Simple Microdetermination of Kjeldahl Nitrogen in Biological Materials CALVIN A. LANG
Division of Medical Entomology, Department of Pathobiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore 5, Md.
Many procedures for the microdetermination of Kjeldahl nitrogen have been devised, most of which require specialized equipment and involved techniques. A simple method
Anal. Chem. 1958.30:1692-1694. Downloaded from pubs.acs.org by UNIV OF KANSAS on 01/04/19. For personal use only.
using commonly available laboratory apparatus has been developed which
involves acid digestion, nesslerization, and measurement of the resultant color. The range of this method is from 1 to greater than 1000 y of nitrogen. Good recoveries of nitrogen in various refractory compounds of obtained. biochemical interest were This method is especially suited to routine analyses, because large numbers of samples can be analyzed with greater ease of technical manipulation.
modifications of the classical
Many Kjeldahl method have been de-
vised to determine micro quantities of nitrogen in biological materials. In general, the specialized and unwieldy equipment required is not suited to routine analyses. The simpler and more appropriate Nessler modification has found disfavor because of the frequent appearance of turbidity during color development which is caused principally by the presence of certain multivalent cations, and improper pH (8-5). The simplified procedure, as presented, is a composite of several conventional techniques, and has the following advantages: extreme sensitivity, accuracy, and range, use of inexpensive, commonly available equipment, ease of technical manipulation, capacity for large numbers of samples, and the possible determination of other elements in the same sample digest. REAGENTS
All chemicals used
analytical reagent grade, low nitrogen, or the best available commercially. Deionized or distilled water of low conductivity is used throughout the procedure. Digestion Mixture. The following chemicals are combined in the order given: potassium sulfate, 40 grams, selenium oxychloride, 2 ml., water to 250 ml., and sulfuric acid, 250 ml.
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ANALYTICAL CHEMISTRY
Although copper sulfate (20 ml. of a 1.0M solution) is often added to this mixture, the formulation given is equally effective and can be used as a digesting agent for trace element
and Coleman colorimeters, the Coleman Jr., Coleman Senior, Beckman DU, and Spectronic 20 spectrophotometers are all satisfactory.
son
analyses.
Nessler Reagent. This reagent is prepared by dissolving 68 grams of Nessler Granules (Tenso-Lab, Irvington-on-Hudson, N. Y.) in 100 ml. of water, followed by the additions of 850 ml. of an aqueous 10% sodium hydroxide solution and of water to a final volume of 1000 ml. This is allowed to stand several days before using. This preparation is indistinguishable from that prepared by the method of Koch and McMeekin (2). The reagent should be protected from light and is stable for at least 1 year. Standard Solutions. Nitrogen Ammonium Sulfate Standard. This is prepared by dissolving 1.179 grams of ammonium sulfate, previously dried for several days in a desiccator, in 250 ml. of 0.21V sulfuric acid to give a final concentration of 1000 y of nitrogen per ml. Lysine Standard Solution. L-Lysine monohydrochloride is recrystallized twice from alcohol-water and dried, and 65.2 mg. are dissolved in 100 ml. of water to give a final concentration of 100 y of nitrogen per ml. Aliquots of these stock solutions are diluted with water to obtain working standards.
EQUIPMENT
Digestion Apparatus. An electric heating disk (375 watts), controlled by a voltage regulator, is embedded into sand contained in an insulated metal pan (20.5 cm. square by 5.0 The pan is covered by a cm. deep). Transite sheet (hard asbestos) into which rows of slanting holes have been bored to support the digestion tubes (15 X 125 mm. borosilicate glass bacteriological test tubes). This arrangement (1) enables each digestion tube to serve as its own condenser, because the perforated sand bath cover insulates the upper portion of the tubes, and provides an effective yet restricted This apparatus permits zone of reflux. the simultaneous digestion of about 40 samples and can be easily modified for greater capacity. Volumetric Glassware. Pipets are the only volumetric ware necessary to perform this determination. Colorimeter. The Klett-Summer-
PROCEDURE
In each digestion tube sample of less than 5-mg. dry weight, or 1.0-ml. volume, and 0.20 m^ of digestion mixture. These tubes, with others containing appropriate amounts of nitrogen standard, are inserted into the holes of the sand bath cover, and embedded to a depth of about 2.5 cm. in the sand. A tube containing sulfuric acid and a thermometer is placed in one of the center holes. Heating for about 1.5 hours raises the temperature to 310° to 320° C., which is below the boiling point of the digestion mixture. After a 1-hour digestion at this temperature, the tubes are removed and cooled to 20° to 27° C. The volume in each tube is now 0.1 ml. Digestion.
is placed
a
Nesslerization. Dilution Method, The acid digest is diluted to 10 ml. with water, and an aliquot of 3.0 ml. or less is transferred to another tube. To this is added water bringing the volume to 4.0 ml., and 2.0 ml. of Nessler reagent are then added. Thorough mixing should follow each addition. Dikect (Method. For samples containing 1 to 10 7 of nitrogen, 1.4 ml. of water are added to the acid digest, followed by the rapid addition of 5.0 ml. of Nessler reagent. The tubes are protected from light after nesslerization. Color Measurement. With the dilution method, maximal color development is achieved in 10 minutes; with the direct method, 30 minutes. The tubes are read in a colorimeter set at 420 m^ for levels less than 15 7 for higher of nitrogen and at 500 levels. The color is stable for 1.5 to 2.5 hours after nesslerization. VALIDATION OF PROCEDURE
Digestion Rate. To determine the of digestion, aliquots of the standard lysine solution containing 100 7 of nitrogen were digested for different lengths of time. The results (Table I) indicate that complete digestion is obtained when the temperature reaches 310° C., and no loss of nitrogen rate
when the sample is digested 3 occurs hours longer. It has been the practice to digest 1 hour after reaching 310° C.
Color Development and Stability. Quantities of ammonium sulfate standard ranging from 1 to 10 7 of nitrogen were nesslerized by both the dilution and the direct methods, and their absorbances were determined at given time intervals after nesslerization. Using the dilution method, the readings at all nitrogen levels changed less than 2% from 5 minutes to 2.5 hours after nesslerization. With the direct method, the color was stable from about 30 minutes to 1.5 hours. Other evidence indicated that the latter method was extremely sensitive to light. Range and Sensitivity. Table II presents data of standard curves for ammonium sulfate standards that were digested and nesslerized by both methods. The average absorbance value of three replicates at each level is given, and the slope of the curve at each point is expressed as the ratio, absorbance units per microgram. In general, this ratio is constant for the levels studied, except for slight discrepancies at 1 and 2 7 in the direct method. A more sensitive spectrophotometer might resolve these small differences. The dilution method is applicable for samples from 10 to greater than 1000 7 of nitrogen. Using the direct method, samples as small as 1 7 of nitrogen can be determined. Nitrogen Recovery. Lysine, tryptophan, and arginine, because of their known refractoriness to acid digestion, and also uric acid, another hetero-
Table
I.
Digestion
Rate
Standard
of Lysine
Digestion Time, Hours After % Total 310° C. Theory Sample 1.6 0 99.3' Lysine standard 100 7 of nitrogen 2.6 1.0 98.9 3.8 2.2 102 4.6 3.0 101 “
Averages of three determinations.
cyclic nitrogen compound, and human serum albumin (five times recrvstallized) were examined. Table III shows that the recovery of 1 to 1670 7 of nitrogen from samples ranging from 6.5 to 5000 7 was essentially complete. Although only 90% of the tryptophan nitrogen was determined, it is sufficient for most biochemical purposes, because the proportion of tryptophan to other nitrogenous compounds in a biological sample is small, and the error due to biological variation far exceeds this analytical error. Excellent agreement in the results of nitrogen analyses of casein and brain homogenates were obtained by Boell and Shen, who compared their deter-
Table II.
Standard Curves for Nessler Nitrogen”
Nitrogen, Method
Digested
Dilution
420 Mm
20 40 20
0
~933
0
0
1
1
2
2
5 10
5 10
47 92 215 432
20
20 a
Spectronic 20 spectrophotometer using 15-mm.
7
1
2 5
10
652
100
34.3
250 1000
L-Tryptophan
l-Arginine (free base) Uric acid albumin (5X
a
a
Av.
7
6.52 13.0 32.6 65.2
L-Lysine. HC1
procedure to cedure (7).
284 608 285
Nitrogen Recovery in Various Chemical Compounds % Theory Xitrogen,
Compound
mination involving
13.6 13.7 14.2 15.2 14.3
68 137
47.0 46.0 43.0 43.2 ~40.8
~815
Determinations were made with diameter round cuvettes. “
Human serum recryst.)
0
47.5 47.2 48.6 48.8 ~46.7
95 118 243 488
10
Table III.
Absorbance X 1000 at 500 Mm Absorbance Per 7 N
0
2.0 2.5 5.0
25 50 100 200 400 1000
Per 7 N
Absorbance
0
0 10
Direct
Absorbance X 1000 at
7
Nesslerized
137
97.0 95.1 98.1 98.9
Replicates
97.0,97.0
91.3 90.0
90.0, 94.5, 100 97.1, 97.1, 100 96.7, 101 98.9, 99.3, 102 90.8,90.8, 92.4 90.0, 90.0, 90.0
100
1000
322
96.8
96.1,97.1, 97.1
5000
1670
97.7
96.1, 97.1, 100
626
100
similar digestion
classical Kjeldahl pro-
DISCUSSION
A nesslerization method has been successfully in this institution for over 8 years on a variety of biological materials ranging from mosquito carcasses to mammalian tissues and excreta. The original digestion procedure involved heating to boiling and digesting for 1 hour after clearing, and only a limited number of samples could be digested at any one time. With the present modification, digestion is carried out below the boiling point, avoiding spatter and nitrogen loss, and much larger numbers of samples can be handled. The digestion apparatus is used
simple and compact, and compared to other Kjeldahl and Nessler methods, this procedure is less complicated technically, because test tubes and pipets are employed in place of more cumbersome Kjeldahl and volumetric flasks. Equally the nesslerized samples are clear and completely devoid of precipitates, and occasional turbidity has been traced to heavy metals in the water, or to an imbalance of digestion mixture and Nessler reagent. The
(
100
99.7, 99.7, 101
effect of interfering cations is minimized by the small amounts of sample required. To reduce variations caused by other factors such as time and temperature, nitrogen standards are always included in each digestion. Because only a fraction of the diluted digest is needed for nesslerization, the remainder can be used for the analysis of other metal constituents—e.g., in studies on the zinc content of individual mosquitoes, 1.0 ml. of the diluted acid digest was nesslerized, and the other 9.0 ml. were used for the determination of zinc using dithizone. Thus it was possible to obtain the ratio, micrograms zinc per milligram nitrogen per mosquito, which is regarded as a more valid expression of zinc concentration than zinc per dry weight. The sensitivity of the described method could be increased by means of a spectrophotometer equipped with a photomultiplier attachment in which event the approximately tenfold increase would enable the estimation of 0.05 7 of nitrogen. The method has proved to be satisfactory and useful for the routine analyses of biological tissues and fluids. However, this modification, like many of the Kjeldahl methods, will deterVOL. 30, NO. 10, OCTOBER 1958
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mine only part of the nitrogen in certain types of compounds, and in in these few instances that occur biological materials, a Dumas nitrogen or similar total nitrogen procedure is indicated. The determination of Kjeldahl nitrogen is one of the most important and widely used chemical analyses in biology. This modification may be of value in those laboratories where the
of the classical method has been discouraged because of the expensive, elaborate apparatus required as well as the involved manipulation. use
(3) Shaffer, F. L., Sprecher, J. C., Anal. Chem. 29, 437 (1957). (4) Thompson, J. F., Morrison, G. R., Ibid., 23, 1153 (1951). (5) Yuen, S. H., Pollard, A, G., J. Sci. Food Agr. 3, 441 (1952). '
LITERATURE CITED
(1) Boell, E. J., Shen, S. C., Exptl. Cell Research 7, 147 (1954). (2) Koch, P. C., McMeekin, T. L., J. Am. Chem. Soc. 46, 2066 (1924).
Received for review November 11, 1957. Accepted May 20, 1958. Work supported in part by a research grant from the Public Health Service.
Microsampling for Small Infrared Spectrophotometers J. U. WHITE, SEYMOUR WEINER, and N. L. ALPERT
The White Development Corp., Stamford, Conn.
W. M. WARD Beckman Instruments, Inc., Fullerton, Calif.
ploying the standard 60 X 75 mm. prisms or larger, where there are instrumental adjustments that can easily be changed to correct for energy losses or other difficulties resulting from the small sample size. Combining them with the recently announced (22) small infrared spectrophotometers requires changes in the methods and equipment. They must be made compatible with the basic philosophy of the simple instruments that:
A three-lens beam condenser has been built to frt in the sample space of the Beckman infrared spectrophotometers. It passes 75% of the light through a 0.5 X 4 mm. sample area by forming a fivefold reduced image of the slit and then remagnifying it. Small capillary absorption cells of this the size have been used to measure spectra of a number of essential oil samples on a small double beam Potassium brospectrophotometer. mide pellets, 1 X 5 mm., have been pressed for solid samples. Polarization measurements are possible on the small samples using a rotatable polarizer.
have recently for measuring the infrared spectra of very small samples. Micro liquid cells (11) have been made to fit where the light beam is small, near the entrance slits of spectrometers. Silver chloride lens systems' (1, 2) have been built to pass most of the light through a small sample at a reduced image of the slit. For further reduction of sample size, a number of reflecting microscopes and sampling systems (8-10, 13-15, 23) have been devised. Special absorption cells in conjunction with a microscope (5, 8) make it practical to measure the spectrum of approximately 1 7 of material in solution. Less than 1 ,ul. of pure liquid may be measured without a microscope (12). With the potassium bromide pellet technique and freeze drying of the samples (17, 19-21), the size of solid samples is reduced to 10 7 without the use of a microscope (18). methods
been developed Numerous
All these methods have been used on commercial infrared spectrographs em1694
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ANALYTICAL CHEMISTRY
,
Accurate spectra be recorded as graphs of transmittance vs. wave length. All spectra be made directly comparable by recording them on identical charts at the same speed, response time, and slit width. Other types of measurements be possible by connecting additional equipment. The operation be as simple and reliable as possible. The cost be held to a minimum. The basic unit on which most of the methods depend is a lens-type beam condenser. The device described here
a unit that fits in the instrument’s normal sample space. It uses three potassium bromide lenses to make an intermediate image of the entrance slit reduced five times and to reimage this again five times enlarged at the original focal point. All the light leaving the unit is then going along the same lines toward the same image points as it was when it entered. When the slits are widest, essentially all the rays pass through an opening 0.5 X 4 mm., giving over-all transmission of about 75%, including reflection losses. Figure 1 shows how the beam condenser fits in the sample beam of the instrument. Figure 2 shows its optical layout, BC, in relation to the rest of the instrument. The three lenses are adjustably mounted in the three upright webs of the base. The middle and lefthand lenses condense the beam to the reduced image where the sample, S, is placed, near the center of the righthand open space. Next to the monochromator cover at the right is the large lens that remagnifies the image and brings it back to its original focal point, which is now virtual. Holders for small samples fit into this space on adjustable
is such
Figure 1. Beam condenser and micropellet holder in Beckman Model IR-5 spectrophotometer
