Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography

A spiral disk assembly composed of five single-channel units was designed for high-speed counter-current chromatography (HSCCC). The retention of different solvent systems ranging from moderately polar to polar organic-aqueous systems to aqueous two-phase systems (ATPs) was investigated under different elution modes. The results indicated that the spiral disk assembly can produce excellent retention of stationary phase for moderately polar organic-aqueous solvent systems, such as chloroform-methanol-water (4:3:2) and hexane-ethyl acetate-methanol-water (1:1:1:1) by pumping lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a high flow-rate (8 mL/min, Sf>70%), regardless of whether the inlet is at the inner or outer terminal of the channel. This makes it possible for fast analysis of some small molecular compounds. This has been proved in the separation of mixtures of three flavones, including isorhamnetin, kaempferol, and quercetin. The spiral disk assembly can also provide satisfactory retention for polar to ATPS such as 1-butanol-acetic acid-water (4:1:5) (<3 mL/min, Sf>70%), 12.5% poly(ethylene glycol) (PEG) 1000-12.5% K2HPO4-75% water (< or =1 mL/min, Sf>70%) and 4% PEG 8000-5% Dextran T500-91% water (< or =0.5 mL/min, Sf>50%) by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a low flow-rate, while this is not possible with the multilayer coil column. Acceptable resolutions were achieved when it was used for the separation of peptides such as Leu-Tyr and Val-Tyr, and proteins including cytochrome c and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins.     

Flat-twisted tubing: Novel column design for spiral high-speed counter-current chromatography

The original spiral tube assembly for high-speed counter-current chromatography (HSCCC) is further improved by a new tube configuration called “flat-twisted tubing” which was made by extruding the tube (1.6 mm I.D.) through a narrow slot followed by twisting along its axis forming about 1 cm twisted screw pitch. This modification interrupts the laminar flow of the mobile phase through the tube and continuously mixes the two phases through the column. The performance of this spiral tube assembly was tested by three types of two-phase solvent systems with different polarities each with a set of suitable test samples such as DNP-amino acids, dipeptides and proteins at the optimal elution modes. In general all these test samples yielded higher resolution with the lower mobile phase than the upper mobile phase. In the most hydrophobic two-phase solvent system composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1, v/v/v/v), DNP–amino acids were separated with Rs-a (peak resolution based on the same column capacity adjusted for comparison) at 4.40 and 73% of stationary phase retention at a flow rate of 0.5 ml/min with the lower mobile phase. In the polar solvent system composed of 1-butanol–acetic acid–water (4:1:5, v/v/v), dipeptide samples were resolved with Rs-a at 4.06, compared to 2.79 with the cross-pressed tube assembly at 45% stationary phase retention, each at a flow rate of 1 ml/min. Finally in the aqueous–aqueous polymer phase systems composed of polyethylene glycol 1000 – dibasic potassium phosphate each 12.5% (w/w) in water, protein samples were resolved with Rs-a at 2.53 compared to 1.10 with the cross-pressed tube assembly at 52% of stationary phase retention, each at a flow rate of 1 ml/min. These results indicate that the present system substantially improves the partition efficiency with a satisfactory level of stationary phase retention by the lower mobile phase.     

Partition Efficiency of High-Pitch Locular Multilayer Coil for Countercurrent Chromatographic Separation of Proteins Using Small-Scale Cross-Axis Coil Planet Centrifuge and Application to Purification of Various Collagenases with Aqueous-Aqueous Polymer Phase Systems

Partition efficiency of the high-pitch locular multilayer coil was evaluated in countercurrent chromatographic (CCC) separation of proteins with an aqueous-aqueous polymer phase system using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The separation column was specially made by high-pitch (ca 5 cm) winding of 1.0 mm I.D., 2.0 mm O.D. locular tubing compressed at 2 cm intervals with a total capacity of 29.5 mL. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate system (pH 9.2) under 1000 rpm of column revolution. This high-pitch locular tubing yielded substantially increased stationary phase retention than the normal locular tubing for both lower and upper mobile phases. In order to demonstrate the capability of the high-pitch locular tubing, the purification of collagenase from the crude commercial sample was carried out using an aqueous-aqueous polymer phase system. Using the 16.0% (w/w) PEG 1000 - 6.3% (w/w) dibasic potassium phosphate - 6.3% (w/w) monobasic potassium phosphate system (pH 6.6), collagenase I, II, V and X derived from Clostridium hystolyticum were separated from other proteins and colored small molecular weight compounds present in the crude commercial sample, while collagenase N-2 and S-1 from Streptomyces parvulus subsp. citrinus were eluted with impurities at the solvent front with the upper phase. The collagenase from C. hystolyticum retained its enzymatic activity in the purified fractions. The overall results demonstrated that the high-pitch locular multilayer coil is effectively used for the CCC purification of bioactive compounds without loss of their enzymatic activities.     

Improved spiral tube assembly for high-speed counter-current chromatography

The original spiral tube support (STS) assembly is improved by changing the shape of the tubing, with 1-cm presses perpendicularly along the length. This modification interrupts the laminar flow of the mobile phase. The tubing in the four return grooves to the center of the rotor is flattened by a specially made pressing tool to increase the number of spiral layers and decrease the dead space volume, thus increasing the column efficiency. The performance of this spiral tube assembly was tested in separations of dipeptides and proteins with suitable polar two-phase solvent systems. The results revealed that the present system yields high partition efficiency with a satisfactory level of stationary phase retention in a short elution time. The present high-speed counter-current chromatographic (HSCCC) system will be efficiently applied to a broad spectrum of two-phase solvent systems including aqueous–aqueous polymer phase systems (TPAS) which are used for separation of biopolymers such as proteins and nucleic acids.     

Protein separation by nonsynchronous coil planet centrifuge with aqueous-aqueous polymer phase systems

Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous-aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000-dibasic potassium phosphate, and PEG 8000-dextran T500 in 5 mM potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000-12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous-aqueous polymer phase systems.     

Studies on the performance of different coiled column configurations for compact type-I countercurrent chromatography

Three types of novel coiled column configurations, i.e. a triangular coiled column and elliptical coiled columns I and II, were designed for type-I countercurrent chromatography and their performances were evaluated with two solvent systems each with suitable test samples. Three dinitrophenyl (DNP) amino acids (DNP-DL-glu, DNP-β-ala and DNP-L-ala) were separated with a moderately hydrophobic two-phase solvent system composed of hexane-ethyl acetate-metanol-0.1 M hydrochloric acid (1:1:1:1, v/v), while two dipeptides (tryptophyl-tyrosine and valyl-tyrosine) were separated with a polar solvent system composed of 1-butanol-acetic acid-water (4.75:0.25:5, v/v). The overall results indicated that the performance of compact type-I countercurrent chromatography was improved by elliptical coiled column I which was mounted with its maximum coil diameter perpendicular to the surface of the column holder. Hydrodynamic effects involved in these separations were discussed.     

Organic high ionic strength aqueous two-phase solvent system series for separation of ultra-polar compounds by spiral high-speed counter-current chromatography

Existing two-phase solvent systems for high-speed countercurrent chromatography cover the separation of hydrophobic to moderately polar compounds, but often fail to provide suitable partition coefficient values for highly polar compounds, such as sulfonic acids, catecholamines and zwitter ions. The present paper introduces a new solvent series which can be applied for the separation of these polar compounds. It is composed of 1-butanol, ethanol, saturated ammonium sulfate and water at various volume ratios and consists of a series of 10 steps which are arranged according to the polarity of the solvent system so that the two-phase solvent system with suitable K values for the target compound(s) can be found in a few steps. Each solvent system gives proper volume ratio and high density difference between the two phases to provide a satisfactory level of retention of the stationary phase in the spiral column assembly. The method is validated by partition coefficient measurement of four typical polar compounds including methyl green (basic dye), tartrazine (sulfonic acid), tyrosine (zwitter ion) and epinephrine (a catecholamine), all of which show low partition coefficient values in the polar 1-butanol-water system. The capability of the method is demonstrated by separation of three catecholamines.     

Mixer-settler counter-current chromatography with multiple spiral disk assembly

A novel system for performing high-speed counter-current chromatography has been developed for separation of biopolymers using polymer phase systems. The spiral disk assembly consisting of eight units, each equipped with over 300 mixer-settler sets, was constructed and performance evaluated in terms of retention of the stationary phase and separation efficiency. A series of experiments was performed with a polymer phase system composed of polyethylene glycol 1000 (12.5%, w/w) and dibasic potassium phosphate (12.5%, w/w) using two stable protein samples of myoglobin and lysozyme at various experimental conditions of flow rates and revolution speeds. The best results were obtained with revolution speeds of 800-1000rpm at flow rates of 0.25-0.5ml/min where the partition efficiency of several 100 theoretical plates was achieved with over 50% stationary phase retention.     

An improved high-performance liquid chromatographic method for simultaneous determination of tocopherols, tocotrienols and γ-oryzanol in rice

An improved normal phase high performance liquid chromatographic (NP-HPLC) method was developed for simultaneous quantification of eight vitamin E isomers (α-, β-, γ- and δ-tocopherols and α-, β-, γ- and δ-tocotrienols) and γ-oryzanol in rice. A complete separation of all compounds was achieved within 25 min using an Inertsil CN-3, SIL-100A 5 μM (4.6 mm × 250 mm) column and an isocratic elution system of hexane/isopropanol/ethylacetate/acetic acid (97.6:0.8:0.8:0.8, v/v/v/v) at a flow rate varying from 0.7 to 1.5 mL min(-1). A linear correlation coefficient (r(2)>0.99) and high reproducibility were obtained at concentrations ranging 0.05-10 μg mL(-1) for vitamin E isomers and 0.5-500 μg mL(-1) for γ-oryzanol. This method proved to be rapid, accurate and reproducible.     

Advantages of a small-volume counter-current chromatography column

Counter-current chromatography (CCC) works with a support-free liquid stationary phase. This allows for preparative separations and purifications. However, there are serious technical constraints because of the need to keep a liquid stationary phase in a column. Centrifugal fields are used. A new commercial hydrodynamic 18 mL column made with a narrow-bore 0.8 mm Teflon tubing was evaluated by comparing it with older hydrodynamic CCC columns and a similar 19 mL column but made with 1.6 mm Teflon tubing. A small-volume CCC column allows for reliable and fast solute partition coefficient determination. When resolution is required, both high efficiency and liquid stationary phase retention are needed. Unfortunately, these two requirements bear technical contradictions. A column coiled with a narrow tubing bore will provide a high chromatographic efficiency while a column containing wider tubing bore will achieve higher stationary phase retention. In all cases, increasing the magnitude of the centrifugal field also increases the stationary phase retention. The solution is to build centrifuges able to produce high fields that will provide acceptable liquid phase retention with narrow-bore tubes. The new 18 mL 0.8 mm tubing bore column is able to rotate as fast as 2100 rpm generating a 240 × g field. The two older CCC columns cannot compete with the new one. However, the small 19 mL column with 1.6 mm bore tubing can be useful when fast results are desired without top resolution.     

Comprehensive separation of a wide variety of compounds from olive leaves by counter-current chromatography with three-phase solvent system

The three-phase solvent system counter-current chromatography has been of great research interest, because it can separate compounds with a wide range of polarity. The solvent system of n-hexane/methyl tert-butyl ether/acetonitrile/water (5:5:7:5, v/v) was used for counter-current chromatographic comprehensive separation of olive leaves. The study adopted the normal elution mode. The middle phase and the lower phase (at a volume ratio of 7:3) were pumped into the column simultaneously, followed by eluting with the upper, middle, and lower phases in sequence. The retention rate of the stationary phase measured by the experiment was 73.5%. The upper phase was used to elute the nonpolar compounds, then the mobile phase was switched to the middle phase to elute the moderately hydrophobic compounds, finally, the polar compounds were eluted by the lower phase remaining in the chromatographic column. This method successfully separated eight compounds in one step within 270 min and five compounds were identified. The logP values of these five compounds were 7.44, 7.86, 4.16, −0.11, and 0.96, respectively, covering a wide range of polarities. The present study demonstrated that the three-phase solvent has a strong extraction capacity for ingredients from extremely hydrophilic compounds to extremely hydrophobic compounds.     

Development and validation of a rapid and efficient method for simultaneous determination of methylxanthines and their metabolites in urine using monolithic HPLC columns

A new rapid, sensitive and validated HPLC method has been developed for the determination of methylxanthines and their metabolites in asthmatic patients. The method was initiated by using spiked urine samples on a silica monolithic column as a novel packing material. The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer/methanol (87.5:12.5 v/v), at a flow rate 1 mL/min. Detection was set at 274 nm. The LOQ for all the compounds ranged from 14 to 41 ng/mL. Excellent linearity was achieved over the studied range of concentration with correlation coefficients 0.9991–0.9998 (n = 6). The developed method was validated by precision and accuracy with RSD <2.55%. On extraction of the drugs and metabolites from the urine samples high recoveries were achieved ranging from 82.06 to 98.34% w/w on RP18 cartridges and methanol/chloroform (20:80 v/v) as the extraction solvent. This method has advantages over other methods using conventional C18 packings.     

高效液相色谱法测定土壤中香豆素类灭鼠药残留

建立了测定土壤中杀鼠灵、杀鼠醚、溴敌隆、氟鼠灵、溴鼠隆5种香豆素类灭鼠药残留量的柱后衍生荧光检测高效液相色谱方法。样品在加入内标物氯杀鼠灵后用丙酮-氨水-甲醇（体积比为100：3：100）混合液提取,浓缩的提取液用5 mL正己烷-氯仿（体积比为3：1）混合液溶解,NH₂固相萃取小柱净化,用15 mL 50 mmol/L四丁基磷酸二氢铵甲醇溶液洗脱分析物,移除溶剂,用甲醇-0.25%（体积分数）乙酸水溶液（体积比为3：2）混合液溶解,过滤后,经高效液相色谱分离,以甲醇-氨水-水（体积比为1：1：8）混合液为衍生试剂进行柱后衍生,采用荧光检测器检测。杀鼠灵、杀鼠醚、溴敌隆、氟鼠灵、溴鼠隆5种鼠药在0.02～10.00 mg/L范围内线性关系良好,相关系数均大于0.999,定量限（LOQ,S/N=10）为2.2～18.5 μg/L。在0.1～0.3 mg/kg添加水平内,5种灭鼠药的回收率为94.6%～118.0%,相对标准偏差（RSD）为0.8%～10.2% （n=3）。实验结果表明该方法灵敏、准确,重复性好。     

Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor

An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid-liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane-ethyl acetate-methanol-water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol-potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor.     

纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。     

Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems

Dual high-speed countercurrent chromatography (dual CCC) literally permits countercurrent flow of two immiscible solvent phases continuously through the coiled column for separation of solutes according to their partition coefficients. Application of this technique has been successfully demonstrated by separation of analytes by gas–liquid and liquid–liquid two-phase systems. However, the method cannot be directly applied to the system with a set of coiled columns connected in series, since the countercurrent process is interrupted at the junction between the columns. However, this problem can be solved by intermittent dual CCC by eluting each phase alternately through the opposite ends of the separation column. This mode of application has an advantage over the conventional dual CCC in that the method can be applied to all types of CCC systems including hydrostatic equilibrium systems such as toroidal coil CCC and centrifugal partition chromatography. Recently, the application of this method to high-speed CCC (hydrodynamic system) has been demonstrated for separation of natural products by Hewitson et al. using a set of conventional multilayer coil separation columns connected in series. Here, we have developed a mathematical model for this intermittent dual CCC system to predict retention time of the analytes, and using a simplified model system the validity of the model is justified by a series of basic studies on both hydrodynamic and hydrostatic CCC systems with a computer-programmed single sliding valve. The present method has been successfully applied to spiral tube assembly high-speed CCC (hydrodynamic system) and toroidal coil CCC (hydrostatic system) for separation of DNP-amino acid samples with two biphasic solvent systems composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1 and 4:5:4:5, v/v).     

Evaluation on the performance of four different column models mounted on the compact type-I coil planet centrifuge

Optimal positions of coiled separation columns on the type-I centrifuge were determined for four typical two-phase solvent systems to obtain the best separation efficiency (resolution and retention of stationary phase) for each with a suitable set of test samples. A set of short coiled columns is connected in series and mounted around the holder hub in four different ways: (model A) the tail of one unit with left-handedness was connected to the head of the next unit with right-handedness (TL-HR); (model B) the tail of one unit with left-handedness was connected to the tail of the next unit with right-handedness (TL-TR); (model C) the tail of one unit with left-handedness was connected to the tail of the next unit with left-handedness (TL-TL); (model D) the tail of one unit with left-handedness was connected to the head of the next unit with left-handedness (TL-HL). The results indicated that the performance of model D was the best among the four models. High revolution speed (800 rpm) is favorable to separation using the moderately hydrophobic solvent system of hexane-ethyl acetate-methanol-0.1M HCl (1:1:1:1, v/v) (HEMW), while lower revolution speed (600 rpm) is beneficial to the separation with polar solvent system of 1-butanol-acetic acid-water (19:1:20, v/v) (BAW).     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

Studies on the effect of column angle in centrifugal helix counter-current chromatography

The performance of the coiled column of centrifugal counter-current chromatography was investigated by changing the angle between column axis and centrifugal force in the separation of dipeptides or DNP-amino acids each with suitable two-phase solvent systems. In general, retention of the stationary phase (Sf) decreased, and peak resolution (Rs) increased as the column angle was increased. The first series of experiments was performed using a polar two-phase solvent system composed of 1-butanol–acetic acid–water (4:1:5, v/v/v) to separate two dipeptide samples, Trp-Tyr and Val-Tyr, at a flow rate of 1 ml/min at 1000 rpm. When the column angle was changed from 0° to 90°, Rs increased from 1.05 (Sf = 60.1%) to 1.17 (Sf = 38.7%) with the lower phase mobile and from 1.02 (Sf = 67.8%) to 1.14 (Sf = 47.4%) with the upper phase mobile, respectively. The second series of experiments was similarly performed with a more hydrophobic two-phase solvent system composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1, v/v/v/v) to separate three DNP-amino acids, DNP-glu, DNP-β-ala and DNP-ala, at a flow rate of 1 ml/min at 1000 rpm. When the column angle was changed from 0° to 90°, Rs increased from 1.38 (1st peak/2nd peak) and 1.20 (2nd peak/3rd peak) (Sf = 61.1%) to 1.66 and 1.45 (Sf = 34.4%) with the lower phase mobile and from 1.14 and 0.63 (Sf = 72.2%) to 1.53 and 0.87 (Sf = 51.1%) with the upper phase mobile, respectively. The overall results of our studies indicate that increasing the column angle against the radially acting centrifugal force enhances the mixing of two phases in the column to improve the peak while decreasing the stationary phase retention by interrupting the laminar flow of the mobile phase.     

超高效合相色谱-串联质谱法快速测定枸杞中甜菜碱含量（英文）

枸杞作为传统的中药材,具有滋补肝肾、益精明目的功效,亦可作为功能性食品被广泛食用。基于超高效合相色谱-质谱(UPC~2-MS),建立了一种快速、灵敏地定量测定枸杞中甜菜碱(指标化合物)含量的新方法。采用ACQUITY UPC2BEH 2-EP色谱柱(150 mm×2.1 mm,1.7μm),以0.7 mL/min超临界CO_2-甲醇(80∶20,v/v)等度洗脱,成功分离了枸杞中的甜菜碱。在该分离过程中,0.1%(v/v)甲酸作为改性剂;背压为1.31×10~7Pa;柱温为40℃;进样体积为1μL;保留时间为3 min。质谱检测工作于电喷雾(ESI)正离子模式和选择离子监测(SIR)模式。在上述条件下得到线性回归方程。其相关系数为0.999 2,检测范围为0.5～50.0μg/mL,检出限为0.013μg/mL。随后,通过对精度、重复性、稳定性和加标回收率(平均值96.3%)的分析,验证了该方法的有效性。最后,将所建立的方法应用于11批样品的分析。结果表明,该方法可较好地用于枸杞的质量控制与分析评价。