Guide Kjeldahl

8/10/2019 Guide Kjeldahl

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Forward

This boo kle t has been deve loped to se rve as a techn ica l

com panion in the se lec t ion and use o f Kje ldahl n i t rogen

de te rm ina t ion m ethods and ap para tus . This mate r ia l hasbeen prepa red with the he lp of Kje ldahl ch emis ts , consul-

tan ts and Labcon co en gineers . I t wil l no t teach you to pe rform

the Kje ldahl pr ocedu re b ut will he lp you un ders tand s teps in

the pro cess . This gener ic pre senta t ion is des igned to he lp

educa te p er sons unfamil ia r with Kje ldahl m ethodology and

answer frequently asked questions raised by Kjeldahl users .





4

rec overy. Salicylic acid followed by sodium thiosulfate has

been used to pre trea t the mixture to ensure comple te r educ-

t ion . Other r ed uc t ion schem es have been devised . Or o ther

pre trea tmen ts have been used to pr event n i t r a tes from be ing

redu ced a t a l l dur ing the char r ing proce ss , leaving a c lea r

d iges t with n o co ntr ibu t ion from ni t r a te ions .

The Dist i l lat ionP r o c e s s

The acid digestion mixture is diluted

and made strongly alkaline with NaOH,

liberating NH 3 as follows:

Salt Additions

The pro blem with u s ing su lfur ic ac id a lone for d iges -

tion is very long digestion times result with many samples

due to the s low ra te of organic decom pos i t ion . The add i tion

of an inorganic salt to the digest elevates the boiling point of the H 2SO 4. The so lu t ion temper a ture of concen tra ted su lfur ic

ac id a lone is about 330 ° C. Addit ion o f a sa l t such as K2SO 4

can e leva te the so lu t ion tem pera ture of the d iges tion m ixture

to 390° C or m ore , depen ding on the ra t io of sa l t to ac id .

This s ignif icantly increases the rate of organic decomposition

in the digestion mixture, shortening the length of time

required for d iges tion .

There a re severa l p recaut ions to keep in m ind con-

cerning salt addition. First, it is possible to raise the solution

temper a ture of the d igest ion m ixture too m uch. I f the tem-

p e r a tu r e g o e s mu c h a b o v e 4 0 0 ° C d u r in g a n y p h a s e o f th e

diges tion , vo la t ile n i t rogen com poun ds m ay be los t to thea tmo s p h e r e .

Remem ber tha t as ac id is g radua l ly consum ed dur ing

the d iges tion pr ocess , for the var ious rea sons m entioned

abo ve, the salt acid r atio of the digest gradu ally r ises. This

mean s tha t the hot tes t so lu t ion tempera tures a re a t ta ined a t

the end of the d iges t ion . Hea t input , consum ption of ac id by

organic mater ial and vaporization, salt/acid ratio, digestion

length, and physical design of the Kjeldahl flask, are al l interr e-

lated. Each has an effect on the f inal solut ion temperature.

A second precaution is that if the salt/acid ratio is too

high , a cons iderable am oun t of ma te r ia l will “sa l t ou t” up on

cooling of the d iges t. Concen tra ted ac id pock e ts can be con-tained within the cake. These can react violently when con-

centrated base is added in the distillation process. A certain

amo unt of sa l ting out can be m anaged b y d ilu t ing the d iges t

with water while it is s till somewhat warm, but not too hot.

Catalyst Additions

Severa l catalysts have been em ployed by Kjeldahl

chem is ts over the years to increase the ra te of organic break-

down d ur ing the ac id d iges tion . Mercur ic ox ide has been the

most effective and widely used. However, mercury forms a

com plex with am mo nium ions du r ing d iges t ion . The add i tion

of sodium thiosulfate or sodium sulf ide af ter digestion and

before d is t il la t ion will b reak the com plex and prec ip i ta te

mercuric sulf ide. This is also important f rom a safety point of

view, as mercury vapor might escape to the a tmosphe re dur ing

the distillation process.

Because of environm enta l concern s over the handl ing

and d isposa l of mer cury, o ther ca ta lys ts a re com ing mor e

into favor . Many methods employ copper sulfate. Titanium

oxide and cop per su lfa te in com bina t ion h ave been found to

be more effective than copper sulfate alone. Selenium is fre-

quent ly used . Com mer c ia lly prepa red mixtures of po tass ium

sulfate and a catalyst are available from laboratory chemicalsuppl ie r s . Bulk cus tom mixtures a re a lso ava i lab le .

Nitrate and Nitrite Reductio n

Kjeldahl digestions do not always recover all forms of

n i t rogen in a sam ple . Nitra te and n i t r ite ions in a samp le

must f irs t be reduced prior to acid digestion for quantitative

(NH4)2SO4 + 2NaOH 2NH3↑ + Na2SO4 + 2H2O

ammoniumsulfate heat

ammoniagas

The Kjeldahl f lask is attached to a water condenser and

is heated to b oil off the NH3 gas from the digest. The tip of

the con denser is subme rged in a flask of ac id ic r ece iv ing

solu t ion , e i ther s tandard ac id or bor ic ac id so lu t ion , to aga in

trap the distilled NH 3 in receiving solution.

Digest ion Mixture Di lution

The acid digest ion mixture is usually cooled and di luted

with ammonia-free water . As mentioned above, with digestion

mixtures containing high salt/acid ratios, dilution prevents orminimizes caking. Sometimes this is done while the digestion

mixture is s t il l qu i te warm ( with cau t ion! ) , and the K2SO 4 h as

not yet salted out. With some digestion mixtures, a cake

forms, but dissolves or breaks up if time is allowed following

dilution. Alterna tively, warm ing or sonicating a fter d ilution

ma y b r e a k u p th e c a k e .

Cake m ater ia l may cause low n i t rogen reco ver ies in the

distillation step. Also, entrapped acid in a cake may react

violently with base during the distillation process.

Dilution of the digestion mixture before making it alka-

l ine an d d is ti ll ing a lso red uces the l ike l ihood of bump ing .

Additional boiling chips added just before distillation alsoredu ces bu mp ing , espec ia lly towards the end of the d is t il la -

t ion as the so lu t ion be com es mo re conc entra ted . Two or

three dr ops o f tr ibu tyl c i tr a te m ay be added as an a id to

reduce foaming.

NaOH Add itio n

Concen tra ted NaOH ( usua l ly 50% solu t ion) is added

slowly down the n eck of the flask. Being hea vier , it for m s a

layer underneath the di luted acid digest ion mixture. General ly,

for e ach 5 ml of concen tra ted su lfur ic ac id used in the

diges tion , 20 m l of 50% sodium hydroxide is r equired tomak e the d iges t s trongly a lka l ine ( pH of > 11) . The flask is

connec ted to the condenser and mixed before hea t ing and

distillation b egins.

For samples no t r equir ing a d iges tion s tep , such as

d irec t amm onia de te rm ina t ions in wate r , the samp le is

buf fe red to a p H of 9 .5 with a so lu t ion of sodium te trabor a te

and sodium hydroxide , to decrease hydro lys is of any c om pl e x

organic n i t rogen compounds present .



5

Disti l lation

The m ajority of the NH3 is d is ti lled and t r apped in the

receiving acid solution within the f irs t 5 or 10 minutes of

bo i l ing . But depend ing on the volume of the digest ion mixture

and the m ethod be ing fo l lowed, 15 to 150 m l of conde nsa teshould b e co l lec ted in the r ece iving flask to ensur e com ple te

recovery of nitrogen. Further extension of the distillation

t ime s and volume s co l lec ted s imply resu l ts in mo re wate r

being carr ied over to the receiving solution. Excess water

does n ot change the t i tr a t ion resu l ts .

Distillation times an d d istillate volum es collected should

be stand ard ized for al l sam ples of a given m ethodo logy. Th e

rate of distillation is affected by condenser cooling capacity

and c ool ing wate r temp era ture , bu t pr imar i ly by hea t inpu t .

Typically the h eating elem ents u sed for distillation ha ve vari-

able temperature controllers . A distillation rate of about

7 .5 ml/minute is mos t commonly c i ted in accepted methods .Connec t ing bulbs or expans ion cham bers be tween the

diges tion flask and the con denser is an im por tan t con s idera -

tion to prevent carryover of the alkaline digestion mixture

into the r eceiving f lask. The slightest bit of contam ination o f

the receiving solution can cause signif icant error in the titra-

tion step.

When very low levels of nitrogen are being determined,

it is advisable to “precondition” the distillation apparatus

prior to distillation. This can be done by distilling a 1:1 mix-

ture of amm onia- free wate r and 50% NaOH for 5 minutes

ju st b e for e sa m p le d is ti lla tio n to r ed u ce c o n ta m in a tio n fr o m

a tmo s p h e r ic a mmo n ia .

Receiving So lution s

If the re ceiving solution is s tandar dized HCl or H2SO 4, it

is desirab le to h ave only a s light excess lef t after the NH3 is

d is ti lled and t r apped in the r ece iving so lu t ion to m inimize

the back t it r a t ion . Based on the an t ic ipa ted am oun t of n it ro-

gen in the sample , a ta rge t amoun t of s tandard ac id can be

calculated from the following formula:

The Titra t ion Pro cess

There are two types of titration: backt it r a t ion , com mo nly used in Macro

Kjeldah l ; and d irect t it ra t ion. Both m ethods

ind ica te the am mo nia present in the d is -

tillate with a color change and allow for calculation of

unknown concentra t ions .

Nitrogen Determination by BACK TITRATION

The amm onia is cap tured b y a ca re fu l ly measu red

excess of a s tandardized acid solution in the receiving f lask.

The excess of ac id in the rece iving so lu t ion keeps the pH

low, and the ind ica tor d oes no t change .

mls of standardacidto addto receivingflask =

[(%nitrogen expected in sample) x (sample aliquot used) x gramsample wt.] +2

(normality of standard acid) x 1.4007 x digestion dilution volume

I f bor ic ac id is used , the exac t con centra t ion is no t

needed because the t i tr a t ion d irec t ly measu res the am ount of

amm onia in the d is ti lla te by neutra l izing the 1 :1 com plex

form ed by amm onia and bor ic ac id . Large quan t it ies of bor ic

ac id ma y be ad ded to the rece iving so lu tion so co mp le te

absorp t ion of the ammonia is assured .

Receiving solution volumes may be increased by the

addition of ammonia-free water so that the tip of the delivery

tube is immersed. Delivery tubes should always be allowed to

dra in mo men tar i ly in to the rece iving flask before r emo val

from the distillation apparatus. The receiving solution should

rem ain be low 45° C dur ing d is t il la t ion to pr event loss of

a mmo n ia .

ammonia

standardsulfuricacid

ammoniumsulfate

excesssulfuricacid

2NH3 + 2H2SO4 (NH4) 2SO4 + H2SO4

(no color change)

The excess acid solution is exactly neutralized by a

care fu lly measu red s tandard ized a lka l ine b ase so lu t ion such

as sodium hydroxide . A color ch ange is produc ed a t the end

point of the titration.

ammoniasulfate

measuredexcessacid

measuredsodiumhydroxide

ammoniumsulfate

(NH4)2SO4 +H2SO4 +2NaOH (Na)2SO4 +(NH4)2SO4 +2H2O

(color change)

Nitrogen Determination by DIRECT TITRATION

If boric acid is used as the receiving solution instead of

a s tandard ized m inera l ac id , the chem ica l r eac t ion is :

ammonia

gas

boric

acid

ammonium-borate

complex

excessboric

acidNH3 + H3BO3 NH4+:H2BO3

- + H3BO3

(color change)

The bor ic ac id captures the amm onia gas , form ing an

amm onium -bora te com plex . As the amm onia co l lec ts , the

color of the rece iving so lu t ion changes .

ammoniumborate complex

sulfuricacid

ammoniumsulfate

boricacid

2NH4H2BO3- + H2SO4 (NH4)2SO4 + 2H3BO3

(color change in reverse)

The ad dition of sulfur ic acid exac tly neu tralizes the

ammonium bora te complex , and a r everse co lor change is

p r o d u c e d .

The bor ic ac id m ethod ha s two advantages : on ly one

s tandard so lu t ion is necessary for the d e te rmina t ion and the

solution h as a lon g shelf life .



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Indicator So lution s

Many different indicators have been used to provide a

“sharp end point” color change. The analyst’s use of specif ic

types o f ind ica to rs c an be a pe r sona l cho ice . The com bina t ion

of methyl red and methylene b lue indica tors i s frequ ent ly usedin man y metho ds . The co lor ch ange of the ind ica tor m us t fa l l

within the equivalent point of the reaction. For s tandard

acid/base titrations, methyl orange is usually the preferred

indicator . I f color change end points are diff icult to detect,

r e fe rence so lu t ions m ade f rom a b lank with an ind ica tor can

be very help ful.

Calculations

The ca lcu la tions for % ni t rogen or % pro te in m us t take

into account which type of receiving solution was used and

any dilution factors used during the distillation process. The

equa t ions g iven h ere a re in long form . They a re o ften s impli-

fied in the publ ished s tandard m ethods . In the equa t ions

below, “N” represents normality. “ml blank” refers to the

mil l il ite r s of base need ed to back t it r a te a r eagent b lank i f

s tandard acid is the receiving solution, or refers to milliliters

of s tandar d ac id n eeded to t it r a te a r eagent b lank i f bor ic

acid is the receiving solution. When standard acid is used as

the receiving solution, the equation is :

Equipment and Apparatus

Very ear ly Kjeldahl digestions a nd distillations wer e

per forme d us ing s tone fume ho ods and gas mantles as a hea t

source . In the 1920 ’s these were re p laced by what is nowknown as classical macro-Kjeldahl digestion and distillation

appa ra tus . Mac ro se tups u se Kje ldahl f la sks from 500 to 800 ml

volume and h andle sam ple s izes from 0 .5 to abou t 5 .0 g .

A smaller version of this apparatus is referred to as

micro -Kje ldahl equipm ent . The equipment consists of smaller

bench top hea te r u n i ts, and Kje ldahl flasks of 30 to 100 ml

volume.

A th ird mo re recen t var ia t ion in equ ipment m akes use

of ce ram ic or a luminum hea t ing b locks des igned to accept a

num ber of s tr a ight d iges t ion tubes a t once . “Block d iges tor s”

are o ften used in conjunc t ion with b enchtop d is t il la t ion uni ts

with s team genera tor s to shor ten the d is t il la t ion t ime .Each of these three types of appar a tus a re descr ibed in

more detail below. In all cases, s ince the Kjeldahl process

involves signif icant corrosive fumes, appropriate attention

mu s t be g iven to fume r emo val . Equipm ent mu s t be su i tab ly

cons truc ted of cor ro s ion- res is tan t mate r ia ls .

Class ical Macro-Kjeldah l Apparatus

Many governmental and regulating agencies have devel-

oped methodologies that specify the classical macro-Kjeldahl

appar a tus . For exam ple , s tandar d m ethods for low leve l

n i t rogen de te rmina t ions in wate r (0-10 mg/1) r equire asamp le s ize of 250 to 500 ml, and there fore la rge Kje ldahl

f lasks. Methodologies in the agricultural industry of ten

requ ire la rger sam ple s izes and involve m atr ices which can

significantly foam.

Eq u ip me n t e n g in e e r e d to a c c o mm o d a te m a c r o m e t hods

can b e subs tan t ia l. Appar a tus des igned to pro cess 2 to 12

samples s imultaneously is typical, and involves a bank of

hea te r s and a fum e m anifo ld for d iges t ion , and a second

bank of heaters with condensers for dist i l lat ion. The apparatus

is available freestanding or contained within an integral

h o o d . Freestanding ins ta l la t ions require addi t ional laboratory

roo m mo di fi ca tions to hand le fum es and hea t . I n e i the r case

the eq uipm ent is large, e xpen sive, involves significant instal-

lation considerations, and ongoing utility and reagent costs .

The d iges tion app ara tus has separ a te hea t ing mantles

to retain each f lask. Typically each mantle has an individual

temperature controller . The long necks of the Kjeldahl f lasks

are po s i tioned to re lease d iges tion fum es in to a comm on

man ifo ld . Manifo ld asp ira t ion ca n b e p rovided e i ther by a

mech anica l b lower with exhaus t th rough d uc twork to the

outs ide , o r b y a la rge wate r asp ira tor tha t p rovides a wate r

spray to d i lu te the fum es for d isposa l down a dra in .

Like the digestion apparatus, the distillation apparatushas separate heating mantles to retain each f lask. With the

flask in place on the mantle, the neck of each f lask is

a t tached to a “connec t ing bulb” or an expans ion chamber

tha t ac ts as a t r ap to keep any of the concen tra ted l iqu id

diges t from mech anica l ly ca r rying over th ro ugh the co n-

densers and in to the rece iving so lu t ion .

% Nitrogen=

[(ml standard acid x N of acid) - (ml blank x N of base)] -

(ml std base x N of base) x 1.4007weight of sample in grams

If the sample weight is in milligrams, the molecular

weight of n it rogen should b e changed to 1 400.67 .

When bor ic ac id is used as the rec e iving so lu tion the

equa t ion is :

% Nitrogen=

(ml standard acid - ml blank) x N of acid x 1.4007

weight of sample in grams

If it is desired to determine % protein instead of

% nitrogen, the calculated % N is multiplied by a factor , the

magnitude of the fac tor d epend ing on the samp le matr ix .

Many protein factors have been developed for use with vari-

ous types of samples. The lis t below repr esents jus t a few of

the factors descr ibed in the s tandard method s of ana lys is pub-

lished b y the Am erican Association of Cerea l Chem ists

(AACC) and AOAC International.

PROTEIN FACTORS PROTEIN SOURCES

6 .3 8 m ilk a n d d a ir y

6 .2 5 o th er gr a in s

5 .9 5 r ice

5 .7 0 wh ea t flo u r



7

Downs tream f rom the conne c t ing bu lb i s the conden ser ,

which is a tube of s ta in less s tee l sur rou nded by a second

water jacket tube. Typically multiple condenser/jacket units

are ganged to gether in a s ingle assem bly. A glass delivery

tube is a t tached to the end of the co nden ser . The d e l iverytube has a ba l l -shaped t ip with small ho les to he lp d isperse

la rge bubbles and e l imina te pr essure f luc tua t ions dur ing d is -

tillation. The t ip is submerged in the f lask of receiving solut ion

to ensur e tha t the d i s ti ll ing am mo nia i s com ple te ly cap tu red .

Micro- Kjelda hl Apparatus

These a re m inia ture ver s ions of the m acro-d iges tion

appar a tus which a re movable and in tended for use in a labo-

ra tory hood. They a re d es igned to d iges t small samples in 30

or 100 ml digestion f lasks. Individually controlled heaters

allow multiple f lasks to be handled simultaneously. Some

micro-d iges tor s inc lude a o ne-p iece g lass manifo ld for fumerem oval . The g lass man ifo ld is conn ec ted to a s tandard wate r

asp ira tor to d i lu te and re mo ve fumes tha t do n ot r e f lux in

the flask necks . Whether o r n o t a g lass man ifo ld is used , the

diges tion appar a tus should be oper a ted ins ide a labora tory

h o o d .

Diges ted samples can b e d i lu ted and an a l iquot taken

for distillation on a micro steam distillation unit. Micro

steam distillation units are available to accept digested sam-

ples of up to 4 ml con centra ted ac id and volumes of about

55 m l. An e lec tr ic imm ers ion hea te r pr oduc es s team h ea t

tha t causes the sample to boi l and re lease am mo nia gas . Thevapor s pass in to a con denser where co ntac t with wate r-

cooled g lass conden ses the vapor s , which dr ip in to the

rec eiving solution via a delivery tube. Distillation time is

appr oximate ly 5 m inutes .

Methodologies have been specif ically developed for

micro -Kje ldahl app ara tus . These mos t of ten a r e m ethods tha t

involve homogeneous samples, relatively high nitrogen levels

in the sam ple , and small samp le s izes nor mally less than

0 .25 g .

The installation considerations, initial expense, space

requ iremen ts , and o ngoing u t il ity and r eagent cos t a re mu ch

less with m icro equipm ent .

Block Digestors

Block d iges tor s a re h igh temp era ture ce ra mic or a lu-

minum blocks with wells to accept s traight walled digestion

tubes . They can accom mo date from 2 to 25 tubes a t once .

Block digestors are heavy but movable self-contained units

des igned to be u sed in a labo ra tory hood. Diges tion tubes

range u p to 300 ml cap ac i ty, su i tab le for samp le s izes up to

a b o u t 2 g o r 7 5 ml .

Since narrow, straight-walled digestion tubes are used

instead of traditional Kjeldahl f lasks with a bulb and neck,

and s ince a n um ber o f tubes a re grou ped c lose ly toge ther ina ver t ica l conf igura t ion in the b lock , ac id re f lux param eter s

a re d iffe ren t . This mean s sa l t /ac id ra t ios and hea t inputs

appr opr ia te for m acro-Kje ldahl m ethods do not exac t ly

transfer to digestion in a block. Methods have been devel-

oped specif ically for block digestors s imilar , but not exactly

ident ica l, to macr o-Kje ldahl m ethods .

The RapidSti ll I is a microdisti llati on unit.

Macro-Kjeldahl Combination Digesti on/Disti llation

Apparatus.

The Micro Digestor uses a glass manifold to remove fumes.



8

Typically block digestors have one controller that

ad jus ts the tem pera ture of the en t ir e b lock . The contro l le r

can be au tomated , a l lowing for t imed r amp ing and m ult ip le

temper a ture se t t ings dur ing the cour se of a d iges tion .

Mos t often b lock d iges tor s a r e used toge ther with fume

rem oval devices , even thou gh they a re o pera ted in a hood .

One such system consists of a glass manifold with individual

bulb edu c tor s in a r ack des igned to res t on top of a se t o f

digestion tubes in the block. Another system uses individual

exhaust caps with f lexible tubing. In either case the manifold

is connected to a water aspirator . Or, instead of a water aspi-ra tor , there a re a l te rna t ive un i ts tha t em ploy mechan ica l

asp ira t ion an d inc lude bu bbling the ac id fumes throu gh a

neutralizing base solution.

Block digestion followed by steam distillation is some-

t ime s re fe r red to as “rap id Kje ldah l” pa r t ly because a n umb er

of b lock d iges tion m ethodologies a re shor te r than c lass ica l

macro-Kjeldahl digestions and benchtop steam distillation

units in use are faster than classical distillations.

Rapid Steam Disti l lation Apparatus

The development of block digestors as an alternative tomacr o-Kje ldahl d iges t ion has b een para l le led by the deve lop-

me nt of benc htop steam distillation un its . Basically, this

equipm ent cons is ts of a s team gener a tor to in jec t s team in to

the a lka l ine d iges tion m ixture an d an evapor a tor- type con-

denser , where contact with water-cooled glass coils causes

the vapor s to cond ense and dr ip in to the rece iving so lu tion

via a delivery tube. Usually the u nit is designed to accep t

s tra ight d iges tion tubes from block d iges tor s , thou gh there is

no r eason in theor y why any d iges tion techniques co uld not

be combined with any distillation technique. Steam distilla-

t ion is much mo re ra p id than c lass ica l macro-d is t il la t ion ,

typ ica l ly tak ing from 3 to 8 m inutes . On the o ther hand , on lyone d is ti lla tion a t a t ime can be do ne .

Rapid steam distillation un its are available with d iffer-

ent features, ranging from relatively manual models to highly

automated m odels . Basic mod els d ispense d i lu t ion wate r and

base und er con tro l of a push bu t ton . Some m odels inc lude a

timer to control the length of distillation, shutting down auto-

matically. Some units automate the entire distillation process

once the d iges tion f lask is in p lace , an d o ther s au tom atica l ly

titrate to end point af ter distillation, and calculate and dis-

p lay the resu l ts in a p r in ted repo r t .

Titrators

Of course, receiving solutions may be individually hand

t it r a ted us ing an ind ica tor so lu t ion and bure t , bu t severa l

mo dels of bench top au tom atic t i tr a tor ins trum ents a re a lso

available. Some units will titrate one rece iving solution a t a

time to a set end point. Others will automatically titrate a

number of receiving f lasks sequentially. Still other models

provide a pr intout of results along with digital readout.

Other Equipment and Proce dures

Following Kjeldahl digestion, there are other proce-

dure s ava i lab le to de te rmine n i t rogen ( or pr o te in) in the

diges tion m ixture . The m os t pop ula r involves the spec-

t r o p h o to me t r ic m e th o d fo r a m mo n iu m d e te r min a tio n s o f

d iges tion m ixtures . The Ness le r and Phena te co lor d eve lop-

men t methods a r e the m os t wide ly used . The Biure t m ethod,

a d ye b ind ing proced ure , is another type . Automa ted ana lyz-

e r s which u se co lor d eve lopmen t chem is try a re ava i lab le and

can p er form la rge nu mb ers of assays . However , the app ara-

tus and ma in tenance cos ts a re h igh .

Manual Ness le r co lor imetr ic m ethods a re of ten used inammonia de te rmina t ions . Diges ted samples a re prepared for

a co lor d eve lopm ent and the ir absor bency is r ead with a

spec trophotometer a t a r ange of 400 to 500 nm. Standards

are a lso run an d a s tandard cu rve is p repar ed which p lo ts

absorb ency versus m ill ig rams o f amm onia . The s tandard

curve is then com pared to the sample resu l ts .

The RapidSti ll I i s a steam disti llation apparatus.

Rapid Digestor-4 with fume removal system



9

The condi t ions requ ired for op t imal co lor d eve lopmen t

and s tab i li ty can be r igorou s for a l l o f these pro cedur es .

However, spectrophotometr ic analysis has the ability to

de te rm ine very small quant i t ies of amm onia — as low as 20

micro-grams per l i te r under opt imal condi t ions .

Ammonia specif ic ion electrodes give the analyst anothermean s for the de te rm ina t ion of amm onia . The e lec trode uses

a h y d r o p h o b ic g a s p e r me a b le me m b r a n e to s e p a r a te th e

sample so lu t ion f rom an in te rna l ammonium chlor ide so lu-

t ion . Disso lved am mo nia d iffuses through the m emb rane an d

changes the in te rna l pH, which is sensed by a pH m eter tha t

has an expanded mil l ivo l t sca le . Amm onia con centra t ions of

s tandards versus potential in millivolts are plotted on a semi-

logar i thm ic graph , and samp le concentra t ions a re r ead f rom

the s tandard cu rve . The amm onia spec ific ion e lec trode can

be p rec ise and has few in te r fe rences .

In ad di t ion , there is a l te rna t ive n i trogen de te rm ina t ion

equipm ent which involves techniques o ther than Kje ldahld iges tion . There a re ins trumen ts which incorp ora te tech-

n iques of Dumas co mb us t ion , neu tron ac t iva t ion , near

infrared ref lectance spectrometry or direct distillation. The

fir s t two ins trum ent/ techniques d e te rmine to ta l n i trogen

regardless of its form within the sample. Their acceptance in

mo s t labor a tor ies has been l imited due to the necessary

changes involved in reporting these types of nitrogen values.

These changes would involve broad multi- industr ial re-evalu-

ations of comparative nitrogen-protein analysis .

Near inf ra red r e flec tance spec trom etry and d irec t d is -

t il la t ion u se method s based on appro ximate re la t ionships

be tween n i t rogen content and pro te in . Both methods dependon ca l ibra t ions with s tandard s which have been de te rm ined

by trad ition al Kjeldahl analysis .

Most Common Problems

Like any o ther m ethod, the Kje ldahl pro cedur e is

subjec t to pr oblems with method ology, technique and eq uip-

men t . Under s tanding the pr inc ip les behind the Kje ldahl

process and us ing appropr ia te qua l i ty contro l procedures

should pr event mo s t problems f rom occur r ing . When d eve l-

oping a new o r m odifying an ex is ting method, o ne sho uld

proce ed with cau t ion and exper imenta l ver i fica tion .Should nitrogen results differ f rom those expec ted,

kno wledge of comm on p roblems m akes finding and cor rec t ing

the e r ror ea s ie r . Com mo n p roblems inc lude the fo l lowing:

sample size and type incompatibility with amounts and types

of acid, sal t or catalyst used; contaminated samples, s tandards,

reagents or equipm ent; inadequa te or excess ive d iges tion

time; inadequate or excessive distillation dilution volume or

a l iquot vo lume; foam ing or bu mp ing dur ing d iges tion ;

uneven d iges tion ; inadequa te o r excess ive sam ple temper a-

ture ; p rec ip i ta tion of sa l t o r cak ing or d iges ted sam ple ; leaks

in appara tus conn ec t ions ; inadequa te sodium hydroxide

addi t ion o r sod ium h ydroxide car r yover ; and ca lcu la tione r r o r s .



1 0

Glossary

Acid Normality The measu rem ent of an acid st rength , ie : the lowerthe norm al ity the weaker the a cid .Aliquot A part which is a definite fraction of a whole, ie: as taking a

por t ion of a sample.Alundum (Al20 3) A granular m ater ia l that ma y be added to thedigestion m ixture to decr ease b um ping dur ing the in i tia l d igest ionphase .Back Titratio n Method of t it ra t ion to determ ine the amo unt of excess acid that was consum ed in the rece iving solution dur ingKjeldah l distil lation.Base Normality The mea surem ent of base st rength , i.e . : the lowerthe nor mal i ty, the weaker the base.Biuret Method A dye-binding pro cedur e using an analyt ical reagentespecial ly designed for proteins.Blank The amount of n i t rogen contained in the reagents used. InKjeldahl calculat ions, the b lank is subtracted so that i t does no tfalsely increase the % ni t rogen o r % pro tein of the sam ple.

Catalyst Any substance o f which a fract ional perc entage notablyaffects the ra te of a chem ical react ion wi thout i tsel f being con sume dor undergoing a chemical change. Most catalysts accelerate reac-t ions, bu t a few retard them .Classical Macro-Kjeldahl A method designed to d igest or d isti llmul t ip le samples that contain h igh levels of n i t rogen.Connecting Bulb A glass component of a classical Kjeldahl distil la-t ion system. Separates l iquid from vapor s pr ior to the vapors enter -ing the cond enser and ef fect ively t raps ca ust ic mist that may car ryover to the receiving solution.Deli very Tube A glass component of a Kjeldahl system that deliversdist il la te to a r eceiving flask . The long ne ck, r oun d b ot tom an d th ickwal ls he lp el iminate pre ssure fluctuat ions in the d isti lla t ion phase.Digest ion In Kjeldahl m ethods, the dest ruct ion of organ ic mat terwi th an acid . Also used as a nou n m eaning digestion mixture .Direct Disti l lation A method based on approximate re la t ionshipsbetween ni t rogen content and protein wi thout per forming a digestionstep .Direct Titration Method of t it ra t ion to de termine the am ount of ammonia that has been absorbed in to the receiving solut ion dur ingKjeldah l distil lation.Disti l lation A separa t ion p roce ss in which a l iquid i s conver ted tovapor and the vapor then condensed to a l iquid .Dumas Combustion A combust ion method measur ing ni t rogen gasinvolving in the total destruction of the sample.End Point The point dur ing a t i tra t ion at which a ma rked colorchange is observed.

H o m o g e n e o u s Adject ive desc r ib ing a m ixture co mpr ised of two orm or e com pou nds o r e l em en t s un i fo r m l y d ispe r sed i n each o t he r.Hydrolysis A chem ical react ion in which water rea cts wi th an othersubstance to form two or more new substances.Hydrophobic Incapable of dissolving in water.Indicator Solution A solut ion a dded to a sam ple dur ing t it ra t ionthat produces a color change indicat ing end point .Kjeldahl Method An a nalyt ical m ethod for determining n i trogencontent in cer ta in organic and inorganic compounds. The methoduses d igestion, d ist i lla tion an d t i t ra t ion to ar r ive a t the n i t rogen co n-tent of the sam ple.Macro-Kjeldahl A Kjeldahl method designed to handle high levelsof nitrogen; classical Kjeldahl.

Micro-Kjeldahl A Kjeldahl m ethod that r equires smal ler volumesof chemical reagents and smal ler sample sizes than macro or c lassicKjeldahl.Molecular Weight The sum of the a tom ic weights of the a tom s in amolecule .Near Infrared Reflectance Spectrometry Protein identificationmethod involving the measu rem ent of the radiant ener gy ref lected bythe sam ple in inf rared wavelengths.

Nessler Method Manual color imetr ic method where digested sam-ples are prepared for color development and thei r absorbency readwith a spect rophotometer .Neutron Activation A sensitive technique for identifying and mea-sur ing very smal l amoun ts of var ious e lements. A sample i s exposedt o neu t r on bom bar d m en t i n a nuc l ea r r eac t o r fo r t he pu r pose o f producing radioisotopes f rom the stable e lements.

Nitrate [NO3- ] Form of nitrogen usually found in fertil izers. I t must

be re duced before d igest ion in the Kjeldahl m ethod.

Nitrite [NO2- ] Form of nitrogen usually found in fertil izers. I t must

be re duced before d igest ion in the Kjeldahl m ethod.pH A mea surem ent that repr esents the ac idi ty or a lkal in i ty of anaqueous solut ion.Phenate Method Manual color imetr ic method where digested sam-ples are prepared for color development and thei r absorbency readwith a spect rophotometer .Precipitate Small particles that have settled out of a liquid orgaseous su spension by gravi ty, or that resul t from a ch emicalreact ion.Rapid Kjeldahl Same m ethod as c lassical Kjeldahl , except , throughef fic ient heat ing and com pactness, re quires less t ime to co mplete thedigest ion step . Digest ion t imes range f rom 6-60 minutes comparedto up to two h our s for c lassical d igest ion.Receiving Solution Solut ion used to capture ammonia separatedthrough distil lation.Reflux The l iquid con densed from the r i s ing vapor .Spectrophotometry Identification method involving an opticaldevice that measu res radiant en ergy within selected wavelengths inthe visual , u l t raviolet or infrared regions of the spe ct rum.Steam Disti l lation Distillation us ing steam to dr ive o ff NH3 gas inKjeldahl determ inat ions.Titration A quantitative analytical process that is basically volumet-r ic . General ly, a s tand ard solut ion, that i s , on e co ntaining a knownconce ntrat ion of substance X ( t i trant ) , i s progressively added to amea sured volume o f a solut ion of a substance Y ( t it rand) that reactswi th the t it rant . The add i tion is con t inued unt i l the e nd point i sr eached .

Water Aspi rator A device that pro vides suct ion throu gh the use of f lowing water past a small orifice.

Bibl iography

American Association of Cereal Chemists. Approved Methods of Analysis. St. Pau l, MN: Ame rican Assoc iation of Cereal Chem ists,1 9 9 5 .

American Public Health Association, American Water Works

Association, Water Pollution Control Federation. Standard Methods for the Examination of Water and Wastewater.Washington, DC:Amer ican Pu bl ic Heal th Associat ion, 1992 .

AOAC Intern ational ( forme rly the Assoc iation of Official AnalyticalChemists) . Official Methods of Analysis.Arlington, VA: AOACInternat ional , 1995.

Bradstreet, R. B. The Kjeldahl Method for Organic Nitrogen. NewYork , NY: Academ ic Press Incorp orated, 1965 .

Jones, J . Benton. Kjeldahl Method for Nitrogen Determinati on.Athens, GA: Micro-Macro Publishing, 1991.

Kjeldahl , Johan Z. “A new m ethod for the determ inat ion of n i trogenin organic bodies.” Analytical Chemistry 2 2 ( 1 8 8 3 ) : 3 6 6 .

Meloan , Clifton E., an d Po me ran z, Y. Food Analysis: Theory & Practice . Westport, CT: AVI Publishing Company, 1978.

United States Environmental Protection Agency. Methods for Chemical Analysis of Water and Wastes. Cincinn ati, OH:Environmental Protection Agency, EPA-600/4-79-020, EnvironmentalMoni tor ing and Suppo r t Labor atory, 197 9.





Documents

Guide Kjeldahl