Orthogonal Separation System Research Articles

Nanoparticle Analysis by Online Comprehensive Two-Dimensional Liquid Chromatography combining Hydrodynamic Chromatography and Size-Exclusion Chromatography with Intermediate Sample Transformation

Polymeric nanoparticles have become indispensable in modern society with a wide array of applications ranging from waterborne coatings to drug-carrier-delivery systems. While a large range of techniques exist to determine a multitude of properties of these particles, relating physicochemical properties of the particle to the chemical structure of the intrinsic polymers is still challenging. A novel, highly orthogonal separation system based on comprehensive two-dimensional liquid chromatography (LC × LC) has been developed. The system combines hydrodynamic chromatography (HDC) in the first-dimension to separate the particles based on their size, with ultrahigh-performance size-exclusion chromatography (SEC) in the second dimension to separate the constituting polymer molecules according to their hydrodynamic radius for each of 80 to 100 separated fractions. A chip-based mixer is incorporated to transform the sample by dissolving the separated nanoparticles from the first-dimension online in tetrahydrofuran. The polymer bands are then focused using stationary-phase-assisted modulation to enhance sensitivity, and the water from the first-dimension eluent is largely eliminated to allow interaction-free SEC. Using the developed system, the combined two-dimensional distribution of the particle-size and the molecular-size of a mixture of various polystyrene (PS) and polyacrylate (PACR) nanoparticles has been obtained within 60 min.

Simultaneous determination of vitamins A, D3 and E in infant formula and adult nutritions by online two-dimensional liquid chromatography

A rapid method for the simultaneous determination of vitamins A, D3 and E in infant formula and adult nutritions has been developed using online two-dimensional liquid chromatography (2D-LC). First of all, C8 and polar embedded C18 columns were chosen as the first and second dimensional column respectively according to hydrophobic-subtraction model, which constituted excellent orthogonal separation system. The detection wavelengths were set at 263 nm for vitamin D3, 296 nm for vitamin E and 325 nm for vitamin A. The purification of vitamin D3 and quantifications of vitamins A and E were completed simultaneously in the first dimensional separation using the left pump of Dual Gradient LC (DGLC) with methanol, acetonitrile and water as mobile phases. The heart-cutting time window of vitamin D3 was confirmed according to the retention time of vitamin D3 in the first dimensional separation. The elute from the first dimensional column (1-D column) which contained vitamin D3 was collected by a 500 µL sample loop and then taken into the second dimensional column (2-D column) by the right pump of DGLC with methanol, acetonitrile and water as mobile phases. The quantification of vitamin D3 was performed in the second dimensional separation with vitamin D2 as internal standard. At last, this method was applied for the analysis of the three vitamins in milk powder, cheese and yogurt. The injected sample solution with no further purification was pre-treated by hot-saponification using 1. 25 kg/L KOH solution and extracted by petroleum ether solvent. The recoveries of vitamin D3 spiked in all samples were 75.50%-85.00%. There was no statistically significant difference for the results between this method and standard method through t-test. The results indicate that vitamins A, D3 and E in infant formula and adult fortified dairy can be determined rapidly and accurately with this method.

Separation of polyphenols from leaves of Malus hupehensis (Pamp.) Rehder by off-line two-dimensional High Speed Counter-Current Chromatography combined with recycling elution mode

In this study, off-line two-dimensional High Speed Counter-Current Chromatography (2D HSCCC) strategy combined with recycling elution mode was developed to isolate compounds from the ethyl acetate extract of a common green tea – leaves of Malus hupehensis (Pamp.) Rehder. In the orthogonal separation system, a conventional HSCCC was employed for the first dimension and two recycling HSCCCs were used for the second in parallel. Using a solvent system consisting of n-hexane–ethyl acetate–methanol–water (1:4:0.6:4.4, v/v) in the first and second dimension, four compounds including 3-hydroxy-phlorizin (1), phloretin (2), avicularin (3) and kaempferol 3-O-β-d-glucoside (4) were obtained. The purities of these four compounds were all over 95.0% as determined by HPLC. And their structures were all identified through UV, MS and 1H NMR. It has been demonstrated that the combination of off-line 2D HSCCC with recycling elution mode is an efficient technique to isolate compounds with similar polarities in natural products.

Systematic and efficient separation of 11 compounds from Rhizoma Chuanxiong via counter-current chromatography–solid phase extraction–counter-current chromatography hyphenation

A counter-current chromatography (CCC)–solid phase extraction (SPE)–CCC system with high preparative capacity was used to realize rapid one-run systematic separation of natural products, in which two six-port valves and the SPE cartridge served as the interface. In the orthogonal separation system, equal column volumes of TEB-300A and TEB-300B were employed for the first dimension (1st-D) and second dimension (2nd-D), respectively. An optimized solid-phase column (25mm×10mm i.d.) packed with Oasis HLB materials acted as the trapping SPE column. The analyte-focusing effect of the trapping column associated with the considerable preparative capacities of 1st-D and 2nd-D using totally different solvent systems significantly facilitated this one-run systematic separation of natural product. Therefore, this proposed approach was successfully applied to isolate chemical compounds from the crude extract of Rhizoma Chuanxiong. As a result, 11 compounds with widely different polarities were separated by running CCC for only one time. More importantly, this hyphenated strategy could serve as a rapid and efficient systematic pathway for the separation of natural products.

On‐line two‐dimensional LC: A rapid and efficient method for the determination of enantiomeric excess in reaction mixtures

A novel LC-based method for the determination of enantiomeric excess (ee) in a sample mixture has been developed by employing on-line 2D LC. The orthogonal separation system is composed of an NH2 column as the first dimension to elute the target chiral compound from the crude mixture and a chiral column as the second dimension to determine the ee of the target chiral product. A series of crude mixtures from asymmetric reactions have been directly analyzed without prepurification. Good reproducibility (intra- and interday precisions were all under 1.33%) and good accuracy (deviations from ee values determined by 1D HPLC were all <1.03%) have been obtained. Compared with the traditional method for the determination of ee, on-line 2D HPLC can be used in real time and holds great potential in the time-saving determination of ee in asymmetric synthesis.

Separation and purification of bovine serum albumin binders from Fructus polygoni orientalis using off-line two-dimensional complexation high-speed counter-current chromatography target-guided by ligand fishing

In this study, off-line two-dimensional (2D) complexation high speed counter-current chromatography (HSCCC) was developed for the separation of bovine serum albumin (BSA) binders from the ethyl acetate extract of Fructus polygoni orientalis. Target-guided strategy of BSA functionalized iron oxide magnetic nanoparticles coupled with high performance liquid chromatography–tandem mass spectrometry ((BSA-Fe3O4 MNPs)-HPLC–MS/MS) experiment was proposed. In the orthogonal separation system, a Normal-Phase HSCCC with 0.01mol/L copper ion as complexation agent in the aqueous phase was employed for the first dimension and Recycling HSCCC, Reverse-Phase HSCCC with 0.1mol/L copper ion were used for the second dimension in parallel. Including two pairs of cis-trans isomers, seven BSA binders including 3,5,7-Trihydroxychromone (1), taxifolin (2), N-cis-paprazine (3), N-cis-feruloyltyramine (4), N-trans-paprazine (5), N-trans-feruloyltyramine (6) and an unidentified compound (7) were obtained. The purities of these seven compounds were all over 95.0% as determined by HPLC. The complexation HSCCC behaviors of seven compounds were also investigated by studying their relationship with copper ion. Results showed that the combinative method using (BSA-Fe3O4 MNPs)-HPLC and HSCCC is a quick, efficient, and reproductive technique to isolate potentially bioactive compounds from the complex mixture system of natural products. And the usage of off-line 2D-HSCCC and introduction of chelating metal ion into solvent system are effective ways to implement HSCCC separations in complex samples.

Separation and identification of flavonoids from complex samples using off-line two-dimensional liquid chromatography tandem mass spectrometry

In this study, a LC-based identification strategy was proposed and off-line two-dimensional liquid chromatography was developed for the separation and identification of flavonoids from complex samples. The highly orthogonal separation system was composed of a CD column, an OEG column and an XTerra C(18) column. The CD column was employed for the first dimensional separation and the OEG column and XTerra C(18) column were used for the second dimensional separation in parallel. A mixture of six traditional Chinese medicines was used as a complex sample in testing this method. Unknown peaks were identified by matching their retention times and accurate mass molecular weights with those of references. The identification efficacy of parallel unidimensional liquid chromatography and off-line two-dimensional liquid chromatography was compared, and the latter was demonstrated to be more efficient for the identification of target compounds-especially co-eluted compounds or minor compounds from complex samples.

Characterization and analysis of soil humic acids by off-line combination of wide-pore octadecylsilica column reverse phase high performance liquid chromatography with narrow bore column size-exclusion chromatography and fluorescence detection

Liquid chromatography method arranged around RP-HPLC using step-wise gradients of dimethylformamide (DMF) in pH 3.0 aqueous phosphate buffered mobile phase and a wide-pore (30 nm) 4mm inner diameter (I.D.) octadecylsilica column was applied to the fractionation separation of soil and peat humic substances (HS), respectively. Combination of acceptable DMF solvating properties for HSs together with wide pore RP sorbent improved surface interactions of the analytes and suppressed influence of size-exclusion effects in RP-HPLC. Individual fractions collected from the RP-HPLC were off-line analyzed by size-exclusion chromatographic method (SEC) using 99/1 DMF/aqueous phosphate buffer pH 3.0 with Spheron HEMA 100 stationary phase filled in a 2.2 mm I.D. column. Both methods provided reproducibility of characterization profiles and robustness resulting from excellent reproducibility of HSs fraction retention times (±0.5% RSD) of peaks enforced by the step gradient shape. Obtained results indicate that the methods can be combined in a compatible, compact, automatic, 2D orthogonal separation system for fluorimetric characterization of such complicated natural substances as are examined humic acids and obtain so more information about their character.

A new method for chemical identification based on orthogonal parallel liquid chromatography separation and accurate molecular weight confirmation

Recent advances in the theory and application of orthogonal LC separation have allowed for the establishment of a more effective method for the chemical identification of target compounds in complex samples, especially structurally similar compounds. In this study, a new chemical identification method based on orthogonal parallel separation and accurate molecular weight confirmation was developed. An orthogonal separation system consisting of an XTerra MS C 18 column, a home-made Click OEG column, and a Click CD column was established for separation and identification. In addition, 82 flavonoids were selected as references, to be used for the construction of a library. Retention times of each reference flavonoid on each column and accurate molecular weights were recorded and imported into a searchable library as “tags” for the unknown screening. For the method validation, two complex mixtures, fractions of Dalbergia odorifera T. Chen and Scutellaria baicalensis Georgi, specifically, were separated and identified. In total, nine compounds were unequivocally identified by retention time and confirmation of accurate molecular weight, demonstrating that this method is suitable and efficient for the chemical identification of complex samples.

Comprehensive two-dimensional gas chromatography: a hyphenated method with strong coupling between the two dimensions

Comprehensive two-dimensional gas chromatography (GC×GC) provides a true orthogonal separation system. It is explained and demonstrated that it generates a peak capacity that is approximately equal to the product of the peak capacities of the two individual separation systems. The resulting peaks are ordered in a two-dimensional plane in bands of compounds with the same characteristics. Quantitation of the separated (groups of) components is fundamentally not different from one-dimensional gas chromatography, but the sensitivity is far better and true baseline is always available. The two co-ordinates of each peak in the plane make the identification more reliable. Instrumental considerations of GC×GC are discussed. The three designs of contemporary GC×GC systems are presented and compared. Although the technique is still very young, a number of applications on complex samples as petroleum and environmental samples have already been reported. Finally, the future perspectives of GC×GC are discussed.

The wave equation and separation of variables on the complex sphere S4

It is shown that every orthogonal separable coordinate system for the Helmholtz equation on S 4 leads to an R-separable system for the complex wave equation. All orthogonal separable systems on S 4 are classified and each is characterized by a commuting triplet of operators from the enveloping algebra of o(5). A consequence of the classification is that the most general cyclidic coordinates for the wave equation arise from ellipsoidal coordinates on S 4.