the two lines intersect. For greatest accuracy, points should be plotted as close to the true to as possible. RESULTS AND DISCUSSION
?‘he value of to obtained is, of course, independent of the homologous series used. When a thermal conductivity detector is used, the calculated to corr t q o n d s to the observed air peak time (Table I). While variations in a.pplicd helium pressure affect t o in all cases tried, the product of Pto m s constant at constant temperature (differenem not statistically significant). Raising the column temperature increases the value of Pto.
A useful parameter was obtained by dividing Pto by the absolute temperature. The resulting quotient, p, was constant and independent of temperature in the case of the two packed columns used (polypropylene glycol and Craig succinate polyester, each 30y0 on Chromosorb), but inversely proportional to temperature over most of the operating range for the capillary column used (squalane and polypropylene glycol). It has been possible to characterize columns with regard to t o by determining p a t various temperatures over the expected operating range and plotting the results, thus eliminating the nccessity of experimentally redetermining t o n-hen
operating conditions are changed. This process should be repeated from time to time as the column ages. LITERATURE CITED
(1) Keulenians, A. I. M., “Gas Chromatography,” 2nd ed., p. 27, Reinhold, New York, 1959. (2) Miwa, T. K., Mikolajczak, K. L.,
Earle, F. R., Wolff, I. A., ANAL. CHEM.
32. 1739 (1960’1. (3) Peterson, M.’ L., Hirsch, J., J . Lipid Research 1, 132 ( 1959). (4) Walsh, J. T., hlerritt, C., Jr., ANAL. CHEM.32, 1378 (1960).
THEU. S. Fruit and Vegetable Products Laboratory is a part of the Southern Ctilization Research and Development Division, Agriculture Research Service, U. S.Department of Agriculture.
Elution Concentration of Paper Chromatogram Spots Frances Davis, Clyde A. Dubbs, and William S. Adams, Veterans Administration Center and University of California Medical School, Los Angeles 24, Calif.
proecdure has been devised for the simultaneous elution and concentration of any spot or band that separates on the paper chromatogram of a complex misture. During the past four years, our laboratory has extensively applied this procedure to many purine and pyrimidine compounds from normal and leukemic urines (1). Most workers extract chromatogram spots by simple elution alone, and the rcsulting dilution requires a separate concentration step such as lyophilization, Thus Dent (6) cuts from the chromatogram an elongated paper strip that includrs the dcsired spot, pinches one end of this strip betu een two glass slides that are dipped into a dish of elution solvent. and allows the frre end of the strip to hang down outside the dish. By capillary and siphon action, the solvent nioves up and over and, carrying the eluting substance nith it, drips from the papcr tip into a rollection vessel. Consden, Gordon, aiid Martin ( 3 ) also use descending olution, but place one end of a capillary tube against each tip to collect the eluate. RIodifications of these original elution techniques are described by ITrork (10) and Lewis ( 7 ) . Evaporative concentration has been introduced by other n orkcrs Thus Winteringham (9) dips a paper strip containing the sample into solvent within a flask, lightly enclosing an upper portion of the strip betu een the groundglass neck and stopper; the eluate rises in the strip and, by evaporating as it cinerges into the open, can concentrate the sample within a 0.5-cm. zone on the
‘‘
N IRIPROIED
protruding paper. Others have used a similar idea to spot paper chromatograms. Thus, after cutting a tip a t one end of a paper strip containing the sample, Moore and Boylen ( 8 ) , using descending elution, and Gregory (6), using ascending elution (which permits a simpler apparatus), touch the tip to a large paper sheet, thereby spotting it by transfu and evaporation. All these
A
elution-concentration techniques suffer from a common fault-spreading of the eluate on the final paper, which can be only partially controlled by rapid evaporation induced by a fan. In contrast, our procedure takes advantage of the confining edges of the tip itself; the eluting solvent pushes relentlessly toward these edges and the apex, evaporates, and finally forces the eluting substance to deposit within an apex area of only several square millimeters. Although Decker (4) has also provided this feature in his effective (but apparently little known) method, our procedure offers greater simplicity and versatility; and the apparatus can be readily assembled from commonplace items. PROCEDURE
The small unit (Figure 1,A) is useful for eluting and concentrating one spot.
Figure 1. Two elution-concentration units assembled with enclosed paper chromatogram cutouts A.
Small unit (secured with soft wire loop) 6. l a r g e unit Each arrow paints to small apex area of final Concentration
Cut from the paper chromatogram the desired spot, forming a pointed tip on one side. Gently clamp or otherwise secure this paper cutout between two small glass plates (half microscope slides) with only the tip protruding from the top, and stand the unit vc.rtically in a shallow (0.5-cm.) layer of deionized water or other selected eluting solvent. A standard histological slide holder in a Petri dish can be conveniently adapted t o support 20 or more of these units. After complete concentration to the apex (overnight is convenient), stand the vertical slides upon an absorbent tissue to drain and dry. Finally cut off each dry apex for immediate processing or storage in small vials. VOL. 34, NO. 1, JANUARY 1962
175
ination with soluble impurities that would also be concentrated to the apex.
Figure 2. Battery of large elutionconcentration units after operation for only 10 minutes
As suggested by Bailey (%), contamination by residual paper impurities would be minimized (along with the time for concentration) by making available to each paper strip only the amount of solvent found by experience to provide complete recovery of the sample. Although we have found that the routine use of washed paper and exhaustive overnight concentration is satisfactory, this further precaution would be useful for the handling of very small samples that require the ultimate reduction of impurities.
Small unit
Spot cutouts in front two units, bond cutouts in r e m units
The large unit (Figure 1,B) is useful for eluting and concentrating a large number of similar spots or hands in one hatch. From prewashed filter paper (Whatman No. l), cut an approximate 6 X 8.5 em. rectangle with an appended tip about 4 em. high (dashed line tip, Figure 1,B). Secure the paper, tip alone protruding, between two 8 X 10 em. glass plates (ordinarylantern slides), and stand the unit vertically in a rectangular dish containing the layer of solvent (Figure 2). (If clothespin clamps are used, the lower pair, resting on opposite lips of the dish, can provide convenient support for each unit.) Ordinarily a small brown spot (residual paper impurities) will be concentrated to the apex. Snip off, cut a new tip, and repeat this washing procedure a t least two more times. Finally cut a sharper tip with concave sides, cover the 6 X 8.5 em. paper area with chromatogram cutouts, secure within the glass slides, and proceed with the overnight elution-concentration procedure. PRECAUTIONS
Table I.
Taken 2.00 4.00
6.00 8.00 10.00
Recovery Data
Adenine, pg. Recovered2.04 2.11 4.08 4.06 5.94 5.62 6.92 8.16 10.17 10.20
Amount recovered was determined by ultraviolet absorbance measurement.
176
Assure that the lower edge of the enclosed paper is above the solvent level. Otherwise losses of substance from the paper and cross contamination between individual units can result from side or hackdiffusion into the solvent layer. Do not clamp directly over enclosed paper, since solvent flow and elution are restricted within a locally compressed area. In the large unit, do not place paper cutouts on top of one another, nor closer t o the paper edge than 0.5 em. In humid atmospheres which inhibit evaporation, or under any circumstances where faster concentration is desired. d a c e a small electric hair drver at one’iide to direct a warm air &w slightly above the tips (Figure 2). (However, do not allow a strong breeze to blow directly upon the exposed tips, drying them out and preventing concentration to the apex.) To minimize solvent evaporation from the dish, cover with plastic film, slitted for insertion of slides. For colorless substances in amounts insufficient to give a visible deposit, confirm localization at the apex hy other suitable means. For purines, pyrimidines, and a number of other substances, visualization under the ultraviolet lamp is excellent. RESULTS AND DiSCUSSlON
Use acid-washed paper and scrupulously clean technique to avoid contam-
ANALYTiCAL CHEMISTRY
Figure 3. Final concentration and crystallization of cupric sulfate at apex
Figure 2 shows that, within 10 minutes after operation has begun, the soluble dye has formed well delineated paths towards the apes in every case; later the dye will he intensely concentrated at the apex. With the small unit, better than 100-fold concentrations can be routinely accomplishedLe., the substance can be transferred onto less than 1/100 as much paperand a considerable greater degree of concentration is possible with the large unit. Thereby minute amounts of a valuable substance can he concentrated on a small and handy paper carrier, facilitatmg storage, handling, and approximate measurement of small quantities without physical loss; yet quantitative elution by a few drops of solvent is easily accomplished when desired. [To determine absorption spectra, add an excised apes (several if desired) t o
1 ml. of solvent, agitate mildly, and transfer the solution directly to the cuvet.] With sufficient substance, a solid deposit or crystalline mass (Figure 3) may be built up on the apex, permitting physical separation of most of the substance from the paper without any need for solvent elution. This crystallization action also deserves special consideration by workers who often have trouble crystallizing natural products and their derivatives. Such difficulties should be minimized by the circumstances prevailing at the apex: a persistently concentrating solution t o initiate the early formation of small “seed” crystals, then a steady arrival of additional concentrating solution t o sustain crystalline growth. (This approach may be even more attractive should any crystallization inhibitor in the solution he selectively retarded by chromatographic action. Seed crystals might then begin to form at the aDex hefore an effec&e concentration of t h e inhibitor could arrive and interfere.) Concentration to the apex is quantitative, as confirmed by recovery of known amounts of adenine (2 to 10 pg.). For 10 runs on the small units (Table I), an average recovery of 99.6% was found (standard deviation 5.5%). LiTERATURE CITED
(1) Adams, W. S.,Davis, F., Nakatani, M., Am. J . Med. 28, 726 (1960). (2) Bailey, G. F., private communication. (3) Consden, R., Gordon, A. H., Martin, A. J. P., Biochem. J . 41, 590 (1947). (4) Decker, P., Nuturwissensehajten 38, 287 (1951). (5) Dent, C. E., Bioeha. J. 41, 240 (1947). (6) Gregory, G. F., Science 121, 169 (1955).
WOREsupported in part by U. S.Public Health Service Grant CY-2433.
