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J C Giddings

Publications and source records attributed to J C Giddings.

At least 19 recordsLinked to original sources
PubMed1992

Rapid breakthrough measurement of void volume for field-flow fractionation channels.

A peak breakthrough technique is described and evaluated for measuring the void volume of field-flow fractionation (FFF) channels, particularly those used for flow FFF. This technique uses a high-molecular-mass macromolecular or particulate probe that can be displaced rapidly by flow through the FFF channel with minimal transverse diffusion. The particles that emerge first are those carried through the entire length near the channel centerline at the apex of the parabolic flow profile. These particles generate a sharp breakthrough profile. The measured breakthrough time is two thirds of the void time, thus making it possible to calculate both the void time and the associated void volume. This method, although applicable to all FFF channels (and capable of extension to open tubes), is particularly useful for flow FFF because conventional low-molecular-mass void probes can diffuse into the permeable walls and thus distort void measurements. The theoretical basis of the breakthrough technique and an explanation for the sharpness of the breakthrough front are given. A method for compensating for deviations from perfect sharpness is developed in which the breakthrough time is identified with the time needed to reach 85-88% of the breakthrough peak maximum. Preliminary experimental results are shown using various protein probes in four different FFF channel systems.

Chemical Fractionation↗
PubMed1992

Dual-field and flow-programmed lift hyperlayer field-flow fractionation.

Field and flow programming and their combination, dual programming, are shown to extend the particle size range to which a single flow/hyperlayer field-flow fractionation (FFF) run is applicable to approximately 1-50 microns. The rationale for programming flow/hyperlayer FFF (or other forms of lift hyperlayer FFF) is to expand the diameter range of micron size particles that can be resolved in a single run. By contrast, the reason for programming normal-mode FFF, the only kind of programming previously realized in FFF, is to reduce the analysis time of submicron particle samples of considerable size variability. These differences are explained in detail in relationship to the basic mechanisms governing retention in normal, steric, and lift hyperlayer FFF. Experiments are described in which field, flow, and dual programming are used to expand the accessible diameter range of flow/hyperlayer FFF. An example is shown in which 11 sizes of latex microspheres in the 2-48-microns diameter range are separated by dual programming in 11 min.

Chemical Fractionation↗
PubMed1991

Hydrodynamic relaxation in flow field-flow fractionation using both split and frit inlets.

Two means are described for achieving hydrodynamic relaxation and thus avoiding the stopflow injection procedure in field-flow fractionation (FFF): split flow injection and frit inlet injection. The advantages, disadvantages, and the theoretical basis of these procedures are discussed. Incremental band broadening due to the final relaxation step is examined theoretically and shown to be negligible when the flow rate of the sample inlet substream is small compared to the total channel flow rate. The optimization of the sample inlet flow rate is discussed. Experimental results for both injection procedures are reported for flow/steric (or hyperlayer) FFF applied to latex standards, confirming the expected trends. However, closer examination shows that the observed incremental band broadening associated with hydrodynamic relaxation is somewhat larger than the value predicted.

Chemical Phenomena↗
PubMed1991

Comparison of power and exponential field programming in field-flow fractionation.

Field programming in field-flow fractionation has the purpose of expanding the molecular weight or particle diameter range subject to a single analytical run. The two most widely used field programs are those in which the field strength decays with time according to an exponential function and a power function, respectively. The performances of these two programming functions are compared by obtaining limiting equations showing how retention time tr, standard deviation in retention sigma t, and fractionating power Fd vary with particle diameter d. It is shown that uniform fractionating power (Fd independent of d) can be obtained with power programming but that in exponential programming Fd is always non-uniform, varying as d-1/2. In exponential programming a linear relationship arises between tr and log d. This particular relationship is impossible to realize in power programming but an alternative linear relationship can be obtained by plotting tr versus dt/3. These results are made more concrete by plotting and comparing field strength, relative field strength, Fd and tr for specific programming cases.

Chemical Fractionation↗
PubMed1991

Particle size distribution by sedimentation/steric field-flow fractionation: development of a calibration procedure based on density compensation.

Because of the important but mathematically complex role played by hydrodynamic lift forces in sedimentation/steric FFF, applied generally to particles greater than 1 micron in diameter, retention cannot readily be related to particle diameter on the basis of simple theory. Consequently, empirical calibration is needed. Unfortunately, retention is based on particle density as well as size so that a purely size-based calibration (e.g., with polystyrene latex standards) is not generally valid. By examining the balance between driving and lift forces, it is concluded that equal retention will be observed for equal size particles subject to equal driving forces irrespective of particle density. Therefore by adjusting the rotation rate to exactly compensate for density, retention can be brought in line with that of standards, a conclusion verified by microscopy. Linear calibration plots of log (retention time) versus log (diameter) can then be used. This approach is applied to two glass bead samples (5-30 and 5-50 microns) using both a conventional and a pinched inlet channel. The resulting size distribution curves are self consistent and in good agreement with results obtained independently.

Calibration↗
PubMed1991

Continuous separation of particles from macromolecules in split-flow thin (SPLITT) cells.

A split-flow thin (SPLITT) cell with a perpendicular driving force of one gravity has been utilized for the rapid separation of micron-sized particles from macromolecules. The procedure involves the simultaneous use of two transport mechanisms and thus two operating modes: a sedimentation process controls the displacement of the particles across the thickness of the thin channel, while diffusion controls the displacement of macromolecules. The theoretical equations for these two operating modes are summarized and it is shown how the two modes can be combined to yield specified recovery factors. The theory was tested on a mixture of 10 microns polystyrene latex beads and three different proteins. The observed separation was in excellent agreement with theory. Attempts to fractionate red blood cells and plasma proteins from whole blood were only partially successful as a consequence of the weak sedimentation of red blood cells. Various remedies to this problem are suggested, the most promising of which is the use of a SPLITT cell subject to mild centrifugal forces.

Cell Fractionation↗
PubMed1990

Hydrodynamic relaxation and sample concentration in field-flow fractionation using permeable wall elements.

The advantages of hydrodynamic relaxation in field-flow fractionation, in which an injected sample is driven rapidly toward its equilibrium distribution by flow, are described relative to conventional field-driven relaxation. A new concept for achieving hydrodynamic relaxation, based on the use of permeable wall elements (or frit elements) embedded in the channel walls, is introduced. Here an auxiliary substream of carrier fluid, permeating uniformly into the FFF channel near the inlet, drives the sample, entrained in its own substream, close to its equilibrium configuration. Such frit elements can also be used to enrich the sample at the outlet. Equations are derived and plots are provided for the position of the splitting plane dividing the two substreams; this position defines the strength of the hydrodynamic relaxation. Variations in shear through these frit-modified end regions are also formulated and plotted. The effects of frit elements on band broadening are discussed. It is concluded that permeable wall elements in many configurations may be broadly applicable to FFF and related methods for improved sample introduction, increased separation speed, reduced risk of sample adhesion to the wall, improved flow stability, and sample enrichment.

Chemistry Techniques, Analytical↗
PubMed1990

Evaluation of pinched inlet channel for stopless flow injection in steric field-flow fractionation.

In this article the concept of utilizing a pinched inlet channel for field-flow fractionation (FFF), in which the channel thickness is reduced over a substantial inlet segment to reduce relaxation effects and avoid stopflow, is evaluated for steric FFF using one conventional channel and two pinched inlet channels. It is shown that with the proper adjustment of flow-rate, the stopflow process in FFF can be completely avoided, thus bypassing the flow interruption associated with stopflow and reducing separation time. The maximum flow-rate that can be used for stopless flow operation without incurring zone distortion is shown to agree reasonably well with simple theory; slight departures from theory are attributed to the existence of reduced transport rates of large particles through thin channel structures.

Centrifugation↗
PubMed1990

Two-dimensional field-flow fractionation.

Multidimensional field-flow fractionation (FFF) is described in two major forms: one in which different separative stages are coupled together and one in which two independent displacements, at least one of them FFF, are carried out in a generally planar channel structure. The latter, the subject of this paper, is relatively promising for implementation in FFF systems because in most cases the geometry of the FFF channel is already planar; the channel structure needs mainly to be broadened along the second dimension and modified with different inlets and outlets for this two-dimensional use. The large number of potential two-dimensional FFF systems is discussed. These systems are described at greater length in four categories: (1) FFF displacement used in both dimensions, (2) FFF along one axis and chromatography along another, (3) FFF along one axis and a field-induced displacement along the other, and (4) FFF separation combined with bulk or flow displacement at right angles. Finally, theoretical equations are obtained for the deflection of the trajectories away from the main flow axis z. The sensitivity of deflection to component properties is described in terms of the deflection selectivity. Several examples are discussed in which the deflection selectivity is remarkably high.

Chemical Fractionation↗
PubMed1989

Hydrodynamic relaxation using stopless flow injection in split inlet sedimentation field-flow fractionation.

In this paper relaxation effects in both the normal and steric operating modes of sedimentation field-flow fractionation are examined by using three different injection procedures: stop flow, stopless flow, and a new stopless flow procedure employing an inlet splitter. In the usual operation of field-flow fractionation (FFF), a stop procedure is used in which the channel flow is halted for an adequate period of time after injection for sample relaxation (in which the sample particles approach equillibrium near one wall) before the resumption of channel flow. If the channel flow is not stopped (stopless flow procedure), the elution profile is shifted and distorted due to the downstream migration of the particles during the relaxation process. To avoid peak distortion while retaining the advantages of the stopless flow procedure, a physical splitter at the channel inlet divides the entering flow stream into two substreams; sample is injected into only one of these. In this way a rapid (although not complete) hydrodynamic relaxation is realized. This stopless split flow injection procedure is compared to ordinary stop and stopless flow procedures using both submicrometer (normal FFF) and supramicrometer (steric FFF) polystyrene latex particles. It is found that much of the distortion normally accompanying stopless flow injection is eliminated by this new procedure. However, further optimization is needed to match the high resolution of the stop flow method.

Chemical Fractionation↗
PubMed1989

Harnessing electrical forces for separation. Capillary zone electrophoresis, isoelectric focusing, field-flow fractionation, split-flow thin-cell continuous-separation and other techniques.

A simple analysis, first presented twenty years ago, showed that the effectiveness of a field-driven separation like electrophoresis, as expressed by the maximum number of theoretical plates (N), is given by the dimensionless ratio of two energies N = -delta mu ext/2RT in which -delta mu ext is the electrical potential energy drop of a charged species and RT is the thermal energy (R is the gas constant and T is the absolute temperature). Quantity -delta mu ext is the product of the force F acting on the species and the path length X of separation. The exceptional power of electrophoresis, for which often N approximately 10(6), can be traced directly to the enormous magnitude of the electrical force F. This paper explores the fundamentals underlying several different means for utilizing these powerful electrical forces for separation, including capillary zone electrophoresis, gel electrophoresis, isoelectric focusing, electrical field-flow fractionation and split-flow thin continuous separation cells. Remarkably, the above equation and its relatives are found to describe the approximate performance of all these diverse electrically driven systems. Factors affecting both the resolving power and separation speed of the systems are addressed; from these considerations some broad optimization criteria emerge. The capabilities of the different methods are compared using numerical examples.

Chemical Fractionation↗
PubMed1989

Field-flow fractionation of macromolecules.

Field-flow fractionation (FFF) is a versatile family of techniques, applicable to macromolecules, colloids, and cell-sized particles. This paper focuses specifically on the applicability of FFF to macromolecules. Following a brief description of the principles of FFF, the characteristics of FFF that bear on its efficacy in separating macromolecules are summarized. The basis of selectivity is established. The general applicability of FFF to macromolecules is then surveyed. For this purpose macromolecular substances are divided into four classes, distinguished by a molecular weight cutoff of 10(6) and by aqueous versus organic solubility. The capabilities of different FFF subtechniques in fractionating these classes of macromolecules is then discussed.

Chemical Fractionation↗
PubMed1989

Separation of B and T lymphocytes by a hybrid field-flow fractionation/adhesion chromatography technique.

A hybrid of the techniques of cellular adhesion chromatography and field-flow fractionation has been used for the effective separation of rat mesenteric B and T lymphocytes with nearly complete recovery of both cell species. Use of this hybrid technique also allows the relative binding strengths of cells to biomaterials and other surfaces to be rapidly and simply estimated. For the copolymer surface used here, B cells appear to bind with a force five times greater than T cells.

Animals↗
PubMed1989

Factor IX Cardiff: a variant factor IX protein that shows abnormal activation is caused by an arginine to cysteine substitution at position 145.

Crude barium chloride eluates prepared from 12 unrelated patients with cross-reacting material positive (CRM+) haemophilia B were activated with celite eluate, the reaction products resolved after reduction by 13% SDS-PAGE, and factor IX antigenic material detected by probing with radiolabelled immunopurified rabbit anti-factor IX antiserum followed by autoradiography. Out of the 12, one sample showed faulty activation with the production of a stable reaction product with a MW compatible with that of a putative light chain-activation intermediate. In order to confirm this, two oligonucleotide primers that bracketed exon 6 of the factor IX gene were constructed and used to prime a polymerase chain reaction on DNA isolated from the patient's peripheral blood leucocytes. A single 489 nucleotide DNA fragment was obtained, gel purified, subcloned into M13, and DNA sequencing carried out on both strands. A single C to T transition was discovered that changed the Arg residue at position 145, the first residue of the first bond in the activation peptide, to a Cys, a result that confirmed the inferences drawn from the activation studies.

Antigens↗
PubMed1989

Protocol for the evaluation of automated blood coagulation instruments (coagulometers) for determination of the international normalised ratio.

Results of prothrombin time tests for the control of oral anticoagulant therapy, when using automated blood coagulation instruments (coagulometers), may not be the same as those obtained when using manual methods, and variation in coagulometer performance may affect clinical interpretation of data. The haematology advisory committee of the Institute of Medical Laboratory Sciences has recommended that coagulometers should be used for determination of prothrombin times only if it can be demonstrated that the results are valid when compared with those of a standardised manual method. A protocol for the evaluation of coagulometers, and details of the procedures, are given in this report, the main emphasis of which is on the assessment of instruments for determination of the International Normalised Ratio. Guidelines are also provided for the general use of machines of this type and for the assessment of safety. The principles of the protocol are applicable to other automated tests of blood coagulation.

Autoanalysis↗
PubMed1988

Hereditary combined deficiency of clotting factors V and VIII with involvement of von Willebrand factor.

A family is described in which two brothers, with a significant haemorrhagic disorder, are affected by combined factor V/VIII deficiency. In one of these patients an abnormal decrease of von Willebrand factor was also observed. Family studies suggest that both of the brothers are homozygous for a recessive gene. Normal laboratory results were found in eight other family members although seven of them had reported a mild bleeding tendency. The results indicate that hereditary combined factor V/VIII deficiency is a heterogeneous disorder and that defects of von Willebrand factor might be involved in the aetiology of the disease in some families.

Adult↗