Preparation of high-efficiency HILIC capillary columns utilizing slurry packing at 2100 bar.

Complex mixture analysis requires high-efficiency chromatography columns. Although reversed phase liquid chromatography (RPLC) is the dominant approach for such mixtures, hydrophilic interaction liquid chromatography (HILIC) is an important complement to RPLC by enabling the separation of polar compounds. Chromatography theory predicts that small particles and long columns will yield high efficiency; however, little work has been done to prepare HILIC columns longer than 25 cm packed with sub-2 µm particles. In this work, we tested the slurry packing of 75 cm long HILIC columns with 1.7 µm bridged-ethyl-hybrid amide HILIC particles at 2,100 bar (30,000 PSI). Acetonitrile, methanol, acetone, and water were tested as slurry solvents, with acetonitrile providing the best columns. Slurry concentrations of 50-200 mg/mL were assessed, and while 50-150 mg/mL provided comparable results, the 150 mg/mL columns provided the shortest packing times (9 min). Columns prepared using 150 mg/mL slurries in acetonitrile yielded a reduced minimum plate height (hmin) of 3.3 and an efficiency of 120,000 theoretical plates for acenaphthene, an unretained solute. Para-toluenesulfonic acid produced the lowest hmin of 1.9 and the highest efficiency of 210,000 theoretical plates. These results identify conditions for producing high-efficiency HILIC columns with potential applications to complex mixture analysis.

Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process.

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.

Daptomycin: A Novel Macrocyclic Antibiotic as a Chiral Selector in an Organic Polymer Monolithic Capillary for the Enantioselective Analysis of a Set of Pharmaceuticals.

Daptomycin, a macrocyclic antibiotic, is here used as a new chiral selector in preparation of chiral stationary phase (CSP) in a recently prepared polymer monolithic capillary. The latter is prepared using the copolymerization of the monomers glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in the presence of daptomycin in water. Under reversed phase conditions (RP), the prepared capillaries were tested for the enantioselective nanoliquid chromatographic separation of fifty of the racemic drugs of different pharmacological groups, such as adrenergic blockers, H1-blockers, NSAIDs, antifungal drugs, and others. Baseline separation was attained for many drugs under RP-HPLC. Daptomycin expands the horizon of chiral selectors in HPLC.

Methoxy terminated poly dimethylsiloxane bonded stationary phase for reversed-phase liquid chromatography.

In this work, a methoxy terminated poly dimethylsiloxane modified silica gel material was proposed as a novel stationary phase for reversed-phase liquid chromatography. With 5 µm silica gel as matrix, methoxy terminated poly dimethylsiloxane polymer was grafted by one step chemical bonding reaction. The obtained stationary phase was characterized by scanning electron microscopy, fourier transform infrared spectroscopy and element analysis. To our knowledge, this is the novel methoxy terminated poly dimethylsiloxane bonded stationary phase with good separation efficiency (42107-46988 plates/m for benzene homologues) and high stability (RSD is 0.08-5.09%). Comparing to other traditional columns of the same type, the proposed stationary phase has a wider polarity separation scale and shorter analysis time. In addition to the rapid separation of hydrophobic compound, such column also exhibited great potential in the separation of hydrophilic analytes.

Characterization and comparison of mixed-mode and reversed-phase columns; interaction abilities and applicability for peptide separation.

In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX.

Selection of calibration compounds for selectivity evaluation of siloxane-bonded silica columns for reversed-phase liquid chromatography by the solvation parameter model.

For the faster evaluation of selectivity in reversed-phase liquid chromatography of siloxane-bonded silica columns using the solvation parameter model a minimal set of calibration compounds is described suitable for mobile phase composition from 20-70% (v/v) methanol-, acetonitrile-, or tetrahydrofuran-water. The Kennard-Stone uniform mapping algorithm is used to select the calibration compounds from a larger database of compounds with known retention properties used earlier for column selectivity evaluation. Thirty-five compounds are shown to be necessary to minimize the standard deviation of the system constants and to minimize the difference between the system constants determined by conventional calibration and the values obtained for the reduced calibration compounds. The models for SunFire C18 with methanol-, acetonitrile- and tetrahydrofuran-water mobile phase compositions and XBridge Shield RP18, XBridge C8, XBridge Phenyl and Discovery HS F5 with methanol- and acetonitrile-water mobile phase compositions had an average coefficient of determination of 0.996 (standard deviation = 0.003, n = 11) and average standard error of the estimate 0.025 (standard deviation = 0.005, n = 11) for the reduced calibration compounds. Some octadecylsiloxane-bonded silica stationary phases with a high bonding density and methanol-water mobile phase compositions containing ≤ 30% (v/v) methanol exhibit extreme retention factors (log k > 2.5) for the low-polarity, two-ring aromatic compounds in the thirty-five compound calibration set. Alternative calibration compounds with more favorable retention properties are suggested as replacements in these cases. The predictive capability of the calibration models is validated using external test sets characterized by the average error, average absolute error and root mean square error of prediction. For the thirty-five calibration compounds sets the average absolute error 0.026 (standard deviation = 0.009, n = 11) and root mean square error of prediction 0.032 (standard deviation = 0.010, n = 11) confirm the suitability of the calibration models for column selectivity evaluation. System maps for XBridge Shield RP18 for 20-70% (v/v) methanol-water and Synergi Hydro-RP and 50% (v/v) methanol-water at temperatures from 25-65 °C together with a correlation diagram for XBridge Shield RP18 and SunFire C18 are presented as representative applications of the reduced calibration compounds for column selectivity evaluation.

Selectivity evaluation of core-shell sislica columns for reversed-phase liquid chromatography using the solvation parameter model.

For the selectivity evaluation of core-shell silica, siloxane-bonded reversed-phase columns by the solvation parameter model a minimal set of thirty-five calibration compounds were identified for applications with mobile phases containing from 20-70 % (v/v) methanol- or acetonitrile-water. The Kennard-Stone uniform mapping algorithm was employed to select the calibration compounds from a larger database of compounds with known retention properties used previously for column selectivity evaluation. The number and identity of the calibration compounds was optimized by considering the statistical quality of the calibration models, the absolute value for the standard deviation of the individual system constants, and the closeness of the estimated system constants for the calibration models to the values for the full data set on seven core-shell columns with different morphology for mobile phases containing 50 % (v/v) methanol- or acetonitrile-water. The predictive capability of the calibration models was evaluated for an external test set for 50 % (v/v) methanol-water for the same columns with a typical root mean square error of prediction (log k) of 0.028 for the thirty-five calibration compounds (n = 35) models. For all columns and mobile phase compositions the calibration models with n = 35 had a range for the coefficient of determination from 0.999 to 0.970 with < 5 % smaller than 0.99 (84 models) and a standard error of the estimate from 0.011 to 0.057 with 75 % < 0.030. System constants determined at 10 % (v/v) increments of organic solvent were utilized for the construction of system maps and correlation diagrams on Kinetex columns with octadecylsiloxane-bonded (C18 and EVO C18), diisobutyloctadecylsiloxane-bonded (XB-C18) octylsiloxane-bonded (C8), biphenylsiloxane-bonded (Biphenyl), phenylhexylsiloxane-bonded (Phenyl-Hexyl) and pentafluorophenylpropylsiloxane-bonded (F5) silica stationary phases for 20-70 % (v/v) methanol- and acetonitrile-water mobile phases. System maps for the Kinetex Phenyl-Hexyl column and correlation diagrams for columns of similar selectivity (Kinetex C8 and Phenyl-Hexyl) and dissimilar selectivity (Kinetex EVO C18 and Biphenyl) is used to illustrate the general approach to selectivity evaluation.

Integration of Solid-Phase Extraction and Reversed-Phase Chromatography in Single Protein-Coated Columns for Direct Injection of Bupivacaine in Human Serum.

A rapid, reliable and precise integrated solid-phase extraction (SPE) and reversed-phase liquid chromatography method was developed and validated to determine bupivacaine in human serum using single protein-coated analytical columns. The protein-coated columns were packed with four different sorbents: TSK-ODS, LiChrosorb RP-8, LiChrosorb RP-2 and µ-Bondapak CN-bonded silica. The method involved direct injection of serum sample onto the columns for trapping of the analyte, clean-up from weakly retained serum endogenous components, as well as the final separation. The protein-coated columns operated in two different chromatographic modes. Serum proteins were extracted and cleaned up by SPE, whereas the final separation of bupivacaine was based on reversed-phase chromatography. The protein-coated TSK-ODS column resulted in more accurate peak integration and more reproducible results. A linear relationship between the concentrations of drug and peak areas was confirmed in the range of 100-2000 ng/mL. Detection and quantification limits were 24.85 and 85.36 ng/mL, respectively. The average recovery for bupivacaine ranged from 96.48% to 98.81%. The present methodology was successfully applied, with a high degree of confidence, to analyze clinical samples obtained from patient receiving 0.5% bupivacaine therapy.

Comprehensive online multicolumn two-dimensional liquid chromatography-diode array detection-mass spectrometry workflow as a framework for chromatographic screening and analysis of new drug substances.

The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-µm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-µm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. Graphical Abstract.

In-column dilution: Improving volume loadability in reverse phase gradient chromatography through the use of a silica pre-column.

Volume overload is a critical limitation in Reversed Phase (RP)-HPLC purification of pharmaceutical compounds. Limited solubility of these materials in most injection solvents leads to large injection volumes in order to maximize throughput. However, peak distortion due to volume overload limits injection amounts, and results in suboptimal use of chromatographic instruments. Volume loading for RP gradient separations was significantly increased by inserting a silica column ahead of the RP separation column. The sole purpose of this column is to dilute the plug of strong injection solvent so that the actual sample constituents are retained when the diluted injection plug arrives at the RP column. This is similar to the concept of a "retention gap" in GC, yet this has never been applied to liquid chromatographic separations. Injection volumes were increased by almost a factor of 3 when using appropriately sized silica columns. A discussion of critical parameters that determine the effectiveness of this approach is provided. The concept is easily applied and does not require any system modifications. It is therefore well suited for open access applications where more instrument intensive approaches, such as "At-Column Dilution", would be less desirable. We will also show that the generic concept which we have titled "In-Column Dilution" can easily be applied to increase the detection sensitivity for analytical application as well by allowing injection of larger sample volumes without peak deterioration for purifications.

A comprehensive investigation of the peak capacity for the reversed-phase gradient liquid-chromatographic analysis of intact proteins using a polymer-monolithic capillary column.

The present study reports on the analysis of different factors affecting the magnitude of the peak capacity for intact protein separations conducted in gradient reversed-phase liquid chromatography. Experiments were conducted using a 200 µm i.d. capillary styrene-co-divinylbenzene monolithic column that was developed in-house and was characterized by a mode globule cluster size of 1.2 µm and a mode macropore size of 1.0 µm (based on scanning electron microscopy). The monolith yielded a minimum plate-height value of 13.3 µm for uracil. The use of trifluoroacetic acid instead of formic acid as ion-pairing agent generally led to better peak symmetry, narrower peak widths which effect is protein-dependent, and improved loadability characteristics. The peak capacity has been systematically assessed at different flow rates and gradient duration. The highest peak capacity of 247 was obtained at a flow rate of 1 µL min-1 and a gradient time of 120 min, which corresponds to an optimal tG/t0 ratio of ∼60. While the optimum van Deemter velocity for intact proteins was approximated to be 0.065 µL min-1, the highest peak capacity was achieved at approximately 20-fold higher flow rate, depending on the gradient duration applied and the molecular weight of the proteins. The optimum velocity increased with decreasing gradient time and is a compromise between the magnitude of the mass-transfer contribution (decreasing the peak capacity with velocity) affected by molecular diffusion, and the increase in peak capacity induced by the more favorable gradient-volume ratio.

Enhancing online protein isolation as intact species from soy flour samples by actively modulated two-dimensional liquid chromatography (2D-LC).

In this study, an enhanced fully automated approach is described for the protein isolation from soy flour samples by two-dimensional liquid chromatography with active modulation interface. The use of two multi-port switching valves is proposed to on-line connect the first to the second dimension column, thus overcoming the problems associated with the re-mixing effects and incompatibility of eluent composition and pH. A 5-cm long C4 analytical column installed in the interface device allows to focus the proteins coming from the first column (size exclusion chromatography), before their selective elution in the second column (reversed-phase). A trap washing step was included in the total workflow, as a desalting step to remove buffer residues from the eluent of the first column and to enhance the chromatographic performances of the second column. The experimental conditions were optimized by analyses of mixed standard solutions of bovine serum albumin, glucose oxidase, immunoglobulin A, thyroglobulin and myoglobin. Then, the optimized 2D-LC method was applied to the protein analysis in extracts of soy flour, known worldwide as one of the major food allergen sources, with the final aim to recovery sufficient protein amounts for the molecular characterization and the assessment of the pattern of allergenic components.

The correctness of van 't Hoff plots in chiral and achiral chromatography.

van 't Hoff plots (logarithm of the retention factor, ln k, vs. the reciprocal of absolute temperature, 1/T) are commonly used in chromatographic studies to characterize the retention mechanisms based on the determined enthalpy (ΔH∘) and entropy (ΔS∘) change of analyte adsorption. In reversed phase liquid chromatography, the thermodynamic parameters could help to understand the retention mechanism. In chiral chromatography, however, the conclusions drawn based on van 't Hoff plots can be deceptive because several different types of adsorption sites are present on the surface of stationary phase. The influence of heterogeneity, however, cannot be studied experimentally. In this study, we employed two reversed phase columns with different retention mechanisms to show that by serially coupling the columns, the obtained thermodynamic parameters are not related to the results obtained on the respective individual columns. Furthermore, our results show that the experimental conditions - such as flow-rate or choice of instrument - will strongly influence the calculated enthalpy and entropy values.

Purification of tertiary and quaternary alkaloids from Rhizoma Corydalis using reversed-phase/weak cation-exchange mixed-mode class separation combined with preparative C18 and silica based strong cation-exchange chromatography.

A new optimization strategy for purification of alkaloids from Rhizoma Corydalis using preparative liquid chromatography was developed, featuring a selective separation of different types of alkaloids into different parts by a reversed-phase/weak cation-exchange mixed-mode column (named C18WCX) at first. The total alkaloids of Rhizoma Corydalis were divided into four fractions with fraction III and IV corresponding to the tertiary type medium bases and the quaternary type strong bases, respectively. For fraction III, a conventional C18 column was used to isolate tertiary alkaloids using acetonitrile and 0.1% phosphoric acid (adjusted with triethylamine to pH 6.0) as mobile phases. High selectivity and symmetrical peak shapes of tertiary alkaloids were obtained, resulting in six main tertiary alkaloids isolated in a single run. As strong bases, quaternary alkaloids often suffer from serious peak tailing problem on conventional C18 columns. Therefore, a silica-based strong cation-exchange (SCX) column was used for purification of fraction IV. On the SCX column, good peak shapes in high sample loading were achieved. Five main quaternary alkaloids were isolated and identified from the fraction in one-step. The procedures presented effective for the preparative isolation and purification of alkaloids from Rhizoma Corydalis.

Timed relay contact closure controlled system for parallel second dimensions in multi-dimensional liquid chromatography.

OBJECTIVE: Short-chain triacylglycerols (TAGs) in lipid extracts of biological samples are not sufficiently resolved using conventional reversed-phase separation on two C18 columns in series, or using a two-dimensional chromatographic separation with a silver ion column as the second dimension (2D). An additional dimension of separation was required. RESULTS: The hardware and software components to allow a second second-dimension (2D) separation and three total separation dimensions were developed. Two contact closure (CC) activated 4-port, 2-position valves (4P2PVs) for ultra-high performance liquid chromatography (UHPLC) were joined together and used for one of two second dimensions in comprehensive two-dimensional liquid chromatography (2D-LC) coupled to four mass spectrometers simultaneously in parallel in an LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4 configuration. A timed contact closure circuit (TCCC) controlled the two UHPLC valves, operated by repetitive CCs for the 4P2PVs. The TCCC-controlled 4P2PVs were used to direct a portion of the 1D eluent to one of the two 2D's for separation by a quaternary UHPLC system that was not allowed by the commercial 2D-LC system. The 1D separation was a non-aqueous reversed-phase HPLC instrument used for separation of TAGs; the commercial 2D-LC 2D binary UHPLC was used for silver-ion chromatography of unsaturated TAGs; and the CC-controlled second 2D was used for separation of short-chain (SC) saturated TAGs.

Preparative HPLC Separation of Underivatized Amino Acids for Isotopic Analysis.

Single-compound analysis of stable or radioactive isotopes has found application in a number of fields ranging from archaeology to forensics. Often, the most difficult part of these analyses is the development of a method for isolating the compound(s) of interest, which can derive from a wide range of sample types including the hair, nails, and bone.Here we describe three complementary preparative HPLC techniques suitable for separating and isolating amino acids from bone collagen and hair keratin. Using preparative reversed-phase, ion-pair, or mixed-mode chromatography in aqueous carbon-free mobile phases, or those from which carbon can easily be removed, underivatized single amino acids can be isolated and further analyzed using mass spectrometric techniques.

Amino Acid Analysis in Physiological Samples by GC-MS with Propyl Chloroformate Derivatization and iTRAQ-LC-MS/MS.

Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography-quadrupole mass spectrometry (GC-qMS) analysis in a single-ion monitoring mode. Derivatization is carried out directly in the aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC-MS. The method delivers the quantification of 26 amino acids. The iTRAQ method employs the labeling of amino acids with isobaric iTRAQ tags. The tags contain two different cleavable reporter ions, one for the sample and one for the standard, which are detected by fragmentation in a tandem mass spectrometer (MS/MS). Reversed-phase liquid chromatography (RP-LC) of the labeled amino acids is performed prior to mass spectrometric analysis to separate isobaric amino acids. The commercial iTRAQ kit allows for the analysis of 42 physiological amino acids with a respective isotope-labeled standard for each of these 42 amino acids.

Methods for Absolute Quantification of Human Plasma Free Amino Acids by High-Performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry Using Precolumn Derivatization.

Plasma free amino acid (PFAA) concentrations in humans are affected by various diseases. However, the variations caused are not dramatic, so a high accurate and precise method for analyzing PFAAs is required. The PFAA analysis protocol described in this chapter covers blood sampling, sample pretreatment, amino acid derivatization, and LC-MS analysis. Each procedure is important for accurate and precise quantification.In the protocol, a human blood sample is collected using an EDTA-2Na or 2K vacuum collection tube and then immediately cooled in water mixed with crushed ice. The sample is then centrifuged on cooling to allow a plasma sample to be removed. A stable-isotope-labeled internal standard solution is added to the plasma, and then the plasma is deproteinized with acetonitrile. The amino acids in the plasma are then derivatized using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS) reagent which is designed for LC-MS analysis. The derivatized amino acids are separated by reverse-phase HPLC and detected by electrospray ionization mass spectrometry. Using this method, 21 amino acids in human plasma can be analyzed with a 12 min cycle. The accuracy and precision are both better than the required criteria given by the US Food and Drug Administration in guidance of Bioanalytical Method Validation.

DL-Amino Acid Analysis Based on Labeling with Light and Heavy Isotopic Reagents Followed by UPLC-ESI-MS/MS.

L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d5]-OSu) were synthesized and used as the chiral labeling reagents for the enantioseparation of amino acids by reversed-phase UPLC-ESI-MS/MS. The enantiomers of amino acids were labeled with the reagents at 60 °C for 10 min in an alkaline medium. The resulting diastereomers were well separated by the reversed-phase chromatography using an ODS column, packed with small particles (1.7 µm) (Rs = 1.95-8.05). A highly sensitive detection at a low-fmol level (0.5-3.2 fmol) was obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy variants for the differential analysis of the DL-amino acids in different sample groups is also presented in this paper. The ratios of D/L-alanine in different yogurt products were successfully determined by the proposed method. The D/L ratios were almost comparable to those obtained from only using light reagent (i.e., L-PGA-OSu). Therefore, the proposed strategy seems to be useful for the differential analysis of DL-amino acids, not only in food products but also in biological samples.

Enhanced Separation Capability of Sequential Injection Chromatography for Fluorimetric Determination of Intracellular Dissolved Free Amino Acids in Marine Microalgae.

This chapter describes improvements in a sequential injection method to automate the fluorimetric determination of amino acids by pre-column derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol. Separation is achieved by reversed-phase liquid chromatography in a 50 × 4.6 mm C18 silica-based monolithic column. The method is low-priced, and the separation is performed by stepwise gradient elution using six mobile phases. The mobile phase used for the first elution step is composed of methanol/tetrahydrofuran/10 mM phosphate buffer (pH 7.2) at volumetric ratio 8:1:91. Additional elution steps use mobile phases containing methanol and 10 mM phosphate buffer at volumetric ratios of 17.5:82.5, 25:75, 35:65, 50:50, and 65:35. Nineteen chromatographic peaks are observed in a mixture of twenty amino acids. The only complete co-elution is between tryptophan and methionine. The entire cycle of amino acid derivatization, chromatographic separation, and column conditioning at the end of separation lasts for 30 min. The method is successfully applied to quantify the major intracellular dissolved free amino acids in the marine microalgae Tetraselmis gracilis, Phaeodactium tricornutum, and Synechococcus elongatus.