Analysis of global components in Ganoderma using liquid chromatography system with multiple columns and detectors

In present study, a multiple columns and detectors liquid chromatography system for analysis of global components in traditional Chinese medicines was developed. The liquid chromatography system was consist of three columns, including size exclusion chromatography column, hydrophilic interaction chromatography column, and reversed phase chromato‐graphy column, and three detectors, such as diode array detector, evaporative light scattering detector, and mass spectrometry detector, based on column switching technique. The developed multiple columns and detectors liquid chromatography system was successfully applied to the analysis of global components, including macromolecular (polysaccharides), high (nucleosides and sugars)‐, and low (triterpenes)‐polarity small molecular compounds in Ganoderma, a well‐known Chinese medicinal mushroom. As a result, one macromolecular chromatographic peak was found in two Ganoderma species, 19 components were identified in Ganoderma lucidum (two sugars, three nucleosides, and 14 triterpenes), and four components (two sugars and two nucleosides) were identified in Ganoderma sinense. The developed multiple columns and detectors liquid chromatography system was helpful to understand comprehensive chemical characters in TCMs.     

High-resolution separation of polychlorinated biphenyls by comprehensive two-dimensional gas chromatography

Comprehensive two-dimensional gas chromatography (GC x GC) with micro electron-capture detection (microECD) has been optimised for the separation of polychlorinated biphenyl congeners with emphasis on the separation of 12 toxic non- and mono-ortho chlorinated biphenyls (CBs), viz. CBs 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169 and 189. The selection of the first- and second-dimension columns and the temperature programme optimisation were carried out with a mixture of 90 CBs and the results are compared with those of one-dimensional GC. A complete separation of all 12 priority CBs was obtained with two column combinations, HP-1-HT-8 and HP-1-SupelcoWax-10. With the HP-1-HT-8 column set, ordered structures show up in the two-dimensional plane, with the number of chlorine substituents and their position (ortho vs. non-ortho) being the main parameters of interest. This can help with congener identification. Estimated detection limits are excellent, i.e. about 10 fg. To illustrate the potential and the versatility of GC x GC-microECD, a cod liver extract and a standard mixture of the 17 most toxic polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans together with 90 CBs were analysed as an application.     

Porous structure of the monolithic sorbent and separation properties of monolithic capillary columns in gas and liquid chromatography

Macroporous polymer based on polydivinylbenzene was used for the preparation of monolithic capillary columns with the diameter from 0.01 to 0.53 mm for separations by gas and liquid chromatography. The separation properties of the columns were studied by analysis of model systems of aromatic (in liquid chromatography) and light (in gas chromatography) hydrocarbons. The permeability was determined and the C parameter of the Van-Deemter equation was found for each column. The permeability of the majority of columns determined by gas chromatography is independent of the column diameter. The permeability of the same columns in liquid chromatography is also almost constant for the columns 0.53–0.1 mm in diameter; however, the permeability decreases sharply on going to columns of smaller diameter. In gas chromatography the value of the C parameter reflecting the effect of the mass transfer of the sorbate between the mobile and stationary phases on the smearing of a chromatographic peak in the column approximately the same for all columns. In liquid chromatography the value of the C coefficient in the Van-Deemter equation for the same capillary columns changes with a change in the column diameter and reaches a minimum for the columns 0.1 mm in diameter. The differences observed for the characteristics of the columns in gas and liquid chromatography are due to different structures of the macroporous monolith formed in columns of different diameter and to the effect of solvation of the monolith by the mobile phase under the conditions of liquid chromatography.     

Observations of focal conic domains in smectic liquid crystals aligned on patterned self‐assembled monolayers

Patterned Self‐Assembled Monolayers (SAMs) promoting both homeotropic and planar degenerate alignment of nCBs in their smectic‐A phase were created using microcontact printing of functionalized organothiols on gold films. By patterning the surface with homeotropic and planar aligning SAMs, the location and formation of the focal conic domains (FCDs) can be controlled. Polarizing microscopy was used to study the formation of FCDs in circle, stripe and checkerboard pattern geometries. Fluorescent confocal microscopy (FCM) was used for the first time to measure the eccentricity of FCDs that form along a stripe pattern.     

Evaluation of gas chromatographic methods for the determination of <Emphasis Type="Italic">trans</Emphasis> fat

Consumption of trans fat has been associated with increased risk of coronary heart disease. For nutrition labeling purposes, the US Food and Drug Administration (FDA) defines trans fat as the sum of all the fatty acids with at least one nonconjugated double bond in the trans configuration. The FDA regulation states that label declarations of trans fat are not required for products that contain less than 0.5 g of trans fat per serving if no claims are made about fat, fatty acids or cholesterol. While attenuated total reflection Fourier-transformed infrared spectroscopy (ATR-FT-IR) provides reproducible measurements for samples containing more than 5% trans fat, methods based on gas chromatography (GC) are needed to measure lower trans fat levels. Trans fat quantitation by GC has recently been updated by considering more fatty acids, focusing more attention on fatty acids present in low amounts, and by using 100-m high-polarity capillary columns for optimal separation. The consistently high interlaboratory relative standard deviations (RSD, e.g., 21% at 1% trans fatty acids (TFA), 60% at 0.17% TFA), and intralaboratory RSD values (e.g., 10% at 1% TFA, 16% at 0.17% TFA) for trans fat at 1% or less of total fat reported in the collaborative study data for American Oil Chemists Society Official Method Ce 1h-05 suggest the need to carefully define the parameters associated with GC analysis of fatty acids.     

Determination of mono-ortho substituted chlorobiphenyls by multidimensional gas chromatography and their contribution to TCDD equivalents

The mono-ortho chlorobiphenyls (CBs) 60, 74, 114, 123, 157, 167 and 189 were determined in Aroclor mixtures and aquatic organisms by multidimensional gas chromatography with electron capture detection (MDGC/ECD), using a combination of an Ultra 2 and an FFAP column. MDGC/ECD is recommended as the most suitable technique for direct determination of these CBs, without a liquid chromatographic (LC) pre-separation of mono-ortho CBs from the other CBs. Dependent, to some extent, on the stationary phase used, single-column determinations of these CBs easily yield too high results due to the presence of interferences. The contribution of the mono-ortho CBs studied to the total 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs) from CBs in fish is highly dependent on the toxic equivalency factors (TEFs) used and varies between 0.02 and 22%. This contribution is mainly due to CBs 74, 114, 157 and 167 which may easily be determined by taking three heart-cuts and combining them into one MDGC/ECD run. The analytical error is negligible compared with the huge uncertainty of the TEFs. A need for more precise TEFs is emphasized.     

Evaluation and application of a mixed‐mode chromatographic stationary phase in two‐dimensional liquid chromatography for the separation of traditional Chinese medicine

In this study, two mixed‐mode chromatography stationary phases (C8SAX and C8SCX) were evaluated and used to establish a two‐dimensional liquid chromatography system for the separation of traditional Chinese medicine. The chromatographic properties of the mixed‐mode columns were systematically evaluated by comparing with other three columns of C8, strong anion exchanger, and strong cation exchanger. The result showed that C8SAX and C8SCX had a mixed‐mode retention mechanism including electrostatic interaction and hydrophobic interaction. Especially, they were suitable for separating acidic and/or basic compounds and their separation selectivities could be easily adjusted by changing pH value. Then, several off‐line 2D‐LC systems based on the C8SAX in the first dimension and C8SAX, C8SCX, or C8 columns in the second dimension were developed to analyze a traditional Chinese medicine—Uncaria rhynchophylla. The two‐dimensional liquid chromatography system of C8SAX (pH 3.0) × C8SAX (pH 6.0) exhibited the most effective peak distribution. Finally, fractions of U. rhynchophylla prepared from the first dimension were successfully separated on the C8SAX column with a gradient pH. Thus, the mixed‐mode stationary phase could provide a platform to separate the traditional Chinese medicine in practical applications.     

Determination of chlorobenzenes in pure,tap, and sea water by static headspace gas chromatography‐electron capture detection

A simple, efficient, solvent‐free, and commercial readily available approach for determination of 11 chlorobenzenes (CBs) in water samples using the static headspace (HS) sampling and gas chromatography‐electron capture detector has been described. The proposed static HS sampling method was initially optimized and the optimum experimental conditions found were 10 mL water sample containing 20% (w/v) sodium chloride placed in a 20 mL vial and stirred at 70°C for 30 min. The linearity of the method ranged from 0.16 to 8.0 μg/L for dichlorobenzene isomers, 0.0176～0.88 μg/L for trichlorobenzene isomers, 0.004～0.2 μg/L for tetrachlorobenzene isomers, and from 0.001 to 0.05 μg/L for pentachlorobenzene and hexachlorobenzene, with correlation coefficients ranging between 0.9992 and 0.9999. The limits of detection were in the low μg/L level, ranging between 0.0002 and 0.04 μg/L. The relative recoveries of spiked CBs with external calibration or standard addition method at different concentration levels in pure, tap, and sea water samples were 83～116%, 89～108%, and 93～112%, respectively, and with relative standard deviations of 1.9～6.3%, 1.6～5.4%, and 2.5～5.7% (n = 5), respectively. It is concluded that this method can be successfully applied for the determination of CBs in pure, tap, and sea water samples.     

Comprehensive two-dimensional gas chromatography of the 209 polychlorinated biphenyls

Comprehensive two-dimensional gas chromatography (GC x GC) of the 209 polychlorinated biphenyls (CBs) was carried out using a longitudinally modulated cryogenic system (LMCS) and liquid carbon dioxide as cryogen. The effluent from a non-polar column was modulated and further separated on either a polar or a shape-selective second-dimension column. Five GC x GC column combinations were evaluated, with DB-XLB as the first column in each case. DB-XLB separates more congeners than any other GC column currently available. When combined with a biscyanopropyl siloxane (SP-2340 or BPX70) or smectic liquid crystal (LC-50) second-dimension column in a GC x GC system many additional CBs can be separated. In total, 176 and 181 of the 209 congeners were separated (Rs = 0.5) on the column combinations DB-XLB/SP-2340 and DB-XLB/LC-50, respectively. Of the 136 CBs present in any Aroclor mixture at concentrations greater than 0.05% (w/w), 126 were resolved using either of these two column combinations. The seven frequently measured CBs 28, 52, 101, 118, 138, 153, 180, and the WHO-PCBs 77, 81, 105, 114, 118, 123, 126, 156. 157, 167, 169 and 189 were all separated from Aroclor CBs on the DB-XLB/LC-50 column set, whereas CBs 118 and 131 coeluted on the DB-XLB/SP-2340 column set. In addition, three technical CB formulations (Aroclors 1232, 1248 and 1260) and a seal blubber sample (Halichoerus grypus) from the Baltic Sea were analyzed. Similar peak patterns were found for Aroclor 1260 and the CBs in the seal blubber, facilitating use of this technical CB formulation to identify the CBs in the blubber by GC x GC. Individual CBs in environmental samples, such as seal blubber, may be identified semi-automatically by matching the samples GC x GC profiles to a template generated using a standard containing all 209 CBs. Using such a template, 64 CBs were identified in the grey seal blubber sample.     

Advances in the liquid chromatography of hop bitter compounds

High efficiency liquid chromatography (LC) on both coupled conventional columns and long micro-LC columns, both with diode array detection, has revealed the presence of some hitherto unknown compounds in hop extracts. Proof is advanced that ethanol extracts of hops contain iso-alpha acids. The elution order of the cis-trans pairs of the iso-alpha acids, and the separation of the alpha acids from the deoxyalpha acids, is reversed by switching from methanol to acetonitrile in the reversed phase solvent system. An LC solvent system giving higher resolution (higher selectivity factor) of the iso-alpha acid cis and trans stereoisomers is obtained by adding magnesium salts to the eluent.     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.     

Determination of atropisomeric and planar polychlorinated biphenyls, their enantiomeric fractions and tissue distribution in grey seals using comprehensive 2D gas chromatography

High prevalence of uterine occlusions and sterility is found among Baltic ringed and grey seal. Polychlorinated biphenyls (CBs) are suspected to be the main cause. The CB concentrations are higher in affected than in healthy animals, but the natural variation is considerable. Thus, it might be possible to assess the health status of seals by CB analysis. The ratios of chiral compounds (enantiomeric fractions (EFs)) such as atropisomeric CBs are of particularinterest, since these may reflect differences in metabolic rates. An analytical procedure was developed and used to determine the levels of atropisomeric CBs, planar-CBs (WHO-PCBs) and total CBs in seals of different health status. Comprehensive 2D gas chromatography (GC x GC) was used to separate the target analytes from other CBs and interferences and a micro electron-capture detector (microECD) was used for detection. EFs of the atropisomeric CBs were difficult to determine as the levels were low and the interferences many. Two column combinations bad to be used to avoid biased results-both had a chiral column as first-dimension column. The second-dimension column was coated with either a high-polarity cyanopropyl or a liquid crystal phase. EFs were determined for five atropisomeric CBs, i.e. CBs 91,95, 132, 149 and 174. The results were verified by GC x GC-time-of-flight mass spectrometry (TOF-MS). Some atropisomeric CBs had EFs that deviated strongly from the racemic-mixture value. The deviations were larger in liver than blubber, which indicates enantioselective metabolism. However, there was no selective passage of the studied atropisomeric CBs across placenta and no selective blood-brain barrier. Similarly, no correlation between EFs and health status was observed, although there was a correlation between the total CB levels and health status.     

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

Applications of hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs, i.e., glycosylated proteins represented by monoclonal antibodies are discussed in the manner of glycoproteomics. They can be analyzed using hydrophilic interaction chromatography in five different stages as (1) their intact forms, (2) their subunits, (3) N‐ and O‐glycopeptides digested by proteases, (4) N‐ and O‐glycans released from the glycoproteins or glycopeptides, and (5) monosaccharides. Hydrophilic interaction chromatography is a more useful tool in the order of (1) to (5). At the stages (4) and (5), quantitation of glycans and saccharides are also reported. Hydrophilic interaction chromatography is employed not only for analytical uses, but also pretreatment items as solid phase extraction, followed by reversed‐phase liquid chromatography separations. Comprehensive search results of these application of hydrophilic interaction chromatography are summarized in tables to show what kind of hydrophilic interaction chromatography columns are suitable for each step of analysis.Relationship of favored and less favored hydrophilic interaction chromatography columns and their separation characteristics such as hydrophilicity, and selectivity for structural difference, is also discussed. Analysis of the therapeutic peptides (not glycosylated) using hydrophilic interaction chromatography is summarized, too.     

Turbidimetric titration and gradient high-performance liquid chromatography of polystyrene samples

Summary Polystyrene samples of narrow molecular-weight distribution have been eluted according to their molecular weight from columns packed with bare silica Si50, phenyl, or C18 bonded phase by gradients of methanol and tetrahydrofuran (THF) or ofiso-octane and THF. Among the six combinations investigated,iso-octane/THF with a silica column formed a proper normal-phase system whereas methanol/THF with a C18 column formed a proper reversed-phase system. The combinations of C18 column andiso-octane/THF or of Si50 column and methanol/THF gradient did not correspond to the approved polarity rules in high-performance liquid chromatography but were nevertheless effective in separating polystyrene mixtures by molecular weight. Methanol andiso-octane are nonsolvents for polystyrene whereas THF is a solvent. The solubility of polystyrene as a function of molecular weight and concentration was determined by means of turbidimetric titration of solutions in THF with the nonsolvents used in the gradients. The solubility and elution characteristics were almost identical on C18 columns or in methanol/THF combinations. The elution from phenyl bonded phase and Si50 columns usingiso-octane/THF gradients required more THF than the solubility experiments. Information is also given on the occurrence of multimodal elution patterns.     

Multidimensional capillary gas chromatography of polychlorinated biphenyl marker compounds

Multidimensional chromatography was used to resolve the specific chlorobiphenyl (CB) congeners 28, 52, 101, 118, 138, 153, and 180 in technical aroclor standards. Single column analysis of polychlorinated biphenyls (PCBs) results in co-elution of key congeners with other components in the mixture; therefore using two columns of different selectivity was necessary to enhance the resolution achievable on just one column. The HT8 column (8% phenylpolycarborane-siloxane phase) has been reported to have specific selectivity characteristics for improved PCB separation. When coupled with a BPX5 column (5% phenylpolysiloxane-silphenylene phase), it has been shown here to provide unambiguous identification of 7 marker compounds which are used to monitor PCB occurrence and distribution. All seven marker CBs are present in aroclor 1254, and by adjusting the size of the heartcut window, it was possible to obtain resolution of the marker congeners from other congeners. Single column analysis is unable to achieve this result. This offers an alternative to GC-MS analysis.     

Optimization of the mobile phase conditions for the purification ofTaql endonuclease

Summary Taql endonuclease was purified by high performance ion exchange liquid chromatography with linear gradient elution. Of the chromatographic media tested as mobile phases for the HPLC purification ofTaql endonuclease, sodium acetate (pH 5.0) and phosphate (pH 7.0) buffers were appropriate for use with cation exchange columns, and L-histidine (pH 6.0) and Tris (pH 8.0) buffers with anion exchange columns.     

Some theoretical considerations on preparative separation with orthogonal pressurised planar electrochromatography

In this work, separation of multicomponent mixtures containing components with the same and different electrophoretic mobility by using orthogonal pressurised planar electrochromatography is studied theoretically. Additionally, a simple way for determination of a maximum amount of mixture causing volume overload of flat/planar columns used in this technique is presented. In the next stage, effects of change in different parameters on process performance by simulation case studies are investigated. A comparative study of separation productivity of orthogonal pressurised planar electrochromatography with the continuous and periodic modes of mixture delivery and column chromatography is carried out.     

The development of an evaluation method for capture columns used in two-dimensional liquid chromatography

Capture columns are important interface tools for on line two-dimensional liquid chromatography (2D-LC). In this study, a systematic method was developed to evaluate and optimize the capture ability of capture columns by off-line method. First, the parameter Δt_R (Δt_R = t₂−t₁−t₀−W) was introduced to quantitatively represent the capture ability of the capture column by connecting a capture column behind the first dimensional column. Based on the value of Δt_R, an appropriate capture column was selected after the first dimensional column was fixed. Then, the capture ability of the selected column was promoted by adjusting the mobile phase of the first dimensional column. Capture ability was also optimized using complex sample analysis software system (CSASS) software. Second, the elution mode of the trapped compounds on the capture column was investigated by connecting the capture column before the second dimensional column. More specifically, in mode I, capture column was connected to the second dimension without changing the flow rate direction and the trapped compounds must pass through the capture column and be eluted into the second dimensional column. The contrary connection mode was mode II. It was found that mode I is more suitable method for 2D-LC. Finally, an off-line reversed-phase/hydrophilic interaction liquid chromatography two-dimensional liquid chromatography (RP/HILIC 2D-LC) system with a C18 capture column was developed to demonstrate the practical application of this method.     

Application of ultra-performance columns in high-performance liquid chromatography for determination of albendazole and its metabolites in turkeys

Methods for determination of albendazole (ALB), albendazole sulfoxide (SOX) and albendazole sulfone (SON) in turkey blood plasma, using high‐performance liquid chromatography (HPLC) with fluorescence detection, were developed. Moreover, comparison of HPLC columns with ultra‐performance liquid chromatography (UPLC) columns was performed. Albendazol was administered orally in 5‐week‐old birds (n = 18) at a dose of 25 mg/kg b.w. Accuracy and precision of the developed method were satisfactory and stability studies showed acceptable variation (below 15%) in ALB, SOX and SON concentrations when the samples were stored at –75°C for 15 days. UPLC® columns gave higher peaks from typical HPLC columns retaining high quality of analysis. Pharmacokinetic analysis indicated quick elimination of ALB from turkey blood plasma. The mean residence time of SON was at least two times longer than that of SOX and four times longer than that of ALB. The elimination half‐lives for ALB, SOX and SON were 0.7 ± 0.27, 5.37 ± 6.03, 9.17 ± 5.12 h, respectively. The obtained results indicate that the described method allows for precise determination of albendazole and its metabolites in turkey plasma. Moreover, using UPLC columns in HPLC apparatus results in higher sensitivity as compared with the classical HPLC columns. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison of packed and capillary columns for quantitative gas chromatography of triglycerides in milk fat

Summary Quantitative gas chromatography of triglycerides in conjunction with established triglyceride formulae can be used to determine various milk fat parameters. Since the evaluation of, for example, iodine number or content of non-milk fats (foreign fats) in milk fat requires only the separation of triglycerides by carbon number and since repeatabilities, especially of the highboiling triglycerides, have been less acceptable with capillary columns in the past, packed columns have been used exclusively. There is, however, an increasing demand for the use of capillary instead of packed columns and to that end the present investigation has been carried out. To achieve a suitable resolution, with this particularly exacting high-temperature application, a short 5 m capillary column of extreme temperature stability has been used. As well as modification of various analytical conditions different injection techniques have been investigated. On-column, PTV and split injection were compared with regard to repeatability. The cold-on-column injection technique was found to produce the best results, being comparable to the excellent precision of a packed column. Thus, a method is now available by means of which the determination of milk fat parameters by the triglyceride formulae, such as the amount of foreign fats in milk fat, can now be carried out with a standardized capillary column.