Improved Peak Shape Research Articles

Selectivity of a Native β‐Cyclodextrin Column in the Separation of Catechins

Catechins are most commonly analyzed by liquid chromatography using a C18 column and reversed mobile phases, involving hydrophobic interactions. It is showed that a β‐cyclodextrin (β‐CD) column can separate five catechins (catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate) as well as gallic acid in the reversed phase mode with methanol‐water mobile phases and in the polar organic mode with acetonitrile‐methanol mobile phases. 0.1% v/v of acetic acid had to be added to all mobile phases to improve peak shapes. The selectivity (order of retention of the catechins) is completely different between classical C18 columns and the β‐CD column. This is due to a classical hydrophobic mechanism on C18 column compared to a H‐bond dominated mechanism with β‐CD column in both RPLC and polar organic mode. It is shown that the catechin solutes could be analyzed ten times faster using the low viscosity polar organic mode and a β‐CD column compared to RPLC with a C18 column.

The Effect of Chromatographic Conditions on the Separation of Selected Alkaloids in RP-HPTLC

Selected alkaloid standards were chromatographed on C18 W layers using various aqueous eluents containing an organic modifier and pH 3 buffer to suppress silanol ionization or an organic modifier and pH 8 buffer to suppress alkaloid ionization. Anionic ion pairs such as sodium dodecyl sulfate, octane-1-sulfonic acid sodium salt, pentane-1-sulfonic acid sodium salt, and bis(2-ethylhexyl)ortho-phosphoric acid are used to improve peak shape, efficiency, and selectivity. Amines (e.g., diethylamine, triethylamine, and tetrabutylamonium chloride) are incorporated into mobile phases to block surface silanols. The effect of chromatographic conditions on the separation of the investigated alkaloids is analyzed by the comparison of particular densitograms, asymmetry factor, or theoretical plate number. The best efficiency, peak symmetry, and separation selectivity of the investigated compounds is obtained through the addition of amine (especially diethylamine) to the mobile phases.

A Deoxynucleotide Derivatization Methodology for Improving LC-ESI-MS Detection

We have developed a novel LC-UV-MS derivatization method for the analysis of deoxyguanosine monophosphate adducts that demonstrates enhanced signal intensities relative to underivatized analytes in positive ion mode electrospray ionization MS. Detection of DNA nucleotide adducts is normally conducted in negative ion mode, which requires basic mobile phases that make chromatographic separations difficult and reduce MS sensitivity. Utilizing coupling reagents typically employed in peptide synthesis, several different deoxyguanosine nucleotide phosphoramidates and phosphomonoesters were synthesized in high conversion yield and under mild reaction conditions. The derivatives were characterized by MS/MS and reaction conversion yields determined from the DAD-UV traces. The derivatives were evaluated for ionization efficiencies, fragmentation patterns, and reversed-phase chromatographic properties by LC/ESI-MS/MS. Overall, the hydrophobic derivatives showed increases in ionization efficiency and improved peak shape. Rank ordering of the derivatizing agents was initially established using the dGp-modified derivatives. The best derivatizing agent, hexamethyleneimine, showed a 3-4-fold signal enhancement compared to underivatized dGp and was selected for additional evaluation. A model system using the carcinogen, N-acetoxy-2-acetylaminofluorene (AAAF), was used to synthesize a N-acetyl-(2-aminofluorenyl)-guanosine 5'-monophosphate (dGpAAF) adduct, which was subsequently derivatized with hexamethyleneimine. Detection limits for dGphex and dGpAAFhex, purified by HPLC, were 10- and 3-fold higher (S/N) than their respective underivatized analogues. Practical applicability, with similar improvements in sensitivity, was established by derivatizing adducts isolated from calf thymus DNA exposed to AAAF. Our results demonstrate the utility of simple reactions for the enhanced detection of a mononucleotide in positive ion mode ESI MS and the application of this technique for the detection of dGp-DNA adducts at the low-femtomole level.

Behaviors of the MS2 virus and related antibodies in capillary isoelectric focusing with whole-column imaging detection

Capillary isoelectric focusing (CIEF) has potential importance for the study of viruses. CIEF with whole-column imaging detection (WCID) is a novel CIEF mode, providing the advantages of high resolution, high speed, and easy method development. To facilitate the application of CIEF-WCID to the immunoassay of viruses, a basic knowledge of related aspects is necessary. In this study, the MS2 bacteriophage was used as a virus model, and the behaviors of MS2 and related antibodies in CIEF were investigated with UV absorbance-WCID and laser-induced fluorescence (LIF)-WCID. The adsorption of the virus and antibodies on the capillary wall was found to be the critical issue in method development. Addition of salt was found to be an effective way to reduce the adsorption and to improve peak shape. The formation of an immunocomplex, which forms the basis of an immunoassay, was monitored with CIEF-WCID. In comparison with UV-WCID, LIF-WCID was advantageous due to its higher detection sensitivity and the elimination of precipitation. Utilization of the noncovalent fluorescent dye, NanoOrange, was demonstrated to be a potential approach for the fluorescent labeling of the virus model and antibody and the associated immunocomplex. The change in microheterogeneity during the immune interactions at different ratios was also observed.

Comprehensive two-dimensional gas chromatography of complex samples by using a ‘reversed-type’ column combination: application to food analysis

The practicability and potential of a non-orthogonal approach in comprehensive two-dimensional gas chromatography (GC × GC) were studied and compared to those of the orthogonal approach for two different complex matrices, and using conventional flame ionisation (FID) and time-of-flight mass spectrometry (ToF MS) detection. The separation of a diesel oil showed that the non-orthogonal approach also provides interesting, but completely reversed, ordered structures. For the more extensively studied flavour analysis in food samples, improved peak shapes and, also, different types of ordered structures and retention behaviour, and improved detectability for polar compounds make the two approaches complementary to each other. As a consequence, identification and/or determination of targets and/or unknowns can be performed more reliably. Analytical performance (close to three-order linearity; LODs, 2–30 pg injected in most cases; R.S.D.s, 1–6% (n = 6)) was fully satisfactory.

Electrothermal Atomic Absorption Spectrometric Determination of Gold by Vapor Formation and in situ Trapping in Graphite Tubes

Gold was determined in ore samples following generation, separation, collection on a graphite cuvette inner wall, and atomization of its volatile species formed by combining an acidified sample solution with an aqueous sodium tetrahydroborate solution at room temperature. A detection limit of 2.6 microg L(-1) (3sigma) was obtained with a 5.0 mL sample volume. Precision of replicate measurements was typically 10% RSD. The overall efficiency of the volatile species generation, transport, and trapping process was 0.4%. Atomization of gold from Pd, Ir, Cu, Ag, and W coated graphite cuvettes, following by trapping of the gaseous volatile gold species on these surfaces, has been investigated. Pd coating was found to decrease the sensitivity. Five microg of Ir, 20 microg of Cu, 20 microg of Ag, and 5 microg of W were chosen as optimum masses. W treatment was the best one for sensitivity enhancement, having an improvement factor of 2.4. Silanization of glass surfaces significantly decreased memory effects; improved peak shapes were thus obtained for flow injection vapor generation atomic absorption spectrometry (FI-VGAAS). The method described in this study was used for the determination of gold in an ore reference material, Gold Ore (MA-1b), Canadian Reference Materials Program.

Comprehensive two-dimensional gas chromatography of complex samples by using a ‘reversed-type’ column combination: application to food analysis

The practicability and potential of a non-orthogonal approach in comprehensive two-dimensional gas chromatography (GC × GC) were studied and compared to those of the orthogonal approach for two different complex matrices, and using conventional flame ionisation (FID) and time-of-flight mass spectrometry (ToF MS) detection. The separation of a diesel oil showed that the non-orthogonal approach also provides interesting, but completely reversed, ordered structures. For the more extensively studied flavour analysis in food samples, improved peak shapes and, also, different types of ordered structures and retention behaviour, and improved detectability for polar compounds make the two approaches complementary to each other. As a consequence, identification and/or determination of targets and/or unknowns can be performed more reliably. Analytical performance (close to three-order linearity; LODs, 2–30 pg injected in most cases; R.S.D.s, 1–6% ( n = 6)) was fully satisfactory.

Selectivity differences in the separation of amphipathic α-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: Effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of α-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac–EAEKAAKEXEKAAKEAEK–amide (with position X in the centre of the hydrophobic face of the α-helix), where position X is substituted by l- or d-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.

Selectivity differences in the separation of amphipathic $alpha;-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0Effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of α-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac–EAEKAAKEXEKAAKEAEK–amide (with position X in the centre of the hydrophobic face of the α-helix), where position X is substituted by l- or d-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.

Analysis of iso-α-acids and reduced iso-α-acids in beer by direct injection and liquid chromatography with ultraviolet absorbance detection or with mass spectrometry

A liquid chromatography (LC) method is described for the simultaneous analysis of iso-α-acids and reduced iso-α-acids in beer. Volatile mobile phase additives were selected to enable hyphenation to mass spectrometric (MS) operated in the atmospheric pressure chemical ionization (APCI) mode. Contrary to other recent LC optimization procedures for the same compounds, an alkaline pH was selected hereby improving peak shape and selectivity. Both UV and MS detection are sensitive enough to analyze beers without sample pre-concentration. All major bitter acids are separated within 65 min with exception of cis-dihydroisoadhumulone, which co-elutes with trans-isocohumulone. Due to the selectivity of the MS, these compounds could be differentiated according to their m/ z value. The performance in terms of quantification of bitter acids by LC–UV and LC–MS are compared for standard solutions and a selection of 14 beers.

Effect of Weak Electrolytes on Electromigration Dispersion in Capillary Zone Electrophoresis

In this paper, simple equations are introduced to predict the electromigration dispersion (EMD) in weak background electrolytes (BGEs) at high pH in capillary zone electrophoresis (CZE) separations. Analytical solutions have been developed in the case of a basic BGE constituted of a neutral acid in equilibrium with its anionic conjugate base, which acts as co-ion to the analytes, and the treatment has been extended to other buffering systems. The treatment assumes that the concentration of the neutral form of the buffer species remains constant throughout the separation and that the pair of ionic species, hydroxide ion and co-ion, effectively act as a single co-ion. Hydroxide ion is shown to have a significant effect on EMD for pH values ≥10. The change in hydroxide ion concentration in the sample zone can compensate for changes in conductivity due to the difference of mobility between the analyte and the co-ion. Under appropriate conditions, the hydroxide species can correct the distortion due to the difference of mobility, and a good peak shape can be obtained, even with a high analyte concentration. This effect has been modeled using numerical treatments and experimentally validated using benzoate and 4-hydroxybenzoate as test analytes in a BGE constituted of a 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer with sodium as counterion. At constant co-ion concentration, the peak efficiency improved from 5.1 × 104 to 1.3 × 105 by changing the pH from 10.85 to 11.00. At a constant pH of 10.94, the peak distortion reversed in sign on increasing the concentration of CAPS co-ion from 15 to 25 mM. All experimental electropherograms were successfully fitted using the Haarhoff−Van der Linde function and distortion parameters extracted. Experimental and predicted distortion parameters are in good agreement. This work offers the potential to improve peak shapes in CZE by taking into account the effect of an ion involved in the water autoprotolysis equilibrium, and could lead to improvement in formulation of low and high pH buffers for CZE.

Improvement of peak shape and separation performance of beta-blockers in conventional reversed-phase columns using solvent modifiers.

A comparative study of peak shape, elution behavior, and resolution of 16 beta-blockers (acebutolol, alprenolol, atenolol, bisoprolol, carteolol, celiprolol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, practolol, propranolol, sotalol, and timolol) chromatographed with hybrid mobile phases of triethylamine (TEA)-acetonitrile and sodium dodecyl sulfate (SDS)-propanol is performed using conventional reversed-phase columns and isocratic elution. Both solvent modifiers (TEA and SDS) prevent the interaction of the basic drugs with the alkyl-bonded phase. However, the protection mechanisms of silanols on the packing are different. Whereas TEA associates with the silanol sites (blocking ion-exchange processes or repelling the solutes), the long hydrophobic chain of SDS is inserted in the bonded organic layer with the sulfate group protruding outside, which makes the stationary phase negatively charged. The effects of TEA, acetonitrile, SDS, and propanol on the elution strength, efficiency, peak asymmetry, and resolution are examined under an experimental design basis that is assisted by computer simulation to reach more general conclusions. The combination of improved peak shapes, larger selectivity, and a smaller range in retention among compounds of extreme polarity leads to the observation that a greater number of beta-blockers can be resolved with a hybrid micellar system.

Preserving the chromatographic integrity of high-speed supercritical fluid chromatography separations using time-of-flight mass spectrometry.

The advantage of high-speed time-of-flight-mass spectrometry (TOF-MS) detection for ultrafast qualitative supercritical fluid chromatography/mass spectrometry (SFC/MS) applications allows the superior resolving power of SFC to be exploited in high-throughput analysis. A chromatographic comparison of quadrupole MS and TOF-MS shows high-speed TOF total ion current data point sampling to be more indicative of fast SFC separations and corresponding short (1-2 s) baseline peak widths. Results shown for analysis of a six-compound mixture with two peaks eluting at 0.86 and 0.89 min exhibit >50% resolution by high-speed TOF data sampling, whereas the same peaks appear to coelute using quadrupole MS data sampling. Additionally, a marked improvement in the peak baseline widths is afforded by fast TOF data acquisition of 0.1 s/spectrum, resulting in a reduction in the baseline width, 1.6 s, of sulfanilamide in a four-compound mixture that is more than 2-fold greater than that achieved at the slower data acquisition of 0.5 s/spectrum. The resulting increase in resolution and improved peak shapes allow automatic integration routines to perform more effectively. For most classes of compounds amenable to high performance liquid chromatography, including druglike species, steroids, and polymers, the union of SFC with TOF-MS provides the maximum density of chemical information per unit time available with any high-speed chromatographic/mass spectrometric method.

Comparison of peak shapes obtained with volatile (mass spectrometry-compatible) buffers and conventional buffers in reversed-phase high-performance liquid chromatography of bases on particulate and monolithic columns

Retention factor, column efficiency and asymmetry factor were recorded for nine basic compounds on a number of RP-HPLC columns using phosphate and a variety of (MS-compatible) volatile mobile phase buffers of acid and neutral pH, in order to assess any effects of the buffer on performance. With formic or acetic acid, some phases gave partial or complete solute exclusion effects (reduced or negative k) compared with results using phosphate buffers at low pH. Despite its possible suppression of mass spectrometer sensitivity, trifluoroacetic acid was useful in enhancing retention times of relatively hydrophilic protonated bases, due to ion-pair effects. Peak shape was relatively poor on some pure silica-based ODS phases at pH 7 compared with results at acid pH. At low pH and at pH 7, ammonium and potassium phosphate gave very similar k, but the former may be preferable due to its volatile cation. Improved peak shapes, attributed to superior silanol masking effects, were obtained with ammonium phosphate at pH 7, but not at acid pH. Ammonium acetate gave acceptable peak shape at pH 7, but due to very limited buffer capacity, poor results were obtained for solutes having a p K a close to the mobile phase pH. Due to its instability, ammonium hydrogen carbonate is not a viable alternative buffer at pH 7.

Vacancy ion-exclusion chromatography of carboxylic acids on a strongly acidic cation-exchange resin

A new approach to ion-exclusion chromatography (IEC) is proposed to overcome the relatively poor conductivity detection response which occurs in IEC on when acids are added to the eluent in order to improve peak shape. This approach, termed vacancy IEC, requires the sample to be used as mobile phase and a sample of water to be injected onto a polystyrene/divinylbenzene (PS/DVB)-based strongly acidic cation-exchange column (TSKgel SCX). Vacancy peaks for each of the analytes appear at the retention times of these analytes. Highly sensitive conductivity detection is possible and sharp, well-shaped peaks are produced, leading to efficient separations. Retention times were found to be affected by the concentration of the analytes in the eluent, and also by the presence of an organic modifier, such as butanol in the mobile phase. Detection limits for oxalic, formic, acetic, propionic, butyric and valeric acids at S/N=3 were 0.04, 0.1, 0.47, 0.66, 0.71 and 0.82 μM, respectively, and linear ranges for some acids extended over two-orders of magnitude. Precision values for retention times were <0.57% and for peak height were <2.06%.

A gradient anion exchange chromatographic method for the speciation of arsenic in lobster tissue extracts

A gradient anion exchange chromatographic technique was developed for the separation of arsenobetaine (AsB), arsenocholine (AsC), arsenite (As III), arsenate (As V), monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) in one chromatographic run. This technique used low residue ammonium carbonate buffer and the inductively coupled plasma-mass spectrometry (ICP-MS) chromatograms showed little baseline drift. Gradient elution improved peak shape and peak separation. The separation was completed in approximately 27 min with low detection limits (0.017–0.029 μg As kg −1). Baseline resolution of all the arsenic species evaluated was achieved when the concentration of AsC was less than approximately 12.5 μg As kg −1. This technique was successfully applied to different extracts of a standard reference material, TORT-2, and lobster tissue. AsB was found to be the major arsenic species present. AsC, DMAA, MMAA and As V were also found, although MMAA was not detected in all of the TORT-2 extracts. Two unknown peaks found may be due to the presence of arsenosugars or other arsenic species. Discrepancy between extraction recoveries previously determined using flow injection–ICP-MS and the high-performance liquid chromatography–ICP-MS was observed in some cases. The differences may be due to the extraction technique and/or conditions at which the extractions were performed.

Separation of banned amine isomers in relation to german ban on azo dyes by derivatization on GC-MS

The German Ban on azo dyes prohibits the use of such dyes that release carcinogenic amines on reduction with sodium dithionate in the presence of citrate buffer at pH 6. Of the amines in the list seven exist in isomeric forms. Isomers, being structurally similar, show similar chromatographic properties and coelute, also, in most cases, mass spectra are identical. This can lead to errors as only specific isomers of the amines are banned. A GC-MS method is described for identification of amines and their isomers by derivatization with pentafluoropropionic anhydride (PFPA). Derivitization is by treating a solution of the amine in tetrahydrofuran with PFPA at 60 °C for 10 min. The amide derivatives formed are analyzed on a DB-5MS column in temperature programming mode. The amines and their isomers show good resolution. From retention times and mass spectral data the various amines and their isomers are identified and quantified. A total of 20 amines have been studied, limits of detection and relative standard deviations (RSD%) for amines and derivatives are reported. The method gave enhanced sensitivity, selectivity, improved peak shape, improved resolution and better detection limits.

Capillary electrochromatography of basic compounds in pharmaceutical analysis

Capillary electrochromatography (CEC) shows promising results in the separation of basic drugs. Traditional reversed-phase systems, with or without amine additives to the mobile phase to improve peak shapes, are the most commonly employed. Alternative useful stationary phases, such as strong cation exchangers and polymer-based continuous beds, are also discussed, as are methods to improve sensitivity by sample preconcentration. So far, studies performed on CEC have mainly been fundamental, but the technique is rapidly maturing and has many potential applications in the pharmaceutical field.

Enantioseparation of vesamicol in human serum by capillary electrophoresis with solid phase extraction and sulfated-β-cyclodextrin

An enantioseparation of racemic vesamicol in human serum by capillary electrophoresis with solid phase extraction and sulfated B-cyclodextrin (S-B-CD) is presented The separation was achieved on an uncoated 72 cm×50 μm id fused silica capillary maintained at 30 °C and +15 kV applied voltage using a run buffer of 128 μM S-B-CD in 50 mM phosphate buffer at pH 5. The detection wavelength was 260 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of the vesamicol samples from serum. Among the CDs studied, the migration order of the enantiomers was reversed in CM-B-CD compared to S-B-CD. Increases in migration time and differences in time between enantiomers was observed with increasing concentrations of S-B-CD. Baseline separation was achieved in the 2–20 μg/ml range of enantiomer concentration ( r>0.996). A sample stacking technique was used to improve peak shape and LOD. LODs were 0.5 μg/ml for each enantiomer. Studies of various factors and CE conditions showed the effect of CD type, CD concentration, buffer type, buffer concentration and pH on stability and resolution.

Rapid separation of pharmaceutical enantiomers using electrokinetic chromatography with a novel chiral microemulsion.

A novel oil-in-water microemulsion incorporating the chiral surfactant dodecoxycarbonylvaline (DDCV) was used to achieve the rapid enantiomeric separation of pharmaceutical drugs by electrokinetic chromatography (EKC). Incorporation of DDCV into a microemulsion resulted in an elution range more than double that provided the micellar form of the surfactant aggregate. Interestingly, for the same compounds the enantioselectivity provided by the chiral DDCV microemulsions ranged from 1.06-1.30 for the neutral and cationic drugs, which was slightly higher than that provided by chiral DDCV micelles. The use of a low surface tension oil (ethyl acetate) permitted a much lower concentration of chiral surfactant to be employed; this, together with the use of a zwitterionic buffer (ACES) resulted in a very low conductivity microemulsion that allowed a higher separation voltage to be utilized, resulting in rapid enantiomeric separations (< 8 min.). Mobility matching of the buffer cation(s) was used to improve peak shape and efficiencies. In our limited survey of the phase diagram, the optimum composition of the microemulsion buffer was 1.0% (w/v) DDCV (30 mM), 0.5% (v/v) ethyl acetate, 1.2% (v/v) 1-butanol and 50 mM ACES buffer at pH 7.