Centrifugal Packing Research Articles

Mass Fabrication of Capillary Columns Based on Centrifugal Packing.

Packed capillary columns have become the standard front-end separation device for mass spectrometry-based proteomics. The development of simple, fast, and robust capillary column technology, especially that with mass-fabrication capacity, can greatly improve analytical throughput and reproducibility in omics research. In this technical note, we report a centrifugal packing technology, which has the capability to mass fabricate high quality capillary columns with a 2886 columns/day fabrication throughput. The centrifugally packed columns presented significantly improved efficiency (reduced plate height hmin = 1.6, 37%-40% improvement compared with slurry packed columns), advanced kinetic performance limit, and excellent column-to-column reproducibility (2.0% RSD for retention time, 50 columns). Such columns enabled ∼5300 HeLa proteins identified in single-shot proteomic analysis, displaying both intercolumn and inter-run retention time stability (retention time RSD = 0.94% between nine replicates on three columns for probing peptide sequence). The mass-fabrication technology reported in this technical note may support disposable use of high quality chromatographic columns in large-scale bioanalysis.

Effect of hematocrit on the erythrocyte susceptibility to centrifugal packing: Authors’ reply

Effect of hematocrit on the erythrocyte susceptibility to centrifugal packing: Authors’ reply

Susceptibility of erythrocytes to centrifugal packing: Effect of hematocrit

Susceptibility of erythrocytes to centrifugal packing: Effect of hematocrit

Susceptibility of erythrocytes to centrifugal packing

Susceptibility of erythrocytes to centrifugal packing

Determination of the wettability of powders by the Washburn capillary rise method with bed preparation by a centrifugal packing technique

The Washburn capillary rise method is a standard technique for determining the wettability of powders expressed as a contact angle. The method requires the preparation of two identical beds of powder. One of these beds is used to follow the capillary rise with a perfectly wetting liquid (contact angle = 0) giving access to a bed structure parameter. The other bed is used with the liquid of interest (contact angle ≠ 0) and the capillary rise data is analysed using the previously determined structure parameter to obtain the contact angle. In the experiments reported here we have used a centrifugal packing technique to prepare beds of powder. This gives reproducible packings and also allows a certain degree of control of the bed porosity. In addition the air permeability of the beds is also determined prior to the capillary rise experiments. The results show that the value of the contact angle of a powder determined by the Washburn method depends on the porosity of the powder bed, and that the structure parameter can be determined from the air permeability using the Kozeny–Carman expression.

Ｘ線マイクロＣＴスキャン装置の粉体工学への応用

X-ray computed tomography is widely used for the observation of internal organs in medical diagnosis. Recently, X-ray micro computer tomography with a micrometer scale resolution has been developed and is now being applied in the field of engineering including powder and particle technology. X-ray micro CT can be used not only for the observation of internal structure of a particle or a powder bed but also for the quantitative measurement of a particle or powder packed bed characteristics. For example, the volume and the surface area of a particle can be calculated by integrating the cross sectional area and the perimeter of particle images captured by X-ray micro CT. Based on the measured volume and the surface area of a particle, we obtained the 3-dimensional particle shape factor such as Wadell's sphericity and the sphericity was compared with traditional 2-dimensional shape indices such as circularity. We also applied X-ray micro CT to the measurement of the void fraction distribution near the bottom of a spherical particle packing bed and the change in void fraction profils of a fine powder packed bed during the compression by a piston and a centrifugal force. Our experimental results showed that the local void fraction in fine powder packed bed increases with the depth of powder layer when compressed by a piston while it decreases with the depth of powder layer in the case of centrifugal packing. X-ray micro computed tomography is a new tool for powder characterization and will be widely used for various powder processes and powder product inspections in the near future.

Ｘ線マイクロＣＴスキャン装置を用いた粉体充填層内の空間率分布測定―ピストン圧縮充填，遠心充填の比較―

The void fraction in a powder packed bed is not uniform and the vertical distribution of void fraction varies with the packing method. However, detailed profile of void fraction distribution and effect of packing method on the distribution have not been discussed. Recently, X-ray micro Computed Tomography was developed and applied to the observation of powder packing structure. We utilized X-ray micro CT Scan to measure the void fraction profiles of powder layer formed by piston compression and centrifugal packing. Our experimental results showed that void fraction increases with the depth of powder layer in piston compression while it decreases with the depth of powder layer in centrifugal packing. The theoretical void fraction profiles, calculated by Janssen's equation with boundary condition of each packing method, well expressed the measured voidage distribution by X-ray micro CT scanning.

Effects of density and of dehydration of sickle cells on their adhesion to cultured endothelial cells

Abnormal adhesion of sickle cells to vascular endothelium may be a factor in the initiation of painful vaso-occlusive crisis. The sickle cell population contains an unusually large number of less dense reticulocytes that are known to be more adhesive than mature red cells, but there is contradictory evidence regarding the adhesiveness of dense sickle cells. We used a flow-based assay of adhesion to cultured human umbilical vein endothelial cells to test the properties of density fractions of sickle cells, prepared either by density gradient or by centrifugation of packed cells. We also examined the effects of incubating sickle cells with or without cyclical deoxygenation on their adhesion. After fractionation on a Percoll-Isopaque gradient, the less dense 10% (reticulocyte-rich) cells and the most dense 10% cells adhered in greater number than the remainder (by about twofold). However, after centrifugation of packed cells, the less dense 10% were again more adhesive than the “middle” cells, but the most dense were not. Exposing sickle cells to constituents of the gradient had no consistent effect on adhesion, while centrifugal packing induced a degree of hemolysis, and tended to reduce adhesiveness of the dense fraction previously obtained from a gradient. Incubation in air at 37°C for 15 hr reduced the number of reticulocytes and the adhesiveness of less dense sickle cells compared to those held at 4°C. On the other hand, incubation at 37°C for 15 hr with cyclical deoxygenation caused formation of dense cells and increased adhesiveness compared to incubation without cyclical deoxygenation. We conclude that young, less dense sickle cells are unusually adhesive, but that this adhesiveness is reduced during maturation. However, repeated sickling in vivo causes formation of an abnormally dense subpopulation of cells which either redevelop an increased tendency to adhere to endothelial cells or preserve their initial adhesiveness. Both adhesive cell populations may be implicated in promoting vascular obstruction. © 1996 Wiley-Liss, Inc.

Effects of density and of dehydration of sickle cells on their adhesion to cultured endothelial cells.

Abnormal adhesion of sickle cells to vascular endothelium may be a factor in the initiation of painful vaso-occlusive crisis. The sickle cell population contains an unusually large number of less dense reticulocytes that are known to be more adhesive than mature red cells, but there is contradictory evidence regarding the adhesiveness of dense sickle cells. We used a flow-based assay of adhesion to cultured human umbilical vein endothelial cells to test the properties of density fractions of sickle cells, prepared either by density gradient or by centrifugation of packed cells. We also examined the effects of incubating sickle cells with or without cyclical deoxygenation on their adhesion. After fractionation on a Percoll-isopaque gradient, the less dense 10% (reticulocyte-rich) cells and the most dense 10% cells adhered in greater number than the remainder (by about twofold). However, after centrifugation of packed cells, the less dense 10% were again more adhesive than the "middle" cells, but the most dense were not. Exposing sickle cells to constituents of the gradient had no consistent effect on adhesion, while centrifugal packing induced a degree of hemolysis, and tended to reduce adhesiveness of the dense fraction previously obtained from a gradient. Incubation in air at 37 degrees C for 15 hr reduced the number of reticulocytes and the adhesiveness of less dense sickle cells compared to those held at 4 degrees C. On the other hand, incubation at 37 degrees C for 15 hr with cyclical deoxygenation caused formation of dense cells and increased adhesiveness compared to incubation without cyclical deoxygenation. We conclude that young, less dense sickle cells are unusually adhesive, but that this adhesiveness is reduced during maturation. However, repeated sickling in vivo causes formation of an abnormally dense subpopulation of cells which either redevelop an increased tendency to adhere to endothelial cells or preserve their initial adhesiveness. Both adhesive cell populations may be implicated in promoting vascular obstruction.

Erythrocyte centrifugal packing in sickle cell anemia

Differential erythrocyte packing at two different centrifugal forces has been proposed as a measure of erythrocyte deformabilitylflexibility.(I,2) The method is based on the assumption that more effective (increased) packing during the initial-slowercentrifugation phase is dependent on erythrocyte deformability. (3) Increased relative packing during this phase should indicate more easily deformable cells. Therefore, the difference between the slower speed centrifugal erythrocyte packing and the final hematocrit reading should correlate with the degree of deformability of the erythrocytes in the sample. This erythrocyte packing difference (EPD) has been found to be altered in diabetes mellitus, peripheral vascular disease, angina and in-vitro manipulations such as glutaraldehyde fixation. (4)

The effect of donor age on the packing susceptibility of erythrocytes

The study of 411 healthy persons indicates that erythrocyte susceptibility to centrifugal packing at 200 g increases with age, evidencing an age-relaled decrease in red cell deformability. Positive correlations between donor age and blood plasma fibrinogen concentration and erythrocyte sedimentation rate were confirmed. The impairment of red cell deformability may affect the circulatory efficiency in the aged.

Influence of cell concentration on the contribution of unfrozen fraction and salt concentration to the survival of slowly frozen human erythrocytes

We have shown previously that the survival of human red blood cells during slow freezing at 2% hematocrit is dependent more on the magnitude of the unfrozen fraction than on the salt concentration in that unfrozen fraction. In parallel, first Nei and more recently Pegg and colleagues have shown that survival is affected by the hematocrit of the suspension. Freezing at hematocrits above 30% becomes increasingly damaging. The present studies were designed to see whether there is a link between the two phenomena. Cells were suspended at nominal hematocrits of 0.4, 2, 8, 40, or 60% in five test solutions of glycerol-NaCl. The test solutions were of such composition that when frozen to a specified temperature, the magnitude of the unfrozen fraction differed but the NaCl concentration ( m s) remained constant. At low hematocrits (0.4 to 8%), red cell survival was dependent predominantly on the unfrozen fraction and was relatively independent of the salt concentration in that fraction. This we term the “rheological” effect because injury appears to be related to interaction with the ice walls and perhaps is due to shearing forces or cell deformation. But at high hematocrits (40 or 60%), cell survival became dependent on both the unfrozen fraction and the salt concentration in that fraction. When freezing occurs at high hematocrits, increasing numbers of cells are presumably brought into contact with their neighbors. Furthermore, they are increasingly shrunken cells, for the progressive removal of liquid water, which is responsible for the crowding, also causes a rise in m s and the consequent osmotic shrinkage of cells. Our data suggest that at unfrozen fractions above those producing injurious rheological forces, the tight packing of less shrunken cells (i.e., high hematocrit, low m s) and the extensive shrinking of loosely packed cells (high m s, low hematocrit) are both quite innocuous. Injury becomes substantial only when extensively shrunken cells are brought into close contact (i.e., high m s, high hematocrit). At high hematocrit the cells occupy a substantial fraction of the unfrozen space, and the water that they lose during slow freezing adds substantially to the volume of extracellular ice. Accordingly, we defined other measures of unfrozen fraction that include these perturbations. However, we found that the conclusions on the relation between survival, unfrozen fraction, and hematocrit were not affected by the method of expressing the unfrozen fraction. Freezing at high hematocrit to high m s and low values of unfrozen fraction is one way to produce contact between shrunken cells at low temperatures. Another way is to subject cells in totally unfrozen solutions at high m s and low hematocrit to centrifugal packing at low temperatures. The centrifugal packing of shrunken cells was not damaging, but their redispersion was. Moreover, the more the cells were shrunken prior to centrifugation (i.e., the higher m s), the greater was the damage during their redispersion.

Leghaemoglobin within bacteroid-enclosing membrane envelopes from soybean root nodules

Methods are reported for the preparation from soybean (Glycine max (L.) Merr.) root nodules, of well-washed, intact membrane envelopes containing bacteroids. The intact envelopes are of much lower density than the bacteroids within and therefore only low speed centrifugation (approx. 150 g) may be used. The optimum osmotic strength is 600 mOsm/kg H2O. The envelope contents were recovered following mild osmotic shock and-or hard centrifugal packing at >10,000 g. Extracts prepared in this way contained leghaemoglobin (identified spectrophotometrically), low-molecular-weight fluorescent materials and other components which are yet to be identified. Envelope leghaemoglobin did not react with specific antibody until the envelopes were ruptured. (131)I-Labelled leghaemoglobin or bovine serum albumin, added during initial breakage of nodule cells, was not released when envelopes were ruptured to release leghaemoglobin. It is therefore concluded that this leghaemoglobin is located within the envelope space and did not arise from adhering or occluded cytosol leghaemoglobin. Based on the number and dimensions of microscopically intact envelopes in these preparations, the concentration within that space was in the range 178-523 μM. Based on these estimates, leghaemoglobin within envelopes represented about one third of the total amount present in the nodule cells. Flat-bed isoelectric focusing of partially-purified envelope leghaemoglobin demonstrated that the latter contained all of the leghaemoglobin components previously reported for soybean nodules and an additional minor component focusing between leghaemoglobins a and b.

A comparison of the effects of incubation on cells containing haemoglobin S or haemoglobin A.

1. A centrifugal packing technique has been used to investigate the effect of incubation on the flexibility of erythrocytes containing normal (Hb A) or sickle-cell (Hb S) haemoglobin. The effects on cell morphology and on the reversibility of morphological changes were also investigated. 2. The two types of cell reacted similarly to incubation. Sickling of the Hb-S was shown not to be responsible for the production of irreversibly deformed cells in these circumstances. 3. It is concluded that for both cell types the membrane inflexibility, cellular deformation and morphological irreversibility induced by the incubation were due to metabolic depletion.

Centrifugal packing of erythrocytes hardened with acetaldehyde.

Summary and ConclusionsThe centrifugally packed column of human or canine RBC hardened with acetaldehyde consists of only 60% cells, in contrast to the near 100% packing for normal RBC. This incomplete packing of hardened RBC was demonstrated with the isotope dilution method using three different macromolecules (albumin, dextran, and PVP) and by following the hematocrit values during the hardening process, and the results agree with theoretical calculations. The striking difference in the centrifugal packing of hardened vs normal RBC indicates that normal RBC are deformable during centrifugal packing and that the low degree of packing must be considered in any study on hardened RBC.

Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles.

A method is described for preparing, by filtration on Millipore filters, very thin (about 10 micro) pellicles of packed particles. These pellicles can be embedded in Epon for electron microscopic examination. They are also suitable for cytochemical assays. The method was used with various particulate fractions from rat liver. Its main advantages over the usual centrifugal packing techniques are that it produces heterogeneity solely in the direction perpendicular to the surface of the pellicle and that sections covering the whole depth of the pellicle can be photographed in a single field. It thus answers the essential criterion of random sampling and can be used for accurate quantitative evaluations.

Osmotic properties of mitochondria isolated from Acanthamoeba sp

Osmotic properties of mitochondria isolated from Acanthamoeba sp. were studied. Included were volumes of amoeba mitochondria in a wide variety of suspending media, a comparison of actual mitochondrial volumes determined by centrifugal packing with volumes determined by the optical density method, and mitochondrial metabolism at different volumes. Amoeba mitochondria were found to have many properties similar to those from mammalian tissues. Particle volume changes were likened to biocolloid effects rather than to osmotic phenomena. The unreliability of the optical density method in determining mitochondrial volumes was demonstrated. The role of intracellular tonicity and the site of citrate and succinate oxidation were discussed.