- Email: [email protected]
Ammonia determination: Reagent modification and interfering compounds
SHORT
243
COMMGKICATXOXF
ACKNOWLEDGMENT This investigation was supported the National lnstitutrs of Health.
in part by U. S. P. H. S. Grant ES-00141 from
REFERENCES 1. PETROW.
C. P., PATT, H. H.. ANLI K~.~IR,P. P., In.ter~z. J. Appl.
Radiatiow
Isotope
16, 599 (1965). 2. DULCIXO,
J., Bosco,
R., \'ERLT, R. G., .IND M.4rsrs.
J. I~., C&L C&m. Acta 8, 58
(1963). 3. MEaDE, R.. C.. AND SITGLITZ, R.> Intern. J. Appl. Rudtition Isotopes 13, 11 (1962). 4. HANSEN, D. L., AND BUSH, E. T.. A~nl. Biochem. 18, 320 (1967). 5. EDWARDS, B., AND KITCHENER. J. ,4.. Intern. J, Appl. Rntlintion Isotopes 16, 445 (1964). 6. WOELLER, F. H., Anal. B&hem. 2, 508 (1961). 7, BOGGIOLINI, M., AKD BICGEL, M. H., A&. Biochem.. 14, 290 (1966). 8. DUNCOMMBE,W. G., ASD RISING, T. J., Anal. Biochwr~. 30, 275 (1969). 9. BRAP, G. A., Ad. Rio&em.. 1, 279 (1660). 10. PROCKLOP,D. J.. AND EBERT, P. S,, Anrrl. Bioch.em. 6, 263 (1963). 11. PATTERSON, M. S., .~ND GREEN, R. C., Awl. Chem. 37, 854 (1965). 12. MOHIN, R. J., AND CARRION. M., Lipi& 3, 349 (1968). I.
I'.
Af0sTA~h
ELIZEBETH
S. C.
FALLIN
FANG
Deparbment of Agticulbural Chemistry Oregon State University CorvalZk, Oregon $7531 Rrceir~rJ Jnnrcary %, 1970
Ammonia Reagent
Modification
Determination: and
tnterfering
Compound,s’
The phenol-hypochlorite reaction for the determination of ammonia is rapid and easy, and requires only two solutions (1, 2). Weatherburn (2) recently reported optimum reagent concentrations and time-temperature color development conditions for the procedure. Using his procedure (2) to determine the ammonia concentration in biological saSmples,we ob’ Contribution No. 105, Department of Biochemistry, Kansas Agricultural ment Station, Manhattan, Kansas 66502. This investigation was supported by Natioral Institutes of Health Grant FR07036.
Experiin part
FIG.
1. Effect. of pH on a~bsorbanw
of ammonia
nitrogen
color
complex
nitrogen). ’ Lab-Line Instruments, Inc., Melrose Park, Illinois. 3 Bausch & Lomb, Inc., Rochester, New York. 4 Model 12 research pH meter, Corning Glass Works. Corning.
Nrw
York.
(3 pg
SHORT
AMMONIA-
245
COMMUNICATION8
NITROGEN CONCENTRATION
(FQ)
FIG. 2. dmmonis
nitrogen standard curves without phosphate buffer in reagent B (a> with phosphate buffer in reagent I3 (O), and with phosphate buffer in reagent B in the presence of 50 @moles HEPES (0).
tories to calorimetrically quantitate total nitrogen as ammonia in small samples of biological material (20 mg muscle) after digestion with sulfuric acid (18 ;I’,) and hydrogen peroxide, neutralization of 90% of the sulfuric acid with sodium hydroxide, and appropriate dilut’ion of the digest. Many reagents commonly used as buffers in biological research were found to influence tile absorbanceof the nitrogen color complex (Table 1). In addition to the reagents listed in Table 1, 250 pmoles of sucrose, 175 pmoles of KCI, and 1 ml of Krebs Ringer bicarbonate (KRB) buffer (4) inhibited the color development of 3 pugof ammonia nitrogen 6,-9, and a%,, respectively. Trichloroacetic acid (50 mg per reaction) and methyl orange (1 pg per reaction) had no effect, whereas hydrogen peroxide (2.5 pmolea per reaction) completely inhibited the color development of the reaction. In practice, the hydrogen peroxide used during the digestion of the biological material was dissipated by hea,ting the digest 30 min after it had turned colorless, HEPES (50 pmoles per react,ion) did not affect, the linearity of t.he st,andard curve (Fig. 1)) even though the sensitivity of the react,ion in the presence of HEPES (50 pmoles per reaction) decreased 22% (Table 1). A similar phenomenon was observed in the presenceof 1 ml of KRB buffer. Reagent A was found to be stable for at least 4 weeks whereas reagent B was stable for only 2 weeks when stored in amber bottles a.t refrigera.tor temperatures (2-4°C). These findings support t’hc recommendations of Weatherburn (2)”
Hydrasine Monoetharlolarrline m!sJ TES” ‘I‘ricene’j Triethanolamine
!r\-l iis --ti IO0 100 I
B. K.
WHITTEN
243
COMMGKICATXOXF
ACKNOWLEDGMENT This investigation was supported the National lnstitutrs of Health.
in part by U. S. P. H. S. Grant ES-00141 from
REFERENCES 1. PETROW.
C. P., PATT, H. H.. ANLI K~.~IR,P. P., In.ter~z. J. Appl.
Radiatiow
Isotope
16, 599 (1965). 2. DULCIXO,
J., Bosco,
R., \'ERLT, R. G., .IND M.4rsrs.
J. I~., C&L C&m. Acta 8, 58
(1963). 3. MEaDE, R.. C.. AND SITGLITZ, R.> Intern. J. Appl. Rudtition Isotopes 13, 11 (1962). 4. HANSEN, D. L., AND BUSH, E. T.. A~nl. Biochem. 18, 320 (1967). 5. EDWARDS, B., AND KITCHENER. J. ,4.. Intern. J, Appl. Rntlintion Isotopes 16, 445 (1964). 6. WOELLER, F. H., Anal. B&hem. 2, 508 (1961). 7, BOGGIOLINI, M., AKD BICGEL, M. H., A&. Biochem.. 14, 290 (1966). 8. DUNCOMMBE,W. G., ASD RISING, T. J., Anal. Biochwr~. 30, 275 (1969). 9. BRAP, G. A., Ad. Rio&em.. 1, 279 (1660). 10. PROCKLOP,D. J.. AND EBERT, P. S,, Anrrl. Bioch.em. 6, 263 (1963). 11. PATTERSON, M. S., .~ND GREEN, R. C., Awl. Chem. 37, 854 (1965). 12. MOHIN, R. J., AND CARRION. M., Lipi& 3, 349 (1968). I.
I'.
Af0sTA~h
ELIZEBETH
S. C.
FALLIN
FANG
Deparbment of Agticulbural Chemistry Oregon State University CorvalZk, Oregon $7531 Rrceir~rJ Jnnrcary %, 1970
Ammonia Reagent
Modification
Determination: and
tnterfering
Compound,s’
The phenol-hypochlorite reaction for the determination of ammonia is rapid and easy, and requires only two solutions (1, 2). Weatherburn (2) recently reported optimum reagent concentrations and time-temperature color development conditions for the procedure. Using his procedure (2) to determine the ammonia concentration in biological saSmples,we ob’ Contribution No. 105, Department of Biochemistry, Kansas Agricultural ment Station, Manhattan, Kansas 66502. This investigation was supported by Natioral Institutes of Health Grant FR07036.
Experiin part
FIG.
1. Effect. of pH on a~bsorbanw
of ammonia
nitrogen
color
complex
nitrogen). ’ Lab-Line Instruments, Inc., Melrose Park, Illinois. 3 Bausch & Lomb, Inc., Rochester, New York. 4 Model 12 research pH meter, Corning Glass Works. Corning.
Nrw
York.
(3 pg
SHORT
AMMONIA-
245
COMMUNICATION8
NITROGEN CONCENTRATION
(FQ)
FIG. 2. dmmonis
nitrogen standard curves without phosphate buffer in reagent B (a> with phosphate buffer in reagent I3 (O), and with phosphate buffer in reagent B in the presence of 50 @moles HEPES (0).
tories to calorimetrically quantitate total nitrogen as ammonia in small samples of biological material (20 mg muscle) after digestion with sulfuric acid (18 ;I’,) and hydrogen peroxide, neutralization of 90% of the sulfuric acid with sodium hydroxide, and appropriate dilut’ion of the digest. Many reagents commonly used as buffers in biological research were found to influence tile absorbanceof the nitrogen color complex (Table 1). In addition to the reagents listed in Table 1, 250 pmoles of sucrose, 175 pmoles of KCI, and 1 ml of Krebs Ringer bicarbonate (KRB) buffer (4) inhibited the color development of 3 pugof ammonia nitrogen 6,-9, and a%,, respectively. Trichloroacetic acid (50 mg per reaction) and methyl orange (1 pg per reaction) had no effect, whereas hydrogen peroxide (2.5 pmolea per reaction) completely inhibited the color development of the reaction. In practice, the hydrogen peroxide used during the digestion of the biological material was dissipated by hea,ting the digest 30 min after it had turned colorless, HEPES (50 pmoles per react,ion) did not affect, the linearity of t.he st,andard curve (Fig. 1)) even though the sensitivity of the react,ion in the presence of HEPES (50 pmoles per reaction) decreased 22% (Table 1). A similar phenomenon was observed in the presenceof 1 ml of KRB buffer. Reagent A was found to be stable for at least 4 weeks whereas reagent B was stable for only 2 weeks when stored in amber bottles a.t refrigera.tor temperatures (2-4°C). These findings support t’hc recommendations of Weatherburn (2)”
Hydrasine Monoetharlolarrline m!sJ TES” ‘I‘ricene’j Triethanolamine
!r\-l iis --ti IO0 100 I
B. K.
WHITTEN