Carbon Stationary Phase Research Articles

Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase

IntroductionAccurately identifying and quantifying polar metabolites using untargeted metabolomics has proven challenging in comparison to mid to non-polar metabolites. Hydrophilic interaction chromatography and gas chromatography–mass spectrometry are predominantly used to target polar metabolites.ObjectivesThis study aims to demonstrate a simple one-step extraction combined with liquid chromatography–mass spectrometry (LC–MS) that reliably retains polar metabolites.MethodsThe method involves a MilliQ + 10% trichloroacetic acid extraction from 6 healthy individuals serum, combined with porous graphitic carbon liquid chromatography–mass spectrometry (LC–MS). The coefficient of variation (CV) assessed retention reliability of polar metabolites with logP as low as − 9. QreSS (Quantification, Retention, and System Suitability) internal standards determined the method's consistency and recovery efficiency.ResultsThe method demonstrated reliable retention (CV < 0.30) of polar metabolites within a logP range of − 9.1 to 5.6. QreSS internal standards confirmed consistent performance (CV < 0.16) and effective recovery (70–130%) of polar to mid-polar metabolites. Quality control dilution series demonstrated that ~ 80% of annotated metabolites could be accurately quantified (Pearson’s correlation coefficient > 0.80) within their concentration range. Repeatability was demonstrated through clustering of repeated extractions from a single sample.ConclusionThis LC–MS method is better suited to covering the polar segment of the metabolome than current methods, offering a reliable and efficient approach for accurate quantification of polar metabolites in untargeted metabolomics.

Linear solvent strength model on porous graphitic carbon stationary phase using high temperature liquid chromatographic method for allopurinol related substances analysis

A high-performance liquid chromatography (HPLC) method was developed for the analysis of Allopurinol and its Ph.Eur. impurities using a porous graphitic carbon (PGC) stationary phase. Retention behavior of solutes was studied across a wide temperature range (30–90 °C) and various gradient times (5–20 min). Analysis of the data revealed distinct retention mechanisms between reversed-phase and PGC phases. However, it was proved that the retention of Allopurinol and its Ph.Eur. impurities on PGC stationary phase can be effectively modeled using the linear solvent strength (LSS) theory. This allows for the utilization of LSS-based method development software to optimize methods under these conditions. By using commercial chromatographic modeling software, separation of Allopurinol and Ph.Eur. impurities was optimized within a large design space. At the optimized operating conditions (pH = 2.0, tG = 6 min, T = 60 °C), all solutes were separated within 6 min with baseline resolution. Comparison between predicted and experimentally measured chromatograms further confirmed the applicability of LSS theory in developing analytical methods for PGC-based HPLC systems. The presented approach offers a general framework for method development on PGC phases.

Insights into the Shape-Selective Retention Properties of Porous Graphitic Carbon Stationary Phases and Applicability for Polar Compounds

In this article, the ability of porous graphitic carbon (PGC) to discriminate between structurally similar compounds is described. After a brief overview of the particle technology and its use in liquid chromatography (LC), two case studies are presented using small-molecule probes to illustrate the importance on how molecular shape and the ability to interact with the graphitic surface lead to the resolution of structural isomers. Finally, an application of this technology in the area of glycan analysis is showcased where released N-glycans from human immunoglobulin G (IgG) were resolved without derivatization, enabling the glycan profile of a biotherapeutic to be determined.

In-situ carbonizing of coal pitch on the surface of silica sphere as quasi-graphitized carbon stationary phase for liquid chromatography

A novel chromatography stationary phase with a quasi-graphitized carbon modified shell has been developed. Coal pitch was directly carbonized on the surface of porous silica with in-situ carbonization. The carbonized coal pitch coating exhibits some degree of graphitization with a 78 nm-thick layer on the surface of silica and a 0.5 nm-thick layer on the inner surface of the mesopores. Based on the special structure of the graphitized carbon coating, the novel stationary phase can provide multiple interactions such as hydrophobic interaction, π-π interaction and dipole-dipole interaction. The novel composite material exhibited unique separation selectivity and excellent separation efficiency for polar compounds, including imidazoles, nucleosides and pesticides. Besides, the packed column also exhibited great repeatability with the RSDs of the retention time of nucleosides between 0.07%-0.50% (n = 5). Finally, satisfied result was achieved in the separation of fullerenes on the new column, suggesting the great potential in the industrial-scale purification of fullerenes.

Evaluation of the Tasso-SST® capillary blood microsampling device for the measurement of endogenous uracil levels

BackgroundUracil (U) plasma or serum levels can be used as surrogates of dihydropyrimidine dehydrogenase (DPD) activity, which is strongly related to the occurrence of severe or fatal toxicity after administration of fluoropyrimidines (FP) chemotherapy. Obtaining blood plasma or serum for U measurement usually requires a phlebotomy procedure by a qualified professional. An alternative to conventional blood drawn is the use of the Tasso-SST® device, which allows the collection of a small blood volume from skin capillaries. This study aimed to implement a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of U in small serum samples and to compare U concentrations measured in venous plasma, obtained after phlebotomy, and serum obtained with the Tasso-SST® device. MethodsFifty microliter samples were prepared through simple protein precipitation with trichloroacetic acid. Chromatographic separation was performed with a porous graphitic carbon stationary phase and mass spectrometric detection used positive electrospray ionization. The assay was validated according to international guidelines. ResultsThe linear range of the assay was 5–250 ng/mL. Measurement accuracy was in the range of 98.8–108.2%, inter-assay precision was 4.3–7.3%, and intra-assay precision was 3.4–6.1%. The average matrix effect was −6.42%. The extraction yield was 95–103.3%. U concentrations measured in serum obtained with the Tasso-SST® device and venous blood plasma were highly correlated (rs = 0.910, P < 0.0001), and no systematic or proportional bias between U levels measured in both matrices was found. ConclusionsThe use of blood microsampling with the Tasso-SST® device is a useful alternative for the measurement of U and the identification of patients with DPD deficiency.

Improving temperature gradient interaction chromatography of polyolefins by simultaneous use of different stationary phases

Temperature gradient interaction chromatography (TGIC) at high temperatures is a powerful method for the chemical composition separation of polyolefins. TGIC is a two-step process where the sample is crystallized on the stationary phase at low temperature followed by the elution of the sample components using a temperature gradient towards high temperatures. For TGIC typically a porous graphitic carbon (PGC) stationary phase is used. The separation mechanism is based on crystallization and adsorption/desorption phenomena and it has been shown that co-crystallization and co-adsorption may affect the separation.The present study reports on the simultaneous use of a non-adsorptive and an adsorptive stationary phase (column) in series to utilize both crystallization and adsorption for improved separation in TGIC. A silica column is used as the non-adsorptive support to allow for the crystallization of the polyolefin sample in the absence of an adsorptive force followed by the typical PGC column for adsorption/desorption. Accordingly, the loci of crystallization and adsorption/desorption are well separated from each other and can be adjusted independently.This novel column setup allows the sample to be introduced slowly onto the second (adsorptive) column eliminating possible co-adsorption and poor selectivity. Low molar mass polyethylene comprising of oligomers with approximately C30C130 was used to illustrate the importance of a non-adsorptive column for improved separation. Utilizing a non-adsorptive silica column allows for higher dynamic flow rates during crystallization, which improves separation. Shorter adsorptive columns are found to be more efficient in this experimental protocol as compared to standard TGIC experiments. Smaller PGC column sizes result in reduced longitudinal and Eddy diffusion and, hence, higher resolution of low and high molar mass polyolefins.

Advanced Liquid Chromatography of Polyolefins Using Simultaneous Solvent and Temperature Gradients.

Olefin copolymers are complex polymer materials that exhibit multiple distributions in molecular properties such as molar mass, chemical composition, and branching. To address the multivariate molecular compositions, chromatographic protocols have been developed that synergistically combine solvent and temperature gradients. As representative examples, blends of olefin copolymers have been fractionated on porous graphitic carbon stationary phases. This is the first study that makes complementary use of solvent and temperature gradient interaction chromatography (SGIC and TGIC, respectively) to capitalize on the advantages of both techniques. In a first experimental setup, solvent and temperature gradients were used simultaneously and complex blends of low molar mass polyethylene and ethylene-co-1-octene copolymers were separated with high efficiency. The separation of oligomers was observed to be significantly better in SGIC as compared to TGIC, while comonomer blends could be separated in either TGIC or SGIC mode. In another innovation, a two-column setup was employed where the columns were placed in different temperature zones. It was demonstrated that the separation of both low and high comonomer content blends was improved significantly when the temperatures of the two zones were manipulated reasonably.

Improving chromatographic separation of polyolefins on porous graphitic carbon stationary phases: effects of adsorption promoting solvent and column length.

The chromatographic separation of complex polyolefins on porous graphitic carbon stationary phases is strongly influenced by the composition of the mobile phase. Of particular interest is the effect of the chemical structure of the adsorption promoting solvent as this component of the mobile phase determines the adsorption–desorption behavior of the polyolefin molecules. In a systematic study, alkyl alcohols and linear alkanes are used as adsorption promoting solvents and the effect of the molecules' carbon chain length on chromatographic resolution is investigated. As representative examples, solvent gradient interaction chromatography experiments on polypropylene stereoisomers and ethylene-co-1-octene copolymers are presented. In a further study, the effect of increasing chromatographic column length on the solvent gradient separation of ethylene-co-1-octene copolymers is investigated. In summary, it is shown that the polypropylene stereoisomers are retained in 1-octanol as well as in n-decane and n-dodecane, allowing for identification of the individual stereoisomers in complex blends. For ethylene-co-1-octene copolymers it is shown that separation improves with increasing carbon chain length of the adsorption promoting solvent. Maximum resolution is obtained when a column length of 300 mm is used with 1-dodecanol as the adsorption promoting solvent.

A positively charged porous graphitic carbon stationary phase for hydrophilic interaction liquid chromatography

High chemical inertness of porous graphitic carbon particles (PGC) makes their surface modification rather difficult. Here a facile way is proposed to prepare a PGC stationary phase with quaternary ammonium-polyvinyl alcohol mixed functional groups for hydrophilic interaction liquid chromatography (HILIC) by generating a thin layer of polyvinyl alcohol (PVA) and poly(diallyldimethylammonium chloride) (DDAC) copolymer onto PGC particles. The phase shows obvious positively electrostatic character and typical HILIC character, exhibiting different selectivity relative to several HILIC phases and bare PGC. To the best of our knowledge, this is the first fully pH-stable, quaternary ammonium functionalized HILIC phase. It is much superior to silica-based HILIC phases in terms of wide pH tolerance (2.1–12.7) and low bleeding.

Separation of Isomers of JWH-122 on Porous Graphitic Carbon Stationary Phase with Non-Aqueous Mobile Phase Using Intelligent Software.

Cannabimimetic compounds have gained an increasing attention from the forensic community during the past few years. The present study was aimed to develop a liquid chromatographic separation method for the analysis of JWH-122 and its methyl isomers. In Hungary, JWH-122 is scheduled as a narcotic compound and its methyl isomers fall into the new psychoactive substance category, attracting significantly milder punishment than JWH-122 does. According to our best knowledge, gas chromatography or reversed phase liquid chromatography coupled with mass spectrometry could not be applied for separation and selective determination of methyl-naphthoyl indol isomers. In this study, we aimed to develop a high performance liquid chromatography method with UV and mass spectrometric detection for the separation of JWH-122 and all its possible isomers, depending on the position of methyl group on the naphthyl frame. Different reversed phase columns were used. Alkyl-modified silica with different selectivity and morphology with different mobile phase composition cannot be applied for separation of JWH-122 isomers. Porous graphitic carbon (PGC) column was used for separation of banned JWH-122 and each of its methyl isomers. In method development, a Quality by Designapproach is presented for modeling the retention of the compounds. According to our knowledge, this is the first time reporting the use of intelligent software to estimate the retention on PGC material and using non-aqueous conditions. Retention times predicted by two program packages (STATISTICA® and DryLab®) are compared. The possibilities and limitations of the software modeling in the conditions described above are also evaluated.

Liquid Chromatography with a Fluorimetric Detection Method for Analysis of Paralytic Shellfish Toxins and Tetrodotoxin Based on a Porous Graphitic Carbon Column.

Paralytic shellfish toxins (PST) traditionally have been analyzed by liquid chromatography with either pre- or post-column derivatization and always with a silica-based stationary phase. This technique resulted in different methods that need more than one run to analyze the toxins. Furthermore, tetrodotoxin (TTX) was recently found in bivalves of northward locations in Europe due to climate change, so it is important to analyze it along with PST because their signs of toxicity are similar in the bioassay. The methods described here detail a new approach to eliminate different runs, by using a new porous graphitic carbon stationary phase. Firstly we describe the separation of 13 PST that belong to different groups, taking into account the side-chains of substituents, in one single run of less than 30 min with good reproducibility. The method was assayed in four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Pecten maximus), scallop (Ruditapes decussatus) and oyster (Ostrea edulis). The results for all of the parameters studied are provided, and the detection limits for the majority of toxins were improved with regard to previous liquid chromatography methods: the lowest values were those for decarbamoyl-gonyautoxin 2 (dcGTX2) and gonyautoxin 2 (GTX2) in mussel (0.0001 mg saxitoxin (STX)·diHCl kg−1 for each toxin), decarbamoyl-saxitoxin (dcSTX) in clam (0.0003 mg STX·diHCl kg−1), N-sulfocarbamoyl-gonyautoxins 2 and 3 (C1 and C2) in scallop (0.0001 mg STX·diHCl kg−1 for each toxin) and dcSTX (0.0003 mg STX·diHCl kg−1 ) in oyster; gonyautoxin 2 (GTX2) showed the highest limit of detection in oyster (0.0366 mg STX·diHCl kg−1). Secondly, we propose a modification of the method for the simultaneous analysis of PST and TTX, with some minor changes in the solvent gradient, although the detection limit for TTX does not allow its use nowadays for regulatory purposes.

Towards a continuous adsorption process for the enrichment of ACE-inhibiting peptides from food protein hydrolysates

Bioactive peptides such as Ile-Trp show great potential as natural ACE (angiotensin-converting-enzyme)-inhibitors. A continuous process for the up-scaled enrichment of ACE-inhibiting peptides based on a columnar adsorber system with an activated carbon stationary phase was developed. The particle size of the adsorbent and the flow rate was investigated as key factors affecting adsorption kinetics and separation performance. Batch and column adsorption experiments of model adsorbate systems were successfully transferred to a more complex system of an α-lactalbumin hydrolysate containing a multitude of species. Ile-Trp was successfully enriched from the hydrolysate by a factor of 4 using the optimized carbon column. The enrichment was more selective using smaller adsorbent particles due to improved adsorption kinetics.

Quantification and structural characterization of raffinose family oligosaccharides in Casuarina glauca plant tissues by porous graphitic carbon electrospray quadrupole ion trap mass spectrometry

Raffinose family oligosaccharides (RFOs) are non-structural, water-soluble carbohydrates widely distributed in the plant kingdom, and include the trisaccharide raffinose, the tetrasaccharide stachyose and the pentasaccharide verbascose. RFOs have been suggested to play a major role in storing carbohydrates in seeds and vegetative tissues, and in conferring osmoprotection against abiotic stresses, such as high salinity and drought. However, the high polarity nature of RFOs makes their analysis with typical reversed phase liquid chromatography (RP-LC) generally difficult, with these compounds eluting very close to the void volume with minimal retention. Moreover, carbohydrate-related compounds lack an inherent chromophore in the ultraviolet-visible (UV) region to make them suitable for UV detection. To overcome these issues, alternative analytical methods based on LC coupled to mass spectrometry (MS) have been developed and porous graphitic carbon (PGC) stationary phases described in the literature as good alternative to typical RP columns for retaining these highly polar compounds were used. In addition, PGC stationary phases allow the use of MS-compatible mobile phases for efficient on-line coupling with electrospray ionization (ESI), and when coupled to tandem MS (MSn) techniques, powerful structural information can be obtained. The present work focuses on the application of a PGC-ESI-QIT-MSn method for the quantitative analysis of RFOs in Casuariana glauca plant tissues under salt stress conditions. In addition, we describe the structural characterization of RFOs through collision induced dissociation (CID) and MSn experiments using a quadrupole ion trap mass spectrometer (QIT-MS) in the ESI positive ion mode.

Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase × reverse phase chromatography in conjunction with porous graphitic carbon stationary phase

A two dimensional-liquid chromatography (2D-LC) based approach was developed for the identification and quantification of histone post translational modifications in conjunction with mass spectrometry analysis. Using a bottom-up strategy, offline 2D-LC was developed using reverse phase chromatography. A porous graphitic carbon stationary phase in the first dimension and a C18 stationary phase in the second dimension interfaced with mass spectrometry was used to analyse global levels of histone post translational modifications in human primary monocyte-derived macrophages. The results demonstrated that 84 different histone peptide proteoforms, with modifications at 18 different sites including combinatorial marks were identified, representing an increase in the identification of histone peptides by 65% and 51% compared to two different 1D-LC approaches on the same mass spectrometer.The use of the porous graphitic stationary phase in the first dimension resulted in efficient separation of histone peptides across the gradient, with good resolution and is orthogonal to the online C18 reverse phase chromatography. Overall, more histone peptides were identified using the 2D-LC approach compared to conventional 1D-LC approaches. In addition, a bioinformatic pipeline was developed in-house to enable the high throughput efficient and accurate quantification of fractionated histone peptides. The automation of a section of the downstream analysis pipeline increased the throughput of the 2D-LC–MS/MS approach for the quantification of histone post translational modifications.

Poly(vinyl alcohol) Modified Porous Graphitic Carbon Stationary Phase for Hydrophilic Interaction Liquid Chromatography.

We report a poly(vinyl alcohol) (PVA)-coated porous graphitic carbon (PGC, Hypercarb) packing as a novel stationary phase for hydrophilic interaction liquid chromatography (HILIC). The exterior and the pores of the PGC particles are coated with a thin layer of PVA by soaking the particles in a PVA solution, filtering, and thermally cross-linking the PVA. Such PVA coated PGC particles (5.7 μm diameter), hereinafter called PVA-PGC are stable at least through pH 1.0-12.7, can be made in <2 h, and exhibit different selectivity relative to six commercial HILIC phases and bare PGC. To our knowledge, this is the first fully pH-stable, completely neutral HILIC phase. Excellent efficiency stable is observed for polar analytes (∼70 000 and 118 000 plates/m for cytosine and resorcinol, respectively). Retention closely resembles standard HILIC behavior. Other substances can also be easily incorporated in the PVA layer; an anion exchange column can be readily made by incorporating diallyldimethylammonium chloride in the PVA coating solution. The ease of preparation without the requirement of synthetic skills or paraphernalia and the possibility of incorporating a variety of modifiers makes this a particularly versatile approach.

Tandem mass spectrometry of isomeric aniline-labeled N-glycans separated on porous graphitic carbon: Revealing the attachment position of terminal sialic acids and structures of neutral glycans.

Quantitative monitoring of changes in the N-glycome upon disease has gained significance in the context of biomarker discovery. Separation and quantification of isobaric glycan isomers can be attained by using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Collision-induced dissociation (CID)-based fragmentation of separated isobaric glycans is evaluated in respect to its potential of providing fragment ions specific for the linkage positions of terminal sialic acids and the presence of intersecting GlcNAc moieties, respectively. N-Glycans were labeled via reductive amination using (12)C6-aniline and (13)C6-aniline as isotope-coded labeling reagents. The differently labeled glycans were merged and separated into various species using a porous graphitic carbon (PGC) stationary phase. Identification of structural features of separated isobaric isomers was performed by CID-based tandem mass spectrometry (MS/MS) carried out in a quadrupole time-of-flight (QqTOF) or a quadrupole ion-trap (IT) mass spectrometer. Working in the negative ion mode, new diagnostic CID fragment ions could be found that are indicative for the α2,6-type linkage of sialic acids. Other diagnostic ions, identified before as being indicative for the substitution of the 6-antenna, could be confirmed as being of relevance also in the case of aniline labeling. In the positive ion mode, CID fragment ions indicative for the structure of short neutral N-glycans were identified. One new diagnostic ion specific for the linkage position of the terminal sialic acids and one for the presence of bisecting GlcNAc in N-glycans were identified. The aniline label introduced for improved relative quantitation in MS(1) was found not to significantly alter the CID fragmentation patterns that were reported previously by other authors for unlabeled/reduced glycans or for glycans with more polar labels.

Improved determination of uracil and dihydrouracil in plasma after a loading oral dose of uracil using high-performance liquid chromatography with photodiode array detection and porous graphitic carbon stationary phase

ObjectivesThe aim of this study was to develop and validate a high-performance liquid chromatographic method for the measurement of plasma concentrations of uracil and dihydrouracil after administration of an oral loading dose of uracil in the context of evaluation of DPD enzyme activity. Design and methodsAnalytes were extracted from 500μL plasma sampler with a mixture of ethyl acetate isopropanol (85:15, v/v) after protein precipitation with solid ammonium sulfate. The extract was inject in the porous graphitic carbon stationary phase, eluted with water and acetonitrile in gradient mode, allowing complete separation of uracil, dihydrouracil and the internal standard (5-fluorouracil). Chromatograms were monitored at 210 and 260nm. ResultsTotal chromatographic run time, including reequilibration, was 30min. The assay was linear in the concentration range of 0.2 to 20μgmL−1. Accuracy was 98.4–105.3%, intra-assay precision was 5.1–12.1% and between-assay precision was of 5.3–10.1%. Analytes were stable in plasma at room temperature up to 6h and for three freeze and thaw cycles. Processed samples are stable up to 12h. ConclusionsThe developed method was fully validated and has significantly reduced running time when compared to previous assay using porous graphitic stationary phase, allowing complete resolution of uracil, dihydrouracil and internal standard. This assay might be suitable to investigate the eventual correlation between concentrations of uracil and dihydrouracil in plasma after an oral loading dose and DPD enzyme activity, with potential contribution to therapeutic drug monitoring.

Homogeneous edge-plane carbon as stationary phase for reversed-phase liquid chromatography.

Carbon stationary phases have been widely used in HPLC due to their unique selectivity and high stability. Amorphous carbon as a stationary phase has at least two sites of interaction with analytes: basal-plane and edge-plane carbon sites. The polarity and adsorptivity of the two sites are different. In this work, the edge-plane carbon stationary phase is prepared by surface-directed liquid crystal assembly. Specific precursor polymers form discotic liquid crystal phases during the pyrolysis process. By using silica as the substrate to align the discotic liquid crystal, edge-plane carbon surfaces were formed. Similar efficiencies as observed for Hypercarb were observed in chromatograms. The column efficiency was studied as a function of linear flow rate. A minimum reduced plate height of 6 was observed in these studies. To evaluate the performance of the homogeneous edge-plane carbon stationary phase, linear solvation energy relationships were used to compare these ordered carbon surfaces to commercially available carbon stationary phases, including Hypercarb. Reversed-phase separations of nucleosides, nucleotides, and amino acids and derivatives were demonstrated using the ordered carbon surfaces, respectively. The column batch-to-batch reproducibility was also evaluated. The retention times for the analytes were reproducible within 1-6% depending on the analyte.

Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography–electrospray ionization-mass spectrometry with graphitic carbon stationary phase

Glycan reductive isotope labeling (GRIL) using 12C6-/13C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins. The method was validated with respect to mobile phase parameters, reproducibility, accuracy, linearity and limit of detection/quantification (LOD/LOQ) using test glycoproteins. It is shown that the developed method is capable of determining relative amounts of N-glycans (including isomers) comparing two samples in one single HPLC-MS run. The analytical potential and usefulness of GRIL in combination with PGC-ESI-TOF-MS is demonstrated comparing glycosylation in human monoclonal antibodies produced in Chinese hamster ovary cells (CHO) and hybridoma cell lines.

Analysis of non-phthalates plasticizers on porous graphitic carbon by supercritical fluid chromatography using evaporative light scattering detection

The analysis of several plasticizers, widely used in the production of medical devices, was investigated on porous graphitic carbon (PGC) stationary phase in supercritical fluid chromatography (SFC) with an evaporative light scattering detector (ELSD). Due to strong interaction of compounds with the PGC support, solvents of strong eluotropic strength were added to the CO2 supercritical fluid. The effect of alkyl chain (pentane, hexane, heptane) and chlorinated (CH2Cl2, CHCl3, CCl4) solvents was studied on the retention and on the ELSD detection of plasticizers. A co-solvent mixture composed of CHCl3/heptane, eluted under gradient mode, allowed a significant improvement of the ELSD response compared to the use of each solvent individually. Then, a central composite design (CCD) was implemented to optimize both the separation and the detection of plasticizers. The parameters involved were the outlet pressure, the gradient slope, the co-solvent composition and the drift tube temperature of the ELSD. After optimization, baseline separation of plasticizers was achieved in 7min and best signal-to-noise ratios were obtained with outlet pressure and drift tube temperature of ELSD set at 200bar and 31°C, respectively. The co-solvent mixture was also composed of CHCl3/heptane (35/65 v/v) and a gradient from 15 to 60% of co-solvent in 2.2min was employed. The results demonstrated that CCD is a powerful tool for the optimization of SFC/ELSD method and the response surface model analysis can provide statistical understandings of the significant factors required to achieve optimal separation and ELSD sensitivity.