Column Overload Research Articles

Batatins VIII–XI, Glycolipid Ester-Type Dimers from Ipomoea batatas

Sweet potato (Ipomoea batatas) is native to the tropics of Central and South America, where many varieties have been consumed for more that 5000 years. In developing countries, this crop is a recognized effective food for fighting malnutrition. Purification of the minor lipophilic glicolipids found in the n-hexane-soluble resin glycosides from the white-skinned variety was performed by preparative-scale recycling HPLC. Application of column overload, peak shaving, heart cutting, and recycling techniques permitted the purification of four new oligosaccharide ester-type dimer derivatives of jalapinolic acid, batatins VIII-XI (1-4). The structural characterization of these complex lipo-oligosaccharides was performed through NMR spectroscopy and MS, indicating that batatins VIII-XI (1-4) possess an oligomeric structure consisting of two pentasaccharide units of the known simonic acid B.

The Association of Metatarsus Adductus to the Proximal Fifth Metatarsal Jones Fracture

This retrospective study aimed to determine if a transverse plane forefoot relationship exists in patients with Jones fractures. Anteroposterior radiographs of 30 acute Jones fractures were compared with radiographs of 30 asymptomatic control subjects. Radiographic metatarsus adductus angle measurements were analyzed with a digital software program. The reliability of the measurements was evaluated with an intra-rater study. An independent t test was used to compare the metatarsus adductus angle between the Jones fracture group and the control group. Radiographic analysis revealed the mean metatarsus adductus angle for the Jones fracture group to be 20.22° (± 6.79°) compared with a mean of 14.27° (± 4.60°) for the control group. This difference between the groups was found to be statistically significant (p < .005). The intraclass reliability coefficient (ICC = 0.9396) confirmed there was no bias in the radiographic measurements. The metatarsus adductus angle in this group of patients with Jones fractures was increased compared with the nonpathologic control group. The Jones fracture mechanism of injury and resultant lateral column overload may be intensified with an increased metatarsus adductus. This relationship should be considered a risk factor for Jones fractures and taken into consideration with respect to bone healing, treatment, and prevention.

Host cell protein adsorption characteristics during protein a chromatography

Protein A chromatography is a critical and 'gold-standard' step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI-TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back-bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D-PAGE was then used to determine individual components associated with resin back-bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs.

New perspective for third generation percutaneous vertebral augmentation procedures: Preliminary results at 12 months

Introduction:The prevalence of osteoporotic vertebral fractures (OVF) increased in the last years. Compression fractures promote a progressive spine kyphosis increase, resulting in a weight shift and anterior column overload, with OVF additional risk (domino effect). The aim of this study is to evaluate the OVF treatment outcome using Spine Jack®, a titanium device for third generation percutaneous vertebral augmentation procedures (PVAPs).Materials and Methods:From February 2010, a prospective randomized study was performed examining 300 patients who underwent PVAP due to OVF type A1 according to Magerl/AO spine classification. Patients enrolled in the study were divided in two homogenous groups with regards to age (65-85 years), sex, and general clinical findings. Group A included 150 patients who underwent PVAP using Spine Jack® system; the second, group B (control group), included 150 patients treated by conventional balloon kyphoplasty. Patients underwent a clinical (visual analogue scale and Oswestry disability index) and radiographic follow-up, with post-operative standing plain radiogram of the spine at 1, 6, and 12 months. The radiographic parameters that were taken into account were: Post-operative anterior vertebral body height, pre-operative anterior vertebral body height, cephalic anterior vertebral body height, and caudal anterior vertebral body height.Results:Compared to the Spine Jack® group, the kyphoplasty group required a little longer operation time (an average of 40 min–group A vs. 45 min–group B, P < 0.05) and a greater amount of polymethylmethacrylate (4.0 mL–group A vs. 5.0 mL–group B, P < 0.05;). The post-operative increase in vertebral body height was greater in the Spine Jack® group than in the kyphoplasty group (P < 0.05).Discussion:PVAP are based on the cement injection into the vertebral body. Vertebroplasty does not allow the vertebral body height recovery. Balloon kyphoplasty allows a temporary height restoration. Spine Jack® has some new features compared to other systems: It is equipped with a mechanical and not a hydraulic opening control; this ensures a gradual and controlled vertebral fracture reduction.Conclusions:In our study, we demonstrated that the third generation PVAP with Spine Jack® is able to determine a safe vertebral body height restoration compared to the conventional balloon kyphoplasty.

Validation of a gas chromatography–mass spectrometry isotope dilution method for the determination of 2-butoxyethanol and other common glycol ethers in consumer products

A gas chromatography–mass spectrometry isotope dilution (GC–MS ID) method was developed and tested for the determination of 14 common glycol ethers in consumer products. Stable isotope labelled standards, 2-methoxyethanol-D 7 and 2-butoxyethanol- 13C 2 (CDN isotopes) were employed to enhance the accuracy and precision of the glycol ethers determination. A 1000-fold sample dilution with methanol was applied to avoid column overload and contamination. At this dilution matrix effects were in most cases negligible and did not interfere with the analysis. The instrument detection limit (IDL) for analysed compounds varied from 0.01 to 1 μg/mL; while the estimated limit of quantification (LoQ) varied between different glycol ethers from 0.02 to 3.4 μg/mL. Calibration was tested in the range of 0.1–200 μg/mL and showed that the linear fit is upheld from 0.1 to 10 μg/mL, and extends beyond this range for some of the analytes. Recoveries of glycol ethers from products with different matrices were similar. The recoveries varied from 87% to 116% between the analysed compounds, while measurements precision varied between 2% and 14%. The method is applicable to products with glycol ether concentrations above 0.002–0.2% (w/w). The concentration range can be extended below the specified limits by decreasing the dilution factor; however, with lower dilution the sample matrix effect is expected to be stronger. Products with very high concentrations of glycol ether (>20%) may need to be further diluted prior to injection to avoid column overload. The method can be used for testing liquid and aerosol products designed for household use, such as cleaners, paints, solvents and paint stripers, for compliance and enforcement of regulations which limit glycol ethers content.

Surgical Strategies: Use of the Cuboid Osteotomy in Combination with the Triple Arthrodesis with Lateral Column Overload

This issue marks the return of the Surgical Strategies section to Foot & Ankle International. For future articles, authors will be asked to not only submit a technique they use to correct a problem, but also outline the scope of the problem itself. As a more comprehensive strategy section, articles will explore the multiple techniques as a uniform whole required to achieve the goal of a successful surgical outcome. If only one technique is required to achieve the surgical strategy (as in this month's article), that technique will be explored in depth. This section will be rich in photographic explanation as well, encouraging intraoperative photography to allow the reader to master the techniques employed. We hope you enjoy it and find it a useful adjunct to your practice.

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 the properties of stationary phases for liquid chromatography in aqueous mobile phases using aromatic sulphonic acids as the test compounds

We investigated the effects of the concentration of naphthalene sulphonic acids (NSAs) as anionic test compounds in the injected sample and of the salt additives to the mobile phase on ion-exclusion. The retention behaviour of NSAs sensitively reflects even minor changes in the ionic and hydrophobic interactions and can be useful for predicting the effects of the stationary phases in reversed-phase chromatography of polar and ionic compounds, both small ones and biopolymers, e.g., oligonucleotides. We studied chromatographic properties of several stationary phases intended for separations in aqueous mobile phases: a C18 column end-capped with polar hydrophilic groups, a densely bonded C8 column doubly end-capped with short alkyl groups, a short alkyl stationary phase designed to keep full pore accessibility in highly-aqueous mobile phases and a Bidentate column with “bridged” C18 groups attached to the silica hydride support. The chemistry and pore structure of various types of column packing materials and of the salt additives to the mobile phase affect the proportion of the pore volume non-accessible to anions due to ion-exclusion and consequently the peak asymmetry and hydrophobic selectivity in reversed-phase chromatography of organic acids. We also addressed the problems connected with the determination of column hold-up volume in aqueous mobile phases. The accessibility of the stationary phase for anionic compounds in contact with the sample zone is affected by ion-exclusion due to repulsive interactions with the negatively charged surface in the pores of the stationary phase. The accessible part of the stationary phase increases and consequently the migration velocity along the column decreases with increasing concentration of the sample in the zone moving along the column. Because of a limited access to the stationary phase, its capacity can be easily overloaded. The combination of the column overload and ion-exclusion effects may result in fronting or tailing peak asymmetry. To explain this behaviour, we proposed a modified Langmuir model, respecting the variation of the column capacity due to the effects of sample concentration on ion-exclusion.

Radiographic and Pedobarographic Comparison of Femoral Head Allograft versus Block Plate with Dorsal Opening Wedge Medial Cuneiform Osteotomy: A Biomechanical Study

The effect of the Cotton osteotomy has not been studied in isolation, and no alternative to bone graft has been investigated for this osteotomy. We hypothesized that there would be no difference in radiographic and pressure findings using the Cotton osteotomy with bone graft or an opening wedge block plate. Each specimen of eight matched pairs of lower extremities was loaded in simulated double-leg stance via pneumatic cylinders as described previously. Weightbearing lateral and anteroposterior radiographs and medial and lateral pressure measurements were obtained for all intact specimens. Specimens were randomly assigned to receive a Cotton osteotomy with a dorsal opening wedge allograft or an opening wedge plate. Each specimen was cycled at 3 Hz to 720 N for 5000 cycles and measurements were repeated. Calcaneal pitch was lower after the block plate procedure (mean +/- standard error of the mean) (intact, 23.4 +/- 1.2 degrees versus post-procedure, 21.8 +/- 1.1 degrees; p = 0.05). There was a significant difference (p < 0.05) in percentage of total plantar pressure medially and laterally between the intact specimen and the specimen after osteotomy with both methods. Pressure increased medially and decreased laterally. With the numbers available, these methods for performing a Cotton osteotomy did not differ in addressing lateral column overload. Dorsal opening wedge medial cuneiform osteotomy performed with femoral head allograft or a block plate may be effective both in reducing lateral column pressures and increasing medial column pressures when they are deficient preoperatively.

Countercurrent Chromatography and the Journal of Liquid Chromatography: A Love Story

Countercurrent chromatography (CCC) is a separation technique that works with a support‐free liquid stationary phase. Since the mobile phase is also a liquid, biphasic solvent systems are used. The problem with CCC is due to its nature: it is difficult to maintain a stable liquid stationary phase when a liquid mobile phase is pushed through it. All modern CCC columns use centrifugal fields. The hydrodynamic CCC columns use open tubes coiled on spools that rotate in a planetary manner. The hydrostatic CCC columns use channels interconnected by ducts, mounted in a centrifuge rotor. It is difficult to work with a support‐free liquid stationary phase, but the advantages of this chromatographic configuration are so important that it is worth finding better ways and to invent better CCC columns. The main advantage is that all injected compounds can access the whole volume of the liquid stationary phase, compared to the necessary solid‐liquid interphase contacts with classical solid stationary phases. Saturation of the stationary phase, or column overload, is much more difficult in CCC than in classical preparative LC with silica gel based stationary phases. CCC is definitely a preparative technique. The other advantages of a liquid stationary phase include the wide range of polarities that can be obtained using the numerous available solvents and mixing them. No irreversible adsorption is possible in CCC, since the liquid stationary can always be recovered. These advantages and the modern commercially available CCC columns are presented in this article celebrating the 30th birthday of the Journal of Liquid Chromatography that always, with its editor, strongly supported the technique by publishing, at certain occasions, half of the whole scientific CCC production of the world.

Lateral Column Calcaneal Lengthening, Flexor Digitorum Longus Transfer, and Opening Wedge Medial Cuneiform Osteotomy for Flexible Flatfoot: A Biomechanical Study

Lengthening of the lateral column is commonly used for reconstruction of the adult and pediatric flatfoot, but can result in supination of the foot and symptomatic lateral column overload. The addition of a medial cuneiform osteotomy has been used to redistribute forces to the medial column. The combined use of a lateral column lengthening and medial cuneiform osteotomy in a reproducible cadaver flatfoot model was evaluated. Twelve cadaver specimens were physiologically loaded and each was evaluated radiographically and pedobarographically in the following conditions: 1) intact, 2) severe flatfoot, 3) lateral column lengthening with simulated flexor digitorum longus transfer, and 4) lateral column lengthening and flexor digitorum longus (FDL) transfer with added medial cuneiform osteotomy. The lateral column lengthening was performed with a 10-mm foam bone wedge through the anterior process of the calcaneus, and the medial cuneiform osteotomy was performed with a dorsally placed 6-mm wedge. Lateral column lengthening with simulated FDL transfer on a severe flatfoot model resulted in a significant change as compared with the flatfoot deformity in three measurements: in lateral talus-first metatarsal angle (-17 to -7 degrees; p<0.001), talonavicular angle (46 to 24 degrees; p<0.001), and medial cuneiform height (16 to 20 mm; p<0.001). Lateral forefoot pressure increased from 24.6 to 33.9 kPa (p<0.001) after these corrections as compared with the flatfoot. Adding a medial cuneiform osteotomy decreased the lateral talar-first metatarsal angle from -7 to -4 degrees, decreased the talonavicular coverage angle from 24 to 20 degrees, and increased the medial cuneiform height from 20 to 25 mm. After added medial cuneiform osteotomy, lateral pressure was significantly different from that of the flatfoot (p=0.01) and was not significantly different from that of the intact foot (p=0.14). Medial forefoot pressure was overcorrected as compared with the intact foot with added medial cuneiform osteotomy. Lateral column lengthening increased lateral forefoot pressures in a severe flatfoot model. An added medial cuneiform osteotomy provided increased deformity correction and decreased pressure under the lateral forefoot.

Plantar Pressures in the Forefoot After Lateral Column Lengthening: A Cadaver Study Comparing the Evans Osteotomy and Calcaneocuboid Fusion

Excessive varus alignment of the forefoot after lateral column lengthening has been reported to lead to overloading of the lateral foot. The purpose of this study was to investigate whether there is a difference between the Evans opening wedge calcaneal osteotomy (Evans) and the calcaneocuboid distraction arthrodesis (CCDA) with respect to lateral forefoot loading. In each of 12 matched pairs of cadaver feet, plantar pressure measurements of the intact specimens were obtained during simulated foot-flat and early heel-rise phases of gait and again after randomly performing the Evans procedure on one foot and the CCDA on the other foot. Cervical plate fixation was used for immediate stability. Both procedures resulted in statistically significant increased loading of the lateral forefoot and decreased loading of the medial forefoot compared with the preoperative status. The relative increase in lateral pressures was statistically greater with the CCDA than with the Evans. The average increase in pressure under the fifth metatarsal head in the foot-flat phase was 46% +/- 42% (range-4% to 141%) with the Evans and 104% +/- 58% (range 9% to 216%) with the CCDA (p = 0.003). In the early heel-rise phase, the increase in pressure was 50% +/- 43% (range 2% to 108%) and 96% +/- 65% (range 12% to 263%), respectively (p = 0.02). The experimental data suggest that lateral column overload may be more likely with the CCDA than with the Evans. Physicians should be aware of the likelihood of increasing lateral column loads with both the CCDA and the Evans procedure. It may be possible to avoid this problem by using less lateral column lengthening than the 1 cm used in this study or by considering a medial column arthrodesis or plantarflexion osteotomy to balance forefoot loading.

Analysis of microsomal metabolic stability using high-flow-rate extraction coupled to capillary liquid chromatography–mass spectrometry

A method is described for on-line high-speed extraction of microsomal samples and analysis by capillary liquid chromatography–mass spectrometry (LC–MS) for the determination of metabolic stability in connection with the development of positron emission tomography (PET) tracers. The method allowed direct injections of large sample volumes at a fast extraction rate, providing a gain in both sensitivity and sample preparation time. The calibration curve of the test compound flumazenil (Ro 15–1788) was linear in the concentration range of 1–150 nM, with a correlation coefficient exceeding 0.999. The accuracy of the method ranged from 98 to 101%. A high precision was obtained, with mean intra-assay and inter-assay relative standard deviations of at most 1.4 and 1.5%, respectively, for quality control (QC) samples. The extraction efficiency was determined to be 99.4%, the total recovery 96% and the carryover to ≤0.23%. Extractions were performed in a concentration interval of 30–3000 nM without any sign of column overload. The method was successfully used for determining the microsomal metabolic stability of flumazenil. As a result, the described analysis system is currently used for metabolic screening of PET tracer candidates in our laboratory.

Evaluation of the human serum albumin column as a discovery screening tool for plasma protein binding

A total of 69 compounds with a variety of chemical structures were assayed using a human serum albumin column in combination with UV and mass spectrometric detection. A moderate correlation, R 2=0.661, between the plasma protein binding, determined by traditional techniques of equilibrium dialysis or ultrafiltration, and chromatographic retention factor ( k′/ k′+1) was observed. Disparity between the regression line and numerous samples was observed across the entire range of plasma protein binding. Attempts to discriminate between compounds from the data set to achieve better correlation based physico–chemical properties were unsuccessful. Good agreement was observed for retention times obtained with UV detection with mobile phase containing phosphate buffer and mass spectrometric detection with mobile phase containing acetate buffer. Essentially identical data were obtained for compounds analyzed in singlet or cassette for minimally or highly bound (>90% bound) compounds. Analysis of cassettes containing compounds with plasma protein binding greater than 90% did not cause column overload, even at analyte concentrations up to 100 μg/ml. Diverse results were obtained when chromatographic retention was used to rank order various classes of compounds. Better correlation with ordering from known binding was obtained when a compound class contained a wide range of protein binding, in contrast to when compounds within a given class were all highly bound.

Shape analysis of DSC ice melting endotherms: Towards an estimation of the instrumental profile

DSC endotherms of ice melting in hermetically sealed aluminum pans are analyzed by means of fitting functions derived for spectroscopy and chromatography signal processing. Two sets of experiments are performed: 1) variable heating rate, investigated between 1-5 K min−1 for a small sample of 0.3 mg, and 2) variable sample mass, for different samples within 2-11 mg. Variations of both broadness and shape of the corresponding endotherms are observed and analyzed. Heating rate influence is best described by an intermediate form of the Pearson IV function, derived and widely used in statistics and also in chromatography, and found to accurately account for the instrumental profile. Thermal transfer through the outer (calorimeter/pan) and inner (pan/sample) interfaces of hermetical Al pans is suspected as the main contributor to the shape of the observed endotherms. An important threshold in their shape is noticed between heating rates of 1 and 2 K min−1, where peaks plotted in temperature scale change from fronted to tailed. For time scale plotted peaks the same change occurs between 3 and 4 K min−1. Symmetrical spectroscopic functions are not suitable for the correct description of the observed shape effects. At least in a formal manner, mass influence closely resembles the column overload effects in gas chromatography, where axial diffusion and dispersion take place. The GC derived HVL (Haarhoff-Van der Linde) function offers an excellent fit of endotherms with mass overload. This apparent coincidence is ascribed to an initial partial melting that makes most of the remaining melting process to be controlled by thermal transfer across the liquid interface layer thus formed.

Reduction in Matrix-Related Signal Suppression Effects in Electrospray Ionization Mass Spectrometry Using On-Line Two-Dimensional Liquid Chromatography

The effect of liquid chromatographic separation on matrix-related signal suppression in electrospray ionization mass spectrometry (LC-ESI-MS) was investigated. A method incorporating on-line two-dimensional liquid chromatography mass spectrometry (LC/LC-MS) was developed to compensate for matrix effects and signal suppression in qualitative and quantitative analysis. The LC/LC-MS(MS) approach was successfully applied for single-component and multicomponent analysis in a variety of complex matrixes. It was demonstrated that matrix-related signal suppression could be induced solely by (i) column overload, (ii) matrix component-analyte coelution, or a combination of each. Application of on-line orthogonal LC/LC separations can be effective in reducing both causes of matrix-related signal suppression effects i.e., column overload and matrix-analyte coelution for a variety of LCn/MSn applications.

Preparation of highly selective stationary phases for high-performance liquid chromatographic separation of enantiomers by direct copolymerization of monomers with single or twin chiral ligands

Uniformly sized macroporous polymer beads, which can be used as chiral stationary phase (CSP), have been prepared by the staged templated suspension polymerization process using chiral monomer as one of the copolymerization components. This approach enables the preparation of CSPs for which properties such as pore size, pore volume, surface area, chemistry, and chiral ligands can be tuned over a broad range. Several types of well-defined chiral monomers were prepared and allowed to assess synergistic effect of multiple selectors attached to a branched linker as well as the effect of the length and chemistry of the linker. Microscale batch screening was used for simple and rapid evaluation of selectivity. The most promising candidate CSPs were prepared on a larger scale and packed into HPLC columns. Their performance was demonstrated on the separation of racemic N-(3,5-dinitrobenzoyl)-α-amino acid alkylamides. The highest separation factors α of up to 27 were observed for CSPs prepared from monomers containing the branched spacer. These highly selective CSPs also enabled the separation of larger amounts of the target racemates upon column overload conditions.

Analysis of the band profiles of the enantiomers of phenylglycine in liquid chromatography on bonded teicoplanin columns using the stochastic theory of chromatography

The retention behaviour of the enantiomers of underivatized phenylglycine was studied on a Chirobiotic T column packed with amphoteric glycopeptide teicoplanin covalently bonded to the surface of silica gel. The retention and the selectivity of separation of the enantiomers increase with rising concentration of ethanol or of methanol in aqueous–organic mobile phases. The band profiles of the less retained l-phenylglycine are symmetrical, but the band profiles of the more strongly retained d-phenylglycine are tailing in all mobile phases tested. The band broadening does not diminish even at very low concentrations of phenylglycine, so that it cannot be attributed to possible column overload. The analysis of the band profile using the stochastic theory of chromatography suggests that the broadening can be attributed to at least two additional chiral centres of adsorption in the stationary phase contributing to the retention of the more strongly retained enantiomer in addition to the adsorption of the less retained one. This behaviour can be explained by the complex structure of the teicoplanin chiral stationary phase.

Validation of a chromatography data analysis software

The performance of chromatography data analysis software packages is of cardinal importance when the precision and the accuracy of a chromatographic system are evaluated. Users cannot rely on a procedure generating chromatographic data of known accuracy. Holistic approaches cannot always be entirely trusted. We propose a new method consisting in validating a data analysis package against computer generated chromatograms of exactly known characteristics by feeding these chromatograms into the vendor supplied software and comparing the results supplied by the software and the exact answers. We simulated symmetrical and tailing chromatograms and processed these signals with the Agilent Technologies (formerly Hewlett-Packard) ChemStation software. The noise profile (i.e. the power spectrum of the baseline) was determined for a HPLC UV detector prior to the calculations, and chromatograms of different signal-to-noise ratios were used for the analysis. For every chromatogram, we simulated 25 replicates with identical signal-to-noise ratios but different noise sequences. In this manner, both the random and the systematic errors of the retention data and peak shape characteristics can be evaluated. When analyzing tailing peaks, we simulated the effects of extra-column band broadening and those of column overload. Our calculations show that the general performance of the data analysis system studied is excellent. The contribution of the random error originating from the data analysis procedure is in most cases negligible compared to the repeatability of the chromatographic measurement itself.

Statistical determination of the proper sample size in multicomponent separations

The Fourier analysis of multicomponent chromatograms is applied to chromatograms of diesel fuels. The concentration of the injected sample was varied over several orders of magnitude. The number of components, the average peak width, as well as the retention pattern were determined for all sample concentrations. The lower edge of the practical sample size is limited by the baseline noise, whereas the upper limit is set by column overload. By means of statistical analysis, those two boundaries were isolated and a method is proposed to determine the useful sample size for the analysis of any multicomponent mixture.