Clarification of the Limit of Detection in Chromatography 1)
J. P. Foley 21 / J . G. D o r s e y *
Department of Chemistry, University of Florida, Gainesville, F L 32611, USA
Key Words
Chromatography
Limit of detection
Calculation of limit
Summary
Current problems with the limit of detection (LOD)
concept in chromatography are reviewed. They include
the confusion of the LOD with other separate, distinct
concepts in trace analysis such as the minimum detect-
ability (MD); the use of arbitrary, unjustified models
for the calculation of the LOD; the use of concentration
units instead of units of amount; and the failure to
account for differences in chromatographic conditions
when comparing LODs.
Solutions to these LOD problems are discussed. Two
models are proposed for calculating the chromatographic
LOD. A new concept, the standardized chromatographic
LOD, is introduced to account for differences in chro-
matographic bandwidths of experimentally measured
LODs. The standardized chromatographic LOD is
shown to be a more reliable parameter than the con-
ventional (non-standardized) chromatographic LOD.
Introduction
fhe limit of detection (LOD) is generally defined as the
smallest concentration or amount of analyte that can be
detected with reasonable certainty for a given analytical
procedure. Though arguably the most important figure
of merit in trace analysis, the LOD remains an ambiguous
quantity in the field of chromatography. Detection limits
differing by orders of magnitude are frequently reported
for very similar (sometimes identical!) chromatographic
systems. Such huge discrepancies raise serious questions
about the validity of the LOD concept in chromatography.
To eliminate any confusion at the outset, we must emphasize
that the "limit of detection" (or "detection limit") concept
~'e have just described is vastly different from the univer-
~Uy recognized minimum detectability (MD) concept
1) This paper is published as a discussion material. We welcome
commentsfrom our readers.
2) Present Address: National Bureau of Standards, Building 222,
RoomAll3, Washington, D.C. 20234
ChromatographiaVol. 18, No. 9, September 1984 Originals
0009-5893/84/9 0503--09 $ 03.00/0 9 1984 Friedr. Vieweg & Sohn VerlagsgesellschaftmbH
developed by the American Society for Testing and Mate-
rials (ASTM) [1-4]. The limit of detection characterizes
an overall trace analytical procedure consisting of one or
more steps, whereas the minimum detectability describes
only one step in a chromatographic analysis: detection.
(We further differentiate these concepts in a later section,
Eliminating Mistaken Identities.)
As part of our continuing effort to accurately measure and
interpret chromatographic figures of merit [5], our goal in
this report is to transform the LOD in chromatography
from an ill-defined, ambiguous concept to a reliable,
meaningful parameter. We attempt to accomplish this by
first identifying the current problems with the chromato-
graphic LOD concept and then discussing possible solutions
to these problems, with emphasis given to those problems
which are the major sources of the observed discrepancies
in chromatographic detection limits. Especially significant
is a method we propose which accounts for the different
amount of chromatographic dilution that an analyte
undergoes in different chromatographic systems (or more
rigorously, for differences in the chromatographic band-
widths - see Converting to chromatographic reference
conditions).
It is beyond the scope of this report to introduce or review
the limit of detection (LOD) from a historical or theoretical
point of view; such discussions and references to additional
discussions may be found elsewhere [6-9]. We assume
some prior knowledge of the LOD and focus on solving
the problems associated with this concept in chror[aato-
graphy. Most of our discussion is written from the perspec-
tive of peak height as the analytical signal, although two
sections are equally pertinent to analyses using peak area
(Eliminating mistaken identities and Using the correct
units). Gaussian peak profiles are assumed throughout.
Identifying Current Problems
Literature survey results
Initially, to determine the sources of discrepancy in chro-
matographic detection limits, we conducted a limited
survey of analytical textbooks, chromatographic mono-
graphs, and the primary chromatographic literature. This
survey revealed two mistakes of omission, as well as four
major sources of discrepancies. All six are summarized
in Table I. The first two problems are omissions which
discredit the work reported, at least to some degree. They
can be eliminated, however, if more attention is given
503
Table I. Problems with the limit o f detection (LOD) concept in during manuscript preparation, and thus may be dismissei
chromatography
without further discussion. The remaining four probler:
mistakes of omission in Table I comprise the major sources of the discrepancie~
1. Detection limits were not reported, although trace analysis in observed chromatographic detection limits and are th~
was stressed. topics to be addressed in this report.
2. Detection limits were reported, but no f o r m u l a f o r calcu-
lating the LOD was given.
Numerical example
sources of discrepancies
3. The LOD concept was confused with other concepts used in
Before proceeding, however, a numerical example whic!
trace analysis, particularly the m i n i m u m detectability (MD). incorporates problems 4 - 6 in Table I will be given. TI~
4. A r b i t r a r y LOD models were used with little or no justifica- example will demonstrate the magnitude of these probler~
tion. and will facilitate later discussion. Although designed wit~
5. Detection limits were reported as concentrations instead o f liquid chromatography in mind, the points made by tl~
amounts.
example apply equally well to gas and supercritical flui~
6. Differences in chromatographic conditions were not taken
into account. chromatography.
The initial assumptions, experimental conditions, an~
results of this example are shown in Table II. To avoi~
the confusion which it would certainly have caused, problez
3 of Table I was not incorporated into this example. Ifi'.
Table II. Example o f widely differing detection limits
had been, the results might have been even more discordan~
A ssump tions Nevertheless, as seen in the bottom row of Table II, th~
1. Conventional liquid chromatograph with U V detector LODs for the two systems employing identical detectors
2. Beer's Law applies, i.e., A = ebc. differ by 3.3 orders of magnitude!
3. Analytical sensitivity. S = eb = 1 0 , 0 0 0 A U Lmo1-1
Although a detailed explanation of this example is bey0ni
4. Peak-to-peak noise, N p . p = 2 X 1 0 - S A u
5. Root-mean-square noise, Nrm s = 1/5 Np,p the scope of the present discussion, the huge discrepan~
in the two LODs will be reconciled after solutions to tk.~
Varl'ables
Experiment A Experiment B last three problems in Table I are discussed. This examp',~
Vin j 5uL 20uL should awaken the reader to the seriousness of the~
LO D clef n 10 N p.p/S 3 N r ms/S problems and demonstrate why they must be elkninate~
VM(mL) 2.5 0.75 if the chromatographic LOD is to be a meaningful figure
k 10 3 of merit.
N (plates) 1000 10,000
ov(mL) a 0.87 0.03
Results
Solving the Problems
LODS reported b 8.7 X 1 0 - 6 M 4.5 X 1 0 - 9 M
log ( L O D A / L O D B) = 3.3 orders o f magnitude difference!
Eliminating mistaken identities
a the bandwidth o f the analyte peak, calculated f r o m
The limit of detection has unfortunately been confu~
o v = V M ( 1 + k)/N 1/2
with three other concepts - particularly the minimu~
b reported to more than one significant figure f o r use in Table Vl. detectability (MD) - which are also used in characterizir,i
chromatographic trace analyses. Table III includes syrnb01~l
and definitions for all four of these concepts.

Table III. Trace analysis concepts in chromatography

terms applicable to other multi-step analytical procedures

1. limit o f detection ( L O D )
pseudo-synonyms b
2. (analytical) sensitivity (S)
-- smallest concentration o r amount o f analyte that can be detected with reasonable certainty for a give~
analytical procedure a
-- m i n i m u m absolute quantity, m i n i m u m detectable amount, sensitivity
- slope o f the calibration curve - signal o u t p u t per unit concentration o r a m o u n t o f analyte introducec
in a given analytical procedure c

terms applicable only to the detection step in chromatography

3. (minimum) detectability (MDI
pseudo-synonyms b
4. detector sensitivity (S d)
-- m i n i m u m concentration or mass f l u x o f analyte (in a solvent) which gives a detector signal that can be
discerned from the noise with reasonable certainty, generally recognized to be twice the peak-t0-peak
noise [1--4].
m i n i m u m detectable level, m i n i m u m detectable concentration
slope o f the detector response curve -- signal o u t p u t per unit concentration or mass f l u x of analyte
introduced to a detector

a In the section Using the Correct Units, we show that concentration units are inappropriate f o r the LOD in chromatography.
b We recommend that the use o f these pseudo-synonyms be discontinued immediately.
c In Using the Correct Units, we show that the independent variable o f a chromatographic calibration curve is the a m o u n t o f analyte injects:
and not the concentration o f analyte injected.

504 Chromatographia Vol. 18, No. 9, September 1984 Originals

 One reason that the LOD is confused with the other con-
cepts in Table III, particularly the MD, is the redundant
nomenclature for the LOD and the MD, as evidenced by
the partial, but representative list of pseudo-synonyms
for these concepts (given in Table III) which appear fre-
quently in the literature. This redundant terminology has
0nly confused the chromatographic community, particularly
since the pseudo-synonyms for the two different concepts
are themselves quite similar. We recommend that the use
ofall such pseudo-synonyms be discontinued immediately.
Even if the confusion resulting from the redundant termi-
n01ogycould be eliminated, the LOD might still be confused
with the MD by the apprentice chromatographer because
their definitions, as shown in Table III and in eqs. (1) and
(2), are so similar in appearance (cf. meaning, however).
LOD = arbitrary detector signal level/
(analytical) sensitivity (1)
MD = arbitrary detector signal level/ (2)
detector sensitivity
] Furthermore, as can be inferred from eqs. (1) and (2), the
L0D and MD can be related mathematically. Assuming an
analytical signal in terms of peak height, the relationship
fora concentration sensitive chromatographic system is [9]
L0D = (27r) 1/2 [VM(1 + k)/N 1/2] bMD (3)
where VM represents the corrected gas holdup volume in
GC and the column void volume in LC; k is the capacity
factor; N is the number of theoretical plates; and b is a
unitless parameter which permits the LOD and the MD
to be defined independently of one another. For a mass
sensitive chromatographic system, the relationship between
the LOD and the MD is [ 10]
L0D = (2rr) l/z [tM (1 + k)/N l/z ]bMD (4)
where tM is the retention time of an unretained solute,
corrected for gas compressibility in GC.
Yet despite their similarities, the LOD and MD are distinct
concepts, as a closer scrutiny of Table III shows. The LOD
isa general concept characterizing any overall trace analytical
procedure consisting of one or more steps, whereas the MD
is a specific term characterizing only one step in a chro-
matographic analysis: detection. For example, the LOD
must, by det'mition, include the chromatographic dilution
of the analyte, whereas the MD cannot. Furthermore, a
chromatographic LOD is measured experimentally with a
complete chromatographic system (including a column)
under the specific conditions of a given trace analysis; the
MD, in contrast, is measured (without a column) under
specified conditions [ 1-4] that in general do not correspond
to those of the analysis.
Finally, one last difference should be noted: Referring to
eqs. (1) and (2), an arbitrary detector signal level of twice
the peak-to-peak detector noise [1-4] has been universally
agreed upon for MD calculations. No such consensus has
beenreached for the LOD.
ChromatographiaVol. 18, No. 9, September 1984 Originals
Choosing a model
As discussed earlier, the LOD may be defined as the quo-
tient of an arbitrary detector signal and the analytical
sensitivity (DS/S). In our survey of the chromatographic
trace analysis literature, we found a multitude of arbitrary
DS/S levels used, ranging from 2 to 10. To further com-
plicate matters, neither the measures of the signal (peak
height, peak area, etc.) or the noise (peak-to-peak, root
mean square, etc.) were specified in many instances.
These inconsistencies and ambiguities are not surprising
since (to our knowledge) no standard model for the LOD
has ever been proposed, much less adopted, by any rec-
ognized organization for the field of chromatography!*
We note specifically the omission of an LOD definition
in chromatography by the American Society for Testing
and Materials (ASTM) and by the International Union
of Pure and Applied Chemistry (IUPAC) in their respec-
tive publications on gas and/or liquid chromatography
nomenclature [ I - 4 , 13-16]. The omission by these and
other organizations is also substantiated in two reviews
[17,18].
More importantly, however, the inconsistencies and ambi-
guities in the chromatographic LOD can be eliminated
completely if a clearly stated LOD model is adopted.
We therefore propose the adoption of, with minor re-
interpretation, the IUPAC model for spectrochemical
analysis [11] or a model based on first order error propaga-
tion [8]. These models are given in eqs. (5a) and (5b),
respectively,
LOD = 3SB/S (5a)
LOD = 3[s 2 + s2 + (ilS)2s2]1121S (5b)
where S, i, Ss, and si are the analytical sensitivity (slope),
intercept, and their respective standard deviations of the
calibration curve obtained via linear regression; and sB is
the standard deviation of the spectroscopic blank signal,
calculated from 20 or more measurements.
The factor of 3 in the numerator of the right-hand ex-
pressions of eqs. (5a) and (5b) gives a practical confidence
level of 90% to 99.7%, depending on the probability
distribution of the blank signal and the accuracy of SB
[8, 11, 19]. Though smaller or larger factors could be
used instead of 3, the resulting confidence levels would
be too low or too high for practical use in most cases.
Both the original proponents of these models [8, 11] and
others [19] strongly recommend the use of the factor 3.
We concur.
Both models are proposed for adoption because it seems
preferable to let the chromatography community judge
their respective merits. Indeed, strong arguments can be
made for each. The IUPAC model, on the one hand, is
computationaUy simpler and has already been employed,
though infrequently, in the chromatographic literature.
* The model which the International Union of Pure and Applied
Chemistry (IUPAC) adopted in 1975 [111 was chosen specifically
for spectrochemical analysis. Though the ACS Subcommittee on
Environmental Chemistry reaffirmed this model in 1980 [12], it
did so for the area of environmental chemistry and not specifi-
caUy for the field of chromatography.
505
On the other hand, the error propagation model is not
really all that complicated; many pocket calculators with
linear regression capability can be easily programmed
for the error propagation model. Furthermore, the error
propagation LOD model takes uncertainties of the slope
and intercept of the calibration curve into account, result-
ing in a more realistic numerical estimate.
Interpretation. The IUPAC and error propagation LOD
models were developed originally for spectroscopic trace
analysis. Nearly all the associated concepts, [e.g., the
calibration curve, the sample (analyte + matrix), etc.]
have identical, straightforward interpretations in chro-
matography. One aspect, however, does not: the inter-
pretation and measurement of sB.
Intuitively it is clear that the chromatographic baseline
is analogous to the blank signal in spectroscopy. More-
over, just as SB represents a measure of the noise in spectro-
scopy (the standard deviation of the blank), sB must also
represent an analogous measure of the noise (baseline
fluctuations) in chromatography. A useful mnemonic
would be to refer to Su as the "standard deviation" of
the baseline.
Given this chromatographic interpretation of Sn, how
should Su be measured? One possible procedure would
be to estimate SB from 20 or more measurements of only
that portion of the noise observed at the analyte's reten-
tion time in the absence of the analyte (when a blank
solution is injected). This is directly analogous to the
measurement of the blank signal (at the analytical wave-
length) in spectroscopy. But this procedure would require
20 or more injections of blank solution (~> 20 blank chro-
matograrns!) and is obviously impractical:
1. Most importantly, a true blank solution (sample -
analyte) would be difficult if not impossible to make!
2. It would be much too time consuming.
3. Variables which affect retention would require strict
control.
4. The retention time of the analyte would need to be
known very precisely.
A much more practical procedure for measuring SB becomes
apparent if one remembers that the standard deviation
(root mean square) of a random (periodic) signal can be
closely approximated by the quotient o f the range of
signal values observed and a parameter, r, dependent on
the type of signal, i.e.,
Ssignal = range/r (6a)
Assuming that the signal of interest is the short-term
noise (baseline fluctuations) on a chromatogram, the
(short-term) peak-to-peak noise, Np_p, if measured over
a sufficiently wide region of the chromatogram, is equiv-
alent to the range in eq. (6a). Thus, the standard deviation
of the noise, SB (equivalent to the root mean square noise,
N r m s ) , is given by
SB = N r m s = Np.p/r (6b)
Furthermore, if the noise is sufficiently random and nor-
mally distributed (Gaussian), then r = 5 (ref. 20) and SB
506
can be estimated from one-fifth of the peak-to-peak n0i~
i.e.,
SB = NR~/5 (6cl
which is dearly more practical than the procedure involve!
20 or more injections of blank solution.
Two additional comments regarding the practical procedure
for measuring SB should be noted:
1. It is beyond the scope of this report to discuss the
measurement of Np.p in detail, except to recommene
that the "sufficiently wide region of the chromatogram
over which Np_p is measured be at least as wide as 3
base widths of the analyte peak, and that this regi0"
contains the analyte peak. Various procedures have
already been described for measuring Np_p in the presence
of long-term noise or drift [ 1-4].
2. If periodic fluctuations in the baseline are present, z
value (of r) less than 5 should be used in eq. (6b). The
values of r may be determined from previously derive~
relationships between Nrms and Np_p for various periodic
signals [21]. If, for example, a triangular baseline
observed (possibly resulting from flow pulsations in the
detector cell due to an insufficiently dampened solvent
delivery system), then r = 3.5 and SB = Np.p/3.5.
Using the correct units
A common misconception about chromatographic detec
tion limits is that it is equally correct to report them ~,
relative values with units of concentration as it is to rep0n
them as absolute quantities with units of amount. Wha~
follows in this section is an attempt to clarify this misc0n
ception.
Dimensional analysis. One way of deducing the correcl
units of the chromatographic LOD is by dimensi0na'
analysis. Referring to the definition of the MD in Table li
it is clear that if the appropriate units of concentration
and mass flux are used for the MD in eqs. (3) and (4j
for the concentration sensitive and mass sensitive detector
cases, respectively, the units for the LOD in eqs. (3)an~
(4) must be in terms of an amount (i.e., moles, grams
or some fraction thereof).
Another approach via dimensional analysis is to consider
the right-hand expression of eq. (1). Given the defmiti0n
for the analytical sensitivity, it is clear that the units for
this term (denominator of eq. (1) should be the quotient
of the units of the measured signal and the units of the
independent variable. Therefore, since the units for the
noise expression (numerator of eq. (1)) are the same
those for the measured signal, the units for the detecti0r
limit should be the same as the units of the independen~
variable of the calibration curve. Thus, to decide whict
units are correct for the LOD, we need only to identif!
the independent variable of the calibration curve, i.e
to determine whether the chromatographic signal depends
on the concentration or amount of analyte injected.
Equations (7) [9, 10] and (8) (inferred by analogy) bel0~
show that the signal (peak height, hp) is directly propor-
tional to the maximum concentration, C m a x , d e t , or the
Chromatographia Vot. 18, No. 9, September 1984 Originals
maximum mass flux, Fmax, det, of the chromatographic
peak flowing through the detector, which in turn are
directly proportional to the amount of analyte injected,
qinj:
hp oc Cmax ,det = qinj NI/2/( 27"01/2 VR (7)
hp cc Fmax, det = qiniNl/2/(21r) 1/2 tR (8)
Thus the independent variable for a chromatographic
calibration curve is the amount (not concentration) of
analyte injected and once again the same conclusion is
reached: Chromatographic LODs should be reported as
amounts, (i.e., in moles, grams, or some fraction thereof)
andnot as concentrations!
Identifying faulty logic. Despite the above arguments,
some researchers insist on reporting their chromatographic
LODs as concentrations. Assuming splitless injection,
theirrationale might be as follows:
1. The amount of analyte injected, qini, is the product of
the concentration of analyte in the sample, Cinj, and
the volume of sample injected, Vinj :
qinj = Cinj Vinj (9)
2. The concentration of analyte injected, Cinj, is directly
proportional to the amount of analyte injected (via
rearrangement of eq. (9)):
Cinj = qinj/Vinj (10)
3. Conclusion: the relative LOD (in units of concentration),
CL, is proportional by 1/Vinj to the absolute LOD, qL
(in units of amount):
CL = qL/Vinj (11)
Though reached in a straightforward manner, the above
conclusion is nevertheless false. The error in reasoning is
best described as an improper or incomplete analogy. In
going from a true expression, eq. (10), to a false state-
ment, eq. (11), Cinj and qinj were replaced by two limiting
quantities CL and qL, respectively. No analogous substitu-
tion was made for Vini, however, and therein lies the error.
Vinj may vary continuously over 0 < Vinj < Vinj, max, where
Vinj,max is a limiting, maximum injection volume to be
discussed momentarily. Unless Vin j = Vinj,max, eq. (11) is
false.
A numerical example will help demonstrate the absurdity
of eq. (11). Suppose the absolute LOD (qL) for an analyte
had been determined independently by two scientists
using the same LC system to be 1 x 10 -~2 mol. If the
scientists had used different injection volumes of 5pL and
50pL, then according to eq. (11) the relative LODs (CL'S)
for the same chromatographic system would be 2 x 10 -6 M
and 2 x 10 -7 M, respectively. Clearly eq. (11) is inappro-
priate.
The correct expression, eq. (12) below, is obtained by
using Vinj,max in place of Winj. This expression is of little
value, however, because of the difficulty in obtaining an
accurate, precise estimate of Vinj, max :
EL = qL/Vinj, max (12)
Chromatographia Vol. 18, No. 9, September 1984 Originals
Problems with estimating the maximum injection volume.
Experimentally, Vini,max can be determined by increasing
Vini until column overloading or some other adverse
phenomenon is observed. This operational definition of
Vinj,max is unsatisfactory, however, because the injection
volume at which these events occur is too dependent on
the experimental conditions, such as the sample matrix,
the percent loading of the column, etc.
Alternatively, numerous theoretical expressions may be
developed for the estimation of Vinj,max. Although many
are only applicable for special cases, a few are completely
general. Perhaps the best is one which relates Vinj,ma x to
the maximum tolerable loss in resolution [22]. Our extension
of this expression is reported below as eq. (13), and is
derived in the Appendix to this Report.
Vini, max = 2 (3) qz (y2 + 2 y) t/2 VR/N l/z (13)
where y is the maximum tolerable loss in resolution due to
a finite injection volume.
Despite the appeal of eq. (13) (or other theoretical ap-
proaches), all theory-based estimations of Vinj,ma x have
some serious shortfalls which may be summarized as
follows:
First, it seems unlikely that expressions based on differing
criteria will predict the same or even similar values for
Vinj,max- We would not expect theoretical expressions
based on such divergent criteria as resolution loss and
column overloading, for example, to yield convergent
values for Vinj, max-
Second, the assumptions made for a given approach may
not always be valid. The constant "2(3)q2" in eq. (13)
resulted from the assumption of plug injection. Experi-
ments have demonstrated, however, that this assumption
is frequently invalid and that the value of this numerical
"constant" varies considerably [9].
Finally, Vini, max may be highly sensitive to changes in a
parameter related to the criterion itself. Referring again to
eq. (13), if y = 0.04 (4 % loss in resolution), eq. (13) be comes
Vinj, max ~ VR/N 1/2 (14)
If y were chosen to be 0.01 or 0.10, then Vinj,ma x would
be approximately equal to 0.5 or 1.5 times the value
predicted by eq. (14).
Conclusion. Although the calculation of a relative detection
limit from an absolute detection limit may be rationalized
by eq. (12), the experimental and/or theoretical difficulties
in obtaining an accurate estimate of the maximum injection
volume preclude the use of this approach. In addition,
dimensional analysis shows that chromatographic LODs
are properly reported only as amounts (absolute quantities)
and not as concentrations (relative quantities). From these
perspectives it is clearly more meaningful to report chro-
matographic LODs as amounts rather than as concentra-
tions.
Converting to chromatographic reference conditions
As seen from eqs. (3) and (4), the LOD depends not only
on the detector characteristics (MD) but also on three
507
chromatographic parameters [k, N, and VM (or tM) ]
which characterize the column and the solute. In this
section a method is proposed for taking the effects of
these parameters into account, thereby solving the final
problem associated with the chromatographic LOD (see
Table I).
For chromatographs with concentration-sensitive detectors,
the relationship between the LOD and the chromatographic
parameters is given by
qL = dVM (1 + k)/N 1/2 (15)
where terms of eq. (3) [(2rr) 1/2, b, MD] have been incorpo-
rated into a single proportionality constant, d. Alternatively,
since Va = VM (1 + k) and N = (VR/OV) 2, we may write
qL = d V R / N 1 / 2 (16)
or
qL = dov (17)
where VR is the retention volume (corrected for com-
pressibility in GC) and Ov is the bandwidth of the chro-
matographic peak, in volume units.
Equations (15)-(17) are equivalent, and any one of them
may be used to derive an expression which accounts for the
effects of these chromatographic parameters on the LOD.
For simplicity, we shall use eq. (17).
Consider an LOD, qL1, obtained under one set of chromato-
graphic conditions, i.e., crv = oVl. By analogy with eq. (17),
QL1 = d o v t (18)
Likewise, for qL2, an LOD obtained under a second set of
chromatographic conditions,
qL2 = d O v 2 (19)
Dividing eq. (19) by eq. (18) and solving for qL2 yields
qL2 = [OV2/OV 1 ] q L l (20a)
which can also be written as
qL2 = [VR2/VR1 ] IN1/N2 ] 1/2 qLl (20b)
or
qL2 = [VM2/VM1 ] [(k2 + 1)/(k1 + 1)1
(20c)
[N1/N2 ]1/2 qLl
Eqs. (20a)-(20c) are important for two reasons: First,
they can be used to predict the change in the detection
limit when switching from one set of chromatographic
conditions to another. Thus-they are useful to the analyst
interested in lowering the detection limits via improve-
ments in the chromatographic (rather than in the detec-
tion) aspects of the trace analysis. It should be noted,
however, that the detection limits cannot be lowered
ad infinitum by such improvements. Eventually the analysis
will be optimized to the point where further improvements
in the chromatography will necessitate a reduction in
sample volume which offsets the chromatographic improve-
ments [9]. Of course, in situations where very little sample
is available, this reduction in sample size while maintaining
a constant detection limit will be helpful.
508
Second, eqs. (20a)-(20c) can be used to compare detecti0:
limits obtained under different chromatographic conditi0r.
(bandwidths). Consider two such detection limits obtaine:
using different concentration sensitive detectors. "[he~
detection limits can be compared only after one of t~
two detection limits is converted from its present yak:.
obtained under a certain chromatographic bandwidth (z
of conditions) to a value corresponding to the bandwidt!
of the other LOD, or after both LODs are converted t:
values corresponding to a ttdrd bandwidth.
Whichever way is chosen, it should be recognized that t~,e
final bandwidth chosen in the LOD conversion procedur~
serves as a reference state and that the initial bandwidth(s
is(are), by definition, (an) experimental state(s). Th~
from eq. (20a) we may write, for chromatographs empl0!
ing concentration sensitive detectors,
qL, ref = [ OV,ref/Ov, exp ] qL, exp (2h
where the subscripts "ref" and "exp" refer to reference
and experimental, respectively. Similar equations resultiri
from the incorporation of this notation into eqs. (20b) ani
(20c) are easily inferred.
The above derivation can be repeated in an analog0~
fashion for chromatographs with mass sensitive detectors
The result is
qL, tee = [ O't, ref/O't, exp ] qL, exp (22,
where ot is the bandwidth of the peak in time units.
Given eqs. (21) and (22), the analyst now has a method for
comparing detection limits obtained under different ckr~
matographic conditions (states). Given two or more LOI)~
measured under different chromatographic states, the
analyst merely converts all of them to values correspondini
to a single, arbitrary, reference state.
Though the choice of the reference state is arbitrary, thi
selection of the reference state is nonetheless important
For example, it would be counterproductive for an analys:
to employ a different chromatographic reference state
each time a new group of experimental LODs (measurei
under different experimental states) were to be compared
since LODs in separate groups could not be compared 1
this was done. For similar reasons, it would also be counteI
productive if each analyst or group of analysts used differeni
reference states.
On the other hand, the reference states by definition mus~
be different for concentration sensitive and mass sensitive
chromatographic systems. And in addition, typical chr~
matographic states (bandwidths) vary considerably depen6
ing on the physical state of the mobile phase (gas, super.
critical fluid, or liquid) and on the type of column used
(packed or open tubular).
The logical compromise which we suggest is a fixed refer.
ence state (bandwidth) to be used exclusively for each0!
four chromatographic areas - conventional liquid chr~
matography (LC), microbore liquid chromatograph!
(MLC), packed column gas chromatography (PGC), an~
open tubular column gas chromatography (OTGC)-
with each of the two types of detectors [concentrati0:
Chromatographia Vol. 18, No. 9, September 1984 Originals
sensitive (eq. (21)) or mass sensitive (eq. (22))], giving
a total of eight fixed references states. Furthermore, we
propose that the LODs resulting from the conversion
to these fixed reference states be referred to as standardized
chromatographic LODs. To emphasize this, eqs. (21) and
(22) may be rewritten as
qL,std = [ OV,ref/OV, exp ] qL, exp (23)
and
qL,std = [Or,ref/Ot, exp ] qL, exp (24)
respectively, where the standardized LODs are indicated
bythe subscript "std".
Note that we have purposely omitted supercritical fluid
chromatography (SFC) and open tubular liquid chromato-
graphy (OTLC) from this discussion both for the purpose
of simplicity and because these areas have not matured
sufficiently to lend themselves to this reference state
treatment. We also chose not to have a separate category
for high-speed liquid chromatography because this area
employs concentration-sensitive detectors almost exclusive-
ly, and the volume bandwidths important for these detec-
tors will usually be within a factor of two of the con-
ventional LC reference volume bandwidth.
Differences in opinion regarding these omissions may exist,
of course. It may become necessary to develop chromato-
graphic reference states for these and other areas we have
chosen to omit. Our focus, however, is not on the number
of chromatographic reference states that should be em-
ployed, but on the reference state concept itself and the
resulting standardized chromatographic LODs, the benefits
0fwkich will be discussed presently.
Advantages o f the chromatographic reference state concept
and the resulting standardized chromatographic LODs. If
the values for the reference states are chosen judiciously,
the resulting standardized LODs will be superior to experi-
mental (hereafter termed conventional) LODs in several
ways:
First, standardized LODs generally represent a more realistic
measure of the trace analysis capabilities of a given chro-
matographic system, thus permitting an analyst to decide
whether or not a particular application reported in the
literature will be feasible with his or her system. Conven-
tional LODs, on the other hand, may be very misleading.
As evidenced in the literature, many trace analyses have
been performed using chromatographic systems which
have only been partially optimized, if at all. As a result,
unusually pessimistic (very high) LODs are obtained. In
contrast, some overly optimistic (very low) LODs have
been reported in some instances where the particular
chromatographic systems have been optimized more than
the typical system could have been.
Second, the use of standardized LODs permits a fair com-
parison of trace analysis chromatographic systems in
different areas, e.g., OTGC vs. LC, or with different types
of detectors (mass vs. concentration sensitive). Such a
comparison is not valid if conventional LODs are used,
evenwithin a given area using the same type of detector.
Chr0matographia Vol. 18, No. 9, September 1984 Originals
Third, the standardized LOD facilitates the prediction
of the value of the LOD under experimental chromato-
graphic conditions. Rearranging eqs. (23) and (24), one
obtains
qL, exp = [OV,exp/~ ref] qL,std (25)
and
qL,exp = [Ot, exp/Ot,ref] qL, stcl (26)
Assuming qL, sta has been reported, one merely needs to
substitute values for Ov, exp(Or Ot.exp) in eq. (25) or (26)
in order to calculate qL,exp.
Fourth, the standardized LOD can serve not only as a
figure of merit for the overall chromatographic analysis,
but as a figure of merit for the detector as well. The need
to measure the minimum detectability (i.e., to independ-
ently characterize the detector) would therefore be elimi-
nated, resulting in a considerable savings of time.
Numerically defining the reference states. Values for the
proposed chromatographic reference states (bandwidths)
were determined by assigning reference values to the com-
ponent parameters [VM (or tM), k, and N] and then calcu-
lating the reference bandwidths using eq. (27) or (28):
OV,ref = VM,ref(1 + kref)/(Nref) 1/2 (27)
Ot, ref = tM, ref(1 + kref)/(Nref) 1/2 (28)
The suggested values for kref, Nref, VM,ref, and tM,re f are
listed in Table IV. They were selected according to one or
more of the following criteria:
1. The value represents an intermediate chromatographic
performance which is easily achieved except under
unusual circumstances.
2. The value falls within a range of values reported directly
in the scientific and trade literature.
3. The value falls within a range of values calculated from
column manufacturer specifications.
4. The value fails within a previously recommended range
(k only - see ref. [23]).
Despite our best attempts to use logical criteria in choosing
appropriate values for kref, Nref, VM,ref, and tM.ref, we
recognize that differences in opinion concerning the criteria
or the values selected may exist. We welcome suggestions
for adjustments to these values, particularly for those
changes which significantly affect the values of the overall
chromatographic reference states (bandwidths), i.e., for
those changes in kref, Nref, VM,ref, and tM,re f which do
not offset themselves in eq. (27) or (28).
Table IV. Suggested values for the component parameters of the
chromatographic reference states a
parameter LC MLC PGC OTGC
kre f 4 4 4 4
Nre f 10,000 10,000 10,000 90,000
V M , re f (mL) 1.5 0.2 3 2
tM,ref (min) 1 1 0.5 1
LC = conventional liquid chromatography, MLC = microbore liquid
chromatography, PGC = packed column gas chromatography, and
OTGC = open tubular column gas chromatography.
509
Table V. Proposed standardized LOD equations and the suggested
chromatographic reference states
concentration
q L,std = [GV,ref/UV ,exp] q L,exp sensitive detector
mass sensitive
qL,std = [at,ref/Ot,exp] q L , e x p detector
LC a MLC a PGC a OTGC a
OV,ref(mL) b 0,075 0,010 0.150 0.033
Ut,ref (min.) c 0.050 0.050 0.025 0.017
key to abbreviations given in Table IV.
b calculated via equation (27) using values from Table IV.
c calculated via equation (28) using values from Table IV.
The proposed chromatographic reference states and corre-
sponding standardized LOD equations are shown in Table
V. Table V and the advantages enumerated earlier con-
veniently summarize the bulk of the material presented
in this section. Only one additional point needs to be
made: experimental bandwidths are best measured directly
using equations such as
Oexp = Wb/4
or
O~xp = Woa/4.292
where Wb and Wo. 1 represent the base width and the width
at 10% peak height, in units of volume or time, whichever
is appropriate. Alternatively, the bandwidths can be calcu-
lated from their component parameters iN, k, and V M (or
tM)], but this is disadvantageous in two respects. First, it
requires at least three measurements instead of one. Second,
different expressions and/or different interpretations are
required for gradient (temperature or mobile phase) elution
conditions.
The numerical example - revisited
We return to our hypothetical LOD example (Table 1I and
associated text) to reconcile the large differences in the
reported detection limits. Recall that since identical chro-
matographic detectors were employed, the huge discrep.
ancies were attributed solely to problems 4 - 6 of Table I.
These problems will now be eliminated one at a time
using solutions proposed in this report. Identical detection
limits will be obtained at the conclusion of this reconcila-
tion, thus validating quantitatively our LOD example and
demonstrating the efficacy of our solutions.
Table V l . Reconciling the differences in the detection limits
from the numerical example in Table II a
510
The reconciliation is summarized in Table Vl, th0u~
readers who wish to perform the calculations will needl
refer to conditions specified in Table II. The progress oftL
LOD reconciliation in Table VI may be noted by inspect~
either the LOD values themselves in the second and tt/
columns, or their ratio in the fourth column, given:
orders of magnitude. The degrees to which the gi~:
problems are responsible for the initial discrepancy betwee:
the LODs are shown in the fifth column; they are 0~
rained by subtracting successive values in the fourth colun~
Three steps were performed in the reconciliation. First, ti
LOD values reported incorrectly in units of concentratic:
(molL-1 ) are converted to the appropriate units of am0u:
(mol) by multiplying by the corresponding sample injectic:
volumes (Vinj'S). Second, these unit-corrected LODs a:
then converted to values consistent with the IUPAC m0d~
previously discussed (qL = 3sB/S). For case A, since Np(
5s B (as discussed earlier in Choosing a Model), the t02,
must be reduced by a factor of 50/3. For case B, sine:
Nrms = SB, no adjustment is necessary. Finally, using tk:
appropriate equation and reference state in Table V a~:
the experimental bandwidths given in Table II, the IUPAI
(29)
consistent LOD values are converted to standardize:
LODs, thereby adjusting for differences in the experimentz
(30) chromatographic conditions.
As seen in Table VI, the discrepancy between the LODs:
reduced significantly with every successive stage. It sh0~:
be noted that the largest source of discrepancy is due t
differences in the experimental chromatographic conditi0:
(bandwidths). This demonstrates the need for a standardize!
chromatographic limit of detection. Finally, the LODst
the bottom row of Table VI are identical, indicating th
the reconciliation has been completely successful.
Conclusion
Our comments and reconunendations for obtaining meanir!
ful chromatographic detection limits are summarized beI0'~
We hope that some, if not all, of these guidelines willb.
put into practice in order to make the limit of detecti0:
a more reliable figure of merit in chromatography.
1. The limit of detection (LOD) should not be confu~:
with the minimum detectability (MD), the (analytic/
sensitivity (S), or the detector sensitivity (Sd).
Step LOD A LOD B
[LOOA7 &10~
log L L O D B j
0 Initial 8.7 X 1 0 - 6 M 4.5 X 1 0 - 9 M 3.3
0,6
1 Amount 44 pmoi 90 fmoi 2.7
1,2
2 IUPAC def'n 2.6 pmol 90 fmol 1.5
1.5
3 qL,std 225 fmol 225 fmol 0.0
a Values in this table are reported to more than one significant figure for
purposes of illustration only. Detection limits are normally reported to
only one significant figure.
Chromatographia Vol. 18, No. 9, September 1984 Originals
2. Chromatographic detection limits should be calculated
using the IUPAC and/or the error propagation model(s).
The calibration curve should be constructed from a
plot of signal versus amount (not signal versus con-
centration) of analyte injected, thus ensuring that the
resulting LODs will be reported properly as amounts
(and not as concentrations)!
3. If obtained under non-standard chromatographic con-
ditions, the detection limits should be standardized
using the equations in Table V. Standardized chromato-
graphic LODs are superior to their conventional (non-
standardized) counterparts for several reasons, in par-
ticular because they permit the valid comparison of
trace analysis chromatographic systems in different
areas (conventional LC, microbore LC, packed column
GC, and open tubular column GC) and/or with different
types of detectors (mass and concentration sensitive).
Appendix - D e r i v a t i o n o f Vinj, ma x
We begin with a previously derived expression [16] which
relates the peak volume observed for a finite size sample,
Vw, to the volume of injected sample, Vinj, and to the
peak volume observed for a very small sample, Vp
Vw
2 = V~ + 4/3(Vini) 2 (A.1)
Let Vw = (y + 1)Vp (A.2)
where y = loss in resolution due to a finite injection volume.
Substituting eq. (A.2) i n t o eq. (A.1) and solving for Vinj
yields
Vinj = [ 3 / 4 ( y 2 + 2y)] 1/2 Vp (A.3)
We now express Vp in terms of N, k, and VM using eqs.
(A.4)-(A.6), assuming Vp = Vb, the volume corresponding
to the base width of the peak.
N = (VR/oV) 2 (A.4)
v~ -- 4 o v (A.5)
VR = VM (1 + k) (a.6)
lho result is
Vp = 4VM(1 + k)/N u2 (1.7)
Substitution of eq. (A.7) into eq. (A.3) yields the expression
Vinj = [3/4(Y 2 + 2y] u2 4VM (1 + k)/N u2 (A.8)
If y is re-designated to be the maximum tolerable loss in
resolution, then eq. (A.8) becomes, upon rearrangement,
Vinj,max = 2 ( 3 ) 1/2 (y2 + 2y)1/2 VM (1 + k)/N 1/2 (A.9)
which is the desired expression (eq. (13)).
Chromatographia Vol. 18, No. 9, September 1984 OriginaLs
Acknowledgement
JPF gratefully acknowledges partial support of this work
by a 1983 ACS Analytical Division Fellowship sponsored
by the Society of Analytical Chemists of Pittsburgh. JGD
gratefully acknowledges Eli Lilly and Company and the
Alcoa Foundation for support of this research.
This work was presented in part at the 186th National
Meeting of the American Chemical Society, Washington,
DC, September 1, 1983.
References
111 ASTM E516-74, Testing Thermal Conductivity Detectors
Used In Gas Chromatography. American Society for Testing
& Materials, Philadelphia, Pa., 1974.
121 ASTM E 594-77, Testing Flame Ionization Detectors Used In
Gas Chromatography. American Society for Testing &
Materials, Philadelphia, Pa., 1977.
[3] ASTM E685-79, Testing Fixed-Wavelength Photometric
Detectors Used In Liquid Chromatography. American Society
for Testing & Materials, Philadelphia, Pa., 1979.
[4] ASTM E 697-79, Use of Electron-Capture Detectors Used In
Gas Chromatography. American Society for Testing &
Materials, Philadelphia, Pa., 1979.
151 J.P. Foley, J. G. Dorsey, Anal. Chem. 55,730-737 (1983).
[6] L.A. Currie, Anal. Chem. 40,586-593 (1968).
[71 H. Kaiser, Pure Appl. Chem. 34, 35-61 (1973).
[81 G.L. Long, J. D. Winefordner, Anal. Chem. 55,712A-724A
(1983).
[9 ] B.L. Karger, M. Martin, G. Guiochon, Anal. Chem. 46,
1640-1647 (1974).
1101 N.H.C. Cooke, B.G. Archer, K. Olsen, A. Berick, Anal.
Chem. 54, 2277-2283 (1982).
[11] Nomenclature, Symbols, Units and Their Usage in Spectro-
chemical Analysis-lI. Data Interpretation, International
Union of Pure and Applied Chemistry. Spectrochimica
Acta 33B, 241-245 (1978); rules approved 1975.
[121 Guidelines for Data Acquisition and Data Quality Evaluation
in Environmental Chemistry. Anal. Chem. 52, 2242-2249
(1980).
[13] ASTM E 260-73, General Gas Cttromatography Procedures.
American Society for Testing & Materials, Philadelphia, Pa.;
originally published 1965; latest revision 1973.
1141 ASTM E 355-77, Gas Chromatography Terms and Relation-
ships. American Society for Testing & Materials, Philadelphia,
Pa.; originally published 1968; latest revision 1977.
[15] ASTM E 682-79, Liquid Chromatography Terms and Rela-
tionships. American Society for Testing & Materials, Phila-
delphia, Pa., 1979.
[161 Recommendations on Nomenclature for Chromatography.
International Union of Pure and Applied Chemistry, Pure
Appl. Chem. 37,445-462 (1974).
1171 L.S. Ettre, J. Chromatogr. 165,235-256 (1979).
[181 L.S. Ettre, J. Chromatogr. 220, 29-63 (1981).
[191 1t. Kaiser, Anal. Chem. 42 (4), 26A-59A (1970).
[20] .I.D. Winefordner (Ed.), Trace Analysis, Vol. 46: Spectro-
scopic Methods for Elements, Wiley Chemical Analysis
Series, Wiley-Interscience, New York, 1976, p. 28.
[21 ] H. V. Malmstadt, C. G. Enke, S. R. Crouch, Electronics and
Instrumentation for Scientists, Benjamin/Cummings Pub-
lishing, Inc., Menlo Park, CA, 1981, pp. 31-32.
[22] L.R. Snyder, J.J. Kirkland, Introduction to Modern Liquid
Chromatography, 2nd ed., Wiley, New York, 1979, p. 289.
123] P.T. Kissinger, L.J. Feliee, D.J. Miner, C.R. Reddy, R.E.
Shoup, "Detectors for Trace Organic Analysis by Liquid
Chromatography: Principles and Applications"; in Con-
temporary Topics in Analytical and Clinical Chemistry,
Vol. 2. D.M. Hercules, G.M. Hieft]e, L.R. Snyder, and
M. A. Evenson, Eds. Plenum Press, New York, 1978, p. 67.
Received: Jan. 13, 1984
Revised manuscript
received: April 25, 1984
Accepted: May 19, 1984
A
511