Apparatus and method for the determination of water by liquid chromatography

The invention is the novel combination of liquid chromatographic separation of water from other components of an injected sample followed by electrochemical detection and quantitation of the separated water.

FIELD OF THE INVENTION 
The invention is in the field of analytical chemistry and is directed 
towards a method for the determination of water using a liquid 
chromatography system. 
BACKGROUND OF THE INVENTION 
Aquametry or the determination of water is an important branch of 
analytical chemistry. Many analytical systems have been developed to 
determine water in solids, liquids and gases. Most of these systems are 
described in 3 volumes of Aquametry, Part I, II and III, J. Mitchell, Jr. 
and D. M. Smith; Wiley - Interscience; 1977, ISBN-0-471-02264-0 (Part I); 
1984, ISBN-0-471-02265-9 (Part II); and 1980, ISBN-0-471-02266-7 (Part 
III). 
Most determinations for water are easily made by a Karl Fischer titration. 
However, interferences are known including oxidizing agents, unsaturated 
compounds and thio compounds, see Aquametry, Part III, supra. Thermal 
conductivity detection gas chromatography (GC) is probably the second most 
used method often resulting in a water peak that elutes rapidly, e.g., 1 
to 2 minutes, and with good sensitivity, e.g., 1 ppm, see Aquametry, Part 
I, supra. However, with GC the other components of a sample can take much 
longer to elute than water and can even decompose on-column and interfere 
with the analysis. 
The present inventors were faced with the need to determine water in 
commercial formulations of dibromonitrilopropionamide (DBNPA), and 
antimicrobial product of The Dow Chemical Company. DBNPA is an oxidizing 
agent and reacts with iodide to yield iodine, and thus interferes with the 
Karl Fischer method. DBNPA is thermally labile and decomposed on-column in 
a GC. The products of the decomposition (believed to include HBr) corroded 
and eventually severed the filaments of the GC detector. 
The present inventors thus considered high performance liquid 
chromatography (HPLC). Blasius et al. determined water by HPLC using a 
cyclic polyether column with a refractive index detector but water and 
other interfering components eluted without retention, Blasius et al., 
Talanta, 27:127, 1980. Fehrman et al. determined water by size-exclusion 
chromatography using a refractive index detector. Fehrman et al. used 
toluene as the eluent (rather than the more commonly used tetrahydrofuran) 
which significantly improved separation of water from other low molecular 
weight interfering components, Fehrman et al., Z. Fur Anal. Chem., 
269(2):116, 1974. However, the DBNPA formulation was not miscible in 
toluene, and water itself has a limited solubility in toluene. Bjorkquist 
et al. reacted phenyl isocyanate with water to form N,N'diphenylurea 
(NN'DPU), with a total reaction time of about 1/2 hour, and then analyzed 
the NN'DPU by reverse phase HPLC, Bjorkquist et al., J. Chrom., 178:271, 
1979. The present inventors wanted a simpler and faster procedure than 
this. Roof et al. used an anion-exchange column with a refractive index, 
ultraviolet absorption or differential density detector to determine water 
in a fluorination process stream in about 12 minutes, but with relatively 
poor column efficiency, i.e., about 30 effective theoretical plates and 
with poorer sensitivity than the present inventors desired, Roof et al., 
U.S. Pat. No. 3,935,097. 
The determination of water without a prior separation by electrochemical 
means (for example by electrical conductivity measurement, dielectric 
constant measurement [dielometry] or oxidation/reduction reactions at 
electrodes) is extensively discussed in the volume titled Aquametry Part 
II, supra. However, such direct measurements can be seriously inaccurate 
due to variations in the sample composition unrelated to variations in 
water concentration. The foregoing patent and literature publications are 
fully incorporated herein by reference. 
It is, accordingly, an objective of this invention to provide a liquid 
chromatographic system for the determination of water generally applicable 
but not limited to samples containing oxidizing agents, unsaturated 
compounds, thio compounds and thermally labile compounds, said system to 
be relatively rapid and accurate, said system to use an electrochemical 
detector. 
SUMMARY OF THE INVENTION 
The invention is the novel combination of liquid chromatographic technology 
for effectively separating water from other components of a sample and 
electrochemical detection technology for effectively measuring the 
separated water. 
The invention relates to an apparatus for the determination of water by 
liquid chromatography comprising an eluent reservoir containing a 
nonaqueous eluent; said reservoir in fluid communication with a sample 
injection means; said injection means in fluid communication with a 
chromatographic separation means; said separation means in fluid 
communication with a nonreactive electrochemical detector. Nonreactive 
electrochemical detectors are conductivity detectors and dielometry 
detectors but not oxidation/reduction detectors. 
The invention also relates to a method for the determination of water by 
liquid chromatography comprising eluting a sample through a separating 
medium effective to separate water from other components of the sample 
using a nonaqueous eluent, said method including the further step of 
effectively electrochemically detecting the separated water of an injected 
sample in the effluent eluent from said separating medium. 
The detector comprises a detector based on the principle of measuring 
dielectric constant (dielometry), on the principle of measuring electrical 
conductivity, and on the principle of measuring oxidation/reduction at 
electrodes. 
The invention also comprises the further step of adding an electrolyte to 
the eluent stream before said stream reaches said detector, said 
electrolyte being at least partially dissolved before passing through the 
detector. 
The elecrolyte added to the eluent comprises acids such as H.sub.2 
SO.sub.4, HCl and paratoluenesulfonic acid. 
The invention alternatively comprises the further step of placing an 
immobilized electrolyte between the electrodes of the detector, said 
electrolyte in contact with said effluent. The immobilized electrolyte 
comprises, for exemplary purposes, gelled electrolyte, liquid 
ion-exchangers and solid ion-exchangers.

DETAILED DESCRIPTION OF THE INVENTION 
Ion-exchange resins have a known affinity for water, see for example 
DOWEX:: Ion Exchange, published by The Dow Chemical Company, 1964, 
specifically page 33, and Roof, supra. A preferred separating medium is a 
chromatographic column of sulfonated styrene-divinylbenzene 
copolymer/acid-type ion-exchange resin such as Bio-Rad Laboratories (P.O. 
Box 4031, Richmond, Calif. 94804) Aminex.RTM. 50WX4, 20 to 30 micron size, 
catalog nuber 147-4203, packed in a Cheminert.RTM. Model L9-9-MA-13 column 
available from The Anspec Company, P.O. Box 7730, Ann Arbor, Mich. 48107, 
catalog number H7224. 
Also preferred are other ion-exchange mediums such as quaternized 
styrene-divinylbenzene copolymer base-type ion-exchange resins such as 
Bio-Rad Laboratories, supra, AG.RTM. 1.times.2, 200 to 400 mesh size, 
catalog number 745-1251. Also useful in the invention are silica based 
ion-exchange columns such as Whatman Corporation's Partisil.RTM. SAX anion 
exchange column or Partisil SCX cation exchange column, available from The 
Anspec Company, supra, catalog number H6303 and H6311, respectively. 
Size-exclusion columns have a known ability to separate water in HPLC, for 
example, see Fehrman et al., supra. Size-exclusion columns having 
effective pore sizes such as the silica based Brownlee Aquapore.RTM. 
OH-100 column or the TSK Sphereogel 2000 SW column available from The 
Anspec Company, supra, catalog numbers H1474 and H4548, respectively are 
believed useful in the invention. 
Size-exclusion columns using porous polymer separation media, such as 
Waters Associates .mu.-Styragel.RTM., are also believed to be useful in 
the invention. However, as with the sulfonated or quaternized styrene 
divinylbenzene acid- or base-type ion-exchange resins, which swell 
varyingly depending on the specific eluent, many porous polymer 
size-exclusion media must be equilibrated with the eluent before packing 
the chromatographic column. 
The separating media believed to be effective for the invention comprise a 
packed-type chromatographic column and a capillary-type chromatographic 
column. 
Silica based normal phase columns such as the Du Pont Zorbax.RTM. SIL 
column are useful in the invention and are believed to work as 
size-exclusion columns. 
Silica based reverse phase columns such as Whatman Partisil ODS-1 and ODS-3 
columns are useful in the invention and are also believed to work as 
size-exclusion columns. 
The essential feature of the separating medium of the invention is that it 
effectively chromatographically separates the water of an injected sample 
from other components of the sample using a nonaqueous eluent. 
Specifically, if the electrochemical determination of water is not 
seriously interfered with by the other components of the sample, then 
there is little compulsion to use this invention. However, when one or 
more of the other components of a sample do interfere, then separating 
them from the water of the sample and presenting this water to the 
detector in the matrix of the eluent can be an effective means to 
eliminate serious interferences with detection and analysis. Therefore, 
the specific chromatographic column used is not critical as long as it 
performs the above-mentioned effective separation function in an otherwise 
operable system. 
A preferred eluent comprises methanol or acetonitrile. Eluents believed to 
be effective in the invention comprise ethanol, propanol ethylene glycol, 
benzene, toluene, carbon tetrachloride, chloroform, cyclohexane, heptane, 
tetrahydrofuran and toluene. The specific eluent used is not critical as 
long as it effectively interacts with the chromatographic media to 
separate water from other components of the sample and as long as the 
detector used will function to effectively detect the separated water in 
the eluent in an otherwise operable system. 
Ideally, the concentration of water in the eluent is zero. However, some 
water can be tolerated. Preferably, the concentration of water in the 
eluent is no more than 100 times the concentration of water in the sample 
and more preferably the concentration of water in the eluent is no more 
than 10 times the concentration of water in the sample e.g., for a sample 
containing 30 ppm of water, the eluent preferably will contain less than 
about 300 ppm of water. Most preferably, the concentration of water in the 
eluent is less than the concentration of water in the sample e.g., for a 
sample containing 5000 ppm (0.5 percent) of water, the eluent most 
preferably will contain less than about 5000 ppm of water. 
Optimally, the eluent should not react with the other components of the 
sample to produce significantly interfering amounts of water. For example, 
ketones and aldehydes can react with methanol to form ketals and acetals 
with the production of water as a by-product. Some organic acids will 
react with methanol to form esters with the production of water as a 
by-product. These interferences are well known with the Karl Fischer 
method for the determination of water and are eliminated by replacing the 
methanol in the Karl Fischer reagent with another non-reactive solvent. In 
this invention the same can be done, for example by using an acetonitrile 
based eluent instead of a methanol based one. 
The sample should be preferably miscible in the eluent. Thus, for many 
samples methanol or acetonitrile based eluents are preferred due to the 
excellent ability of these solvents to form homogeneous solutions with 
other solvents and components. However, it is not critical that the sample 
dissolve in the eluent. For example, the invention is used to determine 
water in clay based agricultural formulations by first shaking said 
formulation with HPLC grade methanol to extract water in the formulation 
and then injecting the methanol extract after said extract is filtered to 
remove the clay. It is also believed to be possible to determine water in 
a gas sample by, for example, contacting the gas with a liquid that would 
extract the water and then injecting said liquid. 
A highly preferred detector of the invention is an electrical conductivity 
detector such as the Wescan Model ICM (Wescan Instrument Inc., 3018 Scott 
Blvd., Santa Clara, Calif. 95050). 
The test of effective electrochemical detection of the separated water of 
the injected sample in the effluent from the separating medium comprises 
the well-known signal-to-noise ratio, where the signal relates to the 
detectors' response to said water and the noise relates to the variation 
in response seen at the baseline of the chromatogram. The signal-to-noise 
ratio must be greater than 2 for effective detection. The signal-to-noise 
ratio can be improved, as is well known in the art, by employing active or 
passive filters in the electronic circuits of HPLC systems. The 
signal-to-noise ratio can also be improved (made larger) by injecting a 
larger volume of sample or by employing a more efficient column in an 
otherwise operable system. However, all of these techniques are limited in 
their beneficial effects and thus the detection limit of the invention is 
also limited. On the other hand, samples containing relatively high 
concentrations of water, e.g., 20 percent water, may require system 
modifications to prevent overloading such as using a smaller injection 
volume. 
The sensitivity of detection of water using an electrical conductivity 
detector is significantly increased when an electrolyte is present in the 
eluent. The conductivity detector responds to the electrolyte and the 
difference in response, between a matrix of eluent and a matrix of eluent 
and the separated water, is greater when an electrolyte is present in the 
eluent. This is an example not of direct but rather indirect measurement 
of water concentration. A highly preferred electrolyte is an acid such as, 
but not limited to, H.sub.2 SO.sub.4, HCl or paratoluenesulfonic acid. 
However, samples containing hydroxide ion, for example, will react with 
acid to form water. Also preferred as an electrolyte to be present in the 
eluent is a salt such as, but not limited to, NaCl, KCl or LiBr. The 
sensitivity of detection of water using the preferred conductivity 
detector is not as good with said salt as with said acid, but the use of 
said salt does eliminate the interference from hydroxide ion and is one 
means of avoiding said interference in the event said interference is 
significant. 
An electrolyte believed useful in the invention when using a conductivity 
detector is a base such as sodium hydroxide or tetrabutylammonium 
hydroxide. The specific electrolyte used, whether acid, base or salt, 
organic or inorganic or a mixture thereof is not critical. Optimally, the 
electrolyte used does not result in significantly interfering reactions 
with sample components and does not significantly degrade the eluent. It 
is believed that the most preferred acid and base electrolytes are strong 
acids and bases, that is acids with pKa.sub.1 values less than 1 and bases 
with pKb.sub.1 values less than 1. However, H.sub.3 PO.sub.4 with a 
published pKa.sub.1 value of 2.12 is quite useful and to a lesser extent 
even acetic acid with a published pKa.sub.1 value of 4.73. The 
concentration of electrolyte added to the eluent is optimized by testing 
for optimum signal-to-noise ratio, supra, for a given system. However, it 
can be desirable to have a relatively high electrolyte concentration in 
the eluent when using an ion-exchange column, if the sample contains one 
or more electrolytes, to maintain the ion form of the column. 
Where the optionally employed electrolyte is added to the eluent is not 
critical as long as the addition step occurs before the eluent passes to 
the detector and as long as the electrolyte is at least partially 
dissolved in the eluent. For example, it should also be possible to add 
the electrolyte to the eluent after it emerges from the chromatographic 
media and before the eluent flows to the detector. 
Postcolumn reagent addition is well known to the art of HPLC. One advantage 
contemplated in this invention with the use of postcolumn electrolyte 
addition is the potential elimination of interferences. For example, when 
a sample contains hydroxide ion and the eluent contains an acid, the acid 
reacts with the hydroxide ion upon injection producing water, said water 
then a potential interference. On the other hand, if the chromatographic 
media separates water from hydroxide ion and the acid is added to the 
eluent following the chromatographic media, then two water peaks will be 
seen by the detector. One water peak will result from the water originally 
in the sample at the standard retention time of water. The other water 
peak from the water produced by the reaction of acid with hydroxide will 
be at a different nonstandard retention time and will not interfere. 
Any solvent used as a carrier with postcolumn electrolyte addition ideally 
has a water concentration of zero. However, some water can be tolerated. 
Preferably, the concentration of water in a postcolumn addition 
electrolyte solvent is no more than 100 times the concentration of water 
in the sample and more preferably no more than 10 times the concentration 
of water in the sample. Most preferably, the concentration of water in a 
postcolumn addition electrolyte solvent is less than the concentration of 
water in the sample. 
Examples of other detectors believed to be useful in the invention comprise 
those measuring dielectric constant and those incorporating 
oxidation/reduction reactions at electrodes. 
It is believed to be preferred to add an electrolyte to the eluent before 
the eluent reaches the detector when the detector is an 
oxidation/reduction detector since such detectors generally require a 
supporting electrolyte as is well known in the art. However, said 
detectors do not always require a supporting electrolyte. It is also 
believed to be preferred to add an electrolyte to the eluent before the 
eluent reaches the detector when the detector is a dielometry detector in 
this invention since the dielectric constant of a solvent can be 
significantly altered when it contains an added electrolyte as is well 
known in the art. 
Alternatively, the electrolyte can be added to the eluent between the 
sample injection means and the chromatographic separation means or at any 
other point before the eluent reaches the detector. A highly preferred 
embodiment of the invention is to mix the electrolyte with the eluent in 
the eluent reservoir 1 of FIG. 1. 
The electrolyte need not be mixed with a carrier solvent and pumped into 
the eluent but can also diffuse into the eluent across a membrane. In 
other words, how the electrolyte gets into the eluent before the eluent 
reaches the detector is not critical to the invention. 
When an immobilized electrolyte is effectively employed within the 
detector, it should be placed between the electrodes of the detector and 
said electrolyte should contact said effluent. When thus employed, it is 
believed that water, separated from other components of the sample and 
entering the detector, can interact with said immobilized electrolyte to 
enhance the detector's direct or indirect response to said water. The 
above reference to placing the immobilized electrolyte between the 
electrodes should not be construed to mean directly and exactly between 
them as it may be preferable to effectively dispose the immobilized 
electrolyte away from a point equidistant from the electrodes but still 
within or adjacent the space measured by the electrodes. The immobilized 
electrolyte can contact one or more of the electrodes. 
The following examples further illustrate the various aspects of the 
invention. 
EXAMPLE 1 
An HPLC system generally similar to FIG. 1 (except that no postcolumn 
reagent addition system 15 to 20 is used) is assembled including an LDC 
Constametric.RTM. III pump, a Rheodyne.RTM. 7120 sample injection valve, a 
9.times. 54 mm column of Bio-Rad Aminex 50WX4 ion-exchange resin, in the 
Na.sup.+ ion form packed in a Cheminert LC-9-MA-13 column, a 
Chromatronix.RTM. CMA-1 conductivity meter with associated MCC-75 
flow-through cell and a Sargent.RTM. SRG-1 strip chart recorder. The 
eluent is composed of HPLC grade methanol containing 0.14 g of NaCl per 
liter. The pump is set to deliver 2 ml of eluent per minute. The injection 
valve is fixed with a predetermined loop size to deliver about 10 .mu.l of 
sample. The detector is set to a sensitivity of 7.5 micro mho per cm for a 
10 mv output. The recorder is set at a full scale response of 10 mv. 
Three successive injections of standards of known amounts of water in 
methanol is then made and the chromatograms shown in FIG. 2 result. 
In FIG. 2, two dips ("peaks") are seen in the chromatogram for each 
injection. The one at about 0.8 minutes is explained as the void volume 
upset. The one at about 2.1 minutes is explained as the water "peak" and 
its size is generally proportional to the amount of water in the injected 
standard. Doubling the amount of NaCl in the eluent doubles the water 
"peak" height and also doubles the background conductivity of the eluent 
from about 250 micro mho per cm to about 500 micro mho per cm. As 
expected, the baseline noise increases with increased background 
conductivity, and the limit of detection observed with either eluent is 
about 0.1 percent water (in a standard) with a 500 .mu.l injection. Using 
LiBr in the eluent instead of NaCl results in similar system performance. 
Reversing the recorder polarity results in upscale "peaks" and this 
becomes standard operating procedure for this mode of the invention. 
EXAMPLE 2 
The system of Example 1 is exactly reproduced except that the column used 
in Example 1 is replaced with a Whatman Partisil 10-ODS-3 column, or a Du 
Pont Zorbax SIL column, or a Whatman Partisil SCX column and the eluent 
flow rate is changed to 1 ml per minute. FIG. 3 shows chromatograms 
resulting from the injection of a standard containing 10 percent water in 
methanol for each column. 
With the use of each column of FIG. 3, a retained water peak resulted whose 
height is generally proportional to the amount of water injected. This 
example demonstrates the wide range of column types useful in the 
invention. 
EXAMPLE 3 
The system of Example 1 is exactly reproduced except that the column is 
shortened to 9.times.21 mm and the NaCl in the eluent is replaced with 
paratoluenesulfonic acid (PTSA), HCl or H.sub.2 SO.sub.4 and the flow rate 
of the eluent is changed to 1.5 ml per minute as described in detail in 
FIG. 4. 
As shown in FIG. 4, the use of HCl, H.sub.2 SO.sub.4 or PTSA in an eluent 
of methanol using a 9.times.21 mm column of H.sup.+ ion form Aminex 50WX4 
results in improved detection sensitivity. Using the NaCl containing 
eluent of FIG. 2, a 50 .mu.l injection of 1 percent H.sub.2 O in methanol 
results in a water peak 0.11 micro mho per cm tall. Using any of the above 
acids (also of a concentration sufficient to give an eluent background 
conductivity of about 250 micro mho per cm) results in a water peak about 
18 micro mho per cm tall (see FIG. 4), an increase in sensitivity of about 
160 fold. This example demonstrates the improvement in sensitivity 
observed with the addition of acid to the eluent versus the addition of a 
salt to the eluent when using a conductivity detector. 
EXAMPLE 4 
The system of Example 3 is exactly reproduced except that the eluent was 
0.05 ml of 96 percent H.sub.2 SO.sub.4 mixed with 800 ml of HPLC grade 
methanol, the injection valve is changed to inject about 1 .mu.l of 
sample, the detector sensitivity is changed to 60 micro mho per cm per 10 
mv output and the recorder sensitivity is changed to 8 mv full scale 
response. When an injection of 20 percent dibromonitrilopionamide (DBNPA) 
in a water/glycol based formulation is made, the chromatogram shown in 
FIG. 5 results. 
Based on the water peak size for injections of known standards of water in 
methanol (not shown) the concentration of water in the DBNPA sample of 
FIG. 5 is estimated to be 20.1 percent (20.1 g H.sub.2 O per 100 ml of 
sample). When the sample is injected 10 times, a statistical evaluation of 
the data indicates a standard deviation of the water concentration of 0.22 
percent. This example demonstrates the utility of the invention for a 
sample that interferes with the Karl Fischer method and with the gas 
chromatographic method for the determination of water. 
EXAMPLE 5 
The system of Example 4 is exactly reproduced except that the sample 
injection volume is changed to about 50 .mu.l, the detector sensitivity is 
changed to 7.5 micro mho per cm per 10 mv output and the recorder span is 
changed to 10 mv. When an injection of Telone.RTM. II soil fumigant (mixed 
isomers of dichloropropenes, a product of The Dow Chemical Company) is 
made, the chromatogram shown in FIG. 6 results. 
Based on the water peak size for injections of known standards of water in 
methanol (not shown) the concentration of water in the Telone II sample of 
FIG. 6 is estimated to be 67 parts per million (ppm) (67 mg of water per 
liter of sample). A GC analysis of the same sample estimates a water level 
of 71 ppm. In the GC analysis, the water elutes at about 2 minutes 
followed by dichloropropene peaks for the next 45 minutes. Thus, the HPLC 
procedure is faster overall. Trace levels of water can not be determined 
in Telone II soil fumigant by the Karl Fischer method since iodine adds 
across the double bonds of the dichloropropenes. 
As a rule, water elutes as the last peak in the chromatogram with this 
embodiment of the invention, well resolved from the other components of a 
sample. However, dimethylsulfoxide (DMSO) elutes just before water and 
interferes. Nevertheless, it is clear that the invention can be used to 
determine DMSO and perhaps other compounds in addition to water. A 
compound that responds similarly as water responds and is effectively 
separated from water is contemplated as a candidate as an internal 
standard with the invention. 
EXAMPLE 6 
The system of Example 5 is exactly reproduced except that the eluent is 
changed to 0.05 ml of 96 percent H.sub.2 SO.sub.4 in 800 ml of 
acetonitrile (at a flow rate of 1 ml per minute), the column is changed to 
a 9.times.7 mm one packed with Bio-Rad AG 1.times.2, SO.sub.4.sup.-2 ion 
form, 200 to 400 mesh, ion-exchange resin, the injection volume is changed 
to about 100 .mu.l, the detector sensitivity is changed to 15 micro mho 
per cm per 10 mv output and the recorder polarity is reversed. When an 
injection of 0.5 percent water in acetonitrile is made, the chromatogram 
shown in FIG. 7 results. 
The water peak in FIG. 7 is positive not negative. In addition, the 
baseline conductivity in FIG. 7 is about 36 times lower than in FIG. 6 
while the response for the same amount of water injected with the system 
of FIG. 6 or 7 (in units of micro mho per cm.times.ml of the water peak 
width at half height) is about the same. Therefore, the use of a more 
efficient chromatographic column with this embodiment of the invention is 
expected to result in potentially increased detection sensitivity over the 
embodiment of FIG. 6. One such column contemplated is a size-exclusion 
column of selected exclusion characteristics specifically designed for 
separation of relatively low molecular weight components such as water. 
Another such column contemplated is an ion-exchange column packed with 
partially sulfonated or partially aminated styrene-divinylbenzene 
copolymer beads. 
EXAMPLE 7 
The system of Example 5 is exactly reproduced except that the column length 
is changed to 18 mm, the injection volume is changed to about 100 .mu.l, 
the recorder sensitivity is changed to 2 mv full scale and the 
conductivity detector is changed to a Wescan Model ICM, set at range 1 and 
for 10 mv output. The concentration of water in the eluent is about 100 
ppm. When an injection of carbontetrachloride containing 30 ppm of water 
is made the chromatogram of FIG. 8 results. 
This example demonstrates the high sensitivity of this highly preferred 
embodiment of the invention. 
EXAMPLE 8 
The system of Example 4 is exactly reproduced except that a KRATOS Post 
Column Reagent Addition Device, Model PCR-1 is added (as generally shown 
by elements 15 to 20 of FIG. 1), the eluent is HPLC grade methanol 
containing no added H.sub.2 SO.sub.4 at a flow rate of about 1.5 ml per 
minute. The post column reagent is 0.1 Ml of 96 percent H.sub.2 SO.sub.4 
dissolved in 800 ml of HPLC grade methanol. The post column reagent flow 
rate is about 1.5 ml per minute. When an injection of DBNPA in a 
water/glycol based formulation is made (same sample as Example 4) a 
chromatogram generally similar to FIG. 5 is believed to result except that 
the peak height is believed to be about one-half of that in FIG. 5. 
EXAMPLE 9 
The system of Example 1 is exactly reproduced except that the eluent is 
composed of HPLC grade methanol containing 0.14 g of sodium hydroxide per 
liter. When an injection of a sample containing water is made, a water 
response is believed to result that is generally proportional to the 
amount of water injected. 
EXAMPLE 10 
The system of Example 4 is equivalently reproduced except that the eluent 
stream exiting from the detector is not directed to waste but is instead 
directed back to the eluent reservoir to be reused for an extensive length 
of time in the laboratory of a DBNPA production plant. The eluent 
reservoir is sealed to prevent the absorption of water from the laboratory 
air into the eluent. The HPLC system is used to determine water in DBNPA 
formulations for production control purposes and the HPLC system continues 
to function for more than 1 month without maintenance despite the buildup 
of sample (including water) in the eluent. It is contemplated that in some 
systems using recycled eluent that an eluent drying means could be 
employed to control the buildup of water in the eluent, e.g., by placing a 
column filled with drying agent between the detector and the eluent 
reservoir. A drying column can also be used between the eluent pump and 
the injection valve. 
This example shows the economy of operation using a recycled eluent and the 
long term ruggedness of the system routinely used in a chemical production 
plant.