Column Overloading Research Articles

Development of a GC-FID method for quantitative characterisation of polyglycerols in polyglycerol polyricinoleate (PGPR) present in a lipid matrix

PGPR is an emulsifier (E476) widely used in the food industry. In this study, a gas chromatography–flame ionisation detection (GC-FID) method was developed for the quantitative characterisation of the polyglycerol composition of PGPR. The method was validated to analyse quantitatively the polyglycerol species in neat PGPR products and in PGPR samples present in a lipid matrix. This method consists of saponification, acidification and petroleum ether extraction to remove interfering fatty acids, neutralisation, silylation and finally GC-FID analysis. Phenyl β-D-glucopyranoside was used as internal standard as sorbitol proved unsuitable due to its susceptibility to interference from Na/K chloride during silylation. The response factors of glycerol and diglycerol towards phenyl β-D-glucopyranoside were determined using pure standards, while response factors of polyglycerols with a degree of polymerisation of at least 3 could be reliably estimated according to an effective carbon number (ECN) approach. The validity of the method applied to PGPR samples was further supported on the basis of a mass balance considering the experimentally determined polyglycerol and fatty acid content. Moreover, recoveries of di-, tri-, tetra- and pentaglycerol were more than 95% for various PGPR samples added to two different lipid matrices at 2 wt% and 5 wt% concentrations. Furthermore, the method proved to be very repeatable (with relative standard deviation values below 2.2%). On the other hand, the inevitable presence of glycerol in the lipid samples caused fouling of the detector and column overloading, requiring frequent cleaning of the detector and trimming off part of the column.

The Role of the Plantar Fascia, Flexor Hallucis Longus, and Fiberosseous Tunnel in Functional Hallux Rigidus

Category: Midfoot/Forefoot Introduction/Purpose: Functional hallux rigidus is characterized by limited first metatarsophalangeal (MTP) dorsiflexion during weight bearing conditions, but normal motion when the metatarsal is allowed to plantarflex. This phenomenon alters the mechanics of the foot, potentially leading to lateral column overloading and dynamic cavovarus. Furthermore, it is suspected that the abnormal forces across the first MTP alters its biomechanics, leading to cartilage destruction and eventual structural hallux rigidus. The purpose of this study is to assess the role of the flexor hallucis longus (FHL) and plantar fascia in functional hallux rigidus and to determine the best surgical treatment. Methods: Six cadaveric lower limbs disarticulated at the hip were analyzed. The feet were positioned manually in the same manner that would be used in a clinical exam of patients. The examiner's thumb was used to stabilize the first metatarsal head and prevent plantarflexion to simulate the closed-chain kinetics of weight bearing. The same hand was used to hold the ankle neutrally aligned to the leg. The other hand was used to maximally dorsiflex the great toe. X-ray was used to obtain images of the feet. The plantar fascia was transected with a Z cut, shortened approximately 5 mm, then repaired. The fiberosseous tunnel was released. The FHL was transected with a Z cut then shortened approximately 5 mm. Great toe dorsiflexion was assessed and X-rays taken after each of the manipulations. Range of motion of the first MTP was measured on Xrays and analyzed using Student's T-test. Results: Average passive dorsiflexion of the first MTP is 29.8° (range 17-46°) prior to manipulation. Shortening the plantar fascia and FHL did not decrease the MTP dorsiflexion significantly beyond baseline (P = 0.08, 0.09 respectively). Transecting the plantar fascia increased the dorsiflexion by 14° (standard deviation (SD) 9.5) and transecting the FHL increased dorsiflexion by 13.7° (SD 4.0). Dorsiflexion increased significantly beyond baseline with both these methods (P = 0.02 and P < 0.001 respectively), but the difference between the two methods was not significant (P = 0.93). Releasing the fiberosseous tunnel alone increased the dorsiflexion 8.8° (SD 5.7). Increased dorsiflexion beyond baseline was significant (P = 0.03). The increase in dorsiflexion with fiberosseous tunnel release was not significantly different than transecting the plantar fascia or the FHL (P = 0.40 and 0.42 respectively). Conclusion: By definition, functional hallux rigidus is diagnosed when first MTP dorsiflexion cannot reach 60°. Its prevalence may be more widespread than previously identified. Both the FHL and plantar fascia are implicated in functional hallux rigidus, as transection of either improves the dorsiflexion of the great toe. However, accessing the plantar fascia comes with significant risks and complete transection of the FHL is not feasible. As an alternative, releasing the fiberosseous tunnel is a simple, minimally morbid procedure that can also significantly improve functional hallux rigidus to a similar degree.

A Novel Numerical Treatment of Nonlinear and Nonequilibrium Model of Gradient Elution Chromatography considering Core-Shell Particles in the Column

An extended method of semidiscrete high-resolution finite volume is used in this paper to obtain numerical solutions for a formulated nonlinear lumped kinetic model of liquid chromatographic process to examine the effect of chromatographic column overloading gradient elution considering core-shell particles. The model constitutes linear solvent strength (LSS), Henry’s constant, coefficient of nonlinearity, and coefficient of axial dispersion. The effects of modulator concentration changes for the elution of single and two components are analyzed. The advantages of introducing gradient elution against isocratic elution in terms of core radius fraction are investigated intensively. Numerical temporal moments are generated from the solutions obtained for a more in-depth examination of the considered model. Moreover, multiple forms of a single- and two-component mixture are generated to analyze the influences of core radius fractions on gradient elution. For example, the obtained results are utilized to investigate the effects of the slope of gradient, concentration of modulator, solvent strength parameter, coefficient of nonlinearity, coefficient of mass transfer, and coefficient of axial dispersion on the profiles of concentration in order to improve the process performance using optimal core radius fraction parameter values.

Isobaric 4-Plex Tagging for Absolute Quantitation of Biological Acids in Diabetic Urine Using Capillary LC-MS/MS.

Isobaric labeling in mass spectrometry enables multiplexed absolute quantitation and high throughput, while minimizing full scan spectral complexity. Here, we use 4-plex isobaric labeling with a fixed positive charge tag to improve quantitation and throughput for polar carboxylic acid metabolites. The isobaric tag uses an isotope-encoded neutral loss to create mass-dependent reporters spaced 2 Da apart and was validated for both single- and double-tagged analytes. Tags were synthesized in-house using deuterated formaldehyde and methyl iodide in a total of four steps, producing cost-effective multiplexing. No chromatographic deuterium shifts were observed for single- or double-tagged analytes, producing consistent reporter ratios across each peak. Perfluoropentanoic acid was added to the sample to drastically increase retention of double-tagged analytes on a C18 column. Excess tag was scavenged and extracted using hexadecyl chloroformate after reaction completion. This allowed for removal of excess tag that typically causes ion suppression and column overloading. A total of 54 organic acids were investigated, producing an average linearity of 0.993, retention time relative standard deviation (RSD) of 0.58%, and intensity RSD of 12.1%. This method was used for absolute quantitation of acid metabolites comparing control and type 1 diabetic urine. Absolute quantitation of organic acids was achieved by using one isobaric lane for standards, thereby allowing for analysis of six urine samples in two injections. Quantified acids showed good agreement with previous work, and six significant changes were found. Overall, this method demonstrated 4-plex absolute quantitation of acids in a complex biological sample.

Column Overload in PLOT Columns

Injecting too much sample onto a chromatographic system will eventually overload the column. Analytical chemists are concerned about column overloading because it reduces column efficiency, and, as a result, the resolution is sacrificed. Surprisingly, there is little data in the literature about the overloading of the porous layer open tubular (PLOT) columns. This month’s article looks at the basic types of column overload, volume and mass, and defines the variables that most affect the sample loading capacity in PLOT column chromatography.

Numerical approximation of nonlinear and non-equilibrium model of gradient elution chromatography

A nonlinear lumped kinetic model of liquid chromatography is formulated and solved numerically to theoretically investigate the effect of column overloading on gradient elution. Linear solvent strength (LSS) model is utilized for Henry’s constant, non linearity coefficient and axial dispersion coefficient. A semi-discrete high-resolution finite volume scheme is extended and applied to obtain the approximate solutions of the governing model equations. The effects of changing modulator concentration are examined on the single and two-component elution. The benefits of gradient elution over isocratic elution are thoroughly discussed. The influences of optimizing free-parameters available in gradient chromatography are analyzed on the efficiency of the column and on the production of targeted components. For instance, the results obtained are used to study the effects of gradient slope, modulator concentration, solvent strength, nonlinearity coefficient, mass transfer coefficient, and axial dispersion coefficient on the concentration profiles.

Unraveling peak asymmetry in chromatography through stochastic theory powered Monte Carlo simulations

An overarching theory of chromatography capable of modeling all analyte-stationary phase interactions would enable predictive design of pharmaceutically relevant separations. The stochastic theory of chromatography has been postulated as a suitable basis to achieve this goal. Here, we implement Dondi and Cavazzini's Monte Carlo framework that utilizes experimentally accessible single molecule kinetics and use it to correlate heterogenous adsorption statistics at the stationary phase to shifts in asymmetry. The contributions cannot be captured or modeled through ensemble chemometrics. Simulations reveal that peak asymmetry scales non-linearly with longer analyte-stationary phase interactions and migrates towards symmetry across the column length, even without column overloading.

A strategy for screening bioactive components from natural products based on two-dimensional cell membrane chromatography and component-knockout approach

Cell membrane chromatography (CMC) is a bioaffinity chromatographic method used to screen active compounds from natural products. However, since the receptor capacity of CMC column is limited, high content/affinity compounds may cause column overloading and thus lead to ignorance of other positive candidates. For avoiding this effect and comprehensively discovering bioactive components, a strategy based on two-dimensional CMC and component-knockout approach was proposed. As an illustrative case study, red yeast rice (RYR), a rice product with good myocardial protective effect in clinical studies, was selected as the model experimental sample. For discovering its potential cardioprotective compounds, a CMC model with H9c2 rat cardiac myoblasts (H9c2/CMC) with good selectivity, stability and reproducibility was established. By using two-dimensional H9c2/CMC-HPLC coupled with QTOF MS system, three components were firstly screened out. After knocking out high content/affinity compound, another four bioactive compounds were then found. By this two-round screening, column overloading caused by high concentration or infinity compounds was avoided, and trace compounds were enriched. As a result, one pigment and six monacolins from RYR were fished out. The results indicate the proposed strategy might be used to discover active compounds from complex matrix.

Total peak shape analysis: detection and quantitation of concurrent fronting, tailing, and their effect on asymmetry measurements

Most peak shapes obtained in separation science depart from linearity for various reasons such as thermodynamic, kinetic, or flow based effects. An indication of the nature of asymmetry often helps in problem solving e.g. in column overloading, slurry packing, buffer mismatch, and extra-column band broadening. However, existing tests for symmetry/asymmetry only indicate the skewness in excess (tail or front) and not the presence of both. Two simple graphical approaches are presented to analyze peak shapes typically observed in gas, liquid, and supercritical fluid chromatography as well as capillary electrophoresis. The derivative test relies on the symmetry of the inflection points and the maximum and minimum values of the derivative. The Gaussian test is a constrained curve fitting approach and determines the residuals. The residual pattern graphically allows the user to assess the problematic regions in a given peak, e.g., concurrent tailing or fronting, something which cannot be easily done with other current methods. The template provided in MS Excel automates this process. The total peak shape analysis extracts the peak parameters from the upper sections (>80% height) of the peak rather than the half height as is done conventionally. A number of situations are presented and the utility of this approach in solving practical problems is demonstrated.

A New Approach for the Characterization of Organic Residues from Stone Tools Using GC×GC-TOFMS

Headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) have traditionally been used, in combination with other analyses, for the chemical characterization of organic residues recovered from archaeological specimens. Recently in many life science fields, comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) has provided numerous benefits over GC-MS. This study represents the first use of HS-SPME-GC×GC-TOFMS to characterize specimens from an experimental modern reference collection. Solvent extractions and direct analyses were performed on materials such as ivory, bone, antlers, animal tissue, human tissue, sediment, and resin. Thicker film column sets were preferred due to reduced column overloading. The samples analyzed by HS-SPME directly on a specimen appeared to give unique signatures and generally produced a higher response than for the solvent-extracted residues. A non-destructive screening approach of specimens may, therefore, be possible. Resin and beeswax mixtures prepared by heating for different lengths of time appeared to provide distinctly different volatile signatures, suggesting that GC×GC-TOFMS may be capable of differentiating alterations to resin in future studies. Further development of GC×GC-TOFMS methods for archaeological applications will provide a valuable tool to uncover significant information on prehistoric technological changes and cultural behavior.

Anomalous temperature dependence of gas chromatographic retention indices of polar compounds on nonpolar phases

The character of the temperature dependences of the retention indices RI(Т) of polar sorbates on nonpolar stationary phases was found to depend on the dosed amounts of sorbates, but not on column overloading. A physicochemical model was suggested to explain the observed anomalies in RI(Т).

Liquid chromatographic method for toxic biogenic amines in foods using a chaotropic salt

Direct separation of biogenic amines by reversed-phase liquid chromatography (RPLC) is not an easy task because their basic and hydrophilic characteristics can lead to poor retention, column overloading, peak tailing, and hence low efficiency. Rather than routinely resorting to derivatization or using classical hydrophobic ion-pair reagents (IPR), this work proposes a new RPLC method making use of the chaotropic salt KPF6 as inorganic additive to an acidic acetonitrilic eluent to remedy the difficulties. Amine retention, overload behavior, peak shape, and column efficiency were significantly improved. The use of excess KPF6 led to a very slight decrease of amine retention. Depending on amine, the dependence of the logarithmic retention factor on the volume percent of acetonitrile could be reasonably linear or quite convex. Coupled with UV detection, the method was applied to trace analysis for six biogenic, aromatic or heterocyclic amines in three types of food after a sample cleanup, as necessary, by ion-pair extraction. The reliability of the whole analysis was demonstrated to be satisfactory. The proposed method outperforms existing methods in that it eliminates the need for long and cumbersome derivatization procedures without losing sensitivity; it also represents a good surrogate for classical ion-pair chromatography (IPC) because of the desirable hydrophilicity of chaotropic salts.

The isolation and characterisation of the synthetic cannabinoid AM-2201 from commercial products using purification by HPLC-DAD

AbstractA total of six products containing legal highs were purchased via the internet from the UK- based retailers and screened for the presence of synthetic cannabinoids using a fast GC-MS method and identified, in the absence of reference materials, by comparing the mass spectra with the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) mass spectral library. Four synthetic cannabinoids were detected: RCS-4, CP-47, 497, UR-144 and AM-2201. The active ingredient (1-(5-fluoropentyl)-3-(1-naphthoyl) indole), with the street name AM-2201, detected in the product named Doob was isolated and purified from the methanolic extract of the product using preparative HPLC with analytical column (column overloading method). The structure of the substance was confirmed using NMR. This approach used common analytical equipment found in forensic and other analytical laboratories (except for the NMR), therefore can be useful for the identification of unknown psychoactive substances in drugs of abuse.

Overload behavior and apparent efficiencies in chromatography

Over the last 50 years, many analysts have attempted to analyze column overloading by measuring the drop of the column HETP with increasing sample size and to predict elution band profiles from overloaded columns by combining the influences of the thermodynamic overloading and of axial dispersion, using a simplistic perturbation model. This approach violates the principle of mass conservation. The results of the predictions do not agree with those of exact calculations made with the equilibrium-dispersive (ED) model of chromatography for constant axial dispersion. The plots of the reduced apparent column efficiency N/Nkin, versus the injected concentration, logC0, or the injected mass, logm0, may provide useful information only regarding the onset of overloading of any particular column but they are meaningless to compare the overloading behavior of columns packed with different packing materials, unless the columns used satisfy impractical requirements (same efficiency, same retention factors, and sample sizes used proportional to the volume of stationary phase in the column).

Multi‐dimensional fractionation and characterization of crude protein mixtures: Toward establishment of a database of protein purification process development parameters

A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.

Development and validation of an analytical method for hydrocarbon residues using gas chromatography-mass spectrometry

This work outlines the development of a gas chromatographic mass spectrometric (GCMS) method for the analysis of hydrocarbon residues such as those encountered in the examination of fire debris. The work compares favourably against an existing ASTM E-1618-06 method for ignitable liquid analysis currently used for the development of the National Centre for Forensic Science (NCFS) ignitable liquid database. A test mixture comprising fourteen hydrocarbons as recommended by the ASTM-1618-06 method was used to evaluate and validate the separation conditions together with a matrix matched ignitable liquid mixture diluted to 2 µL mL−1 in dichloromethane. Optimised conditions were as follows: injection volume of 1 µL with split ratio 50 to 1 at inlet temperature 280 °C, carrier gas flow rate was set as 1.0 mL min−1, the column temperature was maintained at 40 °C for 3 minutes, then increased at a rate of 12.5 °C min−1 to 290 °C, and held for 4 minutes. Undiluted ignitable liquid samples (50% gasoline:diesel) were also analysed with a high split ratio (300:1) and low injection volume (0.2 µL) to avoid column overloading and to eliminate any difficulties associated with solvent masking. This method provided improved separation and sensitivity compared to the ASTM method. Excellent precision both intra-day and inter-day (<3% RSD for peak area) was also obtained. The stability of ignitable liquid storage was also investigated over a two week period for various types of vials and septa.

Protein identification from two-dimensional gel electrophoresis by connecting ZipTip with a home-assembled nanoflow system

Mass spectrometry (MS) is a powerful technique for protein identification in proteomic research. Two-dimensional gel electrophoresis (2-DE) combined with MS is a significant method for protein separation and identification. For protein identification, peptide sequencing is usually carried out by an effective but expensive nano-flow liquid chromatographic system combined to tandem mass spectrometry (MS/MS). However, protein identification based on such method is time-consuming, and contamination may occur as a result of column overloading. In this study, we establish an alternative nanoscale system for protein identification using MS/MS. The system consists of several devices that can be purchased from commercial sources and can be connected to an electrospray ionization quadrupole-time of flight (ESI-Q-TOF) MS in order to analyze proteins from 2D gels. This inexpensive strategy provides an attractive alternative method for rapid identification of proteins using a nanospray source. In addition, the device is disposable so that contamination is avoided. It is shown that peptide sequencing based on this device using ESI-Q-TOF MS is accomplished within 10 min.

Influence of the diameter of monolithic capillary columns on their gas chromatography characteristics

Divinylbenzene polymer monolithic capillary columns were prepared on the basis of capillaries 0.01 to 0.53 mm in diameter. Separation properties of the columns were investigated with the use of a test mixture of light hydrocarbons. The permeability and C parameter in the Van Deemter equation were determined for all the columns. For the most part, the columns had similar characteristics: permeability was in the range (2.2 ± 0.2) × 10−9 cm2, with parameter C in the range (0.7 ± 0.2) × 10−3 s (with n-butane as a sorbate). It was thus established that capillary diameter has only a slight effect on the efficiency of monolithic capillary columns (unlike packed capillary columns and microcolumns, whose properties, according to the literature data, depend strongly on the column diameter). The difference in properties between the narrowest monolithic column (capillary diameter 0.01 mm) and the others is explained by column overloading.

A new approach to the determination of column overload characteristics in reversed-phase liquid chromatography

Column overloading is very common during the separations of basic analytes in analytical scale reversed-phase liquid chromatography (RPLC). Due to the complex interactions of ionic analytes with stationary and mobile phases, only a very small amount of ionized sample compared to the amount of nonpolar solute can be injected before the peak shape is distorted by non-linear chromatographic processes. Often the amount that can be injected before overload is observed is so small that the signal is quite noisy, thereby making the measured plate count imprecise and possibly inaccurate. The purpose of the present study was to develop a practical method for the precise measurement of the plate count and a column overload parameter using a simple but mathematically rigorous model of Langmuirian non-linear chromatography. An “overload profile”, i.e. a plot of apparent plate count versus amount injected, is characterized by two parameters: the limiting plate count ( N 0) and the column sample loading capacity ( ω 0.5). The limiting plate count is the plate count that should be observed when the amount of sample injected is so small that a linear isotherm pertains. The column sample loading capacity, which is taken as the sample load that leads to a plate count equal to half of the limiting plate count, is a measure of the maximum amount of sample that can be injected into that column. The approach was tested by applying it to the study of cationic analytes in RPLC. We show that N 0 under constant conditions (column length, flow rate, mobile phase composition, etc.) is almost independent of column type (manufacturer); however, different column types (at the same length, diameter and flow rate) exhibit clear differences in their sample loading capacity ( ω 0.5). We believe that for most well packed type B columns, the column sample loading capacity and not the limiting plate count is the more important property that accounts for most of the apparent differences in peak width when different types of columns are examined.

Characterization of polymethoxylated flavones in Fructus aurantii by off-line two-dimensional liquid chromatography/electrospray ionization-ion trap mass spectrometry

Off-line two-dimensional reversed-phase liquid chromatography (2D-RPLC) coupled to electrospray ionization-ion trap mass spectrometry (ESI-ITMS) was operated in positive mode (PI) to characterize polymethoxylated flavonoids (PMFs) in botanical sample. The fragments of [M+H− n×15] + produced by loss of one or more methyl group from the protonated molecules, as well as [M+H−14] +, [M+H−29] +, [M+H−33] +, [M+H−43] +, [M+H−46] + and [M+H−61] + fragments formed the multiple MS (MS n ) “fingerprint” of PMFs. 42 target compounds were tentatively identified from the extract of Fructus aurantii ( F. aurantii) based on this “fingerprint” . Experimental outcomes indicated that the application of 2D separation method can reduce the matrix suppression of analytes caused by the coelution with interferential components and the column overloading of interferential components. 42 versus 23 target compounds were detected through 2D versus 1D method, which confirm the superiority of 2D coupled to MS in elimination of matrix effects.