Measurement of Osmotic Pressure of Haemoglobin 



cause errors. In this work it was found that reproducible results could 

 be obtained if the toluene-buffer interface was in the form of a hanging 

 drop of toluene. The surface area is large and the volume relatively 

 small, and errors caused by temperature fluctuations are minimized. 

 The expansion of toluene is compensated by the contraction of water 

 between 0°C and 4°C. Figure 2 shows the simplest type of toluene 

 manometer, adapted for the measurement of pressures up to 15 cm 

 toluene. A graduated pyrex capillary tube of 0-5 mm internal diameter 

 is fused to a flattened bulb of 16 mm diameter. R t is a soft rubber 

 connection ; G x a glass tube 3 cm long, which contains buffer solution 

 in the upper part ; M a collodion membrane as in Figure 1. L=toluene 

 meniscus ; W = meniscus level of dialysate ; B — lowest point of 

 toluene-buffer interface. This interface is in contact with glass at the 

 level B lt at about 5 mm above B. 



The diameter of the outer tube containing buffer is important 

 because of capillarity effects at the meniscus W. There are measure- 

 able variations (0-2 mm) even if the tube be as wide as 35 mm. A 

 diameter of 45 mm is advisable to obtain capillarity readings constant 

 within 0-1 mm. 



In order to prepare the manometer tube for use, a rubber tube, 

 fitted with a screw-clamp and closed at one end with a small glass rod, 

 is filled with buffer and connected to the osmometer below the bulb. 

 By turning the screw, buffer is forced slowly to the level B v The 

 device shown in Figure 2a is then put on to the top of the graduated 

 end of the manometer tube. R 3 is a 

 rubber bung, G 3 a short, wide glass tube. 

 Water is put in G 3 to the level W 1 and 

 a layer of toluene coloured with Sudan 

 III added up to the level T 1 . The screw- 

 clamp is manipulated to draw toluene 

 down the capillary and then to drive air 

 bubbles upwards until all the air is 

 expelled. The buffer-toluene interface 

 is of the form shown in Figure 2. Toluene 

 and water are then removed from G 3 , 

 and R 3 and G 3 are replaced by a short 

 rubber tube which can be closed when 

 necessary with a screw-clamp. 



Before the membrane which contains 

 the protein is connected to the osmo- 

 meter, the rise of toluene due to capillarity is determined. The 

 osmometer is immersed to the appropriate depths in buffer solu- 

 tion contained in the tube of 45 mm diameter and left for at least 



Figure 2. 

 To luene 

 osmometer. 



193 



H— 13 



