Chiral separation of fluorescamine-labeled amino acids using microfabricated capillary electrophoresis devices for extraterrestrial exploration

Chiral separations of fluorescamine-labeled amino acids are characterized and optimized on a microfabricated capillary electrophoresis (CE) device. A standard mixture of acidic and neutral amino acids is labeled with fluorescamine in less than 5 min and the hydroxypropyl-beta-cyclodextrin (HPbetaCD) concentration, temperature, and pH are optimized (15 mM HPbetaCD, 6 degrees C, pH < 9) to achieve high-quality and low background chiral separations in less than 200 s. All four stereoisomers formed in the labeling reaction of the chiral dye with the chiral amino acids are typically resolved. At pH > 9, isomerization of the dye chiral center is observed that occurs on the time scale of the chip separation. Typical limits of detection are approximately 50 nM. These results demonstrate the feasibility of combining fluorescamine labeling of amino acids with microfabricated CE devices to develop low-volume, high-sensitivity apparatus and methods for extraterrestrial exploration.     

Characterisation and identification of proteinaceous binding media (animal glues) from their amino acid profile by capillary zone electrophoresis

A capillary electrophoretic method for identifying different species of proteinaceous binders--collagen, egg white, and milk casein--is described. It allows characterisation of the proteins on the basis of the amino acid profiles obtained after their acidic hydrolysis. The profiles of the underivatised amino acids are recorded directly by capillary zone electrophoresis at pH 2.26 with the aid of a conductivity detector, thus eliminating the need for a derivatisation step. Identification is carried out by means of different relative peak areas of the amino acids. A scheme is given for identification, which is based on the main markers, hydroxyproline, proline, glycine, glutamic acid, and serine and valine.     

Enantioseparation of underivatised amino acids by ligand exchange capillary electrophoresis in a counter-electroosmotic mode

Enantiomer separations of underivatised amino acids were carried out by using ligand exchange capillary electrophoresis (LECE). Chiral discrimination is based on the formation of ternary complexes between copper(II), a chiral selector (L-proline or trans-4-hydroxy-L-proline) and an amino acid. All amino acids containing aromatic moieties or not were detected at 214 nm because of their interactions with copper(II). In order to reduce copper(II) adsorption onto capillary walls, we used hexadimethrine bromide to reverse the electroosmotic flow. Using this original strategy, the studied enantiomers migrated in the opposite direction of the anodic electroosmosis. After optimising the analytical conditions taking into account the chiral resolution and the detection sensitivity, we performed very satisfactory enantioseparations not only of aromatic amino acids (tryptophan, tyrosine, phenylalanine and histidine) but also of aliphatic amino acids (threonine, serine, isoleucine and valine). These enantioseparations were performed in a short analysis time at 35 °C. In order to rationalise the obtained results, we evaluated the complexation constants corresponding to the formed ternary complexes by capillary electrophoresis and we used molecular mechanics modelling.     

Development of a temperature gradient focusing method for in situ extraterrestrial biomarker analysis

Scanning temperature gradient focusing (TGF) is a recently described technique for the simultaneous concentration and separation of charged analytes. It allows for high analyte peak capacities and low LODs in microcolumn electrophoretic separations. In this paper, we present the application of scanning TGF for chiral separations of amino acids. Using a mixture of seven carboxyfluorescein succinimidyl ester-labeled amino acids (including five chiral amino acids) which constitute the Mars7 standard, we show that scanning TGF is a very simple and efficient method for chiral separations. The modulation of TGF separation parameters (temperature window, pressure scan rate, temperature range, and chiral selector concentration) allows optimization of peak efficiencies and analyte resolutions. The use of hydroxypropyl-beta-CD at low concentration (1-5 mmol/L) as a chiral selector, with an appropriate pressure scan rate ( -0.25 Pa/s) and with a low temperature range (3-25 degrees C over 1 cm) provided high resolution between enantiomers (Rs >1.5 for each pair of enantiomers) using a short, 4 cm long capillary. With these new results, the scanning TGF method appears to be a viable method for in situ trace biomarker analysis for future missions to Mars or other solar system bodies.     

Protein mapping by two-dimensional high performance liquid chromatography

Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D-HPLC system described in this work consisted of anion- or cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. We used a comprehensive two-dimensional approach based on different separation speeds. In the first dimension 2.5 microm polymeric beads bonded with diethylaminoethyl and sulfonic acid groups, respectively, were applied as ion exchangers and operated at a flow-rate of 1 ml/min. To achieve very high-speed and high-resolution separations in the second dimension, short columns of 14 x 4.6 mm I.D. with 1.5 microm n-octadecyl bonded, non-porous silica packings were chosen and operated at a flow-rate of 2.5 ml/min. Two reversed-phase columns were used in parallel in the second dimension. The analyte fractions from the ion-exchange column were transferred alternatively to one of the two reversed-phase columns using a 10-port switching valve. The analytes were deposited in an on-column focusing mode on top of one column while the analytes on the second column were eluted. Proteins, which were not completely resolved in the first dimension can, in most cases, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeatability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were less than 1% (n = 15), while the RSDs of the peak areas were less than 15% (n = 15) on average. The limit of detection was 300 ng of protein on average and decreased to 50 ng for ovalbumin. The 2D-HPLC system offered high-resolution protein separations with a total analysis time of less than 20 min, equivalent to the run time of the first dimension.     

Understanding chiral molecular micellar separations using steady-state fluorescence anisotropy, capillary electrophoresis, and NMR

Chiral separations employing four diastereomers of poly sodium N-undecanoyl leucylvalinate (p-SULV) as chiral selectors are probed by use of MEKC, steady-state fluorescence anisotropy, and NMR. By employing diastereomers and thus altering the stereochemistry of a single amino acid in a systematic way, one may control the enantiorecognition ability of the chiral selector. As a result, one can gain a better understanding of the mechanisms of chiral recognition for the two classes of neutral or anionic chiral analytes studied. Evaluation of the chiral interactions leading to chiral separations confirmed our earlier observation of a strong relationship between the selectivity (alpha) observed using a chromatographic separation technique (MEKC) and that determined from the spectroscopic parameter, beta. A linear alpha versus beta relationship was observed for the molecular micelle p-(L)-SULV with all eight analytes included in this study. However, as we earlier predicted, different groups of analytes had different slopes, i.e., values of m, suggesting different chiral separation mechanisms. Evaluation of the data allowed a grouping of the analytes according to the primary site of chiral interaction with the leucine or valine moiety of molecular micelle chiral headgroup.     

Poly(ethylene glycol)-coated microfluidic devices for chip electrophoresis

Herein, we report on a strategy for durable modification of the channel surface in microfluidic glass chips with the neutral hydrophilic-coating material poly(ethylene glycol) PEG-1M-100. Applied in microchip electrophoresis such PEG-coated devices exhibit a suppressed electroosmotic flow and reduced analyte adsorption. The PEG-coated chips were successfully applied in chip electrophoresis of FITC-labelled amines and amino acids and native proteins as well as in chiral separations. The performance of the coated chips was found to be superior compared with uncoated microchips. The coated chips exhibited high stability and the relative standard deviation of migration times in PEG-coated devices was less than 2%.     

Fast, comprehensive two-dimensional HPLC separation of tryptic peptides based on high-temperature HPLC

Two-dimensional HPLC (2D-LC) has recently received considerable attention, and is being used as an alternative to 2D gel electrophoresis in proteomics research. The greatest impediment to the widespread use of 2D-LC is the long analysis time ranging up to days per analysis, making the technique impractical for many jobs. Here we focus on improving the speed of gradient separations since these are typically used as the second dimension in peptide separations by 2D-LC. Specifically we describe high-temperature, ultrafast HPLC conditions, along with the instrument modifications needed to reduce the analysis time of each complete second-dimension gradient separation to tens of seconds. Most importantly, this system is capable of generating a high peak capacity (1350) characteristic of comprehensive 2D-LC in a relatively shorter analysis time (20 min) with a sampling rate sufficient to minimize information loss with simpler instrumentation than currently used; this is equivalent to one unit of peak capacity per second.     

Reduced surface adsorption in 3D printed acrylonitrile butadiene styrene micro free-flow electrophoresis devices

We have 3D printed and fabricated micro free-flow electrophoresis (µFFE) devices in acrylonitrile butadiene styrene (ABS) that exhibit minimal surface adsorption without requiring additional surface coatings or specialized buffer additives. 2D, nano LC–micro free flow electrophoresis (2D nLC × µFFE) separations were used to assess both spatial and temporal broadening as peaks eluted through the separation channel. Minimal broadening due to wall adsorption was observed in either the spatial or temporal dimensions during separations of rhodamine 110, rhodamine 123, and fluorescein. Surface adsorption was observed in separations of Chromeo P503 labeled myoglobin and cytochrome c but was significantly reduced compared to previously reported glass devices. Peak widths of < 30 s were observed for both proteins. For comparison, Chromeo P503 labeled myoglobin and cytochrome c adsorb strongly to the surface of glass µFFE devices resulting in peak widths >20 min. A 2D nLC × µFFE separation of a Chromeo P503 labeled tryptic digest of BSA was performed to demonstrate the high peak capacity possible due to the low surface adsorption in the 3D printed ABS devices, even in the absence of surface coatings or buffer additives.     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

毛细管电动色谱带电环糊精拆分N-FMOC氨基酸对映体

 将负电性磺丁基 -β -环糊精手性添加剂应用于毛细管电泳氨基酸对映体的拆分研究中 ,对 8种氨基酸对映体与 9-芴甲基氧基甲酰氯 (FMOC-Cl)生成的衍生物进行了分离 ,其中 5种得到了基线分离。考察了背景电解质 p H值及磺丁基 -β -环糊精的浓度对 N-FMOC氨基酸对映体拆分的影响。     

Chiral separation of amino acids and glycyl dipeptides by chiral ligand-exchange capillary electrophoresis comparing Cu(II), Co(II), Ni(II) and Zn(II) complexes of three different sugar acids

This paper deals with comparative studies on the use of copper(II), cobalt(II), nickel(II) and zinc(II) complexes of d-gluconic acid, d-saccharic acid and l-threonic acid as chiral selectors for the enantioseparation of aromatic amino acids and glycyl dipeptides using the principle of ligand-exchange capillary electrophoresis. Although copper(II) is the most frequently used central ion in ligand-exchange capillary electrophoresis, in the case of d-gluconic acid cobalt(II) was shown to be an alternative for the enantioseparation of amino acids. Glycyl dipeptides, however, were resolved only with copper(II) complexes. Zn(II) as a central ion was not effective in all cases and with Ni(II) only some partial separations were achieved.     

The impact of sampling time on peak capacity and analysis speed in on-line comprehensive two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (2DLC) offers a number of practical advantages over optimized one-dimensional LC in peak capacity and thus in resolving power. The traditional “product rule” for overall peak capacity for a 2DLC system significantly overestimates peak capacity because it neglects under-sampling of the first dimension separation. Here we expand on previous work by more closely examining the effects of the first dimension peak capacity and gradient time, and the second dimension cycle times on the overall peak capacity of the 2DLC system. We also examine the effects of re-equilibration time on under-sampling as measured by the under-sampling factor and the influence of molecular type (peptide vs. small molecule) on peak capacity. We show that in fast 2D separations (less than 1 h), the second dimension is more important than the first dimension in determining overall peak capacity and conclude that extreme measures to enhance the first dimension peak capacity are usually unwarranted. We also examine the influence of sample types (small molecules vs. peptides) on second dimension peak capacity and peak capacity production rates, and how the sample type influences optimum second dimension gradient and re-equilibration times.     

Linear peak capacity of a comprehensive multi-dimensional separation

In order to resolve (quantifiably and identifiably separate) the same number of peaks in the analysis of the same mixture yielding statistically uniform peak distribution, a comprehensive 2-D separation needs a two times larger peak capacity than a 1-D separation does. Each additional dimension further reduces the utilization of the peak capacity of comprehensive multi-dimensional (MD) separation by a factor of two per dimension. As a result, the same peak capacity means different things for separations with different dimensionalities. This complicates the use of the peak capacity for comparison of the potential separation performance of the separations with different dimensionalities. To facilitate the comparison, a concept of a linear peak capacity has been proposed. The linear peak capacity of an MD separation is the peak capacity of a 1-D separation that, in the analysis of the same mixture, is statistically expected to resolve the same number of peaks as the MD separation is. There are other factors that differently affect the performance of the separations that have different dimensionalities. Peak capacity of a 2-D separation with a rectangular separation space is 27% larger than the product of the peak capacities of its first and second dimension. This advantage of a 2-D separation is essentially nullified by the fact that the peak capacity of the first dimension of an optimized 2-D separation cannot be higher than 80% of the peak capacity of its first dimension standing alone. All in all, the incremental peak capacity gained from addition of a second dimension will not exceed 50% of the peak capacity of the added second dimension. All results are valid for arbitrarily shaped (not necessarily Gaussian) peaks.     

Chiral separations on multichannel microfluidic chips

Chiral separations of FITC-labeled basic drugs on multichannel microfluidic chips with LIF detector were investigated. A preliminary screening procedure for seven neutral CDs was performed under optimized conditions for chiral separations of three FITC-labeled drugs (baclofen, norfenefrine, and tocainide) on a mono-channel microfluidic chip. According to the results of screening, FITC-baclofen and FITC-norfenefrine as well as two chiral selectors including gamma-CD and dimethyl-beta-CD (DM-beta-CD) were selected as models to perform chiral separations on a two-channel chip. FITC-baclofen enantiomers were separated completely by gamma-CD in one channel, while resolution of FITC-norfenefrine enantiomers was achieved by DM-beta-CD in the other channel in the same run. Furthermore, the feasibility of using one chiral selector to separate multiple chiral samples was studied on a four-channel chip. These results show that multichannel chip has a potential for chiral high-throughput screening.     

Comparison of standard capillary and chip separations of sodium dodecylsulfate-protein complexes

Conditions for converting a set of five standard proteins to electrochemically active sodium dodecylsulfate (SDS) complexes were worked out with the aim of using such complexes for conductivity detection with a a chip electrophoresis system. The results obtained were compared with standard capillary electrophoresis (37 cm (effective length 30 cm)×75 μm I.D. capillary, 10 kV, negative polarity at the inlet). The chip separations were run at 500 V per chip (100 V/cm) as compared to the standard capillary arrangement, which was run at 266.6 V/cm. For the capillary set-up the protein complexes were prepared in aqueous solution (Milli-Q water) made 10 mM with respect to SDS. If the SDS concentration was increased to 50 mM, the separation in the capillary was incomplete. On the other hand with the chip system both approaches yielded acceptable results. The chip separations were slightly (but not distinctly) shorter and offered better separations than the standard set-up. The concentration of the surfactant used for the preparation the complexes results in alternations of the elution sequence, which is preserved if the chip separation is used instead of the capillary set-up. Apparently the full capacity of protein–SDS binding is not exploited for the preparation of the adducts.     

Quantification of D‐Asp and D‐Glu in rat brain and human cerebrospinal fluid by microchip electrophoresis

A microchip electrophoresis (MCE) method with LIF detection was presented for quantification of D ‐aspartic acid (D ‐Asp) and D ‐glutamate (D ‐Glu) in biological samples. D ‐Asp and D ‐Glu were determined after precolumn derivatization with FITC. The chiral separation was performed on a glass/PDMS hybrid microfluidic chip using γ‐CD as chiral selector in the running buffer. High sensitive detection was obtained by the LIF detection. The LODs (S/N = 3) for D ‐Asp and D ‐Glu were 6.0×10^–8 and 4.0×10^–8 M, respectively. Using this method, the levels of D ‐Asp and D ‐Glu in rat brain and human cerebrospinal fluid (CSF) were determined.     

毛细管电泳柠檬酸-Zn2+体系对异亮氨酸对映体的手性分离

应用毛细管电泳/电容耦合非接触式电导（CE-C⁴D）分离检测技术,研究了柠檬酸-Zn²⁺体系对异亮氨酸对映体的手性识别行为。结果表明,采用未涂层石英毛细管（45 cm×50 μm,L_eff=40 cm）,以2.8 mmol/L NaOH+0.8 mmol/L柠檬酸+2.0 mmol/L乙酸锌为非手性介质电泳运行液,分离电压+13 kV,D,L-异亮氨酸对映体得到了良好的手性识别,对映体分离度达到2.0。线性检测范围为1.0~20 mg/L,检出限（S/N=3）为0.40 mg/L。对影响分离度的因素（Zn²⁺的浓度、电泳运行液的组成、分离电压以及其他氨基酸的干扰情况等）进行了详细的讨论,并对手性识别机理作了初步探讨。     

Azithromycin as a new chiral selector in capillary electrophoresis

In capillary electrophoresis (CE), separation of enantiomers of a chiral compound can be achieved through the chiral interactions and/or complex formation between the chiral selector and the enantiomeric analytes on leaving their diastereomeric forms with different stability constants and hence different mobilities. A great number of chiral selectors have been employed in CE and among them macrocyclic antibiotics exhibited excellent enantioselective properties towards a wide number of racemic compounds. The use of azithromycin (AZM) as a chiral selector has not been reported previously. This work reports the use of AZM as a chiral selector for the enantiomeric separations of five chiral drugs and one amino acid (tryptophan) in CE. The enantioseparation is carried out using polar organic mixtures of acetonitrile (ACN), methanol (MeOH), acetic acid and triethylamine as run buffer. The influences of the chiral selector concentration, ACN/MeOH ratio, applied voltage and capillary temperature on enantioseparation are investigated. The results show that AZM is a viable chiral selector in CE for the enantioseparation of the type of chiral drugs investigated.