Syringe Pipets AUGUSTKROGH,Laboratory of Zo8physiology, University of Copenhagen, Copenhagen, Denmark
I
N SEVERAL cases the author has been confronted in the laboratory with the problem of measuring off small volumes of fluids which must not come in contact with aire. g., barium hydroxide for microtitration-and this led to an attempt to use the record syringes well known in medical technic. The somewhat surprising result of these attempts was that a syringe with the piston working between two fixed stops was not only much quicker than a pipet of the same volume, but also definitely more accurate. On the basis of this observation several types of measuring syringes or syringe pipets have been constructed (1). The first step was to discard the metal piston which limits the applicability to relatively few solutions, An all-glass syringe of 2-cc. capacity was found suitable and was mounted as shown in Figure 1 with an adjustable stop and a cannula which is generally of stainless steel, but can be made of glass or o t h e r s u i t a b l e material. This 5 syringe will measure any desired volume between 0.1 and 1.5 ml., and an accuracy of *0.0001 ml. is easily obtained. With the very best syringes and the utmost care the error can be reduced to +0.00002 ml. With pipets of similar size it is very difficult to obtain an accuracy of 0.001 ml. and usually the error will be even larger. The correction for temperature is an important consideration when the accuracy is of the order of 0.0001 ml. Within the temperature interval from 15" to 25" C. the temperature correction of these syringes is negligible (less than 0.0001 ml.), because the steel rods carrying the adjustable stop were selected to compensate the change in the glass parts. Somewhat larger syringe pipets being desirable for several purposes, while a somewhat FIGURE1 lower standard of precision is in most cases sufficient, a syringepipet of simpler design was constructed as shown in Figure 2. This is relatively shorter and has no separate cannula, but ends in a capillary tube which can be drawn out as desired and is fairly easy to replace. Syringe pipets are made in lo-, 5-, 2-, and 1-ml. sizes and by means of the screw a t the top can be adjusted to deliver any volume between 10 and 0.1 ml. The adjustment and calibration of a syringe pipet are performed by delivering and weighing distilled water, and it has been found convenient to use one weighing bottle in each pan of the balance into which the volume is delivered aIternately. On a modern damped balance one has then only to move a single weight back and forth. The 10-ml. syringe is exceptionally accurate, while the 1ml. syringe is of average accuracy. The pistons fit so snugly that the increase in resistance produced by pressing the cylind e r between two fingers is generally very pronounced. This makes the fitting of the frame for the top screw difficult, but on the other hand i t makes the accuracy of the measurement quite independent of the viscosity of the fluid. A definite source of error is due to the fact that the lower surface of the piston is not as a rule absolutely perpendicular to the axis. If the piston is turned when in the bottom posi.tion, a meniscus in the cannula is seen to move slightly for-
ward and backward. In accurate measurements the piston therefore must not be turned, the square top of the piston being kept parallel to the square top of the cylinder. The temperature sensitivity of these syringes is greater than that of the precision syringes, but still so small that it can in most cases be disregarded. For instance, a syringe delivering 5.000 ml. a t 20" C. was reduced to 4.996 ml. a t 4" C., while another was reduced from 10.000 to 9.994 ml.
TABLEI. CALIBRATIONS SYRINQ A~
Water delivered
Difference from mean
Grams
9.9680 9.9690 9.9684 9.9683 9.9683 9.9689 Mean 9.9685
SYRINQB B Water Difference delivered from mean Grams
-0.0005
+O ,0005 -0.0001 -0.0002 -0.0002 +0.0004
0.9966 0.9967 0.9965 0.9966 0.9962 0.9963 0.9964 Mean 0.9965
f0.0001
+0.0002 $0:0001 -0.0003
-0.0002 -0.0001
When the same syringe has to be used for different solutions in succession, the dead space from the tip of the cannula to the bottom position of the piston is a drawback. Most of the fluid in it can be removed as a spray of fine drops by rapidly moving the piston up and down. After using a syringe for 20 per cent hydrochloric acid, the author has emptied it in this way and washed twice with about one-third volume of water which was removed from the dead space in the same manner; he has then taken in one-third volume of water to fill up the dead space and thereupon used the syringe for water which was shown by the addition of an indicator to have a pH of 5.5. The acid left in the syringe raised the p H to about 5. The total time for the washing was just under 1 C minute. f When a fluid is sucked up too rapidly, so that a considerable force must be used to overcome the resistance in the cannula, bubbles of air are likely to become liberated in the syringe. S m a l l b u b b l e s which remain in the syringe - - do not interfere with the accuracy as measured by the delivery. It has been pointed out as a possible source of error in measuring solutions that water wiIl evaporate from the upper part of the piston when this becomes exposed in filling the syringe and the concentraFIGURE 2 tion of the content is thereby gradually raised. It is true, of course, that a little fluid is transported along the piston and becomes concentrated even to dryness in the top of the cylinder. When a syringe has been used for some days or weeks, a noticeable amount of salt may accumulate in this place. Fluid is carried, however, from within outward only, by reason of the capillarity between the piston and syringe. When the piston is pushed down, even after having become completely dry by evaporation, fresh fluid is sucked in by capillarity from below and no mixture takes place through a capillary space 1.5 to 3 cm. in length. Even when the space between the collar of the syringe and the piston is filled with fluid, admixture of this fluid with that measured off below the piston is negligibly small, as shown by the following experiment:
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March 15, 1935
ANALYTICAL EDITION
The space between the collar and the piston in a 2-ml. syringe was filled with 20 per cent hydrochloric acid. The syringe was filled and emptied 20 times in 2.5 minutes from 10 ml. of distilled water in a basin. An indicator would show the addition of 1 volume of the acid in 100,000 volumes of water, The acidification observed was 1 in 50,000. A similar experiment with a 10-ml. syringe filled 20 times in 5 minutes from 20 ml. showed no detectable acidification. Syringes which are not used constantly should be washed out with water to prevent the piston’s sticking fast. T h e author has found it possible, however, in such cases to loosen it by treatment with hot water. It is possible with syringe pipets to work several times more rapidly and at the same time with much greater pre--
1 The Laboratory of Zoophysiology is prepared to supply precision syringes mounted with a n ordinary stainless-steel cannula at a price of 25 Danish kroner; glass cannula, 2 Danish kroner; and oannula made entirely of stainless steel, 12 Danish kroner. The prices of the ordinary syringe pipets here described are: for 1 and 2 ml., 10 Danish kroner; 5 ml., 12 Danish kroner; 10 ml., 15 Danish kroner, st the laboratory. The 10-ml. pipet is provided with an extra movable stop on one of the guide roda for preparing definite mixtures.
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cision than with pipets of the same volume, but the higher price will prevent their being generally used.’ They have been found especially suitable for the following purposes: Serial measurements of the same fluid, for which purpose it i8 often advantageous that the syringe can be easily arranged to deliver any desired volume. Measurement of viscous fluids. Delivery of fluids which must not come into contact with air. All measurements in which more precision is desired than can be obtained by means of ordinary pipets. (The author has made many determinations of specific gravity of solutions by measuring with a syringe and weighing as described for the calibration, and has used syringes in combination with a small buret for precision titrations.) In many cases chemical reactions can with advantage be arranged to take place in syringes, especially when it is essential to exclude atmospheric air. LITERATURE CITED (1) Krogh and Keys, J . Chem. SOC.,1931, 2436. R E C ~ I Y ESeptember D 18, 1934.
Precise Determination of Oxygen in Water by Syringe Pipets AUGUSTKROGH,Laboratory of Zotiphysiology, University of Copenhagen, Copenhagen, Denmark
0
XYGEN titration in water is carried out according to Winkler by adding a known small amount of alkali containing potassium iodide (reagent I) and then a
corresponding volume of manganous chloride (reagent 11). Manganous hydroxide is thereby formed as a voluminous precipitate which absorbs the free oxygen. After acidification with hydr&hloric acid, iodine equivalent to the oxygen is liberated and is titrated with thiosulfate. It is customary to utilize water samples of 100 ml. or more, measured in glassstoppered bottles. For years in the author’s laboratory a micro-Winkler method has been used, in 7- to 15-ml. bottles titrating 5-ml. samples from a 2-ml. buret with 0.005 N thiosulfate. A micromethod described b y v a n Dam (3) utilizes a 1-ml. syringe and a Rehberg microburet. In oxygen determinations as hitherto performed there are two main s o u r c e s of e r r o r which should be eliminated. One is the diffusion of oxygen that takes place whenever the sample is not in equilibrium with the atmosphere, and the other is the uncertainty of the correction for oxygen d i s s o l v e d i n the reagents. These sources of error are completely eliminated in the procedure d e s c r i b e d below.
n
The water sample is collected and t h e reaction carried out in a 10-ml. s y r i n g e pipet, slightly modified as shown in Figure 1, by arranging a fixed stop on one of the guide rods. This stop
is placed so as to correspond as nearly as possible to 10.00 ml. and the screw stop is set a t a definite distance above, corresponding to about 0.1 ml. more. For a determination, the dead space of the syringe is first washed out and filled with reagent I, care being taken that no air bubbles remain in the syringe or cannula. As much water as possible is removed from the syringe before taking in the reagent, but since this cannot be done completely the reagent in the bottle becomes slowly diluted. The tube through which the reagent is taken in therefore reaches only halfway down the bottle and the rest of the content is discarded. As pointed out by Yoder and Dresher (4), rubber may give off sulfur and a ground joint in the top of the bottle will be preferable to the rubber stopper shown. The sample of water is drawn in slowly until the top of the piston comes against the fixed stop, and finally the piston is turned free of the stop and reagent I1 drawn in quickly. The tip of the cannula is wiped off between each intake and is finally closed by means of a small piece of rubber tubing with a glass stopper. It is convenient to put this on filled with water. The syringe is well shaken and laced with the tip upward, so that the precipitate collects on t\e piston. The syringe should be left in this position for 0.5 hour to allow the precipitate to settle. If left longer a firm clot of precipitate may form in the cannula, which may have to be pushed down by means of a fine wire before the syringe can be emptied. It is now possible to drive out almost all the fluid without losing any of the precipitate, to take hydrochloric acid into the syringe, and titrate in a small volume of fluid, but the manganous hydroxide is not sufficiently insoluble to make this procedure advisable (except perhaps when the amount of oxygen is very small). It is therefore necessary to titrate on the total volume of fluid. A suitable quantity of hydrochloric acid (0.4 ml., 20 per cent) is measured off into a flat-bottomed test tube of 30 X 100 mm. (as used for Hagedorn blood-sugar determination). Some fluid from the syringe is driven out into this and taken back again, so that the main reaction takes place in the syringe. The content of the syringe can be titrated from a 2-ml. buret with 0.01 N thiosulfate and, in comparative determinations an accuracy of 0.005 ml., corresponding t o about 0.04 ml. of oxygen per liter, is easily obtained. The author has endeavored t o obtain a definite improvement in the accuracy of this determination and has come across several sources of error which can be overcome by a suitable technic.
