US 3926809 A
Description (OCR text may contain errors)
United States Patent [1 1 Jones 1 1 MULTl-COLUMN FRACTIONATOR  Inventor: Roosevelt J. Jones, 3854 Blodgett,
Houston, Tex. 77004  Filed: Oct. 10, 1973  Appl. No.: 396,356
 US. Cl. 210/198 C; 55/386  Int. Cl. B011) 15/08  Field of Search 55/386; 210/198 C, 31 C  References Cited UNlTED STATES PATENTS 2,935,908 5/1960 Phillips, Jr. 210/198 C X 3,030,798 4/1962 Lichtenfels 55/386 X 3,150,517 9/1964 Kuffea et a1. 55/386 X 3,156,548 11/1964 Perry 55/386 3,164,980 l/196 5 55/386 X 3,169,389 2/1965 Green et a1. .1. .z 55/386 X Dec. 16, 1975 3,373,872 3/1968 Habina 210/198 C 3,504,799 4/1970 Ogle 210/198 C 3,518,874 7/1970 Habina... 210/198 C X 3,653,840 4/1972 Silas 55/386 X Primary Examiner.lohn Adee  ABSTRACT A chromatography apparatus for the simultaneous fractionation of two, three and four different mixtures of nucleotides, each on a separate ion-exchange column, using a stepwise elution procedure. Each elution solvent, used in a stepwise elution procedure, is forced by air pressure from a single interchangeable reservoir flask on to and through each of two, three or four chromatography columns at the same moment after having been dispersed equally by a multi-valve control. 1
3 Claims, 3 Drawing Figures US. Patent Dec. 16,1975 Sheet10f3 3,926,809
U.S. Patent Dec. 16, 1975 Sheet 3 of3 3,926,809
MULTI-COLUMN FRACTIONATOR BACKGROUND OF THE INVENTION This invention relates in principle to known liquid 5 ion-exchange chromatography apparatus (Cohn, 1950. .lour. of Amer. Chem. Soc. 72: 1471-1478) but differs in that four columns, rather than one, are employed to permit the fractionation of four separate and different mixtures of nucleotides during a single operation. 19
This apparatus requires a manual placing of each sample or mixture of nucleotides on to each of four ion-exchange resin columns prior to forcing the elution solvent of a given pH through the resin columns. The
elution solvent of a given pH, when forced through the resin column, will selectively remove certain of the nucleotides of each nucleotide mixture. The apparatus was designed to permit a partial separation of the numerous (more than 20) nucleotides which may be present in two, three or four nucleotide mixtures in a rapid fractionator was designed primarily for the fractionation of Phosphorus-labeled nucleotide mixtures of biological origin where small volumes of several nucleotide mixtures are to be analyzed. To study the synthesis of one or several different nucleotides, in vivo, as
many as 12 or more biological samples labeled with 30 phosphorus must be quantitatively and qualitatively analyzed within a period of 4 to 6 weeks, since the half-life of phosphorus is 14.5 days. An advantage of this apparatus is that it permits the simultaneous fractionation of two, three or four different nucleotide 35 mixtures in less time than is required to fractionate two different nucleotide mixtures when two independent single column chromatography apparatus are employed successively.
SUMMARY This invention is based on the novel concept that the elution of nucleotide mixtures from different chromatography columns in parallel would permit the quantitative elution of the same nucleotide when a given elution solvent and flow rate is employed. This invention was devised to permit the elution solvent of a given pH to be forced by air pressure from the reservoir flask through a multi-valve control on to and through each of two, three or four chromatography columns and into collecting tubes. The interchangeable reservoir flasks provide simplicity and save time during shifting from one elution solvent of one pH to another. A prescribed stepwise elution procedure requires eluting with several elution solvents of different pH during a single operation or complete fractionation. The multi-valve control makes it possible to disperse equally each elution solvent of a given pH on to and through each of two, three or four chromatography columns at the same moment. The multi-valve control pennits the regulation of flow rate for each column. The eluent which flows from each chromatography column is collected and measured in burettes before transferring into collecting tubes. The collecting of eluent is done manually. The number of elution solvents of different pH and the specific pH of each elution solvent can be prescribed at the discretion of the operator. The size of the resin bed, type of ion-exchange resin used, and
eluent flow rate are not limited to those described herein.
DESCRIPTION OF THE INVENTION FIG. 1 is a schematic four-column liquid chromatography system in accordance with the present invention in one embodiment.
FIG. 2 shows the component parts of the multicolumn support stand and the compressed air source viewed from the front.
FIG. 3 is a schematic of the individual components and connections of the chromatography apparatus with one of its four chromatography columns illustrated.
MODE OF OPERATION OF THE INVENTION By way of example and with reference to the accompanying drawings the embodiments of the present invention will now be described. As shown in FIG. 2 the multi-column support stand is assembled by fastening, with nut and bolt, the support rod 12 to the support stand base 11. The multi-valve support 15 and the column clamp support 13 are clamped on to the support rod 12. The multi-valve support 15 hold the multivalve control 3 in place. The column clamp support 13, with four spring clamps 14, holds the chromatography glass columns 6 upright and in place. The ion-exchange resin (Dowex 1X8 formate) is prepared as described by Hurlbert, et al. 1957. .lour. of Biol. Chem. 209: 23-39. Each chromatography glass column (28 X 0.8 mm) 6 is partially filled with the Dowex-l resin (bed size: 15 X 0.5 mm) and rinsed with deionized water prior to and after applying the nucleotide mixture. The nucleotide mixture. The nucleotide mixture can be a phosphorus-labeled nucleotide extract, that contains 100,000 to 800,000 counts per minute, or anucleotide mixture, that contains 24 mM of nucleotides. Stepwise elution of nucleotides, as described by Hurlbert, et al. 1957. .lour. of Biol. Chem. 209: 23-39. involves making several graduated shifts in the concentration (from 0.01 M to 4 M formic acid; and 4 M formic acid plus varying concentrations, 0.1 to l M, of ammonium formate) of the elution solvent during a single operation. The various nucleotides (mono-, diand triphosphates of the nucleosides of purines and pyrimidines) which posses different dissociation constants (pK values) are eluted individually as a function of the pH of the eluent (Cohn, 1950. Jour. of Amer. Chem. Soc. 72: 1471-1478). The elution volume for each concentration of the elution solvent varied from 5 to 20 ml between shifts, being collected in 5 ml fractions. To detect the presence of phosphorus-labeled nucleotides in each eluent fraction a 1 ml aliquot is added to 15 ml of a scintillation fluid (POPOP-toluene and Trition X-100, 2:1) and analyzed with a Packard Tri-Carb Scintillation Counter for phosphorus radioactivity. If the eluted nucleotides are unlabeled (containing no phosphorus) their presence in each eluent fraction is determined using a Beckman Model DB Spectre-photometer, which detect ultraviolet-absorbing compounds. Final separation and identification of individual nucleotides, present in each eluent fraction or in pooled fractions, are accomplished by paper chromatography (Wyatt, 1955. The Nucleic Acids of Chemistry and Biology, Acad. Press, New York. Vol. 1: 243-265) or thin-layer chromatography (Randerath and Randerath, 1964. .Iour. of Chromatography 16: 111-125.
To initiate the elution of nucleotides from the resin columns the reservoir jar 1 containing 400 ml of 0.01
M formic acid is connected to the compressed air tank '18 via the inlet glass tube 2B and plastic tubing 2, and to the multi-valve control 3 via the outlet glass tube 2B and a plastic tubing 2. The dripmeters 5' are disconnected from the chromatography glass columns 6 before air pressure is permitted to force the elution mixture from the reservoir jar 1, through the multi-valve control 3 and into the dripmeters 5. Adjustment of the gas regulator 17 and the valve control knobs l6 regulate the flow of the elution solvent. Using the dripmeters 5 the space above the resin bed of each chromatography glass column 6 is filled with the elution mixture before dripmeters 5 are fitted tightly into each chromatography glass column 6. Final adjustment with the valve control knobs 16 is necessary to obtain the desired eluent flow rate of 0.75 ml per minute. Eluent volume is measured in a burette 7, which fits into a burette plastic cap 8, and collected in test tubes 9, which are held upright in test tube holders l0.
To shift from one elution solvent to the next elution solvent, whose pH concentration is higher, the following steps are followed. Air pressure is turned off with the gas regulator l7 before disconnecting the plastic tubing 2 that joins the gas regulator 17 to the reservoir jar l. The dripmeter's 5 are disconnected from the chromatography glass columns 6 and held elevated above the multi-valve support to permit the elution solvent to flow back into the reservoir jar 1 once the valve outlets 19 have been opened using the valve control knobs 16. The previously used reservoir jar 1 is replaced with a reservoir jar 1 containing 400 ml of elution solvent of the next higher pH concentration. The elution solvent above the resin bed in the chromatography glass columns 6 is removed and replaced with the elution solvent of the next higher pH concentration.
The elution procedure is resumed after replacing each drip-meter 5 into their respective chromatography glass columns 6. The presence of the plastic joint connector 4 which joins the connecting tube 2, leading 5 from the valve outlet 19, and the dripmeter 5 permits disconnection of the apparatus at this point.
What is claimed is:
1. In an apparatus for analyzing samples of nucleotides using a plurality of chromatograpic columns the improvement comprising in combination:
interchangeable reservoir means. each said reservoir holding an elution solvent of a different ph concentration;
a set of four chromatographic columns; a four-way multi-valve control which disperses equally elution solvent of the same pH concentration on to each of said four chromatography columns at the same moment;
said columns containing ion-exchange resin capable of separating a micture of nucleotides; means connecting said valve between said reservoir and said columns; a burette for measuring eluent volumecollected from each of said chromatographic columns; a set of collecting tubes held upright in test tube holder means; pressure means connected to said reservoir means for forcing said elution solvent through said valve means and into each of said columns. 2 The apparatus of claim 1 wherein said reservoir means consist of wide mouth jar, clamp and rubber stopper.
3. The apparatus of claim 2 further including said columns, jar and tubes being constructed of glass.