Fast GC analysis with a 50 microm ID column: theory, practical aspects, and application to a highly complex sample

This research focuses on the minimization of GC analysis times through the use of a 5 m x 0.05 mm ID x 0.05 microm (film thickness) column. Experimental minimum plate height (Hmin) and optimum linear velocity values were derived from standard compound applications, under various analytical conditions, and then related to classical chromatographic theory. Deviations from the latter are measured and discussed. Practical aspects linked to the use of such capillaries, such as column sample capacity and detector acquisition rates, are also considered. Furthermore, a fast, and what can be considered a very fast method, were applied to the separation of a fuel sample. Coefficients of variation of elution times and relative peak areas were calculated in the very fast application. All analytical results are compared with those obtained by conventional 0.25 mm ID column applications.     

Fast capillary GC using a low thermal mass column oven for the determination of residual solvents in pharmaceuticals

A low thermal mass column oven was used for fast capillary GC analysis (high throughput) of residual solvents in pharmaceutical products. A dedicated capillary column, 20 m L x 180 microm ID x 1 microm DB-624 was programmed from 35 degrees C (30 s) to 150 degrees C at 100 degrees C/min and to 250 degrees C (30 s) at 200 degrees C/min, resulting in a total GC cycle time of less than 4 min. Complete separation of a target 20-component mixture was achieved, while method performance in terms of repeatability, sensitivity, and linearity was maintained in comparison to the generic method currently applied in our laboratories.     

Conventional and narrow bore short capillary columns with cyclodextrin derivatives as chiral selectors to speed-up enantioselective gas chromatography and enantioselective gas chromatography-mass spectrometry analyses

The analysis of complex real-world samples of vegetable origin requires rapid and accurate routine methods, enabling laboratories to increase sample throughput and productivity while reducing analysis costs. This study examines shortening enantioselective-GC (ES-GC) analysis time following the approaches used in fast GC. ES-GC separations are due to a weak enantiomer-CD host-guest interaction and the separation is thermodynamically driven and strongly influenced by temperature. As a consequence, fast temperature rates can interfere with enantiomeric discrimination; thus the use of short and/or narrow bore columns is a possible approach to speeding-up ES-GC analyses. The performance of ES-GC with a conventional inner diameter (I.D.) column (25 m length x 0.25 mm I.D., 0.15 microm and 0.25 microm d(f)) coated with 30% of 2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl-beta-cyclodextrin in PS-086 is compared to those of conventional I.D. short column (5m length x 0.25 mm I.D., 0.15 microm d(f)) and of different length narrow bore columns (1, 2, 5 and 10 m long x 0.10 mm I.D., 0.10 microm d(f)) in analysing racemate standards of pesticides and in the flavour and fragrance field and real-world-samples. Short conventional I.D. columns gave shorter analysis time and comparable or lower resolutions with the racemate standards, depending mainly on analyte volatility. Narrow-bore columns were tested under different analysis conditions; they provided shorter analysis time and resolutions comparable to those of conventional I.D. ES columns. The narrow-bore columns offering the most effective compromise between separation efficiency and analysis time are the 5 and 2m columns; in combination with mass spectrometry as detector, applied to lavender and bergamot essential oil analyses, these reduced analysis time by a factor of at least three while separation of chiral markers remained unaltered.     

Comparison of the use of single capillaries and coupled capillaries based on micellar electrokinetic chromatography (MEKC) and sweeping-MEKC modes

The use of single capillaries (25 and 50 microm inner diameter (ID)) and coupled capillaries of different diameters (100-50 and 75-25 microm ID) based on micellar electrokinetic chromatography (MEKC) and sweeping-MEKC modes is compared and reported. Naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized dopamine was selected as the model compound by examining the fluorescence intensity when a violet (410 +/- 7 nm, 2 mW) light-emitting-diode (LED) was used as the light source. When a single capillary (50 microm ID) was used, the detection limit for NDA-derivatized dopamine was determined to be 2.0 x 10(-7) M (Signal-to-nose ratio S/N = 3) based on the MEKC mode. This was improved to 4.0 x 10(-9) M when the sweeping-MEKC mode was applied. In addition, this can be further improved to 1.0 x 10(-9) M and 5.6 x 10(-10) M when 100-50 and 75-25 microm ID coupled capillaries are used. The use of the coupled capillary is also helpful for improving the separation efficiency. Based on the sweeping-MEKC mode, the number of theoretical plates (N) for the detected peaks were determined to be 6.3 +/- 2.7 x 10(5) by means of a single capillary (50 microm ID). This can be improved to 9.4 +/- 3.6 x 10(5) and 9.4 +/- 0.9 x 10(6) when the 100-50 and 75-25 microm ID coupled capillaries were applied.     

Considerations on the possibilities and limitations of comprehensive normal phase-reversed phase liquid chromatography (NPLC x RPLC)

A comprehensive normal phase system LC-reversed phase LC (NPLC x RPLC) was evaluated for the separation of a pharmaceutical mixture and citrus oil extracts. NPLC was performed on a 25 cm x 1 mm ID x 5 microm dp diol phase. In the second dimension, an RP 18 monolithic column (10 cm L x 4.6 mm ID x 2 microm macropore size) and an octadecyl silicagel-packed column (5 cm L x 4.6 mm ID x 3.5 microm dp) were applied for the analyses of the pharmaceutical sample and the citrus oil extracts, respectively. A two-position/ten-port switching valve was used as interface. Under optimised LC conditions, the high degree of orthogonality between NP and RP resulted in peak capacities of 300 for the pharmaceutical sample and of 450 for the citrus oil extract composed of lemon and orange oil. Despite the features of NPLC x RPLC, several shortcomings related with the solvent incompatibility between the two LC modes were identified and the practical consequences were discussed.     

Capillary electrochromatography using fibers as stationary phases.

Fiber-packed capillary columns have been evaluated in chromatographic performance in capillary electrochromatography (CEC). The change of electroosmotic flow (EOF) velocity and selectivity using different kinds of fiber materials was examined. Although the EOF velocity among the different fiber packed columns was almost the same, retention of parabens was larger on the Kevlar-packed column than on the Zylon-packed one, and was larger on the as-span-type fiber-packed column than on the high-modulus-type packed one. Using 200 microm ID x 5 cm Kevlar packed column combined with a 100 microm ID x 20 cm precolumn capillary and a 530 microm ID x 45 cm postcolumn capillary, the separation of three parabens within 30 s was achieved. Other compounds were also separated in a few minutes by the fiber-packed CEC method.     

Fast separation of UV absorbing anions using ion-interaction chromatography

Ion-interaction chromatography on a short (30 x 4.6 mm) 3 microm ODS column has been investigated with the aim of developing fast chromatographic separations of selected inorganic anions. Tetrabutylammonium chloride (TBA-Cl) was used as the ion-interaction reagent in mobile phases that also contained up to 20% methanol. Separations of simple test mixtures of up to eight UV absorbing anions illustrated how excellent efficiencies (>50,000 plates/m) could be obtained under optimized conditions. The use of an optimised mobile phase containing 20 mM TBA-Cl and 20% methanol resulted in the baseline separation of five important anions (iodate, bromate, nitrite, bromide and nitrate) in a separation window of just 28 s, with a shortest total analysis time of 50 s. The method was briefly applied to the rapid analysis of nitrite and nitrate in both a drinking water and a river water sample with a view to future on-line monitoring.     

Fast analysis of decabrominated diphenyl ether using low-pressure gas chromatography-electron-capture negative ionization mass spectrometry

This paper reports the applicability of low-pressure gas chromatography-mass spectrometry operated in electron-capture negative ionization mode (LP-GC-ECNI-MS) for the analysis of decabrominated diphenyl ether (BDE-209). Particular attention was paid to find optimal injector and oven conditions for minimal thermal degradation of BDE-209. The analytical characteristics were compared for LP-GC columns (10 m x 0.53 mm) with different film thicknesses (d(f) 0.15 microm versus 0.25microm) and for a conventional GC column (15 m x 0.25 mm, 0.10 microm d(f)). Short residence times (6.5 and 9.8 min) of BDE-209 were found for the LP-GC systems with 0.15 and 0.25microm d(f), respectively, resulting in a low elution temperature and minimal degradation. Additionally, baseline separation of 22 polybrominated diphenyl ether (PBDE) congeners (major components of PBDE technical mixtures) was possible in less than 12 min using the LP-GC-ECNI-MS system with 0.15microm d(f). The optimized method was applied for the determination of PBDEs in Belgian indoor dust samples. The obtained concentrations of BDE-209 (range 8-292 ng/g dry weight) were in the same range or lower than concentrations in dust from other European countries.     

Comparison of sub-2-microm particle columns for fast metabolite ID

The use of sub-2-microm particle columns for fast high throughput metabolite ID applications was investigated. Three LC-MS methods based on different sub-2-microm particle size columns using the same analytical 3 min gradient were developed (Methods A, B, and C). Method A was comprised of a 1.8 microm particle column coupled to an MS, methods B and C utilized a 1.7 microm particle column (BEH 50 x 2.1 mm2 id) and 1.8 microm particle column coupled to a Q-TOF MS. The precision and the separation efficiency of the methods was compared with repeated standard injections (N=10) of reference compounds verapamil (VP), propranolol, and fluoxetine. Separation efficiency and MS/MS spectral quality were also evaluated for separation and detection of VP and its two major metabolites norverapamil (NVP) and O-demethylverapamil (ODMVP) in human-liver microsomal incubates. Results show that 1.8 microm particle columns show similar performance for separation of VP and its major metabolites and comparable spectral quality in MS(E) mode of the Q-TOF instrument compared to 1.7 microm particle columns. Additionally, the study also confirmed that sub-2-microm particle size columns can be operated with standard analytical HPLC but that performance is maximized by integrating column in UPLC method with reduced void volumes. All the methods are suitable for the determination of major metabolites for compounds with high metabolic turnover. The high throughput metabolite profile analysis using 384-well plate format of up to 48 compounds in incubates of human-liver microsomes was discussed.     

Separation of related opiate compounds using capillary electrochromatography

Capillary electrophoretic separations have been investigated for six controlled narcotic analgesic compounds having related structures. Owing to the similar charge-to-mass ratios of these compounds, capillary zone electrophoresis failed to provide a satisfactory separation, whereas a baseline-resolved separation was achieved in 10 min using micellar electrokinetic chromatography. Column efficiencies of 40,000-150,000 plates/m were obtained with a 50 cm long, 50 microm inner diameter (ID) capillary using 50 mM sodium dodecyl sulfate (SDS) in a 50 mM borate solution containing 12% isopropanol. In contrast, separation of this mixture by capillary electrochromatography proved to be significantly superior. The capillary was 15 cm long, with an ID of 75 microm, and was packed with 1.5 microm nonporous octadecyl silica (ODS) particles. The mobile phase consisted of 80% 10 mM tris(hydroxymethyl)aminomethane (Tris) and 20% acetonitrile, and contained 5 mM SDS. A complete separation was obtained in 2.5 min with an efficiency of 250,000-500,000 plates/m.     

Qualitative evaluation of thermal desorption-programmable temperature vaporization-comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry for the analysis of selected halogenated contaminants

The separation of 38 toxic and predominant polychlorinated biphenyl (PCB) congeners, 11 persistent halogenated pesticides, 1 brominated biphenyl (BB), and 8 polybrominated diphenyl ethers (PBDEs) has been optimized using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC x GC-TOFMS). A thermal desorption-programmable temperature vaporization (TD-PTV) step was used for the injection. Different column sets were investigated, and a 100% dimethylpolysiloxane (15 m x 0.25 mm i.d. x 0.25 microm film thickness) narrowbore capillary column coupled to a high temperature (8% phenyl)-polycarborane-siloxane (2 m x 0.10 mm i.d. x 0.10 microm film thickness) microbore column set was selected. Of the 58 compounds investigated, only one pair of PCBs was not resolved. All other analytes were either baseline separated into the chromatographic plane or were virtually separated using the deconvolution capability of the TOFMS.     

Ultra-fast essential oil characterization by capillary GC on a 50 microm ID column

A 5 m x 50 microm capillary column with 0.05 microm stationary phase film thickness, with a calculated efficiency of almost 20,000 plates per metre (under optimum conditions), was used for very fasthigh resolution GC analysis of lime essential oil. The total analysis time of this volatile essential oil was less than 90 s. Fast GC is shown to be appropriate for essential oil quality assurance analysis, and quantitative results of key components are comparable with those obtained by using conventional GC analysis. The fast GC analysis is approximately 33 times faster than the conventional GC method.     

Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat (Triticum aestivum L.) and related species with phosphate-based buffers

This study focused on optimizing phosphate-based buffers and other capillary electrophoresis (CE) parameters for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in bread wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), emmer (Triticum dicoccum, AABB, 2n = 4x = 28) and Aegilops tauschii (DD, 2n = 2x = 14). The fast and high-resolution separation of HMW-GS was achieved using 0.1 M phosphate-glycine buffer (pH 2.5, containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose) at 12.5 kV and 40 degrees C with 25 microm inside diameter (ID)x27 cm uncoated fused-silica capillary. In general, one sample separation can be analyzed in 15 min. The good run-to-run repeatable separation of HMW-GS could be obtained with a relative standard deviation of less than 1% when capillaries were rinsed with 1 M phosphoric acid for 2 min, followed by separation buffer for 2 min after each separation. The HMW-GS from some bread wheat cultivars as well as tetraploid and diploid accessions was separated by the CE method described above, and all subunits detected were well characterized and readily identified. Some HMW-GS showed reversed mobilities and elution order compared to the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-CE. Particularly, most of the HMW-GS analyzed with the CE buffer used were separated into multiple peaks, generally a high peak plus a minor peak. CE appears to be capable of separating and characterizing HMW-GS with fast and high-resolution features, therefore it is expected to be useful for specific germplasm screening and desirable HMW-GS identification in wheat quality improvement.     

Ruggedness and other performance characteristics of low-pressure gas chromatography-mass spectrometry for the fast analysis of multiple pesticide residues in food crops

Low-pressure gas chromatography-mass spectrometry (LP-GC-MS) using a quadrupole MS instrument was further optimized and evaluated for the fast analysis of multiple pesticide residues in food crops. Performance of two different LP-GC-MS column configurations was compared in various experiments, including ruggedness tests with repeated injections of pesticides in matrix extracts. The tested column configurations employed the same 3 m x 0.15 mm i.d. restriction capillary at the inlet end, but different analytical columns attached to the vacuum: (A) a 10 m x 0.53 mm i.d., 1 microm film thickness RTX-5 Sil MS column; and (B) a 10 m x 0.25 mm i.d., 0.25 microm film thickness DB-5MS column. Under the optimized conditions (compromise between speed and sensitivity), the narrower analytical column with a thinner film provided slightly (<1.1-fold) faster analysis of <5.5 min separation times and somewhat greater separation efficiency. However, lower detection limits for most of the tested pesticides in real extracts were achieved using the mega-bore configuration, which also provided significantly greater ruggedness of the analysis (long-term repeatability of analyte peak intensities, shapes, and retention times). Additionally, the effect of the increasing injection volume (1-5 microl) on analyte signal-to-noise ratios was evaluated. For the majority of the tested analyte-matrix combinations, the increase in sensitivity caused by a larger injection did not translate in the same gain in analyte detectability. Considering the costs and benefits, the injection volume of 2-3 microl was optimal for detectability of the majority of 57 selected pesticides in apple, carrot, lettuce, and wheat extracts.     

Optimization of separation and detection conditions for the multiresidue analysis of pesticides in grapes by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry

A comprehensive GCxGC-TOFMS method was optimized for multiresidue analysis of pesticides using a combination of a non-polar (RTX-5MS, 10 m x 0.18 mm x 0.2 microm) and a polar capillary column (TR-50MS, 1 m x 0.1 mm x 0.1 microm), connected in series through a dual stage thermal modulator. The method resolved the co-elution problems as observed in full scan one-dimensional GC-MS analysis and allowed chromatographic separation of 51 pesticides within 24 min run time with library-searchable mass spectrometric confirmation. Four pesticides, viz. chlorpyrifos-methyl, vinclozoline, parathion-methyl and heptachlor could be baseline separated on GCxGC, which were otherwise closely eluting and interfering each other's detection in 1D GC-MS run. Similarly, it could be possible to separate myclobutanil, buprofezin, flusilazole and oxyfluorfen on GCxGC. Although in 1D GC-MS, these closely eluting compounds could be identified through deconvolution algorithm and 'peak-find' option of the Chromatof software but the spectral purity significantly improved on GCxGC analysis. Thorough optimization was accomplished for the oven temperature programming, ion source temperature and GCxGC parameters like modulation period, duration of hot pulses, modulation-offset temperature, acquisition rate, etc. to achieve best possible separation of the test compounds. The limit of detection significantly improved by 2-12 times on GCxGC-TOFMS against GC-TOFMS because of sharper and narrower peak shapes. The method was tested for grape matrix after preparing the samples using previously described method and recoveries of the entire test pesticides were within 70-110% at 10 ng/g level of fortification. GCxGC-TOFMS was found to be an excellent technique for library-based screening of pesticides with high accuracy and sensitivity.     

Separation and determination of four active anthraquinones in Chinese herbal preparations by flow injection-capillary electrophoresis

A simple, rapid, and accurate method for the separation and determination of physcion, chrysophanol, aloe-emodin, and emodin in Rhubarb, Juemingzi, and Chinese herbal preparations was developed by combination of flow injection-capillary zone electrophoresis for the first time. The analysis was carried out using an unmodified fused-silica capillary (75 mm x 50 microm ID x 375 microm OD, effective separation length of 48 mm) and direct ultraviolet detection at 254 nm. By a series of optimization, the sample solvent consisted of NaOH (100 mmol/L) and ACN (1:1 v/v), and a running buffer composed of 15 mmol/L sodium borate - 12.5 mmol/L sodium dihydrogen phosphate - 42% v/v ACN (pH 10.1) was applied for the separation of the four anthraquinones. The separation was rapid and highly reproducible, with complete resolution of all four compounds within 6 min. The sample throughput rate could reach up to 12 per h. The repeatability (defined as relative standard deviation) was 4.45, 4.44, 4.34, 0.61% with peak height evaluation and 1.62, 0.89, 2.49, 2.19% with peak area evaluation for physcion, chrysophanol, aloe-emodin, and emodin, respectively.     

Chiral separation of synthetic vicinal diol compounds by capillary zone electrophoresis with borate buffer and beta-cyclodextrin as buffer additive.

The investigation on capillary electrophoretic enantioseparation of six synthetic compounds containing vicinal diol groups has been undertaken to acquire the optimum conditions using native beta-cyclodextrin (beta-CD) as chiral selector and borate as a background electrolyte. The separation was carried out in an uncoated capillary (58.5 cm x 75 microm i.d., effective length 48.5 cm) and the effects of several important factors were investigated in detail. The results showed that beta-CD as a chiral selector exhibited good enantioselectivity and that the enantioseparation was greatly influenced by the structure of the diols, the borate concentration and the buffer pH. The optimum performance was obtained for the chiral vicinal diols under the conditions of 200 mM borate buffer of pH 9.8 containing 1.7% beta-CD at an applied voltage of 15 kV and a capillary temperature of 20 degrees C. Under the conditions, four diols were baseline separated with fast analysis time and the good theoretical plate numbers (above 10 x 10(4)) and favorable migration-time reproducibilities (RSDs below 3.0%) were obtained. The separation results were satisfactory.     

Online trace enrichment to determine pyrethroids in river water by HPLC with column switching and photochemical induced fluorescence detection

The potential of online trace enrichment on a highly apolar short column in LC was evaluated for the determination of pyrethroids in river water. Twelve millilitres of water samples, modified with 8 mL ACN (ACN/water 40:60, v/v), were passed through 50 x 4.6 mm ID first separation column packed with 5 microm Hypersil Elite C18. Pesticides were preconcentrated in this column while the matrix background was eluted to waste. Separation of pesticides was performed on a 3.5 microm symmetric C18 column (250 x 4.6 mm ID) with an ACN step gradient as mobile phase and fluorescence detection was used after postcolumn derivatization by using UV light. The use of photochemically induced fluorescence for detection improved sensitivity and selectivity. Quantification limits ranged from 0.05 to 0.1 microg/L and pesticide recoveries at two concentration levels (0.1 and 0.5 microg/L) were between 93.1 and 118.6%, with RSD between 2.5 and 7.5% (n = 3) in river water samples. No matrix effect was detected.     

Fast and sensitive determination of pesticide residues in vegetables using low-pressure gas chromatography with a triple quadrupole mass spectrometer

In this study, the feasibility of low-pressure gas chromatography (LP-GC) in conjunction with a triple quadrupole mass spectrometer, as a route towards fast pesticide residue analysis, was investigated. A Varian GC-MS system equipped with a mass spectrometer model 1200 was used. LP-GC-MS experiments were performed on a HP-5 10 m x 0.32 mm x 0.25 microm analytical column connected to a 2.5 m x 0.15 mm non-coated restriction precolumn at the inlet end. For comparison purposes conventional GC-MS analysis was performed on a RTX-5 30 m x 0.25 mm x 0.5 microm column. Under the optimized conditions the analysis time was reduced to 13.3 min with the LP-GC approach which corresponds to an almost threefold gain in speed versus the conventional GC (37 min). Despite the poorer separation power of the LP-GC column, the experiments conducted with tomato and onion extracts spiked with 78 pesticides proved that LP-GC-MS is of practical value to perform full scan screening analysis. Moreover, the rate of false negative results was higher in the case of conventional GC-MS while the LP-GC-MS enabled correct identification of pesticides at lower levels since the peaks were improved in both size and shape. Validation experiments were performed on a sample of 12 representative pesticides for comparison of performance characteristics of the LP-GC and GC approaches with mass spectrometer operated in scan, SIM and MS/MS mode. The LP-GC column set-up interfaced to the MS detector was found to be superior to the conventional GC with respect to obtained linearity, accuracy and precision parameters. Also, lower limits of detection in real extracts were achieved using the LP-GC approach. Finally, the LP-GC-MS/MS analysis of tomato samples with incurred pesticide residues demonstrated the applicability of the developed method for analysis of real samples.     

Capillary electrophoresis in the evaluation of ischemic injury: simultaneous determination of purine compounds and glutathione

An understanding of tissue energy metabolism and antioxidant status is of major interest in the field of organ preservation for transplantation. Nucleotide and glutathione are indicators of cell damage occurring during ischemia and reperfusion. A high performance capillary electrophoresis (HPCE) method with UV detection (185 nm) for the simultaneous analysis of intracellular free ribonucleotides, nucleosides, bases and glutathione (oxidized and reduced form) in myocardial tissues is described. The method does not involve thiol derivatization. The separations were carried out in an uncoated fused-silica capillary, 60 cm long, 52.5 cm to detector, 75 microm ID, with 20 mM Na-borate buffer, pH 10.00, at 20 kV voltage and reading at 185 nm. Injection was hydrostatic for 12 s and total analysis time was 20 min. The technique enables optimum separation of all the compounds examined and has a resolution similar to that of HPLC analysis, with the advantage of fast simultaneous measurement of cell nucleotide metabolism and redox state, not possible with HPLC.