A rapid and precise cryoscopic method applicable to biological fluids

Abstract

Beckmann’s classical method of chryoscopic dcscent is modified in order to obtain the greatest accuracy in the resuljts using the mínimum amount of time and sample.

The theorctical principie of the modified method lies in the production of a slight supercooling of the sample (perfectely measured) before the analysis. As a consequence when the liquids are passed into the chryoscopic chambér and the coid thermometer is introduced in, crystalization of ice takcs place immediately. The quantity of ice formed is very small, and for that reason the concentration of the sample is not changed. There is a very little difference in the temperaturebetween the refrigerant mixture and the freezing sample, for that reason chryoscopic temperature is not changed during the time of the analysis.

To carry out the determination, three Dewar Vessels are employed (fig. 1), and a refrigerant mixture composed of ice, water, and salt in different proportions is placed in each one. In Vessel I the obtained temperature is about —1.0° C (aproximately 0,5° C below the freezing point of the liquids to be analized.

Two mililiters of sample are dropped into each standard test-tube (13 mm in diameter), and it is cooled in Vessel I to the same temperature as that of the refrigerant mixture. In Vessel II, the temperature is lowred to —1.5° C. In this Vessel there are two concentric tubes forming a double air- pocket in order to avoid any heat irradiation. In Vessel III in which the temperature has been lowered to —18° or —20° C, a dry test-tube (with some filter paper in the bottom) is placed in it in order to cool the Beckmann’s thermometer. For the

reading of the freezing-point of the samples, the coid tcst-tubes are passed from I to II; then the coid thermometer, which has cought microcrystals of ice from the air, is put into the liquid, and the sample is well shaken until the temperaturegoes up to a máximum where it remains for a certain length of time (fig. 2). The thermometer is dried with filter paper, then it is introduced in III for the next reading. The total time for the analysis of uriníƒe is less than 90 seconds for each sample ; consequently more then 40 analysis can be done in one hour.

When plasma or serum in needed for further analysis it should be mclted by hand-heat before taking the thermometerout, thus a change in its concentration is avoided. In this case the required time is little more (about 3 minutes) than in the other.

Many concentrations of puré substances have been determined using this method, giving resulting values in satisfactory concordance with the theoretical ones (obteined by weighing) in the zone of physiological concentrations.

Urea, glucose, and NaCl (registered in fig. 3) present an absoulte identity of behaviour in low concentrations. On the other hand in higher concentrations the osmotic activity of NaCl is decreased, while the glucose activity increases.

Table I and II register the osmotic behaviours of glycine and ethyl-alcohol which agree with the theoretical predictions, according to their structure. The equation A = 1.858 c is applied, c being the concentration expressed in miliosmols/Kg of water.