Gas chromatography of retinol and α-tocopherol without derivatization

An improved gas chromatographic method for the analysis of retinol and α-tocopherol in biological samples is described. The use of cold on-column injection in combination with wall coated open tubular column gas chromatography eliminates thermal decomposition of vitamin A and yields efficient separations of fat-soluble vitamins (A, D₂, D₃, and E) without derivatization. Peak tailing was judged to be minimal. Vitamins were quantified by flame ionization detection responses down to 3.5 ng injected, and their identifies were confirmed using gas chromatography-mass spectrometry. Extracts of biological samples were saponified, and sterols were removed using digitonin-impregnated celite chromatography before analysis by gas chromatography and gas chromatography-mass spectrometry. Recoveries of vitamins from a test diet ranged from 89 to 103%.     

Separation of prostaglandins using the new horizontal flow-through coil planet centrifuge

Prostaglandins and their metabolites have been separated using the new horizontal flow-through coil planet centrifuge to perform countercurrent chromatography. The system is a continuous-flow system which provides separations similar to column and thin-layer chromatography without the use of solid supports. Either phase of a two-phase solvent system can be used to elute compounds with the low pressure produced by a metering pump. Separations were produced in PTFE tubing, 0.55 mm i.d. (wound in 0.68 cm helical diameter) with a total capacity of 24 ml. A solvent system of chloroform/acetic acid/water (2 : 2 : 1, v/v) was used at flow rates of 2.4 and 6 ml/h and at a rotation of 500 rev./min. Microgram quantities of prostaglandins E₂, F_2α, A₂, and B₂ and thromboxane B₂ can be separated. This method of can be scaled up to 260-ml columns and thromoboxane B₂ can be separated. This method can be scaled up to 260-ml columns with comparable results.     

Chiral separation of neonicotinoid insecticides by polysaccharide‐type stationary phases using high‐performance liquid chromatography and supercritical fluid chromatography

The enantiomeric separations of three neonicotinoid insecticides (identified as compounds 1 , 2 , and 3 ) were performed on three polysaccharide‐type chiral columns, that is, Chiralcel OD‐H, Chiralpak AD‐H, and Chiralpak IB, by high‐performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Effects of the modifier percentage and column temperature on chiral recognitions of chiral stationary phases were also studied. Both 1 and 2 could be resolved on all three columns selected, with the highest R_s values obtained on Chiralpak AD‐H and Chiralcel OD‐H, respectively. However, satisfactory separation of the four stereoisomers of 3 was only achieved on Chiralcel OD‐H. Considering the effects of ethanol on the values of k, α, and R_s, we concluded that hydrogen bonding, π–π, and/or dipole–dipole interactions might be all responsible for the chiral separation. In comparison to HPLC, a shorter run time was achieved for 1 and 2 by SFC. However, 3 could not be stereoselectively resolved using SFC. On the basis of the calculated thermodynamic parameters, we found that the separation processes of enantiomers of 1 and 2 were entropy controlled and enthalpy controlled, respectively. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

NADP phosphatase as a marker in free-flow electrophoretic separations for cisternae of the Golgi apparatus midregion

Based on cytochemical analysis, the enzyme NADP phosphatase is most concentrated in the so-called intercalary cisternae from the mid-region of the Golgi apparatus stack. Using free-flow electrophoresis to separate different Golgi regions of rat liver Golgi apparatus, the NADP phosphatase activity, based on estimation of the rate of release of inorganic phosphate from NADP under standard conditions, was similarly localized to membrane fractions from the center of electrophoretic separations. Peak specific activities for both a putative cis marker (NADH-cytochrome c reductase) and an established trans marker (galactosyltransferase) coincided with minima in NADP phosphatase activity, in agreement with the cytochemical observations. The pattern of distribution of enzyme activity for NADP phosphatase differed from that of both acid phosphatase and glucose-6-phosphatase. The pH optimum was 5.0, the K_m for NADP was 0.6 mM and a corresponding production of NAD and inorganic phosphorus was shown. Taken together with other markers for free-flow electrophoresis separation, the NADP phosphatase will provide considerable utility as a specific market to help identify intercalary cisternae of the mammalian Golgi apparatus and to monitor electrophoretic separations.     

Chiral 3D Open‐Framework Material Ni(D‐cam)(H2O)2 Used as GC Stationary Phase

Metal‐organic frameworks (MOFs) have been explored for analytical applications because of their outstanding properties such as high surface areas, flexibility and specific structure features, especially for chromatography application in recent years. In this work, a chiral MOF Ni(D‐cam)(H₂O)₂ with unusual integration of molecular chirality, absolute helicity, and 3‐D intrinsic chiral net was chosen as stationary phase to prepare Ni(D‐cam)(H₂O)₂‐coated open tubular columns for high‐resolution gas chromatographic (GC) separation. Two fused‐silica open tubular columns with different inner diameters and lengths, including column A (30 m × 250 µm i.d.) and column B (2 m × 75 µm i.d.), were prepared via a dynamic coating method. The chromatographic properties of the two columns were investigated using n‐dodecane as the analyte at 120 °C. The number of theoretical plates (plates/m) of the two metal–organic framework (MOF) columns was 1300 and 2750, respectively. The racemates, isomer and linear alkanes mixture were used as analytes for evaluating the separation properties of Ni(D‐cam)(H₂O)₂‐coated open tubular columns. The results showed that the columns offered good separations of isomer and linear alkanes mixture, especially racemates. Chirality 26:27–32, 2013. © 2013 Wiley Periodicals, Inc.     

Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry

As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.     

Countercurrent chromatography with the flow-through centrifuge without rotating seals

The seal-free centrifuge device is applied to helix countercurrent chromatography. Capability of the scheme is demonstrated on DNP-amino acid separation on a CHCl₃/CH₃COOH/0.1 n HCl (2/2/1) phase system.     

Separation of Tryptophan Enantiomers by Ligand‐Exchange Chromatography With Novel Chiral Ionic Liquids Ligand

Chiral ionic liquids (CILs) with amino acids as cations have been applied as novel chiral ligands coordinated with Cu²⁺ to separate tryptophan enantiomers in ligand exchange chromatography. Four kinds of amino acid ionic liquids, including [L‐Pro][CF₃COO], [L‐Pro][NO₃], [L‐Pro]₂[SO₄], and [L‐Phe][CF₃COO] were successfully synthesized and used for separation of tryptophan enantiomers. To optimize the separation conditions, [L‐Pro][CF₃COO] was selected as the model ligand. Some factors influencing the efficiency of chiral separation, such as copper ion concentration, CILs concentration, methanol ratio (methanol/H₂O, v/v), and pH, were investigated. The obtained optimal separation conditions were as follows: 8.0 mmol/L Cu(OAc)₂, 4.0 mmol/L [L‐Pro][CF₃COO] ,and 20% (v/v) methanol at pH 3.6. Under the optimum conditions, acceptable enantioseparation of tryptophan enantiomers could be observed with a resolution of 1.89. The results demonstrate the good applicability of CILs with amino acids as cations for chiral separation. Furthermore, a comparative study was also conducted for exploring the mechanism of the CILs as new ligands in ligand exchange chromatography. Chirality 26:160–165, 2014. © 2014 Wiley Periodicals, Inc.     

Non-aqueous capillary electrophoresis chiral separations with sulfated β-cyclodextrin

This paper reports the application of an anionic cyclodextrin (CD), sulfated β-cyclodextrin with a degree of substitution of four (β-CD-(SO₄⁻)₄, in chiral separations of pharmaceutical enantiomers by non-aqueous capillary electrophoresis (NACE). Upon complexation with the anionic CD, electrophoretic mobilities of the basic enantiomers decreased, however, both separation selectivity and resolution were enhanced. The advantage of NACE chiral separations over the aqueous CE with the charged CD is that higher electric field strength and higher ionic strength could be applied due to the characteristics of the solvent formamide. The higher ionic strength leads to stacking of peaks and reduces the electrodispersion caused by the mobility mismatch between β-CD-(SO₄⁻)₄–analyte complexes and the co-ions in the running buffer. As a result, better peak shapes and higher separation efficiency were obtained. Comparing with NACE chiral separations with neutral CDs, lower concentration of β-CD-(SO₄⁻)₄ was needed due to the fact that the electrostatic attraction caused stronger binding between β-CD-(SO₄⁻)₄ and the enantiomers. The effects of the experimental parameters, such as concentration of the CD, apparent pH (pH*), degree of substitutions of the CDs, percentage of water in mixed solvent systems, and type of solvents were also studied.     

Complete separation by high performance liquid chromatography of metabolites of arachidonic acid from incubation with human and rabbit platelets

Separations of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C₁₈ reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-¹⁴C]arachidonic acid was quantitative. HPLC also resolved several minor metabolites that were not detected by scanning of TLC separations.     

Rapid separation of anionic oligosaccharide species by high performance liquid chromatography

We have developed a method for the rapid separation of anionic oligosaccharide species by high-performance liquid chromatography utilizing a MicroPak AX-10 ion-exchange column (Varian Associates) with the mobile phase consisting of 25–500 mm KH₂PO₄, pH4.0. Separation of oligosaccharides bearing zero, one, two, three or four sialic acid residues requires less than 45 min. Oligosaccharides containing mannose-6-PO₄ moieties in monoester or diester linkage can also be analyzed in this system. Preparative separations of as much as 20 mg of oligosaccharide can be accomplished in a single chromatographic analysis with quantitative yields of oligosaccharide. This method should prove useful for the rapid isolation and characterization of anionic oligosaccharide species.     

Retention factors and resolutions of amino benzoic acid isomers with some lonic liquids

Ionic liquids in the form of organic salts are being widely used as new solvent media. In this paper three positional isomers,o-amino benzoic acid,m-amino benzoic acid, andp-amino benzoic acids were separated with four different ionic liquids as mobile phase additives using high performance liquid chromatography (HPLC). The following ionic liquids were used: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]). The effects of the alkyl group length on the imidazolium ring and its counterion, and the concentrations of the ionic liquids on the retention factors and resolutions of amino benzoic acid isomers were tested. The results of the separations with ionic liquids as the eluents were better than those without ionic liquids. Excellent separations of the three isomers were achieved using 2.0≈8.0 mM/L [OMIm][MS] and 1.0≈8.0 mM/L [EMIm][MS] as the eluent modifiers.     

High-performance liquid chromatographic determinations of disaccharides resulting from enzymatic degradation of isomeric chondroitin sulfates.

The use of Whatman Partisil-10 PAC and Altex LiChrosorb NH₂ bonded phase columns for the high-performance liquid chromatographic separation of the disaccharides obtained from digestion of isomeric chondroitin sulfates with chondroitinases has been investigated. The substituted unsaturated disaccharides in the enzymatic degradation mixtures undergo rapid isocratic separations on both bonded stationary phases. A complete separation can be established within 10 min. The development of these procedures has expedited enzymatic studies of isomeric chondroitin sulfates.The rate of depolymerization of chondroitin sulfates by the action of chondroitinases based on quantitative high-performance liquid chromatographic separations and detection of the disaccharide products was studied.     

Computational screening of ZIFs for CO2 separations

Using molecular simulations, we studied a diverse collection of zeolite–imidazolate frameworks (ZIFs) to evaluate their performances in adsorption- and membrane-based gas separations. Molecular simulations were performed for both single-component gases (CH₄, CO₂, H₂ and N₂) and binary gas mixtures (CO₂/CH₄, CO₂/N₂, CO₂/H₂ and CH₄/H₂) to predict the intrinsic and mixture selectivities of ZIFs. These two selectivities were compared to discuss the importance of multi-component mixture effects on making predictions about the separation performance of a material. Gas separation performances of ZIFs were compared with other nanoporous materials and our results showed that several ZIFs can outperform well-known zeolites and metal–organic frameworks in CO₂ separations. Several other properties of ZIFs such as gas permeability, working capacity and sorbent selection parameter were computed to identify the most promising materials in adsorption- and membrane-based separation of CO₂/CH₄, CO₂/N₂, CO₂/H₂ and CH₄/H₂.     

Direct chiral separations of the enantiomers of phenylpyrazole pesticides and the metabolites by HPLC

The enantiomeric separation of the enantiomers of three phenylpyrazole pesticides (fipronil, flufiprole, ethiprole) and two fipronil metabolites (amide‐fipronil and acid‐fipronil) were investigated by high‐performance liquid chromatography (HPLC) with a CHIRALPAK^® IB chiral column. The mobile phase was n‐ hexane or petroleum ether with 2‐propanol or ethanol as modifier at a flow rate of 1.0 mL/min. The influences of mobile phase composition and column temperature between 15 and 35°C on the separations were studied. All the analytes except ethiprole obtained complete enantiomeric separation after chromatographic condition optimization. Fipronil, flufiprole, amide‐fipronil, and acid‐fipronil obtained complete separation with the best resolution factors of 2.40, 3.40, 1.67, and 16.82, respectively, but ethiprole showed no enantioselectivity under the optimized conditions. In general, n‐ hexane with 2‐propanol gave better separations in most cases. The results showed decreasing temperature and content of modifier in the mobile phase resulted in better separation and longer analysis time as well. The thermodynamic parameters calculated according to linear the Van't Hoff equation indicated the chiral separations in the study were enthalpy‐driven. Fipronil and its two chiral hydrolyzed metabolites obtained baseline separation simultaneously under optimized conditions.     

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Summary Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-¹⁴C-glucose or UDP-¹⁴C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl₂ was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl₂ the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10M UDP-glucose in the presence of 3 mM MnCl₂ was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 M UDP-xylose in the presence of 3 mM MnCl₂, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-¹⁴C-glucose, and fractions enriched in Golgi apparatus, both (13)- and (14)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (13)-glucosyl. With the substrate, UDP-¹⁴C-xylose, mostly (14)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.Abbreviations ATP adenosine-5-triphosphate - CR crude fraction from downward centrifugation - FL purified fraction from flotation centrifugation - GC gas chromatography - GC-RPC gas chromatography-radiogas proportional counting - IDP inosine-5-disphosphate - NPA naphthylphthalamic acid - UDP uridine-5-diphosphate - TEM transmission electron microscopy     

Enhanced thin-layer chromatographic separation of GM1b-type gangliosides by automated multiple development

Enhancement in separation of gangliosides on silica gel precoated high-performance TLC plates has been obtained by automated multiple development chromatography. A less polar mixture of the standard solvent chloroform-methanol-20 mM aqueous CaCl₂ (120:85:20, v/v) was used. Lowering the water content achieved separation of two complex monosialoganglioside fractions, isolated from murine YAC-1 T lymphoma and MDAY-D2 lymphoreticular cells. Three-fold chromatography in the solvent chloroform-methanol-20 mM aqueous CaCl₂ (120:85:14, v/v) resulted in TLC separation of G_M1b-type gangliosides, substituted with C₂₄ and C₁₆ fatty acids and with Neu5Ac and Neu5Gc as well, which could not be achieved by undirectional standard chromatography. Compared to conventional single chromatography, the technique described allows high-resolution separation of extremely heterogenous ganglioside mixtures and offers a convenient tool for both analytical and preparative TLC.     

A general method for quantitative separation of prostaglandins by paper chromatography.

Although prostaglandin (PG) mixtures have previously been resolved by chromatography on silica-impregnated paper, drawbacks inherent in each technique have kept them from becoming generally accepted for routine analytical separations. Singh and co-workers (1,2) obtained excellent separation of prostaglandin mixtures on silica-impregnated glass fiber paper. However, this paper was not commercially available and its preparation is tedious. On the other hand, Stamford and Unger (3) separated PGE and PGF on commercially available paper using benzene/chloroform/acetone/methanol/acetic acid as developing solvent. Nevertheless, this solvent does not resolve less polar prostaglandins and fatty acids. More generally acceptable solvent systems cannot be used quantitatively with Stamford and Unger's technique due to irreversible binding of prostaglandins at the origin. Tobias and Paulsrud (11) have separated prostaglandins on commercial silicic acid-impregnated glass fiber sheets, but these are extremely brittle and difficult to accommodate to standard paper radiochromatogram scanners.This communication describes the quantitative chromatographic separation of PGF_1α, PGE₂, PGA₁, and arachidonic acid on commercially available Whatman SG-81 silica-impregnated paper using a wide variety of developing solvents. Irreversible binding of prostaglandins at the origin, previously a serious drawback, has been eliminated by applying the sample onto premoistened paper. This method is quantitative, sensitive, reproducible, and applicable to a variety of solvent systems. In addition, it is simple and inexpensive. Although loading capacity is somewhat limited, this is no problem with prostaglandins since they can be readily concentrated in organic solvents.     

LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes

15-series prostaglandins (PGE₂s) and isoprostanes (isoPGE₂s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE₂/D₂ and isoPGE₂ without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE₂/PGD₂/isoPGE₂ such as PGE₂, entPGE₂, 8-isoPGE₂, 11β-PGE₂, PGD₂, and 15(R)-PGD₂ to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE₂ in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE₂ levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE₂s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.     

Preparative solvent partition chromatography of butyrylated cyclic AMP analogues

A simple and effective separation of cyclic adenosine-3′,5′-monophosphate (cAMP) and its butyryl-substituted analogues using partition chromatography on columns of Sephadex gel in isopropanol/0.5 m ammonium acetate (4:1) is described. The technique is suitable for preparative separations as demonstrated by revised uv spectral data obtained on butyrylated cAMP's purified by this technique. In addition, it has analytical utility in that it allows complete separation of N⁶-monobutyryl cAMP from O^2′-monobutyryl cAMP, thereby permitting simultaneous and independent assessment of the rate of acyl substituent hydrolysis from the disubstituted derivative (N⁶,O^2′-dibutyryl cAMP), and this is demonstrated under several conditions.