Gas chromatography of plasma amino acids using a wide bore column: Comparison with packed and capillary columns

A procedure is described in which a wide bore glass capillary column is used as an alternative to the more traditional packed column in the analysis of amino acid levels in plasma. The coefficients of variation for all amino acids (with the exception of aspartic acid) were better than 11% with recoveries ranging from 81% to 122%. The data are compared with the corresponding results obtained using a packed column and show significant differences (p < 0.005) between values for glycine, serine, isoleucine, proline, methionine, aspartic acid, phenylalanine, and lysine. A similar comparison between results from the wide bore and the fused-silica open tubular (FSOT) column shows better agreement. Adjustment of chromatographic conditions for the wide bore analysis yields results in good agreement with those from FSOT analysis but which still differe significantly from the backed column data.     

Determination of amino acids in human cerebrospinal fluid by gas chromatography

In the present study, the use of gas chromatography (GC) for the determination of amino acids in human cerebrospinal fluid (CSF) is described. Although some amino acids may be determined using a packed column following the removal of glucose, the major interfering component, the inadequate resolution of other amino acids from remaining unidentified components results in poor quantitation. The use of wide bore columns improves reproducibility considerably, but still it does not provide sufficient resolution to enable quantitative determination of all amino acids in human CSF. Good reproducibility data, with CV values for all amino acids of 7% or less and recoveries generally between 80% and 100%, can only be obtained using the fused silica open tubular (FSOT) column. Normal amino acid levels are presented for 10 samples of human CSF, which compare well with data previously reported in the literature.     

Low flow rate modifier addition in packed capillary column supercritical fluid chromatography

A new method to accurately deliver small amounts (0.5 to 20 mol%) of modifier into CO₂ was used to study the effects of three different modifiers (methanol, water, and formic acid) in packed capillary column SFC. The method allows the use of different modifiers, with minimal instrument modification. The effects of the different modifiers at different concentrations on retention and peak shape are shown by analyzing a polarity test mixture and a sample of free fatty acids.     

Determination of amino acids in the hepatic and brain tissue of the rat by gas chromatography

A procedure is described for the quantitative determination of amino acids in hepatic and brain tissue samples from the rat. Because the presence of certain matrix components in the tissue material led to interference with chromatographic analysis they were removed by a prechromatographic “clean-up” step. Quantitative analysis of amino acids, as their N-heptafluorobutyryl iso-butyl ester derivatives, was achieved by high resolution gas chromatography on an apolar fused silica open tubular column. Reproducibility data from the complete procedure are presented; coefficients of variation for arginine and histidine in hepatic tissue varied between 7.1 and 10.1% whereas those for most other amino acids were better than 5%, with a mean recovery of 90%.     

Capillary gas chromatographic analysis of amino acids in geological environments with electron capture detection

Studies have been performed on the analysis of 21 amino acids using a fused-silica open tubular (FSOT) capillary column, and electron-capture detection (ECD) or flame-ionization detection (FID). It was shown with the N(O)-heptafluorobutyryl (HFB) amino acid isobutyl esters that the ECD response was several hundred times more sensitive than the FID response. The relative standard deviation (RSD) of retention relative to norleucine is determined with the ECD. RSD values for all N(O)-HFB amino acid isobutyl esters are relatively small (≦ 0.5%). This method has been successfully applied to trace analysis of most of the amino acids from fossil brachiopods and black shales.     

Gas chromatography of amino acids: Use of a fused-silica capillary column in the determination of plasma amino acids

A capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre-chromatographic “clean-up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on-column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p < 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p < 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.     

Enantiomeric separation of amino acids and nonprotein amino acids using a particle-loaded monolithic column

A solution is prepared of 5 microm silica particles modified with (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine (particle 1) or (S)-N-3,5-dinitrophenylaminocarbonyl-valine (particle 2) suspended in liquid tetraethylorthosilicate, ethanol, and aqueous hydrochloric acid. This solution is injected under pressure into a 30 cm long, 75 microm inner diameter capillary column and heated for 1 h at 120 degrees C after which the modified particles are embedded in a monolithic column of sol gel. The packed column measures approximately 15 cm from the inlet to the window used to view the laser-induced fluorescence. Thirteen different amino acids and three nonprotein amino acids are derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) before injection onto the column for capillary electrochromatographic separation. The enantiomeric separation of the monolithic column packed with particle 1 results in a resolution ranging from 1.14 to 4.45, whereas that packed with particle 2 results in a resolution ranging from 0.79 to 1.17. On the basis of resolution and amount of chiral packing material the enantiomeric separation obtained by capillary electrochromatography is judged to be superior to that obtained previously with high performance liquid chromatography (HPLC).     

Enantiomeric Separation of Non-Protein Amino Acids by Chiral Ligand-Exchange High-Performance Liquid Chromatography

Abstract

The enantiomeric separation by high-performance liquid chromatography of underivatized non-protein amino acids was investigated by using a column packed with octadecylsilanized silica coated with N,S-dioctyl-D-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000). Excellent to good separations of enantiomers were achieved with a variety of nonprotein amino acids carrying aliphatic or aromatic side-chains by optimizing the amount (0-20%, v/v) of the organic component (2-propanol) and the concentration (1-5 mM) of the complexing metal ion (Cu²⁺) in the hydro-organic eluent.     

GC with LC-like packed capillary columns

Capillary columns of 0.3–0.35 mm internal diameter and 0.3–7.7 m length, packed with 3 to 30 μm octadecylsilica stationary phases as used for liquid chromatography, were applied to gas chromatographic separation of low boiling hydrocarbons. Van Deemter plots for these columns showed the optimum column efficiency to occur at linear velocities of 4–5 cm/s. A short column was applied to the rapid separation of components of a natural gas and impurities in standard gases, while a long column was applied to the separation of complex mixtures.     

A new type of capillary column for gas chromatography

A new type of capillary column for gas chromatography was proposed. A sorbent layer (for example, stationary liquid phase) is supported on the internal capillary surface, and the internal (interstitial) volume is packed with nonporous large particles of a sorbent (particle diameter is 0.1—0.6 of the capillary internal diameter). The external surface of the particles can also be coated with the sorbent layer (for example, stationary liquid phase). The specific separation efficiency (number of separation) on the new type column is by 1.6—2.3 times higher than that of the initial classical capillary column.     

Steroid analysis by gas chromatography with SCOT and wide-bore WCOT columns

Glass capillary columns are ideal for analysis of urinary steroid profiles (i.e. the total steroid neutral fraction without prior separation into sub-groups). In this paper performance of both SCOT and wide-bore WCOT columns has been compared, resulting in no significant quantitative difference on urine extracts run as trimethylsilyl ether derivatives. However, because of increased efficiency the WB-WCOT column exhibited more sensitivity in measurement of small amounts of 11-ox-aetiocholanolone. Sensitivity of other steroids was greatly enhanced by use of either SCOT or WB-WCOT, leading to an almost tenfold increase over conventional packed columns (e.g. androsterone and aetiocholanolone < 40 μg per 24 h urine sample). A simple splitless injection system (based on a design of Dr. W. Greenaway, Oxford University) is presented. Experiments were also carried out using a SCOT column in conjunction with an FM-electron capture detector. Whilst efficiency was impaired due to the design of the detector, use of He (carrier) and N₂ (make up gas) showed that several steroid derivatives could be easily measured at well below the 10^?10 g level with much more rapid retention times than with a 1½m packed column. Highly successful separation of 19 amino acids (as fluoro-acyl derivatives) was achieved in 35 minutes using a SCOT (30 m) SP2100 column.     

毛细管电泳-激光诱导荧光用于中药制剂中氨基酸成分的分析

建立了一种用于测定中药制剂中氨基酸成分的毛细管电泳-荧光检测方法. 用含有α-环糊精(α-CD)的硼砂缓冲溶液为背景电解质, 经异硫氰酸荧光素(FITC)衍生的5种氨基酸在50 min内可以得到很好的分离和测定. 考查了各个分离参数对分离的影响, 得到的优化条件为: 含45 mmol/L的α-环糊精的80 mmol/L硼砂缓冲溶液(pH值9.2)作为背景电解质, 分离电压20 kV; 柱温22 ℃. 衍生试剂FITC与单个氨基酸的化学计量比为4∶1时, 能够获得稳定荧光强度的氨基酸衍生物. 在优化条件下, 各氨基酸成分在73.5～2900 nmol/L 的浓度范围内呈良好的线性关系(相关系数r2为0.9906～0.9998). 保留时间和峰面积的相对标准偏差分别为0.8%～3.0%和0.7%～5.7%, 检测限(3倍信噪比)为3.5～35 nmol/L. 该方法准确可靠, 可用于质量控制为目的的中药制剂中氨基酸成分的定量测定.     

Capillary Electrophoretic Resolution of Enantiomers of Aromatic Amino-Acids with Highly Sulfated α-, β- and γ-Cyclodextrins

Using cyclodextrin capillary electrokinetic chromatography (CD-EKC), baseline separation of aromatic amino acids was achieved. The variations in amino acids side chain, in the nature of the aromatic moiety and its substitution, and their derivatives at the amino or carboxylic terminal provide an ideal model for the systematic study of the structural interactions with the chiral selectors:anionic cyclodextrins (highly sulfated-CD or highly S-CD). The use of negatively charged cyclodextrins provides a driving force in the opposite direction of the positively charged or neutral molecules in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. The complete resolution was obtained using 25 mM phosphate buffer at pH 2.5 containing 3 (w/v) of highly S-CD at 25 °C with a applied field of 0.30 kV cm⁻¹ with capillaries dynamically coated with polyethylene oxide (PEO). A comparison was possible to investigate the structural arrangement responsible for regiospecific interactions with the enantiomers of the different amino acids.     

用离子交换树脂脱除氨基酸与盐混合液中的盐

在用蛋白质酸性水解备氨基酸时,因中和残留的酸会使水解液中带入大量的盐。在进一步用离子交换色谱法分离混合氨基酸时,首先需脱掉其中的盐。本文用苯乙烯系强酸性阳离子交换树脂的盐型柱,根据氨基酸与苯乙烯系强酸性阳离子交换树脂之间既存在离子间的静电作用,又存在疏水作用,且二者之间存在协同作用,而盐在盐型苯乙烯系强酸性阳离子交换树脂柱上不保留的原理,用水作为洗脱剂,使盐和氨基酸（配制的盐和氨基酸混合液及含盐的毛发水解液）得到分离,本方法脱除氨基酸中的盐简单易行,用水作为洗脱剂即廉价由不造成污染,盐型树脂不用再生即可用于下次运行,研究了各种条件对分离性能的影响。     

带喷头混合型毛细管色谱柱的制备及其在蛋白质组分离分析中的应用

本研究制备了一种应用于液-质联用（LC-MS）系统中的带喷头混合型毛细管色谱柱．用标准蛋白和酵母蛋白胰蛋白酶酶切溶液考察了其在LC—MS中的离子化效率、毛细管色谱柱分离性能和寿命,并与直接填充型毛细管色谱柱的色谱性能进行比较,未见显著性差异．将制备的带喷头混合型毛细管色谱柱应用于鼠肝蛋白组的分析,在假阳性率为1％的条件下鉴定到1262个肽段,归属于513个蛋白簇．通过对鉴定蛋白质理化性能的统计分析,其等电点和分子量分布高于一般两维凝胶电泳的范围．实验结果还表明该混合型毛细管色谱柱在肽段的理化性能上没有偏性,可以广泛应用于蛋白质组学的研究中．     

一种多通道匀浆装填毛细管色谱柱的新装置及应用

针对目前毛细管色谱柱装柱效率低、不同批次装填的毛细管色谱柱之间性能差异大的问题,我们发展了一种多通道匀浆装填毛细管色谱柱的新装置。该装置以液相色谱泵提供压力、采用磁力搅拌保持匀浆液均匀分散,一次可装填多达6根毛细管色谱柱。以牛血清白蛋白(BSA)的胰蛋白酶酶切肽段混合物为样本,选择峰容量、蛋白覆盖率、3个特定离子的保留时间以及毛细管色谱柱柱压为指标,在毛细管液相色谱-质谱联用系统上对装填的反相毛细管色谱柱的性能进行了评价。分别考察了一次装填的6根毛细管色谱柱、两次装填的12根毛细管色谱柱以及一次装填1根与一次装填6根毛细管色谱柱的性能及稳定性。实验结果表明:同一批次装填的6根毛细管色谱柱的性能相近;不同批次装填的12根毛细管色谱柱的峰容量和覆盖率没有明显的区别,但保留时间和毛细管色谱柱柱压的稳定性较差;一次装填1根和一次装填6根毛细管色谱柱柱性能的稳定性与两次分别装填6根毛细管色谱柱的稳定性相近,即采用本装置可显著提高毛细管色谱柱的装填效率且每次装填毛细管色谱柱的数量不会对柱性能产生影响。     

毛细管胶束电动色谱法分析PTH氨基酸

建立了用毛细管胶束电动色谱法（MEKC）分离19种PTH氨基酸的方法,并探讨了电压、pH值、温度、胶束浓度对氨基酸迁移时间的影响。方法具有速度快、灵敏度高、样品用量少的优点。     

Separation of complex sugar mixtures on a hydrolytically stable bidentate urea‐type stationary phase for hydrophilic interaction near ultra high performance liquid chromatography

A stationary phase bearing both bridged bis‐ureido and free amino groups (USP‐HILIC‐NH₂–2.5SP) for high‐speed hydrophilic interaction liquid chromatography separations was prepared using a one‐pot two‐step procedure starting from 2.5 μm totally porous silica particles. Highly polar compounds, such as polyols, hydroxybenzoic acids, and sugars, were successfully analyzed in shorter times and with higher peak efficiency, when compared to results obtained with a bidentate urea‐type column packed with 5 μm particles. Increased sugarophilicity and better peak shape were attested for the USP‐HILIC‐NH₂–2.5SP column (100 × 3.2 mm id) when compared with two commercially available UHPLC columns, namely an acquity BEH amide packed with totally porous 1.7 μm microparticles and a HILIC Kinetex column packed with core–shell 2.6 μm particles. Finally, the new column was employed in the separation of complex mixture of sugars (mono‐, di‐, and oligosaccharides) and in the analysis of beer samples. The resulting chromatograms showed good selectivity and overall resolution, while the catalyzing effect of the free amino moieties resulted in excellent peak shapes and in the absence of split peaks due to sugar anomerization phenomena.     

Electrochromatographic performance of graphene and graphene oxide modified silica particles packed capillary columns

Graphene oxide functionalized silica microspheres (GO@SiO₂) were synthesized based on condensation reaction between amino from aminosilica particles and carboxyl groups from GO. Reduction of GO@SiO₂ with hydrazinium hydroxide generated graphene modified silica particles (G@SiO₂). GO@SiO₂ and G@SiO₂ packed capillary columns for capillary electrochromatography were thereafter fabricated by pressure slurry packing with single‐particle frits. GO of 0.3 mg/mL in dispersion solution for GO@SiO₂ synthesis was considered as a compromise between retaining and column efficiency whereas GO@SiO₂ of 20 mg/mL in slurries for column packing was chosen for a homogenous and tight bed. Optimum mobile phases were acquired considering both electroosmotic flow and resolution at an applied voltage of −6 kV as the following: acetonitrile/phosphate buffer (10 mM, pH 7.0), 75:25 (v/v) for polycyclic aromatic hydrocarbons and 50:50 (v/v) for aromatic compounds. A comparison was made between electrochromatographic performances for three PAHs (naphthalene, fluorene and phenanthrene) and three aromatic compounds of various polarities (toluene, aniline and phenol) on bare aminosilica, GO@SiO₂ and G@SiO₂ packed columns, which proved the contribution of alone or combinational actions of solvophobic effect and π‐π electron stacking as well as hydrogen bonds to retaining behaviors by GO@SiO₂ and G@SiO₂. Well over‐run, over‐day and over‐column precisions (retention time: 0.3–1.4, 1.1–3.8 and 2.8–5.2%, respectively; peak area: 2.6–6.5, 4.8–8.3 and 6.5–12.6%, respectively) of GO@SiO₂ packed columns were a powerful proof for good reproducibility. Analytical characteristics of GO@SiO₂ packed capillary columns in CEC analysis of fresh water were evaluated with respect to linearity (R² = 0.9961–0.9989) over the range 0.1 to 100 mg/L and detection limits of 9.5 for naphthalene, 12.6 for fluorene and 16.2 μg/L for phenanthrene. Further application to fresh water increased the visibility of the proposed material, where good spike recoveries in the range 89–96% were offered.     

Fast supercritical fluid chromatography of polymers using packed capillary columns

Summary Fast separations of perfluorinated polyethers and polymethylsiloxanes that are composed of 50–80 oligomers were demonstrated in packed capillary column supercritical fluid chromatography (SFC) using a carbon dioxide mobile phase. Separations were accomplished within 10 min using a 13 cm×250 μm i.d. column packed with 2 μm porous octadecyl bonded silica (ODS) particles. Effects of particle diameter of the packing material and pressure programming on separation were investigated, and packed column SFC was compared with open tubular column SFC. Results show that as the particle diameter was decreased from 5 to 3 to 2 μm and the column length was reduced from 85 to 43 to 13 cm, the separation time could be reduced from 70 to 20 to 10 min while still maintaining similar separation (resolution). Short columns packed with small porous particles are very suitable for fast SFC separations of polymers.