On the phase transition in a monolayer adsorbed on graphite at temperatures below the 2D-critical temperature

Monte Carlo simulations in the grand ensemble and meso-canonical ensemble in which the adsorbent is connected to a finite reservoir have been used to study adsorption isotherms for monolayer argon adsorption on graphite at temperatures below the 2D-critical temperature in order to elucidate the microscopic details of the 2D-transitions: vapour–solid, vapour–liquid and liquid–solid. An S-shaped van der Waals (vdW) loop was found when a small square surface was used; however, for large square surfaces and rectangular surfaces the isotherms exhibit a vdW-type loop with a vertical segment which indicates the coexistence of two phases separated by a boundary that changes its shape with the loading. This coexistence occurs at the same chemical potential as determined by the mid-density scheme, developed by Do and co-workers (Z. Liu, L. Herrera, V.T. Nguyen, D.D. Do, and D. Nicholson, A Monte Carlo scheme based on mid-density in a hysteresis loop to determine equilibrium phase transition. Mol Simul. 37(11):932–939, 2011; Z. Liu, D.D. Do, and D. Nicholson, A thermodynamic study of the mid-density scheme to determine the equilibrium phase transition in cylindrical pores. Mol Simul. 38(3):189–199, 2011).     

A Monte Carlo study of equilibrium transition in finite cylindrical pores

In this paper, we report a comprehensive analysis of the adsorption of argon in cylindrical pores of finite length having different dimensions and adsorbent energies. We determine the mechanisms of adsorption and desorption in the hysteresis region, and use the recently introduced mid-density scheme, as an approximate method, to determine the equilibrium transition, which is found to lie wholly within the loop and closer to the desorption branch. For a given loading in the hysteresis region, we determine the microscopic behaviour of two metastable states (one on the adsorption branch and the other on the desorption branch) and the stable equilibrium state. The adsorption metastable state is characterised by a bulging adsorbed layer, while the desorption metastable state is characterised by a liquid bridge with a meniscus at each end in the shape of a long elliptical cone. The equilibrium state has a configuration that displays a liquid bridge with hemispherically shaped menisci having areas less than those of the two menisci in the desorption metastable state.     

A Monte Carlo scheme based on mid-density in a hysteresis loop to determine equilibrium phase transition

A new and simple method to determine equilibrium phase transition in adsorption systems exhibiting a hysteresis loop is presented as an alternative to methods such as multiple histogram reweighting, gauge cell method and thermodynamic integration. This method is based on the NVT-grand canonical Monte Carlo mid-density scheme to determine the coexistence chemical potential and coexistence densities of an adsorption system. We illustrate this new scheme with argon and methane adsorption in a number of model solids having slit and cylindrical pores. This method does not have a strong basis on thermodynamic ground, but it does provide a simple heuristic approach that is simpler to understand physically.     

On the existence of a hysteresis loop in open and closed end pores

We have studied the adsorption of argon at 87 K in slit pores of finite length with a smooth graphitic potential, open at both ends or closed at one end. Simulations were carried out using conventional GCMC (grand canonical Monte Carlo) or kMC (kinetic Monte Carlo) in the canonical ensemble with extremely long Markov chain, of at least 2 × 10⁸ configurations; selected simulations with much longer Markov chains do not show any change in the results. When the pore width is in the micropore range (0.65 nm), type I isotherms are obtained for both pore models and for both simulation methods. However, wider pores (1, 2 and 3 nm in width) all exhibit hysteresis loops in the GCMC simulations, while in the canonical ensemble simulations, the isotherms pass through a sigmoid van der Waals type loop in the transition region. This loop locates the true equilibrium transition. For the pores with one closed end, this transition is close to, or coincides with, the adsorption branch of the GCMC hysteresis loop, but for the open-ended pores, it is more closely associated with the desorption branch. In a separate study of adsorption hysteresis in an infinitely long slit pore, using both simulation techniques, the van der Waals loop follows the adsorption branch of the GCMC isotherm to the transition, then reverts to a long vertical section that falls midway between the two hysteresis branches and finally moves to the desorption transition close to the evaporation pressure. An examination of molecular distributions inside the pores reveals two coexisting phases in the canonical simulations, whereas in the grand canonical simulations, the molecules are uniformly distributed along the length of the pores.     

Hydrophobic chromatography of β-galactosidase

The hydrophobic interaction of β-galactosidase with Sepharose 4B substituted with 3,3′-diaminodipropylamine was studied in both batch and column experiments. The equilibrium and the binding rate constants were determined for different phosphate buffer concentrations. The equilibrium constants exhibit a hysteresis effect, i.e., desorption constants are less than adsorption constants, and the higher the ionic strength to start the desorption, the larger the effect. The rate data are not satisfactorily described by a simple reversible first-order model. The column chromatographic data are semiquantitatively described by a local equilibrium theory without axial dispersion or intraparticle diffusion.     

Electrostatic and hydrophobic effects in affinity chromatography

A semi–quantitative theory is developed to explain the nonspecific binding of proteins to substituted affinity chromatography supports due to electrostatic and hydrophobic interactions. The equilibrium constant for the absorption of an enzyme to a solid support, and the rate of desorption of the enzyme are studied as functions of ionic strength. Experimental measurements were taken of the adsorption equilibrium constant and rate of desorption of E. coli β–galactosidase on Sepharose 4B substituted with 3, 3,-diaminodipropylamine in batch systems. It was found that the enzyme adsorption exhibits a hysteresis effect as the ionic strength is increased and then decreased. Furthermore, the adsorption of theenzyme becomes more reversible at the lower ionic strengths, while at the higher ionic strengths it is essentially irreversible. Using the measured equilibrium constants, and knowing the region of ionic strength where the adsorption becomes reversible, we were able to predict the desorption of enzyme in a continuous stirred tank as a function of time when a decreasing linear gradient of ionic strength was introduced into a slurry. It was found that the presence of another protein, hemoglobin, does not affect these results, and therefore can be separated from the enzyme.     

Properties of Confined Square-well Fluids using the Gibbs Ensemble Simulation Technique

In this work we have used the extension of the Gibbs ensemble simulation technique to inhomogeneous fluids [Panagiotopoulos, A.Z. (1987) "Adsorption and capillary condensation of fluid in cylindrical pores by Monte Carlo simulation in the Gibbs ensemble", Mol. Phys. , 62 (3), 701-719], which has been applied to adsorption phenomena of confined fluids. Fluid molecules are described by spherical particles interacting via a square-well potential. The fluid is confined in two types of walls: symmetrical (two hard walls) and non-symmetrical (one square-well wall and one hard wall). In order to analyze the behavior of the confined fluid by varying the potential parameters, we evaluated the bulk and confined densities, the internal energies and the density profiles for different supercritical temperatures. A variety of adsorption profiles can be obtained by using this model. The simulation data reported here complements the available simulation data for this system and can be useful in the development of inhomogeneous fluid theories. Since the square-well parameters can be related to real molecules this system can also be used to understand real adsorption systems.     

Protein binding to two-dimensional hydrophobic binding-site lattices: adsorption hysteresis on immobilized butyl-residues

Long-lived metastable states involving multiple binding sites of a protein ligand with immobilized alkyl residues on a solid phase can be observed at high ionic strength between butyl agaroses (5.21 μ mol/ml packed gel) and phosphorylase b by perturbations enforcing either the on-reaction (adsorption) or the off-reaction (desorption). These apparent equilibrium states are suggested because the adsorption isotherms of phosphorylase b on butyl agaroses are not retraced by the desorption isotherms. In this first example of macromolecular adsorption hysteresis on immobilized alkyl residues, it can be shown that the irreversible entropy (Δ_iS) produced in an adsorption-desorption cycle lies between 6 (5 μ mol/ml packed gel) and 40 (21 μ mol/ml packed gel) J mol ¹ K⁻¹. For the latter gel the apparent standard entropy of adsorption (Δ_aS_i⁰′) is 160 J mol⁻¹ K⁻¹. The metastable state observed during adsorption is probably due to an energy barrier which must be overcome for the nucleation of protein binding on the matrix. Other metastable states may possibly be encountered during desorption when the adsorbed enzyme resists the breakage of hydrophobic interactions. In the transition from the adsorption branch to the desorption branch of the hysteresis loop, the apparent affinity of the enzyme-matrix interaction is enhanced. For the desorption branch, the apparent association constant of half-maximal saturation corresponds to K_d,0.5′ = 4.2 × 10⁹ ]m⁻¹ as compared to the respective constant of adsorption K_{a, 0.5}′ = 1.6 × 10⁵m⁻¹ (gel: 21μ mol/ml packed gel). Since the area of the hysteresis loops (see also Δ_iS) depends strongly on the density of butyl residues on the gel, it is concluded that the number of alkyl residues interacting with the protein molecule is crucial for the metastable states and hysteresis. It is unlikely that hysteresis is due to the pore structure of the agarose or to nearest neighbour interactions of ligand molecules. Since thermodynamic irreversibility and hysteresis may be encountered when macromolecules, such as proteins, are adsorbed to cell membranes or cell organelles: an analysis and understanding of these phenomena should be of general biological significance.     

Editorial

Abstract

Grand canonical molecular dynamics (GCMD) simulations are used to study the adsorption and desorption of Lennard-Jones nitrogen in three slit pore junction models of microporous graphite. These networks consist of two narrow pores separated by a wider (cavity) pore. We report results for cases where the narrow pore has a width of only two or three molecular diameters. Using the GCMD technique, a novel freezing transition is observed which results in pore blocking in the narrow pores of the network, which are less than 1 nm wide. This freezing results from the adsorption energy barrier at the junction between the narrow and wider pores. This type of pore blocking could account for the apparent increase in pore volume with increasing temperature that has been experimentally observed in microporous graphite systems. For networks in which the narrower pores are somewhat larger, with a width of 1.28 nm, this pore blocking effect is much reduced, and adsorbate molecules enter and fill the central cavity. In such cases, however, desorption is incomplete, some residual adsorbate remaining in the central cavity even at the lowest pressures.     

Structural Relaxation During Drying and Rehydration of Food Materials��the Water Effect and the Origin of Hysteresis

The state of water in foodstuffs is a guiding principle in food design, and the equilibrium concept of water activity (Aw) is ubiquitous. It is regarded as a primary variable or “hurdle” in preservation technology, and a key variable influencing chemical reaction during storage. However, the amount of water in any system differs as function of water activity depending whether it is determined by water sorption or desorption. Even though this hysteresis behaviour has already been described in the literature, no physical interpretation of its origin has yet been proposed with respect to detailed molecular organisation. This work shows, for two different food powders, gluten and a milk-based product that the hysteresis disappears when either go through their glass transition. A more complete DSC analysis for gluten during different sorption/desorption cycles demonstrates that the hysteresis is dependent on the ageing of the material, which evolves in the glassy state and is induced by structural relaxation.     

The thermodynamics of water-protein interactions

Information about the effects of water on protein structure and function can be obtained from studies on freeze dried protein powders of varying water content. Sorption isotherms of water on proteins can be used to obtain thermodynamic quantities for water-protein interactions. Since such isotherms show hysteresis, there is doubt in regard to their interpretation.General expressions for the thermodynamic quantities of sorption are derived. If isotherms represent data at equilibrium, it is possible to calculate these thermodynamic quantities.There are two types of hysteresis, non-equilibrium hysteresis and equilibrium hysteresis. Absorption and desorption isotherms can show equilibrium hysteresis if different protein conformations, which are only slowly interconvertible, can be present. In this case valid thermodynamic quantities can be obtained. Experimental tests for equilibrium hysteresis are presented. More experiments are needed before definite conclusions can be drawn in regard to isotherms in the literature.If the protein conformation in a protein powder is similar to the protein conformation in aqueous solution, equilibrium data obtained from sorption isotherms can be used to approximate thermodynamic quantities for the interaction of water with proteins in aqueous solution. Examination of what experimental evidence is available indicates that the protein in powders prepared by desorption of water should have a conformation similar to that in solution. Further study of such samples will help to clarify the thermodynamics of water-protein interactions in aqueous solution.     

Fluid adsorption in linear pores: a molecular simulation study of the influence of heterogeneities on the hysteresis loop and the distribution of metastable states

Porous materials are known to adsorb fluid and can be characterised by measurement of fluid adsorption isotherms. Many nanoporous materials exhibit linear pores such as MCM-41 and porous silicon or alumina. In such systems, data adsorption analysis is considered to be straightforward within the approximation of independent domains. This article, which reviews previous molecular simulation works, aims at showing that the presence of heterogeneities within the pores actually invalidates this hypothesis, with consequences for porosity characterisation. To enlighten the effects, starting from perfect cylinders, the number of heterogeneities is progressively increased, up to large numbers, for which specific simulation tools are used to take into account the interdependence between the domains. The adsorption/desorption isotherms are calculated and correlated to the appearance of an exponentially large number of metastable states.     

Interfacial properties of an amphipathic alpha-helix consensus peptide of exchangeable apolipoproteins at air/water and oil/water interfaces

Amphipathic alpha-helices are the main structure and the major lipid binding motif of exchangeable apolipoproteins. To understand how these apolipoproteins behave at an hydrophobic lipoprotein interface, the interfacial properties of a consensus sequence peptide (CSP) derived from three exchangeable apolipoproteins (A-I, A-IV, and E) were studied using an oil drop tensiometer at air/water (A/W) and dodecane/water (DD/W) interfaces. CSP ((PLAEELRARLRAQLEELRERLG)2-NH2) contains two 22-amino acid tandem repeat sequences that form amphipathic alpha-helices. CSP, when added into the aqueous phase, lowered the interfacial tension (gamma) of A/W and DD/W in a concentration-dependent fashion. The gammaA/W was lowered approximately 24 mn/m, and gammaDD/W approximately 31 mn/m, indicating a greater affinity of CSP for DD/W. Using the Gibbs equation for surface, the surface area per CSP molecule was estimated at approximately 702 A2 ( approximately 16 A2/amino acid) on A/W and approximately 622 A2 on DD/W ( approximately 14 A2/amino acid) suggesting that adsorbed CSP lies flat with alpha-helices in the plane of both interfaces. At equilibrium gamma, CSP desorbed from the interface when compressed and re-adsorbed when expanded. The adsorption rate was concentration-dependent, but the desorption rate was not. Less CSP desorbed from DD/W than A/W indicating that CSP has higher affinity for DD/W. Dynamic analysis of elasticity shows that the faster the oscillation period (4, 8 s) and the lower the oscillation amplitude the more elastic the surfaces. CSP can be compressed 6-12% while remaining on the surface, but large increases in pressure eject it from the surface. We suggest that surface pressure-mediated desorption and readsorption of amphipathic alpha-helices provide lipoprotein stability during remodeling reactions in plasma.     

Adsorption of Five Model Organic Compounds on a Peat at Different Stages of Drying

Acridine orange (AO), dinitrobenzoic acid (DNB), bromocresol green (BCG), bromophenol blue (BPB), and methylene blue (MB) were chosen as model aromatic compounds of different polarity, charge, and solubility in water to examine the effects of solute properties on hydrophobic adsorption. These compounds show strict structural similarities to some herbicides and other potential xenobiotic pollutants and exhibit distinct absorption maxima in the visible region, which allows for their easy determination. A well-decomposed peat (medisaprist) at four different stages of drying was used to determine compound adsorption/desorption influences based on the degree of hydrophobicity and charge density of an organic surface. Adsorption and desorption isotherms were investigated using the batch equilibration method and determining the concentration of free chemicals by UV-Vis spectrophotometry. AO had a high tendency of adsorption and was strongly sorbed on peat samples that had been air-dried for 12 months. The lower Freundlich coefficient values found for MB when compared with AO at all the drying stages of the peat indicated that electrostatic attraction has a secondary contribution to sorption. On the contrary, the higher energy that must be spent to break solute-solvent interactions in the case of charged or polar molecules is one of the main factors in determining the position of the equilibrium. For a given solute, K_f values varied with the degree of hydrophobicity and the charge density of the surface, but again solute-solvent interactions appear to be much more important in the overall energy balance of hydrophobic pollutants than the electrostatic sorbate-sorbent interactions. A change in the solution pH does not improve the adsorption of the relatively polar DNB molecule, but sorption increases strongly for BCG and BPB when these molecules are in non-dissociated forms. The larger increase in BPB sorption observed on H⁺ saturated peat suggests that the degree of interaction increases with the suppression of the negative charge, but charge repulsion has a small effect in preventing adsorption of molecules bearing hydrophobic groups such as BCG. Desorption results differed depending on the chemical structure of the compound examined. For example, with AO there was no desorption from the more hydrophobic peat surfaces. A negative hysteresis was observed for DNB; the magnitude of hysteresis, evaluated using the ratio of Freundlich coefficients for adsorption and desorption, increased with the drying stage of the sorbent and was larger on oven-dried samples.     

四个牦牛品种MC1R基因部分序列的多态性研究

为探索4个牦牛品种MC1R基因多态性的相关信息,选取甘南牦牛、天祝白牦牛、青海高原牦牛、大通牦牛4个品种共408头个体为研究对象,采用PCR-SSCP方法分析牦牛MC1R基因部分序列的基因多态性。结果表明,与GenBank中牛MCIR基因序列(登录号:AF445641.1)比对发现,该扩增片段在3 891 bp处发生C→G的突变,在3 912 bp处发生T→C的突变,共发现CC、DD、EE、CD、CE和DE 6种基因型。4个牦牛品种中CD、CE和DE 3种基因型在青海高原牦牛和大通牦牛中占主要优势,这3种基因型频率总和在青海高原牦牛和大通牦牛群体中分别是0.778和0.781。DD和CD两基因型是甘南牦牛群里中的优势基因型,其基因型频率分别是0.351和0.328。天祝白牦牛中优势基因型是DD,其基因型频率是0.500。D等位基因是4个地方品种牦牛中的优势等位基因。4个地方品种在该基因座上都处于Hardy-Weinberg平衡状态(P>0.05)。青海高原牦牛和大通牦牛两个群体处于高度多态(PIC>0.5),甘南牦牛和天祝白牦牛处于中度多态(0.25相似文献   

Kinetics of adsorption, desorption, and re-adsorption of a commercial endoglucanase in lignocellulosic suspensions

This study conducted quantitative kinetic modeling and in situ and temporally resolved measurements of adsorption, desorption, and re-adsorption of a commercial endoglucanase in lignocellulosic suspensions. The study defined a cellulase adsorption and desorption competition parameter, a pseudo rate of binding and desorption, binding and desorption capacity, as well as cellulase-binding reversibility (a thermodynamic property) and recyclability (a engineering parameter). The results indicate that both substrate chemical and physical structures play important roles in cellulase binding and desorption. Binding of a commercial cellulase onto a cellulosic substrate was reversible. Bindings to two different lignocellulosic substrates were almost irreversible. While lignin and its structure positively affect binding capacity to substrate, they negatively affect cellulase recyclability. Collapsing of substrate pores reduced cellulose accessibility and cellulase-binding capacity and increased reversibility and recyclability. Increasing temperature and pH increase cellulase desorption and increased binding reversibility and capacity. This study lays the foundation for developing effective cellulase recycling strategies.     

Dosimetric impact of hysteresis on lung cancer tomotherapy: A moving phantom study

PurposeTo investigate the dosimetric impact of hysteresis on lung cancer tomotherapy.MethodsMeasurements were acquired using MapCheck with an XY4D motion simulation table. Six hysteresis states (0, π/32, π/16, π/8, 3π/16, and π/4) were considered with sinusoidal motions in the superior–inferior and left–right orientations. The measured data were analyzed both globally (from all detectors) and structure-by-structure in the measurement plane. The dose difference (DD) analysis method with local normalization in the absolute dose mode with a DD threshold of 6 cGy was adopted to analyze each hysteresis vs. static state (H(1)S) and nonzero vs. zero hysteresis (H(1)0). The threshold was 10% for all analyses. Wilcoxon signed rank tests with significance level p = 0.05 were used for statistical analysis.ResultsThe DD analysis of each H(1)S mostly indicated that the passing rate differed between structures but was similar between hysteresis states. The DD analysis of H(1)0 showed that the passing rate decreased with increasing hysteresis. The differences between larger hysteresis (≥3π/16) and other states were significant for comparisons between global, left lung, chest wall, and target. Both analyses showed that the DD distribution changed with hysteresis.ConclusionsHysteresis difference causes the DD distribution to change. Structural difference had more impact than hysteresis state difference on hysteresis motion vs. static comparisons. Remarkable effects on nonzero vs. zero hysteresis comparisons were only seen for structures closely related to the target at large hysteresis. Small organs at risk that are close to the target need to be considered further.     

Kinetics of protein adsorption and desorption on surfaces with grafted polymers

The kinetics of protein adsorption are studied using a generalized diffusion approach which shows that the time-determining step in the adsorption is the crossing of the kinetic barrier presented by the polymers and already adsorbed proteins. The potential of mean-force between the adsorbing protein and the polymer-protein surface changes as a function of time due to the deformation of the polymer layers as the proteins adsorb. Furthermore, the range and strength of the repulsive interaction felt by the approaching proteins increases with grafted polymer molecular weight and surface coverage. The effect of molecular weight on the kinetics is very complex and different than its role on the equilibrium adsorption isotherms. The very large kinetic barriers make the timescale for the adsorption process very long and the computational effort increases with time, thus, an approximate kinetic approach is developed. The kinetic theory is based on the knowledge that the time-determining step is crossing the potential-of-mean-force barrier. Kinetic equations for two states (adsorbed and bulk) are written where the kinetic coefficients are the product of the Boltzmann factor for the free energy of adsorption (desorption) multiplied by a preexponential factor determined from a Kramers-like theory. The predictions from the kinetic approach are in excellent quantitative agreement with the full diffusion equation solutions demonstrating that the two most important physical processes are the crossing of the barrier and the changes in the barrier with time due to the deformation of the polymer layer as the proteins adsorb/desorb. The kinetic coefficients can be calculated a priori allowing for systematic calculations over very long timescales. It is found that, in many cases where the equilibrium adsorption shows a finite value, the kinetics of the process is so slow that the experimental system will show no adsorption. This effect is particularly important at high grafted polymer surface coverage. The construction of guidelines for molecular weight/surface coverage necessary for kinetic prevention of protein adsorption in a desired timescale is shown. The time-dependent desorption is also studied by modeling how adsorbed proteins leave the surface when in contact with a pure water solution. It is found that the kinetics of desorption are very slow and depend in a nonmonotonic way in the polymer chain length. When the polymer layer thickness is shorter than the size of the protein, increasing polymer chain length, at fixed surface coverage, makes the desorption process faster. For polymer layers with thickness larger than the protein size, increases in molecular weight results in a longer time for desorption. This is due to the grafted polymers trapping the adsorbed proteins and slowing down the desorption process. These results offer a possible explanation to some experimental data on adsorption. Limitations and extension of the developed approaches for practical applications are discussed.     

Breakthrough performance of linear-DNA on ion-exchange membrane columns

Breakthrough performance of linear-DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves were not significantly affected by flow-rate or inlet solution concentration. In the theoretical analysis a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine coupled to the solution of the partial differential model equations was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant and the forward interaction rate-constant, which are the parameters of the membrane transport model. Through this approach a good prediction of the adsorption phenomena is obtained for inlet concentrations and flow rates greater than 0.2 mg/ml and 0.16 ml/min.     

Characterization of a solid-phase extraction device for discontinuous on-line preconcentration in capillary electrophoresis-based peptide mapping

Peptide mapping by capillary electrophoresis (CE) with UV detection is problematic for the characterization of proteins that can only be obtained at low micromolar concentrations. Dilution of peptide fragments during digestion of the protein can further reduce the detection sensitivity in peptide mapping to the point where analysis at sub-micromolar concentrations is not possible. A remedy to this problem is preconcentration (sample enrichment) of the proteolytic digest by solid-phase extraction (SPE). To minimize non-specific adsorptive losses during sample handling, on-line SPE–CE is preferred. However, packed-inlet SPE–CE is not always feasible due to either instrument or sample limitations. We describe here a simple method of preconcentration by discontinuous on-line SPE–CE, specifically applied to peptide mapping in low-pH separation buffer after protein digestion in a solid-phase enzyme microreactor. The SPE–CE system does not require application of a low pressure during electrophoretic separation to overcome reversed electroosmotic flow because the preconcentrator device is disconnected from the separation capillary before the electric field is applied. Up to a 500-fold preconcentration factor can be achieved with this device, which can be reused for many samples. Parameters such as the volume of desorption solution, the adsorption/desorption (chromatographic) process, reproducibility of packing the SPE preconcentrator and effects of sample concentration on the peptide map are investigated.