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A

Gu i de
To
Kje l da h l
Ni t r o ge n
De t e r mi n a t i o n
Me t h o ds a n d
Appa r a t u s
An Indus try Se rvi ce Publ i cati o n
Fo r wa r d
This booklet has been developed to ser ve as a technical
companion in the selection and use of Kjeldahl nitr ogen
deter mination methods and appar atus. This mater ial has
been pr epar ed with the help of Kjeldahl chemists, consul-
tants and Labconco engineer s. It will not teach you to per for m
the Kjeldahl pr ocedur e but will help you under stand steps in
the pr ocess. This gener ic pr esentation is designed to help
educate per sons unfamiliar with Kjeldahl methodology and
answer fr equently asked questions r aised by Kjeldahl user s.
3
Hi s t o r y
Nitr ogen deter mination has a long histor y in the ar ea of
analytical chemistr y. Johan Kjeldahl fir st intr oduced the
Kjeldahl nitr ogen method in 1883 at a meeting of the Danish
Chemical Society. As chair man of the chemistr y depar tment
of the Car lsber g Labor ator ium near Copenhagen, Kjeldahl
was assigned to scientifically obser ve the pr ocesses involved
in beer making. While studying pr oteins dur ing malt pr oduc-
tion, he developed a method of deter mining nitr ogen content
that was faster and mor e accur ate than any method available
at the time. His method used simple equipment and could be
per for med by an inexper ienced technician.
Appl i ca t i o n s
Since 1883, the Kjeldahl method has gained wide
acceptance and is now used for a var iety of applications.
Kjeldahl nitr ogen deter minations ar e per for med on food and
bever ages, meat, feed, gr ain, waste water, soil and many
other samples. The method has been r efined and tested for a
wide var iety of substances and appr oved by var ious scientific
associations including:
AOAC Inter national ( for mer ly the Association of Official
Analytical Chemists)
Association of Amer ican Cer eal Chemists
Amer ican Oil Chemists Society
Envir onmental Pr otection Agency
Inter national Standar ds Or ganization
United States Depar tment of Agr icultur e
Wh a t i s t h e Kje l da h l me t h o d?
The Kjeldahl method is a means of deter mining the
nitr ogen content of or ganic and inor ganic substances.
Although the technique and appar atus have been alter ed
consider ably over the past 100 year s, the basic pr inciples
intr oduced by Johan Kjeldahl endur e today.
The Kjeldahl method may be br oken down into thr ee
main steps:
Di ge s ti o n - the decomposition of nitr ogen in or ganic
samples utilizing a concentr ated acid solution. This is
accomplished by boiling a homogeneous sample in
concentr ated sulfur ic acid. The end r esult is an
ammonium sulfate solution.
Di s ti l l ati o n - adding excess base to the acid digestion
mixtur e to conver t NH4
+
to NH3, followed by boiling
and condensation of the NH3 gas in a r eceiving solution.
Ti trati o n - to quantify the amount of ammonia in the
r eceiving solution.
The amount of nitr ogen in a sample can be calculated
fr om the quantified amount of ammonia ions in the r eceiving
solution.
Th e Di ge s t i o n Pr o ce s s
A gener al equation for the digestion
of an or ganic sample is shown below as
one basic example:
OrganicN +H2SO4
(NH4)2SO4 +H2O+CO2 +other sample matrix by-products
A number of inter r elated digestion conditions deter mine
the r ate of r eaction and the completeness of the br eakdown
of nitr ogen to ammonium sulfate. Among these ar e heat
input to the acid digestion mixtur e, amount of inor ganic salt
added to elevate the acid boiling temper atur e, r eflux r ate of
H2SO4 in the neck of the digestion flask, length of digestion,
and catalyst addition. Adjusting any one of these factor s has
an influence on the other s. Pr oper digestion conditions for a
given sample matr ix ar e achieved thr ough establishing a bal-
ance of these factor s in a contr olled and r epeatable fashion.
In addition, if the sample contains nitr ate or nitr ite nitr ogen,
it is possible to chemically pr etr eat the digest to include or
exclude this nitr ogen sour ce fr om the analysis as desir ed in a
par ticular situation.
Aci d Co ns i de rati o ns
Sulfur ic acid has been used alone for the digestion of
or ganic samples. ( As a convenience to r educe bumping of
the digestion mixtur e, Alundum boiling chips or pumice ar e
often added.) The amount of acid r equir ed is influenced by
sample size and r elative amount of car bon and hydr ogen in
the sample, as well as amount of nitr ogen. A ver y fatty sample
consumes mor e acid. Also, heat input and digestion length
influences the amount of acid loss due to vapor ization dur ing
the digestion pr ocess. Remember that a Kjeldahl flask is
essentially a bulb with a condenser neck off to the side for
r efluxing of the acid.
He at Input and Di ge s ti o n Le ngth
Typically the heating elements used for Kjeldahl diges-
tions have var iable settings. Heat input is fr equently specified
as that setting which br ings 250 ml of water at 25 C to a
r olling boil in 5 minutes.
Initially an or ganic sample usually char s and blackens.
The r eaction may at fir st be ver y vigor ous depending on the
matr ix and the heat input. With or ganic decomposition the
digestion mixtur e gr adually clear s as CO2 evolves. Metallic
ions might tint the clear digestion mixtur e. Note that solution
clear ing in itself is not an indication that all or ganic nitr ogen
has been br oken down. Digestion length must be deter mined
by r ecover y studies on known mater ials of similar matr ix if a
new method is being developed.
4
r ecover y. Salicylic acid followed by sodium thiosulfate has
been used to pr etr eat the mixtur e to ensur e complete r educ-
tion. Other r eduction schemes have been devised. Or other
pr etr eatments have been used to pr event nitr ates fr om being
r educed at all dur ing the char r ing pr ocess, leaving a clear
digest with no contr ibution fr om nitr ate ions.
Th e Di s t i l l a t i o n
Pr o ce s s
The acid digestion mixtur e is diluted
and made str ongly alkaline with NaOH,
liber ating NH3 as follows:
Sal t Addi ti o ns
The pr oblem with using sulfur ic acid alone for diges-
tion is ver y long digestion times r esult with many samples
due to the slow r ate of or ganic decomposition. The addition
of an inor ganic salt to the digest elevates the boiling point of
the H2SO4. The solution temper atur e of concentr ated sulfur ic
acid alone is about 330 C. Addition of a salt such as K2SO4
can elevate the solution temper atur e of the digestion mixtur e
to 390 C or mor e, depending on the r atio of salt to acid.
This significantly incr eases the r ate of or ganic decomposition
in the digestion mixtur e, shor tening the length of time
r equir ed for digestion.
Ther e ar e sever al pr ecautions to keep in mind con-
cer ning salt addition. Fir st, it is possible to r aise the solution
temper atur e of the digestion mixtur e too much. If the tem-
per atur e goes much above 400 C dur ing any phase of the
digestion, volatile nitr ogen compounds may be lost to the
atmospher e.
Remember that as acid is gr adually consumed dur ing
the digestion pr ocess, for the var ious r easons mentioned
above, the salt acid r atio of the digest gr adually r ises. This
means that the hottest solution temper atur es ar e attained at
the end of the digestion. Heat input, consumption of acid by
or ganic mater ial and vapor ization, salt/ acid r atio, digestion
length, and physical design of the Kjeldahl flask, ar e all inter r e-
lated. Each has an effect on the final solution temper atur e.
A second pr ecaution is that if the salt/ acid r atio is too
high, a consider able amount of mater ial will salt out upon
cooling of the digest. Concentr ated acid pockets can be con-
tained within the cake. These can r eact violently when con-
centr ated base is added in the distillation pr ocess. A cer tain
amount of salting out can be managed by diluting the digest
with water while it is still somewhat war m, but not too hot.
Catal ys t Addi ti o ns
Sever al catalysts have been employed by Kjeldahl
chemists over the year s to incr ease the r ate of or ganic br eak-
down dur ing the acid digestion. Mer cur ic oxide has been the
most effective and widely used. However, mer cur y for ms a
complex with ammonium ions dur ing digestion. The addition
of sodium thiosulfate or sodium sulfide after digestion and
befor e distillation will br eak the complex and pr ecipitate
mer cur ic sulfide. This is also impor tant fr om a safety point of
view, as mer cur y vapor might escape to the atmospher e dur ing
the distillation pr ocess.
Because of envir onmental concer ns over the handling
and disposal of mer cur y, other catalysts ar e coming mor e
into favor. Many methods employ copper sulfate. Titanium
oxide and copper sulfate in combination have been found to
be mor e effective than copper sulfate alone. Selenium is fr e-
quently used. Commer cially pr epar ed mixtur es of potassium
sulfate and a catalyst ar e available fr om labor ator y chemical
supplier s. Bulk custom mixtur es ar e also available.
Ni trate and Ni tri te Re ducti o n
Kjeldahl digestions do not always r ecover all for ms of
nitr ogen in a sample. Nitr ate and nitr ite ions in a sample
must fir st be r educed pr ior to acid digestion for quantitative
(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O
ammonium
sulfate heat
ammonia
gas
The Kjeldahl flask is attached to a water condenser and
is heated to boil off the NH3 gas fr om the digest. The tip of
the condenser is submer ged in a flask of acidic r eceiving
solution, either standar d acid or bor ic acid solution, to again
tr ap the distilled NH3 in r eceiving solution.
Di ge s ti o n Mi xture Di l uti o n
The acid digestion mixtur e is usually cooled and diluted
with ammonia-fr ee water. As mentioned above, with digestion
mixtur es containing high salt/ acid r atios, dilution pr events or
minimizes caking. Sometimes this is done while the digestion
mixtur e is still quite war m ( with caution!) , and the K2SO4 has
not yet salted out. With some digestion mixtur es, a cake
for ms, but dissolves or br eaks up if time is allowed following
dilution. Alter natively, war ming or sonicating after dilution
may br eak up the cake.
Cake mater ial may cause low nitr ogen r ecover ies in the
distillation step. Also, entr apped acid in a cake may r eact
violently with base dur ing the distillation pr ocess.
Dilution of the digestion mixtur e befor e making it alka-
line and distilling also r educes the likelihood of bumping.
Additional boiling chips added just befor e distillation also
r educes bumping, especially towar ds the end of the distilla-
tion as the solution becomes mor e concentr ated. Two or
thr ee dr ops of tr ibutyl citr ate may be added as an aid to
r educe foaming.
NaOH Addi ti o n
Concentr ated NaOH ( usually 50% solution) is added
slowly down the neck of the flask. Being heavier, it for ms a
layer under neath the diluted acid digestion mixtur e. Gener ally,
for each 5 ml of concentr ated sulfur ic acid used in the
digestion, 20 ml of 50% sodium hydr oxide is r equir ed to
make the digest str ongly alkaline ( pH of > 11) . The flask is
connected to the condenser and mixed befor e heating and
distillation begins.
For samples not r equir ing a digestion step, such as
dir ect ammonia deter minations in water, the sample is
buffer ed to a pH of 9.5 with a solution of sodium tetr abor ate
and sodium hydr oxide, to decr ease hydr olysis of any complex
or ganic nitr ogen compounds pr esent.
5
Di s ti l l ati o n
The major ity of the NH3 is distilled and tr apped in the
r eceiving acid solution within the fir st 5 or 10 minutes of
boiling. But depending on the volume of the digestion mixtur e
and the method being followed, 15 to 150 ml of condensate
should be collected in the r eceiving flask to ensur e complete
r ecover y of nitr ogen. Fur ther extension of the distillation
times and volumes collected simply r esults in mor e water
being car r ied over to the r eceiving solution. Excess water
does not change the titr ation r esults.
Distillation times and distillate volumes collected should
be standar dized for all samples of a given methodology. The
r ate of distillation is affected by condenser cooling capacity
and cooling water temper atur e, but pr imar ily by heat input.
Typically the heating elements used for distillation have var i-
able temper atur e contr oller s. A distillation r ate of about
7.5 ml/ minute is most commonly cited in accepted methods.
Connecting bulbs or expansion chamber s between the
digestion flask and the condenser is an impor tant consider a-
tion to pr event car r yover of the alkaline digestion mixtur e
into the r eceiving flask. The slightest bit of contamination of
the r eceiving solution can cause significant er r or in the titr a-
tion step.
When ver y low levels of nitr ogen ar e being deter mined,
it is advisable to pr econdition the distillation appar atus
pr ior to distillation. This can be done by distilling a 1:1 mix-
tur e of ammonia-fr ee water and 50% NaOH for 5 minutes
just befor e sample distillation to r educe contamination fr om
atmospher ic ammonia.
Re ce i vi ng So l uti o ns
If the r eceiving solution is standar dized HCl or H2SO4, it
is desir able to have only a slight excess left after the NH3 is
distilled and tr apped in the r eceiving solution to minimize
the back titr ation. Based on the anticipated amount of nitr o-
gen in the sample, a tar get amount of standar d acid can be
calculated fr om the following for mula:
Th e Ti t r a t i o n Pr o ce s s
Ther e ar e two types of titr ation: back
titr ation, commonly used in Macr o
Kjeldahl; and dir ect titr ation. Both methods
indicate the ammonia pr esent in the dis-
tillate with a color change and allow for calculation of
unknown concentr ations.
Ni tro ge n De te rmi nati o n by BACK TITRATION
The ammonia is captur ed by a car efully measur ed
excess of a standar dized acid solution in the r eceiving flask.
The excess of acid in the r eceiving solution keeps the pH
low, and the indicator does not change.
mls of standardacidtoaddtoreceivingflask =
[(%nitrogenexpectedinsample) x (samplealiquot used) x gramsamplewt.] +2
(normality of standardacid) x 1.4007 x digestion dilution volume
If bor ic acid is used, the exact concentr ation is not
needed because the titr ation dir ectly measur es the amount of
ammonia in the distillate by neutr alizing the 1:1 complex
for med by ammonia and bor ic acid. Lar ge quantities of bor ic
acid may be added to the r eceiving solution so complete
absor ption of the ammonia is assur ed.
Receiving solution volumes may be incr eased by the
addition of ammonia-fr ee water so that the tip of the deliver y
tube is immer sed. Deliver y tubes should always be allowed to
dr ain momentar ily into the r eceiving flask befor e r emoval
fr om the distillation appar atus. The r eceiving solution should
r emain below 45 C dur ing distillation to pr event loss of
ammonia.
ammonia
standard
sulfuric
acid
ammonium
sulfate
excess
sulfuric
acid
2NH3 + 2H2SO4 (NH4)2SO4 + H2SO4
(no color change)
The excess acid solution is exactly neutr alized by a
car efully measur ed standar dized alkaline base solution such
as sodium hydr oxide. A color change is pr oduced at the end
point of the titr ation.
ammonia
sulfate
measured
excess
acid
measured
sodium
hydroxide
ammonium
sulfate
(NH4)2SO4 +H2SO4 +2NaOH(Na)2SO4 +(NH4)2SO4 +2H2O
(color change)
Ni tro ge n De te rmi nati o n by DIRECT TITRATION
If bor ic acid is used as the r eceiving solution instead of
a standar dized miner al acid, the chemical r eaction is:
ammonia
gas
boric
acid
ammonium-borate
complex
excess
boric
acid
NH3 + H3BO3 NH4
+
:H2BO3
-
+ H3BO3
(color change)
The bor ic acid captur es the ammonia gas, for ming an
ammonium-bor ate complex. As the ammonia collects, the
color of the r eceiving solution changes.
ammonium
borate complex
sulfuric
acid
ammonium
sulfate
boric
acid
2NH4H2BO3
-
+ H2SO4 (NH4)2SO4 + 2H3BO3
(color change in reverse)
The addition of sulfur ic acid exactly neutr alizes the
ammonium bor ate complex, and a r ever se color change is
pr oduced.
The bor ic acid method has two advantages: only one
standar d solution is necessar y for the deter mination and the
solution has a long shelf life.
6
Indi cato r So l uti o ns
Many differ ent indicator s have been used to pr ovide a
shar p end point color change. The analysts use of specific
types of indicator s can be a per sonal choice. The combination
of methyl r ed and methylene blue indicator s is fr equently used
in many methods. The color change of the indicator must fall
within the equivalent point of the r eaction. For standar d
acid/ base titr ations, methyl or ange is usually the pr efer r ed
indicator. If color change end points ar e difficult to detect,
r efer ence solutions made fr om a blank with an indicator can
be ver y helpful.
Cal cul ati o ns
The calculations for % nitr ogen or % pr otein must take
into account which type of r eceiving solution was used and
any dilution factor s used dur ing the distillation pr ocess. The
equations given her e ar e in long for m. They ar e often simpli-
fied in the published standar d methods. In the equations
below, N r epr esents nor mality. ml blank r efer s to the
milliliter s of base needed to back titr ate a r eagent blank if
standar d acid is the r eceiving solution, or r efer s to milliliter s
of standar d acid needed to titr ate a r eagent blank if bor ic
acid is the r eceiving solution. When standar d acid is used as
the r eceiving solution, the equation is:
Equ i pme n t a n d Appa r a t u s
Ver y ear ly Kjeldahl digestions and distillations wer e
per for med using stone fume hoods and gas mantles as a heat
sour ce. In the 1920s these wer e r eplaced by what is now
known as classical macr o-Kjeldahl digestion and distillation
appar atus. Macr o setups use Kjeldahl flasks fr om 500 to 800 ml
volume and handle sample sizes fr om 0.5 to about 5.0 g.
A smaller ver sion of this appar atus is r efer r ed to as
micr o-Kjeldahl equipment. The equipment consists of smaller
benchtop heater units, and Kjeldahl flasks of 30 to 100 ml
volume.
A thir d mor e r ecent var iation in equipment makes use
of cer amic or aluminum heating blocks designed to accept a
number of str aight digestion tubes at once. Block digestor s
ar e often used in conjunction with benchtop distillation units
with steam gener ator s to shor ten the distillation time.
Each of these thr ee types of appar atus ar e descr ibed in
mor e detail below. In all cases, since the Kjeldahl pr ocess
involves significant cor r osive fumes, appr opr iate attention
must be given to fume r emoval. Equipment must be suitably
constr ucted of cor r osion-r esistant mater ials.
Cl as s i cal Macro - Kje l dahl Apparatus
Many gover nmental and r egulating agencies have devel-
oped methodologies that specify the classical macr o-Kjeldahl
appar atus. For example, standar d methods for low level
nitr ogen deter minations in water ( 0-10 mg/ 1) r equir e a
sample size of 250 to 500 ml, and ther efor e lar ge Kjeldahl
flasks. Methodologies in the agr icultur al industr y often
r equir e lar ger sample sizes and involve matr ices which can
significantly foam.
Equipment engineer ed to accommodate macr o methods
can be substantial. Appar atus designed to pr ocess 2 to 12
samples simultaneously is typical, and involves a bank of
heater s and a fume manifold for digestion, and a second
bank of heater s with condenser s for distillation. The appar atus
is available fr eestanding or contained within an integr al
hood. Fr eestanding installations r equir e additional labor ator y
r oom modifications to handle fumes and heat. In either case
the equipment is lar ge, expensive, involves significant instal-
lation consider ations, and ongoing utility and r eagent costs.
The digestion appar atus has separ ate heating mantles
to r etain each flask. Typically each mantle has an individual
temper atur e contr oller. The long necks of the Kjeldahl flasks
ar e positioned to r elease digestion fumes into a common
manifold. Manifold aspir ation can be pr ovided either by a
mechanical blower with exhaust thr ough ductwor k to the
outside, or by a lar ge water aspir ator that pr ovides a water
spr ay to dilute the fumes for disposal down a dr ain.
Like the digestion appar atus, the distillation appar atus
has separ ate heating mantles to r etain each flask. With the
flask in place on the mantle, the neck of each flask is
attached to a connecting bulb or an expansion chamber
that acts as a tr ap to keep any of the concentr ated liquid
digest fr om mechanically car r ying over thr ough the con-
denser s and into the r eceiving solution.
% Nitrogen=
[(ml standard acid x Nof acid) - (ml blank x Nof base)] -
(ml std base x Nof base) x 1.4007
weight of sample in grams
If the sample weight is in milligr ams, the molecular
weight of nitr ogen should be changed to 1400.67.
When bor ic acid is used as the r eceiving solution the
equation is:
% Nitrogen=
(ml standard acid - ml blank) x Nof acid x 1.4007
weight of sample in grams
If it is desir ed to deter mine % pr otein instead of
% nitr ogen, the calculated % N is multiplied by a factor, the
magnitude of the factor depending on the sample matr ix.
Many pr otein factor s have been developed for use with var i-
ous types of samples. The list below r epr esents just a few of
the factor s descr ibed in the standar d methods of analysis pub-
lished by the Amer ican Association of Cer eal Chemists
( AACC) and AOAC Inter national.
PROTEINFACTORS PROTEINSOURCES
6.38 milk and dair y
6.25 other gr ains
5.95 r ice
5.70 wheat flour
7
Downstr eam fr om the connecting bulb is the condenser,
which is a tube of stainless steel sur r ounded by a second
water jacket tube. Typically multiple condenser / jacket units
ar e ganged together in a single assembly. A glass deliver y
tube is attached to the end of the condenser. The deliver y
tube has a ball-shaped tip with small holes to help disper se
lar ge bubbles and eliminate pr essur e fluctuations dur ing dis-
tillation. The tip is submer ged in the flask of r eceiving solution
to ensur e that the distilling ammonia is completely captur ed.
Mi cro - Kje l dahl Apparatus
These ar e miniatur e ver sions of the macr o-digestion
appar atus which ar e movable and intended for use in a labo-
r ator y hood. They ar e designed to digest small samples in 30
or 100 ml digestion flasks. Individually contr olled heater s
allow multiple flasks to be handled simultaneously. Some
micr o-digestor s include a one-piece glass manifold for fume
r emoval. The glass manifold is connected to a standar d water
aspir ator to dilute and r emove fumes that do not r eflux in
the flask necks. Whether or not a glass manifold is used, the
digestion appar atus should be oper ated inside a labor ator y
hood.
Digested samples can be diluted and an aliquot taken
for distillation on a micr o steam distillation unit. Micr o
steam distillation units ar e available to accept digested sam-
ples of up to 4 ml concentr ated acid and volumes of about
55 ml. An electr ic immer sion heater pr oduces steam heat
that causes the sample to boil and r elease ammonia gas. The
vapor s pass into a condenser wher e contact with water-
cooled glass condenses the vapor s, which dr ip into the
r eceiving solution via a deliver y tube. Distillation time is
appr oximately 5 minutes.
Methodologies have been specifically developed for
micr o-Kjeldahl appar atus. These most often ar e methods that
involve homogeneous samples, r elatively high nitr ogen levels
in the sample, and small sample sizes nor mally less than
0.25 g.
The installation consider ations, initial expense, space
r equir ements, and ongoing utility and r eagent cost ar e much
less with micr o equipment.
Bl o ck Di ge s to rs
Block digestor s ar e high temper atur e cer amic or alu-
minum blocks with wells to accept str aight walled digestion
tubes. They can accommodate fr om 2 to 25 tubes at once.
Block digestor s ar e heavy but movable self-contained units
designed to be used in a labor ator y hood. Digestion tubes
r ange up to 300 ml capacity, suitable for sample sizes up to
about 2 g or 75 ml.
Since nar r ow, str aight-walled digestion tubes ar e used
instead of tr aditional Kjeldahl flasks with a bulb and neck,
and since a number of tubes ar e gr ouped closely together in
a ver tical configur ation in the block, acid r eflux par ameter s
ar e differ ent. This means salt/ acid r atios and heat inputs
appr opr iate for macr o-Kjeldahl methods do not exactly
tr ansfer to digestion in a block. Methods have been devel-
oped specifically for block digestor s similar, but not exactly
identical, to macr o-Kjeldahl methods.
TheRapidStill I is a microdistillation unit.
Macro-Kjeldahl Combination Digestion/Distillation
Apparatus.
TheMicro Digestor uses a glass manifold to removefumes.
8
Typically block digestor s have one contr oller that
adjusts the temper atur e of the entir e block. The contr oller
can be automated, allowing for timed r amping and multiple
temper atur e settings dur ing the cour se of a digestion.
Most often block digestor s ar e used together with fume
r emoval devices, even though they ar e oper ated in a hood.
One such system consists of a glass manifold with individual
bulb eductor s in a r ack designed to r est on top of a set of
digestion tubes in the block. Another system uses individual
exhaust caps with flexible tubing. In either case the manifold
is connected to a water aspir ator. Or, instead of a water aspi-
r ator, ther e ar e alter native units that employ mechanical
aspir ation and include bubbling the acid fumes thr ough a
neutr alizing base solution.
Block digestion followed by steam distillation is some-
times r efer r ed to as r apid Kjeldahl par tly because a number
of block digestion methodologies ar e shor ter than classical
macr o-Kjeldahl digestions and benchtop steam distillation
units in use ar e faster than classical distillations.
Rapi d Ste am Di s ti l l ati o n Apparatus
The development of block digestor s as an alter native to
macr o-Kjeldahl digestion has been par alleled by the develop-
ment of benchtop steam distillation units. Basically, this
equipment consists of a steam gener ator to inject steam into
the alkaline digestion mixtur e and an evapor ator-type con-
denser, wher e contact with water-cooled glass coils causes
the vapor s to condense and dr ip into the r eceiving solution
via a deliver y tube. Usually the unit is designed to accept
str aight digestion tubes fr om block digestor s, though ther e is
no r eason in theor y why any digestion techniques could not
be combined with any distillation technique. Steam distilla-
tion is much mor e r apid than classical macr o-distillation,
typically taking fr om 3 to 8 minutes. On the other hand, only
one distillation at a time can be done.
Rapid steam distillation units ar e available with differ -
ent featur es, r anging fr om r elatively manual models to highly
automated models. Basic models dispense dilution water and
base under contr ol of a push button. Some models include a
timer to contr ol the length of distillation, shutting down auto-
matically. Some units automate the entir e distillation pr ocess
once the digestion flask is in place, and other s automatically
titr ate to end point after distillation, and calculate and dis-
play the r esults in a pr inted r epor t.
Ti trato rs
Of cour se, r eceiving solutions may be individually hand
titr ated using an indicator solution and bur et, but sever al
models of benchtop automatic titr ator instr uments ar e also
available. Some units will titr ate one r eceiving solution at a
time to a set end point. Other s will automatically titr ate a
number of r eceiving flasks sequentially. Still other models
pr ovide a pr intout of r esults along with digital r eadout.
Othe r Equi pme nt and Pro ce dure s
Following Kjeldahl digestion, ther e ar e other pr oce-
dur es available to deter mine nitr ogen ( or pr otein) in the
digestion mixtur e. The most popular involves the spec-
tr ophotometr ic method for ammonium deter minations of
digestion mixtur es. The Nessler and Phenate color develop-
ment methods ar e the most widely used. The Biur et method,
a dye binding pr ocedur e, is another type. Automated analyz-
er s which use color development chemistr y ar e available and
can per for m lar ge number s of assays. However, the appar a-
tus and maintenance costs ar e high.
Manual Nessler color imetr ic methods ar e often used in
ammonia deter minations. Digested samples ar e pr epar ed for
a color development and their absor bency is r ead with a
spectr ophotometer at a r ange of 400 to 500 nm. Standar ds
ar e also r un and a standar d cur ve is pr epar ed which plots
absor bency ver sus milligr ams of ammonia. The standar d
cur ve is then compar ed to the sample r esults.
TheRapidStill I is a steamdistillation apparatus.
Rapid Digestor-4 with fumeremoval system
9
The conditions r equir ed for optimal color development
and stability can be r igor ous for all of these pr ocedur es.
However, spectr ophotometr ic analysis has the ability to
deter mine ver y small quantities of ammonia as low as 20
micr o-gr ams per liter under optimal conditions.
Ammonia specific ion electr odes give the analyst another
means for the deter mination of ammonia. The electr ode uses
a hydr ophobic gas per meable membr ane to separ ate the
sample solution fr om an inter nal ammonium chlor ide solu-
tion. Dissolved ammonia diffuses thr ough the membr ane and
changes the inter nal pH, which is sensed by a pH meter that
has an expanded millivolt scale. Ammonia concentr ations of
standar ds ver sus potential in millivolts ar e plotted on a semi-
logar ithmic gr aph, and sample concentr ations ar e r ead fr om
the standar d cur ve. The ammonia specific ion electr ode can
be pr ecise and has few inter fer ences.
In addition, ther e is alter native nitr ogen deter mination
equipment which involves techniques other than Kjeldahl
digestion. Ther e ar e instr uments which incor por ate tech-
niques of Dumas combustion, neutr on activation, near
infr ar ed r eflectance spectr ometr y or dir ect distillation. The
fir st two instr ument/ techniques deter mine total nitr ogen
r egar dless of its for m within the sample. Their acceptance in
most labor ator ies has been limited due to the necessar y
changes involved in r epor ting these types of nitr ogen values.
These changes would involve br oad multi-industr ial r e-evalu-
ations of compar ative nitr ogen-pr otein analysis.
Near infr ar ed r eflectance spectr ometr y and dir ect dis-
tillation use methods based on appr oximate r elationships
between nitr ogen content and pr otein. Both methods depend
on calibr ations with standar ds which have been deter mined
by tr aditional Kjeldahl analysis.
Mo s t Co mmo n Pro bl e ms
Like any other method, the Kjeldahl pr ocedur e is
subject to pr oblems with methodology, technique and equip-
ment. Under standing the pr inciples behind the Kjeldahl
pr ocess and using appr opr iate quality contr ol pr ocedur es
should pr event most pr oblems fr om occur r ing. When devel-
oping a new or modifying an existing method, one should
pr oceed with caution and exper imental ver ification.
Should nitr ogen r esults differ fr om those expected,
knowledge of common pr oblems makes finding and cor r ecting
the er r or easier. Common pr oblems include the following:
sample size and type incompatibility with amounts and types
of acid, salt or catalyst used; contaminated samples, standar ds,
r eagents or equipment; inadequate or excessive digestion
time; inadequate or excessive distillation dilution volume or
aliquot volume; foaming or bumping dur ing digestion;
uneven digestion; inadequate or excessive sample temper a-
tur e; pr ecipitation of salt or caking or digested sample; leaks
in appar atus connections; inadequate sodium hydr oxide
addition or sodium hydr oxide car r yover ; and calculation
er r or s.
10
Gl o s s a r y
Aci d No rmal i ty The measur ement of an acid str ength, ie: the lower
the nor mality the weaker the acid.
Al i quo t A par t which is a definite fr action of a whole, ie: as taking a
por tion of a sample.
Al undum ( Al 2 0 3 ) A gr anular mater ial that may be added to the
digestion mixtur e to decr ease bumping dur ing the initial digestion
phase.
Back Ti trati o n Method of titr ation to deter mine the amount of
excess acid that was consumed in the r eceiving solution dur ing
Kjeldahl distillation.
Bas e No rmal i ty The measur ement of base str ength, i.e.: the lower
the nor mality, the weaker the base.
Bi ure t Me tho d A dye-binding pr ocedur e using an analytical r eagent
especially designed for pr oteins.
Bl ank The amount of nitr ogen contained in the r eagents used. In
Kjeldahl calculations, the blank is subtr acted so that it does not
falsely incr ease the % nitr ogen or % pr otein of the sample.
Catal ys t Any substance of which a fr actional per centage notably
affects the r ate of a chemical r eaction without itself being consumed
or under going a chemical change. Most catalysts acceler ate r eac-
tions, but a few r etar d them.
Cl as s i cal Macro - Kje l dahl A method designed to digest or distill
multiple samples that contain high levels of nitr ogen.
Co nne cti ng Bul b A glass component of a classical Kjeldahl distilla-
tion system. Separ ates liquid fr om vapor s pr ior to the vapor s enter -
ing the condenser and effectively tr aps caustic mist that may car r y
over to the r eceiving solution.
De l i ve ry Tube A glass component of a Kjeldahl system that deliver s
distillate to a r eceiving flask. The long neck, r ound bottom and thick
walls help eliminate pr essur e fluctuations in the distillation phase.
Di ge s ti o n In Kjeldahl methods, the destr uction of or ganic matter
with an acid. Also used as a noun meaning digestion mixtur e.
Di re ct Di s ti l l ati o n A method based on appr oximate r elationships
between nitr ogen content and pr otein without per for ming a digestion
step.
Di re ct Ti trati o n Method of titr ation to deter mine the amount of
ammonia that has been absor bed into the r eceiving solution dur ing
Kjeldahl distillation.
Di s ti l l ati o n A separ ation pr ocess in which a liquid is conver ted to
vapor and the vapor then condensed to a liquid.
Dumas Co mbus ti o n A combustion method measur ing nitr ogen gas
involving in the total destr uction of the sample.
End Po i nt The point dur ing a titr ation at which a mar ked color
change is obser ved.
Ho mo ge ne o us Adjective descr ibing a mixtur e compr ised of two or
mor e compounds or elements unifor mly disper sed in each other.
Hydro l ys i s A chemical r eaction in which water r eacts with another
substance to for m two or mor e new substances.
Hydro pho bi c Incapable of dissolving in water.
Indi cato r So l uti o n A solution added to a sample dur ing titr ation
that pr oduces a color change indicating end point.
Kje l dahl Me tho d An analytical method for deter mining nitr ogen
content in cer tain or ganic and inor ganic compounds. The method
uses digestion, distillation and titr ation to ar r ive at the nitr ogen con-
tent of the sample.
Macro - Kje l dahl A Kjeldahl method designed to handle high levels
of nitr ogen; classical Kjeldahl.
Mi cro - Kje l dahl A Kjeldahl method that r equir es smaller volumes
of chemical r eagents and smaller sample sizes than macr o or classic
Kjeldahl.
Mo l e cul ar We i ght The sum of the atomic weights of the atoms in a
molecule.
Ne ar Infrare d Re fl e ctance Spe ctro me try Pr otein identification
method involving the measur ement of the r adiant ener gy r eflected by
the sample in infr ar ed wavelengths.
Ne s s l e r Me tho d Manual color imetr ic method wher e digested sam-
ples ar e pr epar ed for color development and their absor bency r ead
with a spectr ophotometer.
Ne utro n Acti vati o n A sensitive technique for identifying and mea-
sur ing ver y small amounts of var ious elements. A sample is exposed
to neutr on bombar dment in a nuclear r eactor for the pur pose of
pr oducing r adioisotopes fr om the stable elements.
Ni trate [ NO3
-
] For m of nitr ogen usually found in fer tilizer s. It must
be r educed befor e digestion in the Kjeldahl method.
Ni tri te [ NO2
-
] For m of nitr ogen usually found in fer tilizer s. It must
be r educed befor e digestion in the Kjeldahl method.
pH A measur ement that r epr esents the acidity or alkalinity of an
aqueous solution.
Phe nate Me tho d Manual color imetr ic method wher e digested sam-
ples ar e pr epar ed for color development and their absor bency r ead
with a spectr ophotometer.
Pre ci pi tate Small par ticles that have settled out of a liquid or
gaseous suspension by gr avity, or that r esult fr om a chemical
r eaction.
Rapi d Kje l dahl Same method as classical Kjeldahl, except, thr ough
efficient heating and compactness, r equir es less time to complete the
digestion step. Digestion times r ange fr om 6-60 minutes compar ed
to up to two hour s for classical digestion.
Re ce i vi ng So l uti o n Solution used to captur e ammonia separ ated
thr ough distillation.
Re fl ux The liquid condensed fr om the r ising vapor.
Spe ctro pho to me try Identification method involving an optical
device that measur es r adiant ener gy within selected wavelengths in
the visual, ultr aviolet or infr ar ed r egions of the spectr um.
Ste am Di s ti l l ati o n Distillation using steam to dr ive off NH3 gas in
Kjeldahl deter minations.
Ti trati o n A quantitative analytical pr ocess that is basically volumet-
r ic. Gener ally, a standar d solution, that is, one containing a known
concentr ation of substance X ( titr ant) , is pr ogr essively added to a
measur ed volume of a solution of a substance Y ( titr and) that r eacts
with the titr ant. The addition is continued until the end point is
r eached.
Wate r As pi rato r A device that pr ovides suction thr ough the use of
flowing water past a small or ifice.
Bi bl i o gr a ph y
Amer ican Association of Cer eal Chemists. Approved Methods of
Analysis. St. Paul, MN: Amer ican Association of Cer eal Chemists,
1995.
Amer ican Public Health Association, Amer ican Water Wor ks
Association, Water Pollution Contr ol Feder ation. Standard Methods
for theExamination of Water and Wastewater. Washington, DC:
Amer ican Public Health Association, 1992.
AOAC Inter national ( for mer ly the Association of Official Analytical
Chemists) . Official Methods of Analysis. Ar lington, VA: AOAC
Inter national, 1995.
Br adstr eet, R. B. TheKjeldahl Method for Organic Nitrogen. New
Yor k, NY: Academic Pr ess Incor por ated, 1965.
Jones, J. Benton. Kjeldahl Method for Nitrogen Determination.
Athens, GA: Micr o-Macr o Publishing, 1991.
Kjeldahl, Johan Z. A new method for the deter mination of nitr ogen
in or ganic bodies. Analytical Chemistry22 ( 1883) : 366.
Meloan, Clifton E., and Pomer anz, Y. Food Analysis: Theory &
Practice. Westpor t, CT: AVI Publishing Company, 1978.
United States Envir onmental Pr otection Agency. Methods for
Chemical Analysis of Water and Wastes. Cincinnati, OH:
Envir onmental Pr otection Agency, EPA-600/ 4-79-020, Envir onmental
Monitor ing and Suppor t Labor ator y, 1979.
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