Accred Qual Assur (1999) 4:504-510

© Springer-Verlag 1999

Thomas Anglov Uncertainty of nitrogen determination

Inge M. Petersen
Jesper Kristiansen by the Kjeldahl method

Abstract The uncertainty of the largest contribution to the uncer-

J. Kristiansen (lEI)
The National Institute of Occupational
Health, LerSI/l Parka lie 105,
DK-2100 Copenhagen, Denmark
e-mail: jkr@ami.dk
Tel.: +45-39-165200
Fax: +45-39-165201
T. Anglov, I.M. Petersen
Department of Metrology, Novo Nordisk
A/S, Krogshl/ljvej 51, DK-2XXO Bagsvrerd,
Denmark
Kjeldahl method for determination
of nitrogen in insulin was evalu-
ated according the procedure de-
scribed in the Guide to the Expres-
sion of Uncertainty in Measure-
ment. The relative standard uncer-
tainty of the method was found to
be 0.19%, compared to the relative
intermediate precision experimen-
tally found to be 0.085%. The un-
certainty components were organ-
ized in Tables, which allowed an
easy overview and evaluation. The
tainty came from volumetric equip-
ment. Systematic uncertainty bud-
gets such as the design presented
here facilitate the uncertainty eval-
uation process and makes it easier
to compare uncertainty evaluations
performed by different analysts.
Key words Uncertainty budget·
Uncertainty evaluation .
Uncertainty component·
Traceability . Reference standards

Introduction
Determination of nitrogen content plays a key role in
assigning values to insulin reference materials. The ref-
erence materials in question serve as reference stand-
ards when measuring insulin in drug products. Thus,
the Kjeldahl nitrogen determination is a crucial link in
the traceability chain. The uncertainty budget for the
Kjeldahl method published in this paper has three ob-
jectives: Firstly, to estimate the uncertainty of results
obtained by the Kjeldahl method; secondly, to identify
steps in the analytical procedure that may be targets for
improvement; and thirdly, to contribute a generally ap-
plicable procedure for evaluating an uncertainty budget
for a chemical analytical method to the current litera-
ture. The need for such schemes or procedures is ur-
gent as accredited laboratories in the near future will
be required to state their uncertainty of measurement
[1]. The uncertainty budget published in this paper is
based on existing guidelines [2, 3].
Methods
Kjeldahl method
The nitrogen content of dry insulin was determined by
the Kjeldahl method, which consists of three steps, di-
gestion, distillation and titration. In brief, approximately
50 mg of the sample was weighed accurately and trans-
ferred to a digestion test tube. Concentrated sulphuric
acid was added and the mixture was heated to 390°C
for 4 h (digestion). The tube with the digested sample
was placed in the Kjeldahl apparatus, sodium hydrox-
ide and steam was added, and ammonia was distilled
off. The ammonia-rich steam was condensed in the re-
ceiving flask (distillation). The content of ammonia in
the receiving flask was determined by end-point titra-
tion to pH 4.5 with 0.1 molll hydrochloric acid (titra-
tion). The normality of the acid (N HCh in mol/I) was de-
termined by titration of tris-(hydroxymethyl)-amino-
methane (Tris). Blanks consisted of empty digestion
274 T. Anglov . I.M. Petersen' 1. Kristiansen
tubes treated as samples. Samples were measured in
duplicate and blanks in triplicate.
Let a (mg) denote the amount of sample. If b (ml)
and c (ml) denote the volume of hydrochloric acid used
for titration of the blanks and the sample, respectively,
then the relative nitrogen content of the sample (Ntota, )
can be calculated as:
N _ 14.01 g/mol· (c - b)· N HC1
total - (1)
a + (N to tal)2 u(a)2 + (N to tal)2 u(N HCI )2
The method was validated using two insulin drugs. The
relative standard deviation under intermediate preci-
sion conditions was found to be 0.085%.
Uncertainty budgets
The uncertainty of Ntotal was estimated by combining
standard uncertainties of N HC ]' a, band c. Uncertainties
were combined by using the rule of "error propaga-
tion" [3, 4]. In general, when a result of measurement
(y) is determined from other quantities, the relation-
ship between y and the values of these quantities (input
estimates) can be expressed by a function, / [2]:
y = f(Xb X2 ••• ,Xj ••• ,XN) (2)
where XI .•. Xj ...XN represents N input estimates. The un-
certainty of y (u(y)) is related to the uncertainty of the
input estimates by the equation:
(3)
where U(XJ2 is the standard uncertainty variance and
u(xJ the standard uncertainty of the uncertainty com-
ponent number i, and where a/ is the partial deriva-
aXj
tive of the function, f, with respect to the uncertainty
component number Xj. The partial derivative is often
called the sensitivity coefficient because it describes
how the measurement result varies with changes in the
value of the input estimates [2]. It should be noted that
Eq. (3) is an approximation that is valid if there are no
correlated input estimates [2]. The result of measure-
ment of the Kjeldahl method is N total as given by
Eq. (1), and the expression for N total is the function/in
Eq. (2). Thus, the following expression for the uncer-
tainty of Ntotal is obtained:
7f
( U(C))2 (U(b))2
( U(Ntotal))2 = if + urement should be reported as:
N total (c-b)
(4a)
u(a))
+ (U(N HCI ))2 + ( £
N HC1 a
The factors 1Iy2 and 1/y3 account for the number of
replicate determinations (2 and 3, respectively). The
expression may be rearranged in order to emphasize
the sensitivity coefficient of each uncertainty compo-
nent:
(N .)2 = ( Ntotal )2 ( )2 + ( N total )2 (b)2
U totdl y2 . (c _ b) u C y3 . (c _ b) u
(4b)
y2·a N HCI
The standard uncertainties u(N HCI ), u(a), u(b) and u(c)
are themselves composed from various uncertainty
components. These uncertainties were likewise ob-
tained from uncertainty budgets (Appendix Bl-4).
Relative uncertainty variance contributions
In this paper the relative uncertainty variance contribu-
tions are used to illustrate the relative significance of
different uncertainty components [5]. The relative con-
tribution (rJ of an uncertainty component Xj to the
combined uncertainty, is defined here as the standard
uncertainty variance of the component multiplied with
its squared sensitivity coefficient divided by the com-
bined standard uncertainty variance [5]:
(5)
where xj=N HC ], a, band c. It follows from Eqs. (3) and
(5) that L rj = 1 (assuming the absence of correlated in-
put estimates).
Results
Uncertainty of Ntotal
The values of N HC ]' a, b, c and Ntotal as well as the re-
spective standard uncertainties u(N HCI ), u(a), u(b), u(c)
and u(Ntotal) and the corresponding sensitivity coeffi-
cients are given in Table 1. The combined standard un-
certainty u(Ntota') was estimated to 0.00023, corre-
sponding to a relative standard uncertainty of 0.19%.
U sing an coverage factor k = 2 the result of the meas-
Ntotal ± V(N total ) =0.1233 (±0.00046)
where V(Ntotal) is the expanded standard uncertainty.
Details on the uncertainty budgets for u(N HCI ), u(a),
u(b) and u(c) are given in Appendix B.
 Uncertainty of nitrogen determination by the Kjeldahl method 275

Table 1 Values of uncertainty components, their standard uncertainty and sensitivity coefficient

Component Symbol Value Standard Sensitivity coefficient Reference

uncertainty

Normality of hydrochloric acid N Hcl 0.1 molll 1.1 x 10-4 molll (N ,o,al) = 1.23 llmol Appendix B1
N Hcl

Amount of sample a 50mg 0.059 mg (NTo,al) = 1.74 X 10-3 mg- I Appendix B2

V2'a
Volume of titrant used on blank sample b 0.1 ml 4.0 x 10-3 ml ( N'o'a' )=1.fl2XI0- 2 ml- 1 Appendix B3
tHe-b)
Volume of titrant used on the sample e 4.5 ml 7.2x10 3 ml ( N'o'a' )=1.9HXI0- 2 ml- I Appendix B4
V2·(e-b)
Result of measurement Niota' 0.12329 0'()()()23 Eq. (4b)

Relative contributions to the uncertainty
The relative contributions (r;) from U(N HCI ), u(a), u(b)
and u(e) to the combined standard uncertainty variance
u(Ntotal)2, are shown in Fig. 1. The two largest contribu-
tions come from U(NHCI) and u(e). Both contribute
35-38% to the combined standard uncertainty var-
iance. The uncertainty budget for U(NHCI) (Appendix
Bl) shows that the uncertainty of this component is
composed mainly of uncertainty contributions from the
temperature of the titrant, the weighing of the Tris base
and the volume of the titrant. The primary contribu-
tions to a(e) (see Appendix B4) come from the uncer-
tainty of digesting the sample (which consists of the un-
certainty of the amount of sample transferred to the di-
gestion tube) and from the uncertainty of the volume of
the titrant.
In Fig. 2 the relative contributions to the uncertainty
variance are summarized for various types of analytical
steps and equipment used in the Kjeldahl method. The
contribution from volumetric equipment (Fig. 2) in-
cludes the contributions from uncertainty of the vol-
ume of the titrant and from uncertainty of the tempera-
ture of the titrant (Appendix Bl, 3 and 4). Weighing
(Fig. 2) includes first and second weighing of both Tris
base and the sample (Appendix Bl and 4). Digestion
(Fig. 2) includes all uncertainty components denoted in
digestion in Appendix B3 and 4. Lastly, Tris-purity
(Fig. 2) denotes the uncertainty component associated
with the purity of the Tris base evaluated in Appendix
B1. From Fig. 2 it can be seen, that the largest uncer-
tainty contribution comes from the use of volumetric
equipment, i.e. burettes used for titration of hydro-
chloric acid and for titration of samples and blanks.
This uncertainty component is composed of the preci-
sion of titration and of the uncertainty of the tempera-
ture. The latter factor influences the volume of titrant
used for titration. The second largest contribution
comes from weighing (of Tris base and of the sam-
ple).

~
c
0
~ i
.c
c
0
i.c i
..
;:
c
0
8.:
!J
u
..:
::

Fig.2 Relative contributions from volumetric equipment, weigh-

Fig. 1 Relative contributions from N He," a, band e to the com- ing, digestion and the purity of the Tris base to the combined
bined standard uncertainty variance u(N'o,al)2 standard uncertainty variance
276 T. Anglov . I.M. Petersen' J. Kristiansen
Discussion
The uncertainty budget presented in Table 1 indicates
that a typical result obtained by the Kjeldahl method
(as described in this paper) will have a relative standard
uncertainty of around 0.19%, which is approximately
twice as large as the experimentally derived relative
standard deviation estimated under intermediate preci-
sion conditions. This is not surprising as some of the
uncertainty components included in the uncertainty
budget are difficult to evaluate experimentally, e.g. pur-
ity of the Tris base, accuracy of the pH meter, etc.
The uncertainty evaluated for the Kjeldahl method
should be propagated to the uncertainty of the insulin
reference standards as well as to the analytical methods
that are calibrated with these standards. Failure to con-
sider the uncertainty of the reference standards or of
the analytical method, or to base the uncertainty on less
encompassing estimates (e.g. intermediate precision),
may lead to an excess number of nonconforming results
when evaluating the fulfilment of acceptance criteria
for insulin drug products.
The present uncertainty budget demonstrates that
the Kjeldahl method may be improved. The most
promising target for improvement is the titration. Ac-
cording to the analysis of the uncertainty contributions,
this can be accomplished by improving the temperature
control during titration or by improving the accuracy of
the volume of titrant. The latter goal may be achieved
by using more accurate titration equipment, or, at least
partly, by increasing the number of replicate determi-
nations. Increasing the number of replicates will im-
prove the precision, but will not influence the trueness
of the volumetric equipment, nor does it compensate
for long-term fluctuations that may affect the equip-
ment.
The uncertainty budget in this study was organized
to make it easy to review the evaluation of individual
uncertainty components. The uncertainty budget is or-
ganized in three evaluation steps: In the first step, the
uncertainty is evaluated based on the input estimates
that are used directly in the calculation of Nlolal (i.e.
N HCb a, band c in Eqs. 1 and 4b) (results in Table 1). In
the second step, the uncertainty of each of the input
estimates (i.e. N HCb a, b and c) is evaluated and calcu-
lated. Correction factors are applied to correct for ef-
fects such as the influence of temperature on the vol-
ume, inaccuracy of the pH meter, etc. (equations and
results in Appendix B). Where necessary, a third step
of uncertainty evaluation was applied. An example is
air bubbles in the burette, water in the Tris base and
other effects that are evaluated and included in the un-
certainty of qconlT (Appendix B1). Generally evalua-
tions in the third step should only be carried out if the
uncertainty components contribute significantly to the
combined uncertainty.
It was not found necessary to consider the correla-
tion between input estimates because the contribution
from covariances was judged to be minor in the uncer-
tainty budget presented above. Examples are c and b in
Eq. (1) which are determined using the same burette.
This means that uncertainties of c and b originating
from, e.g. long-term variations of volume (delivered by
the burette), wear of the burette, etc. are positively cor-
related. In other words, the uncertainty of the differ-
ence (c - b) tends to be unaffected by these effects. The
same reasoning can be used for m1 and m2, which are
determined for calculation of a (Appendix B2).
The structure of this uncertainty budget for the Kjel-
dahl method may be used as a paradigm for other ana-
lytical methods. However, it must be understood that
an uncertainty budget is designed for a specific method,
and is therefore only valid for a set of specified analyti-
cal conditions (including the analytical method, type of
sample and its concentration level). For highly auto-
mated (i.e. computerized) analytical methods, the result
of measurement is often calculated by specialized soft-
ware, and this makes the interdependency of various
analytical steps less obvious. For such systems other ap-
proaches for making uncertainty budgets must be ap-
plied [5, 6].
Conclusions
A structured evaluation of uncertainty components was
applied for the Kjeldahl method for nitrogen determi-
nation. The design offers a systematic approach for
making uncertainty budgets for analytical chemical
methods, and it may facilitate comparison of uncertain-
ty budgets made by different analysts or laboratories.
The evaluation of the contribution from individual or
grouped uncertainty components made it possible to
suggest specific improvements of the analytical meth-
od.
Appendix A: Definitions, abbreviations and equations
1. Definitions and abbreviations
Terms and definitions used in accordance with GUM
and VIM [2, 7].
a: mg sample used in the measurement
b: ml titrant (hydrochloric acid) used on the blank
sample (average of three determinations)
c: ml titrant (hydrochloric acid) used on the sam-
ple
k: Correction factor 14.01 g/mol containing the
atomic weight of nitrogen
MPE: Maximum permissible error
 Uncertainty of nitrogen determination by the Kjeldahl method 277

Table 81 Estimation of the uncertainty of NHC1

No. Input Symbol Value Stated or Type Standard Sensitivity coefricient Contribution to Reference
quantiliy (x,) of input evaluated (AlB) and uncertainty ar the standard
estimate uncer- type of u(x,) ax, uncertainty
tainty distribution of the output
estimate
ar
u(x,)~
ax,

various
contri-
q~ulltr
lxlO-4, B
rectangular
CX~)-4)=5.xX 10-' N lln =0.1 molll
qC~lnlr
5.X x 10-6 molll Own estimate

butions'
2 MPE of qrll '" Oh Instrument
the pH- specification.
meter own estimate
3 Purity of qpu, 0.9989 (UKW)SC B 2.9 x 10-' molll Supplier
Tris (99.X9%) liJ- .x 0
0.005 _ 2 9 1 -4 Nlln=O.1 molll
qpur
certificate
4 First m, 11K) mg 0.1 mg B O.lmg 5.9 x 10-' molll Instrument
rectangular Nlln = 1.02 x 10-' moll(mg·l) specification
weighing
of Tris base 7~
5.8x 10-" mg
m,-III,

5 Second m, 2mg 0.02mg B 0.02 mg 1.2 x 10-' molll Instrument

weighing rectangular yr= ~= 1.02 x 10-' moll(mg·l) specification
ln2-n1 1
of Tris base 1.2 x 10-' mg
6 Volume of v 8.06 ml 0.004ml" A 0.004ml Nlln 5.0xI0-' molll Instrument
titrant normal - - = 0.012 moll(L'ml) qualification
v
report
Temperature O.lX1l2 6.9 x 10-' molll Own estimate
7 (1.2~~0-')=6.l)XIO-4 Nlln =0.1 molll
C
qlcmp
B
of titrant rectangular r J q'cmr Laboratory
temp.
15-25°C
Molar mass M ...,;, 12l.l4 ",Og/mol' - Own estimate
of Tris base glmol
u (XN",) =Y(5.8 x 10 ")' + (2.9 x 10 Sf + (5.9 x 10 'f + (1.2 x 10 ')'+ (5.0 x 10 ')'+(6.9 x 10 ')' = l.l x 10 -4 molll

a Contributions from: The water content of the silica gel; lack of C The stipulated purity of (hydroxymethyl)-aminomethane (Tris)

complete drying of the Tris base; variation in the time of drying; is 99.H9% with an uncertainty of (l.05%
variation in the time of dissolution of the Tris base; the hygrosco- d A standard deviation of 0.004 mL was found experimentally
py of the Tris base; air bubbles in the burette: Combined uncer- C Water density at 15°C is 0.99913 glmL, at 20°C 0.99H23 glml and

tainty estimated to 0.01 mg Tris base or 0.01 % of approximately
100 mg Tris base
h The pH-meter specifications stipulate a MPE of 0.02 pH units.
The form of the titration curve indicate that an uncertainty of 0.02
pH corresponds to a negligible uncertainty in the volume of ti-
trant. Thus, the value of this uncertainty component is not in-
cluded in the uncertainty budget
at 25°C 0.99707 glmL. It is assumed that the temperature in the
laboratory (and the temperature of the acid) in average is 20°C,
but in worst case may vary ±5°C. The largest change in water
density from 20 to 25°C is 0.00116 g/ml, or 0.12%
f The uncertainty of the molar mass is assumed to give a negligi-
ble contribution to the combined uncertainty

Table 82 Estimation of the uncertainty of a

No. Input quantity Symbol Value of Stated or Type Standard Sensitivity Contribution Reference
(x,) input evaluated (AlB) uncertainty coefficient to the standard
estimate uncertainty and type of u(x,) ~r uncertainty
distribution ax, of the output
estimate
ar
u(x,)~
ax,

First weighing III, 52mg 0.1 mg B 0.1 mg 5.8x 10-' mg Instrument

of sample rectangular y1 specifications
2 Second weighing 2mg 0.02mg B 0.02 mg 1.2 x 10-' mg Ins(rument
vr
III,
of sample rectangular specifications

u(a)=Y(5.8XIO ')'+(1.2xlO ')'=0.059mg

278 T. Anglov . I.M. Petersen' J. Kristiansen

Table 83 Estimation of the uncertainty of b

No. Input Symhol Value of Stated or Type Standard Sensitivity Contrihution Reference
quantity (x,) input evaluated (AlB) uncertainty coefficient to the standard
estimate uncertainty and type of u(x,)
i!L uncertainty
distrihution ax, of the output
estimate
It (x,) af
ax,
Digestion' qJip.cst N/A N/A N/A N/A N/A Digestion not
performed
2 Distillation" qdistil Own estimation
3 Volumen of v 0.1 ml O.(X)4 ml A O.!X)4 ml e 4 x 10-' ml Instrument
titrant normal !!. = 1 qualification
v
report
4 Temperature qtcmp 1.2x 10-'" B
_b_=O.1 ml 6.9xlO-' ml Own estimation
of titrant rectangular qtcmp
Lahoratory
temp. 15-25°C
5 MPE of the qpH Instrument
pH meter specifications.
Own judgement

a No Kjeldahl mixture or acid was used. The digestion was not laboratory (and the temperature of the acid) on average is 20°C,
performed but in worst case may vary ± 5°C. The largest change in water
h Addition of water and NaOH; distillation time; leaks; incom- density from 20 to 25°C is 0.001 Hi glml, or 0.12%
plete transmission of NH3 to the receiving vessel; temperature of C The pH-meter specifications stipulate a MPE of 0.02 pH units.

the vapour: All components are assumed to be negligible The form of the titration curve indicates that an uncertainty of
C A standard deviation of 0.004 ml was found experimentally 0.02 pH corresponds to a negligible uncertainty in the volume of
d Water density at 15°C is 0.99913 glml, at 20°C 0.99823 glml and titrant. Thus, the value of this uncertainty component is not in-
at 25°C 0.99707 g/m\. It is assumed that the temperature in the cluded in the uncertainty budget

Table 84 Estimation of the uncertainty of c

No. Input Symhol Value of Stated or Type Standard Sensitivity Contrihution Reference
quantity (x,) input evaluated (AlB) uncertainty coefficient to the standard
estimate uncertainty and type of u(x,) af uncertainty
distrihution ax, of the output
estimate
u(x,)~
at"
ax,
Digestion' (Um B c 5.2 X 10-3 ml Own estimation
qdigcst
rectangular (O~2)= 1.2 x 10-' --=4.5ml
qdigcst

2 Distillation b qJistii Own estimation

3 Volume of v 4.5 ml O.(Xl4 ml A 0.004 ml' 4.0x 10-' ml Instrument
titrant normal ~= 1 qualification
v
report
4 Temperature (W1l2 B c 3.0x10-3 ml Own estimation
of titrant
qtemp
rectangular (().~12) =6.9 x 10-' --=4.5 ml Lahoratory
qJistil
temp. 15-25°C
5 MPE of the qpll zO ml" Instrument
pH meter speci[ications.
Own judgment
u(c)=V(5.2xlO ')'+(4xlO .l)'+(3.0xlO .l)2=7.22x 10-' ml

"The uncertainty components: Amount of catalysator, the
amount of Kjeldahl mixture and H 2S0 4 , block temperature, di-
gestion time and boiling are assumed not to contribute signifi-
cantly to the uncertainty. Transfer of sample to digestion vessel is
assumed to contribute with 0.1 mg or 0.2%
h Addition of water and NaOH; destillation time; leaks; incom-
plete transmission of NH3 to the receiving vessel; temperature of
the vapour: All components are assumed to be negligible
C A standard deviation of O'()04 ml was found experimentally
d The pH-meter specifications stipulate a MPE of 0.02 pH units.
The form of the titration curve indicates that an uncertainty of
0.02 pH corresponds to a negligible uncertainty in the volume of
titrant. Thus, the value of this uncertainty component is not in-
cluded in the uncertainty budget
 Uncertainty of nitrogen detennination by the Kjeldahl method 279

N: Number of quantities or uncertainty compo- rection factors for the water content of Tris, the accura-
nents cy of the pH meter, the temperature of the titrant, and
N HC (: The normality of the hydrochloric acid the purity of Tris (Table BI):
N tota (: Content of nitrogen in the sample (average of 2
samples)
qi: Correction factors
u(x;): Standard uncertainty of the input estimate Xi
U(y): Expanded uncertainty of y
u(y): The combined standard uncertainty of y 2. Estimation of the uncertainty of a
Xi: Input estimates
y: Output estimate (the result of a measurement) The input estimate a is obtained from weighing the
aJ. Sensitivity coefficient for Xi
sample (m2) and sample cup alone (m(), i.e.
aXi a = J(x;) = m2 - mi' Thus, the standard uncertainty of a
is given by the equation u,7 = u;', + u,;", (Table B2).

2. Equation for the standard uncertainty

In addition to Eq. (3) the following equations evaluat-
ing the standard uncertainty were applied: If the input
estimates Xi in Eq. (2) is related to y only by multiplica-
tion's and divisions, Eq. (3) can be simplified to [2, 4]:
(AI)
with sensitivity coefficients given by:
3. Estimation of the uncertainty of b
In evaluating the uncertainty of b it was assumed that
the volume of titrant used for titration of a blank de-
pends on factors associated with digestion, distillation,
pH measurement and the assessment of the volume of
titrant used. The combination of uncertainty compo-
nents was based on the following relation between the
volume (b) and the various factors:

aJ y (A2)
aXi Xi where v is the estimated volume of titrant read from
If the input estimates Xi is related to y by additions and the burette and all the correction factors (qi) are equal
subtractions, Eq. (3) becomes: to I (Table B3).

U(y)2 = U(X()2 + U(X2)2 + ... + U(XN)2 (A3)

with all sensitivity coefficients equal to 1. 4. Estimation of the uncertainty of c

The uncertainty components and equations used are

Appendix B the same as for b (Appendix B3), i.e.

1. Estimation of the uncertainty of N He(

where v is the estimated volume of titrant read from
N Hc( is calculated from the amount of Tris base used, the burette and the correction factors (q;) are all equal
the molar mass of Tris, the volume of titrant, and cor- to I (Table B4).

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