chapter 4 spectrophotometric determination cot·ip...

1 05

CHAPTER 4

SPECTROPHOTOMETRIC DETERMINATION OF NITROGEN IN ORGANIC

COt·iP OUN DS

4.1 Introduction

Elemental analysis is the first step in the 1 uci~~

tion of the structure of a~ of~anic compound. l

yeare.,it has been supplemented by various spectr

studies such as UV, IR, NMR, mass epectra,etc. 6 Vt: l ;

the instrumental techniques have not yet been dev£- 3pe~

sufficiently enough to alter radically the deper

the organic chemist on the classical methods.

unusual for new compounds to be described in the litera-

ture without at least three elemental or functional

group analyses being quoted, regardless of the information

supplied by instrumental techniques. A rapid elemental

or functional group determination may provide all the

data necessary for routine control purposes.

At one time, reliable methods for the determination

of functional groups were few. However, a whole range of

the methods for functional group analysis is now available.

When these methods are applied in combination wi\h

1 06

elemental analysis and molecular weight determinations,

it is possible to assign structure to most of the organic

compounds without the help of instrumental methods.

Earlier deve~oped procedures of macroanalysis were

put on a sound basis for the determination of carbon end

hydrogen by Liebig and Glasner, for estimation of nitrogen

by Dumas and Kjeldahl and thei·determination of halogen

and sulphur by Carius in organic compounds. The principle

of these methods ~emains the same, although their

applications have been altered greatly.

The introduction of micromethods is the greatest

advance made in organic analysis. The elemental and

group analysis by macromethods is tedious end time

consuming. It requires large amount of sample for the

analysis. The advantages of the micromethods are the

small amount of sample (ca.20mg) for complete analysis,

as well as its rapidity.

Discussion still centres on whether the micro,

semimicro or macro scale of working is suitable for

routine analysis. Nowadays,rnacrornethods ere mainly used

in industry where sample homogeneity is doubtful.

Generally_, semimicro techniques requires less stringent

107

attention than the micromethods. The semimicro

procedures are often preferred over macro procedures

because of lesser space requirements, reduced equipment

and reagent costs. However, materials analyzed by

semimicro techniques must be finely ground to improve

the reproducibility of results. for the research

establishments, the micro technique is virtually indi-

spensible·.

Most of the methods for the estimation of nitrogen

154 155 in organic compounds are derived from Dumas, ' the

Kjeldah1156

and to a lesser extent the Ter Meulen157

•158

methods of analysis• In Dumas method, nitrogen in the

compound is oxidized in en atmosphere of carbon dioxide,

reduced to elementary nitrogen by heated copper and the

nitrogen measured gasanetrically in nitrometer. In

Kjeldahl procedure, nitrogen in the compound is converted

into ammonium sulphate by digestion with sulphuric acid

and a catalyst. Ammonia is determined by various methods

from the digest. Nitrogen in the compound is converted

to ammonia by hydrogenation with hydrogen and catalyst

is the basis of Ter Meulen technique and ammonia is

determined similarly as in the Kjeldahl method. Unfortu-

natelY, there is no single method which is applicable to

108

all organic compounds, because the type of nitrogen

compounds governs the products of degradation. For

example, nitro group in organic compounds will not be

converted quantitatively into ammonium sulphate by

I

Kjeldahl digestion and requires reduction prior to

digestion. Certain nitrogen containing hetercyclic

compounds form a nitrogenous charcoal in the Dumas f'

method, end thus, give low results for nitrogen content.

While compound with a fairly high boiling point

(over 360°C) cannot be successfully analyzed by Ter Meulen

method, since hydrogenation reactions are best carried out

below this temperature.

The exhaustive literature is available on develop-

rnents made in both the Kjeldahl and Dumas methods. In

many cases, the aim of these developments has been to

extend the scope of the methods. Inspite of these modi-

fications_,most of the laboratories still find it necessary

to use both the methods. Hydrogenation by the Ter Meulen

method seems to have received less attention.

4.2 ~ielda!!!_ ~itrogen Determination

In 1663, Kjeldah1156 described a procedure for

determination of nitrogen in a variety of substances.

This procedure involves destructive digestion of the

109

sample in hot concentrated sulphuric acid. The "organic

nitrogen 11 is converted into ammonium sulphate, followed

by distillation and titration of the ammonia produced.

Later, several modifications have been carried out in

the original method. The literature on the subject is

. d h . 1 159-163 Th d" reviewe ex austive y. e mo ified procedures

are applied to the analysis of a wide spectrum of

nitrogenous materials.

Kjeldahl method has a number of advantages over

the classical Dumas method, ~., speed and simplicity

of operation for application to aqueous solutions of

nitrogenous compounds e.g. serum, urine, plant extracts

etc., and accuracy with certain compounds which form

nitrogenous charcoals in the Dumas procedure. However,

there are certain disadvantages of this procedure,for

example, many compounds, particularly those in which

nitrogen is linked to oxygen or to nitrogen, need

special treatment which makes the Kjeldahl procedure

itself tedious and time consuming.164

Further there

are few compounds which do not give correct values for

· d · 164 Th f . l nitrogen content, e.g. pyri ine. ere ore, universe

applicability has never been claimed for the Kjeldahl

110

method. Several of the modifications have been

described which contribute to a better appreciation of

the best conditions to be use in order to obtain

satisfactory results for nitrogen content of more

refractory compounds.

In general, Kjeldahl procedure involves three

steps :

(i} Digestion of sample,

(ii) Separation of ammonia from digest,

(iii) Determination of ammonia.

(i) Digestion of sample

In the digestion step, the organic compound is

decomposed by treatment with hot concentrated sulphuric

acid and the nitrogen is converted to ammonium sulphate. 165

Extensive investigation by Friedrich et al., have

indicated that no single digestion method of universal

application will ever be found. The nature of sample

material defines largely the digestion technique and the

conditions to be applied in the particular case.

111

In early work, sulphuric acid wes used as the

digestion reagent by Kjeldahl.156 A mixture of

sulphuric acid and phosphoric acid in proper proportion

is used in the digestion of proteins.166

For digestion

of plant materials, a mixture of sulphuric acid and

perchloric acid is found ta b,. the most suitable digestion

reagent. However, care should be taken to avoid conditions

of high temperature and a high localized concentration of

hl . "d 167 perc ar1c ac1 • The addition of a dilute solution

of perchlori~ acid in concentrated sulphuric acid is a

convenient approach to overcome this difficulty.

A mixture containing hydrofluoric acid, phosphoric

acid, and potassium dichromate has been employed to

168 dissolve refractory metals and alloys. Most of the

hydrofluoric acid is removed by volatilisation prior to

distillation of ammonia.

Salt addition

The addition of neutral salt to raise the boiling

point of digestion mixture was introduced first by

Gunning169 in 1889. It is effective in increasing the

1 1 2

rate of digestion and is almost universally used.

High temperature digestion ensures recovery of nitrogen

in compounds which decompose at the boiling point of

concentrated sulphuric acid, and reduces markedly the

time required for complete digestion of samples. The use

of potassium sulphate in digestion is now widely accepted.

Salt : acid ratios (more~than 0.69 K2 so4 per ml of

sulphuric acid) gives high digestion temperature and

reduce digestion time. However, sa~ple handling diffi-

culties increase with high salt : acid ratio5, because

mixture tends to bump and splatters during digestion.

Moreover,the digest solidifies on cooling. The solidified

digests are often difficult to dissolve prior to ammonium

analysis. further, the solidification during digestion

results in loss of nitrogen from the digest. The use of

lower salt : acid ratios promotes easier digestion.

The digestion mixture containing 0.33 - D.Sg potassium

sulphate per ml of sulphuric acid is found to be the

b t 170 es •

Various other salts employed in digestion mixture

include sodium sulphate, dipotassium hydrogen phosphate

and phosphorus pentoxide.

113

Quantity of sulphuric acid required for digestion

is related to the amount of compound to be digested end

the salt added. This was investigated extentsively by

Self.171

i

It has been recognized that a mixture of sulph~ric

acid end potassium sulphate i' not sufficiently drastic

reagent for many compounds especially when the digestion

is carried out in open vessel. The severity of the reaction

has been increased by the addition of oxidizing agent or

catalysts or in some cases both.

A variety of substances have been investigated aa

possible catalysts in the Kjeldahl digestion process for

the purpose of decreasing the digestion time.

There exists a lot of confusion and lack of agreement

in literature on the role of catalysts. Osborn and Wilkie172

have studied extensively the role of catalysts in Kjeldahl

digest~on. They found mercury to be superior for protein

digestion, followed by tellurium, titanium, iron, copper,

selenium, molybdenumi vanadium, tungsten and silver in

that order. Out of these, only three metals Y!_!., mercury,

copper and selenium are widely employed in the Kjeldahl

digestion.

11 4

Mercury (as metal oxide or sulphate) has been used

since the origin of the Kjeldahl method.159

The prevail-

ing opinion is thet mercury is the best single

catalyst. 162 • 173 On the basis of a critical study, Hiller

174 et al., concluded "that mercury is the only Kjeldahl

catalyst, capable of yielding protein nitrogen values

comparable to those obtained by Dumas method. This

catalyst doe_s not cause loss cj:f ammonia and is, therefore,

very reliable. It is useful in the analysis of the

compounds whose behaviour towards more drastic agents is

unknown.

A disadvantage of mercury as catalyst is that the

mercury must be precipitated with alkali sulphide175

•176

or sodium thiosulphate176 • 177 before distilling off the

ammonia from the digest. Further,it increases environ-

mental pollution.

Copper (as metal oxide or sulphate) has been used

widely in the Kjeldehl digestion. However, its efficiency

159 is not as high as that of mercury. It has been claimed

that a mixture of copper sulphate and mercuric oxide is

more efficient than either of the catalysts individually.178

A comparative study for copper and mercury as catalyst

f d . t• f t . 179 d f t•1· 180 h b or iges ion o pro eins an er i izers as een

carried out. Both the catalysts are found to be equally

11 5

ff . . t Y . 1 81 d W 11 1 B2 h d e icien • esui an a ave use copper

sulphate and titanium dioxide as a catalyst. They

obtained satisfactory results in the analysis of foods

with low level of nitrogen.

The use of selenium as a catalyst was introduced

183 by Lauro. Sometimes, it is used in combination with

184 t.' copper and more often withrmercury. The mixture of

mercury and selenium is the most efficient catalyst

known in the Kjeldahl digestion160

•161

and many procedures

185-189 based on its use are reported. The enhancing

effect of mercury oxide on the selenium catalyst has been

. 190 191 explained. ' When the mixed mercury selenium

catalyst is used, there may be a loss of ammonia, if the

digestion is carried out for a longer time.186

Although,

selenium is toxic, it is less hazardous than mercury

and is effective in shortening of the clearing time.

However, the quantity of selenium must be kept as small

as possible. An excess of selenium causes the loss of

ammonia.

Bjarone192 has investigated antimony as catalyst.

He found antimony as effective as the usual mercury

catalyst.

1 1 6

T 11 . 193 d z· . d" .d 194 d e urium an 1rcon1um 1oxi e are use

successfully es catalysts in place of mercuric oxide.

A new and faster digestion procedure is described by

V L k t l 195 . d" . . . on enger en e a ., using igestion mixture contain-

ing hydrogen peroxide, selenium and sulphuric acirl.

Oxidizin~ Agents f'

Oxidizing agent is added into Kjeldahl digest in

order to reduce the digestion time. However_, the indis

criminate use of oxidizing agents to hasten the digestion

may promote the oxidation of ammonia. The use of oxidizing

agents is considered to be safe only in the presence of

sufficient amount of reducing agents. The violent oxidizing

agents such as persulphate, permanganate or perchlorate

f -d b" l" b"l"t 159,196-198 are o u 1ous app ica 1 1 Y•

In the early 1900's, potassium permanganate was

employed successfully as an oxidizing agent but its use

is undesirable due to the loss of ammonia during the

digestion. Perchloric acid is also not employed for the

same reason.

Out of all the oxidizing agents recommended,

hydrogen peroxide has been used widely. Minimal amounts

of hydrogen peroxide have been used successfully in

117

. 199-201 Kjeldahl procedure. Since hydrogen peroxide

contains often nitrogen containing stabilizers, the

nitrogen blank must be determined.

202 Hambleton and Noel reported that the use of

hydrogen peroxide in digestion mixture results in

excessive foaming and loss of sample nitrogen from

digestion tube. There appearsfto be little advantage

to the use of other oxidants in ·conjunction with

sulphuric acid for Kjeldahl digestion.

Re~ing ~~ent~

The Kjeldahl method is not directly applicable to

a number of compounds containing nitrogen in a hetero-

cyclic ring or nitrogen linked to oxygen or to a other

164 nitrogen atom. However, in most of these instances,

Kjeldahl procedure cen be applied after the compound

has been reduced.

A large number of reducing agents have been used

in the analysis of nitro compounds by Kjeldahl method.

I 1 . d.f. t• 203 h 1 dd d t th n an ear 1er mo 1 ica ion, p eno s were a e o e

digestion mixture followed by a reducing agent such as

zinc dust. Cope 204 recommends the use of salicylic acid

1 1 8

in place of phenol. Normally, determination of nitrates

and of organic nitre compounds involves pretreatment with

salicylic acid-sulphuric acid mixture. The resulting

5-nitro salicylic acid is then reduced with sodium thio-

205-208 sulphate to form the •amino compound. Water interferes

with the nitration reaction, and therefore, the samples

must be moisture free. The use of thiosalicylic acid . ,. 209-210

eliminates the need of thiosulphate. The method

is not suitable when the sample contains

211 nitrate ion in 1:3 ratio or greater.

chloride and

The intereference of chloride ion in the above method

h b . d d . 212 R as een overcome by employing re uce iron or aney

nickle alloy213 (containing 403 nickle, 10% cobalt and

50% aluminium) as reducing agent. Both these reducing

agents have given excellent results. The reduced iron

method is slightly shorter than that in which the Raney

nickle alloy is employed as reducing agent.

A chromium reduction method introduced by Gehrke

et al., 214 is short and gives accurate results with sample

containing high chloride-nitrate ratio.

215 AOAC recommends pretreatment of the sample with

chromiurn:hydrochloric acid before proceeding with the us~al

digestion.

119

216 217 . Recently, Devarda's alloy ' is proposed as

reducing agent for total nitrogen determination, including

nitrate in the semimicro Kjeldahl procedure.

for nitrogen determination in compound such as '

azines, hydrazones, oximes and semicarbazones, the sample

dissolved in acetic acid methanol mixture is reduced by

216 zinc-hydrochloric acid follow'd by digestion as usual.

The nitrogen of nitrile compounds has been determined

by the strong reducing action of hydroiodic acid prior to

Kjeldahl digestion.165

The hydroiodic acid formed~~ in the reaction between sulphuric acid and potassium

iodide has also been employed to reduce nitrile. 2 ~ 9

However_, this hydroiodic acid method requires complete

removal of iodine before digestion which makes the procedure

tedious and time consuming. Vanetten and Wiele220 have

reported the successful determination of nitrogen in

nitriles by the use of the ordinary Kjeldahl digestion

using selenium catalyst without the addition of other

reducing agents.

Azide nitrogen has been determined by adding sodium

thiosulphate to sample before digestion. 221

120

Procedures based on digestion of the organic compounds

with concentrated sulphuric acid at high temperatures in

1 d t b h b t d '·'hi"te and Long222

have sea e u e ave een repor e • "

determined nitrogen on the micro-scale in heterocyclic

compounds using mercuric oxide 0

catalyst at 470 c. 223 f

Baker applied ,the method to nitro compounds by introducing

reducing agents such as glucose' or thioselicylic acid into

the digest. The procedure involving digestion in sealed

tubes has been employed in the estimation of nitrogen on

1 224-226 the microgram sea e. The most suitable temperature

c for digestion was found to be in the range of 400-450 c.

Below 38D0 c, the digestion was incomplete, while above

0 . 224 227 500 C, loss of ammonia occurred. '

Mechanism of digestion

Inspite of the lapse of more than hundred years

since the inception of the Kjeldahl method and the hundreds

of paper published on this subject, there is little

accurate information on the nature of the breakdown of

organic compounds taking place in the Kjeldahl digestion.

It is believed 228 - 229 that, the first stage is the

1 21

oxidation of the organic material and the final is

hydrolysis to . 228

ammonia. However, there are instances

where ammonia is formed without oxidation, for example, the

~ction of phosphoric acid on glycine or sulphanilic acid.

Quartaroli229 considers that the digestion includes two

steps (i) oxidation of the organic material and (ii)

reduction of the 'nitrogen' to ammonia. Strong oxidizing

agents are injurious to step (ii). from a study of the

action of concentrated sulphuric acid on some simple

amines in the presence or absence of catalysts, it has

been concluded that the groups attached to nitrogen 230

are successively removed and replaced by hydrogen atom.

~igestion_~

The duration of digestion depends on the size and

chemical character of the sample, and nature of the

catalysts and oxidizing agents. Digestion time may vary

from a few minutes to more than several hours. The organic

nitrogen is not always completely converted into ammonium

sulphate when the digest has become char free. Beet231

has actually isolated certain acids from the char free

digest of coal. Therefore, it should be noted that

clearing time is not the digestion time. In many cases,

a considerable •after-boil' is necessary to obtain

complete conversion of nitrogen to ammonia.

122

(ii) Separation of ammonia

Separation of ammonia from the digest is achieved

by distillation, aeration or diffusion. Distillation is

the conventional method to separate ammonia from the

Kjeldahl digest.

In the macro scale metho,, ammonia after the addition

of excess of alkali to the digest is distilled, using a

.trap to prevent alkali being carried over. The distillate

is collected in an acidic solution. In the semimicro end

micro-scales procedure, steam distillation is generally

carried out for the separation of ammonia from alkaline

digest.

Aeration166 • 232 method for the isolation of ammonia

involves the treatment of the digest with a saturated

potassium carbonate or sodium hydroxide solution and sweep-

ing the liberated ammonia by means of an air current.

The disadvantage of the method is that, the ammonia which

is in the form of amine sulphate in the digest, is not

recovered.

123

The micro-diffusion method of ammonia separation in

a closed system is especially applicable to the spectra-

photometric determination of a small amount of ammonia.

The Conway micro-diffusion technique has been extensively

d 142,233 5 . 1 d"ff . 234,235 d . 1 use • imp e 1 usion proce ure is a so

applied for ammonia separation.

(iii) Determination of Ammonia

Ammonia is determined after its separation from the

digest or directly in the digest.

(a) Distillation method

The distillate of Kjeldahl digest is collected into

standard acid end measuring the excess of acid by titrating

with standard elkali. 236 Originally Kjeldah1156

proposed

the iodometric estimation of the excess of the standard

acid, but due to interference from the carbon dioxide, 237

the method was not favoured. Ballentine and Gregg

used potassium biiodate to determine ammonia by iodo-

metric titration. In another method, the ammonia in the

distillate is oxidized with sodium hypobromite followed

d . d h b . t 189 by etermination of unuse ypo romi Bo

124

Winkler238

collected the distilled ammonia in boric

acid and titrated directly with standard acid. This method

is now commonly used for the estimation of ammonia in

Kjeldahl digest. 239 It requires only one standard solution

instead of the two, necessary for other methods. The only

disadvantage of the method is the buffering action of the f

boric acid which makes the end point less sharp than with

the back titration method.

As an alternative t~ titration method,the ammonia

has been collected in boric acid and the pH of the solution

is meesured.240

241 Appleton collected ammonia in boric acid, and

the conductivity of the resulting ammonium borate-boric

acid solution was measured. The amount of ammonia is

determined by referring to calibration curve. Wise242

distilled the ammonia into a boric acid solution and the

activity of the ammonium borate (NH~H 2B03 ) was measured ..

by using a cation electrode sensitive to ammonium ion and

ani_on electrode sensitive to borate ion (H2

Bo;).

Blom and Schwarz243 collected ammonia in a solution

of nickle ammonium sulphate (NiSD4 (NH 4 ) 2so46H 2 D).

The amine compound formed is titrated directly with

I

~

125

standard acid using methyl red as an indicator. In this

systemJloss of ammonia is negligible and the end point of

the titration is sharp.

Recently, ammonia after separation from Kjeldahl

digest has been determined by ammonia sensing electrode.

140,244-247 The technique is claimed to be relatively

inexpensive. nondestructive, jsnsitive and interference

free. The main advantage of the ammonia electrode is

that a large number of samples can be analysed in a short

time and total nitrogen concentration can be obtained

directly from a potentiometer calibrated for the conditions

used. However, considerable care must be taken to maintain

and to standardize the electrode to ensure reproducible

results.170

The ammonia in the distillate has also been

determined colorimetrically by the use of Nessler's

248 . 249 250 reagent and by the indophenol method. '

Nondistillation Methods

The major disadvantages of Kjeldahl procedure are

the time consuming distillation and titration step,

limiting the number of samples which can be processed

126

in a day. Many attempts have been made to eliminate

the distillation step in the Kjeldahl determination of

nitrogen. However, the presence of metallic ions, either

from the catalyst used in the digestion or present in the

sample material, interfer with the titration reactions.

Further, the type of nitrogen under examination may yield

a mixture of ammonium sulphate and amine sulphate. The

latter is not amenable to titf~tion·in situ~

Marcali and Rieman251

have modified the formal

titration method, in which the digest is neutralized

exactly and the mercury is complexed by the addition of

sodium bromide. Neutral formaldehyde is added to the

reaction mixture and the liberated acid is titrated

against standard alkali. The interference of calcium,

barium, cupric and phosphate ions is eliminated by the

addition of zirconium hydroxide.252

An another modifi

cation in the method has been suggested in which selenium

is employed as catalyst for the digestion.253

Harvey254

described the method in which ammonia

in neutralized digest is oxidized to nitrogen by hypo

bromite and the excess hypobromite is determined

iodometrically.

127

Belcher and Bhotty255

and Ashraf 256

et el., reported

hypochlorite-arsenite titration procedure. The neutralized

digest is treated with sodium bicarbonate end potassium

bromide to dissolve the precipitated mercury compound.

The ammon~a is oxidized with excess standard sodium hypo-

chlorite solution. The excess of hypochlorite is destroyed

with excess standard sodium arsenite. The excess of the

sodium arsenite is titrated with standard hypochlorite

solution using Bordeaux as indicator. This method has been

used on the semi-micro, micro and submicro scales.

Arcand and Swift96 described coulometric titration

of ammonia based on hypobromite oxidation principle.

Mercury and copper ion which are usually present in

Kjeldahl digest do not interfer in the titration.

However, selenium ion is reported to interfer in the

titration. Similar coulometric method is applied for the

. t. f . . t 257 estima ion o protein ni rogen.

The direct determination of ammonia in digest is

carried out colorimetrically by two most popular methods,

viz., Nessler's reagent and indophenol method.

I

~

128

Nessler's Method

N 1 ' . 258,259 . . ess er s reagent reacts with ammonia to

form a reddish-brown colloidal compound. Ammonia nitrogen

in concentration of 0.2 mg or less absorbs sufficiently

in the region from 400-425 nm.

Generally in the Nessleri.reaction,there are two

types of interference ~., (i) the appearance of a yellow

or green color and (ii) onset of turbidity in presence

of chemical substances~ 60 • 261 A number of aliphatic and

aromatic amines gives yellow color with Nessler's

reagent. Nessler reagent when exposed to vapour of

chloroform, ethyl acetate, methanol, n-butanol, isopropanol

acetone, methyl ethyl ketone, methyl isobutyl ketone,

aldehydes or diisobutyl ketone develops varying degree

of cloudiness or turbidity.261

-264

Longer time and

higher temperature of reaction also promotes turbidity. ++ ++ ++ +++

The presence of ions such as Mg , Mn , Fe or Fe

or those giving precipitate with iodide and sulphide

( ) t b"d"t 26\ 263,264 M

e.g. mercury causes ur 1 1 y. noreover,

stability of the reagent is also a matter of critical

importance.

129

Many modifications have been suggested for the

specific applications of Nessler's reagent.264

•265

Alkalinity of the reaction mixture must lie between

D.15 - 0.6N NaDH concentration. Sodium polyacrylate

is used as protective colloid and stabilize~, 266 while

the use of potassium

stabilizes the color

cyanide prevents turbidity

267 for sevefal hours.

and

In order to obtain reprod~cible results base

strength, time, temperature and sequence of the reagent

addition should be maintained properly.

Indophenol Method

The green color produced in the reaction of ammonia

with phenol and hypochlorite was described first by

Berthelot268 in 1859. The observation forms the basis of

indophenol method for the spectrophotometric determine-

tion of ammonia. This method has been studied extensi-

vely and various modifications in the procedure have

b t d f t . t t• R tl Searle269

een sugges e rom 1me o 1me. ecen y,

reviewed the reaction exhaustively.

130

Bolleter et al., 270 proposed a mechanism in which

the first step of the reaction is the reaction between

ammonia and hypochlorite to form monochloramine (25)

which then reacts with phenol to form quinone chloroimine

(26). Thus formed quinone chloroimine (26) reacts with

another molecule of phenol to form the indophenol (27)

which dissociates in alkali medium to give the blue· f

colored chromophore (28).

1 • NH3

+ OCl- --.--.) NH 2Cl

25

>

NCl

0

NCl

OH 26

N

0

27

N

28

1 31

Generally,sodium hypochlorite is used as an oxidizing

agent. Hypobromite, chlorine water or chloramine are also

employed as oxidizing agent. Nitroprusside or manganese

ion are usually present as cetalyst in the reaction

mixture. The color is extractable with organic solvents.

The reaction conditions such as pH, temperature,

reagents concentration, time fnd ord~r of addition of

reagents~etc. affect the sensitivity of the reaction.

In addition, a number of elements, compounds, high salt

concentration and exposure to light interfer in the

reaction. Various metals, nonmetals and ~itrogen compounds

interfering in the reaction are given in Table VIII.

The indophenol method has become popular for the

d t . t. f . . K. ld hl d · t 2 71- 282 e ermine ion o ammonia in JB a iges •

Automated versions of the indophenol method are

widely employed where large number of samples are to be

analyzed for nitrogen content. The majority of the

automated methods are based on the use of continuous

• (CF.) 282-285 . . h . . flow analysis A in whic the reaction vari-

ables are controllable and reproducible. Moreover, the

methods using automated discrete analysis286

and flow

injection anElysis {FIA) 287 methods have been described.

132

TABLE VIII

Compounds/ions interfering_!~ Indophenol Method

A. Compound/ions

1. Allantoin ••

2. Amines ••

3. Amides . f

• •

4 ~ Amino acids ••

5. Aminophenol ••

6. Alloxan ••

7. C~eatine, Creatinine ••

8. Cyanide ••

9. Ethyl carbamate ••

10. Hydrazine sulphate ••

11. Hydroxylamine ••

12. Nitrile ••

13. Protein ••

14. Thiocyanate • •

15. Thiourea ••

16. Urea ••

1 7. Uric acid ••

18. Sulphate ions, thiols, dimethyl Sulphoxide, Ascorbic acid. ••

References

290

270,291-300

290' 301

146,290,296, 300-314

315

290

290,316,317

311,313

290

299

304,318

297,299,300, 311,313,319

296

311,313

299,300

146,290,295, 320

290,317,320

300

133

19. Bromide ion •• 270,304,321

20. Fluoride ion • • 311,313

21 • Sulphide ion • • 295,311,313

22. Thiosulphate ion •• 299,311,313

23. Selenium ion • • 271,322

24. Copper ion •• 299,304,305, 322-329

25. f

Mercury ion • • 282,299,311, 313,319,322,330-332

26. Aluminium ion •• 311,313

27. Calcium, Barium ion • • 304,328

28. Cobalt ion •• 311,313,333

29. Chromium ion • • 300,323,333

30. Iron • • 323

31. Lithium ion •• 300,333

32. Magnesium ion • • 300,328,331

33. Manganese ion •• 270,299,304,323

34. Sodium ion •• 280,300,333

35. Nickle ion •• 311,313,331

36. lead ion •• 331

37. Zinc ion • • 270,304,333

134

Ammonia in digest is directly determined by

bispyrazolone method. 288 The red-violet color produced

is measured at 550 nm.

The other methods for ammonia determination in

Kjeldahl digest includes conductometric,89

polarogre-

h . 289 d t . b . 123 p ic an a omic a sorption spectroscopy.

,. The sensitivity of some of the methods used for

the analysis of ammonia has been recorded in Table IX.

\

1 35

TABLE IX

Ammonia

No. Method

I. Seectrophotometric

(i) Indophenol method

(ii) Nessler's method

(iii) Bispyrazolone method

II. Electrometric method

(i) Coulometric

(ii) Polarography

III. Ammonia sensing electrode

IV. Molecular absorption and emission spectroscopy

v. Atomic absorption spectroscopy

VI. Plasma emission spectroscopy

Sensitivity Reference ~m_c_g~/_m_l~~~~-~~~

0.100

2.000

0.025

10.000

i.000

1.000

0.200

1 .ooo

0.006

268-281

258-267

106,107, 288

96-100, 257

129,130, 289

117-120, 140,224-247

124-127

123

128

136

~~ectrcphotometric Determination of Ammonia Nitrogen-2!!

Kjeldahl Di~est

It is known that in Hantzsch reaction, yellow

colored compound 3,5-diacetyl-1,4-dihydrolutidine is

formed on reacting ammonia with acetylacetone-formal

dehyde reagent (Chapter 3). It was, therefore, thought

of interest to extend the applfcation of the reac~ion

to determine ammonium nitrogen in Kjeldahl digest.

The reaction conditions are modified suitably to

establish a method for the estimation of nitrogen in

Kjeldahl digest. In the present work, various condi

tions of the reaction such as the concentration of

reactants, pH, time and temperature of the reactionJetc.

are standardized to obtain maximum color intensity.

Pure samples of nitrogenous compounds are analysed

by the proposed method. The results are comparable

with those obtained by official method. The proposed

method is applied to assay the nitrogen containing

drugs in the dosage forms.

The application of the method has been extended

to the determination of nitrogen content in arninoacids,

protein formulations, foods, fertilizers and in organic

synthetic compounds.

and accurate.

The procedure is simple, rapid

137

4.3 Experimental

4.3.1 ~parat~

1. Double beam Beckman Model 25 spectrophotometer having

two matched cells of 1 cm. light path.

2. Systronic pH meter.

3. Constant temperature water bath {Townson and Mercer

Ltd., England).

4. Micro Kjeld2hl flask 10 ml and 30 ml capacity.

5. Volumetric flasks of 25, 50 and 100 ml capacity

made of corning glass. f

4.3.2 Reagents ~nd material

Ammonium Sulphate {BDH); Diammonium Phosphate (SD'S);

Ammonium Chloride (BDH); Ammonium Bicarbonate (BDH);

Urea (BDH); Sodium Hydroxide (Pellets) (BDH}; Hydrochloric

Acid (BP); Acetylacetone (SD'S); formaldehyde (BDH);

Sodium Acetate {BDH); Potassium Sulphate (BDH); Mercuric

Oxide (Yellow) (BDH); Selenium Dioxide (Laba); Copper

Sulphate (BDH); Sulphuric Acid (BDH) and double distilled

water were used in the study.

The reagent solution was prepared by mixing

formaldehyde (150 ml) and acetylacetone (78 ml) in

water and the volume was made to 1 litre with water.

138

4.3.2.2 ~reparation of sodi~~ate solution 1.!.t!l

Sodium acetate (77g) was weighed accurately and

dissolved in water and diluted to 1 litre with the same

solvent.

4.3.2.3 P_::eparatio~of standard ~mmoniurn sulphate solution

f' Ammonium sulphate (472 mg), previously dried at

105°C for 2 hours, was weighed 'accurately and dissolved

in and diluted to 100 ml with water.

4.3.3 Procedures

4.3.3.1 Standard procedure of digest!E.!!

To the standard ammonium sulphate solution (5.0 ml)

in a 10-ml microkjeldahl digestion flask was added

potassium sulphate (1g), yellow mercuric oxide (50 mg)

and concentrated sulphuric acid (2 ml). The flask was

heated on a naked flame vigorously for 30 minutes, and

cooled to room temperature. The contents of the flask

were diluted with water (10 ml),( place thin film of

vaseline on rim of flask) and transferred quantitatively

to a 100-rnl volumetric flask ~ith the help of water and

diluted to the mark with water. The final concentration

in the digest was 50 mcg of N per ml.

1 39

4.3.3.2 Determination of wavelen~th of maximum absorbance

To the standard ammonium sulphate digest (2.0 ml)

I

~ in a 50-ml conical flask was added sodium acetate solution

(3ml, 1M) and the reagent solution (4 ml). The flask

containing the reaction mixture was immersed in a boiling

water bath for 15 minutes and cooled to room temperature.

The contents of flask were tr,nsferred quantitatively to

a 25.ml volumetric flask with the help of water. The

volume was adjusted to the mark with water. The absorbance

of the colored solution was scanned on a Beckman Model 25

spectrophotometer in the range 350 to 550 nm against

reagent blank.

Maximum absorbance of the colored solution was

obtained at 412 nm (Fig.28).

4.3.3.3 Lambert-Beer's Curve

The standard ammonium sulphate digest (0.25, 0.5,

1.0, 1.5, 2.0, 2.5, 3.5 and 4.0 ml) were pipetted into

a series of SO.ml conical flask~and analyzed as described

under 4.3.3.2.

The absorbance of the colored product was measured

at 412 nm against blank (fig.29).

140

4.3.4 Factors affecting the reaction

4.3.4.1 Effect of the concentration of sodium acetate --(1M) solution -------

Different volumes of sodium acetate solution

(1 to 6 ml; 1M) were pipetted into a series of 50-ml

conical flaskscontaining standard ammonium sulphate

digest solution (2.0 ml). Th8j·reagent solution (4 ml)

was added to each flask and the reaction mixture was

analyzed as described under 4.3.3.2. Absorbance of the

colored reaction mixture was measured at 412 nm against

blank.

Maximum absorbance was observed in presence of 3 ml

of sodium acetate solution (1M} which remained constant

on increasing the quantity of sodium acetate solution.

In the present work, 3 ml sodium acetate solution was

used (Fig.30).

4.3.4.2 ~ect of concentratio!!._2.!._the reagen~-~tion

The absorbance of the colored products

obtained as above by the reaction of ammonium sulphate

digest solution (2.0 ml) and sodium acetate solution

(3 ml; 1M} with different volume of reagent solution

(1 to 6 ml) was measured at 412 nm against blank.

1 41

Maximum color intensity was obtained in the

presence of 4 ml of the reagent solution which remained

constant with further increase of the volume of reagent

solution (fig. 31 ) •

4.3.4.3 Effect ~f temperature

The color reaction of st~ndard ammonium sulphate f

digest solution (2.0 ml) with reagent solution {4 ml)

in presence of sodium acetate solution (3ml; 1M) was

carried out at different temperature (25°, 30°, 53° and

0 100 C} for 10 and 30 minutes. Absorbance of the reaction

product was measured at 412 nm against blank.

Maximum color intensity was obtained at 100°c

temperature (fig.32).

4.3.4.4 Effect of Time of reaction --The color reaction of standard ammonium sulphate

digest solution (2.0 ml) with reagent solution (4 ml) in

presence of sodium acetate solution (3ml; 1M) was carried

out in boiling water both for different time intervals.

Absorbance of the reaction mixtures was measured at

412 nm against blank.

142

Maximum color intensity was obtained when the

reaction mixture was immersed in boiling water bath for

15 minutes, which remained constant on further heating

(Fig.33).

4.3.4.5 Effect of catal~t used in Kjeldahl di~estion

The standard ammonium sulphate solution (5 ml) was

transferred to different 10-ml Kjeldahl digestion flasks

and treated with sulphuric acid (2 ml); sulphuricacid (2ml)

and potassium sulphate (1 g); sulphuric acid (2 ml),

potassium sulphate (1 g) and yellow mercuric oxide (50 mg};

sulphuric acid (2 ml), potassium sulphate (1 g) and copper

sulphate (50 mg); or sulphuric acid (2 ml), potassium

sulphate (1 g), and selenium dioxide (50 mg). The reaction

mixtures were digested as described under 4.3.3.1. The

different digests were analyzed as described under 4.3.3.2

by measuring the absorbance of the colored product at

412 nm against blank (Table X).

4.3.5.1 Analvsi~f Ammonium Sulphate

Ammonium sulphate equiv8lent to about 5 mg of

nitrogen was weighed accurately and transferred to a

10-ml Kjeldahl digestion flask and digested as described

under 4.3.3.1 and analyzed as described under 4.3.3.2

(Table XI).

143

4.3.5.2 Analysis ~f nitrogenous comeounds

Powdered drugs (Table XII) equivalent to nitrogen

(ca.5mg) was weighed accurately and transferred to a

10-ml micra-Kjeldahl flask. Sulphuric acid (2 ml), pota

ssium sulphate (1 g) and yellow mercuric oxide (50 mg)

' were added to it and digested as described under 4.3.3.1.

Analysis of digest was carried out as described under

under 4.3.3.2.

The amount of nitrogen was calculated by referring

to the standard curve (Table XII).

(a) Tablets

Twenty tablets were weighed and powdered. The

powder equivalent to nitrogen (ca.Smg) was weighed

accurately and transferred to a 10-ml micro-Kjeldahl

digestion flask and digested as described under 3.3.3.1.

The nitrogen content of the digest was analyzed as

described under 3.3.3.2 (Table XIII).

144

(b) Injectio~

Volume equivalent to nitrogen (ca.5 mg) was

pipetted into 30 ml Kjeldahl flask and concentrated

to about ( 5 ml) by heatipg. Sulphuric acid ( 2 ml),

potassium sulphate (1 g) and yellow mercuric oxide

(50 mg} were added to it and digested and analyzed as

described under 4.3.3.2 (Tablef XIII).

(iii) Amino Acids

Samples of amino acid equivalent to (ca,5 mg)

nitrogen were weighed accurately and transferred to a

microKjeldehl flask. It was digested and analysed as

described under 4.3.3.2 (Table XIV).

(iv) Proteins

Accurately weighed powdered sample of protein

equivalent to Cca,5 mg) nitrogen was transferred to a

10-ml microKjeldahl digestion flask and digested as

described under 4.3.3.1. The nitrogen content was

analyzed as described under 4.3.3.2 {Table XV).

145

(v) Fertilizers

Accurately weighed powdered sample equivalent to

(ca.5 mg) nitrogen was transferred to rnicroKjeldahl

flask. The sample was digested and analyzed as described·

under 4.3.3.2 (Table XVI}.

(vi) Anal~sis of s~nthetic o:o/Janic compounds

Sample equivalent to (ca.5 mg) nitrogen was

weighed accurately and digested and analysed as described

under 4.3.3.2 (Table XVII to XXII).

146

4.4 Results and Discussion

Earlier, ammonium nitrogen in Kjeldahl digest

h b d . 271-281 as een etermined colorimetrically using Berthelot

d N 1 258,259 d B th d . an ess er proce ures. o proce ures require

alkaline media for the determination. The metal catalysts

used in the digestion, however, are known to interfer . 1·

in the estimation of ammonium nitrogen by these

th d 261-264,304-333 r · th d·t· f me a s 4oreover, e con 1 ions or

color development have to be rigorously controlled in

order to obtain reproducible results. The colorimetric

methods are desirable for the analysis of ammonium ion

in digest because they do not require the time consuming

distillation step prior to the ammonium determination

by conventional Kjeldahl procedure. They are also

rapid and adaptable to automation.

In the preliminary experiments, the reaction

conditions such as concentration of reagent (Fig.31)

and sodium acetate (Fig.30}, temperature {Fig.32) and

time (Fig.33) of reaction etc. were standardized in

order to obtain maximum color intensity of the reaction

mixture.

147

In the proposed method, ammonium digest was reacted

with acetylacetone-formaldehyde reagent (4 ml) in the

presence of sodium acetate solution (3 ml, 1M) in boiling .

water bath for 15 minutes. The pH of the resulting

mixture is about 5.5 to 6.D. The yellow colored product

formed has maximum absorbance at 412 nm (fig.28). The

color remains stable for 4 hours. The Lambert-Beer's f

law is obeyed in the concentration range of 1 to 6 mcg

of ammonium nitrogen per ml rif the reaction mixture

(fig.29). The optimum concentration range far the deter

mination as evaluated from the Ringbom plot83

was found

to be 1 to 6 ppm of ammonium nitrogen. The molar absorp

tivity in terms of nitrogen was found to be 1.4 x 103

L mal-1 cm- 1 and the photometric sensitivity84

of the . -2

color reaction was found to be D.01 mcg of nitrogen cm

at 412 nm. The standard and percent relative standard

deviation are found to be + D.893. and 0.8943,respectively

(Table XI).

The method was tested for the possible interference

of three catalyst namely mercury, selenium and copper.

None of them interfer in the color development under the

experimental conditions (Table X). The reaction mixture

is slightly acidic, so there is no risk of lass of

ammonia by volatilization. The method is simple, rapid

and accurate.

148

Pure sample of drugs were analysed by the proposed

and official method. The results ere in good agreement·

with those obtained by official methods,334

as well as

with calculated nitrogen content (Table XII). The proposed

method is applied to assay_ the dosage forms of the drugs.

The recovery of the drug by proposed procedure is in

good agreement with the label~~d amounts (Table XIII).

Six amino acids (Table XiV1, three samples of

protein and protein formulation, milk powder and cereals

were analyzed (Table XV) by the proposed procedure. The

results are comparable to those obtained by official

335 method. The proposed method was also applied to

determine total nitrogen content in diammonium phosphate,

urea and their mixtures used as fertilizers. Results

are in good agreement with those obtained by official

335 method (Table XVI).

Heterocyclic compounds containing different ring

systems are analyzed by the proposed method. The esti-

mated nitrogen is in good agreement with the calculated

nitrogen content (Table XVII to XXI).

It is known that the commonly used Kjeldahl

procedures do not completely recover nitrogen from

149

compounds containing N-N and N-0 bonds e.g. nitrites,

164 nitrates, oximes, azo, nitroso and nitro compounds.

Th . d t• . d. t• 203-221 ey require re uc ion prior to iges ion.

However, it was reported that organic nitro compounds

d t ' . d t• . to d' t' 223,256,336 o no require re uc ion prior iges ion.

Similar observation is made in analysis of nitre compounds

by the proposed procedure (Table XXII). They give satis-

f factory results under the experimental conditions.

o.s

0.4

m u D.3 c IO .0 H 0 en .0 <(

0.2

0 .1

350

Figure 28

150

400 450 500 550

Wavelength (nm)

Visible spectrum of the colored product

obtained on reacting Ammonium Sulphate

digest with reagent.

E c

N

«:1

+> ro

(l)

u c ro

.1J H 0 en .0 ~

D.7

0.6

D.5

D.4

D.3

0.2

0 .1

Figure 29

1 51

Nitrogen (mcg/ml)

Lambert-Beer's curve for nitrogen in Ammonium

sulphate digest.

152

o.6

o.s E c

N ..-~ D.4 +> ID

m u c ID D.3 .0 H 0 Ul .0 <(

0.2

0. 1

0 1 2 3 4 5 6

Sodium acetate (in ml)

Figure 30 Effect of sodium ac~tate (1M) concentration

on color intensity.

o.6

o.s E c

N

~ D.4

+> ru

(IJ

u c D.3 rn

.D H (l U)

.D <(

0.2

0. 1

0

Figure 31

1

153

2 3 4 5 6

Reagent (in ml)

Effect of reagent concentration on color

intensity.

154

• • • • After 30 min •

o.s 0 e o o f\fter 10 min.

0,4 E c

N ~

.:;:

+' n.3 IU

Ill 0 c ro .0 ·_;. 2 H 0 (/)

.0 <

u. 1

0 20 40 60 80 100

Temperature (°C)

Figure 32 Effect of temperature on color intensity.

155

\ I

D.6

o.s E c

N

'<::! 0.4

+> 1(1

Ql u c Ill D.3 .n H 0 en .0 <!:

0.2

0 .1

0 5 10 1 5 20 25 30

Time (in minutes)

Figure 33 Effect of heating time on color intensity.

156

TABLE X

I

\

1 H2so4 1 00 + 0.89 -

f 2 H

2so

4 + K2so4

100 +.0.90 -3 H

2so

4 + K2so4

+ H gO (Yellow) 1 DO + D.90 -4 H

2so4 + K

2so

4 + CuS0

4 1 00 + D.92 -

5 H2so4 + K2so4 + Se0 2

100 + 0.91 ---------------

a) Average of nine determinations.

157

TABLE XI

~lysis of Ammonium Nitrogen in Digest

~~--~------~~----~--~---~------~-------·--~-,· 1i Nitrogen Recovery f?L No. Proposed method Off iciel method335

1 • 101.30 101 .60

2. 100.00 f 100.25

3. 98.50 99 .01

4. 98.90 99.23

5. 1 00 .1 0 100.21

6. 100.00 99.90

7. 99.60 100 .15

e. 99.35 98.50

9. 100.20 100.30

10. 101.21 1 DO .20

AV 99.92 99.93

SD + 0.893 + 0.857 - -RSD + 0.8943 + 0.858% - -

-

158

~ TABLE XII

~nal~sis of Nitro~en content of Drugs t

No. Name of drug Calculated ~ Found 8 ) . by it· N

Proposed Official method method334

1 • Paracetamol 9.25 9.28 9.20

2. Bemegride 9.02 B.96 B.98

3. Isoprenaline sulphate 5.03 4.99 5 .02

4. Chlorpropamide 10.12 10.19 10 .17

s. Sulphacetamide sodium 12 .12 11.94

6. Sulphanilamide 16.27 16 .21

7. Acetanilide 10.37 10.22

e. Trimethoprim 19.29 19.39

a) Average value of five determinations.

159

TABLE XIII

~nalysis of Drugs (contain~!!,g_~itrog~n) in~age forms

-~~~----------~-~~~~~~~~~~~~~---r~~---·--~~--

N o. Name Labelled .. ___ R_e_c_o~ry~ (in ma)~

I. Tablets

( i) Primidone

(ii) Imipramine

(iii) Tolbutamide

co~tent ol p d Official name drug repose 334 (in~->--~~~-~m~e_t_h_o_d·--~~~-m-e~t_h_o_d~-

250

25

500

249.22

25.46

487 .so

249.72

(iv) Methoin 100 99.83

249.93

25.22

486.55

1DO.00

250.45 (v) Chlorpropamide 250

II. Injection

( i) Bemegride 5mg/ml 4.97 4.96

a} Average value of five determinations.

160

TABLE XIV '

Anal~sis of Amino Acids .

No. Name Calculaifed % N Found 8 ) b.l__ 1i N Proposed Official

method method335

1 • Glycine 18.65 1 B .86 18.85

2. Lysinemono 15 .33 14.92 14.99 Hydrochloride

J. Glutamic acid 9.52 9.64 9.60

4. Arginine 32.15 29.17 29.11

s. Aspargine 18.65 18.98 16.65

6. Tryptamine 14.24 14 .15 14 .12


161

TABLE XV

Analysis of Proteins and Cereals

No. Name of substance iN fo~ndaJ by

Proposed Official method method335 --

1 • Pep tone 14.68 14.60

2. Casein 13 .so 13 .69

3. Albumin f 11 .26 11.35

4. Protein formulation 3.21 3.27

s. Milk powder 5.89 5.84

6. Wheat flour 2.12 2.13

7. Gramdal (flour) 2.98 3 .01


TABLE XVI

Analysis of Fertilizers

No. Name of compound ~ N founda) by

Proposed Official method method335

--~~-----~~·~~~~------~----~~~--~~

1 •

2.

3.

Diammonium phosphate

Urea

Urea + Diammonium Phosphate

20.95

46.67

25.55


20.98

46.69

25.56

162

TABLE XVII

Analysis of 2-[(4-Substituted ehen~hio or sulphonyleth~lJ

~~eno(2,3-d)p~rimidin-4(3H)-ones 337 for nitrogen content

0

No. R1 R2 R3 x Molecular Formula IJ{i N

Calculated Found by proposey method a

1 • CH 3 CH3 Cl s c16H15N2os2c1 7.99 7.96

2. CH 3 CH 3 CH 3 5 C17H1 BN2052 B.4B B.28

3. CH 3 CH 3 H 502 C16H16N203S2 B.04 B.03

4. CH 3 COOC2H5 CH 3 502 C 1 9 H 2 ON 2 O 5 S 2 6.66 6.83

s. CH 3 COOC2H5 Cl 502 C1eH17N20sS2Cl 6.36 6.57

6·. CH 3 CH 3 Cl 502 C16H15N20352Cl 7.32 7.69

7. - (CH2)4- H 502 C1BH1BN2°352 7.48 7.33

e. - (CH2)4- CH 3 502 C19H20N203S2 1.21 6.94

9. - (CH2)4- H 5 C1BH1BN20S2 B.18 e.19

10. - (CH2)4- CH 3 s C19H20N2052 7.BS e.20

a) Average value of three determinations.

163

TABLE XVIII

Anal:t:sis of 2-D 4-substi tuted~enyl) thio or sulehon~lethyl]

quinazoline-4-(3H)-ona~337 for nitro~en content

No. R x Molecular formula 1i N

Calculated Found by proposey method 9

1 • H s C1BH14N205 9.92 10.16

2. Cl 502 c18H13N203SC1 B.03 B.32

3. H 502 C18H14N203S B.91 9 .01


164

TABLE XIX

~~~~is of 3-Aralk~-2-mercapto-~-substitut~d:~henylp~~azolo

i3,4-d)pyrimidi~-4(3H)-one 338 for n~troge~~nt

~----- --No. R1 R2 R3 Molecular formula % N

Calculated Found by propose~ method 8 -- ----

1 • p-CH 3-c6H4 H H C1BH14N405 16.75 16.69

2. p-OCH3C6H4 H H C1BH14N4025 15.98 16.02

3. p-DC2H5c 6H4 H H C 19H16N4 02 S 15.36 15.33

4. C6H5 H H C17H12N405 1 7 .so 17.61

s. p-Br-C6H4 H H c 17H11 N405Br 14.03 1 3. B9

6 •. c6H5 CH 2- H H C18H14N405 16.75 16.48

7. C6H5 H SCH 3 C1BH14N 4° 52 15.28 15.15

8. m-CH 3-c6H 4 H SCH 3 C19H16N40S2 14.70 14 .89

9. p-OCH 3-c6H 4 H SCH

3 C19H16N402S2 1 4 .1 3 1 4 .14

10. p-OC 2H5-c6H4 H 5CH 3 C20H18N402S2 13 .64 13. 70

----- -- -- -- ---

165

---- ---No. R1 R2 RJ Molecular _-1L!! __

Formula Calculated Found b

\ propose

-- method 8 -------I

11 • p-Cl-C6H4 H SCH 3 c1aH13N4DS2Cl 13.98 1 4. 26

1 2 • p-Brc 6H 4 H 5CH 3 c 18H13N40s2Br 12.57 12. 71

13. o-CHjC 6H4 -CH 2COOH 5CH 3 c~·1H1BN4 03 52 12. 77 12.47

14. p-Cl~C 6H 4 -CH 2COOH SCH 3 C20H1SN40352Cl 12. 21 12.34

1 5. o-OCH3c 6H4 CH 3 5CH 3 C 2 OH 1 8 N 4 O 2 S 2 13.64 1 3. 78

1 6. p-BrC6H 4 CH 3 SCH 3 c19H14N40S2Br 12.23 11 • 91

1 7. C6H5 -CH 2CDOH H C19H14N4035 14. 79 15.21

1 8 • p-DC 2H5C 6H 4 -CH 2CDOH H C21H18N404S 13.25 13.25

1 9 • p-DC 2H5- c6H4 CH3 H C20H18N4025 14.BO 14.92

20. p-CHjC6H4 CH

3 H C19H16N405 16.07 16.39

-- -------------a) Average value of three determinations.

166

TABLE XX

An~!..Y,sis of 2t3-Substituted ~.!:!.2(2,3-d)eyrimidin-4(3H)-ones 339 • 34~for nitrogen content

.. ·

--- -- -- --No. R1 R2 R3 R4 Molecular 3 N

Formula Calculated Fouiidby proposey method 8

-----1 • CH 3 CH

3 -CH 2c6H5 o-CH 3 ~H 4 C22H22N205 7.65 7.83

2. CH 3 CH3 -CH 2-~N-CODC2H 5 H C16H24N40JS 1 6 .16 16.29

3. -(CH2)4- OH C6H5 C16H1402N25 9.39 9.40

4. -(CH ) -2 4 -CH 2 CH

2N(C 2H

5) 2 H C26H230N35 13.77 1 3. 70

5. -(CH2 ) 4- -CH2c

6H5 C6H5 C23H 20N 2 OS 7.52 7.27

----a) Average value of three determinations.

167

TABLE XXI

for nitrogen content

H 5 c 2 o 2 C-CH-f~N

~~

N}R 3 s

No. R1 R2 R3 Molecular % N

Formula Calculated Found by propose~ method 8 -- -----

1 • CH3

CH 3 C6H5 C 1 9 H 1 7N 3 O 2 S 11 • 9 6 11 • 78

2. C6H5 H H C17H13N3025 1 3. DO 12.92

3. C6H5 H C6H5 C23H17N3D2S 10. 52 10.39

4. -(-CH ) -. 2 4 H C15H14N3D2S 13 .95 14.29

-- ---a) Average value of three determinations.

1 68

TABLE XXII

No. Name of compound

1 • Chloramphenicol

2. Metronidazole

3. 3-Amino-5-tert-butylamino-4-carbethoxy-2-nitrothiophene342

f ---~N Calculated

6.70

23.54

14.63


Found by proposey method 8

B.52

24.23

14.49

chapter 4 spectrophotometric determination cot·ip...

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