Separation Of Underivatized Amino Acids Research Articles

Mixed-acidic cation-exchange material for the separation of underivatized amino acids

Mixed-acidic cation-exchange (MCX) columns with both strongly (SCX) and weakly (WCX) acidic functional groups were developed for the separation of standard amino acids. The resins were prepared by carboxylation of highly crosslinked monodisperse poly(styrene-divinylbenzene) copolymer particles with performic acid and subsequent sulfonation with sulfuric acid. The degree of functionalization was varied independently for each processing step and controlled by measuring pH dependent retention of the obtained resins. A series of mixed-acidic resins with different SCX/WCX-ratios was chromatographically characterized by variation of formic acid and acetonitrile concentration in the aqueous eluent. The overall cation-exchange capacity was varied from 33 to 68 µmol/mL. The comparison with two commercial columns (Metrohm Metrosep C6, WCX and Hamilton PRP X-200, SCX) revealed the additive character of the different functional group properties within MCX columns and a unique selectivity which can be adjusted by both eluent composition and SCX/WCX-ratio of the resin. The retention window between neutral and basic amino acids was altered by varying the amount of sulfonic acid groups attached to the polymer. Orthogonality plots demonstrated constant selectivity for neutral amino acids. Correlating the retention data with log P data demonstrated the influence of non-ionic hydrophobic and π-π-interactions for the separation of amino acids on PS/DVB-based cation-exchangers. An isocratic IC-ESI-MS method was developed to separate and quantitate 20 underivatized standard amino acids within 30 min. Limits of detection were between 4 and 64 nmol L−1 and a high linearity of calibration curves was obtained for all analytes. The method was validated by comparing a certified reference standard with external calibration data.

Chiral separation of underivatized amino acids in supercritical fluid chromatography with chiral crown ether derived column.

The supercritical fluid chromatographic separation of underivatized amino acids was explored using immobilized chiral crown ether column CROWNPAK CR-I (+) and mass spectrometric detection. The type of modifier, acidic additives, and the role of water were investigated. Enantioseparation was achieved for all 18 amino acids investigated with short retention times (less than 3 minutes) and average resolution of greater than 5.0. Analysis of enantiomerically pure standards demonstrated the D enantiomer eluted first for all amino acids using a CROWNPAK CR-I (+) column.

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.

Isolation of underivatized amino acids by ion-pair high performance liquid chromatography for precise measurement of nitrogen isotopic composition of amino acids: Development of comprehensive LC × GC/C/IRMS method

Nitrogen isotopic composition of amino acids has been widely applied to biochemical, ecological, archeological, and biogeochemical studies in an attempt to trace nitrogen source and transformation processes. For accurate isotope analysis of individual amino acids, we validated a preparative method involving the isolation of underivatized amino acids by ion-pair chromatographic separation and confirmed the consistency of nitrogen isotope composition. Ion-pair reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS) and gas chromatography/combustion coupled with isotope ratio mass spectrometry (GC/C/IRMS) were conducted for the purpose of separation of underivatized amino acids and nitrogen isotopic analysis, respectively. Firstly, we confirmed the resolution of proteinogenic and non-proteinogenic amino acids by the preparative ion-pair LC separation. Diagnostic product ions determined by mass spectrometry can support the rapid identification of individual amino acids in screening analyses. Secondly, we observed no dependency on nitrogen isotopic composition for the injection amount of underivatized amino acids and even for different chemical formula including neutral, acidic, sulfur-containing, heterocyclic, and aromatic species. The recovery during the LC was 91.7±4.3% (n=3). The present method and strategy of LC coupled with GC/C/IRMS (i.e., comprehensive LC×GC/C/IRMS) are useful for the high precision determination of the nitrogen isotopic composition of amino acids, in conjunction with an appropriate pre-treatment of cation-exchange chromatographic procedures.

Enantiomeric Separation of Underivatized Amino Acids: Predictability of Chiral Recognition on Ristocetin A Chiral Stationary Phase

The present work aimed to investigate the predictability of the chromatographic behavior for the separation of underivatized amino acids on ristocetin A, known as Chirobiotic R, using a DryLab high-performance liquid chromatography (HPLC) method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. After implementing the basic runs, and judging the predictability via the computed resolution map, it can be deduced that the chiral recognition mechanisms tend towards a hydrophilic interaction chromatography rather than the reversed phase mode, which limits the ability of DryLab software to predict separations on Chirobiotic R.

Fast enantioseparation by HPLC on a modified carbon nanotube monolithic stationary phase with a pyrenyl aminoglycoside derivative

This work deals with the development of a carbon nanotube (CNT) monolithic column coated with a pyrenyl derivative as chiral selector. For this, a solution of pyrenyl neomycine A was pumping through a monolithic CNT column previously developed by our group. This coating was stable against the desorption for months when aqueous mobile phases were used. This column was applied to the chiral separation of underivatized amino acids. As well, ultra fast separations in the range of seconds were achieved using high flow-rates.

Compound-Specific Radiocarbon Dating of Essential and Non-Essential Amino Acids: Towards Determination of Dietary Reservoir Effects in Humans

When humans consume foods from different radiocarbon reservoirs offset in age to the atmosphere, inaccuracies in the 14C date of bone collagen can occur. Mesolithic human skeletons from the Iron Gates section of the Lower Danube Valley have yielded reservoir offsets of up to ∼500 yr. This has been demonstrated through direct dating of bulk collagen from human bones and the remains of ungulate bone projectile points that were found embedded in them (Cook et al. 2001). We present improvements to a novel HPLC method for the detection and separation of underivatized amino acids using a water-only mobile phase free of organic or inorganic modifiers, ensuring very low carbon backgrounds. Our hypothesis is that direct 14C dating of single essential and non-essential amino acids might allow an improvement in the dating accuracy for reservoir-affected human bones. The method facilitates separation of less polar amino acids (mostly “essential”), currently not possible in the recently published protocol. We discuss methodological developments, demonstrate carbon backgrounds, and present analytical approaches to minimize their effects. We validate the precision and accuracy of the method by accelerator mass spectrometry (AMS) dating relatively modern and 14C-dead, known-age bone standards. Finally, we apply the method to the dating of single amino acids from bone samples with a proven ∼500–yr carbon reservoir effect from Mesolithic burials at the Iron Gates sites. We investigate whether differences can be found in AMS dates for essential and non-essential amino acids since, although contemporaneous, these are expected to derive from dietary sources with differing 14C reservoirs.

Polyfunctional low-capacity cation-exchange packing material for the separation of underivatized amino acids

A polyfunctional low-capacity cation-exchange column-packing material was developed for the simultaneous separation and determination of underivatized 20 amino acids including 16 proteinogenic ones using a binary gradient HPLC. The new packing material was prepared by sulfoacylating a highly crosslinked macroreticular poly(styrene-divinylbenzene) copolymer of 3μm in diameter commercially available for size-exclusion chromatography. The polyfunctionality could derive from unintentional carboxy groups innately present in the base polymers probably came from polymerization initiators in addition to the intentionally introduced sulfopropionyl groups, which was certified and evaluated by measuring dynamic capacity curves of the cation exchanger. Sixteen underivatized proteinogenic amino acids were separated in 23min using a binary high-pressure gradient elution system with two liquids of 1mmolL−1 H3PO4 and 20mmolL−1 NaH2PO4/30% (v/v) CH3CN, which led to the cycle time of 25min. In the case of separation of 20 amino acids, the cycle time was 27min. The developed low-capacity cation-exchange chromatography with post-column fluorescence detection was sufficiently quantitative, providing linear calibration lines ranged through almost three digit for all analytes. The detection limits were calculated as nmolL−1 order of magnitude with 20-μL injection. The method was applicable to the direct analysis of urinary amino acids of diagnostic markers for inborn errors of metabolism.

Rapid determination of amino acids in foods by hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry

This study describes a rapid and sensitive analytical method for the determination of amino acids in foods and drinks. The method entailed dilution or extraction of amino acids from foods using the mixture of acetonitrile and 0.1% aqueous formic acid (50:50, v/v). Chromatographic separation of underivatized amino acids was performed using a hydrophilic interaction liquid chromatography within a runtime of 6 min. Both hydrophobicity and charge of the side chain played important roles on the elution order of amino acids under the chromatographic conditions. High-resolution mass spectrometry allowed qualitative and quantitative detection of amino acids in complex food matrices. Its response was found linear over a concentration range of 0.25-10 μg/ml. The method could be successfully applied to various foods and drinks to profile individual amino acids. Mean percentage recoveries of amino acids from different matrices were 88.5% or higher with residual standard deviation of less than 5.0%.

Advances in Liquid Chromatographic and Voltammetric Analysis of Underivatized Amino Acids

The problems associated with liquid chromatographic analysis of underivatized amino acids are identified and, through reviewing the recent ten years literature, possible solutions are described. Emphasis has been placed on amperometric as well as on multi mode detection. The advantages offered by preliminary voltammetric studies at microelectrodes (under mass transfer conditions similar to those of a flow cell detector) in optimising the operation of amperometric detectors are discussed and the use of environmentally friendly Bi electrode materials for the stripping voltammetric determination of amino acids is also proposed. This review is also aimed at giving a broad outline of the potential for improvement of the separation of underivatized amino acids offered by multimode gradient elution and/or ion-chromatography provided that the relevant separation theory and optimization algorithms are available. Keywords: Underivatized amino acids, liquid chromatographic separation, ultraviolet, conductivity, evaporative light scattering, mass spectrometry, electrochemical, voltammetric analysis, Conductometric detection, capacitively coupled contactless conduc-tivity detection, C4D, RI, refractive index detector, ELS, ELS detector, Chemiluminescent nitrogen, CLN, CLN detector, Fluorescence (FL) Detection, nuclear magnetic resonance, NMR, Electrochemical (EC) or Amperometric Detection, homocystinuria, sinusoidal voltammetry, SV, Linear Sweep, Differential Pulse Voltammetry, LSV, DPV, Adsorptive Stripping Voltammetry, CSV, AdSV, cathodic stripping voltammetry, square wave cathodic stripping voltammetry, SWCSV, Carbon Paste Electrodes, CPEs, Reversed-phase liquid chromatography, RP-HPLC, reversed phase ion-pair chromatography, IPC, drophilic interaction chromatography, HILIC

Chiral Separation of Underivatized Amino Acids by Reactive Extraction with Palladium−BINAP Complexes

In answer to the need for a more economic technology for the separation of racemates, a novel system for reactive enantioselective liquid-liquid extraction (ELLE) is introduced. Palladium (S)-BINAP complexes are employed as hosts in the separation of underivatized amino acids. The system shows the highest selectivity for the ELLE of tryptophan with metal complexes as hosts reported to date and shows a good selectivity toward a range of natural and unnatural amino acids. Furthermore, the host can be prepared in situ from commerically available compounds. Bulk-membrane transport in the form of U-tube experiments demonstrates the enantioselective and catalytic nature of the transport. The dependency of the system on parameters such as pH, organic solvent, and host-substrate ratio has been established. (31)P NMR spectroscopy has been used to confirm the preferred enantiomer in the extraction experiments. The intrinsic selectivity was deduced by determination of the association constants of the palladium complex with the tryptophan enantiomers.

Analysis of amino acid 13C abundance from human and faunal bone collagen using liquid chromatography/isotope ratio mass spectrometry

The scope of compound-specific stable isotope analysis has recently been increased with the development of the LC IsoLink which interfaces high-performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement of non-volatile compounds previously not amenable to compound-specific analysis or requiring substantial modification for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino acids are an example of such compounds. We present a new chromatographic method for the HPLC separation of underivatized amino acids using an acidic, aqueous mobile phase in conjunction with a mixed-mode stationary phase that can be interfaced with the LC IsoLink for compound-specific delta13C analysis. The method utilizes a reversed-phase Primesep-A column with embedded, ionizable, functional groups providing the capability for ion-exchange and hydrophobic interactions. Baseline separation of 15 amino acids and their carbon isotope values are reported with an average standard deviation of 0.18 per thousand (n = 6). In addition delta13C values of 18 amino acids are determined from modern protein and archaeological bone collagen hydrolysates, demonstrating the potential of this method for compound-specific applications in a number of fields including metabolic, ecological and palaeodietary studies.

Preparative separation of underivatized amino acids for compound‐specific stable isotope analysis and radiocarbon dating of hydrolyzed bone collagen

Analysis of stable and radioactive isotopes from bone collagen provides useful information to archaeologists about the origin and age of bone artifacts. Isolation and analysis of single amino acids from the proteins can provide additional and more accurate information by removing contamination and separating a bulk isotope signal into its constituent parts. In this paper, we report a new method for the separation and isolation of underivatized amino acids from bone collagen, and their analysis by isotope ratio MS and accelerator MS. RP chromatography is used to separate the amino acids with nonpolar side chains, followed by an ion pair separation to isolate the remaining amino acids. The method produces single amino acids with little or no contamination from the separation process and allows for the measurement of accurate stable isotope ratios and pure samples for radiocarbon dating.

Performance of 2-Amino Tetraphenyl Porphyrin as Stationary Phase in RP-HPLC of Amino Acids

The search for new stationary phases has been one of the predominant concerns in high performance liquid chromatography (HPLC) in order to achieve better resolutions, longer column lives, and reduce the time of analysis. A chromatographic packing for separation of underivatized amino acids (AAs) were prepared by dynamically coating 2-amino tetraphenyl prophyrin (atpp) on a C-18 reversed-phase packing and its properties were examined. The retention characteristics of 20 AAs forming the building blocks of proteins were investigated on the atpp coated C-18 column at pH 7. Results obtained seem to confirm a mixed mechanism of retention involving the hydrophobic interaction between the aromatic porphyrin macrocycle and some of the AAs, p-p interaction between the p-electrons of the porphyrin macrocycle and the p-electrons of analyte, and the hydrogen bonding interactions between Ass and the porphyrin nitrogens.

Chiral separation of underivatized amino acids by ligand-exchange capillary electrophoresis using a copper(II)– l-lysine complex as selector

A ligand-exchange capillary electrophoretic method was explored, with l-lysine as the ligand and copper(II) as the central ion. Its applicability was demonstrated with underivatized aromatic amino acids, namely d,l-phenylalanine, d,l-tryptophan, d,l-tyrosine and d,l-β-phenylserine. Optical resolutions of a single pair of amino acid enantiomers, and of mixed amino acids were obtained with a running buffer of 10 m M NH 4Ac, 6.67 m M Cu(II) and 13.33 m M l-lysine, pH 7.0. Sodium dodecylsulfate (SDS) was shown to be necessary for simultaneous separation of the mixed amino acids. The resolution was found to increase with the concentration of the copper(II) complex at a copper(II)-to-lysine ratio of 1:2. If the total concentration of copper(II) and lysine was kept at 20 m M, decreasing the ratio of copper(II) to lysine caused a resolution loss of tryptophan, but a slight resolution improvement of the other three amino acids. The pH of buffer is another important factor controlling the separations. For all the studied amino acids, the optimum pH was 6.0. An interesting phenomenon was observed in this study. SDS induces precipitation at a concentration below 32 m M at room temperature (16±2 °C), possibly due to the formation of neutral substance from the SDS monomer and the copper(II)–lysine complex.

Chiral separation of amino acids by ligand-exchange capillary electrochromatography using continuous beds.

A chiral ligand-exchange phase for capillary electrochromatography based on continuous bed technology was developed. The chiral stationary phase is prepared by a one-step in situ copolymerization procedure using methacrylamide, piperazine diacrylamide, vinylsulfonic acid and N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline. These chiral continuous beds are inexpensive and easy to prepare. They also have several advantages over silica-based packed capillaries. Since the bed is covalently attached to the capillary wall, no frit is required. The applicability of this new approach to the chiral separation of underivatized amino acids is demonstrated.

Molecularly imprinted ligand-exchange adsorbents for the chiral separation of underivatized amino acids

Ligand-exchange adsorbents that are enantioselective for underivatized α-amino acids have been synthesized by molecular imprinting using only achiral monomers. Bulk polymers were prepared by allowing the functional monomer, Cu(II)–N-(4-vinylbenzyl)iminodiacetic acid, to form complexes with the template amino acid in solution, followed by crosslinking with ethylene glycol dimethacrylate. To make supports suitable for chromatography, the imprinted polymer was grafted to derivatized silica particles. Racemic mixtures of various underivatized α-amino acids are resolved on the imprinted adsorbents. Adsorbents prepared from amino acids with larger, aromatic side chains exhibit the highest selectivities (α=1.65 for the enantioresolution of d,l-phenylalanine). Cross-selectivity for similar amino acids also depends on side chain size: materials templated with l- or d-phenylalanine exhibit good enantioselectivity when challenged with racemic tyrosine (α∼1.4) and much reduced enantioselectivities towards d,l-tryptophan or aliphatic amino acids. Materials imprinted with alanine show no selectivity. The ability of a material imprinted with an amino acid enantiomer to resolve an analogous chiral amine is also demonstrated. The mechanisms underlying the observed enantioselectivity are discussed in light of the three-point interaction model for conventional chiral ligand-exchange separations.

Preparation and Characterization of Chemically Bound Tartrate‐Sillca Gel as Sorbent for Ligand Exchange Chromatography

AbstractTartrate‐based sorbents of two kinds for ligand‐exchange chromatography were prepared by covalent bonding of functional groups to silica gel through an amide bond. The structure and conversion of functional groups of the resin were confirmed with IR spectral and elemental analysis. The influence of pH on adsorption of metal ions to the resin was examined. The coordination behaviour of the sorbent was investigated by means of IR, EPR and electronic absorption spectroscopy. The results indicate that the affinity of metal ions toward the synthesized resins decreased in the order Cu(II) > Zn(II) > Cd(II). The conformation of the copper(II)‐sorbent complexes may be distorted tetragonal, whereas those of zinc(II) and cadmium(II) may be tetrahedral. These complexes between metal ion and sorbent were investigated as a stationary phase for enantiomeric separation of underivatized amino acids. Factors affecting the retention and the sample selectivity were examined. The interactions between sorbent metal complexes and analytes in this ternary system were also investigated.