Normal-phase Solvent Research Articles

Development of an On-Line Two-dimensional Normal Phase Liquid Chromatography System for Analysis of Weakly Polar Samples

In this study, a novel on-line two-dimensional (2D) normal phase × normal phase liquid chromatography (NPLC × NPLC) was developed for the separation of weakly polar samples. The 2D NPLC was integrated by a unique designed solvent evaporation (SE) interface, including a 6-port 2-position valve and a 10-port 2-position valve in conjunction with two silica gel-packed enrichment columns. The enrichment columns played a pivotal role in evaporating normal phase (NP) solvent from the first dimensional NPLC under vacuum and elevated temperature condition. The working parameters of the interface were evaluated comprehensively. To demonstrate the resolving powerful 2D system, we analyzed three natural-sourced weakly polar compounds from the extracts of toad venom, dammar resin, and propolis. To this end, we studied the effects of mobile phase combinations, the second dimensional column length for separation of the extracts from toad venom, and propolis, respectively. In general, we have found that excellent separation can be obtained with diversity of NP solvent combinations and longer NPLC column (150 mm long). Moreover, compared with the generally used 2D reversed-phase liquid chromatography (RPLC × RPLC), the NPLC × NPLC method exhibited better separation orthogonality. In conclusion, the new NPLC × NPLC separation method provides potential advantages for analysis of weakly polar samples.

Determining aflatoxins in raw peanuts using immunoaffinity column as sample clean-up method followed by normal-phase HPLC-FLD analysis

This study was aimed at simplifying the conventional aflatoxin HPLC-FLD analysis procedure for determining aflatoxins B1, B2, G1, and G2 by utilizing the advantage of immunoaffinity column (IAC) and normal-phase chromatography. Normally, a derivatization procedure is needed to enhance the fluorescence signal of aflatoxin quenched by the reversed-phase solvent used as the HPLC mobile phase. However, the proposed mobile phase (toluene/ethyl acetate/methanol/formic acid, 90 mL/6 mL/2 mL/2 mL) considerably enhanced the fluorescence signal by eliminating the quenching effect. IAC is a fast and reproducible method for sample clean-up, but the miscibility of the eluate is low in the normal-phase solvent. The miscibility of the eluate in the normal-phase solvent was improved by replacing methanol with acetonitrile as the eluent, dehydrating water after IAC elution, and mixing with toluene before HPLC analysis. The results demonstrate that the time-consuming procedures, such as nitrogen evaporation and iodine-derivatization, in the sample preparation were no longer needed, which improved the recovery and repeatability. The LOQ can be as low as 2.0-0.5-1.0–0.5 ng of aflatoxin B1-B2-G1-G2 per gram of peanut. All linearities (R2) of the four types of aflatoxin were greater than 0.999. The inter-day recovery rates of aflatoxin B1, B2, G1, and G2 at LOQ were 109%, 115%, 113%, and 77%, and the corresponding standard deviations were 12%, 8%, 5%, and 14%, respectively. The performance of the proposed method in analyzing raw peanuts was better than that of the official method (HPLC-FLD combined with post-column iodine-derivatization) and can meet the US FDA's standard for examining human food. The shelf-life of the proposed mobile phase was prolonged to 2 weeks under room temperature, further saving the use of solvent. Each analysis needed less than 25 mL of organic solvent to complete. Furthermore, the method can determine AF concentration in raw peanuts contaminated with more than 4.00 ng/g of total aflatoxins (containing 2.00 ng/g of aflatoxin B1, 0.50 ng/g of aflatoxin B2, 1.00 ng/g of aflatoxin G1, and 0.50 ng/g of aflatoxin G2).

Micro Thin-Layer Chromatography Fingerprints of Selected Basil Species and Their Chemometric Analysis

The dried aerial parts of 12 plants of Ocimum spp. were extracted with the Soxhlet apparatus using dichloromethane and methanol as solvents. A micro-TLC system with silica and a normal-phase solvent system (propan-2-ol-n-heptane-formic acid) was used for the chemometric analysis of 12 selected basil methanolic extracts. Some indices of similarity (Pearson's correlation coefficient, determination coefficient, congruence coefficient, and Euclidean, Manhattan (city-block), Chebyshev, and Hausdorff distances) were calculated on the basis of thin-layer chromatograms using ImageJ software. Principal component analysis was also performed. The method allowed the comparison of the analyzed extracts and confirmed the identities of two selected unknown plants.

Screening approach, optimisation and scale-up for chiral liquid chromatography of cathinones

Screening approaches adopted in pharmaceutical companies for chiral LC method development may be quite complicated and sophisticated in order to guarantee a high success rate. However in other environments it may be of more value to assess how simple a screen might be used to still have a good chance of achieving success. The genuine need to develop chiral separations for the former ‘legal-high’ drug mephedrone and related cathinones of topical interest presented a good opportunity to develop this theme. In initial work on mephedrone itself, no chiral separation was observed on Chirobiotic V, Cyclobond I 2000 DNP, Whelk-O1 and AmyCoat using reversed phase mobile phases. However, using normal phase solvents, chiral separation was observed on all the chiral stationary phases (CSP) used except Chiralcel OJ-H. Of the chiral separations observed on RegisPack, RegisCell and Whelk-O1, some optimisation work was carried out on the latter two which had showed the greatest enantioselectivity. Following optimisation, the best enantioselectivity (1.59) and enantioresolution (5.90) was found with a 250mm×4.6mm I.D. Whelk-O1 column using a propan-2-ol (IPA)–hexane–trifluoroacetic acid (TFA)–triethylamine (TEA) (10:90:0.05:0.05, v/v/v/v) mobile phase. Subsequent screening on other cathinones was restricted to RegisPack, RegisCell and Whelk-O1 or equivalent phases with two mobile phases and this gave a very good success rate. Indeed it was possible to separate all six cathinones on one column, RegisCell, with one mobile phase, propan-2-ol–hexane–TFA (15:85:0.1, v/v/v) but obviously it had been necessary to go through the 3-column screen to arrive at this finding. While Whelk-O1 was not so successful, ease of optimisation on this phase was again a feature. To illustrate the applicability of these separations, it was shown that, as a basis for semi-preparative work, the optimsed mephedrone separation on Whelk-O1 could be scaled-up to a 2000μl injection of a 1.0mgml−1 solution in mobile phase (2.0mg on-column) while still using the 250mm×4.6mm I.D. analytical column.

Atmospheric Pressure Photoionization as a Powerful Tool for Large-Scale Lipidomic Studies

Lipidomic studies often use liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) for separation, identification, and quantification. However, due to the wide structural diversity of lipids, the most apolar part of the lipidome is often detected with low sensitivity in ESI. Atmospheric pressure (APPI) can be an alternative ionization source since normal-phase solvents are known to enhance photoionization of these classes. In this paper, we intend to show the efficiency of APPI to identify different lipid classes, with a special interest on sphingolipids. In-source APPI fragmentation appears to be an added value for the structural analysis of lipids. It provides a detailed characterization of both the polar head and the non polar moiety of most lipid classes, and it makes possible the detection of all lipids in both polarities, which is not always possible with ESI.

An ultraperformance liquid chromatography method for the normal-phase separation of lipids

An ultraperformance liquid chromatography method using normal-phase solvents, a silica column, and evaporative light-scattering detection is presented. The method is based on a quaternary gradient profile and is capable of resolving the major neutral and polar lipids present in plasma and animal tissue in under 5 min, with a total cycle time of 11 min. Limits of quantitation for 7 different lipid classes were on the order of 200 ng of material on column which enables an accurate analysis from as little as 20 μL of plasma or 50 mg of tissue for typical samples. Intraday and interday precision for the determination of the major lipid classes in human plasma ranged from 3.6 to 10.5% CV with a variability in retention time of less than 6%. The utility of the method is demonstrated through the separation and quantitation of lipids in mouse plasma, liver, and heart tissue.

Quantitative analysis of phospholipids containing arachidonate and docosahexaenoate chains in microdissected regions of mouse brain

Phospholipids containing polyunsaturated fatty acyl chains are prevalent among brain lipids, and regional differences in acyl chain distribution appear to have both functional and pathological significance. A method is described in which the combined application of GC and multiple reaction monitoring (MRM) MS yielded precise relative quantitation and approximate absolute quantitation of lipid species containing a particular fatty acyl chain in milligram-sized tissue samples. The method uses targeted MRM to identify specific molecular species of glycerophosphocholine lipids, glycerophospho-ethanolamine lipids, glycerophosphoinositol lipids, glycerophosphoserine lipids, glycero-phosphoglycerol lipids, and phosphatidic acids that contain esterified arachidonate (AA) and docosahexaenoate (DHA) separated during normal phase LC/MS/MS analysis. Quantitative analysis of the AA and DHA in the LC fractions is carried out using negative ion chemical ionization GC/MS and stable isotope dilution strategies. The method has been applied to assess the glycerophospholipid molecular species containing AA and DHA in microdissected samples of murine cerebral cortex and hippocampus. Results demonstrate the potential of this approach to identify regional differences in phospholipid concentration and reveal differences in specific phospholipid species between cortex and hippocampus. These differences may be related to the differential susceptibility of different brain regions to neurodegenerative disorders.

Solvation of the Whelk-O1 chiral stationary phase: A molecular dynamics study

Density functional theory calculations and molecular dynamics simulations are employed to explore the solvation of the Whelk-O1 chiral stationary phase. First, a semi-flexible representation of the Whelk-O1 selective molecule is extracted from an extensive series of B3LYP/6-311+ G(2d,p) calculations. The resulting model is used to build a chiral surface, including end-caps, for molecular dynamics study of the interface between solvent and Whelk-O1. Three solvent environments in common use for Whelk-O1 HPLC have been examined: a normal-phase solvent of n-hexane/2-propanol; a reversed-phase solvent of water/methanol; and a supercritical solvent of CO 2 and methanol. In each case, we analyze the interface with an emphasis on solvent composition and solvent hydrogen bonding to the Whelk-O1 selector.

Atmospheric pressure photoionization mass spectrometry for analysis of fatty acid and acylglycerol lipids

In this work, we optimize parameters and conditions for analysis of fatty acid ester and acylglycerol lipids by atmospheric pressure photoionization-mass spectrometry (APPI-MS). The investigated parameters include atmospheric pressure chemical ionization (APCI) nebulizer/vaporizer physical orientation and APPI lamp face position, solvent selections, mobile phase compositions and flow rates, cone voltages and probe temperatures. APPI sensitivity is found to be highly dependent on mobile phase compositions. Normal phase solvents offer much higher sensitivity and better peak shape than reversed phase for nonpolar lipids. Hexane and isooctane are found to be two solvents generating highest S/N for eicosapentaenoic acid (EPA) methyl ester. The effects of mobile phase flow rates on sensitivity are found to be target analytes and target ions specific. However, the flow rate changes do not significantly affect the sensitivity of three out of four tested analytes under normal phase conditions over tested flow rates of 50–500 μL/min. Cone voltage is found to be one of key parameters affecting sensitivity. Optimum probe temperature is found to be more dependent on mobile phase compositions than on the specific target analytes. Aqueous reversed-phase mobile phase requires higher probe temperature than normal phase for better sensitivity. More volatile mobile phase solvents require lower probe temperature for analyte desolvation. APPI offers four to five decades of linear ranges under normal phase condition. Full scan mass spectra of individual lipid standards, custom lipid mixtures and natural fish oil show that APPI spectra are clean and very easy to interpret. APPI also gives stable, reproducible peak responses with good peak shape. Limits of detection (LODs) by FIA (S/N = 3) are estimated to be 12 pg for EPA methyl ester and monoarachidin, 19 pg for diarachidin and 7 pg for trielaidin. LODs on-column are estimated to be 94 pg for EPA methyl ester, 90 pg for monoarachidin and diarachidin and 24 pg for trielaidin.

Comparison of Atmospheric Pressure Photoionization, Atmospheric Pressure Chemical Ionization, and Electrospray Ionization Mass Spectrometry for Analysis of Lipids

In this work, we compare the quantitative accuracy and sensitivity of analyzing lipids by atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) LC/MS. The target analytes include free fatty acids and their esters, monoglyceride, diglyceride, and triglyceride. The results demonstrate the benefits of using LC/APPI-MS for lipid analysis. Analyses were performed on a Waters ZQ LC/MS. Normal-phase solvent systems were used due to low solubility of these compounds in aqueous reversed-phase solvent systems. By comparison, APPI offers lower detection limits, generally highest signal intensities, and the highest S/N ratio. APPI is 2-4 times more sensitive than APCI and much more sensitive than ESI without mobile-phase modifiers. APPI and APCI offer comparable linear range (i.e., 4-5 decades). ESI sensitivity is dramatically enhanced by use of mobile phase modifiers (i.e., ammonium formate or sodium acetate); however, these ESI adduct signals are less stable and either are nonlinear or have dramatically reduced linear ranges. Analysis of fish oils by APPI shows significantly enhanced target analyte intensities in comparison with APCI and ESI.

Experimental design approach for the optimisation of pressurised fluid extraction of additives from polyethylene films

A pressurised fluid extraction (PFE) and normal-phase-high performance liquid chromatography (NP-HPLC) method is proposed for the determination of additives in polyethylene films. The study of PFE variables was performed using a Plackett–Burman (PB) experimental design for screening and a central composite design (CCD) for optimising the main variables obtained from the Pareto charts. The studied variables were: temperature, time, cyclohexane (CHx) and tetrahydrofuran (THF) as modifiers, flush volume and extraction cycles, and an isopropanol:CHx (92.5:7.5) mixture twice at 105 °C for 15 min were the final conditions selected. The additives in the PFE extracts were separated by NP-HPLC using a silica column and a gradient n-hexane:dichloromethane:acetonitrile mobile phase. Additive solubility is higher in normal-phase solvents; thus, their separation can be carried out at room temperature. Finally, the method was applied to determine additives in several polyethylene films.

Analysis of cell membrane aminophospholipids as isotope-tagged derivatives

Glycerophosphoethanolamine (GPEtn) and glycerophosphoserine (GPSer) lipids were reacted with a multiplexed set of differentially isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, which place isobaric mass labels at a primary amino group. The resulting derivatized aminophospholipids were isobaric and chromatographically indistinguishable but yielded positive reporter ions (m/z 114 or 117) after collisional activation that could be used to identify and quantify individual members of the multiplex set. The chromatographic and mass spectrometric response of N-methylpiperazine amide-tagged aminophospholipids was probed using glycerophosphoethanolamine and glycerophosphoserine lipid standards. The [M+H]+ of each tagged aminophospholipid shifted 144 Da, and during collision-induced dissociation the major fragmentation ion was either m/z 114 or 117. This mode of detecting aminophospholipids was useful for an unbiased analysis of plasmalogen GPEtn lipids. Molecular species information on the esterified fatty acyl substituents was obtained by collisional activation of the [M-H]- ions. The isotope-tagged reagents were used to assess changes in the distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to Cu2+/H2O2 to illustrate the ability of these reagents to aid in the mass spectrometric identification of aminophospholipid changes that occur during biological stimuli.

Evaluation of ethoxynonafluorobutane as a safe and environmentally friendly solvent for chiral normal-phase LC-atmospheric pressure chemical ionization/electrospray ionization-mass spectrometry

Coupling normal-phase LC separation methods to atmospheric pressure ionization (API)-mass spectrometry (MS) for detection can be problematic because of the possible detonation hazard and because nonpolar solvents do not support ionization of the analyte. Unlike achiral separations, enantiomeric separations can be very sensitive to small changes in the separation environment. Thus, completely substituting the main mobile phase component of a normal-phase LC solvent for an environmentally friendly, nonflammable fluorocarbon-ether as a safe and effective solvent must be thoroughly evaluated before it can be recommended for enantioselective separations with API-MS detection. Ethoxynonafluorobutane (ENFB) was used as a normal-phase solvent for the enantioselective separation of 15 compounds on two macrocyclic glycopeptide chiral stationary phases (CSPs) and a new polymeric chiral stationary phase. The chromatographic figures of merit were compared between results obtained with the ENFB mobile phases and traditional heptane-based mobile phases. In addition, the limits of detection (LOD) using the API-MS compatible ENFB were examined, as well as flow rate sensitivities and compatibilities with common polar organic modifier. ENFB is a safe and effective solvent for enantioselective normal-phase/API-MS analyses.

Normal-phase chiral separations by pressure assisted capillary electrochromatography using the Pirkle type stationary phase Whelk-O 1

Using normal phase solvents with small percentages of water, it was shown that an electroosmotic flow (EOF) could be generated and these solvents can be used to separate a normal phase test mixture on a silica stationary phase. Run times were reduced further by pressure assisted capillary electrochromatography (CEC). This knowledge was then transferred to the chiral stationary phase Whelk-O 1 where it was used successfully to separate of a number of chiral analytes. The effects of the different contributions of pressure and voltage on the linear velocity were investigated. Altering the applied pressure and reducing the length of the packed bed then optimized the separation of the enantiomers of the chiral compound benzoin. For the column with the shorter packed bed, run times of 6 minutes were observed, whilst still retaining good resolution.

Optimisation of procedures for the extraction of structural analogues of propranolol with molecular imprinted polymers for sample preparation

A propranolol-derived molecular imprinted polymer (MIP) was prepared using methacrylic acid as monomer and ethylene glycol dimethacrylate as cross-linker. The extraction properties of five compounds structurally related to propranolol were assessed on the MIP and on a blank polymer made under the same conditions but in the absence of an imprint molecule. Using application from aqueous solution with methanol–water–triethylamine (TEA)-based solvents for elution (i.e. reversed-phase conditions) the MIP showed only marginal selectivity for the compounds on the MIP compared to the blank. Despite the limited selectivity there did appear to be a relationship between structure of the compound (relative to propranolol) and the extent of selective retention. Application of the compounds in toluene with elution using toluene–TEA or toluene–trifluoroacetic acid resulted in the MIP showing dramatically enhanced retention and selectivity of the compounds on the MIP compared to the blank. The enhanced selectivity for extraction on to the MIP relative to the blank, for all compounds using normal-phase solvents seem to be a class effect as there was no apparent relationship between compound structure and retention.

Use of fluorine-containing stationary phases for the separation of an alkyl bromide from its hydrocarbon analog by high-performance liquid chromatography

Radiolabelled Sch 13835, an inhibitor of platelet derived growth factor, was prepared by catalytic hydrogenolysis of a benzyl bromide precursor with tritium gas. Regular and deactivated reversed-phase HPLC stationary phases gave poor peak shapes and little resolution of Sch 13835 and the benzyl bromide precursor. Fluorodecyl and fluoroether stationary phases in the analytical reversed-phase mode gave baseline separation using organic-aqueous (no buffers) mobile phases. Elution orders were reversed on either by a change in the organic component from methanol to acetonitrile. The product is unstable in aqueous (or other protic) solvents, forming a ring-opened acid. Conditions were developed to successfully purify the compound by reversed-phase HPLC with minimal decomposition. A second analytical HPLC assay was developed using normal-phase solvents on a fluorether stationary phase.

Hexamethyldisiloxane as a mobile phase component for normal phase liquid chromatography

Hexamethyldisiloxane (HMDX) has been evaluated as a modifier solvent for the normal phase separation of substituted naphthalene componds as well as aromatics such as nitrobenzene, acetophenone, benzonitrile, and aniline. It is found to be a weaker eluting solvent than n-butyl ether and other commonly used normal phase solvents filling a niche in the eluotropic series. HMDX exhibited an unusual retention factor (k′) profile as a function of mobile phase composition in hexane. In a range from 0 to 40 %, thek′ values remained quite constant with increasing HMDX composition. As the concentration of HMDX progressed to 100 %, thek′ values actually increased in a generally linear fashion. This trend ink′ is due to longer solute retention not just a change in the retention time of the unretained component. Although slight silanization of the silica column may occur with initial use of HMDX, retention of these analytes on a commerical C-1 silica column is much lower with no positivek′ slope with increasing HMDX. The chromatographic data provide evidence that HMDX is likely adsorbed to the silica surface and a solute partitioning mechanism may be operative.

Unusual Maxima in HPLC Retention of an Homologous Series Consisting of Longer Alkyl Chain Substitutions

A series of compounds were studied which had increasing alkyl chain length between an aromatic group and a tertiary amine. In this homologous series, a maximum in retention versus alkyl chain length was found for a wide range of mobile phases using either a silica gel column or a cyanopropyl-bonded silica gel column. One nonaqueous mobile phase using an octadecyl-bonded silica gel column also showed this maximum. The selectivity between E and Z geometric isomers found with the shorter chain analogues decreased with increasing chain length greater than about pentyl, with eventual complete loss of resolution. A few normal-phase solvents produced a reversal of the usual E−Z elution order. 1H NMR assignments are described for the individual geometric isomers. NMR studies and ab initio calculations indicate that a folded conformation in solution is possible for the longer chain analogues. The unusual retention patterns are most likely due to a combination of localized adsorption and analyte solution conformation effects.

Optimization of chiral selectivity on cellulose-based high-performance liquid chromatographic columns using aprotic mobile-phase modifiers

Tris-3,5-dimethyl phenyl carbamate-derivatized cellulose HPLC columns and other cellulose-based columns are popular for the direct separation of many racemic drugs and agrichemicals. These columns exhibit good efficiency and stability under well-controlled chromatographic conditions. Cellulose-based chiral columns also exhibit high sample-load capacity with normal-phase solvents, which is particularly advantageous for isolating purified solutes. Several investigators have reported that the stereoselectivity of these columns can be varied by changing the type and composition of alcohol modifiers in the mobile phase, and by altering temperature. We now find that separation selectivity and resolution can be significantly increased, and often optimized, by modifying the nonpolar mobile phase with certain aprotic solvents. The potential of this new separation dimension greatly increases the general applicability of cellulose-based columns for analytical and preparative separations of racemates. A simple, systematic scheme is proposed for optimizing the separation of enantiomeric drugs with the Chiralcel OD column.

Mass fragmentographic assay for 25‐hydroxyvitamin D in plasma without derivatization: Enhanced sensitivity for metabolites of vitamins D2 and D3 after pre‐column dehydration

AbstractA mass fragmentographic method for the measurement in plasma of underivatized, dehydrated 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 was developed. Quantitative dehydration of vitamin D metabolites was achieved prior to gas chromatography using a new high‐temperature injection system and a small precolumn packed with aluminium powder in the injection port, followed by mass fragmentography on an inexpensive bench‐top mass spectrometer. Plasma (2 ml) was incubated with hexadeuteriated 25‐hydroxyvitamin D3 prior to acetonitrile extraction and purification using cartridges pre‐packed with microparticulate silica using reversed‐ and normal‐phase solvent systems. After purification, capillary gas chromatography mass spectrometry was carried out following dehydration of the secosteroids on the aluminium power pre‐column at 400°C. High‐intensity dehydrated molecular ions were produced which were used for selected ion monitoring. The assay sensitivity for 25‐hydroxyvitamin D was approximately 1 ng ml−1. The intra‐assay variation was less than 7% and the recovery of added standard was quantitative. It is suggested that this method may be used to provide target values for much needed quality control schemes to monitor assays routinely used in the estimation of 25‐hydroxyvitamin D. The behaviour of a number of other hydroxylated vitamin D metabolites, when injected without derivatization on to a gas chromatographic column, was also investigated.