included for both powdered and molded PE-1009. The crystallinity figures in Table I appear reasonably close to one another especially if one considers the experimental difficulties. The data are also similar in magnitude to that for the molding grade resin PE-1009. The crystallinit'y of the Poly-Em 10 latex appears to be a little low but this material may not be a t all typical due to the shielding effect of the potassium ions. This effect, is removed akogether in the coagulated materials as it is washed out during the process. Considerable discrepancies are seen in the crystallhe size figures in Table I. The crystallite size of the freeze-dried powder samples appear extremely low with respect to the other forms. A sample of PE-1009 powder was run for comparison purposes and it is apparent that the sample state has some effect on this measurement. A possible explanation for this effect is that the bulk density of the powder is about half that of the molded solid. This leads t'o a lower absorptivity for the powder. The x-ray beam will, therefore, penetrate about twice as far into the powdered sample and results in the so-called transparent sample effect ( 4 ) . The x-rays which are re-
flected from deep in the sample may appear a t some place on the small angle side of the detector. The result is an anomalous broadening of the band and probably a lower intensity a t the center of the peak. In fact, calculations of effective sample thickness indicate that the determination of crystallite size by this technique may not be a t all accurate for organic materials of this type as the thickness "seen" by the radiation is about 10 to 20 times that of the silicon powder sample that n a s used as a standard for this method. Figure 4 illustrates the comparison between three liquid systems; the first, a crystalline polymer emulsion; the second, an amorphous polymer emulsion; and the third, water. It is interesting to note that the polystyrene latex has a broad reflection a t approximately the same angle as the amorphous region in polyethylene. This is logical since amorphous phases of each polymer are essentially a disordered carbon-hydrogen matrix. CONCLUSION
It is felt that a technique has been developed which offers a means for evaluating the crystallinity of polymers in an emulsion or fluid system. Calcu-
lations on the systems studied indicate that the degree and type of crystallinity of the polyethylene particles in emulsion is very similar to that of the isolated materials and also similar to that of a standard solid molding grade polyethylene. It will be interesting to extend this type of measurement to other crystalline polymer emulsion system.;. ACKNOWLEDGMENT
The authors express their appreciation to Sam Baker for carrying out many of the experimental measurements. LITERATURE CITED
(1) Aggarwal, S. L., Tilley, G. P., J . Polymer Sei. 18, 17 (1955). (2) Bunn, C. W., Trans. Faraday SOC.35,
482 (1939).
( 3 ) Helin, A. F., Mantell, G. J., Stryker, H. K., J . A p p l . Polymer Sci. in press. (4)Parrish, W., "Advances in X-Ray
Diffraqtometry and X-Ray Spectrography, p. 63, Centrex Publishing Co., Eindhoven. 1962. (5) Scherrer,' P., Sachr. Ges. Was. Gottingen 1918, 96-100. (6) Taylor, H. S., Glasstone, S., "Treatise on Physical Chemistry," Vol. 2, 3rd Ed., D. 549. Tan Kostrand. Yew York. 1951.'
Sixteenth hlidwest Chemistry Conference, Kansas City, hIo., Sovember 20, 1964.
Improved Method for Microtitration of Fatty Acids Thomas F. Kelley, Bio-Research Institute and Bio-Research Consultants, Inc., Cambridge, Mass.
icrotitration of fatty acids released
M by the action of enzymes, such as
lipases, is frequently used as an assay method. It is a more accurate and direct measure of enzyme activit'y than turbidity measurement or glycerol assay. Microtitration is, however, a time consuming and tedious technique. I n the procedure generally employed, fatty acids are extracted into an organic solvent to exclude water soluble acids ( I , 2 ) , and titrated with dilute aqueous NaOH. Indicator dissolved in 95% ethanol is added. This two-phase system is mixed during the titration by bubbling prepurified nitrogen through it which also acts to exclude atmospheric carbon dioxide. The mixing of the two phases results in a translucent emulsion which acts to mask the end-point color. Thus, t,he titrat,ion must be stopped frequently to check progress toward the end point. Overtitration is a constant danger because of the long time lag in the reaction due to the existence of two phases. &tempts to increase t'he rate of react,ion by accelerating the nitrogen bubbling only result in rapid evaporation of the solvent, and the consequent cooling results in the coating of the tube with a layer of condensed water. The 1078
ANALYTICAL CHEMISTRY
whole procedure is, therefore, very tedious. A microtitration system is described below which, because it operates in a single-phase system, Overcomes these difficulties and decreases the time required for a set of titrations to less than one fourth. EXPERIMENTAL
Reagents. T l T R A N T . ii stock titrant solution is prepared by diluting 15 ml. of tetrabutylammonium hydroxide titrant in methanol (Eastman No. 7774) to 100 ml. with methanol. Fresh working solution (about 0.01N) is prepared daily by diluting the stock solution 1 to 10 with methanol. INDICATOR SOLETIOX. Phenol red indicator solution is prepared in a manner similar to that described by Mosinger ( 3 ) . One milliliter of a 1% aqueous phenol red solution is diluted with 99 ml. of absolute ethanol and then added to 200 ml. of heptane. A concentration curve was plotted for this solution titrated to end point. h quantity of 0.4 ml. of the indicator solution lay in the flat part of the titration curve, and this concentration was chosen to minimize pipetting errors.
02 141
For ease in manipulation, the stock solution is diluted 2.5-fold with heptane so that 1-ml. portions can be used. FATTY A C I D STANDARD. Ninety-two milligrams of oleic acid (99% pure by gas chromatography) are dissolved in 10 ml. of ether. This solution is then added to 50 ml. of 95% ethanol. The alcohol-ether solution is rapidly mixed with about 250 ml. of water, to yield an emulsion which is stable for four to six weeks under refrigeration. The final volume is adjusted to 325 ml. with water t o yield a solution which contains 1 peq./ml. Procedure. F a t t y acids from both enzyme reaction mixtures and samples of standard solution are extracted with isopropyl alcohol, heptane, l N H2S04 (40: 10: 1 v/v) according to Dole ( I ) . Three milliliters each of water and heptane are then added to separate the mixture into two phases. Three milliliters of the top layer are transferred to 18- X 150-mm. tubes. Indicator solution (1.0 ml.) is added, and the solution is titrated to a purple end point with O.Olhr tetrabutylammonium hydroxide. The color changes from a lemon yellow to an orchid (purple). For manual microtitration, a Rehberg microburet of 0.125-ml. capacity with 1-pl. divisions is used. The solution is stirred by bubbling NZ through it.
AUTOMATEDMICROTITRATION PROThe titration is carried out in cuvettes in the well of a colorimeter (Bausch & Lomb Spectronic 20). Titrant is added to the cuvette through a 20-em. length of hypodermic needle (18 gauge) connected with 0.99-mm. plastic intravenous tubing to a buret with a numerical read-out. The tip of the needle is constricted to prevent wash-out of the titrant. Either a digital-type microburet or a syringe microburet with a micrometer gauge can be used. The buret is driven by a small, slow-speed synchronous motor. Any motor (Haydon Co., Torrington, Conn., Hurst Co., Princeton, Ind.) which stops rapidly due to inertia or which incorporates an internal brake can be used. The speed of the motor is selected, depending on the particular buret chosen t o deliver titrant a t a rate of about 1 to 2 pl. per second. An existing motorized buret is not acceptable because at low speed it is sluggish in starting and continues to drive after power cut-off. Adequate mixing to prevent overtitration is the most critical factor in the system. Bubbling NP1which works very efficiently with manual titration, causes instability by interfering with the light path and thus cannot be used. Mixing can be achieved by making the hypodermic needle tube which delivers the titrant act as a vibrating reed. The hypodermic tube is attached t o a small electric vibrator (Type BU, Potter and Brumfield, Princeton, Ind.) by means of epoxy glue. The degree of agitation is controlled by operating the vibrator through a Powerstat voltage regulator. The feedback loop is most simply established by feeding the output of the
Table I.
CEDURE.
Fatty acid added to extraction mixture, req. 0
0.25 0.5 1 .o
2.0 4 Each titrant.
Replicate Values Achieved with Automatic Titrator Av.
1
5.0 15.8 26.2 50.8 98.0 scale division
Numerical micrometer difference Av. of 4 sets from mean, readout of syringe bureta 2 3 4 of titrations % 6.2 5.0 5.2 5.35 8.0 16.2 16.8 17.0 16,45 2.7 2 4 28.2 27.2 27.8 27.35 51.2 51.0 49.0 50 50 1 5 99.0 98.0 100.0 98.75 0 8 of micrometer gauge represented delivery of 2.5 p l . of 0.005,V
colorimeter in parallel through a meter relay (Simpson No. 29XA, Simpson Electric Co., Chicago, Ill.) with a range selected to match the particular colorimeter. The motorized buret is driven through a general purpose relay, wired normally closed, which is opened when the meter relay closes. This systemis pictorially diagrammed in Figure 1. Motors, meters, and relays are generally available through industrial electronics distributors. Operation of this system is as follows: The sample (3.0 ml.) and indicator (1.0 ml.) are added to a cuvette which is placed in the colorimeter. The vibrating reed is positioned out of the light path and started, the colorimeter is set at 570 mp. and zeroed, and the circuit driving the buret motor is closed, starting the titration. When the ammeter needle in the meter relay reaches its set end point, the &volt circuit is closed, activating the holding coil of the general purpose relay and stopping the drive.
YH
RESULTS AND DISCUSSION
A linear relationship was found t o exist between concentration of fatty acid titrated and the amount of titrant required. The method was used to measure from 0 and 3 keq. of fatty acid, but the range could be extended by increasing the concentration of the titrant. Repeatability of the automatic titration is shown in Table I. Four different sets of standards were extracted and titrated as described. Agreement between duplicate samples was excellen€ with the exception of the Idank tubes. Duplicate values for manual titrations agreed to within +2.5y0 of their mean. Although a Rehberg microburet was used for the manual titrations, any buret of accuracy to 0.2 pl. would also be suitable. Bubbling K2 was used to stir the solution, but any other means would also be satisfactory. Atmospheric CO, does not influence the titration because there is no aqueous phase. Because the present technique operates in a single-phase system, the reaction is instantaneous. The solution remains clear while being stirred, and thus the titration need not be interrupted. The color change is dramatic. Whereas a set of 40 titrations formerly required 4 hours to perform, they now can be finished manually in less than 1 hour. Pipetting is now the major time limiting factor. Additional saving in time and increased accuracy is achieved with the automated system. The apparatus is being manufactured by Elec Ser Products, Inc., Maywood,
N. J. 07607.
I
h
U
Figure 1. tion system A. B. C.
D. E. F.
G. H. I.
c
B
A Pictoral
diagram
of
1
ACKNOWLEDGMENT
1
6V
115VAC
automatic microtitra-
Colorimeter Colorimeter output Meter reloy General purpose relay Low-speed drive motor Buret with numerical read-out Plastic intravenous tubing Vibrator 20-cm. length of 1 &gauge hypodermic needle
The author thanks Vincent Korkus for valuable technical assistance. LITERATURE CITED
(1) Dole, V. P., J. Clzn. Invest. 35, 150 (1956). ( 2 ) Gordan, R. S., Zbzd., 36, 810 (1957). (3) Mosinger, F., J . Lzpzd Res. 6 , 157 (1965). Work supported in part by USPHS Research Grant No. HE-07149 from the National Heart Institute and by BioResearch Consultants, Inc. VOL. 37, NO. 8, JULY 1965
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