Various microextraction methods have demonstrated a positive effect when assisted by vacuum. However, working with such systems is often laborious, they often require expensive and non-portable vacuum pumps, and may even suck off some sample vapor or solid particles during the evacuation process. To address these issues, a simple, and affordable vacuum-assisted headspace solid-phase microextraction (HS-SPME) device was developed in this study. The device, named In Syringe Vacuum-assisted HS-SPME (ISV-HS-SPME), utilizes an adjustable 40 mL glass syringe as a vacuum provider and sampling vessel. A new fiber coating, made from a hybrid of covalent triazine-based frameworks and metal-organic frameworks (COF/MOF), was prepared and characterized by Fourier transform infrared spectrometry, field emission scanning electron microscopy, energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis, and Brunauer–Emmett–Teller techniques for use in the ISV-HS-SPME. By optimizing parameters such as extraction temperature, extraction time, desorption temperature, desorption time, and, humidity using a simplex method, the ISV system was found to increase the extraction efficiency of polycyclic aromatic hydrocarbons (PAHs) and benzene, toluene, ethylbenzene, and xylenes (BTEX) in solid samples by up to 175%. The determinations were followed by GC-FID measurements. Compared to three commercially available fibers, the ISV-HS-SPME device with the COF/MOF (2DTP/MIL-101-Cr) fiber exhibited significantly higher peak areas for PAHs and BTEX. The linear dynamic ranges for BTEX and PAHs were 7.1–9000 ng g−1 and 0.23–9000 ng g−1, respectively, with limits of detection ranging from 2.1–5 ng g−1 for BTEX and 0.07–1.6 ng g−1 for PAHs. The relative standard deviation of the method was 2.6–7.8% for BTEX and 1.6–6.7% for PAHs. The ISV-HS-SPME was successfully used to simultaneously determine PAHs and BTEX in polluted soil samples with recoveries ranging from 80.4 to 108%.

The flow reversal (FR) technique consists of reversing the flow direction along a chromatographic column. It is used to reveal the origin (such as poor column packing, active sites, or slow absorption/escape kinetics) for the resolution limit of 4.6 mm × 150 mm long columns packed with 1.7 μm 200 Å Bridge-Ethylene-Hybrid (BEHTM) Particles. These columns are used to separate manufactured monoclonal antibodies (mAb, ∼ 150 kDa) from their close impurities (or IdeS fragments, ∼ 100 kDa) by size exclusion chromatography (SEC). FR unambiguously demonstrates that the resolution limit of these SEC columns is primarily due to long-range flow velocity biases covering distances of at least 500 μm across the column diameter. This confirms the existence of center-to-wall flow heterogeneities which cause undesirable tailing for the mAb peak. Because the transverse dispersion coefficient (Dt=1.1 × 10−6 cm2/s) of mAbs across the column diameter is intrinsically low, the bandspreading of the mAb in a single flow direction is in part reversible upon reversing the flow direction. For the very same residence time in the column, the column efficiency is found to increase by +85% relative to that observed under conventional elution mode. The observed peak tailing of the mAb and its sub-units is not caused by active surface sites or by slow absorption/escape from the BEH Particles. Therefore, the most critical mAb impurities (hydrolytic degradation Fab/c and IdeS F(ab′)2 fragments) can only be successfully separated and quantified with acceptable accuracy by adopting alternate pumping recycling liquid chromatography (APRLC). APRLC enables the full baseline separation of the mAb and 100 kDa mAb fragments and partial separation of Fab/c and F(ab′)2 fragments after increasing the number of cycles to ten. It was made possible to accurately measure the relative abundances of the mAb (99.0 ± 0.1%), F(ab′)2 fragment (0.88 ± 0.03%), and Fab/c immunogenic fragment (0.13 ± 0.02%) in less than 45 min for a total mAb sample load of only 5 μg. Still, further improvements are needed to increase the sensitivity of the APRLC method and to reduce the solvent consumption by adopting narrow-bore 2.1 mm i.d. SEC columns.

The amenability of traveling wave ion mobility spectrometry (TWIMS) to extended separation pathlengths has prompted a recent surge of interest concerning the technique. While promising, the optimization of ion transmission, particularly when analyzing increasingly disparate species, remains an obstacle in TWIMS. To address this issue, we evaluated a suite of dynamic TW profiles using an original TW structures for lossless ion manipulations (TW-SLIM) platform developed at Washington State University. Inspired by the range of gradient elution profiles used in traditional chromatography, three distinct square TW profiles were evaluated: a static approach which represents a traditional waveform, a dual approach which consists of two distinct TW profiles within a given separation event; and a ramp approach which varies TW speed and amplitude at a fixed rate during separation. The three waveform profiles were evaluated in terms of their impact on separation (quantified as resolution) and sensitivity (quantified using signal-to-noise ratio (SNR), and ion abundance). Concerning separation, the highest resolution (R) was observed when operating with the static waveform (R = 7.92); however, the ramp waveform performed comparably (R = 7.70) under similar conditions. Regarding SNR, optimum waveform profiles were species dependent. Bradykinin2+ displayed the largest gains in SNR (36.6% increase) when ramping TW speed, while the gains were greatest (33.5% increase) for tetraoctylammonium when modulating TW amplitude with the static waveform. Lastly, significant (>10%) increases in the abundance of tetraoctylammonium ions were observed exclusively when utilizing a ramped waveform. The present set of experiments outline the results and challenges related to optimizing separations using alternative TW profiles and provides insight concerning TW-SLIM method development which may be tailored to enhance select analytical metrics.

Cationic, anionic, zwitterionic and, partially polar metabolites are very important constituents of blood serum. Several of these metabolites underpin the core metabolism of cells (e.g., Krebs cycle, urea cycle, proteins synthesis, etc.), while others might be considered ancillary but still important to grasp the status of any organism through blood serum analysis. Due to its wide chemical diversity, modern metabolomics analysis of serum is still struggling to provide a complete and comprehensive picture of the polar metabolome, due to the limitations of each specific analytical method. In this study, two metabolomics-based analytical methods using the most successful techniques for polar compounds separation in human serum samples, namely hydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE), are evaluated, both coupled to a high-resolution time-of-flight mass spectrometer via electrospray ionization (ESI-Q-TOF-MS). The performance of the two methods have been compared using five terms of comparison, three of which are specific to metabolomics, such as 1) compounds’ detectability 2) Pezzatti score (Pezzatti et al. 2018), 3) intra-day precision (repeatability), 4) ease of automatic analysis of the data (through a common deconvolution alignment and extrapolation software, MS-DIAL, and 5) time & cost analysis. From this study, HILIC-MS proved to be a better tool for polar metabolome analysis, while CE-MS helped identify some interesting variables that gave it interest in completing metabolome coverage in metabolomics studies. Finally, in this framework, MS-DIAL demonstrates for the first time its ability to process CE data for metabolomics, although it is not designed for it.

In this work, a three-dimensional fluorinated covalent organic frameworks (3D FCOFs) JUC-515 was synthesized from tetra(4-aminophenyl)methane (TAM) and 2,3,5,6-tetrafluoroterephthalol (TFA) by an ionic liquid method. JUC-515 was introduced into the capillary column and bonded to the inner wall of the capillary column by chemical bonding. Through a variety of characterization results, JUC-515 was successfully synthesized and introduced into the capillary column. The effects of buffer solution concentration, organic additive content and pH of the buffer solution on the separation of fluoroquinolones (FQs) were investigated in detail. The JUC-515-coated capillary column showed good resolution (>1.5) and reproducibility. The relative standard deviations (RSDs) of the retention time for intraday, interday, column-to-column and interbatch precision were less than 0.88%, 2.45%, 2.74% and 3.32%, respectively. The RSDs of the peak area for intraday, interday, column-to-column and interbatch precision were less than 3.79%, 4.31%, 3.33% and 5.62%, respectively. The JUC-515-coated capillary column could be used no less than 150 times. The results showed that the JUC-515-coated capillary column had good separation performance. In addition, by separating fluorinated β-phenylalanine analogs, β-phenylalanine and trifluoromethyl β-phenylalanine analogs, the separation mechanism based on fluorine interactions was discussed. In conclusion, JUC-515 had good potential as a stationary phase for capillary electrochromatography.

The residue of carbaryl in food is a threat to human health. In this study, activated soybean shell biochar (A-SBC) was used as a carrier, methacrylic acid (MAA) was used as a functional monomer, and carbaryl was used as a template molecule to synthesize the activated biochar surface molecularly imprinted polymer (A-SBC@MIP). The synthesized A-SBC@MIP was characterized by SEM, FT-IR, XRD and XPS techniques, and then applied as adsorbent for carbaryl removal. The adsorption capacity of A-SBC@MIP for carbaryl was 8.6 mg‧g−1 and the imprinting factor was 1.49 at the optimum ionic strength and pH. The kinetic and isothermal data indicated that it had fast mass transfer rate and high binding capacity(Qmax=47.9 mg‧g−1). A-SBC@MIP showed good regenerative properties and the adsorption of carbaryl was excellent in its structural analogues. A solid-phase extraction (SPE) column composed of A-SBC@MIP was developed for the detection of rice and corn under optimized conditions, with recoveries of 93–101% for the spiked carbaryl. The limit of detection (LOD) of the method was 3.6 μg‧kg−1 with good linearity (R2=0.994) in the range of 0.01–5.00 mg‧L−1. The results show that the developed MIPs-SPE can enrich carbaryl from food samples as a specific and cost-effective method.

The culturable endophytic bacteria from the weeds Cleome rutidosperma of the family Cleomaceae and Digitaria sanguinalis of the family Poaceae obtained from a previous dumpsite in Pampanga, Philippines have been assessed for their anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and the analytes with such activity should be identified. However, due to the limited amounts collected from the isolation process, 1.8 mg yield of compound 1 from the endophyte of C. rutidosperma and 1.2 mg of a mixture from the endophyte of D. sanguinalis were selected for LC-MSE analysis. The production of compounds from the culturable endophytic bacteria Pseudomonas aeruginosa— determined by gene-sequencing, an untargeted and data-independent analysis (DIA) by ultra-high performance liquid chromatography-high resolution-elevated energy mass spectrometry (UHPLC-HR-MSE) technique was employed to profile the metabolites present in the two high-performance liquid chromatography (HPLC) fractions. The analytes present from P. aeruginosa detected by UHPLC-HR-MSE isolated from C. rutidosperma was phenazine-1-carboxylic acid (1), and for D. sanguinalis were chamigrenal (2), dialkyl resorcinol (3), and a pyoverdine elicitor (4). This study proves that UHPLC-HR-MSE could identify the anti-MRSA constituents in P. aeruginosa from commensal weeds C. rutidosperma and D. sanguinalis. The UHPLC-HR-MSE could help strengthen metabolomics antibacterial research and its related applications from a future perspective. Application of metabolomics research using UHPLC-HR-MSE could enhance the rehabilitation of dumpsites by the microbial community present.

The rare presence of circulating tumor cells (CTCs) in the bloodstream has made their recording and separation one of the major challenges in the recent decade. Inertia-based microfluidic systems have received more attention in CTCs separation due to their feasibility and low cost. In this research, an inertial microfluidic system is proposed using a curved expansion-contraction array (CEA) microchannel to separate CTCs from white blood cells (WBCs). First, the optimal flow rate of the proposed microfluidic device was determined to maximize the separation efficiency of the target cells (CTCs) from the non-target ones (WBCs). Then, the efficiency and purity of the straight and curved-CEA microchannels were assessed. The experimental results indiated that the proposed system (curved-CEA microchannel) can offer the highest efficiency (-80.31%) and purity (-91.32%) at the flow rate of -7.5 ml/min, exhibiting ∼11.48% increment in the efficiency compared to its straight peer.

Continuous multi-column chromatography (CMCC) has been successfully implemented to address biopharmaceutical biomolecule instability, to improve process efficiency, and to reduce facility footprint and capital cost. This paper explores the implementation of a continuous multi-membrane chromatography (CMMC) process, using four membrane units, for a large viral particle in just few weeks. CMMC improves the efficiency of the chromatography step by enabling higher loads with smaller membranes for multiple cycles of column use and enables steady-state continuous bioprocessing. The separation performance of CMMC was compared to a conventional batch chromatographic capture step used at full manufacturing scale. The product step yield was 80% using CMMC versus 65% in batch mode while increasing slightly the relative purity. Furthermore, the total amount of membrane area required for the CMMC approach was approximately 10% of the area needed for batch operation, while realizing similar processing times. Since CMMC uses smaller membrane sizes, it can take advantage of the high flow rates achievable for membrane chromatography that are not typically possible at larger membrane scales due to skid flow rate limitations. As such, CMMC offers the potential for more efficient and cost-effective purification trains.

A compact, inexpensive sampler instrument for portable capillary electrophoresis (CE) was developed and tested to monitor common inorganic ions in drinking water samples. The sampler uses peristaltic and vacuum pumps and pinch and check valves to control liquid flows. The paper also addresses various aspects of CE associated with portability, open access instrumentation and prospects of CE for citizen science. The extensive use of items provided by the electronic and computer industry contributes to this trend.

Hydroquinone (HQ), resorcinol (RS), m-aminophenol (m-AMP) and p-phenylenediamine (p-PPD) are aromatic compounds which are generally used in hair dyes to provide different colours to hair. In European Union the concentrations of HQ, RS, m-AMP and p-PPD is regulated in hair dyes and other cosmetic products by EU commission regulation EU/2019/831. This legislation is generally exercised because all these compounds are toxic and may cause severe allergies when used regularly. However in India no such regulations exist to monitor these toxic compounds in hair dyes therefore in this study a simple, rapid, economical and ecofriendly micellar liquid chromatographic (MLC) technique has been developed which can monitor all the selected toxic compounds simultaneously. HQ and RS are positional isomers and are difficult to be separated by HPLC whereas with the developed MLC method it was well separated and detected. The developed MLC technique has been applied to detect and quantify selected analytes in oxidative and non-oxidative hair dyes and swab samples from the scalp. The simultaneous separation of selected analytes was performed in mobile phase 0.09 M SDS, 0.01 M NaH2PO4-2% v/v 1-butanol at pH 7 running through C18 column under isocratic mode at 1 mL/min. flow rate. All the analytes were eluted within 6 min. The present method has been validated following the EURCHEM Guideline, 2014 in terms of calibration range (0.08-15 µg/mL), limit of detection (0.01-0.08 µg/mL), limit of quantification (0.08-0.18 µg/mL), accuracy (<5.6%), precision (91-105%) and robustness (<5.8%). The selected compounds in hair dye formulation were found in the range of 0.09-253 µg/mL. Hair dyes persistence study was conducted up to 10 days from the day of application on the scalp, suggesting that the dyes were not completely washed off and were retained on the scalp for more than one week. SEM analysis of dyed hair revealed that hairs are severely damaged due to use of dyes. The advantage of the developed method is that it could easily be adopted by quality control and cosmetic laboratories for quality control and check for the simultaneous separation of positional isomers together with two other aromatic compounds.

The growing global popularity of traditional Chinese medicine (TCM) has generated a growing interest in the quality control of TCM products. Shuanghuanglian Oral Liquid (SHL) is a commonly used TCM formula for treating respiratory tract infections. In this study, we present a thorough evaluation method for the quality of SHL and its intermediates. We assessed the quality through multi-wavelength fusion high-performance liquid chromatogram (HPLC) fingerprints of 40 batches of SHL samples and 15 batches of intermediates. Meanwhile, we introduced a new method called multi-markers assay by monolinear method (MAML) to quantify ten components in SHL, and revealed quality transmitting of ten components from intermediates to formulations. This information allowed us to establish a quality control system for intermediates, ensuring their quality consistency. Furthermore, we proposed UV quantum fingerprinting as an orthogonal complement to the quality evaluation by HPLC fingerprint. The relationship between fingerprinting and antioxidant capacity was also established. Overall, this study presented a novel and integrated approach for the quality evaluation of TCM products, providing valuable information for ensuring the safety and efficacy of TCM products for consumers.

High-performance liquid chromatography is one of the most important analytical tools for the identification and separation of substances. The efficiency of this method is largely determined by the stationary phase of the columns. Although monodisperse mesoporous silica microspheres (MPSM) represent a commonly used material as stationary phase their tailored preparation remains challenging. Here we report on the synthesis of four MPSMs via the hard template method. Silica nanoparticles (SNPs) which form the silica network of the final MPSMs were generated in situ from tetraethyl orthosilicate (TEOS) in the presence of (3-aminopropyl) triethoxysilane (APTES) functionalized p(GMA-co-EDMA) as hard template. Methanol, ethanol, 2-propanol, and 1-butanol were applied as solvents to control the size of the SNPs in the hybrid beads (HB). After calcination, MPSMs with different sizes, morphology and pore properties were obtained and characterized by scanning electron microscopy, nitrogen adsorption and desorption measurements, thermogravimetric analysis, solid state NMR and DRIFT IR spectroscopy. Interestingly, the 29Si NMR spectra of the HBs show T and Q group species which suggests that there is no covalent linkage between the SNPs and the template. The MPSMs were functionalized with trimethoxy (octadecyl) silane and used as stationary phases in reversed-phase chromatography to separate a mixture of eleven different amino acids. The separation characteristics of the MPSMs strongly depend on their morphology and pore properties which are controlled by the solvent during the preparation of the MPSMs. Overall, the separation behavior of the best phases is comparable with those of commercially available columns. The phases even achieve faster separation of the amino acids without loss of quality.

This study compares different solvent systems with the use of spontaneous fermentation on the phytochemical composition of leaf extracts from a locally grown white variety of common fig (Ficus carica Linn.). The aim was to detects and identify bioactive compounds that are responsible for acetylcholinesterase (AChE), α-amylase and Cyclooxygenase-1 (COX-1) enzyme inhibition, and compounds that exhibit antimicrobial activity. Bioactive zones in chromatograms were detected by combining High-performance thin-layer chromatography (HPTLC) with enzymatic and biological assays. A new experimental protocol for measuring the relative half-maximum inhibitory concentration (IC50) was designed to evaluate the potency of the extracts compared to the potency of known inhibitors. Although the IC50 of the fig leaf extract for α-amylase and AChE inhibition were significantly higher when compared to IC50 for acarbose and donepezil, the COX-1 inhibition by the extract (IC50 = 627 µg) was comparable to that of salicylic acid (IC50 = 557 µg), and antimicrobial activity of the extract (IC50 = 375-511 µg) was similar to ampicillin (IC50 = 495 µg). Four chromatographic zones exhibited bioactivity. Compounds from detected bioactive bands were provisionally identified by comparing the band positions to coeluted standards, and by Fourier transform infrared (FTIR) spectra from eluted zones. Flash chromatography was used to separate selected extract into fractions and isolate fractions that are rich in bioactive compounds for further characterisation with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) analysis.The main constituents identified were umbelliferon (zone 1), furocoumarins psoralen and bergapten (zone 2), different fatty acids (zone 3 and 4), and pentacyclic triterpenoids (calotropenyl acetate or lupeol) and stigmasterol (zone 4).

Micro gas chromatography (μGC) using microfabricated silicon columns has been developed in response to the requirement for portable on-site gas analysis. Although different stationary phases have been developed, repeatable and reliable surface coatings in these rather small microcolumns remains a challenge. Herein, a new stationary phase coating strategy using magnetic beads (MBs) as carriers for micro column is presented. MBs modified with organopolysiloxane (MBs@OV-1) and a metal organic framework (MBs@HKUST-1) are deposited in on-chip microcolumns assisted with a magnetic field with an optimized modification process. MBs@OV-1 column showed a minimum HETP of 0.074 cm (1351 plates/m) of 62 cm/s. Mixtures of volatile organic compounds are successfully separated using MBs carried stationary phase which demonstrates that this technique has good chromatographic column efficiency. This method not only provides a novel coating process, washing and characterization of the stationary phases but also establishes a straightforward strategy for testing new absorbent materials for μGC systems.

In this work, for the first time, magnetic-phthalated maltodextrin nanosponges (M-PAMDNSs) were synthetized and introduced as efficient and green sorbents. The integration of phthaloyl groups as hydrophobic moieties into networks of maltodextrin nanosponges provided good enrichment for hypothalamic-related peptides (HRPs). The synthesized materials were characterized by 1H nuclear magnetic resonance spectroscopy, water contact angle, attenuated total reflection-Fourier transform infrared spectroscopy, dynamic light scattering, zeta potential, pH point of zero charge, acid-base titration, field-emission scanning electron microscopy, Brunauer-Emmett-Teller, and vibrating sample magnetometer. Under the optimized conditions (sorbent amount: 5.0 mg, desorption solvent volume and type: 300 µL of methanol: H2O: trifluoroacetic acid, extraction time: 15 min, and desorption time: 10 min), the developed magnetic solid-phase extraction (MSPE) method in combination with HPLC-UV was used as a novel and sensitive analytical method for the determination of HRPs in plasma samples. The proposed MSPE-HPLC-UV method provided good linearity (1.5-500 ng mL−1 R2 ≥ 0.9988), low limits of detection (0.1-0.2 ng mL−1) and quantification (0.4-0.8 ng mL−1), desirable precision (RSD ≤ 8.8%, n ₌ 5), satisfactory enrichment factor (EFs ≥ 66.0), and well relative recoveries (92.8-108.8%). Overall, the established method effectively expanded the analytical potential of MSPE approach for the quantification of HRPs in biological samples.

The distribution of neutral compounds in biphasic separation systems can be described by the solvation parameter model using six solute properties, or descriptors. These descriptors (McGowan's characteristic volume, excess molar refraction, dipolarity/polarizability, hydrogen-bond acidity and basicity, and the gas-liquid partition constant on n-hexadecane at 298.15 K) are curated in two publicly accessible databases for hundreds (WSU compound descriptor database) or thousands (Abraham compound descriptor database). These databases were developed independently using different approaches resulting in descriptor values that vary for many compounds. Previously, it was shown that the two descriptor databases are not interchangeable, and the WSU descriptor database consistently demonstrated improved model performance for chromatographic systems where the uncertainty in the dependent variable was minimized by suitable quality control and calibration procedures. In this report we wish to evaluate whether the same conclusions are true for models with a dependent variable containing significant measurement uncertainty. To evaluate this hypothesis, we assembled databases for water-air, octanol-air, and octanol-water partition constants reported by multiple laboratories using various measurement methods. It was found that database selection has little effect on model quality or model predictive capability but significantly affects the assignment of the contribution of individual intermolecular interactions to the dependent variable. The latter information is database specific, and a quantitative comparison of system constants should be restricted to models using the same compound descriptor database.

The separation and detection of multi-component mixtures has always been a challenging task. Traditional detection methods often suffer from complex operation, high cost, and low sensitivity. Surface enhanced Raman scattering (SERS) technique is a high sensitivity, powerful and rapid detection tool, which can realize the specific detection of single substance components, but it must solve the problem that multi-component mixtures cannot be accurately determined. Thin layer chromatography (TLC) technology, as a high-throughput separation technology, uses chromatographic plate as the stationary phase, and could select different developing phases for separation experiments. The advantages of TLC technology in short distance and rapid separation are widely used in protein, dye and biomedical fields. However, TLC technology has limitations in detection ability and difficulty in obtaining ideal signal intensity. The combination of TLC technology and SERS technology made the operation procedure simple and the sample size small, which can achieve rapid on-site separation and quantitative detection of mixtures. Due to the rapid development of TLC-SERS technology, it has been widely used in the investigation of various complex systems. This paper reviews the application of TLC-SERS technology in food science, environmental pollution and biomedicine.

Novel grafted anion exchangers with covalently bonded hyperbranched functional layers were prepared and evaluated for the separation of monovalent standard inorganic anions and oxyhalides. Preparation of base coating included grafting highly polar N-vinylformamide to the ethylvinylbenzene-divinylbenzene (EVB-DVB) substrate surface in highly polar solvent (methanol) with subsequent hydrolysis of grafted amide polymer in basic media, which resulted in preparation of polymer chains with multiple primary amino groups. Those amino groups were used as attachment points for forming hyperbranched anion-exchange layers using 1,4-butanediol diglycidyl ether and primary mono- or diamine (methylamine or 1,3-diaminopropane, respectively). The effects of hyperbranching reaction cycles number on selectivity were evaluated which revealed that selectivity and capacity can be controlled independently for the covalently bonded stationary phases in contrast to electrostatically bonded phases. It was demonstrated that unlike for electrostatically bonded phases, the intentional increase of crosslink by using primary diamine instead of primary monoamine doesn't cause the shift of selectivity coefficients. It was also shown that crosslink distribution throughout the hyperbranched layer is an important factor determining selectivity of hyperbranched anion exchangers.

Mobile phase additives are used to improve retention behavior in chromatography. In supercritical fluid chromatography (SFC), for which supercritical fluid carbon dioxide (SF-CO2) is used as the main mobile phase, additives can only be added into the modifier. For that reason, when gradient analysis is performed by changing the modifier ratio to SF-CO2, the additive concentration in the mobile phase increases in parallel with the modifier ratio. In a preliminary study performed using the conventional SFC system, ammonium acetate was necessary to improve the peak shape of a polar steroid, dehydroepiandrosterone sulfate (DHEA-S), while the peak intensity of a non-polar steroid, progesterone, decreased by 78% compared to that in the absence of the additive in mobile phase when gradient elution was performed. Since ammonium acetate had both favorable and unfavorable effects on sensitive and simultaneous analysis of these two steroid compounds, a compromise between these effects had to be sought. A three-pump configuration of SFC was developed by adding a pump unit to SFC instrument, which enabled control of the additive concentration independently of the modifier ratio, for the purpose of investigating the additive effect in detail using both steroids as model compounds. The putative cause of the decrease in peak intensity of progesterone was excessively elevated additive concentration in gradient analysis. When the additive concentration in the mobile phase was controlled to ensure that it did not increase during gradient analysis, the peak intensities of progesterone, cortisol, corticosterone, and testosterone were 55%, 40%, 25%, and 17% higher than when the additive concentration was not controlled, respectively. On the other hand, the peak intensity of DHEA-S was almost identical between the conditions, with an increase of 2% with three-pump instrument. The three-pump configuration showed the potential to solve problems relating to the use of modifier additives by keeping their concentration constant in gradient SFC analysis.