The development of an evaluation method for capture columns used in two-dimensional liquid chromatography

Capture columns are important interface tools for on line two-dimensional liquid chromatography (2D-LC). In this study, a systematic method was developed to evaluate and optimize the capture ability of capture columns by off-line method. First, the parameter Δt_R (Δt_R = t₂−t₁−t₀−W) was introduced to quantitatively represent the capture ability of the capture column by connecting a capture column behind the first dimensional column. Based on the value of Δt_R, an appropriate capture column was selected after the first dimensional column was fixed. Then, the capture ability of the selected column was promoted by adjusting the mobile phase of the first dimensional column. Capture ability was also optimized using complex sample analysis software system (CSASS) software. Second, the elution mode of the trapped compounds on the capture column was investigated by connecting the capture column before the second dimensional column. More specifically, in mode I, capture column was connected to the second dimension without changing the flow rate direction and the trapped compounds must pass through the capture column and be eluted into the second dimensional column. The contrary connection mode was mode II. It was found that mode I is more suitable method for 2D-LC. Finally, an off-line reversed-phase/hydrophilic interaction liquid chromatography two-dimensional liquid chromatography (RP/HILIC 2D-LC) system with a C18 capture column was developed to demonstrate the practical application of this method.     

The flow-through silica as the matrix to immobilize gold nanoparticles for HPLC applications

In the present study, the flow-through silica, featured with hierarchical pores, i.e., tunable mesopores and penetrable macropores, was attempted as the chromatographic stationary phase matrix to immobilize gold nanoparticles (AuNPs). It was first modified by mercapto groups (named as SiO₂-SH), and then by AuNPs (named as SiO₂-S-Au). Thanks to the characteristic macropores, the column backpressure of SiO₂-S-Au was comparable to SiO₂-SH, which effectively overcame the difficulty of high column backpressure upon the nanoparticles were introduced to the chromatographic matrix. Both the reversed-phase and hydrophilic interaction liquid chromatographic performance were observed on these two columns but with different selectivities. Hydrophobic, hydrophilic, hydrogen bond and electrostatic interactions between the SiO₂-S-Au stationary phase and analytes could contribute to the retention. The SiO₂-S-Au column showed excellent aqueous compatibility by “Stop-flow” test with the relative standard deviations (RSD) of analyte’s k (capacity factor) values from 0.59% to 2.88%. The reproducibility of SiO₂-S-Au was acceptable with RSDs of analyte’s k values in the range of 3.13%-5.03%. In addition, compared with the SiO₂-SH column, the SiO₂-S-Au column had better separation performance and selectivity. The results demonstrated that the flow-through silica was a promising matrix for nanoparticles with low backpressure and different selectivities.     

Remediation of lead by pretreated red algae: adsorption isotherm,kinetic, column modeling and simulation studies

Batch and column biosorption experiments were carried on for lead uptake using ecofriendly biosorbents (raw and calcium chloride [CaCl₂] or formaldehyde pretreated form of red marine algae Jania ruben [Linnaeus]). The best-fit (linear and nonlinear) isotherms, kinetics, and column adsorption models were identified using statistical analysis. Freundlich, Langmuir, Temkin, Redlich–Peterson, and Elovich adsorption isotherm models were tested. Highest adsorption capacity (774 mg/g) has been observed with Jania algae pretreated with formaldehyde. Pseudo-first- and second-order adsorption kinetics were applicable. The column capacity for lead removal with Jania pretreated with CaCl₂ was found to be 1089.6 (mg/g) from the breakthrough curve area method. Thomas, Yoon-Nelson, Yan, and Clark column models were tested with regression analysis. Thomas model is the best column model with capacity q_Th = 1092.4 mg/g. Overall, 98.5% regeneration of column was achieved, indicating the suitability of using algae materials in multioperational cycles. Adsorbed lead present in algae material was confirmed by thermal analysis.     

Protein separation on a polar-copolymerized C<Subscript>8</Subscript> stationary phase

A novel polar-copolymerized C₈ stationary phase composed of octyl and chloropropyl (C₈-C₃Cl)-bonded silica has been developed for protein separation. Separation of standard proteins on this homemade column was investigated and compared with separation on a Waters Symmetry 300 C₄ column. Results indicated that the chromatographic performance of the homemade column in the separation of proteins was excellent. The new polar copolymerized C₈ stationary phase enabled glycoprotein separation with good resolution and reproducibility. It was also used for purification of ovalbumin glycoprotein in order to improve identification of the protein.     

Preparation and characterization of an extraction chromatography column for technetium separation based on Aliquat-336 and silica gel support

An extraction chromatographic material based on Aliquat-336 anchored on hydrophobized silica gel support was prepared as an ion exchanger. The prepared material appeared to be suitable for the separation of ⁹⁹Tc from environmental matrices in column application. The properties of the material, sorption characteristic and distribution coefficient of ^99mTcO₄ ^-in various media were studied. The prepared sorbent was conditioned by washing with nitric acid. The solution containing ^99mTcO₄ ^- in 0.1M HNO₃ was passed through the column. Tc was eluted from the column by 8M HNO₃. The flow rate was 0.4 ml/min. The chemical yield of technetium during the separation process was determined using ^99mTc tracer and gamma measurement. The sorption recovery of Tc from the prepared sorbent with 0.1M HNO₃ solution was more than 98%, and the desorption recovery from the column using 8M HNO₃ varied between 92-96%. It was found that the prepared sorbent is suitable for the separation of technetium from environmental matrices and radioactive wastes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Performance of columns packed with the new shell particles,Kinetex-C18

The performance of the new Kinetex-C₁₈ column was investigated. Packed with a new brand of porous shell particles, this column has an outstanding efficiency. Once corrected for the contribution of the instrument extra column volume, the minimum values of the reduced plate heights for a number of low molecular weight compounds (e.g., anthracene and naphtho[2,3-a]pyrene) were between 1.0 and 1.3, breaking the legendary record set 3 years ago by Halo-C₁₈ packed columns. The liquid-solid mass transfer of proteins (e.g., insulin and lyzozyme) is exceptionally fast on Kinetex-C₁₈ much faster than on the Halo-C₁₈ column. The different contributions of dispersion and mass transfer resistances to the column efficiency were determined and discussed. The possible reasons for this extremely high column efficiency are discussed.     

Separation Performance of MOFs Zn(ISN)2·2H2O as Stationary Phase for High-Resolution GC

Metal–organic frameworks (MOFs) as a new type of porous materials have attracted tremendous attention for their potential applications in recent years. Here, we used a simple chiral MOF, Zn(ISN)₂·2H₂O, as chiral stationary phase for high-resolution gas chromatography. A Zn(ISN)₂·2H₂O-coated fused-silica capillary column (3 m long × 75 μm i.d.) was fabricated by using a dynamic coating method with ethanol suspension of Zn(ISN)₂·2H₂O (1 mg mL⁻¹⁾. Then, the coating properties, polarity, and column efficiency of the column were studied. The column efficiency of Zn(ISN)₂·2H₂O-coated column was up to 3,020 plates m⁻¹. The separation performance was investigated by separating a wide range of organic compounds such as Grob test mixtures, alkanes, alcohols, isomers, and racemates. The relative standard deviations for the five replicate separations of alanine were 0.32 and 2.6 % for retention time and peak area, respectively. The experimental results showed that the new stationary phase possesses high column efficiency, good reproducibility, excellent selectivity, and chiral recognition ability.

     

Separation Performance of MOFs Zn(ISN)2·2H2O as Stationary Phase for High-Resolution GC

Metal–organic frameworks (MOFs) as a new type of porous materials have attracted tremendous attention for their potential applications in recent years. Here, we used a simple chiral MOF, Zn(ISN)₂·2H₂O, as chiral stationary phase for high-resolution gas chromatography. A Zn(ISN)₂·2H₂O-coated fused-silica capillary column (3 m long × 75 μm i.d.) was fabricated by using a dynamic coating method with ethanol suspension of Zn(ISN)₂·2H₂O (1 mg mL^?1). Then, the coating properties, polarity, and column efficiency of the column were studied. The column efficiency of Zn(ISN)₂·2H₂O-coated column was up to 3,020 plates m^?1. The separation performance was investigated by separating a wide range of organic compounds such as Grob test mixtures, alkanes, alcohols, isomers, and racemates. The relative standard deviations for the five replicate separations of alanine were 0.32 and 2.6 % for retention time and peak area, respectively. The experimental results showed that the new stationary phase possesses high column efficiency, good reproducibility, excellent selectivity, and chiral recognition ability.     

99mTc generator with hydrated MnO2 as adsorbent of 99Mo

An ^99mTc generator with MnO₂ as adsorbent of ⁹⁹Mo was investigated. Through batch experiments the retention of ⁹⁹Mo onto MnO₂was studied as a function of the shaking time and the pH value of the ⁹⁹Mo solution. It was found that ⁹⁹Mo showed a retention of 100% onto MnO₂ at the pH range from 3 to 11, and the equilibrium distribution was reached in less than 10 minutes. In column experiments the retention of ⁹⁹Mo onto MnO₂ was also high: 99.72%. In this case, the pH of the utilized ⁹⁹Mo solution was 5. The ^99mTc could be eluted from the MnO₂-⁹⁹Mo column by using either distilled water with a pH of 5 or an aqueous solution of 0.9% NaCl. With the saline solution, the ^99mTc elution yields were higher than 80%, and only one aliquot of 5 ml was needed to get these yields. The best results were obtained when the column was packed with 1 g of MnO₂. The water and the saline solution were passed through the column at a rate of 1.25 ml/min.     

A generator system for production of medical alpha-radionuclides Ac-225 and Bi-213

A generator system, consisting of two anion- and one cation-exchange columns for production of medical α-radionuclides ²²⁵Ac and ²¹³Bi, has been developed. The first anion-exchange column was used for periodical separation of ²²⁵Ac from Th with 8 M HNO₃, while the second anion-exchange column was employed for purification of ²²⁵Ac from Ra and inactive impurities in mixed HNO₃/CH₃OH media. From anion-exchange column ²²⁵Ac was stripped with 0.1–0.2 M HNO₃ and loaded directly onto cation-exchange column, used as a generator for repeated isolation of ²¹³Bi. The cation exchange behavior of Bi and other radionuclides of the interest were studied in diluted HCl, HBr and DTPA solutions used as eluents. As result, a simple and effective method for the production and purification of ²²⁵Ac and ²¹³Bi suitable for biomedical studies was elaborated.     

Isolation and purification of phycoerythrin from red algaGracilaria verrucosa by expanded-bed-adsorption and ion-exchange chromatography

Summary R-phycoerythrin was isolated and purified fromGracilaria verrucosa on an expanded-bed adsorption column combined with ion-exchange chromatography, which can effectively solve the problem of blockage of chromatographic column due to polysaccharides during isolation and purification of phycobiliproteins. 0.1 M (NH₄)₂SO₄ proved best to elute R-phycoerythrin from the expanded-bed column, and desalted 0.1 M (NH₄)₂SO₄ eluate was used on an ion-exchange column to purify the R-phycoerythrin. Using this two-stage chromatography, the purity (OD₅₆₅/OD₂₈₀) of the R-phycoerythrin fromG. verrucosa is increased to 4.4, and the yield of purified R-phycoerythrin can reach 0.141 mg·g⁻¹ of the frozen alga.     

High Performance Liquid Chromato-Graphy Separations of Nitrosamines. II. Acyclic Nitrosamines

Abstract

The separation of acyclic nitrosamines by HPLC using a β-cyclodextrin bonded silien gel column and a C₁₈ reversed phase column was evaluated. Four groups of nitrosamines were used to evaluate the utility of both columns: (1.) methylmethyl-, methylethyl-, methylpropyl and methylbutyl-; (2.) ethylmethyl-, ethylethyl-, and ethylpropyl-; (3.) propylmethyl-, propylpropyl-, and propylbutyl-; and (4.) butylmethyl-, butylethyl-, butylpropyl-, and butylbutyl nitrosamines.

The results show that both columns performed well in separating the nitrosamines in each group, with the C₁₈ column requiring a higher percentage of organic modifier (methanol) than the β-cyclodextrin column. The β-cyclodextrin column was superior in separating all the E to Z isomers of short chain alkyl nitrosamines, while the C₁₈ column was superior in separating long alkyl chain nitrosamines and their E and Z isomers.

It, was also found that, the longer the alkyl chain the longer the retention time, while nitrosamines having the same number of carbons, for example ethylbutyl-and propylpropyleluted at virtually the same time, using both columns.     

Fast trypsin digestion of proteins on a cross‐linked [Os(dmebpy)2Cl]+/2+‐derivatized copolymer of acrylamide and vinylimidazole column

Fast digestion of proteins was observed when they were loaded together with trypsin onto the cross‐linked [Os(dmebpy)₂Cl]^+/2+‐derivatized copolymer of acrylamide and vinylimidazole column. The insoluble Os‐complexed polymer particles were packed into an electrospray tip to monitor peptides eluted during loading, washing and elution periods with a mass spectrometer. The proteolytic cleavage of proteins was observed immediately when the mixture of trypsin and substrates in 0.2 mM ammonium bicarbonate 50:50 H₂O/acetonitrile reached the column tip, and continued through the loading period. Some tryptic peptides were released from the column during the loading and following washing periods. The others still stayed on the column until the low pH elution buffer reached the column. If a protein was first loaded onto the column, no tryptic peptides of the protein were observed when trypsin was loaded later for the on‐column digestion. Only the autolysis peptides of trypsin were observed. On‐column digestion of 100 fmol myoglobin was successfully detected with a low sensitivity quadrupole mass spectrometer. A hybrid Os‐polymer/C₁₈ column tip was constructed for the online trypsin digestion of proteins in the aqueous buffers and the following trapping and elution of peptides from the C₁₈ column. The digestion of reduced and alkylated bovine serum albumin and human transferrin in 2.5 mM ammonium bicarbonate and 0.2 M urea buffer was observed on the column, with more peptide coverage than conventional 4 h in‐solution digestion at 37°C. Control experiments without the Os‐polymer in the column tip excluded the spontaneous in‐solution digestion of proteins in the short time window of buffer delivery onto the column, indirectly confirming the contribution of Os‐polymer on the fast trypsin digestion. Copyright © 2010 John Wiley & Sons, Ltd.     

Graphene oxide‐SiO2 hybrid nanostructure as coating material for capillary electrochromatography

Graphene oxide (GO) has been considered as a promising stationary phase for chromatographic separation. However, the very strong adsorption of the analytes on the GO surface lead to the severe peak tailing, which in turn resulting in decreased separation performance. In this work, GO and silica nanoparticles hybrid nanostructures (GO/SiO₂ NPs@column) were coated onto the capillary inner wall by passing the mixture of GO and silica sol through the capillary column. The successful of coating of GO/SiO₂ NPs onto the capillary wall was confirmed by SEM and electroosmotic flow mobilities test. By partially covering the GO surface with silica nanoparticles, the peak tailing was decreased greatly while the unique high shape selectivity arises from the surface of remained GO was kept. Consequently, compared with the column modified with GO (GO@column), the column modified with GO and silica nanoparticles through layer‐by‐layer method (GO‐SiO₂ NPs@column), or the column modified with silica nanoparticles (SiO₂ NPs@column), GO/SiO₂ NPs@column possessed highest resolutions. The GO/SiO₂ NPs@column was applied to separate egg white and both acidic and basic proteins as well as three glycoisoforms of ovalbumin were separated in a single run within 36 min. The intra‐day, inter‐day, and column‐to‐column reproducibilities were evaluated by calculating the RSDs of the retention of naphthalene and biphenyl in open‐tubular capillary electrochromatography. The RSD values were found to be less than 7.1%.     

Residual Monomer Reduction in Polymer Latex Products by Extraction with Supercritical Carbon Dioxide

Summary: Extraction of residual monomer from a latex product with supercritical carbon dioxide ((sc)CO₂) in a column was studied. Operating conditions were chosen at 35 °C and 100 bar. For reducing the residual styrene level in a polystyrene latex from 10⁴ ppm to 100 ppm and from 10⁴ ppm to 10 ppm, a countercurrent bubble column with latex as continuous and (sc)CO₂ as dispersed phase is suggested. Monomer partitioning was demonstrated to be a key parameter in the equipment design. Monomer transport was found to be governed by the shuttle effect, caused by Brownian motion of the latex to and from the H₂O/CO₂ interface. The drift-flux approach was followed to determine the column flooding conditions. Small column volumes are obtained. (sc)CO₂ is a promising extraction medium for residual monomer reduction in latex products. Performance towards steam stripping is better as the final residual monomer level becomes lower.     

Methane separation by a continuous membrane column

A continuous membrane column was employed to separate methane from gas mixtures of CO₂CH₄, CH₄N₂ and CO₂CH₄ N₂ at room temperature. Shell-side composition profiles along the column were taken experimentally and compared with the calculated results. The agreements were excellent, indicating that the model calculation is adequate for a ternary system. Combination of two columns was developed to separate the ternary mixture.     

Ion-exclusion chromatography with the direct UV detection of non-absorbing inorganic cations using an anion-exchange conversion column in the iodide-form

An ion-exclusion chromatographic method for the direct UV detection of non-absorbing inorganic cations such as sodium (Na⁺), ammonium (NH₄⁺) and hydrazine (N₂H₅⁺) ions was developed by connecting an anion-exchange column in the I⁻-form after the separation column. For example, NH₄⁺ is converted to a UV-absorbing molecule, NH₄I, by the anion-exchange column in the I⁻-form after the ion-exclusion separation on anion-exchange column in the OH⁻-form with water eluent. As a result, the direct UV detection of Na⁺, NH₄⁺ and N₂H₅⁺ could be successfully obtained as well as the well-resolved separation. The calibration graphs of the analyte cations detected with UV at 230 nm were linear in the range of 0.001-5.0 mM. The detection limits at S/N = 3 of the cations were below 0.1 μM. This method was applied to real water analysis, the determination of NH₄⁺ in river and rain waters, or that of N₂H₅⁺ in boiler water, with the satisfactory results. This could be applied also to low- or non-absorbing anions such as fluoride or hydrogencarbonate ions by the combination of a weakly acidic cation-exchange resin in the H⁺-form as the separation column and the anion-exchange conversion column.     

Determination of diffusion coefficients by supercritical fluid chromatography: Effects of mobile phase mean velocity and column orientation

The supercritical fluid (SCF) chromatographic technique based on the Taylor dispersion theory has been widely applied in determination of diffusion coefficients of various organic compounds in SCFs. This study was aimed to understand impacts of mobile phase mean velocity (MPMV) and column orientation on diffusion coefficient measurements. The benzene/SCCO₂ system was investigated. Experiments were carried out at 40 and 60 °C and 9–15 MPa over a wide range of CO₂ densities at varying MPMV and repeated in two column orientations, vertical and horizontal. It was found that both MPMV and column orientation significantly affected measurements of diffusion coefficients in SCFs. When the column was installed vertically, apparent diffusion coefficients obtained at relatively low CO₂ density (<580 kg/m³) increased with increasing MPMV over the entire velocity ranges. This results in a conclusion that diffusion coefficients cannot be accurately determined under these conditions using a vertically installed column. Under all other conditions, as MPMV increased, apparent diffusion coefficients initially increased, then remained constant, and finally increased again. The initial increase of apparent diffusion coefficients was associated with significant decline of curve-fitting errors, which indicates that the buoyancy effects are non-negligible and will cause larger errors. Accordingly, a new generalized D₁₂–U diagram comprised of three regions is proposed. Column orientation affected diffusion coefficient measurements mainly by enhancing or weakening the buoyancy effects. When the column was installed vertically, the buoyancy effects were enhanced, leading to lower apparent diffusion coefficients, especially when CO₂ density was relatively low. In addition, it was found that when CO₂ density was below ∼580 kg/m³, diffusion coefficients obtained when the column was horizontally installed were higher than those obtained when the same column was vertically installed. When CO₂ density was above that value, opposite outcomes resulted. Finally, the horizontal orientation of a diffusion column is recommended for diffusion coefficient measurements by the SCF chromatographic technique, especially when densities of SCFs are relatively low.     

Separation of intact proteins on porous layer open tubular (PLOT) columns

Porous layer open tubular (PLOT) polystyrene divinylbenzene columns have been used for separating intact proteins with gradient elution. The 10 μm I.D. × 3 m columns were easily coupled to standard liquid chromatography–mass spectrometry (LC–MS) instrumentation with commercially available fittings. Standard proteins separated on PLOT columns appeared as narrow and symmetrical peaks with good resolution. Average peak width increased linearly with gradient time (t_G) from 0.14 to 0.33 min (t_G 20 and 120 min, respectively) using a 3 m column. With shorter columns, peak widths were larger and increased more steeply with gradient time. Theoretical peak capacity (n_c) increased with column length (tested up to 3 m). The n_c increased with t_G until a plateau was reached. The highest peak capacity achieved (n_c = 185) was obtained with a 3 m column, where a plateau was reached with t_G 90 min. The within- and between column retention time repeatabilities were below 0.6% and below 2.5% (relative standard deviation, RSD), respectively. The carry-over following injection of 0.5 ng per protein was less than 1.1%. The retention time dependence on column temperature was investigated in the range 20–50 °C. Proteins in a skimmed milk sample were separated using the method.     

Determination of radioactive strontium in seawater

This paper describes the procedures of isolating strontium and yttrium from seawater that enable the determination of ^89,90Sr. In one procedure, strontium is directly isolated from seawater on the column filled with Sr resin by binding of strontium to the resin from 3 M HNO₃ in a seawater, and successive elution with HNO₃. In others, strontium is precipitated from seawater with (NH₄)₂CO₃, followed by isolation on a Sr column or an anion exchange column. It is shown that strontium precipitation is optimal with concentration of 0.3 M (NH₄)₂CO₃ at pH = 11. In these conditions, 100% Y, 78% Sr, 80% Ca and 50% Mg are precipitated. Strontium is bound on to Sr column from 5 to 8 M HNO₃, separated from other elements by elution with 3 M HNO₃ and 0.05 M HNO₃. Strontium and yttrium are bound on to anion exchange column from alcoholic solutions of nitric acid. The optimum mixture of alcohols for sample binding is a mixture of ethanol and methanol with the volume ratio 1:3. Strontium and yttrium are separated from Mg, Ca, K, and other elements by elution with 0.25 M HNO₃ in the mixture of ethanol and methanol. After the separation, yttrium and strontium are eluted from the column with water or methanol.In the procedure of direct isolation from 1 l of the sample, the average recovery of 50% was obtained. In the remaining two procedures, the strontium recovery was about 60% for the Sr column and 65% for anion exchange column. Recovery of yttrium is about 70% for the anion exchange column. It turned out that the procedure with the Sr resin (direct isolation and isolation after precipitation) is simpler and faster in the phase of the isolation on the column in comparison with the procedure with the anion exchanger. The procedure with the anion exchanger, however, enables the simultaneous isolation of yttrium and strontium and rapid determination of ^89,90Sr. These procedures were tested by determination of ^89,90Sr on liquid scintillation counter and Cherenkov counting in 5 M HNO₃. Obtained results showed that activity of 50 mBq l⁻¹ of ^89,90Sr and higher can be simultaneously determined.