A Method for the Determination of the Osmotic Pressure of Biological Fluids

BY Einar Blegen and P. Brandt Rehberg

(From the Zoophysiological Laboratory, University of Copenhagen) (With I figure in the text)

Though the introduction of the thermocouple method of Hil l (1930) has made it possible to determine the osmotic pressure of very small samples of biological fluids, the method requires much training and a rather expensive outfit. The method here to be described was worked out in the hope of finding a method, which though using only amounts of fluid, which are available in most biological work, would not require too much outfit.

The principle of the method is to use the initial inflow of water from one solution through a semipermeable membrane into another solution, caused by the difference in osmotic pressure between the two solutions, as a measure of this difference. As semipermeable membranes we have used collodion tubes prepared according to the method described by Blegen. The ordinary collodion tubes prepared according to the method of Krogh (1922) are so permeable to salts that the osmotic pressure of these is only partly effective and rapidly decreasing be- cause of the diffusion of the salts. By drying the tubes for an hour or more it is however possible to prepare tubes, which are so impermeable that the osmotic pressure of the salts becomes effective and because of the slow diffusion of the salts stays constant for a considerable time. By further preparation of the tubes as described in the paper by Blegen it is possible to prepare tubes which are practically ideal.

The tubes we use have been made on glass tubes with an outer diameter of 3-4 mm., they have a surface of 2-4 cm2 and hold about 0,2-0,4 cc. of fluid. They are mounted on a short piece of glass tubing

1 Received for publication June ~ q t h . , 1938.

A METHOD FOR THE DETERMINATION OF THE OSMOTIC PRESSURE ETC. 41

the outer diameter of which is so that the collodion tube will just slip Over it. A piece of rubber tubing is rolled down over the upper part of the collodium tube, so that the connection between the collodion tube and the glass tube is completely covered, a piece of copper wire may be used as additional security. The rubber tubing shall have a free length of a t least I cm. This end of the rubber tube is used to connect the collodion tube with a capillary glass tube in which the movement of the water is measured. This tube shall have a bore between 0.2 and 0.5 mm. It is bent at a right angle with one leg which is only a few centimeters long, while the other is about 15 cm. (See fig.). In selecting the tubes we have used the water permeability test according

@==-

a = rubber tubing b = short glass tube c = collodion tube

to Zsigmondy and Bachmann (1918). Only tubes which have a permeability number of more than 40.000 minutes can be used and only tubes with this low permeability are mounted. The final test is however that the tubes shall show a constant rate of water movement for a sufficient time when tested in the way to be described.

The tubes are used in the following way: a tube with the attached rubber tube is filled completely with the unknown solution. The collodion tube is then connected with the capillary glass tube, care being taken to avoid air bubbles. The rubber tubing is pushed slowly over the short end of the capillary tube so as to avoid undue pressure in the collodion tube. The rubber tubing is pushed as far over the glass tube as possible and is then withdrawn a little, so that the meniscus of water is some centimeters from the end of the tube.

42 EINAR BLEGEN AND P. BRANDT REHBERG

If the collodion tube is now placed in a solution of sodium chloride the osmotic pressure of which is somewhat higher than that of the solution in the collodion tube, it will be observed that the meniscus after the lapse of a few minutes begins to move. After a few more minutes the rate of movement gets constant - if the tube is usable. If the capillary tube is provided with a paper scale with marks ior each centimeter it is possible to determine the time it takes for the meniscus to move from mark to mark. In our determinations we have however observed the movement of the meniscus by means of a microscope with micrometer eyepiece. As it is necessary to follow the meniscus over several runs along the scale it is best to arrange the capillary tube so that it can be moved parallel with the front of the microscope. As container for the outer solution of known composition we have used a cylinder, which holds about IOO cc. I t is closed with a stopper in which is an opening about the double diameter of the collodion tube, which dips down into the fluid through this opening. The fluid shall fill the cylinder so completely that it presents a meniscus in the opening. In this way the surface is kept at a minimum and the evaporation is small, but it is necessary to fill the cylinder with fresh solution from a stock bottle every day. If the temperature of the room is fairly constant, the temperature of the cylinder will only vary slightly during a deter- mination, but if larger temperature differences are met with, it may however be necessary to place the cylinder in a larger water bath.

In order to be able to use the tube it is necessary that the rate of the meniscus is constant for a considerable lenght of time. As an example of the behaviour of these membranes the following series of measurements with same solutions may be used:

I . 11/12 2. 12/12 3. 14112 4. 14/12

15 ,, : 101 ,, 25 ,, : 9 6 ,, 15 ,, :97 ,. 10 ,. : 9 3 ,, 8 min. : IOO sec. 16 min. : 91 sec. 10 min. : gg sec. 7 min. : 93 sec.

24 ., : 100 .. 40 ,, : 9 4 ,, 17 ,. :97 ., I3 : 9 2 ,, 30 ,, : 99 ., 48 ,, : 9 8 ,, 26 :97 , I 50 ,, : 9 3 ,,

70 ., : 9 9 ,, The time given in minutes is the time elapsed since the tube was placed in

the outside solution. The time given in seconds is the time it took for the meniscus to cover the scale.

It will be seen that the results in the individual tests show that the meniscus moves at a constant rate for up to 70 minutes. If the different tests are compared it will be seen that the rate may vary from day to day and even within the same day. Because of this we prefer to alternate constantly between known and unknown solutions.

To determine the osmotic pressure of an unknown solution we proceed in the following way: we determine the number of seconds

A METHOD FOR THE DETERMINATION OF THE OSMOTIC PRESSURE ETC. 43

needed for the meniscus to move the lenght of the scale, when the tube is filled with a solution of sodium chloride of known concentration f. i. 0.900%, while the cylinder is filled with a solution which is somewhat more concentrated f. i. 1.1000,~. 10 minutes are allowed for a steady state to develop before the time is taken by means of a stopwatch and two or more readings are averaged. When this time has been determined the tube is rinsed with the unknown solution, filled with it and the time it takes for the meniscus to cover the same distance noted and the procedure is next repeated with the known solution again - if a series of determinations has to be done one goes on to the next unknown and proceeds in the same way alternating between known and unknown solutions.

Example: As outer solution is used 1.110% NaC1. Known solution: o,goo% NaC1. Unknown solution: serum. Readings started 18 minutes after the tube was placed in the outer solution.

I) known: 62 sec., 2) serum: 71 sec., 3) known: 63 sec., 4) serum: 70 sec., 5) known: 63 sec.

The principle of the calculation is now that the rate of the movement of the meniscus is proportional to the difference in osmotic pressure of the two solutions or as the time is the reciprocal of the rate, the difference must be inversely proportional with the time. The result is expressed in the concentration of sodium chloride with the equivalent osmotic pressure. When the meniscus with a concentration difference of 0.210% moves along the scale in 62 seconds while it in the case of the unknown takes 71 seconds, then the difference in osmotic pressure between the unknown and the outer solution must be 62/,1 x 0.210% or o.1810/,. When alternating fillings of known and unknown solutions are used the results from two determinations on the known solution are incorporated in the calculation. From the example given we get from the first three determinations that the unknown must have an

x 0.210% or 0.9250//;, osmotic pressure equal to 1.110% - - 62 + 63 2 x 71

x 0.210 63+63 NaC1. From the three last determinationsweget 1.110% -- 2x70

or 0.921% NaC1. As examples of the accuracy of the method the following deter-

minations of known solutions of sodium chloride may serve: I) 0.950% determined to: 0.9524.954 -0,954 -0.953 -0.950

-0.958 average: 0.954, mean error on the single determination: 0.0025% NaC1.

2) 0.857% determined to: 0.861 -0.856 -0.859 -0.863. Average: 0.860%.

44 EINAR BLEGEN AND P. BRANDT REHBERG

3) 0.7joX determined to: 0.7j2 -0.7j4 -0.748 -0.748. Average:

4) o.goo% determined to: 0.900 -0.902 -0.900 -0.902. Average:

The re;ults show that the concentration of sodium chloride solutions may be determined with great accuracy by this method, the largest deviation from the known result in these determinations being less than 1% in the single determination. One might however doubt whether results obtained on solutions in which easily diffusing non-electrolytes like urea are responsible for a large part of the osmotic pressure are reliable and the same may be asked for mixtures of salts and solutions containing protein. We have found however in a large series of deter- minations on serum that the results by this niethod are of the same order of magnitude as the results obtained by the Hi l l method. In a series of 58 double determinations on blood serum we have found the standard deviation of the method on the single determination to be 0.0038% NaCl or about 0.4%.

In order to test the method further we have in a few cases made simultaneous determinations with this method and with the vapour pressure method 2.

The results were expressed in equivalent normal NaCl solutions:

0.751 ?A.

0.901 yo.

Solution tested

I. sample of sea water .......

2. sample of dilute urine ....

3. sample of urine.. ........

4. sample of serum from uraemic patient .........

Present method

0.151 n 0.149 n 0.150 n 0.150 n 0.049 n 0.049 n 0.050 n 0.049 n 0.049 n 0.217 n 0.218 n 0.218 n 0.218 n

0.177 n

Vapour pressure method

0.154 n 0.149 n ___- 0.154 n 0.152 n 0.049 n 0.049 n

0.049 n (0.230 n) 0.218 n

0.2 24/11

0.179 n

These results though few show that the osmotic pressure found by this method is very close to that found by the vapour pressure method.

2 The determinations with the vapour pressure method were kindly car- ried out by Dr. R. Conklin.

A 3fETIIOD FOR THE DETERMINATION O F 'THE OSMOTIC PRESSURE ETC. 45

With more concentrated urine we have encountered some difficulty because concentrated urine in some way affects the permeability of the membrane, probably because of adsorption of substances to the mem- brane. Even then it is possible however to get constant results by using the method of alternating between known and unknown solutions. As example may serve:

Outside solution: 2.053% NaC1, known solution: 1.02776. (The tube used was very little permeable so that a large difference between the two solutions was used.) Unknown solution: urine.

I ) known: 68.5 sec., 2) urine: 90.9 sec., 3) known: 69,8 sec., 4) known: 66.7 sec., 5) urine: 91,6 sec., 6) known: 72.3 sec., 7) urine: 95.2 sec., 8) known: 73.6 sec. (Between 3 and 4 was a pause of 2 hours.)

I t will be seen that the tube gets less and less permeable so that the time increases. Nevertheless the results obtained by using the determinations on the known solutions on both side of the individual determinations on the urine we get the following three results: 1.271:,, 1.275:&, 1.268"; or very close agreement.

Summary A method is described by which the osmotic pressure of fluids may

be determined using specially prepared collodion tubes as semipermeable membranes. The rate of water movement between two fluids is used as a measure of the difference in osmotic pressure. The method has been tested on various fluids including serum. It has in a few cases been compared with the Hi l l vapour pressure method and the results were found to agree.

References

Blegen , E., S.kand. Arch. Im Druck. H i l l , A . V., Proc. Roy. SOC. London 1930 (I) B. 106, 252. H i l l , A. V., Proc. Roy. SOC. London 1930 (2) A. 127, 9. K r o g h , A., The Anatomy and Physiology of Capillaries. New Haven 1922. Z s i g m o n d y and Bachmann, Z a n o r g . P. allg. Chem. 1922. 103, 119.