117
V O L U M E 28, NO, 1, J A N U A R Y 1 9 5 6 Forty samples in the concentration range of 20 to 80 y of fluoride and pH range of 2.9 were titrated according to the analytical procedure outlined above. The fluoride content of each sample was then calculated in two manners: The actual volume of thorium nitrate required to titrate each sample was used, regardless of the pH of the final titration volume; and the volume of thorium nitrate was corrected for variation in pH of the final titration volume from the recommended 2.90 and used as the basis for calculation. Table I summarizes the comparative results obtained by the two methods of calculation. The mean per cent recoveries obtained by the two calculation procedures show excellent :tgreement. However, the range when no correction is made for variations in pH of the final titration volume is somewhat greater than that obtained when the suggested correction for pH variation is made. Comparison of the standard deviations obtained by the two calculation procedures indicates a marked increase in precision by correcting the volume of thorium nitrate used to a standard pH according to the proposed procedure. CORRECTIOS O F . w P . m E w FLUORIDE LEVEL
Table I.
Determination of Known Quantities of Sodium Fluoride Corrected for p H 40 20-80 99.7 93.8-103.7 2.8 2.83-2.94
ZTncorrected for pH 40
Number of samples Concentration range, y Mean recovery, Range of recovery. % Standard deviation, R Range of pH
20-80 100.4 92.5-107.8 7 7
2.83-2: 9;
upward or downward, until it intersects the ordinate representing a pH of 2.90. The actual volume of thorium nitrate which would have heen required by the given quantity of fluoride a t a pH of 2.90 is then determined at the vertical intersection of the point :it 2.90 and the abscissa. LITERATURE CITED
Chemists, Washington, D. C., “Official Methods of Analysis,” 7th ed., 1950. Remmert, L. F., Parks, T. D., Lau-rence,A. >I., and McBurney, E. H., ANAL.CHEM.25,450 (1953). Rowley, R. J., Grier, J. G., and Paraons, R. L., Ibid., 25, 1061 (1953). Smith, F. A., and Gardner, D. E., Arch. Biochem. 29, 311 (1950). Smith, F. A., and Gardner, D. E., J . Dental Research 30, 182 (1951). Smith, F. .I.,and Gardner, D. E.. U. S . Atomic Energy Commission, Bull. AECD-2161, 1948. Killard, H. H., and Winter, 0 . B.. ISD. ENG.C m x , ANAL. ED. 5 , 7 (1933). Williams, H. *4., AnaZ?/sf71,l i s (1946).
(1) hssoc. Offic. Agr.
(2)
In order to improve the reproducibility of the modified Williams titrat’ion on replicated samples, the titration volume of
(3)
thorium reagent required for a given sample must be correct’ed for the effect of variations on pH from 2.90 of the final tit’rat’ion solution. A graphic correct,ion may be made by reference to a family of curves such as 8hoJT.n in Figure 1. The point of intersection of the final pH and the observed volume of thorium is first located within this family of curves, and t’he point’ is then moved in a manner parallel with the family of curves, either
(5)
(4)
(6)
(7) (8)
RECEIVED f o r review June 13, 1955. Accepted October 17, 1955.
Velocity Barrier to Eliminate Absorption of Carbon Dioxide during Titrimetric Procedures ARTHUR M. CRESTFIELD Department of Physiological Chemistry, University of California School of Medicine, Berkeley, Calif.
Absorption of acidic and basic gases from laboratory air is a difficulty associated with titrations involving solutions of low buffer capacity. RIethods of enclosing the solution in an inert atmosphere are generally cumbersome and not adaptable to the routine treatment of many samples. A simple and convenient new method permits access to the titration vessel at all times. A flat stream of nitrogen is directed horizontally across the top of the titration vessel so as to form a seal of rapidly moving nitrogen through which the acidic and basic gases may not diffuse. This velocity barrier is useful in the assay of ribonuclease activity.
Nozzle
(J
Vessel
Figure 1.
D
---.
I
Titration
Lucite nitrogen nozzle
URISG the development of a micromethod for the quanti-
tative determination of ribonuclease based upon the acidic groups which are liberated from the substrate (ribonucleic acid), it became necesbar) to carry out titrations on a small scale (2 ml.) on solutions of low buffer capacity ( 0 . 3 ~eq. per pH unit), with the assurance that no absorption of acidic or basic gases from laboratory air would occur. Several arrangements for the use of nitrogen were tried. The most efficient and convenient arrangement was found to be a nozzle constructed from Lucite, as shown in Figure 1. This nozzle fixes the position of the titration vevel with respect to the stream of nitrogen. The stream of nitrogen functions in
two ways to prevent the absorption of laboratory gases: (1) An aspirator-type action removes the gases from between the liquid and the streaming nitrogen, replacing them with nitrogen and water vapor from the sample; and ( 2 ) a velocity barrier action decreases the probability that a molecule of carbon dioxide, for example, could diffuse through the rapidly moving stream and into the titration vessel. The efficiency of the first action depends upon the velocity of the stream and its orientation with respect to the titration vessel. For this reason the titration vessel is shown to have a fixed position with respect to the efferent
ANALYTICAL CHEMISTRY
118
nitrogen (Figure 1). The secoiid action depends upon the thickness and velocity of the moving stream. A flow rate of 7 to 8 cubic feet per hour has been found to be sufficient.
r\
70
1
I
Thirty milligrams of ribonucleic acid are dissolved in 2 nil. of 0.251V sodium sulfate, for optimal ionic strength, and the solution is ad'usted to pH 7.50 by means of a micrometer syringe buret which delivers 0.01N sodium hydroxide. The pH is measured on a Beckman Model G pH meter with shielded external electrodes, The solution is stirred magnetically and absorption of carbon dioxide is prevented by means of the nitrogen barrier applied across the top of the vessel. Ribonuclease (2 to 10 y ) is added to the adjusted nucleic acid solution under the nitrogen barrier and the pH is brought to 7.50 and maintained by continuous addition of 0.01N sodium hydroxide from the syringe buret. After the first 4 minutes of reaction, during which there is no reproducible relationship between acidic groups formed and the amount of enzyme added, the titer for any interval up to an hour is directly proportional to the amount of enzyme present, as shown in Figure 3. Errors due to the setting precision of the meter may be reduced by plotting the titer every 2 minutes and drawing the best straight line through the data for a period of 10 to 15 minutes. A total time of 20 minutes per sample is adequate and after the titration vessel has been emptied by aspiration and washing, the next sample may be started in the 8ame vessel.
1
2
3
4
Time in Minutes Figure 2.
Action of velocity barrier
Figure 2 demonstrates the action of the velocity barrier. If 2 ml. of distilled water are adjusted to p H 8 by the use of 0.01N sodium hydroxide and stirred with no precautions against the uptake of carbon dioxide from the air, a rapid decrease in pH can be shown to occur. However, when the nitrogen barrier is placed in the system the pH remains constant. cs
cc
06
08
of Rlbonucleose Figure 4. Relationship between titer, after 24 hours at 50' C., and quantity of ribonuclease 2 oc
Open circles represent one series of hydrolyses and closed circles are for second series started immediately after first series
In order to conserve ribonucleic acid and to increase the aesa,v sensitivity the enzymatic reaction may be carried out with 5 mg. of nucleic acid and 0.05 to 0.8 y of ribonuclease in 2 ml. of 0.25N sodium sulfate incubated a t 50' for 24 hours by transferring a portion of the adjusted solution to small screw cap vials of N1.9-ml. capacity. The vials are sealed, free of air, with a disk of polyethylene which is held in place by the cap. Titration of an aliquot of each sample under the nitrogen barrier a t a suitable interval of time yields the standard curve which is given in Figure 4.
of R,bonucIeose
These procedures have been used for the assay of ribonuclease activity in samples from the column fractionation method of Hirs, Moore, and Stein (1) in various cellular fractions from pancreas, and in certain studies of inactivation of ribonuclease.
Figure 3. Relationship between titer and quantity of ribonuclease A. B. C.
Titers between 4 a n d 16 minutes Interval of 4 t o 34 minutes Interval of 4 t o 60 minutes
LITERATURE CITED
(1) Him C . H. W., Moore,
S.,and Stein, W. H., J . Bid. Cham. 200,
493 (1953).
The merits of this type of velocity barrier under typical experimental conditions are illustrated in the following analytical procedures for the assay of ribonuclease action.
RECEIVEDfor review J u n e 22, 1954. Accepted October 3, 1955. Work supported in part b y Grant-in-aid RG2496, U. S. Public Health Service, and by Cancer Research Funds of the University of California t o Frank R o r t h inoton Allen.
