of ethanol/n-hexane was applied to provide a stable elution. Prep-RPLC was subsequently adopted to purify the SFC fractions due to its good orthogonality with SFC. Furthermore, chiral SFC was used to separate the structural analogues. As a result, 14 compounds with high purity were obtained from one fraction, including three pairs of diastereoisomers. These data indicate that the separation strategy using prep-SFC combined with prep-RPLC is an effective tool to separate complex samples such as natural products.

Separation and purification of compounds from piper kadsura using preparative reversed-phase liquid chromatography and preparative supercritical fluid chromatography

A method based on preparative reversed-phase liquid chromatography (prep-RPLC) and preparative supercritical fluid chromatography (prep-SFC) was developed for the separation and purification of compounds from piper kadsura. A pretreatment method was first developed, including methanol extraction, water precipitation, petroleum ether extraction, etc. Chlorophyll and other strong polar impurities were removed from the piper kadsura samples, and the target components were enriched in petroleum ether extracts. The piper kadsura samples were separated into 18 fractions on a Unitary C18 column (250 mm×20 mm, 5 μm) with water and methanol as the mobile phases. Then, the SFC parameters, including the column, modifier, temperature, and backpressure were optimized. The optimized conditions for prep-SFC were as follows:XAmide column (250 mm×20 mm, 5 μm), methanol as the modifier, 30℃ column temperature, and 15.0 MPa backpressure. Because of the good orthogonality of RPLC and SFC, six highly pure compounds were isolated, including kadsurenone, wallichinine, denudatin B, pellitorine, 2E-decenoic acid N-isobutylamide, and futoxide.

Identification of triacylglycerols in coix seed extract by preparative thin layer chromatography and liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

Here we reported a methodology for identification of triacylglycerols (TAGs) and diacylglycerols (DAGs) in coix seed by preparative thin layer chromatography (prep-TLC) and non-aqueous reversed-phase liquid chromatography (NARP LC)-atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MS/MS). Lipid components were extracted from coix seed by reflux extraction using n-hexane for 3 hr. TAGs and DAGs in coix seed extract were effectively purified and isolated from matrix interferences by prep-TLC and then analyzed by LC-APCI-MS and MS/MS for identification. TAGs were effectively identified taking into consideration of their LC retention behavior, APCI-MS spectra patterns, and MS/MS spectra of [DAG] + ions. In MS/MS spectra of TAGs, diacylglycerol-like fragment [DAG] + ions were useful to identify TAGs with isobaric fragment ions. Based on an established method, 27 TAGs and 8 DAGs were identified in coix seed extract. Among them, 15 TAGs and 8 DAGs were for the first time observed in coix seed. Interestingly, some of TAGs isolated by prep-TLC were partly converted into DAGs through probably photolysis process during storing in room temperature. Thus, degradation phenomenon of TAGs should be considered in the quality evaluation and nutritional property of coix seed. LC-APCI-MS/MS combined with prep-TLC will be practical method for precise TAG and DAG analysis of other herbal plants.

Purification of high-purity glycyrrhizin from licorice using hydrophilic interaction solid phase extraction coupled with preparative reversed-phase liquid chromatography.

Glycyrrhizin (GA), a major bioactive compound in licorice, has been extensively used throughout the world as a medicine to treat chronic viral hepatitis and allergic dermatitis. In this study, a new method based on hydrophilic interaction solid phase extraction (HILIC-SPE) and preparative reversed-phase liquid chromatography (prep-RPLC) was developed to purify GA with high purity from the complex licorice extract. Via evaluation of retention behavior of GA and flavonoids in different commercially available columns, a hydrophilic column--Click XIon was finally chosen for the purification due to its excellent resolution toward GA and flavonoids under HILIC mode. To optimize the SPE elution conditions, relative factors including water content, pH and ionic strength had been investigated in chromatographic condition. The result indicated that the most appropriate water content was 30% and pH at 4.00, as well as salt concentration should be controlled at 5mM. In addition, the optimization revealed that GA experiences both hydrophilic interaction and ion-exchange interaction on the Click XIon material. According to the chromatographic evaluation, the optimized conditions were applied to HILIC-SPE to enrich GA from licorice, which leads to an increased content of GA from 13.67% to 64.22%. Finally, prep-RPLC was performed to obtain GA with purity higher than 99.00%，which demonstrating great prospect in large-scale preparation of GA.

Identification of Antihyperuricemic Peptides in the Proteolytic Digest of Shark Cartilage Water Extract Using in Vivo Activity-Guided Fractionation

A peptide that exerts antihyperuricemic activity after oral administration was identified from a microbial protease (alcalase) digest of the water extract of shark cartilage by in vivo activity-guided fractionation, using oxonate-induced hyperuricemic rats. Water extract of shark cartilage was first fractionated by preparative ampholine-free isoelectric focusing, followed by preparative reversed-phase liquid chromatography. The antihyperuricemic activity of the alcalse digests of the obtained fractions was evaluated using an animal model. Alcalase digests of the basic and hydrophobic fractions exerted antihyperuricemic activity. A total of 18 peptides were identified in the alcalase digest of the final active fraction. These peptides were chemically synthesized and evaluated for antihyperuricemic activity. Tyr-Leu-Asp-Asn-Tyr and Ser-Pro-Pro-Tyr-Trp-Pro-Tyr lowered the serum uric acid level via intravenous injection at 5 mg/kg of body weight. Furthermore, orally administered Tyr-Leu-Asp-Asn-Tyr showed antihyperuricemic activity. Therefore, these peptides are at least partially responsible for the antihyperuricemic activity of the alcalase digest of shark cartilage.

Comparison of preparative reversed phase liquid chromatography and countercurrent chromatography for the kilogram scale purification of crude spinetoram insecticide

Reversed phase HPLC (RP-HPLC) and high performance countercurrent chromatography (HPCCC) were compared for the pilot scale purification of two semi-synthetic spinosyns, spinetoram-J and spinetoram-L, the major components of the commercial insecticide spinetoram. Two, independently performed, 1kg, purification campaigns were compared. Each method resulted in the isolation of both components at a purity of >97% and yields for spinetoram-J and spinetoram-L of >93% and ≥63% of theoretical, respectively. The HPCCC process produced a 2-fold higher throughput and consumed approximately 70% less solvent than preparative scale RP-HPLC, the volume of product containing fractions from HPCCC amounted to 7% of that produced by HPLC and so required much less post-run processing.

Use of an Orthogonal System, RP-LC–HILIC, for Evaluation of Purity

The reliability of evaluation of compound purity depends on the analytical method selected. A suitable analytical method, including the modes of separation and detection, can provide reliable information about compound purity. Effective separation is the key to achieving precise results. In this work we developed a novel method, based on an orthogonal system, RP-LC-hydrophilic interaction liquid chromatography, for evaluating the purity of compounds isolated by preparative reversed-phase liquid chromatography. Because of its good orthogonality, good separation and more accurate assessment of compound purity could be achieved.

Systematic purification of polydatin, resveratrol and anthraglycoside B from Polygonum cuspidatum Sieb. et Zucc.

For comprehensive utilization of Polygonum cuspidatum Sieb. et Zucc., a systematic, environmental friendly preparative process was developed for purification of polydatin, resveratrol and anthraglycoside B simultaneously from the herb. The process was simple, consisting of macroporous resin adsorption and reversed-phase liquid chromatography. Macroporous resin adsorption separated the solvent extract into fraction I containing polydatin and fraction II containing resveratrol as well as anthraglycoside B. The fractions I and II were further purified by reversed-phase chromatography to get the fractions corresponding to polydatin, resveratrol and anthraglycoside B, respectively. Two polydivinylbenzene microspheres (PDVB-SPG and PDVB-Swelling) were prepared by SPG membrane emulsification and by two-step activated swelling polymerization, respectively. These new media were compared with a commercial C 18 bonded silica medium in the reversed-phase liquid chromatography step. The PDVB-SPG column demonstrated a better separation compared with C 18 and PDVB-Swelling column. The pH value of the mobile phase showed little effect on the separation of polydatin and resveratrol, and large effect on the separation of anthraglycoside B for both HPLC analysis and preparative reversed-phase liquid chromatography. After the chromatographic process and a further crystallization, polydatin, resveratrol and anthraglycoside B were purified from 7.5%, 1.3% and 2.4% to 98.8%, 98.2% and 98.6%, respectively, with total recoveries of 81.3%, 77.4% and 80.2%.

Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates

In the past few decades, marine natural products bioprospecting has yielded a considerable number of drug candidates. Two marine natural products have recently been admitted as new drugs: Prialt (also known as ziconotide) as a potent analgesic for severe chronic pain and Yondelis (known also as trabectedin or E-743) as antitumor agent for the treatment of advanced soft tissue sarcoma. In this protocol, methods for bioactivity-guided isolation, purification and identification of secondary metabolites from marine invertebrates such as sponges, tunicates, soft corals and crinoids are discussed. To achieve this goal, solvent extraction of usually freeze-dried sample of marine organisms is performed. Next, the extract obtained is fractionated by liquid-liquid partitioning followed by various chromatographic separation techniques including thin layer chromatography, vacuum liquid chromatography, column chromatography (CC) and preparative high-performance reversed-phase liquid chromatography. Isolation of bioactive secondary metabolites is usually monitored by bioactivity assays, e.g., antioxidant (2,2-diphenyl-1-picryl hydrazyl) and cytotoxicity (microculture tetrazolium) activities that ultimately yield the active principles. Special care should be taken when performing isolation procedures adapted to the physical and chemical characteristics of the compounds isolated, particularly their lipo- or hydrophilic characters. Examples of isolation of compounds of different polarities from extracts of various marine invertebrates will be presented in this protocol. Structure elucidation is achieved using recent spectroscopic techniques, especially 2D NMR and mass spectrometry analysis.

Modeling and optimization of preparative reversed-phase liquid chromatography for insulin purification

This paper presents a model for reversed-phase purification of insulin from desamido insulin. The system is described by a reaction dispersive model with a competitive Langmuir isotherm. A model building and calibration method is presented and the model's region of validity is defined. The model is calibrated using only two-component experiments on the raw mixture by the inverse method and then experimentally validated. The model is then used to optimize the system's production rate with both purity and yield requirements. The yield requirement is varied between 80 and 95% to study the effect on the production rate and the operating point. The operating points found with the optimization were found outside the model's region of validity, but the experimental validation of the operating points shows that the model can be extrapolated to the interesting operating points.

Improved Detection of Sugar Addition to Maple Syrup Using Malic Acid as Internal Standard and in 13C Isotope Ratio Mass Spectrometry (IRMS)

Stable carbon isotope ratio mass spectrometry (delta13C IRMS) was used to detect maple syrup adulteration by exogenous sugar addition (beet and cane sugar). Malic acid present in maple syrup is proposed as an isotopic internal standard to improve actual adulteration detection levels. A lead precipitation method has been modified to isolate quantitatively malic acid from maple syrup using preparative reversed-phase liquid chromatography. The stable carbon isotopic ratio of malic acid isolated from this procedure shows an excellent accuracy and repeatability of 0.01 and 0.1 per thousand respectively, confirming that the modified lead precipitation method is an isotopic fractionation-free process. A new approach is proposed to detect adulteration based on the correlation existing between the delta13Cmalic acid and the delta13Csugars-delta13Cmalic acid (r = 0.704). This technique has been tested on a set of 56 authentic maple syrup samples. Additionally, authentic samples were spiked with exogeneous sugars. The mean theoretical detection level was statistically lowered using this technique in comparison with the usual two-standard deviation approach, especially when maple syrup is adulterated with beet sugar : 24 +/- 12% of adulteration detection versus 48 +/- 20% (t-test, p = 7.3 x 10-15). The method was also applied to published data for pineapple juices and honey with the same improvement.

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 ≤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 ∼7–10 mg/ml (with sample loads varying from 67 to 200 mg) onto a small C 18 column (150×4.6 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 (≤200 mg) varying by as much as a factor of three.

Preparative reversed-phase liquid chromatography of proteins from rabbit skeletal troponin, a multi-protein complex

A reversed-phase high-performance liquid chromatography protocol for purification of all proteins in a multi-protein (TnI, TnC, TnT, tropomyosin) complex from rabbit skeletal muscle has been developed, enabling efficient purification of sample amounts ranging from 43 mg of protein complex on a standard analytical column, to 1400 mg on a column of 21.2 mm I.D. and finally, to 5700 mg on a column of 50 mm I.D. Due to problems associated with scale-up procedures for these proteins (e.g. aggregation and/or solubility issues), an initial sample fractionation was devised whereby 50% of the TnC component was precipitated with acetonitrile prior to sample introduction on the RPLC column. By subsequently taking advantage of sample overload conditions to enhance the displacement effect between sample components, coupled with very slow gradient conditions (0.1% acetonitrile/min), we were able to achieve excellent protein separations at high yields of purified proteins.

Isolation and preparative purification of microcystin variants

Preparative reversed-phase liquid chromatography was successfully used to purify two microcystins (microcystin LR and microcystin LA) from a cyanobacterial process waste. The separation protocol involved extraction of lyophilized cells by methanol, isolation and concentration by solid-phase extraction, and purification by reversed-phase HPLC. Milligram-level loading of microcystins was obtained on a solid-phase extraction cartridge packed with 0.5 g of C 18 stationary phase. The separations were first carried out on an analytical column and then scaled-up to a preparative column. The microcystins were quantified by HPLC and enzyme-linked immunosorbent assay. A method to remove microcystins rapidly and economically from the cyanobacterial process waste is also described.

Non-flammable preparative reversed-phase liquid chromatography of recombinant human insulin-like growth factor-I

Acetonitrile is used as an eluent for reversed-phase chromatography. However, because it is a flammable solvent, using acetonitrile on a large scale requires expensive equipment and facilities specially designed for flammable solvents. Using a non-flammable solvent as an eluent eliminates this expense. A method was developed to purify recombinant human insulin-like growth factor I by reversed-phase high-performance liquid chromatography using gradient elution with hexylene glycol, a non-flammable replacement for acetonitrile. The separation produced equivalent yield, purity and throughput as reversed-phase chromatography using elution with acetonitrile.