o RO LAN D C. HAW ES AND EDW IN K. SK A V IN SK I*
L ab oratories o f G eorge l ’iiie ss, M .D ., L os A n g e le s, C alif.
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E C E N T L Y it was desired to determine the nitrogen content of a num ber of allergen preparations. Analysis of extractable and “protein” fractions, in duplicate, brought the number of determ inations in prospect to about 1800. A micromethod was definitely indicated by the cost of the materials, even if the advantage of economy of reagents, convenience, and speed had not influenced the decision. In fact, since m any pollen extracts contain less than 0.1 per cent (calculated on a dry pollen basis) of nitrogen precipitable with trichloroacetic or tungstic acid, and since it was desired to do a complete analysis on a total sample not exceeding 0.5 gram, a m ethod adapted to th e determination of 100 micrograms or less was required. Choice of published methods finally lay between colorimetry using direct nessleri- zation, and th e Borsook and Dubnoff (1) adaptation of the Conway and Byrne (3) diffusion micromethod. The advantages of the form er in speed and simplicity were weighed against its more lim ited precision and the occasional presence of interfering substances from these materials.
Experience w ith the two methods showed th a t the advan
tage of the colorimetric method in speed would be overcome to a considerable extent if a satisfactory way could be found of avoiding th e sample transfer from the digestion tube into the diffusion vessel, while a t the same time one of the chief sources of handling difficulties and variation of results would be avoided. Needham and Boell (<?) described apparatus for this purpose, b u t it has some disadvantages. The setup for large scale use is rath er expensive because of the ground joints required, and th e diffusion rate is inconveniently slow.
While this m anuscript was being prepared a paper by Tompkins and K irk (8) appeared, describing apparatus which attains the same object in a similar wi-y, but which is different in several respects from th a t shown below, and especially in th at the digestion tube cannot so readily be used in the cen
trifuge and th a t th e diffusion rate is slower. Their paper reviews the need for an improved method. References in it and in the Conway book (S) include all the previous work considered except the aeration method of Folin (4).
Fi g u r e 1. Se c t i o n a l Vi e w o f Di f f u s i o n Ap p a r a t u s
The essential feature of the present method is that a plain test tube (or conical centrifuge tube, if desired) serves as both digestion and diffusion vessel. The diffusion receiver consists of a helix of platinum wire carrying a drop of absorbing solution, held in the tube by means of a grooved rubber stopper. The apparatus set up for diffusion is shown in Figure 1. ,
With this assembly it was apparent that convenience of manip
ulation would result if a concentrated solution of a weak acid were used for the absorbing solution, since then the volume of the droplet need not be measured precisely and the helix could be adjusted so as to pick up the desired volume when dipped in the solution. However, it was found that boric acid, which is commonly used for the purpose (2), is too weak to be suitable. A 0.02-ml. droplet of saturated solution would provide stoichio
metric absorption of ammonia only up to about 20 micrograms of nitrogen. As this was considered an inconveniently small maximum sample size, search was started for a substitute.
Xn-1 P resen t address, Sou th ern C alifornia G as C om pany, Los Angeles, Calif.
spection of a table of ionization constants showed th at the second hydrogen of phosphoric acid has very nearly the ideal dissociation tendency, while the constant for the first hydrogen is sufficiently largo that its dissociation is nearly complete a t the end point near pH 4.5.
The advantage of 1.0 M sodium dihydrogen phosphate mono
hydrate over boric acid saturated a t 20° C. (0.8 M ) may be seen by inspection of the titration curves of Figure 2. They show ex
perimental titrations of 5 ml. of each absorbent with 10.5 N ammonium hydroxide, delivered beneath the surface of the solu
tion from a buret with a capillary tip. The pH values were ob
tained with a glass electrode pH meter (Beckman laboratory model). Also shown are the pH's of 1 to 50 dilutions, made by removing 0.02-ml. portions of the solutions at the indicated points during titration with the aid of a set of receivers and diluting each with 1.0 ml. of water for the pH reading. Those a t the higher pH values were held only a few moments between removal and dilution, but probably lost appreciable amounts of ammonia in the interval, as suggested by the lines drawn on the plot to indicate various levels of ammonia dissociation in water.
Only about 0.08 mole of ammonia per mole of boric acid initially present was required to raise the pH to 6.4, the level of 0.1 per cent ammonia dissociation. The corresponding ratio for phos
phate is 0.42. Furthermore, by use of a saturated solution of ammonium dihydrogen phosphate monohydrate the sample size could be increased to about 0.5 mg. of nitrogen with, of course, some loss of end-point precision for smaller samples.
In order to determine the time necessary for the diffusion, the rate constant for the apparatus was calculated from measurements on duplicate samples containing 100 micrograms of nitrogen in the form of ammonium sulfate at 0.5, 1, 2, and 3.5 hours. The constant, calculated from the equation
jr ______^______
log A/(A - x)
averaged 120 minutes (minimum 103, maximum 142). A is the total amount of ammonia and x is the amount found in the re
ceiver after t minutes of diffusion. The constant shows the time required to transfer 90 per cent of the ammonia, and may be compared with a constant of about 50 minutes for the standard Conway (3) “unit", 260 minutes for the device of Tompkins and Kirk (S), and 700 minutes for the Needham and Boell (6) apparatus. (Constants were calculated from data given in the references, corrected for solution composition and to a tempéra
ture of 20° C.)
A t 20° C. ammonia diffusion is 99.5 per cent complete in less than 5 hours. Amines (2) wall diffuse more slowly, so th a t in analyzing digests of most substances of biological interest the recommended overnight diffusion is not unneces
sarily long with the present setup. N aturally, the rate can be increased by raising the tem perature, and by other means (5, 8) if desired.
I t seems w orth while, because of the confusion th a t may result from the method used by Tompkins and K irk (8) to determine the length of diffusion required for various size samples, to re-emphasize th a t the above formula has been shown (3) to apply generally to the diffusion of substances having low vapor pressure, such as dilute ammonia. Its significance is, of course, th a t for equal relative accuracy of determ ination the same length of tim e is required for all sample sizes. In unpublished work it was found th a t the equation has the same constant and predicts the diffusion course as well for 10 as for 100 micrograms of nitrogen. I t is, therefore, n o t clear how Tompkins and K irk obtained values usually within 1 per cent of theory on various samples con
taining less than 10 micrograms of nitrogen, when th eir recommended diffusion tim e is sufficient to transfer only about 90 per cent of the ammonia in such samples, as shown by their own ra te curves.
No attem p t was made to determ ine the limits possible w ith
918 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 14, No. 11 P r o c e d u r e
th is diffusion apparatus, b u t it was found th a t 10 micrograms can be determ ined w ithout alteration of the m ethod except to use 0.2 M sodium dihydrogen phosphate, if electrom etric determ ination of the end point is used. A ground-glass joint, or possibly a stopper made of a synthetic elastom er in place of the rubber, and a sm aller titra tio n volume would no doubt perm it fu rth er increases in sensitivity.
P r e p a r a tio n o f R eceivers
Five turns of 22-gage platinum wire are close-wound on a rod 2.4 mm. in diameter (13-gage), leaving a handle of straight wire 1.5 cm. long. The end of the latter is sealed into a short piece of 6-mm. soft-glass tubing inserted through the hole in a No. 0 pure gum rubber stopper.
Stoppers, obtained from the Chemical Rubber Company, Cleve
land, Ohio, are treated before making up the receivers by boiling 15 minutes in 1 N sodium hydroxide, then boiling and rinsing in distilled water. When prepared in this way they contribute the equivalent of less than 0.3 microgram of nitrogen to the value of the blanks. A neat groove in the stopper is most easily made with the aid of an electric drill stock carrying a metal rod to hold the stopper, and a power grinding wheel, but it may also be cut by hand with a sharp knife. The groove is about 1 mm. deep and 6 mm. wide, and starts about 4 mm. from the small end of the stopper. I t is effective in preventing the stopper from forcing itself out of the tube or moving so as to bring the helix in contact with the tube wall.
The helix is cleaned either by immersing for a few minutes in hot cleaning solution, or by heating to incandescence for a second in a flame. It is then adjusted by pulling with fine forceps or spreading the center turns with a knife blade, until the volume of the drop it carries is within 10 per cent of 0.02 ml. (determined by titration). Uniformity is of more importance than the abso
lu te size of the droplet.
The helices are recleaned at frequent intervals in order to main
tain a imiform drop size. Platinum wire was chosen because of the ease of cleaning, although Nichrome wire which was also tried will work, as, no doubt, would other materials, including glass.
R e a g e n ts
Digestion solution, IS N sulfuric acid (nitrogen-free) containing 0.1 gram of selenium dioxide and 0.1 gram of copper sulfate per
100 ml.
Hydrogen peroxide, 30 per cent, conforming to A. C. S. speci
fications.
Sodium hydroxide, 50 per cent by weight, which has stood or been centrifuged until clear.
Absorption solution, 1.0 M monobasic sodium phosphate (Merck reagent).
Standardized acid, of strength suited to the microburet and sample size.
Samples are pipetted (I) or weighed into ordi
nary 125 X 15 mm. Pyrex test tubes taking care to keep the upper wall as clean as possible, 0.2 ml. of acid digestion solution is added to each, and excess water is removed overnight in an oven at 100° C. If the sample volume is less than 0.5 ml., the preliminary drying may be omitted if the first heating is done cautiously.
Digestion is carried out in a sand bath heated by a Tirrill burner so adjusted as to cause refluxing to about 4 cm. from the bottom of the tubes. Ebullition and bumping do not occur.
The tubes are covered with glass caps cut from the bottoms of soft-glass vials 21 mm. in diam
eter, to prevent concentration of the digests and to permit operation outside the fume hood.
No attention is required during digestion as a rule, but if the digests are not nearly colorless in about 4 hours the flame is removed for 10 minutes, and then a small uniform drop of 30 per cent hydrogen peroxide is placed in each tube, including the blanks. Refluxing is con
tinued for a t least 30 minutes after the straw color is gone.
When digestion is complete the tubes are placed in a rack and allowed to cool, then 0.6 ml. of water is added to each, and all are stirred by shaking the rack. After further cooling each in turn is treated as follows:
Three-tenths milliliter of 50 per cent sodium hydroxide is run down the side of the tube without agitation and so as to avoid wetting the rim and 1 cm. of the upper wall of the tube. Although the solutions become warm, the large difference in specific gravity
S
revents complete mixing which would cause loss of ammonia, in warm days it may be necessary to cool the tube in a beaker of ice water. I t is left undisturbed while the receiver is prepared.The latter is removed from the 1.59 X 12.7 cm. (*/* X 5 inch) test tube in which it is kept, and rinsed with distilled water, flamed, and rerinsed. The water is shaken off the helix and wiped off the stopper with cleansing tissue. The helix is dipped in 1.0 M sodium dihydrogen phosphate solution, deeply enough to cover it, but not so deeply th a t the platinum-glass junction is wet with the solution. The stopper is lubricated with a drop of glycerol and then inserted in the tube, taking care th a t the helix does not touch the wall. If it does it must be rerinsed and re
charged. Contact may be avoided by placing the rim of the tube against a notch in the bench edge, holding the receiver in the other hand in a position coaxial with the tube, and sliding the tube up over the receiver, which is held motionless while the tube is twisted to seat the stopper.
The digest is now ovemeutralized by shaking the tube while cooling with a stream of tap water. The entire wall of the tube is coated with a film of alkali by inverting and rotating, and the assembly is left overnight on its side on a paper towel or blotter placed on a level surface.
When ready to titrate, the receiver is carefully removed by re
versing the insertion procedure. The stopper is wiped free of alkali and the helix is immersed in 1 ml. of water containing either indicator or a slight excess of quinhydrone, depending on whether the end point is to be detected colorimetrically or electrometrically.
F i g u r e 3. pH o f D i l u t e d A b s o r b e n t a n d A b s o r p t i o n E f f i c i e n c y f o r V a r i o u s A m o u n t s o f A m m o n iu m I o n
November 15, 1942 A N A L Y T I C A L E D I T I O N 919 The solution is titrated with standardized acid from a niicro-
buret to the pH given by 0.02 ml. of the buffer solution diluted in the same way. The end point lies between pH 4.3 and 4.7.
Blanks are, of course, included with each set of determinations.
With electrometric titration the end point can be determined to 0.02 pH, or 0.2 per cent of the 100 microgram “optimal”
sample. Indicator end point error is about three times as great, but could be decreased by decreasing the titration volume (for a discussion of limitations see Conway, 3). Drying and digestion introduce greater variation, but duplicate determinations on protein solutions almost always agree within 1 per cent and agree with larger samples run by the semimicromethod (7) to
within the same limit. _ . .
An important advantage of electrometric titration, in addition to greater precision, more warning of end-point approach, and no need to back-titrate if the end point is overrun (small excesses can be read from a previously prepared titration curve), is that the size of the droplet of absorbent solution can be checked by comparing the pH before titration with the amount of acid re
quired to reach the end point. [A pH meter with continuous (nonballistic) indication of balance is more convenient and rapid than the ballistic type, especially for titration purposes.] In this way it is easy to be sure that sufficient buffer was present to ensure stoichiometric absorption of the ammonia.
In Figure 3 is shown th e normal relation between pH and sample nitrogen content, also the completeness of absorption, for a considerable range of amounts of ammonia added as ammonium sulfate solution.
Table I gives the results of a series of determinations done on an am monium sulfate solution containing 0.500 gram of nitrogen per liter (2.358 grams of the salt, recrystallized from water, and dried in vacuo over fresh calcium chloride.) A semimicro-Kjeldahl (7) determination done in triplicate on the same am monium sulfate solution and referred to the same independent standardization of acid and alkali gave a value of 0.4982 ± 0.0005 mg. per ml.
Since the main point of novelty in the method and conse
quently of interest in these determinations was the diffusion procedure, th e samples were not carried through the digestion steps prior to th e diffusion or distillation. . are reported, except for a single determination obtained wi i pipet B, which was 163.8 microliter_ and was discarded as probably in error through a fault in pipetting technique.
The difference between the value obtained with pipe tween th e evolving and absorbing solutions.
N o tes an element in the ceiling, resulting in rapid evapora ion
out danger of loss by bumping. _ f sand to make a layer about 10 cm. (4 inches) in diameter is placed in it and the tubes are leaned into the notches with their bottoms immersed to about the level of the digest, around the outer edge of the sand layer.
The sand bath also makes a very satisfactory digestion rack for six 30-ml. Kjeldahl flasks as used in the semimicro or micro- method, but must then be placed in the fume hood and heated with a Meker-type burner.
A titration stand is an even greater convenience for micro- than for macroanalytical work. T h a t used in this laboratory may be of interest because of its simple construction. I t is
I /O Cm. 1
Fi g u r e 4 . Ti t r a t i o n St a n da n d Ca l o m e l Ha l f- Ce l l
The stirring motor is a 1400 r. p. m. shaded-pole induction motor, with a chuck from a small hand drill attached to hold the stirring paddle. The latter is an old platinum foil electrode, 3 mm. wide by 5 mm. high, sealed into a piece of soft-glass tubing. The motor is mounted on twro shelf brackets fastened to a block of wood which also carries a rod to support the electrode terminal block and a piece of Bakelite in which holes for the electrodes and stirrer and a slotted hole to carry the receiver have been cut as shown.
The titration vessel, consisting of a beaker 10 mm. high and 15 mm. in diameter, is supported on a counterweighted hinged shelf of Masonite. Electrodes are held in the holes in the Bake
lite sheet by means of small soft wood wedges.
The construction of the calomel lialf-cell and agar bridge is also illustrated in Figure 4. I t is made of Pyrex glass, except for the internal platinum electrode, which is scaled into a small soft- glass tube. A fiber of acid-washed asbestos sealed into the tip may be substituted for the agar bridge, if desired. When not in use the electrode is stored with the agar bridge immersed in saturated potassium chloride in a squat bottle closed with a one- hole rubber stopper.
The quinhydrone electrode consists of a short piece of 22-gage platinum wire sealed into a piece of 8-mm. soft-glass tubing drawn down a t the tip to about 2-mm. inside diameter. I t was sub
stituted for the small glass electrode used by Borsook and Dubnoff because of the fragility of the latter. For these titrations pure quinhydrone provides an entirely satisfactory half-cell.
An easily constructed m icroburet is illus
tra te d in Figure 5. Its outflow is started and stopped b y immersion and withdrawal of th e tip from th e liquid being titra ted , eliminating th e need for a stopcock or any kind of posi
tive displacement device.
I t is made from a Kahn serological pipet, 0.2 ml. graduated in microliters. A constriction 4 cm. long and 2 mm. in diameter is drawTi, as near the tip as possible, pulling the tip with a handle or forceps. The farthest point of the constriction is further drawn until it has a diameter of about 0.2 mm., and it is broken at the narrowest point. The entire tip is bent at an angle of about 75° away from the side carry
I t is made from a Kahn serological pipet, 0.2 ml. graduated in microliters. A constriction 4 cm. long and 2 mm. in diameter is drawTi, as near the tip as possible, pulling the tip with a handle or forceps. The farthest point of the constriction is further drawn until it has a diameter of about 0.2 mm., and it is broken at the narrowest point. The entire tip is bent at an angle of about 75° away from the side carry