Separation of 50 S ribosomal proteins from sulfolobus acidocaldarius by discontinuous reversed-phase chromatography

Summary The total protein of the 50S ribosomal subunit (TP50) from the archaebacterium Sulfolobus acidocaldarius was prefractionated by discontinuous reversed-phase HPLC with several column combinations. The purity of the eluted fractions was tested by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional PAGE (2D-PAGE). With regard to the load capacity and selectivity, best results were obtained with a semi-preparative three-column combination (total length 67.5cm). Twelve A₂₃₀ units of TP50 were separated into 35 fractions, 19 of which contained nearly pure proteins. When the sample load was increased to 176A₂₃₀ units (109mg), 13 of 28 collected fractions still contained nearly pure proteins. The selectivity of a semi-preparative short-column combination (total length 12cm) was similar to that of the semi-preparative column combination, and separation time could be reduced to one third of that required for the longer column combination. The load capacity of the short-column combination was lower than that of the semi-preparative column combination.     

Separation of chiral primary amino compounds by forming a sandwiched complex in reversed-phase high performance liquid chromatography

Separation of chiral primary amino compounds was efficiently achieved under reversed-phase high performance liquid chromatography (RP-HPLC) conditions using a mixture of non-chiral crown ether (18-crown-6) and dimethyl-β-cyclodextrin (DM-β-CD) in the mobile phase. Under these conditions, the amino group of the chiral compound was protonated in a low pH mobile phase, and then interacted with 18-crown-6 and DM-β-CD to form a sandwiched complex [18-crown-6 + amine + CD]. Enantiomers of the compound in the sandwiched complex were separated with good enantioselectivity. Formation of the sandwiched complex among the chiral compound and additives in the mobile phase is a key step of the chiral separation. Four different chiral amino compounds namely, 1-aminoindan (AI), 1,2,3,4-tetrahydro-1-naphthylamine (THNA), tyrosine (Tyr), and phenylalanine (Phe), were selected to demonstrate the separation using the sandwiched complex mechanism in RP-HPLC.     

Separation of enantiomers by high performance liquid chromatography using chiral eluents

Simple procedures are presented for separating the enantiomers of α-methyldopa, 5-hydroxytryptophan, tryptophan, triiodothyronine and thyroxine, which require neither special sorbents nor difficult-to-obtain or unstable reagents. The method for α-methyldopa, 5-hydroxytryptophan and tryptophan is based on the use of L-phenylalanine copper complex as the chiral constituent of the mobile phase; LiChrosorb® RP-18 serves as the stationary phase. The procedure for triiodothyronine and thyroxine is grounded on the L-proline copper complex as the chiral reagent and LiChrosorb® Si 60 as the stationary phase. In all observed cases, the D-enantiomer is eluted prior to the respective L-enantiomer. Chirality inversion of the mobile phase (application of the D-phenylalanine copper complex) reverses the order of elution; a racemic eluent (DL-phenylalanine copper complex) leads to no separation. In addition to the enantiomers of α-amino acids, the enantiomers of α-hydroxy acids (mandelic acid) can be separated.     

Quick development of an analytical enantioselective high performance liquid chromatography separation and preparative scale-up for the flavonoid Naringenin

The HPLC enantioselective separation of (R/S)-Naringenin, a chiral flavonoid found in several fruits juices and well-known for its beneficial health-related properties, including antioxidant, anti-inflammatory, cancer chemopreventive, immunomodulating and antimicrobial activities, has been performed on both analytical and (semi)-preparative scale using an amylose derived Chiralpak AD chiral stationary phase (CSP). A standard screening protocol for cellulose and amylose based CSPs was firstly applied to analytical Chiralcel OD-H and Chiralpak AD-H, as well as to Lux Cellulose-1, Lux Cellulose-2 and Lux Amylose-2 in order to identify the best experimental condition for the subsequent scaling-up. Using Chiralpak AD-H and eluting with pure methanol (without acidic or basic additives) relatively short retention times, high enantioselectivity and good resolution (α=1.49, R(s)=3.48) were observed. Therefore, these experimental conditions were properly scaled-up to (semi)-preparative scale using both a pre-packed Regispack column and a Chiralpak AD column packed in house with bulk CSP. The developed preparative method proved to be superior to previously published methods in terms of elution times, separation and resolution and is suitable for obtaining a quick access to the desired enantiomers with high enantiomeric excess and amounts sufficient for biological investigations. Future scale-up options (enantioselective supercritical fluid chromatography or HPLC in the Simulated Moving Bed mode) were also evaluated. It could be shown that both methodologies have a high potential for future production of Naringenin enantiomers by enantioselective chromatography.     

反相高效液相色谱法测定川楝树皮中的川楝素

川楝素是从楝属植物川楝树的韧皮或果子中分离出来的呋喃三萜类化合物,在医药上,川楝素主要用作驱蛔药,此外,它还具有明显的抗肉毒效应,能治愈致死剂量肉毒中毒的动物,在农业上,川楝素作为杀虫剂对多种害虫且有很高的活性,可用于防治各种鳞翅目幼虫,对人畜、环境及害虫天敌较为安全,故特别适用于一些重要经济作物的害虫防治,近年来,     

Separation of intact proteins on γ‐ray‐induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high‐performance liquid chromatography with high‐resolution mass spectrometry

Polymethacrylate‐based monolithic capillary columns, prepared by γ‐radiation‐induced polymerization, were used to optimize the experimental conditions (nature of the organic modifiers, the content of trifluoroacetic acid and the column temperature) in the separation of nine standard proteins with different hydrophobicities and a wide range of molecular weights. Because of the excellent permeability of the monolithic columns, an ion‐pair reversed‐phase capillary liquid chromatography with high‐resolution mass spectrometry method has been developed by coupling the column directly to the mass spectrometer without a flow‐split and using a standard electrospray interface. Additionally, the high working flow and concomitant high efficiency of these columns allowed us to employ a longer column (up to 50 cm) and achieve a peak capacity value superior to 1000. This work is motivated by the need to develop new materials for high‐resolution chromatographic separation that combine chemical stability at elevated temperatures (up to 75°C) and a broad pH range, with a high peak capacity value. The advantage of the γ‐ray‐induced monolithic column lies in the batch‐to‐batch reproducibility and long‐term high‐temperature stability. Their proven high loading capacity, recovery, good selectivity and high permeability, moreover, compared well with that of a commercially available poly(styrene‐divinylbenzene) monolithic column, which confirms that such monolithic supports might facilitate analysis in proteomics.     

反相离子对高效液相色谱法分离金属配合物｛Fe［3-(2-吡啶基)-5,6-二苯基-1,2,4-三嗪］3｝2+几何异构体

采用反相离子对高效液相色谱法分离测定了金属配合物｛Fe［3-(2-吡啶基)-5,6-二苯基-1,2,4-三嗪］3｝2+ (［Fe(PDT)3］2+)的两种几何异构体,研究了流动相中有机改性剂(乙腈、甲醇)的含量、不同种类和浓度的离子对试剂(高氯酸钠和十二烷基硫酸钠(SDS))对色谱分离的影响。并在不同的试验条件下,对所获得的色谱参数(保留因子(k)、分离度、选择性因子等)进行了探讨。在不同种类及浓度的离子对试剂条件下,二元流动相中乙腈的含量与两种几何异构体的ln k之间均呈显著的线性关系。研究进一步发现SDS的浓度变化对异构体的保留因子影响程度更为显著。在上述实验的基础上,引入更能灵活调节洗脱强度和分离度的三元流动相(乙腈/甲醇/水),优化选择了三元流动相中有机改性剂的比例以及离子对试剂的种类和浓度,使得异构体的色谱分离得到了满意的结果。实验结果表明,异构体的峰面积(A)和浓度(C)之间的线性关系良好,面式和经式异构体的检测限分别为4.28和3.44 ng/mL (S/N=3)。