Pressure device for displacement of sucrose ... - ACS Publications

Pressure Device for Displacement of Sucrose Gradients. Sherman R, Dickman and Veri J. Perry. Department of Biochemistry, University of Utah College of...
0 downloads 0 Views 2MB Size
Pressure Device for Displacement of Sucrose Gradients iherman R. Dickman and VerI J. Perry leprtment of Biochemistry, University of Utah College of Medicine, Sah Lake City, Utah 84112

TECHNIQUES FOR MEASUREMENT Of Samples separated by Zone velocity centrifugation may be divided into two types: Punct u r e i n which the tube is pierced and the solution is collected into small tubes for subsequent analysis, or is led through a flow cell for optical monitoring. Noll (I)has discussed many of the problems involved in the use of puncture systems. Displacement-in which the solution is displaced from the bottom by a more dense solution. In the latter type procedures, the displacing solution is generally introduced into the system from a syringe which is operated by a gear drive. With the larger sizes of centrifuge tubes, the syringe must be filled and the system purged for each sample. Large glass syringes break easily under these conditions with attendant complications in sample recovery and calculation of S values. Gear drives of suitable accuracy and adaptability to different syringe sizes are quite expensive. For these reasons, a new system for displacement of the gradient solution has been devised. Nitrogen gas provides pulseless pressure which propels the displacing solution from a reservoir through tubing into the bottom of the centrifugetube. The apparatus contains no moving parts and is.readily adjustable under operatirig conditions. A photograph of the device and tube holder is presented in Figure 1.

Figure 1. Double hemisphere reservoir and centrifuge tube holder

FABRICATION The pressure vessel is made from two pieces of acrylic plastic, each measuring 2.500 X 4.675 X 4.675 inches. A hemisphere was hollowed out from each piece on a milling machine. The head of the mill was tilted to a 45" angle and locked. A rotary table was mounted on the milling machine, after which a fly cutter was adjusted to 2.437 inches which was equal to the 90" chord of the hemisphere. The plastic block was centered on the rotary table and secured. The mill was set to 12M) rpm, the table raised until the cutter was cutting 0.100 inch deep, then the rotary table was rotated 360". This manipulation commenced hollowing out the hemisphere. The table was then raised another 0.100 inch and the rotary table again rotated 360" and so on until the cutter had reached the depth described. The same procedure was then followed for the second block. The pieces are held together by %-inch bolts and wing nuts. The seal between the blocks was accomplished by milling a groove 0.215 inch wide and 0.100 inch deep in one block and inserting an 0 ring, Parker No. 2-341. A 0.055-inch (No. 54 drill) hole was drilled in the center bottom piece into which the discharge tube of polyethylene (0.d. 0.060 inch, i.d. 0.034 inch) was pressed. A hole was drilled in the center of the top piece and tapped to *I8-inchpipe size. This is used for fillingthe pressure vessel. It is plugged by a piece of threaded Teflon (Du Pont). Pressure is applied through a hole that is drilled and tapped to '/&nch pipe size. The blocks are easily separated for cleaning. The cost of plastic and fittings was $17.50 and fabrication time was approximately 10 hours. The tube holder is also constructed of acrylic plastic. A hole is bored in the lower portion to accommodate a particular size tube. The cap is constructed of brass or stainless steel and contains two stainless steel tubes; the inlet extends to the bottom of the tube; the outlet is flush with the bottom

(I) H. Noll, Anal. Biochem., 27, 130 (1969). 1302

'7

0.90

~

__

II

....

I .

10-30% linear sucrose gradient from centrifuge tube lycerol "4

-

no"sul 'l..".."lr

tained 0.2 mg/ml of yeast RNA. Flow rate 4.5 ml/nI n . Pressure of N2, 35 psi. Solici line (-), ex. perimental tracing

0.70 0 60

0.

0.

If the cap. The materials 1'01 md required 8 hours for fabr ic;

UP produced in a Britten-Roberts apparatus (2) such that the lighter portion was formed first and was conducted to the bottom of a polyallomer tube. The 30Z sucrose solution contained RNA. On the basis that a linear gradient had been formed in the tube, its displacement at a constant rate should result in a straight line of increasing absorbance. This is illustrated in Figure 2. Such data are highly reproducible. (2) R. J. Britten and R. 8. Roberts, Science, 131, 32 (1960).

ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970

Commercial 2-stage pressure regulators have been used to control the gas pressure. The seat should be leak-proof or a build-up of pressure will ensue. Ambient temperature should be maintained relatively constant (=t1") during a run. Flow rates are constant as long as sufficient Nz remains in the tank to register the required pressure on the gauge. Polyethylene tubing (0.060-inch 0.d. x 0.034-inch i d . ) is used to conduct the solutions. Where necessary, connections are made with pieces of 20-gauge needle. The rate of flow can be altered by variation in gas pressure. Variation in pressure in the range 20 to 40 psi resulted in efflux of 70% glycerol through tubing from the sphere of 4.8 to 9.2 ml/min, respectively. The flow rate was reduced in the complete system, which includes displacement of the gradient from the centrifuge tube and through the flow cell. Thus, 8 ml/min was obtained from the sphere with 35 psi, but this was reduced to 4.5 ml/min in the complete system. Although the device has not been tested with gradients of smaller volumes, we see no reason why equally satisfactory

results should not be achieved. It should also be applicable to puncture type systems in which the gradient is displaced from the bottom (3). One limitation that deserves mention occurs with 10-40 % sucrose gradients. With this solution, 70 glycerol mixes with the 40 sucrose. A clean junction can be obtained with 100% glycerol but this solution requires greater pressure for feasible flow rates than the system can handle.

x

ACKNOWLEDGMENT

We thank Helene Davis for her help in the evaluation experiments.

RECEIVED for review March 5,1970. Accepted June 18,1970. Support for this research was provided by United States Public Health Service Research Grant No. AM-00803. (3) M. K. Brakke, Anal. Biockem., 5,271 (1963).

Atomic Absorption Spectrometry for Direct Determination of Metals in Powders M. A. Coudert and J. M. Vergnaud Dkpartement de Chimie, Facultk des Sciences, Alger, Algbrie

SAMPLES FOR ATOMIC ABSORPTION analysis must usually be in some type of solution. Venghiatis, however, has mixed solid samples with flammable powders, the combustion of which resulted in vapors suitable for atomic absorption analysis (I). In another technique, the metal to be determined is adsorbed from solution into a flammable powder which is then burnt (2). The method presented here, which is quantitative and continuous, consists of a device feeding the powder at a constant rate inside the burner. The powder is then pushed into the flame by the gas mixture. EXPERIMENTAL Apparatus. A Techtron model AA-4 atomic absorption spectrometer was used. The burner was removed and replaced by a round one with a feeding device constructed by the authors (Figure 1). The powder first goes through the spiral conveyor from the hopper to the center burner channel and is moved up to the flame by the gaseous current. The screw rod which is rotated by a motor is kept vibrating in order to improve the flow of the powder and its regularity. A Perkin-Elmer palladium hollow cathode lamp was used. Lamp current was 20 mA. Air flow rate was 4 liters/minute and acetylene flow rate about 0.3 liter/minute (3). Reagents. The powders of the catalysts used were first diluted with calcium carbonate. Regardless of the metal concentration in the powder, it will then behave like pure calcium carbonate through the feeding device. The mass flow rate of the powder will remain constant. (1) A. A. Venghiatis, Ar. Absorption Newsleft., 6(1), (1967). ( 2 ) P. Maud and J. Robin, Bull. SOC.Chim. Fr., 1968,854. (3) M. A. Coudert and J. M. Vergnaud, C. R. Acad. Sci. Paris, Ser. C , 268, 1225 (1969).

I

i

Figure 1. Diagrammatic section of burner and feeding device for powders (1) burner, (2) hopper, (3) powder, (4) screw rod, (5) electric motor, (6) acetylene inlet, (7) air inlet, (8) capillary, (9) drain

tube Procedure. The adjustable prop of the burner and the feeding device for gases are kept in place; however, when powders are used, the capillary of the sprayer and the drain tube (for liquid not transformed to an aerosol) are closed. Damping of the galvanometer is maximum in order to avoid absorbance fluctuations. The amount of powder required for one measurement is about 1 gram-i.e., 10 mg of powder to analyze. The time required is about 10 seconds. Calibration curves were prepared for Pd on activated carbon, Pd on alumina, and the molecular sieve Siliporite for which the sodium had been exchanged for palladium. All materials had been previously diluted 100 times in calcium carbonate.

ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970

1303