Claims
- 1. A procedure for determining the size and size distribution of test particles by using a test sedimentation field-flow fractionation channel through which a liquid carrier stream flows, said procedure comprising
- carrying out at least one calibration run in which at least one calibration sample of particles of known density and size is injected into a liquid carrier stream flowing through a calibration sedimentation field-flow fractionation channel, wherein the acceleration G acting on the calibration sample during the calibration run and the density difference .DELTA..rho. between the particles of the calibration sample and the liquid carrier of the calibration run are known so that the product G.DELTA..rho. for the calibration run has a determined value, and further wherein the calibration channel is the test channel or a duplicate channel having essentially the same dimensions as the test channel or a similar channel having a thickness that is about the same as the thickness of the test channel with the linear velocity of the liquid carrier in the similar channel being maintained essentially the same as the linear velocity of the liquid carrier in the test channel;
- measuring retention time or retention volume of particles of different known sizes in the calibration sample;
- using the measured retention times or retention volumes to develop a calibration relationship expressing retention time or retention volume as a function of particle size;
- in a test run, injecting a test sample of test particles into the liquid carrier stream flowing through said test channel, wherein the density difference .DELTA..rho.' between the test particles and the liquid carrier stream is known, but the test particles have sizes and a size distribution that are unknown;
- adjusting the acceleration G' acting on the test sample during the test run such that the product G'.DELTA..rho.' for the test run is the same as the product G.DELTA..rho. for the calibration run;
- determining the relative concentration of test particles eluted at different times in the test run; and
- combining this relative concentration of determination with the calibration relationship to calculate the particle sizes and the particle size distribution of the particles in said test sample.
- 2. A procedure in accordance with claim 1, wherein a plurality of separate, calibration runs are carried out with a plurality of distinct calibration samples of particles being injected into the carrier stream, with each calibration sample containing a uniform, specified particle size that is different from the particle size of other calibration samples, and the retention time or retention volume is measured for each separate calibration sample.
- 3. A procedure in accordance with claim 1, wherein the calibration relationship is a plot of log t.sub.r versus log d, wherein d is the diameter of the particles of different known sizes in the calibration sample and t.sub.r is the retention time for respective calibration particles measured in the calibration run.
- 4. A procedure in accordance with claim 1, wherein the calibration relationship is a plot of log V.sub.r versus log d, wherein d is the diameter of the particles of different known sizes in the calibration sample and V.sub.r is the retention volume for respective particles of different sizes measured in the calibration run.
- 5. A procedure in accordance with claim 1, wherein the calibration relationship is a mathematical expression relating t.sub.r to d, wherein d is the diameter of the particles of different known sizes in the calibration sample and t.sub.r is the retention time for respective calibration particles measured in the calibration run.
- 6. A procedure in accordance with claim 1, wherein the calibration relationship is a mathematical expression relating V.sub.r to d, wherein d is the diameter of the particles of different known sizes in the calibration sample and V.sub.r is the retention volume for respective calibration particles measured in the calibration run.
- 7. A procedure in accordance with claim 1, wherein the relative concentration of the test particles eluted during the test run is used to plot a fractogram having a relative particle concentration axis and a retention time axis or a retention volume axis, with the relative particle concentration being plotted versus the retention time or retention volume, and the calibration relationship is used to develop particle size as a second parameter along an axis parallel to or coincident with the retention time axis or the retention volume axis.
- 8. A procedure in accordance with claim 1, wherein the test sample is comprised mostly of particles that migrate through the channel according to the steric mechanism of sedimentation field-flow fractionation.
- 9. A procedure in accordance with claim 1, wherein the test sample is comprised mostly of particles having a diameter range from 1 .mu.m to 100 .mu.m.
- 10. A procedure in accordance with claim 2, wherein the calibration relationship is obtained by measuring the retention times or retention volumes of a series of latex standards.
- 11. A procedure in accordance with claim 10, wherein the calibration relationship is obtained by measuring the retention times or retention volumes of a series of polystyrene latex standards.
- 12. A procedure for determining the size and size distribution of test particles using a test sedimentation field-flow fractionation channel through which a liquid carrier stream flows, said procedure comprising
- carrying out at least one calibration run in which at least one calibration sample of particles of known density is injected into a liquid carrier stream flowing through a calibration sedimentation field-flow fractionation channel, wherein the acceleration G acting on the calibration sample during the calibration run and the density difference .DELTA..rho. between the particles of the calibration sample and the liquid carrier of the calibration run are known so that the product G.DELTA..rho. for the calibration run has a determined value, and further wherein the calibration channel is the test channel or a duplicate channel having essentially the same dimensions as the test channel or a similar channel having a thickness that is about the same as the thickness of the test channel with the linear velocity of the liquid carrier in the similar channel being maintained essentially the same as the linear velocity of the liquid carrier in the test channel;
- collecting separate fractions of the carrier stream and calibration sample flowing from said calibration channel;
- measuring retention time or retention volume for the separate fractions;
- experimentally determining an average diameter of the particles in each of said fractions;
- using the measured retention times or retention volumes and the determined diameter of the particles to develop a calibration relationship expressing retention time or retention volume as a function of particle diameter;
- in a test run, injecting a test sample of test particles in the liquid carrier stream flowing through said test channel, wherein the density difference .DELTA..rho.' between the test particles and the liquid carrier stream is known, but the test particles have sizes and a size distribution that are unknown;
- adjusting the acceleration G' acting on the test sample during the test run such that the product G'.DELTA..rho.' for the test run is the same as the product G.DELTA..rho. for the calibration run;
- determining the relative concentration of test particles eluted at different times in the test run; and
- combining this relative concentration determination with the calibration relationship to calculate the particle sizes and the particle size distribution of the test particles in said test sample.
- 13. A procedure for producing a calibration relationship for use with sedimentation field-flow fractionation channels having similar dimensions, said procedure comprising
- carrying out at least one calibration run in which at least one calibration sample of particles of known density and size is injected into a liquid carrier stream flowing through one of the sedimentation field-flow fractionation channels, wherein the acceleration G acting on the calibration sample during the calibration run and the density difference .DELTA..rho. between the particles of the calibration sample and the liquid carrier of the calibration run are known so that the product G.DELTA..rho. for the calibration run has a determined value;
- measuring retention time or retention volume of particles of different known sizes in the calibration sample; and
- using the measured retention times or retention volumes to develop a calibration relationship expressing retention time or retention volume and particle size.
- 14. A procedure in accordance with claim 13, wherein the calibration relationship is a plot of log t.sub.r versus log d, wherein d is the diameter of the particles of different known sizes in the calibration sample and t.sub.r is the retention time for respective particles measured in the calibration run.
- 15. A procedure in accordance with claim 13, wherein the calibration relationship is a plot of log t.sub.r /t.sup.0 versus log d, wherein d is the diameter of the particles of different known sizes in the calibration sample, t.sub.r is the retention time for respective particles measured in the calibration run and t.sup.0 is void time.
- 16. A procedure in accordance with claim 13, wherein the calibration relationship is a plot of log V.sub.r versus log d, wherein d is the diameter of the particles of different known sizes in the calibration sample and V.sub.r is the retention volume for respective particles measured in the calibration run.
- 17. A procedure in accordance with claim 13, wherein the calibration relationship is a plot of log V.sub.r /V.sup.0 versus log d, wherein d is the diameter of the particles of different known sizes in the calibration sample, V.sub.r is the retention volume for respective particles measured in the calibration run and V.sup.0 is void volume.
BACKGROUND OF THE INVENTION
1. Government Support
This invention was made with Government support under Public Health Service Grant GM10851-33 awarded by the National Institutes of Health. The Government has certain rights in the invention.
US Referenced Citations (6)