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An improved ultramicro Kjeldahl technique
Experimental
147
Cell Research, 7, 147-152 (1954)
AN IMPROVED
ULTRAMICRO E. J. BOELL
Osbom Zoological
Laboratory,
Yale
K JELDAHL
TECHNIQUE1
and S. C. SHEN University,
Received January
New Haven,
Conn., U.S.A.
28, 1954
FOR the determination of the amount of tissue used in biological and chemical work on an ultramicro scale, as for example in studies on the early embryo where the total amount of tissue available corresponds to approximately 0.5 to 5.0 pg protein nitrogen, no method has so far been devised which is superior to the conventional Kjeldahl technique. A number of good methods are available for determining total nitrogen in the range from 1 to 10 pg (2, 3), and recently Briiel, Holter, Linderstrsm-Lang, and Rozits (1) outlined a method for use in the range from 0.1 to 1 pg for which an accuracy of ca. 0.005 pg is claimed. There would be little justification for presenting a new technique were it not for the fact that the method herein described possesses certain features which make it superior, in many respects, to existing methods. The chief advantages of our method are: 1) relatively little specialized equipment is required, 2) the volumes of reagents are sufficiently large to make for ease in handling and to permit of duplicate or triplicate determinations on aliquots of the sample tested, 3) the time for digestion and distillation has been reduced so that a determination can be completed in 4 hours or less, and 4) the method is not only extremely sensitive but also extremely accurate. The method as described below is similar in principle to the conventional macro or micro Kjeldahl technique, so that a detailed account of procedure need not be given. Suffice it to say that tissue or tissue homogenate is heated with a sulfuric acid digestion mixture, that aliquots of the diluted digest are neutralized with alkali in specially constructed vessels, and that the ammonia freed is then permitted to diffuse into an acid film of known volume and strength so that the amount of nitrogen in the sample can be determined titrimetrically. Reagents The digestion mixture is prepared by adding to 100 ml of 50 per cent H,SO( (Mallinckrodt’s low nitrogen analytical reagent) one gram each of CuS04, K,S04, and SeO,. 1 Supported in part by a grant from The American mittee on Growth.
Cancer Society
through
the NRC Com-
Experimental
Cell Research’ 7
E. J. Boell and S. C. Shen
148
The receiving acid consists of 0.004 N H&SO,, containing 4 ml of 0.4 per cent bromocresol green per 100 ml. The standard alkali is 0.01 N NaOH. The solution is accurately standardized and protected from contamination and evaporation. A color standard, to serve as a titration endpoint, consists of 4 ml of 0.4 per cent bromocresol green in 100 ml acetate buffer at pH 4.6. In addition, ammonia-free water and saturated NaOH are required.
Digestion Digestion is carried out in straight-walled 10 x 75 mm Pyrex brand ignition tubes (glass No. 172). The nitrogen source, either tissue or tissue homogenate, is delivered to the bottom of each tube by means of a calibrated constriction pipette after which exactly 24 ~1 of digestion mixture are added. Suitable blanks are similarly prepared. The tubes are then pushed to a depth of about 2 cm into an electrically heated sandbath. They are supported in place by a perforated asbestos plate which by insulating the part of the digestion tube above it provides an effective but restricted zone of reflux. With this technique, even when the acid is made to fume strongly, no measurable loss of acid occurs. The time necessary for complete digestion was found to be about three ho,urs.
Diffusion After digestion has been completed, the tubes are cooled and a given volume, usually 200 ~1, of ammonia-free water is added to each. The contents are thoroughly mixed, and aliquots of known volume, about 90 ~1, are then transferred to a series of diffusion vessels. The diffusion vessel consists of a micro-florence flask whose dimensions are: inside diameter of bulb 10 mm, length of neck 22 mm, inside diameter of neck 5 mm. It can be easily constructed from tubing with a 5 mm bore. The neck of the diffusion vessel is coated with a thin film of paraffin wax (M.P. 72°C). Uniform pellets of wax are first prepared by dropping melted paraffin from a tine pipette onto a clean, cold glass plate. One pellet is placed in the neck, and the vessel is then maneuvered so that a rigidly-held heated silver wire can be inserted. The vessel is then rotated by hand with the result that melted paraffin is applied as a uniform coating along the entire length of the neck as far as the vessel bulb. Next 36 ~1 of saturated NaOH solution, giving a final concentration of Experimental
Cell Research 7
149
Ultramicro Kjeldahl technique
about 4 N, are layered below the digest. In order to prevent contact of the alkali pipette with the vessel neck, a perforated glass-thimble is first placed over the vessel mouth. The pipette can then be accurately guided by the edge of the perforation. Finally, by means of a constriction pipette a measured volume, about 45 p.1, of 0.004 N receiving acid is placed in the neck to form a column about 3 mm long. The acid column is positioned in the upper portion of the neck of the diffusion vessel so that following subsidence of the heat of neutralization, it does not move too far down the length of the flask neck. Initially, we used a lower concentration of acid since most of our nitrogen samples were in the range of 0.5 pg or less. We found, however, that the rate of ammonia absorption could be increased appreciably by raising the acid concentration. This also extends the upper limit of the range of the technique. The diffusion vessels are capped with small squares of Parafilm. After each film has been pressed in place, it is perforated with a fine needle to permit pressure equilibration during the diffusion process. The diffusion vessels are then secured in a rotating stand. This stand consists of a thin aluminium disc to the rim of which 18 spring clips have been radially fastened. The clips are mounted so that the vessels are held at right angles to the plane of the disc. The effective diameter of the rotating stand is 25 cm. The vessel stand is next mounted on a horizontal shaft in a constant temperature incubator, at 4O”C, and rotated in a perpendicular plane at 30 rpm. Because of the rotation, the contents of the bulb are spread as a thin film over the bulb surface. Diffusion of the ammonia from bulb to acid is, therefore, very rapid and is complete in as little as one-half hour as compared to two hours required for stationary vessels.
Titration Titration of the receiving acid in the diffusion vessel is done with standard 0.01 N NaOH delivered from a 10 ~1 capacity Greiner microburette graduated in 0.01 ~1. The diffusion vessel is held during the titration in a holder made from a segment of rubber tubing. The rubber holder is mounted on the tongue of an electric vibrating massager, and the amplitude of vibration is controlled by means of a Powerstat voltage regulator. The entire vibrator is fastened to a rack and pinion mount, so that the film can conveniently be brought into contact with the burette tip. The above arrangement provides excellent Experimental
Cell Research 7
150
IL J. Boell and S. C. Shen
stirring by vibration and eliminates the necessity of inserting anything, such as a magnetic flea or glass filament, into the thin film of acid being titrated. A color standard is set up in another diffusion vessel, and the two films are illuminated by a narrow beam of light from a fluorescent source. Color matching with this arrangement is easily done, and the maximum end point error is of the order of 0.03 ~1, corresponding to about 0.004 pg N (Table I). TABLE I Titration
of 44.7 pl ca. 0.002 N H,SO. with ea. 0.01 N NaOH (brom cresol green as indicator).
Sample
~1 NaOH
Deviation
8.57 8.55 8.55 8.53 8.58 8.52 8.53 8.53 8.56 8.55 Mean
I
from
mean
+0.02 0 0 - 0.02 +0.03 - 0.03 - 0.02 - 0.02 f0.01 0
8.55
Standard deviation. of mean & 0.02 ~1.
The nitrogen of the digested sample is obtained by content of the aliquot used for diffusion by a dilution used in calculating the dilution factor represents the to the digestion flask at the completion of digestion in the digestion mixture.
multiplying the nitrogen factor. The total volume sum of the water added and the volume of acid
Tests of the method The effective lower limit of the method was tested by determining the recovery of known amounts of nitrogen from ammonium sulfate solutions (Table II). Th e ammonium salt was treated as organic material and “digested” for 3 hours before dilution and transfer to the diffusion vessels. Recovery of nitrogen source from 0.15 pg up is complete in all cases. Experimental
Cell Research 7
151
Ultramicro Kjeidahl technique TABLE II Recovery of known amounts of nitrogen. (NH&SO,
(0.03 ug N/PI) as N source w N
vol. sample Pl
expected
5 10 20 40
0.150 0.300 0.600 1.200
obtained 0.150 0.298 0.593 1.200
In addition to the test with inorganic nitrogen, parallel determinations of the nitrogen content of a purified caseinl preparation were made using the micro-Kjeldahl method of Parnas and Pregel and the present ultramicro technique. The difference in the amount of material used in the two methods is a hundred fold. Identical results were obtained with both techniques (Table III). TABLE III Comparison of nitrogen determination
by micro (M) and ultramicro
p1 sample
@l-M) Kjeldabl.
pg N recovered/sample
pg N/u1 sample
N source M Casein . . . . . . . . . . . Homogenate whole brain Optic lobe . . . . . . . .
1,000 2,400 1,150
1 ul-M 10 80 11.5
M 216 142 44
)
ul-M
M
2.16 4.75 0.436
0.216 0.0592 0.0383
1 ul-M 0.216 0.0594 0.0379
Similar tests of the method were also made on homogenates of parts of tadpole brain. Appropriate aliquots of the same homogenates were used for the micro and ultramicro methods. Excellent agreement in the results obtained by the two methods is indicated in Table III. For casein as well as tissue homogenate a three hour period of digestion was found to be adequate for complete recovery. 1 We are indebted to Dr. H. B. Vickery, Connecticut Agricultural sample of casein of known nitrogen and moisture content.
Experiment Erperimenfal
Station, for a Cell Research-8
E. J. Boell and S. C. Shen
152
SUMMARY
A modified ultramicro Kjeldahl technique has hfen described which is applicable to the determination of total nitrogen in the range from 0.1 pg and up with an accuracy of 0.005 g. REFERENCES
1.
D.,
BR~EL,
HOLTER,
H.,
LINDERSTRBN-LANG,
K., and
ROZITS,
K.,
Biochim.
et Biophys.
Acta,
1, 101 (1947).
2.
CONWAY,
E. J., Microdiffusion analysis and volumetric error. Crosby Lockwood and Son, Ltd., London. Chapters XIV and XV. 1947. D., Techniques of histo- and cytochemistry. Interscience Publishers, Inc., New York. Pp. 230-239,1949.
3.
CLICK,
Ezperimental
Cell Research 7
147
Cell Research, 7, 147-152 (1954)
AN IMPROVED
ULTRAMICRO E. J. BOELL
Osbom Zoological
Laboratory,
Yale
K JELDAHL
TECHNIQUE1
and S. C. SHEN University,
Received January
New Haven,
Conn., U.S.A.
28, 1954
FOR the determination of the amount of tissue used in biological and chemical work on an ultramicro scale, as for example in studies on the early embryo where the total amount of tissue available corresponds to approximately 0.5 to 5.0 pg protein nitrogen, no method has so far been devised which is superior to the conventional Kjeldahl technique. A number of good methods are available for determining total nitrogen in the range from 1 to 10 pg (2, 3), and recently Briiel, Holter, Linderstrsm-Lang, and Rozits (1) outlined a method for use in the range from 0.1 to 1 pg for which an accuracy of ca. 0.005 pg is claimed. There would be little justification for presenting a new technique were it not for the fact that the method herein described possesses certain features which make it superior, in many respects, to existing methods. The chief advantages of our method are: 1) relatively little specialized equipment is required, 2) the volumes of reagents are sufficiently large to make for ease in handling and to permit of duplicate or triplicate determinations on aliquots of the sample tested, 3) the time for digestion and distillation has been reduced so that a determination can be completed in 4 hours or less, and 4) the method is not only extremely sensitive but also extremely accurate. The method as described below is similar in principle to the conventional macro or micro Kjeldahl technique, so that a detailed account of procedure need not be given. Suffice it to say that tissue or tissue homogenate is heated with a sulfuric acid digestion mixture, that aliquots of the diluted digest are neutralized with alkali in specially constructed vessels, and that the ammonia freed is then permitted to diffuse into an acid film of known volume and strength so that the amount of nitrogen in the sample can be determined titrimetrically. Reagents The digestion mixture is prepared by adding to 100 ml of 50 per cent H,SO( (Mallinckrodt’s low nitrogen analytical reagent) one gram each of CuS04, K,S04, and SeO,. 1 Supported in part by a grant from The American mittee on Growth.
Cancer Society
through
the NRC Com-
Experimental
Cell Research’ 7
E. J. Boell and S. C. Shen
148
The receiving acid consists of 0.004 N H&SO,, containing 4 ml of 0.4 per cent bromocresol green per 100 ml. The standard alkali is 0.01 N NaOH. The solution is accurately standardized and protected from contamination and evaporation. A color standard, to serve as a titration endpoint, consists of 4 ml of 0.4 per cent bromocresol green in 100 ml acetate buffer at pH 4.6. In addition, ammonia-free water and saturated NaOH are required.
Digestion Digestion is carried out in straight-walled 10 x 75 mm Pyrex brand ignition tubes (glass No. 172). The nitrogen source, either tissue or tissue homogenate, is delivered to the bottom of each tube by means of a calibrated constriction pipette after which exactly 24 ~1 of digestion mixture are added. Suitable blanks are similarly prepared. The tubes are then pushed to a depth of about 2 cm into an electrically heated sandbath. They are supported in place by a perforated asbestos plate which by insulating the part of the digestion tube above it provides an effective but restricted zone of reflux. With this technique, even when the acid is made to fume strongly, no measurable loss of acid occurs. The time necessary for complete digestion was found to be about three ho,urs.
Diffusion After digestion has been completed, the tubes are cooled and a given volume, usually 200 ~1, of ammonia-free water is added to each. The contents are thoroughly mixed, and aliquots of known volume, about 90 ~1, are then transferred to a series of diffusion vessels. The diffusion vessel consists of a micro-florence flask whose dimensions are: inside diameter of bulb 10 mm, length of neck 22 mm, inside diameter of neck 5 mm. It can be easily constructed from tubing with a 5 mm bore. The neck of the diffusion vessel is coated with a thin film of paraffin wax (M.P. 72°C). Uniform pellets of wax are first prepared by dropping melted paraffin from a tine pipette onto a clean, cold glass plate. One pellet is placed in the neck, and the vessel is then maneuvered so that a rigidly-held heated silver wire can be inserted. The vessel is then rotated by hand with the result that melted paraffin is applied as a uniform coating along the entire length of the neck as far as the vessel bulb. Next 36 ~1 of saturated NaOH solution, giving a final concentration of Experimental
Cell Research 7
149
Ultramicro Kjeldahl technique
about 4 N, are layered below the digest. In order to prevent contact of the alkali pipette with the vessel neck, a perforated glass-thimble is first placed over the vessel mouth. The pipette can then be accurately guided by the edge of the perforation. Finally, by means of a constriction pipette a measured volume, about 45 p.1, of 0.004 N receiving acid is placed in the neck to form a column about 3 mm long. The acid column is positioned in the upper portion of the neck of the diffusion vessel so that following subsidence of the heat of neutralization, it does not move too far down the length of the flask neck. Initially, we used a lower concentration of acid since most of our nitrogen samples were in the range of 0.5 pg or less. We found, however, that the rate of ammonia absorption could be increased appreciably by raising the acid concentration. This also extends the upper limit of the range of the technique. The diffusion vessels are capped with small squares of Parafilm. After each film has been pressed in place, it is perforated with a fine needle to permit pressure equilibration during the diffusion process. The diffusion vessels are then secured in a rotating stand. This stand consists of a thin aluminium disc to the rim of which 18 spring clips have been radially fastened. The clips are mounted so that the vessels are held at right angles to the plane of the disc. The effective diameter of the rotating stand is 25 cm. The vessel stand is next mounted on a horizontal shaft in a constant temperature incubator, at 4O”C, and rotated in a perpendicular plane at 30 rpm. Because of the rotation, the contents of the bulb are spread as a thin film over the bulb surface. Diffusion of the ammonia from bulb to acid is, therefore, very rapid and is complete in as little as one-half hour as compared to two hours required for stationary vessels.
Titration Titration of the receiving acid in the diffusion vessel is done with standard 0.01 N NaOH delivered from a 10 ~1 capacity Greiner microburette graduated in 0.01 ~1. The diffusion vessel is held during the titration in a holder made from a segment of rubber tubing. The rubber holder is mounted on the tongue of an electric vibrating massager, and the amplitude of vibration is controlled by means of a Powerstat voltage regulator. The entire vibrator is fastened to a rack and pinion mount, so that the film can conveniently be brought into contact with the burette tip. The above arrangement provides excellent Experimental
Cell Research 7
150
IL J. Boell and S. C. Shen
stirring by vibration and eliminates the necessity of inserting anything, such as a magnetic flea or glass filament, into the thin film of acid being titrated. A color standard is set up in another diffusion vessel, and the two films are illuminated by a narrow beam of light from a fluorescent source. Color matching with this arrangement is easily done, and the maximum end point error is of the order of 0.03 ~1, corresponding to about 0.004 pg N (Table I). TABLE I Titration
of 44.7 pl ca. 0.002 N H,SO. with ea. 0.01 N NaOH (brom cresol green as indicator).
Sample
~1 NaOH
Deviation
8.57 8.55 8.55 8.53 8.58 8.52 8.53 8.53 8.56 8.55 Mean
I
from
mean
+0.02 0 0 - 0.02 +0.03 - 0.03 - 0.02 - 0.02 f0.01 0
8.55
Standard deviation. of mean & 0.02 ~1.
The nitrogen of the digested sample is obtained by content of the aliquot used for diffusion by a dilution used in calculating the dilution factor represents the to the digestion flask at the completion of digestion in the digestion mixture.
multiplying the nitrogen factor. The total volume sum of the water added and the volume of acid
Tests of the method The effective lower limit of the method was tested by determining the recovery of known amounts of nitrogen from ammonium sulfate solutions (Table II). Th e ammonium salt was treated as organic material and “digested” for 3 hours before dilution and transfer to the diffusion vessels. Recovery of nitrogen source from 0.15 pg up is complete in all cases. Experimental
Cell Research 7
151
Ultramicro Kjeidahl technique TABLE II Recovery of known amounts of nitrogen. (NH&SO,
(0.03 ug N/PI) as N source w N
vol. sample Pl
expected
5 10 20 40
0.150 0.300 0.600 1.200
obtained 0.150 0.298 0.593 1.200
In addition to the test with inorganic nitrogen, parallel determinations of the nitrogen content of a purified caseinl preparation were made using the micro-Kjeldahl method of Parnas and Pregel and the present ultramicro technique. The difference in the amount of material used in the two methods is a hundred fold. Identical results were obtained with both techniques (Table III). TABLE III Comparison of nitrogen determination
by micro (M) and ultramicro
p1 sample
@l-M) Kjeldabl.
pg N recovered/sample
pg N/u1 sample
N source M Casein . . . . . . . . . . . Homogenate whole brain Optic lobe . . . . . . . .
1,000 2,400 1,150
1 ul-M 10 80 11.5
M 216 142 44
)
ul-M
M
2.16 4.75 0.436
0.216 0.0592 0.0383
1 ul-M 0.216 0.0594 0.0379
Similar tests of the method were also made on homogenates of parts of tadpole brain. Appropriate aliquots of the same homogenates were used for the micro and ultramicro methods. Excellent agreement in the results obtained by the two methods is indicated in Table III. For casein as well as tissue homogenate a three hour period of digestion was found to be adequate for complete recovery. 1 We are indebted to Dr. H. B. Vickery, Connecticut Agricultural sample of casein of known nitrogen and moisture content.
Experiment Erperimenfal
Station, for a Cell Research-8
E. J. Boell and S. C. Shen
152
SUMMARY
A modified ultramicro Kjeldahl technique has hfen described which is applicable to the determination of total nitrogen in the range from 0.1 pg and up with an accuracy of 0.005 g. REFERENCES
1.
D.,
BR~EL,
HOLTER,
H.,
LINDERSTRBN-LANG,
K., and
ROZITS,
K.,
Biochim.
et Biophys.
Acta,
1, 101 (1947).
2.
CONWAY,
E. J., Microdiffusion analysis and volumetric error. Crosby Lockwood and Son, Ltd., London. Chapters XIV and XV. 1947. D., Techniques of histo- and cytochemistry. Interscience Publishers, Inc., New York. Pp. 230-239,1949.
3.
CLICK,
Ezperimental
Cell Research 7