Adjusting selectivity in liquid chromatography by use of the thermally tuned tandem column concept

In this study, we propose the novel "thermally tuned tandem column (T3C)" concept for the optimization of selectivity in LC by continuous adjustment of the stationary phase. Two columns with distinctly different chromatographic selectivities (e.g., polybutadiene- and carbon-coated zirconia) are serially coupled and independently temperature-controlled. Selectivity is "tuned" by adjusting the individual temperatures of the two columns. The effect of changing column temperature is quite analogous to changing the relative column lengths, thereby altering the relative and absolute contribution each column makes to the overall retention time in T3C. The distinct selectivity differences between polybutadiene- and carbon-coated zirconia as well as the extraordinary thermal stability of zirconia-based phases (thermally stable to 200 degrees C) allow us to tune the overall chromatographic selectivity over a very substantial range. We have developed a simplified useful model, which characterizes retention and selectivity for the T3C system as a function of the two column temperatures. The model is in good agreement with the experimental results. We also describe a simple computer-assisted optimization strategy based on the window diagram method, which facilitates the optimization of the T3C system with only four or five initial runs.     

Application of the thermally tuned tandem column concept to the separation of several families of environmental toxicants

Separations of several families of environmental toxicants were optimized by means of the thermally tuned tandem column (T3C) concept. We use a tandem combination of an octadecylsilane (ODS) and a carbon-coated zirconia (C-ZrO2) column; and tune the selectivity by independently adjusting the isothermal temperatures of the two columns. This results in the change in the contribution that each column makes to the overall retention and selectivity. The separation was optimized by locating the optimum pair of column temperatures which give the best separation of the critical solute pair. For both triazine herbicides and carbamate pesticides samples, dramatically different selectivities and different critical pairs were observed for the two types of phases. Although neither individual phase gave adequate separation, the T3C approach provided baseline separations using only four preliminary trial separations. We also showed that, for the triazine samples, the T3C approach gave a better separation than did conventional mobile phase optimization with an ODS column. The combination of superior selectivity of T3C and high flow rate allows the baseline separation of complex mixtures in just a few minutes.     

Separation of selected basic pharmaceuticals by reversed-phase and ion-exchange chromatography using thermally tuned tandem columns

The separation of basic pharmaceuticals is usually performed on C8 or C18 bonded silica supports. Silanolphilic interactions between basic analytes and surface silanol groups often lead to tailed peaks, poor efficiency, and irreproducible retention times. To solve these problems, many new types of silica-, zirconia-, and polymer-based columns, which provide unique selectivities, improved stability at high pH, or both, have been developed for the analysis of basic compounds. The essence of method development for the chromatographic analysis of basic compounds is to choose a system in which the band spacing can be varied dramatically, quickly, and conveniently while minimizing the tailing due to silanolphilic interactions. The thermally tuned tandem column (T3C) approach has been shown to provide an effective way to adjust stationary-phase selectivity for nonionic compounds. In this study, a tandem combination of an octadecylsilane (ODS) and a polybutadiene-coated zirconia (PBD-ZrO2) phase was used to separate nine antihistamines. Selectivity is tuned by independently adjusting the isothermal temperatures of the two columns. We found dramatic differences in the retention factors, elution sequences, and band spacing for the above set of basic drugs on the two types of columns. The T3C model has been used successfully to locate the optimal temperatures based on only four exploratory runs. The nine antihistamines were baseline separated on the tandem column combination even though they could not be separated on the individual phases. The effect of the buffer concentration on retention of the basic antihistamines was also studied. We conclude that cation-exchange interactions predominate on the PBD-ZrO2 phase, while reversed-phase interactions are more important on the ODS phase. Interestingly, an increase in column temperature causes a significant increase in the retention on the ODS column and a decrease of retention on the PBD-ZrO2 column. This can be explained by the change in the analyte's degree of ionization with temperature. The T3C combination of silica- and zirconia-based RPLC columns is demonstrated to be a powerful approach for the separation of this mixture of basic analytes.     

带回流的多级连续电渗析分离氨基酸

以丝氨酸(Ser)和脯氨酸(Pro)为例,采用带回流的多级连续电渗析实验研究近等电点氨基酸的分离,探讨了回流级数、回流氨基酸流量、电流强度对分离性能的影响.结果表明:增加回流级数能在保持脯氨酸产品纯度基本不变的前提下提高丝氨酸产品的纯度.在4级回流时,脯氨酸产品纯度和丝氨酸产品纯度(摩尔分数)分别可以达到94.9%和90.9%.在相同的回流级数和电流强度下,增大回流氨基酸流量,可以提高丝氨酸产品纯度,但会降低脯氨酸产品纯度.在相同回流条件下,增大电流可以提高脯氨酸产品纯度,但会降低丝氨酸产品纯度.     

Complete enantiomeric separation of phenylthiocarbamoylated amino acids on a tandem column of reversed and chiral stationary phases

The enantiomeric separation of phenylthiocarbamoyl derivatives of amino acids (PTC-AAs) was studied on a series of reversed phase HPLC columns coupled to the chiral phase HPLC columns. First, the five chiral phases (native, 0.2, 3.3, 7.5 and 16.9 phenylcarbamoylated/β-cyclodextrins, Ph/CD) were newly prepared by modification of β-cyclodextrin with phenyl isocyanate and were examined for the enantiomeric separation of PTC-AAs. Among them, the 3.3Ph/CD phase gave the best enantiomeric separation (α ≥ 1.04). However, the separation of the individual PTC-AAs was not sufficient. Next, these separations were investigated on various reversed phase HPLC columns, and octyl silica was selected in terms of the suitability of the mobile phase adopted for the enantiomeric separation mentioned above. The effects of the column temperature, the ion-pairing reagent, and the final content of methanol were also studied on the tandem column of octyl silica and the 3.3Ph/CD phase. Under the best conditions (100 mM ammonium acetate (pH 6.5) containing 1 mM butanesulfonate with 0-40% methanol as the mobile phase), all the individual PTC-AAs were well separated within 150 min. The applicability of the method was demonstrated by the sequence/configuration analysis of a peptide containing a d-amino acid ([d-Thr(2)]leucine enkephalin-Thr).     

Characterization and thermodynamic studies of the interactions of two chiral polymeric surfactants with model substances: phenylthiohydantoin amino acids.

Analytical ultracentrifugation is used for determination of the molecular weights and the sedimentation coefficients of poly(sodium undecanoyl-L-valinate) (PSUV) and poly(sodium undecanoyl-L-threoninate) (PSUT) at different temperatures. Plots of absorbance as a function of radius indicates that both PSUV and PSUT are highly monodispersed. A method for evaluating the partial specific volumes using density measurements is presented. The partial specific volumes of PSUV are slightly higher than those of PSUT. In addition, the temperature dependence of the retention factor in electrokinetic chromatography was used to estimate the enthalpy, the entropy, and the Gibbs free energy of the surfactant/analyte complexes. Five phenylthiohydantoin-DL-amino acids were separated and each enantiomeric pair was completely resolved. Comparison of the thermodynamic values obtained with PSUV vs PSUT using a van't Hoff relationship suggests that PSUT, with a less favorable free energy change (i.e., less negative delta (delta G)), generates a more positive entropy change, hence slightly less chiral resolution.     

On-column coaxial flow chemiluminescence detection for underivatized amino acids by pressurized capillary electrochromatography using a monolithic column

A new method for pressurized capillary electrochromatography (pCEC) coupling with chemiluminescence (CL) detection using a modified on-column coaxial flow detection interface was developed. To evaluate the feasibility and reliability of the experimental setup, the typical CL compounds luminol and isoluminol were separated and detected by using this pCEC-CL system. A detailed investigation of CL detection interface and postcolumn CL reagent flow rate parameters was described. The excellent resolution and detection sensitivity was achieved by using 3-microm ODS-C18 packed column with 30% ACN (v/v), 5 mmol/L phosphate buffer (pH 8.0). Moreover, with the presence of Co(II) (1.0 x 10(-4) mol/L) in the mobile phase, the linear range of the concentration for luminol was 2.0 x 10(-9)-2.0 x 10(-6) mol/L with a detection limit (S/N = 3) of 2.0 x 10(-10) mol/L, and 2.5 x 10(4) theoretical plates was achieved. In addition, separation and detection of the underivatized amino acids (l-threonine and l-tyrosine) were accomplished by using a polymerized monolithic column based on the principle of the luminol-H2O2-Cu(II)-amino acid CL system. Under the optimum conditions, the mixture of amino acids was efficiently separated with satisfactory results.     

Quantitative chiral analysis of sugars by electrospray ionization tandem mass spectrometry using modified amino acids as chiral reference compounds

Rapid quantitative enantiomeric analysis of mannose, glucose, galactose, and ribose is achieved using electrospray ionization and cluster ion dissociation with data analysis by the kinetic method. Several modified amino acids (N-Ac-L-Phe, N-benzoyl-L-Phe, N-t-Boc-L-Phe, N-Ac-L-Pro, N-t-Boc-L-Pro, N-Fmoc-L-Pro, N-Ac-L-Tyr, O-Me-L-Tyr) and four transition divalent metal cations (Co2+, Cu2+, Ni2+, and Zn2+) were tested to select the best system for chiral recognition and quantitation of each sugar. Quantitative determinations of the enantiomeric compositions of sugar solutions were achieved using either multiple- or two-point calibration curves; differences between the actual and experimental values were <2% enantiomeric excess (ee).     

液相色谱串联质谱法测定烟草中的游离氨基酸

建立液相色谱串联质谱法测定烟草中20种游离氨基酸的方法。烟草样品经0.1%的盐酸溶液超声萃取并离心后,直接进样测定。色谱柱采用XTerra MS C18(50mm×2.1mm×2.5μm),0.1%甲酸溶液和乙腈为流动相。结果表明:20种氨基酸的检出限为0.001～0.011μg/mL,标准曲线的拟合度均大于0.999,回收率在86.4%～105.9%之间。该方法操作简单,灵敏度高,适用于烟草中游离氨基酸的检测。     

Isolation of amino acids from natural samples using sublimation

Amino acids have appreciable vapor pressures above 150 degrees C and will sublime under partial vacuum at elevated temperatures without any racemization or decomposition. The recoveries of several amino acids including aspartic acid, serine, glycine, alanine, alpha-aminoisobutyric acid, and valine were optimized by varying the temperature and duration of sublimation. Sublimation has been shown to be a rapid and effective technique for the isolation of amino acids from natural samples for enantiomeric analyses and a good substitute for conventional cation-exchange desalting techniques.     

Separation of metal sulfates and nitrates from their acids using nanofiltration

In this study the authors look at fractionation at a very low pH.^[1] The problem was to separate nickel sulfate from sulfuric acid, or from nitric acid, when it was the single salt in solution, or when it was present together with other salts as in the case of a rinsing water used in the mining industry.     

Direct plasma analysis of drug compounds using monolithic column liquid chromatography and tandem mass spectrometry

A monolithic silica column high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed for the high-speed direct simultaneous determination of a drug discovery compound and its major circulating metabolite (M-72) in rat plasma. This methodology makes use of flow programming and an alkyl-bonded silica rod column for fast macromolecule removal and chromatographic separation without the need for significant sample preparation. The matrix ionization suppression effect on the monolithic column HPLC-MS/MS system was investigated using the postcolumn infusion technique. After 200 plasma injections on a 50 x 4.6 mm monolithic silica column, consistent column efficiency of close to 39,000 theoretical plates/m and reproducible retention times for the analytes were observed. The apparent on-column recoveries of 12 test compounds in rat plasma samples were greater than 90%. The proposed fast direct plasma injection method was tested over a 3-day period with the interday coefficient of variation less than 15% for both analytes.     

Modeling of thermally stimulated depolarization current (TSDC) using dipole–dipole interaction concept

The study of thermally stimulated depolarization current (TSDC) using the dipole–dipole interaction model is describedin this work. The dipole–dipole interaction model (DDIM) determines the TSDC peak successfully since it gives significant peak parameters (i.e. activation energy (E{\bf \textit{E}}) and pre-exponential factor (t₀){\boldsymbol\tau}_{{\bf 0}})) in addition to the dipole–dipole interaction strength parameter (d_i){\bf \textit{d}}_{{\bf i}}). Application of this model to study the peak parameters of some polymeric systems is presented.     

Fabrication of capped gold nanoparticles by using various amino acids

The production of gold nanoparticles (GNPs) by amino acid is one of the most attractive and interesting subjects in nanobiotechnology. In this study, amino acids have been utilised as a reducing agent and also an agent for capping GNPs. The GNPs were prepared using a reduction solution containing gold cations with optimum concentration of gold salt (5?mM), and also functionalised by glutamic acid, phenylalanine and tryptophan with optimum concentration of amino acids (25?mM). The optimum condition of gold solution and amino acids were achieved by ultraviolet–visible spectroscopy. The size of nanoparticles was obtained 5–20, 10–20 and 20–30?nm, respectively, by transmission electron microscopy and dynamic light scattering techniques. The results obtained from experimental and quantum calculations confirm that amino acids have strong bond while they have anion binding. Moreover, the free carboxylic groups of capped GNPs are one of the suitable and capable beads for binding biological agents. As a result, the medical applications of amino acids and proteins can be used as a practical method due to the strong interaction of peripheral amine groups with nanoparticles.     

Selective analysis of secondary amino acids in gelatin using pulsed electrochemical detection

A method was developed for selective analysis of the secondary amino acids proline and 4-hydroxyproline from gelatin hydrolysates using anion-exchange high-performance liquid chromatography followed by integrated pulsed amperometric detection (HPLC-IPAD). An extraction scheme was implemented prior to HPLC-IPAD analysis to isolate the secondary amino acids by the removal of primary amino acids through derivatization with o-phthalaldehyde followed by solid-phase extraction with C18 packed columns. The use of the IPAD technique eliminated the need for a second derivatization step to detect secondary amino acids. The removal of interfering primary amino acids prior to chromatographic analysis allowed the use of isocratic mobile-phase conditions to achieve effective and efficient separation of the amino acids. This led to a more precise and accurate quantitation of their content in gelatin hydrolysates. Detection limits approach 10 parts per billion ( approximately 2 pmol/injection) with a chromatographic analysis time under 8 min. The ratios of secondary amino acids, in addition to their abundances, were used to distinguish gelatin manufactured from bovine, porcine, and fish raw material sources.     

Statistical models for protein validation using tandem mass spectral data and protein amino acid sequence databases

The purpose of this work is to develop and verify statistical models for protein identification using peptide identifications derived from the results of tandem mass spectral database searches. Recently we have presented a probabilistic model for peptide identification that uses hypergeometric distribution to approximate fragment ion matches of database peptide sequences to experimental tandem mass spectra. Here we apply statistical models to the database search results to validate protein identifications. For this we formulate the protein identification problem in terms of two independent models, two-hypothesis binomial and multinomial models, which use the hypergeometric probabilities and cross-correlation scores, respectively. Each database search result is assumed to be a probabilistic event. The Bernoulli event has two outcomes: a protein is either identified or not. The probability of identifying a protein at each Bernoulli event is determined from relative length of the protein in the database (the null hypothesis) or the hypergeometric probability scores of the protein's peptides (the alternative hypothesis). We then calculate the binomial probability that the protein will be observed a certain number of times (number of database matches to its peptides) given the size of the data set (number of spectra) and the probability of protein identification at each Bernoulli event. The ratio of the probabilities from these two hypotheses (maximum likelihood ratio) is used as a test statistic to discriminate between true and false identifications. The significance and confidence levels of protein identifications are calculated from the model distributions. The multinomial model combines the database search results and generates an observed frequency distribution of cross-correlation scores (grouped into bins) between experimental spectra and identified amino acid sequences. The frequency distribution is used to generate p-value probabilities of each score bin. The probabilities are then normalized with respect to score bins to generate normalized probabilities of all score bins. A protein identification probability is the multinomial probability of observing the given set of peptide scores. To reduce the effect of random matches, we employ a marginalized multinomial model for small values of cross-correlation scores. We demonstrate that the combination of the two independent methods provides a useful tool for protein identification from results of database search using tandem mass spectra. A receiver operating characteristic curve demonstrates the sensitivity and accuracy level of the approach. The shortcomings of the models are related to the cases when protein assignment is based on unusual peptide fragmentation patterns that dominate over the model encoded in the peptide identification process. We have implemented the approach in a program called PROT_PROBE.     

Simultaneous analysis of amino acids and carboxylic acids by capillary electrophoresis-mass spectrometry using an acidic electrolyte and uncoated fused-silica capillary

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18](+) or [M + 23](+), after protonated molecule detection (m/z = [M + 1](+)) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = [M - 1](-)), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism.