Microgasometric Determination of Nitrogen in Blood and SalivaP. F. Scholander Citation: Review of Scientific Instruments 13, 362 (1942); doi: 10.1063/1.1770060 View online: http://dx.doi.org/10.1063/1.1770060 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/13/8?ver=pdfcov Published by the AIP Publishing Articles you may be interested in White Light Generation in Human Saliva AIP Conf. Proc. 1349, 218 (2011); 10.1063/1.3605814 Conductimetric method of determining the hematocrit value of blood Rev. Sci. Instrum. 54, 1186 (1983); 10.1063/1.1137547 Velocity Profile Determination for Flowing Blood Rev. Sci. Instrum. 36, 625 (1965); 10.1063/1.1719650 Determination of the Acoustic Properties of Blood and its Components J. Acoust. Soc. Am. 25, 286 (1953); 10.1121/1.1907033 Improved Apparatus and Technique for Electromagnetic Determination of Blood Flow Rev. Sci. Instrum. 23, 235 (1952); 10.1063/1.1746234

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. AUGUST, 1942 R. S. 1. VOLUME 13

Physical Instruments for the Biologist

Microgasometric Determination of Nitrogen in Blood and Saliva

P. F. SCHOLANDER

Edward Martin Biological Laboratory, Swarthmore College, Swarthmore, Pennsylvania

(Received July 14, 1942)

T HE. method permits the determination of dissolved nitrogen in about 40 cubic

millimeters of blood and saliva (or other liquids) with an accuracy of 0.02 milliliter nitrogen per 100 milliliters of sample. A single analysis takes 10 minutes; in series each analysis takes 5-6 minutes.

PRINCIPLE

A micrometer burette is provided with a ground glass sleeve into which the extraction tube fits. The tube has a bulb and a fine capil­lary stem. The extraction tube is filled with mercury and some alkaline hydrosulfite which was vacuum extracted in the tube in advance. Blood or saliva is measured into the tube by means of the micrometer. The capillary is sealed free of air with a drop of melted bees' wax and the extraction tube is placed in a centrifuge tube with some mercury at the bottom. It is centrifuged for 3 minutes, whereby the mercury in the bulb of the extraction tube is

. thrown down to the wide lower part of it leaving a large surface and big vacuum space for the extraction of gas. After centrifugation the extraction tube is placed back on the micrometer burette and the volume of the gas bubble in the capillary is measured with the micrometer, or against a millimeter scale.

APPARATUS

The apparatus (Fig. 1) consists of a micrometer burette (A) with the original spindle exchanged for a i-inch drill rod. It has a ground joint for the extraction tube (B). This has a 5-cc pear­shaped bulb and ends in a capillary 8 centi­meters long of i-millimeter bore, which is ground flat at the end. The ground joint is submerged in a wide mercury trap (C) which at the same time takes up spilled mercury. For a better tempera-

ture control, it is an advantage to fill the rest of the trap with water at room temperature. The extraction tube fits into a strong 50-cc centri­fuge tube (D) and is steadied with a loosely fitting cork stopper.

Accessories are straight un calibrated pipettes (E, F) ending in rather fine points which can be fitted into a piece of rubber cylinder made with a cork borer. The pipette for saliva has a flanged­out end (F) which can be placed over the open­ings of the sublingual salivary ducts. For secur­ing blood samples a procedure is used modified after Mook. 1 A 15 millimeter long glass or transparent plastic tube is used, with one end flanged out (G). The tube is held against the finger tip by means of a loop of elastic with one or two knots on it. A spring blood lance is adjusted so that it gives a cut 2 millimeters deep when shot through the tube into the finger tip. Vacuum sealing of the capillary of the extraction tube is done by means of a drop of hot bees' wax which is melted from a stick of wax, using a match, following a most valuable suggestion by Dr. W. J. Scott of this laboratory. The reagent is normal potassium hydroxide solution containing enough of the powder mixture of hydrosulfite (10 units) and anthra quinone beta­sulfonate (1 unit) to give a dark red solution when made up anaerobically in a full flask.

PROCEDURE

Extraction of Reagent

The extraction tube is filled with mercury by suction and is fitted onto the micrometer burette. The mercury is screwed to the top of the capillary which is then provided with a section of a rubber tube 7-8 millimeters long. This is filled with the bydrosulfite solution which is drawn down into

1 H. W. Mook, Biochem. Zeits. 242, 338 (1931).

362

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D E T E R MIN A T ION 0 F NIT R 0 G E N I N B L 0 0 DAN D SAL I V A 363

the capillary and bulb corresponding to a read­ing of 5 millimeters on the micrometer. A small drop of mercury is drawn in on top of the hydro­sulfite in the capillary and the end of the capil­lary is dried off with a piece of filter paper. The mercury is brought flush with the opening of the capillary and a drop of mel ted wax is dropped onto the end, sealing it vacuum tight. The tube is detached while the micrometer is being screwed in to prevent the reduction of pressure in the system which might cause air to leak in around the micrometer spindle. The lower end of the extraction tube is dried off and the bulb is slightly heated with the hand until mercury protrudes from the lower end. It is then placed in a centrifuge tube which contains enough mercury to give the biggest possible surface in the bulb of the extraction tube when the inside and outside mercury surfaces come level during the centrifugation. The tube is steadied with a cork stopper and is centrifuged for 3

minutes at around 1000 r.p.m. This throws the mercury down in the tube and the vacuum so formed extracts the nitrogen out of the reagent completely enough to render the remainder insignificant. After centrifugation a pencil mark is put on the capillary at the end of the gas bubble and the tube is stuck onto the micrometer burette, screwing the micrometer out at the same time so as to keep the gas bubble at its original length. The wax seal is cut off with a knife and the apparatus is ready for the blood or saliva sample. In serial determinations it is necessary to have several extraction tubes prepared in advance with extracted solutions.

Analysis of Blood or Saliva

The blood sample is taken with a minimum contact with air by attaching the little flanged tube on the finger tip by means of an elastic (G). The hand should have been kept for some time in warm water. A few crystals of citrate are placed in the tube arid a tourniquet is wound around the finger towards the tip. The finger is pricked through the tube with the properly adjusted spring blood lance. The tube has to be filled quickly with the blood, which is immedi­ately drawn into the pipette from the bottom of the tube. The little rubber cylinder is stuck

B E F

G

G

A

. FIG. 1. .Apparatus for m!crogasometric determination of mtroge!l In blood and sahva. A, micrometer burette. E, extract1~:m tube. C! mercury trap. D, extraction tube placed In the centrifuge tube. Dotted line indicates the level of. the mercury surface during centrifugation. E, blood pipette and rubber cylinder. F, saliva pipette. G blood cup held by elastic to finger tip. '

onto the pipette tip and is held firmly against the end of the capillary of the extraction tube, and 5 millimeters of blood read on the microm­eter are drawn down immediately on top of the hydrosulfite. Avoid trapping air bubbles. A little mercury is sucked in after the blood, the tube is dried, sealed with wax, and centrifuged for 3 minutes as described for the extraction of the reagent.

For measurement of the gas volume extracted, the tube is put back on the micrometer burette while the micrometer screw is screwed back so as to leave the bubble slightly extended, i.e., at slightly reduced pressure. A short section of a rubber tube, 3-4 millimeters, is put on top of the capillary and is filled with a drop of 5 per­cent Tergitol2 (a commercial wetting solution). The wax is cut away with a knife through the Tergitol solution which seals the gas bubble. All except one or two millimeters of the Tergitol is sucked away and the gas bubble is moved down to a mark on the capillary, e.g., to a hair tied

2 Carbide and Carbon Chemicals Corporation, 30 East 42nd Street, New York, New York.

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364 P. F. SCHOLANDER

around it, and the volume of the bubble can be measured with the micrometer. Less accurately, but more quickly, the bubble can be measured directly in millimeters on the capillary tube by keeping it horizontally against a millimeter scale. The zero point, which is usually not visible on account of the wax seal, is found by fixing a needle with its point right above the zero point of the scale, and then pushing the capillary end up against it, piercing the wax. Using this linear measurement, the conversion factor from millimeters on the capillary to micrometer units must be determined.

Using saliva instead of blood, a pipette is used which has a wide flange at one end (F). The flange is put against the ducts of the sublingual glands immediately after the subject has bitten and swallowed a piece of lemon, pickle, or other stimulating agent. The saliva must fill the tube quickly. The saliva is run over to the pointed end of the pipette and some saliva run out. The rubber cylinder is attached and the proce­dure is the same as described for blood, only it is convenient to use twice as much saliva as blood with the same amount of hydrosulfite reagent.

CALCULATIO N

The amount of blood taken in was 500 microm­eter divisions; if the volume of gas were 5 micrometer divisions, the percentage of nitrogen would be 1 percent, measured moist and at room temperature. For reduction factors to gas volume measured at standard conditions, the simple Table 15, page 129, Peters and Van Slyke3 is used, as reabsorption and unextracted amount of gas with the present procedure are negligible, compared with the measuring ac-

3 J. P. Peters and D. D. Van Slyke, Quantitative Clinical Chemistry, Vol. 2, Methods (Baltimore, 1932).

curacy. If the gas bubble is' measured directly in millimeters on the capillary, the amount found is multiplied with the empirically determined factor, transforming millimeters into micrometer units.

The method described has wider applications. I t has been useful in microdetermination of oxygen in water, and it could be used for other fractional absorption determinations requiring successive measurements, such as are done with the Van Slyke apparatus.

ACCURACY

The accuracy of the method was tested on water samples prepared to contain different amounts of nitrogen by bubbling with oxygen. Two-cc samples were used in the Van Slyke apparatus, together with 1 cc of alkaline hydro­sulfite extracted in advance in the apparatus. The micro method used around 40 cubic milli­meters. Results were calculated after formulas 1 and 2, pages 249 and 282, Peters and Van Slyke3 (see Table I).

TABLE I.

Micro apparatus Van Slyke apparatus

1. Distilled water 1.10 1.11 1.11 1.11 aerated

2. Water aerated 0.77 0.78 0.77 0.80 with oxygen

3. Water aerated 0.16 0.17 0.14 0.15 with oxygen

The agreement between the two methods is good, and duplicates with the micro method in blood, saliva, or water usually check within ±0.01 percent.

I am much indebted to Dr. Laurence Irving for his stimulating support, and to Mr. George Edwards for valuable help.

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