Small Sample Loads Research Articles

A simplified small-scale workflow for determination of complete protein-bound amino acids using pre-column derivatization HPLC method

Existing quantification methods of complete protein-bound amino acids profile of a food or feed often involve complicated hydrolysis setup and multiple hydrolysate treatment steps. Herein, we present a simplified workflow requiring only two steps: hydrolysis (in milligram scale) and direct sample dilution (in microliter scale), well adapted for automated pre-column O-phthaldialdehyde (OPA)/ 9-fluorenylmethyl chloroformate (FMOC) derivatized reverse phase high performance liquid chromatography (HPLC) method (the concentration range was established between 0.005 and 0.50 mM). This new workflow significantly reduces the analysis time (by up to 12 h for every 36 samples) while achieving over 90 % recovery for total protein-bound amino acids, including tryptophan, cysteine, and methionine. The easy hydrolysis setup eliminates the need for special hydrolysis tube or explosive reagents and substitutes laborious sample preparation steps like dry-down, reconstitution, neutralization with voluminous diluents and pH adjustment, with straightforward dilution using 0.1 N hydrochloric acid and deionized water. This method accommodates a small sample load, addressing concerns of limited sample availability. Robustness of this established protocol was validated by bovine serum albumin, and further confirmed by several plant proteins and casein standard. Good correlation of our results with an ISO accredited laboratory was verified (r = 0.992).

Resiliency and Allostatic Load among Veterans at Risk for Adverse Prostate Cancer Outcomes.

To examine the relationships between resiliency, sociodemographic factors, and allostatic load among male Veterans. Cross-sectional study with minority (African American or Hispanic) and non-minority (White) male Veterans undergoing prostate biopsy. Veterans Affairs Medical Center located in Charleston, SC. Self-reported resilience measured using the two item sub-scale from the Connor-Davidson Resiliency Scale and allostatic load determined from biomarkers measured in blood. In this small sample, bounce-back resilience and allostatic load level had a significant negative correlation, while adaptation resilience and allostatic load were slightly correlated, but the association was not statistically significant. Sixty-six percent of participants reported that they were able to adapt and 40% reported they were able to bounce back. Higher income and lower PSA level were significantly correlated with greater adaptation resilience. Minority men were significantly more likely than non-minority men to report that they are able to bounce back. Married men were also significantly more likely than unmarried men to report that they were able to bounce back. It may be important to target resiliency training programs to Veterans based on their social determinants and to examine the effects of these programs on allostatic load.

Polymer dots for quantifying the total hydrophobic pathogenic lysates in a single drop

A selective, rapid, small sample load (2–4μL), and sensitive quantification method for the hydrophobic cellular biomolecules of pathogenic bacteria and their biosensing application were reported. The present approach is based on using polythiophene polymer dots (2.5nm), which were prepared via the oxidation/polymerization reactions and then were characterized using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), ultraviolet spectroscopy (UV), and matrix (surface) assisted laser desorption/ionization mass spectrometry (M(S)ALDI-MS). The present method requires only gentle agitation for a single drop of aqueous bacteria suspension (10μL, Pseudomonas aeruginosa (1×104cfu/mL) and Staphylococcus aureus (1×105cfu/mL)) with 1mL of polythiophene (0.5mg/mL) in chloroform, and the time required for quantifying the total hydrophobic was significantly reduced to less than 3min. The polythiophene polymer dots is also a quantitative assessment of bacteria for aqueous and blood samples if exposed to more than 4–5μL of pathogenic bacteria and thus, it is a new biosensor for quantitative hydrophobic portions. The fluorescence intensity of polythiophene was enhanced after adding different volumes of pathogenic bacteria with low colony units. The standard bacteria suspensions of P. aeruginosa and S. aureus have low LOD (limits of detection) for 2μL (1×104cfu/mL) and 4μL (1×105cfu/mL), respectively. Further, the pathogenic bacteria were spiked into mouse blood and the total hydrophobic biomolecules were quantified. This method is extremely rapid as it does not require any culture steps prior to analysis and also no need for any separation or post sample treatments.

Asymmetric flow field‐flow fractionation of liposomes: 2. Concentration detection and adsorptive loss phenomena

The applicability of different concentration detection methods for online quantification of liposomes upon asymmetric flow field-flow fractionation was investigated. Filter-extruded egg phosphatidylcholine liposomes of different size were used. Online quantification using a differential refractive index (dRI) detector was found feasible for relatively high sample loads in the magnitude of 100 microg lipid (under the chosen fractionation conditions). UV-Vis detection of the turbidity of liposomes was ruled out as online detection method because turbidity increases with particle size and the signal is not only concentration but also particle-size dependent. Staining of liposomes by Rhodamine phosphatidylethanolamine or Sudan Red and subsequent online UV-Vis detection at the absorption maximum of the dye enabled quantification with much higher sensitivity than dRI detection. Furthermore analyte loss and carry-over phenomena upon repeated injection of varying liposome sample loads were studied using regenerated cellulose (RC) membranes as accumulation wall. It could be shown that RC membranes are prone to adsorption in case of very small sample loads (0.5 microg). This effect may be overcome by pre-saturation of the membrane with sample loads of at least 2 microg. For higher sample loads adsorptive losses play a minor role. Recovery from pre-saturated membranes reached approximately 100% and carry-over was found negligible.

Detection of genotoxicity of atmospheric particles using a high-throughput microplate umu-test system.

A previously developed and highly sensitive umu-microplate test system based on the nitroreductase- and O-acetyltransferase-overproducing strain Salmonella typhimurium NM3009 and the O-acetyltransferase-overproducing strain S. typhimurium NM2009 was applied to the detection of genotoxic activity in atmospheric particles in urban areas using a relatively small sample load. The results showed that the test system was able to detect slight increases in induced genotoxicity in atmospheric particles and that genotoxicity was detected mainly in the fine fraction but also partially in the coarse fraction. The present sensitive microplate test system has potential for application to the screening of various other environmental samples.

Detection of genotoxicity of atmospheric particles using a high-throughput microplate umu-test system.

A previously developed and highly sensitive umu-microplate test system based on the nitroreductase- and O-acetyltransferase-overproducing strain Salmonella typhimurium NM3009 and the O-acetyltransferase-overproducing strain S. typhimurium NM2009 was applied to the detection of genotoxic activity in atmospheric particles in urban areas using a relatively small sample load. The results showed that the test system was able to detect slight increases in induced genotoxicity in atmospheric particles and that genotoxicity was detected mainly in the fine fraction but also partially in the coarse fraction. The present sensitive microplate test system has potential for application to the screening of various other environmental samples.

Transverse pore gradient gel electrophoresis, using the PhastSystem.

The application of pore gradient gels prefabricated for the PhastSystem (Pharmacia) to transverse pore gradient gel electrophoresis is demonstrated. It has the twofold advantage of (i) horizontal positioning, avoiding gel stretching during the preparation of these gels and resulting pore size irreproducibility experienced with vertically applied pore gradient gels, which necessitate an orthogonal transfer of spacers, and (ii) miniaturized gel dimensions, which allow a small sample load and a short duration of electrophoresis and staining.

A Specification of the General Theory of Quasi-Static Linear Gradient Chromatography: Relationship with the Overload Chromatography Theory in the Presence of Mutual Molecular Interactions

Abstract The earlier theory of quasi-static linear gradient chromatography with small sample loads is partially specified. On the basis of this study, the relationship between the theory of small load gradient chromatography and the approximate theory of overload gradient chromatography which was developed several years ago is also specified; this will promote a better understanding of the overload theory.

Sample overloading effects in polymer characterization by field‐flow fractionation

AbstractThere are two subtechniques of field‐flow fractionation (FFF), thermal FFF and flow FFF, that have been successfully employed for polymer fractionation and characterization. These techniques are primarily analytical in nature, yielding accurate polymer characteristics from small sample loads (∼ 10 μg or less, depending on detection sensitivity). In this study the effects of increasing sample size are examined. Modest increases in load are found to result in shifts toward higher retention volumes. These modest loads also result in some broadening of the sample peaks without a major loss of peak symmetry. Excessive loading, by contrast, appears to give rise both to skewed peaks and to new artifact peaks at higher levels of retention. These observations are discussed in terms of the concentration dependence of various properties (viscosity, diffusivity, thermal diffusivity) which influence polymer transport through the FFF channel. The results are used to indicate upper limits to suitable sample concentrations.

A Theory of Stepwise Elution Chromatography with Small Sample Loads: Effect of the Top of the Column

Abstract A theory of stepwise elution chromatography was developed on the basis of a continuity equation for a simplest case of linear chromatography with small sample loads. Longitudinal diffusions taken into consideration are limited to both thermal Brownian diffusion occurring in the mobile phase and diffusion caused by the flow heterogeneity. However, account is taken of the existence of the column top, where longitudinal Brownian diffusion can be assumed to occur associated with a diffusion that is provoked by a type of flow heterogeneity. This paper provides a fundamental theory that was necessary for the earlier development of the theory of gradient hydroxyapatite chromatography.

Gradient Hydroxyapatite Chromatography with Small Sample Loads. V. Effect of the Top of the Column

Abstract The earlier theory of gradient hydroxyapatite chromatography with small sample loads is further developed by taking into account the effect of the top of the column. The ambiguity in the theory occurring when the value of the parameter s is extremely small is eliminated. The resolving power of the column is discussed at the limit when the slope of the gradient tends to zero.

Gradient Hydroxyapatite Chromatography with Small Sample Loads. VI. Effect of Thermal Brownian Diffusion Plus Diffusion Due to the Second Type of Flow Heterogeneity Occurring near the Top of the Column

Abstract A theory of gradient hydroxyapatite chromatography with small sample loads is developed by taking into account the associated effect of thermal Brownian diffusion plus diffusion due to the second type of flow heterogeneity occurring near the top of the column. Theoretical chromatograms with slightly asymmetrical shapes similar to those obtained experimentally can be calculated. However, this effect is small. The earlier theory in which the effect is assumed to be infinitesimal is valid from a practical point of view. The possibility of drastically increasing the chromatographic resolution is suggested.

Gradient Hydroxyapatite Chromatography with Small Sample Loads. IV. Gaussian Expression of the Chromatogram and a Further Consideration on the Resolving Power of the Columns

Abstract In earlier papers a pair of equations that represent a theoretical chromatogram using an intermediate parameter was derived. In the present paper the two equations are reduced into a single Gaussian form; essentially the same chromatogram can be calculated from the new equation. For a practical purpose, this equation is much more useful. Introducing a further approximation, the change in the chromatographic resolution occurring with a change in the experimental condition can explicitly be understood. This approximation, with a slight modification, is useful for roughly estimating the optimal chromatographic condition with a very simple calculation. A limit of the application of the theory occurring in an extreme case is discussed.

Gradient Hydroxyapatite Chromatography with Small Sample Loads. III. Fundamental Differential Equation for Gradient Chromatography

Abstract In contrast to stepwise Chromatography, with gradient Chromatography, it is impossible in principle for a chromatographic process to be described on the basis of a continuity equation for the actual molecular flux occurring on the column itself. It is the abstract molecular flux occurring on the mplarity gradient of competing ions that is fundamental. From this point of view it is not the column but rather the molarity gradient of the ions that is fixed; both the sample molecules and the “gradient” which is defined as an assembly of longitudinal positions on the column migrate on the molarity gradient. A continuity equation for the abstract flux is proposed. It is confirmed that the theoretical chromatogram obtained in an earlier paper is a solution of this equation.

Gradient Hydroxyapatite Chromatography with Small Sample Loads. II. Experimental Analysis and the Resolving Power of the Columns

Abstract Several chromatograms of biological macromolecules on hydroxyapatite columns were analyzed on the basis of a theory previously developed. The experimental data confirm the validity of the theory. The optimal chromatographic conditions were explored by introducing experimental data into the theory. In many instances it is indispensable that the slope of the molarity gradient of competing ions used in elution be extremely small to obtain high resolution; this slope generally is much too steep in published chromatograms. The differences in adsorption energies per molecule between the collagen components present in the neighboring peaks of the multipeak chromatogram obtained in an earlier paper were measured. These gave a value of about 0.5 kcal/mol for any pair of peaks. This should correspond to the energy of adsorption for a functional carboxyl group onto a C site. The same value was obtained in an earlier paper by an independent method on the basis of a micro-heterogeneous model of collagen. The agreement strongly supports the micro-heterogeneous model.

Gradient Hydroxyapatite Chromatography with Small Sample Loads. I. Fundamental Theory

Abstract On the basis of experimental data, it can be deduced that, on a hydroxyapatite column, the effect of thermodynamic longitudinal diffusion of molecules is “hidden” within diffusion occurring due to the heterogeneity in the flow rate of the solution. This can be assumed to occur caused by the heterogeneity in interspaces among hydroxyapatite crystals packed in the column. The chromatographic process is virtually a quasi-static process. By taking into account the longitudinal diffusion in the column, a theory of hydroxyapatite chromatography was developed for small sample loads for the linear gradient elution. The chromatographic mechanisms are fundamentally different between gradient and stepwise chromatographies. No theories that have been developed over many years for stepwise chromatography are applicable to gradient chromatography. Relations of the present theory to both classical “equilibrium” and recent “rate” theories are discussed.

Carrier ampholyte distribution in isoelectric focusing

The spatial distributions of individual components of carrier ampholyte mixtures obtained by isoelectric focusing in cylindrical (6 mm × 75 mm) polyacrylamide gels (PAG) and in a thin-layer Sephadex gel (TLSG) were determined by ion-exchange chromatography of the eluates of gel slices. A ca. 1-mm slice of PAG separating the A and B forms of β-lactoglobulin (isoelectric points of 5.21 and 5.34, respectively) was found to contain at least 12–14 ampholyte components out of a total of at least sixty in the ampholyte mixture (LKB Ampholine, pH 3.5–10, Lot No. 17). Fifteen to twenty components were found in each 2-mm slice of a sequence of six slices of PAG subsequent to isoelectric focusing of sperm whale myoglobin with the same ampholyte mixture.An ampholyte mixture prepared by the copolymerization of triethylenetetramine with acrylic acid was focused in TLSG on 20 cm × 5 cm plates and the eluates obtained from ca. 5-mm slices were analyzed by ion-exchange chromatography. The results were compared with the caramelization pattern obtained by the Felgenhauer technique. The spatial distributions of individual ampholyte components were broad with half-band widths of ca. 1 cm.The effects of sample load, ampholyte concentration, and duration of focusing on spatial distribution were investigated in the PAG focusing of [14C]histidine. The narrowest and most symmetrical distributions were obtained with small sample load and high ampholyte concentration.

The choice of carrier gas in preparative gas chromatogaphy

Helium and hydrogen as carrier gases are more suitable than nitrogen for preparative gas chromatography. Having regard, however, to the high price of helium and the danger of hydrogen, nitrogen may be preferred. For small analytical sample loads on large bore columns helium and hydrogen are far superior to nitrogen, but for larger sample loads this difference is not so great. The separations obtained with nitrogen are much better than indicated by the recorded chromatograms because of the non-linearity of the detection.