A simple procedure for eluting nucleotides from thin-layer plates

SHORT

A Simple

Procedure from

169

COMMUNICATIONS

for

Thin-Layer

Eluting

Nucleotides

Plates

A number of solvent systems have been developed by Randerath and co-workers for the separation of nucleotides on thin layers of polyethyleneimine (PEI)-cellulose, and the distinct advantages of this technique have been summarized (1). Generally, in thin-layer chromatography, elution of a compound from a spot of adsorbent may be carried out by scraping the adsorbent into a mound, t,ransferring it to an elution tube by means of further scraping or a microvacuum-cleaner type of device (2-4), followed by the passage of an elution solvent through the adsorbent. With cellulose thin layers, however, scraping is particularly unsatisfactory because of the fibrous nature of the adsorbents. The method of Randerath and Randerath (1) for eluting the compound in situ without prior removal of the spot from the plate avoids scraping, but in our experience with the in situ procedure it is difficult to ensure a uniform treatment of different spots or to prevent occasional floodings and crosscontamination of closely adjacent spots. More recently, Randerath and Randerath (5) introduced the use of thin layers on plastic sheets rather than on glass plates, and this permits the spots to be cut out and eluted individually. However, this method is useful only with adsorbents that can adhere to plastic surfaces. We wish to report here an elution procedure which makes use of the ease of lifting a spot of frozen adsorbent as an icy sheet. The removal of adsorbent in the form of intact sheets rather than loose powder also minimizes cross-contamination. This procedure has been applied to the recovery of ribonucleoside triphosphates from PEI-cellulose plates. After chromatography according to Neuhard, Randerath, and Randerath (6)) the spots of nucleotides were detected by ultraviolet illumination. Each spot was made into an island by scraping out a narrow clearing peripheral to the spot. The island was dampened with a small amount of distilled water, and the plate was placed in a freezer until the water had frozen. The spot was then lifted from the plate using a spatula, broken into pieces, suspended in distilled water, and washed on to an elution column. To prepare an elution column, a disposable Pasteur pipet was plugged with a few strands of glass wool, and a thin layer of Sephadex G-25 was deposited on top of the glass wool. This assembly was washed with 1 ml of 1.2 M LiCl and 1 ml of 2 M LiCl to remove any ultraviolet-absorbing impurities, and then rinsed with distilled water. The layer of Sephadex

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ensured the total retention of adsorbent when the suspension of PEIcellulose was placed on the column. After the PEI-cellulose had packed down, the nucleotide was eluted by 1 ml of 2 M LiCl. Efficiency of the elution procedure is illustrated in Figure 1. For the standard curve, different amounts of adenosine triphosphate (ATP) dissolved in 1 ml of water were read at 260 rnp in a Gilford spectrophotometer. For the test curve, the same amounts were spotted on the PEIcellulose plate, and the spots were removed and eluted with 2 M LiCI. Absorbancies of the test samples were first corrected for a blank ab-

5. 0.3 E 0 m cv ‘0 0.2 %

16

32

48

mu mole ATP FIG.

1. Efficiency

of elution

procedure.

sorbance of 0.031 that resulted from the elution of an empty spot of similar size (2 M LiCl itself had an absorbance of 0.028). In the experiment in Figure 1, the recovery of ATP was 88%. There was more scattering of the single samples along the linear test curve compared to the standard curve, but no systematic deviation was apparent down to an absorbancy of less than 0.05. ACKNOWLEDGMENT This

study

was supported

by

the

Medical

Research

Council

of Canada.

REFERENCES 1. RANDERATB, E., AND RANDERATH, K., Anal. Biochem. 2. GOLDRICK, B., AND HIRSCH, J., J. Lipid Res. 4, 482 3. MATTHEWS, J. S., PEREDA, V. A. L., AND AGUILERA, (1963).

12,

83

(1963). P. A.,

(1965). J. Chromatog.

2, 331

SHORT 4.

RUCHELMAN,

5.

RANDERATH,

6.

NEUHARD,

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M. W., J. Chem. Ed. 44, 110 (1967). K., AND RANDERATH, E., J. Chromatog. 22, 110 (1966). J., RANDERATH, E., AND RANDERATH, K., Anal. Biochem.

13, 211 (1965).

PATRICIA

J. T.

HASTINGS M’ONG

Department of Biochemistry University of Toronto Toronto, Canada Received August 10, 1967

A Simple

Density-Gradient

Large-Capacity

Zonal

Engine Ultracentrifuge

for

Loading Rotors

Many devices for preparing density gradients from sucrose solutions have been described (see, e.g., references l-5). These devices produce gradients of various profiles but are suitable only for the production of small volumes of gradient in tubes for use in swinging-bucket or anglehead rotors. The loading of the large-capacity, high-speed zonal ultracentrifuge rotors that have recently been developed (6) and are now beginning to come into general use presents some difficulties, since these rotors require a gradient engine which can form and pump gradients of volumes from 400-1200 ml in 30-60 min (7). One gradient engine with such capabilities is currently available (Beckman Instruments Inc., Palo Alto, Calif.) but, although it is very versatile, it is also expensive. This note describes a simple gradient engine that is readily constructed from commonly available apparatus and can be used to load zonal rotors with a variety of simple density gradients. A diagram of the apparatus is shown in Figure 1. The peristaltic pump should be capable of delivering at least 2 liters/hr against a back-pressure of 20 psi, and it must be possible to vary the flow rate without stopping the pump. A suitable pump is the Hughes Hiloflow (F. A. Hughes and Co., Epsom, Surrey, England), which was used throughout these experiments. The pressure gauge is calibrated for O-30 psi. The sucrose solutions, which are precooled to 4”, are kept cold by surrounding the two vessels, A and B, wit,h baths of ice. To form a gradient, the volume of heavy sucrose solution required is put in vessel A and the tube from A is filled with the sucrose solution to point C, at which point the tube is clamped. The volume of light