Microchromatographic device and method for rapid determination of a desired substance

A microchromatographic device for use in a rapid quantitative determination of a numerical value as a diagnostic indicator of the blood sugar condition of a specific person, the device comprising the combination of:

• a microcolumn having exterior walls and containing a packing of a set amount of ion exchange resin, said ion exchange material being contained within a predetermined intermediate portion of the microcolumn, the microcolumn containing space for accomodating liquid above an upper boundary of the intermediate portion of the microcolumn;
• an aqueous buffer solution contained in the microcolumn, a first part of the total volume of the buffer solution permeating the packing of the ion exchange resin and being contained in the intermediate portion of the microcolumn, a second part of the buffer solution comprising the remainder of the total volume of the buffer solution and and being contained in the space above the intermediate portion of the microcolumn, said second part of the buffer solution including a predetermined volume which is capable of selectively eluting a glycosylated hemoglobin species from the ion exchange resin, the second part of the buffer solution being at least as much as the predetermined volume of the buffer solution;
• means located on the microcolumn below the intermediate portion for sealing the packing of ion exchange resin in the microcolumn but allowing for outflow of the buffer solution from the microcolumn at the option of an operator, and
• a centrifuge tube having inner walls, the microcolumn being at least partially inserted into the centrifuge tube wherein it is removably but fixedly held in an upright position, a void space being disposed between the inner walls of the centrifuge tube and the exterior walls of the microcolumn, a portion of the void space equalling in volume the predetermined volume of the buffer solution;
• the relative dimensions of the microcolumn, of the intermediate portion of the microcolumn, of the centrifuge tube, and of the second part of the total volume of the buffer solution being adjusted in such a manner that upon passage of the predetermined volume of the buffer solution through the microcolumn into the centrifuge tube the level of the buffer solution in the centrifuge tube substantially coincides with the level of the buffer solution remaining in the microcolumn, said level being disposed not substantially lower than the upper boundary of the intermediate portion of the microcolumn wherein the ion exchange resin is contained, whereby a centrifugation of the assembled microcolumn and centrifuge tube causes the passage of the predetermined volume of the buffer solution into the centrifuge tube, and whereby the glycosylated hemoglobin species is eluted from the ion exchange resin into the centrifuge tube and said glycosylated hemoglobin species is dissolved in the predetermined volume of the buffer solution.

A device for use in a rapid quantitative determination of a numerical value as a diagnostic indicator of the blood sugar condition of a specific person, the device comprising the combination of:

• a microcolumn;
• a settled packing of an ion exchange resin confined in the microcolumn, the settled packing substantially having an upper limit;
• a buffer solution contained within the microcolumn permeating through the packing of the ion exchange resin and having an additional predetermined volume disposed above the upper limit of the packing, the predetermined volume capable of selectively eluting a glycosylated hemoglobin species through the packing of the ion exchange resin;
• a lysing agent dissolved in the buffer solution capable of lysing whole human red blood cells and releasing the glycosylated hemoglobin species and nonglycosylated hemoglobin species therefrom;
• means located below the packing of the ion exchange resin for shutting the flow of the buffer solution through the microcolumn and for allowing said flow at the option of an operator;
• a centrifuge tube adapted for receiving the microcolumn and for holding the same in an upright position, a void space being disposed between interior walls of the centrifuge tube and exterior walls of the microcolumn, the void space being large enough to accommodate the predetermined volume of the buffer solution, the relative dimensions of the centrifuge tube and of the microcolumn including the packing of the ion exchange resin being selected in such a manner that upon passage of the predetermined volume of buffer solution through the microcolumn into the centrifuge tube the level of the buffer solution in the centrifuge tube and in the microcolumn substantially coincide with the limit of the settled packing of the ion exchange resin whereby after an introduction of a sample of red blood cells into the predetermined volume of the buffer solution the glycosylated hemoglobin species is released therefrom by the lysing agent, and whereby after centrifugation of the assembled microcolumn and centrifuge tube only the predetermined volume of buffer solution passes through the microcolumn eluting the glycosylated hemoglobin species which is contained in said predetermined volume.

A method for rapid quantitative determination of a first numerical value as a diagnostic indicator of a cumulative blood sugar condition of a specific person, the method comprising the steps of:

• introducing a sample of whole red blood cells into a top portion of a microcolumn having an intermediate portion which is packed with an ion exchange resin, the ion exchange resin being permeated by a first part of a first buffer solution having a lysing agent dissolved therein, the top portion of the microcolumn containing a second part of the first buffer solution,
• allowing the lysing agent to break up the whole red blood cells and to release therefrom glycosylated hemoglobin species and nonglycosylated hemoglobin species, the ratio of the glycosylated and nonglycosylated hemoglobin species being indicative of the cumulative blood sugar condition of the specific person;
• positioning the microcolumn into a centrifuge tube;
• subjecting the microcolumn to a centrifugal force of 50-1000 g thereby forcing a predetermined volume of the first buffer solution to pass from the top portion of the microcolumn through the ion exchange resin and to be discharged into the centrifuge tube, at least the predetermined volume of the first buffer solution being originally included in the second part of the first buffer solution and being originally contained in the top portion of the microcolumn, the predetermined volume of the first buffer solution reaching a level in the centrifuge tube which coincides with the level of the first buffer solution remaining in the centrifuge tube, said level being positioned in the microcolumn at least as high as a top surface of the ion exchange resin packing whereby further centrifugation fails to force more of the first buffer solution to pass through the ion exchange resin into the centrifuge tube;
• the first buffer solution and the ion exchange resin further being selected so that the predetermined volume of the first buffer solution selectively elutes the glycosylated hemoglobin species through the ion exchange resin;
• spectrophotometrically measuring in the eluted predetermined volume of the first buffer solution a second numerical value proportional to the concentration of glycosylated hemoglobin species therein, and
• preparing a sample of lysed red blood cells of the same specific person in a second buffer solution of the same composition as the first buffer solution and spectrophotometrically measuring a third numerical value proportional to the sum of glycosylated and nonglycosylated hemoglobin species in the second buffer solution, said first numerical value being calculated from the second and third numerical values.