ANALYTICAL BIOCHJZMISTSY 20, 16-23 (1967)

Spectrophotometrical Method for Determination

of Nitrogen in Biological Preparations Based

on Thymol-Hypobromite Reaction

L. 1. GLEBKO, J. I. ULKINA, AND V. E. VASKOVSKY

Institute of Biologically Active Substances, Siberian Department of the Academy of Sciences, Vladivostok 22, USSR

Received November 17, 1966

The determination of nitrogen is a routinous analyses used in the biochemistry of proteins, lipids, nucleic acids, and certain carbohydrates. Calorimetric procedures involving Nessler’s reagent (l-3) are most com- monly applied. The major disadvantage of the method is the fact that the colored complex does not form a molecular solution; moreover, the analysis cannot be performed in the presence of cations forming precipi- tates with alkali. Minute amounts of heavy metals produce a consider- able inhibition.

Other calorimetric techniques have been used in a number of recent studies (47). Among them, the phenol-hypochlorite procedure (8-10) seems the most advantageous; the sensitivity of this method is of the order of that involving Nessler’s reagent.

In 1939, Hansen and Nielsen demonstrated that the thymol-hypo- bromite reaction is more suitable than the phenol-hypochlorite one, and elaborated a procedure involving distillation of ammonia (11) for the determination of nitrogen in plant materials. This method, now widely used in inorganic analysis (12-14)) has not been applied since that time to biochemical analysis.

The present paper is concerned with the study of the effect of experi- mental conditions on the thymol-hypobromite reaction and of its ap- plicability to the analysis of various biological substances.

REAGENTS

All the reagents were of analytical grade. Water was twice glass-distilled in the presence of H,SO, (1 ml/liter). Commercial thymol was purified by vacuum distillation. An alkaline

solution of thymol was prepared daily by dissolving 2.265 gm of thymol in 8 ml 2 N NaOH and adjusting with water to 190 ml.

16

THYMOL-HYPOBROMITE REACTIOS Ii

-0.1 N hypobromite was prepared as follows: 1‘2 ml of water was added to 28 ml of 40% NaOH; the solution was cooled, treated dropwise with continuous stirring with 3.8 ml Br?, and adjusted to 1 liter with water. The concentration of hypobromite was determined as follows: 5 ml of the solution was added to 10 ml water, 0.2 gm KI, and 10 ml 10% H,SO, in a glass-stoppered Erlenmeyer flask, left for 10 min in the dark, and t’he liberated free iodine titrated with 0.1 iV Na,S,O,; 7.3-7.4 ml of t,hiosulfate solution must be consumed. The freshly prepared hypobromite solution was kept in t.he refrigerator for three clays before use; it is stable for about 4 weeks at 65”.

Ammonium sulfate was used as a reference substance; it was twice recrystallized from water and dried to a constant weight at 70-80”.

Standard solutions: Solution A, 100 y N/ml-O.4717 gm (NH,),SO, in 1 liter of water. Stable for 2 months. Solution B, 207 N/ml-5 times diluted solution A; a series of standard solutions were prepared from solution B containing 2.5, 5, 10, 20, 30, and 40 y of nitrogen in 5 ml. Solution B and the standard solutions used for measuring the calibration curve were used immediately after preparation.

EXPERIMENTAL RESULTS

Absorption Spectrw~~ of Indoth,ymoL

The absorption maximum of indothymol dye in t.he mixture n-butyl alcohol/methanol (24/l) ip at about X 686 rnp (Fig. 1).

02

Apel_--_ 500 600 700 000 900 1000 mp

‘iWave length

FIG. 1. Absorption spertnlm OI mdot,hynlt)l

18 GLEBKO, ULKINA, AND VASKOVSKY

The molar extinction coefficient (e) at this wavelength is 6.2 X 103. The range of nitrogen concentrations convenient for direct calorimetric measurements is between 1.12 and 56.25~ N/25 ml; precise measure- ments performed in the range 2.5-40 y provided the best reproducibility.

No changes in optical density of indothymol solution in n-butanol occurred during at least three days storage.

Reagent Ratio

The optimum conditions for quantitative binding of nitrogen in the form of NH,+ in indothymol critically depend on the amounts of thymol and hypobromite, on their ratio, and on the order of addition (Table 1).

TABLE 1 Dependence of Optical Density on Reagent Ratio

Reagent added, ml Optical density Ratio of reagents, thymol to

Thymol Hypobromite Thymol Control Test hypobromite

0.5 0.5 0.5 0.005 0.068 2:l 0.5 1.0 0.5 0.008 0.118 1:l 0.5 1.5 0.5 0.010 0.149 1:1.5 0.5 2.0 0.5 0.013 0.173 1:2

0.5 2.5 0.5 0.016 0.124 1:2.5 0.5 3.0 0.5 0.017 0.74 1:3 1.0 0.5 1.0 0.005 0.37 4:l 1.0 1.0 1.0 0.007 0.64 2:l 1.0 2.0 1.0 0.007 0.127 1:l 1.0 3.0 1.0 0.010 0.157 1:1.5 1.0 4.0 1.0 0.014 0.171 1:2

1.0 5.0 1.0 0.030 0.050 1:2.5

1.5 6.0 1.5 0.015 0.175 1:2

2.0 4.0 2.0 0.032 0.117 I:1 2.0 8.0 2.0 0.020 0.173 1:2

2.5 10.0 2.5 0.21 0.174 1:2

As can be seen in Table 1, the thymol-hypobromite ratio of 1~2 provides the maximum optical density.

The optimum ratio of reagents was determined by adding thymol twice 0.5 ml (Table 1). Addition of the same amount of thymol (once 1 ml) produces nearly a twofold decrease in optical density. Thus, by adding thymol twice 0.5 ml, the optimum density for 10 Y of nitrogen is 0.173 and, by adding thymol once 1 ml, the optimum density is 0.106; for 407 of nitrogen these values are, accordingly, 0.715 and 0.475.

THYMOL-HYPOBROMITE RE.4CTIOIX 19

TABLE 2 Comparat,ive Determinat,ion of Nitrogen Content by Three Independent Methods

Nitrogen content, y.

Jlaterials Dumas method Kjeldahl method Thymol-hypobromite

reaction

Valine Glutamic acid Alanine Sphingosin Cerebroside preparations:

first sample second sample third sample

DNA preparation

12.05 12.05 12.03 9.52 9.41 9.52

15.77 15.75 15.65

3.80 3 79 3 .7 4

2.10 I.95

1.74 1.67

2.08 1.93

15.40 15.32

2 07 I.66 2.00

15.26

Amount of Reagents

The amount of reagents is critical for quantitative yield of the re- action (Fig. 2) : 1 ml of thymol solution and 2 ml of hypobromite solution are enough for nitrogen amounts up to 30 7: at 70 7, this

pg Nitrogen

FIG. 2. Amount of reagents needed for different amounts of nitrogen. Standard solutions containing 2.5 to 100 y nitrogen were prepared (see text). Curve 1 was obtained by addition of 1 ml thymol solution and 2 ml hypobromite solution to standard solutions. Curve 2 was obtained with double the amount of reagents.

amount must be increased twofold. At the optimum ratio specified in the above paragraph, an excess of reagents does not produce an increase of optical density (curves 1 and 2 for amounts from 2.5 to 30 y N overlap, Fig. 2).

GLEBKO, ULKINA, AND VASKOVRKY

Time Required for Formation of Indothymol

Figure 3 shows that the minimum time for color development is 50 min at room temperature.

0.201

0.12

d 20 --L-%i--h 40

Time (minutes)

FIG. 3. Time required for formation of indothymol. Optical density of solution containing 1Oy of nitrogen and reagents was measured at time intervals.

Efiect of Temperature

An increase of temperature from 30” to 40°C results in both optical density increase and higher rate of formation of indothymol.

It is more convenient, however, to work at room temperature (22- 25°C) ; in this narrow temperature interval, temperature has no effect on the results.

Ejfect of Methanol

In the process of extraction of indothymol with n-butanol, drops of lower phase in the upper one produce a turbidity that interferes with calorimetry. The addition of methanol (l-l.5 ml) results in clarification of the solution. Addition of more than 2 ml of methanol, on the other hand, also results in a turbidity due to insolubility of indothymol; the effect increases with increasing indothymol concentration.

RECOMMENDED PROCEDURE

A solid sample (3-5 mg) is placed in a thick-walled combustion tube (1 X 12 cm) and 0.5 ml concentrated H,SO, added. If the substance is in solution, an aliquot of this solution is evaporated in the tube and 0.2 ml concentrated H,SOa is added.

Flame-seal the tubes, place in an oven, and heat at 400-41O”C for a time required for complete mineralization. Cool with solid carbon

THYMOL-HYPOBROMITE REACTIOii 21

dioxide, open the tubes, and heat for 5 min at 80-90°C to remove CO,, SO,, and other combustion gases.

Transfer into a flask and adjust to a known volume to a concentration of 2.5-40~ N/5 ml.

Place 5 ml aliquots into 50 ml extracting funnels and carefully neu- tralize with 2 N NaOH (methyl red as indicator). Add 1 ml of alkaline thymol solution and, immediately thereafter, 4 ml of hypobromite solution dropwise, with agitation. After 5 min add 1 ml of thymol solution and leave for 50 min. Extract indothymol dye repeatedly with 3-4 ml portions of n-butanol and transfer extracts into a 25 ml meas- uring flask containing l-l.5 ml methanol. Thoroughly mix and adjust to 25 ml with n-butanol.

Measure the optical density at 686 1~1,~ !optical cuvet length 1 cm/ with ?l-butanol as reference substance. Under the same conditions, prr- pare a control blank solution.

DISCUSSIOK

Lapin and Gein propose the use of thymol and hypobromite for the microanalytical determination of ammonia (15). Later Lapin studied this reaction in detail (16) and demonstrated that it involves three steps. The first step, more specific and typical, gives rise to p-amino- phenol. The second results in conversion of p-aminophenol to a halo derivative of quinonimide. The third step results in indophenol.

The use of thymol instead of phenol permits extraction of the dye formed into an organic solvent. The reaction is highly specific because, except for some primary amines, other compounds, both organic and inorganic, do not interfere with the determination (15, 16).

At present, the thymol-hypobromite reaction is extensively used fol the quantitative determination of nitrogen in steels (12, 13)) of ammonia in feed water (17)) and of nitrogen in titanium carbide and titanium boridc as well as in other hard-melting materials (14).

However, the papers mentioned specify different conditions of indo- thymol formation and various amounts and concentration of reagents. It seems that the relationship between the amount of reagents and nitrogen concentration has not been studied in detail although it is very important in the analysis of biological materials, in which the amount of nitrogen may widely vary. We have also failed to find the molar extinction coefficient in any paper.

Many authors point to the instability of alkaline hypobromite solution. This complicates the work, the calibration curve of every new batch of reagent must be measured.

Hashmi. et al. 118, 19) have found that hypohromittx solutlot,

22 GLEBKO, ULKINA, AND VASKOVSKY

prepared by mixing bromine and an appropriate amount of NaOH con- tains negligible amounts of bromites and bromates and is stable at an alkali concentration of 0.4-0.5 N. Our solutions prepared on the basis of this observation were stable for 4 weeks.

The alkaline solution of thymol becomes colored during storage and gives poor results. As the solution is simple to obtain, it is advisable to prepare it daily, immediately before use.

Amines, amino acids, albumin, preparations of sphingosins and cere- brosides, DNA, and RNA have been examined by this procedure. The preparations were of varying grades of purity and contained 3-18s of ash. The amount of nitrogen in the above materials was simultaneously determined by three methods: by combustion according to Dumas, by distillation of ammonia according to the classical Kjeldahl procedure, and by the thymol-hypobromite reaction (Table 2). The results show that the thymol-hypobromite reaction can be applied to the analysis of biological substances contaminated with reagents used during chromatog- raphy and does not require preliminary distillation of ammonia.

The method is especially convenient in serial analysis. Due to the stability of indothymol color in n-butanol, the calorimetry may be done the next day.

Statistical treatment of the results shows that the method has the same accuracy as that of Kjeldahl and, like the latter, gives rise to no ap- preciable systematic errors.

SUMMARY

Optimum conditions have been established of the thymol-hypobromite reaction with ammonium ion and the possibility has been demonstrated of its application to the quantitative determination of nitrogen in biological preparations.

A detailed procedure of the analysis is presented. The spectrophotometric method of nitrogen estimation based on the

thymol-hypobromite reaction provides the same accuracy as the classical Kjeldahl procedure and gives rise to no appreciable systematic errors.

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