Application of polymer based stationary phases in high performance liquid chromatography and capillary high performance liquid chromatography hyphenated to microcoil 1H nuclear magnetic resonance spectroscopy

The increased demand for chromatographic materials that are able to achieve a fast separation of large quantities of structure analogues is a great challenge. It is known that polymer based chromatographic materials have a higher loadability, compared to silica based sorbents. Unfortunately these polymer materials cannot be used under high pressure which is necessary in order to obtain high flow rates, and hence long times are needed to perform a separation. However, by immobilizing a polymer on a mechanically stable porous silica core, this problem can be circumvented and higher flows become feasible on these materials. Especially for capillary liquid chromatography hyphenated with nuclear magnetic resonance a high loadability is of great importance in order to obtain sharp, resolved, and concentrated peaks thus resulting in a good signal to noise ratio in the NMR experiment. Therefore, a highly shape selective chromatographic sorbent was developed by covalently immobilizing a poly(ethylene-co-acrylic) acid copolymer (-CH(2)CH(2)-)(x)[CH(2)CH(CO(2)H)-](y) (x=119, y=2.4) with a mass fraction of acrylic acid of 5% as stationary phase on silica via a spacer molecule (3-glycidoxypropyltrimethoxysilane). First, the loadability of this sorbent compared to C(30) is demonstrated by the HPLC separation of two xanthophyll isomers. Subsequently, it has been successfully employed in the hyphenation of capillary HPLC with microcoil (1)H NMR spectroscopy by separating and identifying a highly concentrated solution of the tocopherol homologues.     

Capillary liquid chromatography-microcoil 1H nuclear magnetic resonance spectroscopy and liquid chromatography-ion trap mass spectrometry for on-line structure elucidation of isoflavones in Radix astragali

Miniaturization and hyphenation of chromatographic separation techniques to nuclear magnetic resonance spectroscopy is being increasingly demanded in the field of biomedical, drug metabolite and natural product analysis. Herein, capillary liquid chromatography was coupled on-line to microcoil 1H nuclear magnetic resonance spectroscopy (capLC-NMR) equipped with a 1.5 microL solenoidal probe for structure elucidation of isoflavones in Radix astragali. The extract was screened by HPLC-UV-MS as the preliminary step and four major peaks were identified tentatively by ion trap mass spectrometry molecular weights and characteristic fragments. Then, stopped-flow capLC-UV-NMR was performed using 33 microg extract injected on-column. The four peaks were parked manually in the micro probe one by one and corresponding 1H NMR spectra were recorded with good resolutions under the applied capLC-NMR conditions (120 and 220 ng injected on-column for peaks 2 and 4, respectively). All aromatic regions of 1H NMR spectra correlated well to the characteristic signals of isoflavone aglycone protons. And the signal corresponding to the anomeric proton of the glucopyranoside of isoflavone glycoside was also obtained for peak 1. Therefore, these four peaks are determined as calycosin-7-O-beta-D-glucopyranoside (1), ononin (2), calycosin (3) and formononetin (4) unambiguously. The capLC-NMR results indicate that this hyphenated technique could be used for the determination of a great variety of natural products from small sample amounts, e.g., only 5 g R. astragali in this study.     

Splitless on-line coupling of capillary high-performance liquid chromatography,capillary electrochromatography and pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy

A mixture of unsaturated fatty acid methyl esters was separated with a new splitless capillary set-up. With the employed apparatus configuration different capillary separation techniques such as capillary high-performance liquid chromatography (cHPLC), capillary electrochromatography (CEC) and pressurized capillary electrochromatography (pCEC) could be applied. The detection and identification of the sample compounds were accomplished by hyphenating these capillary separation techniques with nuclear magnetic resonance (NMR) spectroscopy using a novel configuration of the detection capillary set-up. Using modified electrokinetically driven separation techniques, the electric field was applied solely across the separation column. With this improved interface for capillary liquid chromatography-NMR on-line coupling, the stereochemical assignment of the cis and trans configuration of unsaturated fatty acids could be easily accomplished. Finally, the results of cHPLC-NMR, CEC-NMR and pCEC-NMR coupling experiments were compared.Dedicated to Professor Günter Häfelinger on the occasion of his 65th birthday     

Hyphenation of capillary separations with nuclear magnetic resonance spectroscopy

The hyphenation of small-volume separations to information-rich detection offers the promise of unmatched analytical information on the components of complex mixtures. Nuclear magnetic resonance (NMR) spectroscopy provides information about molecular structure, although sensitivity remains an issue for on-line NMR detection. This is especially true when hyphenating NMR to capillary separations as the observation time and analyte mass are decreased to the point where reduced information is obtained from the eluting analytes. Because of these limitations, advances in instrumental performance have a large impact on the overall performance of a separation–NMR system. Instrumental aspects and the capabilities of cLC–NMR, CEC–NMR and CE–NMR are reviewed, and applications that have used this technology highlighted. Recent trends towards small volume capillary scale separations are emphasized, as is the recent success of capillary-isotachophoresis (cITP)–NMR.     

Direct on-line hyphenation of capillary liquid chromatography to nuclear magnetic resonance spectroscopy: practical aspects and application to drug metabolite identification

In combining the high peak concentrations of capillary liquid chromatography (CapLC) with the high mass sensitivity of micro scale nuclear magnetic resonance (NMR) the hyphenation of CapLC to micro NMR offers a substantial gain in overall sensitivity. This paper deals with our experiences gained using a commercial CapLC-NMR system which has very recently become available. The limits of detection (SNR > 3) for a test compound of a molecular weight of M 318 were found to be approximately 100 ng (0.35 nmol) within an hour acquisition time and approximately 25 ng over night (85 pmol). Practical aspects such as the feasibility of stopped-flow experiments and sample handling issues are discussed in detail and first possible drug metabolite applications to hepatocyte incubations and direct analysis of plasma samples are presented.     

Microstructure elucidation of historic silk (Bombyx mori) by nuclear magnetic resonance

¹H NMR cryoporometry and solid-state ¹³C cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy were used to characterize the microstructure of historic and fresh silk samples. Silk is a polymeric bicomponent material composed of fibroin and water located in micropores. According to the ¹H NMR cryoporometry method, the intensity of the water resonance as a function of the temperature was used to obtain the pore size distribution, which was strongly asymmetric with a well-defined maximum at 1.1 nm. Compared with the fresh silk samples, the volume of pores around 1.1 nm decreased distinctly in the historic silk, and more pores larger than 2 nm emerged accordingly. In addition, these results correlated well with solid-state ¹³C CP/MAS NMR spectroscopy as the percentage of random coil in the historic silk sample was much less than that in the fresh silk samples. Therefore, it is suggested that the water-filled microvoids grow larger as the random coil conformation fades away in the degradation process.

The application of two-dimensional nuclear magnetic resonance spectroscopy in computer-assisted structure elucidation

One of the most powerful tools for the determination of an unknown organic structure is two dimensional NMR spectroscopy. In some cases, this information quickly leads to a unique structure, but in many cases there remains a larger number of structural possibilities. A linked system of computer programs has been developed which uses several types of 2-D NMR data and generates all possible topological structures consistent with these data and user-input constraints. When symmetry is present, algorithms based on group theory limit the structures to those consistent with all the different symmetries that are possible. Applications of these programs to problems described in the literature, in which 2-D NMR was used to determine the structures of natural products, are described.     

Retention characteristics of protonated mobile phases injected into deuterated mobile phases in capillary liquid chromatography (LC) using on-line nuclear magnetic resonance (NMR) detection

The behavior of protonated binary solvents injected into deuterated binary mobile phases in capillary LC is studied with NMR. Specifically, the solvent elution is followed on-flow with a capillary LC coupled to a 900 nL volume microcoil NMR probe. A range of identical composition 5% protonated (and 95% deuterated) solvents is injected into composition-matched deuterated mobile phases of CD(3)CN/D(2)O and CD(3)OD/D(2)O. The protonated components separate for all solvent combinations except at 80% CD(3)CN/20% D(2)O and similar to 72% CD(3)OD/28% D(2)O where only a single retention time is observed. The more hydrophilic protonated component, HOD, elutes first with higher percentages of hydrophilic solvent, D(2)O, in the mobile phase whereas retention is reversed with the higher percentage of the more hydrophobic solvent (CD(3)CN and CD(3)OD) in the mobile phase. The hydrophilic/hydrophobic nature of the chromatographic system as a function of mobile phase composition is characterized by following the retention times of protonated solvents.     

Detection and quantification of phenolic compounds in olive oil by high resolution H nuclear magnetic resonance spectroscopy

High resolution ¹H NMR spectroscopy has been employed as a versatile and rapid method to analyze the polar fraction of extra virgin olive oils containing various classes of phenolic compounds. The strategy for identification of phenolic compounds is based on the NMR chemical shifts of a large number of model compounds assigned by using two-dimensional (2D) NMR spectroscopy. Furthermore, 2D NMR was applied to phenolic extracts in an attempt to discover additional phenolic compounds. The ¹H NMR methodology was successful in detecting simple phenols, such as p-coumaric acid, vanillic acid, homovanillyl alcohol, vanillin, free tyrosol, and free hydroxytyrosol, the flavonols apigenin and luteolin, the lignans (+) pinoresinol, (+) 1-acetoxypinoresinol and syringaresinol, two isomers of the aldehydic form of oleuropein and ligstroside, the dialdehydic form of oleuropein and ligstroside lacking a carboxymethyl group, and finally total hydroxytyrosol and total tyrosol reflecting the total amounts of free and esterified hydroxytyrol and tyrosol, respectively. The absolute amount of each phenolic constituent was determined in the polar fraction by using anhydrous 1,3,5-triazine as an internal standard.     

Separation and identification of the Z and E isomers of 2-(3-pentenyl)pyridine by capillary electrophoresis and nuclear magnetic resonance spectroscopy

A mixture of the Z and E isomers of 2-(3-pentenyl)pyridine has been separated with baseline resolution by capillary electrophoresis. Using molecular modelling it was proposed that the smaller more rapidly migrating peak would be the Z isomer. This agreed with a 38:62 (Z/E) composition by nuclear magnetic resonance spectroscopy. The sample was also investigated by gas chromatography coupled to mass spectrometry.     

Determination of enantiomer separation factors by nuclear magnetic resonance spectroscopy and by chiral liquid chromatography

The equilibrium constants K+ and K- for formation of the diastereomeric complexes of the two enantiomers of O,O'-dibenzoyltartaric acid (DBTA) with the chiral selector N,N'-diallyltartardiamide bis-(4-tert.-butylbenzoate) (TBB) have been determined by 1H-NMR. The experiments were performed at different temperatures in CDCl3 or in cyclohexane-d12/2-propanol-d8 mixtures. The equilibrium constants from the 1H-NMR results have been compared with the retention factors (k') obtained from the chromatographic resolution of rac. DBTA on a Kromasil CHI-TBB column with the same solvents as mobile phases. A satisfactory correlation between the 1H-NMR data and the chromatographic data was found.     

Adducts of rhodium(II) tetraacylates with methionine and its derivatives: 1H and 13C nuclear magnetic resonance spectroscopy and chiral recognition

Complexation of rhodium(II) dimeric tetraacylates: tetraacetate Rh₂AcO₄, tetratrifluoroacetate Rh₂TFA₄ , and (S)-Mosher’s acid salt Rh₂MTPA₄ with both enantiomerically pure and racemic methionine and its derivatives: hydrochloric salt of methionine, hydrochloric salt of methionine methyl ester, N-formyl methionine, N-phthaloyl methionine, N-phthaloyl methyl ester of methionine, and methyl ester of N,N-dimethylmethionine has been investigated by means of ¹H and ¹³C nuclear magnetic resonance (¹H and ¹³C NMR) and absorption electronic spectroscopy in the visible range. Complexation processes were investigated in D₂O or CDCl₃ solutions, depending on the ligands’ and rhodium salts’ solubilities. Some supporting measurements were performed in the solid phase, using ¹³C and ¹⁵N CPMAS NMR techniques.All ligands investigated form 1:1 and 1:2 adducts in the solution, depending on the rhodium salt to ligand molar ratios. The complexation site in the ligands (S atom) was deduced on the basis of the NMR parameter adduct formation shift (Δδ = δ_adduct ? δ_ligand) and calculated chemical shifts (DFT, NMR GIAO). In the cases of the Rh₂TFA₄ and Rh₂MTPA₄ adducts, decreasing the temperature within the range 220–254 K slowed down the ligand exchange and allowed us to observe the signals of all diastereoisomers in the ¹H and ¹³C NMR spectra.     

In situ complexation of rhodium(II) tetracarboxylates with some derivatives of cysteine and related ligands studied by 1H and 13C nuclear magnetic resonance spectroscopy

The complexation of N-phthaloyl, N-formyl, and N,N-dimethyl derivatives of S-methylcysteine methyl ester (both racemic and optically pure) with three dimeric rhodium(II) salts, acetate Rh₂AcO₄, trifluoroacetate Rh₂TFA₄, and (R)-(+)-α-methoxy-α-trifluoromethylphenylacetate Rh₂Mosh₄ was investigated by nuclear magnetic resonance spectroscopy (NMR) at room and lower temperatures. The complexation was carried out in situ, in CDCl₃ solution using titration procedure; the results were examined by the analysis of ¹H and ¹³C NMR chemical shift change (Δδ). The complexation of free S-methyl cysteine and hydrochloride salt of its methyl ester was performed in D₂O solution. For comparison, complexation of some derivatives of leucine, phenylalanine, and proline was examined.

N-phthaloyl and N-formyl derivatives of cysteine formed 1 : 1 and 1 : 2 axial complexes with all dirhodium salts. Rhodium substrates were bonded via sulfur. In one case, the complexation of Rh₂TFA₄ by both sulfur and N-formyl oxygen was noted. Similar complexation of Rh₂TFA₄, via CHO group, was found for N-formyl derivatives of leucine, phenylalanine, and proline. For N,N-dimethyl derivative of cysteine, both N and S atoms were involved in bonding. At room temperature, in all cases, ligand exchange was fast on the NMR timescale.     

1H magnetic resonance spectroscopy of some substituted acetophenones

Measurements are reported on the nuclear magnetic resonance spectra of the acetyl protons of a series of substituted acetophenones. Although the extreme values of the chemical shifts, δ for the meta- and para- substituted compounds differ only by 0·2 units, the values themselves are linearly related to the Hammett substituent constants. No such relationship exists for the ortho-substituted compounds. The magnitude of the chemical shift is independent of substrate concentration over a five-fold variation.     

Characterization of sodium laureth sulfate by reversed-phase liquid chromatography with evaporative light scattering detection and H nuclear magnetic resonance spectroscopy

A direct and effective method utilizing reversed-phase liquid chromatography combined with evaporative light scattering detection was developed to determine the relative ratio of different alkyl chain lengths, to quantify the average ethylene oxide (EO) level and to identify EO distribution in the presence of sodium laureth sulfates. A C8 bonded silica gel column and an acetonitrile–water gradient mobile phase containing ammonium acetate were used as the best stationary and mobile phase, respectively. The results were confirmed by nuclear magnetic resonance spectroscopy. The detection limit was 80 μg/mL and the calibration curve, i.e., the log–log plots (peak area vs. concentration), was linear in the working range of 80–4200 μg/mL with R² values of above 0.999 (in the case of 3 mol sodium laureth sulfates). Furthermore, the application of the chromatographic method to a commercial product without pretreatment was presented. The raw material was identified by fast atom bombardment mass spectrometry.     

Union of capillary high-performance liquid chromatography and microcoil nuclear magnetic resonance spectroscopy applied to the separation and identification of terpenoids

This paper describes the first coupling of a commercial capillary HPLC system with a diode array spectrophotometric detector and a custom-built nuclear magnetic resonance (NMR) flow microprobe. The eluent from a 3-microm diameter C18 HPLC column is linked to a 500 MHz 1H-NMR microcoil probe with an observe volume of 1.1 microl. The separation and structurally-rich detection of a mixture of terpenoids under both isocratic and gradient solvent elution conditions is presented. The lowest limits of detection yet reported for capillary HPLC on-line measurement (i.e., 37 ng for alpha-pinene) are achieved with this system. The complementary nature of diode array and NMR detection allows stopped-flow data collection from analytes which would otherwise go unnoticed in continuous-flow NMR. Moreover, stopped-flow NMR data is presented for the detection of a trace (sub-nmol) impurity in the sample mixture. Since NMR signals degrade and shift during solvent gradients, flow injection analysis studies are conducted with injected solvent plugs differing in mobile phase composition. The NMR signal degradation accompanying these injections is largely due to the variance in chemical shift with the solvent composition rather than to changes in magnetic susceptibility of the solvent. Characterization of such effects enables the development of improved NMR probes for the coupling of capillary HPLC and NMR.     

Conformational temperature dependence of a poly(ethylene-co-acrylic acid) stationary phase investigated by nuclear magnetic resonance spectroscopy and liquid chromatography

A polymer-based RP sorbent was prepared by immobilizing a poly(ethylene-co-acrylic acid) copolymer with an acid mass fraction of 5% on silica by using a 3-glycidoxypropyl linkage. 13C cross-polarization/magic angle spinning NMR spectroscopy of the sorbent, either in the dry state or suspended in the mobile phase, showed an increase in mobility at elevated temperatures. Alkyl chain segments with gauche conformations were more mobile than chain segments with trans conformations. The strength of the 13C-1H dipolar couplings in the alkyl chains was measured using the constant time dipolar and chemical shift pulse sequence, revealing less molecular motion for the trans conformation. Non-linear van't Hoff plots were observed for separations of shape-constrained solutes (such as geometric beta-carotene isomers and polycyclic aromatic hydrocarbons). At higher temperatures, the retention behavior was similar to that of monomeric C18 sorbents, whereas at ambient and lower temperatures, enhanced shape-selective properties were exhibited similar to those of polymeric C30 sorbents.     

Soybean biodiesel methyl esters,free glycerin and acid number quantification by 1H nuclear magnetic resonance spectroscopy

Production of alternative fuels, such as biodiesel, from transesterification of vegetable oil driven by heterogeneous catalysts is a promising alternative to fossil diesel. However, achieving a successful substitution for a new renewable fuel depends on several quality parameters. ¹H NMR spectroscopy was used to determine the amount of methyl esters, free glycerin and acid number in the transesterification of soybean oil with methanol in the presence of hydrotalcite‐type catalyst to produce biodiesel. Reaction parameters, such as temperature and time, were used to evaluate soybean oil methyl esters rate conversion. Temperatures of 100 to 180 °C and times of 20 to 240 min were tested on a 1 : 12 molar ratio soybean oil/methanol reaction. At 180 °C/240 min conditions, a rate of 94.5 wt% of methyl esters was obtained, where free glycerin and free fatty acids were not detected. Copyright © 2012 John Wiley & Sons, Ltd.