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B.J. Hoffman and L.T. Taylor..........................................................................................................61
An Isocratic Liquid Chromatographic Method with Diode-Array Detection for
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Evaluation of Select Variables in the Ion Chromatographic Determination of
F
, Cl
, Br
, NO
3
, SO
4
2
, and PO
4
3
in Serum Samples
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and F. Bartoli...............................................................................................................................101
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A High-Pressure Liquid ChromatographicTandem Mass Spectrometric
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J.E. Conte, Jr., E. Lin, Y. Zhao, and E. Zurlinden...........................................................................113
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Contents
C hroma t o gra p h i c S c i enc e
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61
Alkylphenol polyethoxylates (APEs) are a widely used group of
nonionic surfactants in commercial production. Characterization of
the composition of APE mixtures can be exploited for the
determination of their most effective uses. In this study sample
mixtures contain nonylphenol polyethoxylates and octylphenol
polyethoxylates. The separation of individual alkylphenols by
ethoxylate units is performed by supercritical fluid chromatography
(SFC)-UV as well as normal-phase high-performance liquid
chromatographic (HPLC)-UV employing packed columns. The
stationary phase and column length are varied in the SFC setup to
produce the most favorable separation conditions. Additionally,
combinations of packed columns of different stationary phases are
tested. The combination of a diol and a cyano column is found to
produce optimal results. An advantage of using packed columns
instead of capillary columns is the ability to inject large amounts of
sample and thus collect eluted fractions. In this regard, fractions
from SFC runs are collected and analyzed by flow injection
analysiselectrospray ionizationmass spectroscopy in order to
positively identify the composition of the fractions. In comparing
the separation of APE mixtures by SFC and HPLC, it is found that
SFC provides shorter retention times with similar resolution. In
addition, less solvent waste is produced using SFC.
Introduction
Alkylphenol polyethoxylates (APEs) are referred to as nonionic
surfactants. Since the mid 1940s, APEs have been used commer-
cially for their surfactant ability. The term surfactant includes
surface-active compounds characterized by their ability to con-
centrate at surfaces and form micelles in solution (1). They have
been used in a wide variety of applications including industrial
process aids, dispensing agents in paper and pulp production,
emulsifying agents in latex paints and pesticide formulations,
flotation agents, industrial cleaners (metal surfaces, textile pro-
cessing, and food industry), and household cleaners (1). These
compounds are commercially available as oligomeric mixtures
with varying ethoxylate chain lengths as well as varying alkyl
sizes. Certain APEs have been determined to be estrogenic in fish,
birds, and mammals (2).
APEs contain two main molecular regions: the polyethoxylate
(POE) chain (EO) is polar and thus hydrophilic and the alkyl-
phenol is the hydrophobic area. The hydrophilic nature of the EO
is attributed to the hydration of the ether-linked oxygen atoms
(3). A technical synthesis of APEs start with phenol, which is alky-
lated by trimethylpentane and thus produces octylphenol (OP), or
by nonene isomers, which forms nonylphenol (NP) in an acid-cat-
alyzed process. Ethoxylation is performed by using KOHethanol
as a catalyst with a known ratio of ethylene oxide to the
alkylphenol (1). The reaction results in an oligomeric mixture of
the alkylphenol containing an EO chain of varying lengths.
The separation and identification of the components of an APE
mixture can be useful for the determination of their most effec-
tive applications. Several different types of chromatography have
been studied previously in efforts to achieve better separation
conditions. Gas chromatography (GC) coupled with flame ioniza-
tion detection as well as mass spectrometry (MS) has been used in
the analysis of APEs (4). Isomers of each oligomer tend to be sep-
arated into clusters by GC. Usually, it is necessary to derivatize
samples containing APEs for analysis by GC, because the com-
pounds are not very volatile. GC poorly separates higher molec-
ular-weight oligomers because of their lower volatility.
High-performance liquid chromatography (HPLC) has been
used to separate APEs of higher mass oligomers. Both reversed-
phase (3) and normal-phase (57) chromatographic separations
have been performed on solutions containing APEs. Each
oligomer is separated by an ethoxylate unit, and isomers of each
oligomer tend to coelute. Recently, Gundersen used a graphitic
carbon column in research to separate isomers of individual
ethoxylated alkylphenols by HPLC (8). Ferguson et al. used
reversed-phase HPLCelectrospray ionization (ESI)MS to ana-
lyze APEs and their metabolites in aquatic environments (9).
Normal-phase HPLCESIMS was used by Shang et al. to quanti-
tate NPEOs in marine sediment (10).
In addition to traditional forms of chromatography, supercrit-
ical fluid chromatography (SFC) has been employed for APE sep-
aration. SFC has advantages over both HPLC and GC. SFC can
operate at lower temperatures than GC, allowing samples that are
thermally labile to be analyzed. Supercritical fluids have densities
similar to liquids and diffusivities similar to gases. These qualities
allow large molecular-weight molecules that are not volatile to be
Abstract
A Study of Polyethoxylated Alkylphenols by Packed
Column Supercritical Fluid Chromatography
Brian J. Hoffman and Larry T. Taylor
Virginia Tech, Department of Chemistry, Blacksburg, VA 24061-0212
Reproduction (photocopying) of editorial content of this journal is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, February 2002
Journal of Chromatographic Science, Vol. 40, February 2002
62
separated by SFC similar to HPLC but with shorter retention
times because of the physical properties of supercritical fluids.
This reduces solvent waste and decreases the total analysis time.
Capillary-column SFC using flame ionization detection (11,12)
has been used to separate both NPEO and OPEO. Because a
sample is generally destroyed by this method, it is not possible to
directly determine analyte identity. Peak identity can be surmised
by comparing retention times of samples with other APE mix-
tures that contain a large fraction of a known single oligomer. A
disadvantage associated with capillary columns is the inability to
inject large sample volumes, which precludes semipreparative
fraction collection.
In addition, OPEO mixtures have been separated on packed-
column SFC using reversed-phase (13,14) and normal-phase
(15,16) packing material. Both Takeuchi and Saito and Giorgettie
et al. used C18 packed columns to separate OPEO samples by
SFC. Takeuchi and Saito found that a microcolumn (1.0 500
mm) had the best separation performance, but a semimicro-
column (1.7 250 mm) produced the best results. A conventional
column (6.0 250 mm) was used in their research for preparative
purposes. Packed-column SFC allows larger amounts of sample
to be injected into the system for the semipreparative collection
of analyte fractions. Giorgettie et al. studied mixed mobile phases
using the addition of a modifier in order to make their mobile
phase more polar. They used pressure programming and a modi-
fier additon to produce optimum separations. Highly efficient
separations were produced under constant modifier concentra-
tion and pressure programming.
The object of this study was to compare the ability of normal-
phase packed columns to separate APEs on an SFC system.
Individual packed columns as well as stacked packed columns of
different stationary phases were used in the SFC experiments.
Additional goals of this study were to identify the components
that gave rise to the chromatographic peaks in hopes of pro-
ducing individual ethoxylated alkylphenol standards. Fractions
that contain a single ethoxylate compound could later be used as
standards for quantitating APEs in a variety of applications. A
comparison of the ability of SFC and HPLC to separate APEs
using normal-phase packed columns was also studied.
Experimental
Packed-column SFC
A Berger (Newark, DE) SFC system was used in the SFC anal-
ysis. A Berger autosampler with a 10-L injection loop was used
for conventional sample analysis, and a 75-L injection loop was
used for the injection of semipreparative samples. SFC-grade
carbon dioxide (Air Products and Chemicals, Inc., Allentown, PA)
was used with methanol (Burdick & Jackson, Muskegon, MI) as a
modifier. The mobile phase flow rate was 2.0 mL/min. The oven
temperature was set at 60C, and the outlet pressure was kept at
120 atm. Absorbance was read at 225 nm by a diode-array
detector. The detection wavelength was determined by finding the
maximum absorbance of an individual APE sample by obtaining
its UVvis spectrum. Supelcosil LC-Diol, Supelcosil LC-CN
(Supelco, Bellefonte, PA), and Spherisorb NH
2
(Waters, Millford,
MA) columns were used for the chromatographic separation of
the APE mixtures. All columns measured 4.6 250 mm with a
5-m particle size. A diol bonded silica guard column was used.
Normal-phase HPLC
For HPLC analysis, a Hewlett-Packard (Little Falls, DE) 1050
Series HPLC system was used with a variable wavelength detector
(reading 225 nm) and an inline vacuum degasser. Injections were
made manually with a Rheodyne (Rohnert Park, CA) injector
equipped with a 20-L injection loop. Data were collected and
chromatograms were processed by MassLynx software (Fisions
Instruments, Altricham, U.K.). A Supelcosil LC-Diol column (4.6
250 mm, 5 m) was used for the chromatographic separation of
the APE mixtures.
Flow injection analysisMS
A Fisions Instruments VG Platform MS was used for the mass
analysis of collected sample fractions. All samples were analyzed
under positive ESI. A syringe pump (Harvard Apparatus, South
Natick, MA) supplied an 80:20 methanolwater mobile phase to
the probe. Samples were injected by a Rheodyne injector
equipped with a 20-L injection loop. Nitrogen was used as both
the drying and sheath gas. Data were collected and analyzed by
MassLynx software.
Alkylphenol samples
POE-(4)-NP (ChemService, West Chester, PA) and Triton N-101
(Sigma-Aldrich, Milwaukee, WI) were used as NPEO mixtures.
POE-(5)-tert-OP (ChemService) was used as an OPEO mixture.
All of the samples that were analyzed by SFC were dissolved in
methanol, and samples analyzed by normal-phase HPLC were
dissolved in hexane. The Triton N-101 sample that was used for
HPLC was dissolved in 9:1 hexaneacetone in order to increase
solubility. HPLC samples were prepared at approximately 1.0
mg/mL, and SFC samples were prepared at approximately 2.0-
mg/mL concentrations.
Semipreparative SFC
A tee was placed inline between the column and diode-array
detector of the SFC system, splitting effluent approximately 75%
to the collection and 25% to the detector. Eluent was diverted
using a portion of fused-silica capillary tubing. Fractions were
collected in preweighed 16-mL collection vials. Absorbance was
monitored, and fractions were collected manually between min-
imum absorbance values. POE-(4)-NP and POE-(5)-tert-OP were
separated in this fashion. Fractions were evaporated by nitrogen
blow-down on a hot plate. The remaining residue was weighed.
The fractions were then diluted to 10.0 mL with methanol.
Fractions were analyzed by SFC-UV followed by flow injection
analysis (FIA)ESIMS for purity.
FIAESIMS method
SFC-collected fractions were evaporated by nitrogen blow-
down and weighed. Collected fractions were then dissolved in
methanol. Optimal MS settings were found by injecting each frac-
tion and tuning the instrument. Fractions were then reinjected,
and mass-spectral data were recorded and analyzed. The source
temperature was set at 100C. ESI nebulizing gas flow was set at
Journal of Chromatographic Science, Vol. 40, February 2002
63
20 L/h, and the drying gas flow was 300 L/h. Samples were
recorded in full-scan mode from m/z 200 to 700. The cone voltage
ranged from 52 to 75 V, and the high voltage lens and ESI capil-
lary voltage were kept at 0.88 and 3.46 kV, respectively.
HPLC method
Hexane and isopropanol were used as the mobile phase. A linear
gradient was used starting with 100% hexane and then changing
to 70:30 hexaneisopropanol over 30 min. From t = 30 to 35 min,
the mobile phase was returned to 100% hexane and held for
5 min in order to equilibrate. POE-(4)-NP and POE-(5)-tert-OP
were separated in this fashion.
Results and Discussion
APEs are complex mixtures that provide moderate challenges
for chromatographic techniques. Our research studied how the
total column length, stationary phase, and column stacking order
of different stationary phases affect the SFC separation of ethoxy-
late units in APE mixtures. Our goal was to find a setup that pro-
duced the best separation. In order to accomplish this we kept all
system parameters constant throughout the study other than
column setup and modifier gradient. All of the columns used
were uniform in size (4.6 250 mm, 5 m) in order to allow us to
verify the effect of column length and packing material. POE-(4)-
NP was used in all of the diol column studies because of its short
elution time.
POE-(4)-NP was separated on a combination of one-, two-, and
three-packed diol columns connected in series to study the effect
of column length (Figure 1). A single diol column poorly sepa-
rated the sample. Baseline separation was not achieved with a
single column. SFC separation on two diol columns increased
separation, but early eluting peaks were not baseline separated.
Using two diol columns, SFC separation was comparable with
normal-phase HPLC using one diol column. For comparison,
POE-(4)-NP, POE-(5)-tert-OP, and Triton N-101 were separated by
SFC on two diol columns and HPLC on one diol column (Figures
24). The retention time of the chromatographic peaks for SFC
separation using two diol columns was considerably lower than
normal-phase HPLC separation using one diol column (Tables I
and II shows data for the NPEO sample and Table III shows data
for the OPEO sample). The addition of a third diol column to the
SFC system generated a better separation, but later-eluting peaks
began to broaden.
The effect of the stationary phase on separation was sequen-
tially tested using a single diol, amino, and cyano column (Figure
5). The retention of oligomers with longer ethoxylated units
varied with each stationary phase tested. The diol column had the
least retention, the amino column had intermediate retention,
and the cyano column had the greatest retention. It was not pos-
sible to elute all of the compounds off the cyano column using the
corresponding gradient. In general, a larger methanol modifier
concentration was needed to elute longer ethoxylate-chain com-
Figure 2. Chromatograms of POE-(4)-NP using (A) normal-phase HPLC-UV
with one Supelcosil LC-Diol column and (B) SFC-UV with two Supelcosil LC-
Diol columns. The peak annotations represent the number of ethoxylate units.
Figure 1. Packed-column supercritical fluid chromatograms using stacked diol
columns: (A) one Supelcosil LC-Diol column, (B) two Supelcosil LC-Diol
columns, and (C) three Supelcosil LC-Diol columns. The sample used in each
chromatogram was POE-(4)-NP (2.0 mg/mL). A linear modifier gradient was
used by the following program: 10.0% methanol was increased to 26.0% at a
rate of 0.6%/min with a 2.0-min hold and then returned to 10.0% in 4.0 min
followed by a 2.0-min hold.
Journal of Chromatographic Science, Vol. 40, February 2002
64
pounds. Because of this, we can conclude that APEs with a longer
ethoxylate chain are more polar than those with shorter chains.
Following this reasoning, the cyano column must be the most
polar stationary phase because it retained the more polar compo-
nents longer, and the diol column is the least polar.
Columns with different stationary phases were coupled in
series to test how the arrangement would affect the retention of
an APE sample. Two column arrangements were tested. The first
consisted of one diol column followed by one cyano column. The
second setup contained three columns, a diol column, a cyano
column, and an amino column in series (Figure 6). A steeper gra-
dient was needed than previously used in order to elute all of the
compounds because of the presence of the cyano column (as pre-
viously mentioned). The modifier gradient that was used is
described in Figure 6.
One of our goals in this study was to achieve separation that
would allow us to easily collect individual oligomers for use as
standards. The combined diolcyano setup rendered shorter
retention times than the combined diolcyanoamino setup;
therefore, this arrangement was used for preparative fraction col-
lection. In the chromatograms of stacked columns using different
stationary phases, peak splitting was observed for later-eluting
peaks. POE-(4)-NP and POE-(5)-tert-OP were separated, and five
fractions of each sample were collected. A large volume (75 L) of
concentrated sample was injected six to eight times in the collec-
tion process. Isolated fractions were reanalyzed both by SFC for
purity (Figures 7 and 8) and FIAESIMS for identification. The
concentrations used for the semipreparative work caused the
chromatographic peaks to significantly broaden and in some
cases combine. Because of this phenomenon we were not able to
collect individual fractions of the two initial oligomers of POE-
(4)-NP and fractions of the three initial oligomers of POE-(5)-tert-
OP as evidenced by the SFC-UV of the early fractions.
FIAMS was used to identify the components in each fraction.
ESIMS was chosen because it is amenable to high-molecular-
weight analytes and works well with liquid mobile phases.
Samples were dissolved in methanol (a compatible solvent for
ESIMS), which made ESIMS a desirable tool for fraction iden-
tification. It was possible to produce sodium-adducted molecular
ions rather easily. In order to create an optimum response, the
fractions were first injected and the cone voltage varied in order
to produce the greatest response for each individual analyte. After
MS tuning conditions were perfected, the fractions were rein-
jected into the instrument. A spectrum was created between
Figure 4. Chromatograms of Triton N-101 using (A) normal-phase HPLC-UV
with one Supelcosil LC-Diol column and (B) SFC-UV with two Supelcosil LC-
Diol columns. The peak annotations represent the number of ethoxylate units.
Figure 3. Chromatograms of POE-(5)-tert-OP using (A) normal-phase HPLC-UV
with one Supelcosil LC-Diol column and (B) SFC-UV with two Supelcosil LC-
Diol columns. The peak annotations represent the number of ethoxylate units.
Journal of Chromatographic Science, Vol. 40, February 2002
65
m/z 200 and 700 by averaging scans of the injected sample.
Figures 9 and 10 show the average mass spectrum of each fraction.
The spectra confirm that each chromatographic peak varied by
one ethoxylated unit (a separation of m/z 44 represents an ethoxy-
late unit). It was possible to identify NP3EO through NP7EO in
basically pure collected fractions of POE-(4)-NP and OP5EO
through OP8EO in fractions collected from POE-(5)-tert-OP.
Major ion peaks consisted of Na
+
adduct ions, and minor peaks
were produced by K
+
adduct ions under positive electrospray con-
ditions. Trace levels of sodium and potassium must be present in
the mobile phase that was used for FIAESIMS because elec-
trolyte was not added to the solutions. According to Okadas
research (17), APEs have an affinity for alkali metals and have a
flexible structure that allows them to form complexes with alkali
metals. This explains the ion pairing seen in the mass spectra.
Crescenzi et al. performed an experiment to see if the detector
response would decrease because of the complexation of
oligomers competing for the limited metal pool available. When
equivalent amounts of ethoxylated compounds were analyzed by
ESIMS, it was found that the detector response increased expo-
nentially from 1 to 6 EO units and then leveled off at 8 EO units
(the scope of the study) (18). A decrease in signal was most notice-
able for lower ethoxylated oligomers. This can be explained by
noting that ethoxylated compounds can form increasingly stable
complexes with alkali metal ions as the EO unit number increases
(17).
Table I. Chromatographic Peak Retention Times of
POE-(4)-NP (NPEO) Separated by SFC Using Two
Supelcosil LC-Diol Columns and HPLC Using One
Supelcosil LC-Diol Column
EO unit SFC RT* HPLC RT
2 7.18 9.14
3 7.86 9.93
4 8.64 10.66
5 9.68 11.82
6 10.61 13.08
7 11.56 14.46
8 12.49 15.83
9 13.43 17.28
10 14.37 18.74
11 15.16
* RT, retention time.
Table II. Chromatographic Peak Retention Times of Triton
N-101 (NPEOs) Separated by SFC Using Two Supelcosil
LC-Diol Columns and HPLC Using One Supelcosil
LC-Diol Column
EO unit SFC RT* HPLC RT
2 7.29 9.88
3 8.02 10.68
4 8.83 11.84
5 9.75 13.08
6 10.66 14.33
7 11.57 15.55
8 12.48 16.80
9 13.36 18.06
10 14.20 19.39
11 15.03 20.68
12 15.84 22.29
13 16.61 24.11
14 17.37
15 18.10
16 18.81
17 19.58
18 20.10
* RT, retention time.
Table III. Chromatographic Peak Retention Times of
POE-(5)-tert-OP (OPEOs) Separated by SFC Using Two
Supelcosil LC-Diol Columns and HPLC Using One
Supelcosil LC-Diol Column
EO unit SFC RT* HPLC RT
2 6.79 9.28
3 7.48 10.06
4 8.20 10.92
5 9.05 12.08
6 10.00 13.39
7 10.96 14.75
8 11.91 16.10
9 12.88 17.51
10 13.86 18.96
11 14.80
12 15.76
* RT, retention time.
Figure 5. Packed-column supercritical fluid chromatograms using single
columns of different polar packing material: (A) Supelcosil LC-Diol column,
(B) Spherisorb NH
2
column, and (C) Supelcosil LC-PCN column. The sample
used in each chromatogram was POE-(4)-NP (2.0 mg/mL). A linear modifier
gradient was used by the following program: 10.0% methanol was increased
to 26.0% at a rate of 0.6%/min with a 2.0-min hold and then returned to 10.0%
in 4.0 min followed by a 2.0-min hold.
Journal of Chromatographic Science, Vol. 40, February 2002
66
It was important to perform chromatographic separations with
absorbance detection on the fractions as well as MS analysis, thus
allowing us to positively identify sample components because MS
could not detect all of the compounds present. The first fraction
of both POE-(4)-NP and POE-(5)-tert-OP contained more than
one compound (as seen in their SFC-UV chromatograms). The
sodium ion affinity of the smaller ethoxylate chain compounds is
lower than the larger chain oligomers, and because of this they
were not detectable in the mass spectra.
APEs can be categorized by their average ethoxylate unit value.
According to Wang and Fingas (3), all of the oligomers have
almost identical molar absorptivity, which allows integrated chro-
matographic peak areas to be used directly to determine the mole
fraction of each oligomer. POE-(4)-NP contained NP predomi-
nantly with short ethoxylate chains. NP2EO through NP11EO
were observed in its SFC-UV separation. An average ethoxylate
Figure 6. Packed-column supercritical fluid chromatograms using stacked
columns of different polar stationary phases: (A) one Supelcosil LC-Diol
column and one Supelcosil LC-PCN column and (B) one Supelcosil LC-Diol
column, one Supelcosil LC-PCN column, and one Spherisorb NH
2
column.
The sample used in each chromatogram was POE-(4)-NP (2.0 mg/mL). Multiple
linear modifier gradients were used by the following program: 10.0% methanol
was increased to 13.2% by 0.5%/min and then continued to 14.4% at
0.7%/min, 16.6% at 0.8%/min, 20.0% at 1.0%/min, 40.0% at 8.0%/min (held
for 5.0 min), and then returned to 10.0% at 15.0%/min.
Figure 7. Supercritical fluid chromatograms of collected POE-(4)-NP fractions.
Separation was conducted on one Supercosil LC-Diol column and one
Supelcosil LC-PCN column in series (the system settings were the same as
Figure 3).
Figure 8. Supercritical fluid chromatograms of collected POE-(5)-tert-OP frac-
tions. Separation was conducted on one Supelcosil LC-Diol column and one
Supelcosil LC-PCN column in series (the system settings were the same as
Figure 3).
Figure 9. Positive-ion FIAESIMS of POE-(4)-NP fractions operated in full-scan
mode. Ions were in the form of (M+Na)
+
and each were separated by m/z 44
(the mass of one ethoxyl unit): (A) fraction 1, cone voltage of 59 V; (B) fraction
2, cone voltage of 53 V; (C) fraction 3, cone voltage of 62 V; (D) fraction 4, cone
voltage of 65 V; and (E) fraction 5, cone voltage of 67 V. Each spectrum was
averaged over the sample injection peak.
Journal of Chromatographic Science, Vol. 40, February 2002
67
unit value of 4.20 was calculated from peak areas. POE-(5)-tert-
OP had a similar distribution as POE-(4)-NP. Its average ethoxy-
late unit value was calculated as 4.48, and it contained OP2EO
through OP12EO in its SFC-UV separation. Triton N-101 con-
tained a greater range of NPEOs. Its calculated average ethoxylate
unit was 9.97. NP2EO through NP18EO were observed in its SFC-
UV chromatogram. Higher EO peaks were detected in SFC sepa-
rations, which were not detected by HPLC analysis. Wang and
Fingas produced similar average EO unit values from their capil-
lary SFC data. Their analysis of Igepal CO430 (trade name for
POE-(4)-NP), Triton X-45 (trade name for POE-(5)-tert-OP), and
Triton N-101 produced average EO values of 4.14, 4.50, and 9.52,
respectively (11,12). We used the chromatographic data from the
SFC-UV separations on two diol columns to calculate our average
EO values.
Conclusion
Normal-phase packed-column SFC produced a similar separa-
tion of APE mixtures compared with normal-phase HPLC.
Column length, stationary phase, and column combinations with
different stationary phases all affected the separation of the APE
mixtures tested. Longer column lengths increased the separation
of oligomers. More-polar stationary phases retained oligomers
with larger ethoxylate units for a longer time. A combination of
columns with different stationary phases produced separations
combining both the effects of longer columns and the separation
ability of each stationary phase. Retention times for SFC separa-
tions were notably shorter than normal-phase HPLC. One of
SFCs advantages is its ability to use longer combined column
lengths without elevated back pressure, which occurs in HPLC.
Combining multiple columns with different stationary phases
seemed to provide the best separation.
An advantage of using packed columns over the use of capillary
columns is the ability to inject larger amounts of sample and col-
lect eluted fractions. It is possible to isolate and identify individual
APEs. Additionally, it is possible to identify the remaining chro-
matographic peaks because of each peak differing by one ethoxy-
late unit. Our study demonstrated the importance of using both
absorbance detection as well as MS. MS alone did not show all the
components of our initial fractions because of the decreased
detector response.
Less solvent waste was produced using SFC compared with
HPLC. Each SFC separation that used cyano packing as part of its
column arrangement used 6.7 mL of methanol. The remaining
SFC setups (the studies of column length and stationary phase)
used 11.8 mL of methanol. All separations performed by normal-
phase HPLC used 34.75 mL of hexane and 5.25 mL isopropanol
for a combined volume of 40 mL. The HPLC system used almost
600% more solvent than the SFC system using a cyano stationary
phase and over 330% more than the other SFC setups studied
(this is not including the volume of solvent needed to initially
equilibrate the systems). The reduction of solvent waste is an
important step of reducing pollution.
Because of the fact that APEs are used as industrial cleaners and
other processing aids, they enter wastewater and end up in
sewage treatment plants. Some APE waste is transferred into the
environment and metabolized into lower ethoxylated alkylphe-
nols, which are considered endocrine disrupters (2). APEs have
been found in fish, river sediment, and other environmental sam-
ples through analytical techniques (1,4,9,10,1822). The results
of our study could lead to the further use of the method developed
for applications in the analysis of environmental samples.
Additionally, our method could be altered for use in a future
large-scale separation and collection of individual ethoxylated
alkylphenols. Access to standards of individual ethoxylated
alkylphenols is important for their quantitative analysis.
Acknowledgments
We would like to acknowledge Dr. Clifford P. Rice (USDA
ARS/NRI/EQL, Beltsville, MD) for APE information and Air
Products and Chemicals, Inc. for supplying SFC-grade carbon
dioxide.
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Figure 10. Positive-ion FIAESIMS of POE-(5)-tert-OP fractions operated in
full-scan mode. Ions were in the form of (M+Na)
+
and each were separated by
m/z 44 (the mass of one ethoxyl unit): (A) fraction 1, cone voltage of 63 V; (B)
fraction 2, cone voltage of 68 V; (C) fraction 3, cone voltage of 65 V; (D) frac-
tion 4, cone voltage of 75 V; and (E) fraction 5, cone voltage of 75 V. Each spec-
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Journal of Chromatographic Science, Vol. 40, February 2002
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Manuscript accepted December 7, 2001.
69
An isocratic high-performance liquid chromatography (HPLC)
method for the simultaneous determination of -tocopherol,
retinol, and five carotenoids (luteinzeaxanthin, -cryptoxanthin,
lycopene, and - and -carotene) in human serum is described.
Serum samples are deproteinized with ethanol and extracted once
with n-hexane. Resulting extracts are injected onto a C18 reversed-
phase column eluted with methanolacetonitriletetrahydrofuran
(75:20:5, v/v/v), and full elution of all the analytes is realized
isocratically within 20 min. The detection is operated using three
channels of a diode-array spectrophotometer at 290, 325, and
450 nm for tocopherol, retinol, and the carotenoids, respectively.
An internal standard is used for each channel, which improves
precision. The choice of internal standards is discussed, as well as
the extraction protocol and the need for adding an antioxidant
during the extraction and chromatographic steps. The analytical
recoveries for liposoluble vitamins and carotenoids are more than
85%. Intra-assay relative standard deviation (RSD) values (n = 20)
for measured concentrations in serum range from 3.3% (retinol)
to 9.5% (lycopene), and interassay RSDs (n = 5) range from 3.8%
(-tocopherol) to 13.7% (-cryptoxanthin). The present method is
used to quantitate the cited vitamins in healthy subjects (n = 168)
from ages 9 to 55 years old.
Introduction
Retinol (vitamin A) and -tocopherol (vitamin E) are nonen-
zymatic antioxidants (1). Vitamin A acts as a direct scavenger
of reactive oxygen species (ROS) and is also thought to inhibit
free radical synthesis via increasing the activity of detoxifying
systems (2).
Vitamin E protects unsaturated fatty acids located in both cell
and organelle membranes against endo- and exogenous free rad-
icals and ROS, which are involved in the initiation and extent of
membrane damages caused by nonenzymatic lipid peroxidation
(3,4). Carotenoids act as ROS and free radical scavengers (5),
stimulants of immune response (6), and anticarcinogenic agents
(7). Because of their wide variety of functions and biological
roles, clinical interest in the evaluation of retinol, -tocopherol,
and carotenoids has increased in recent years owing to their role
as antioxidants, which may be important in reducing the risk of
numerous diseases including cancer (811), coronary heart dis-
ease (12,13), and diabetes mellitus (1418).
Thus, rapid, simple, sensitive, and selective methods for the
simultaneous determination of these antioxidants in biological
fluids are needed. As a matter of fact, the measurement of an
individual class of antioxidants such as thiols (19), hydrophilic,
or liposoluble vitamins provides more information for the mech-
anistic evaluation of a clinical disease linked to oxidative stress
than a total antioxidant status assay (20).
Numerous spectroscopic and separative methods have already
been reported for the assay of retinol, -tocopherol, and
carotenoids in plasma or serum, and among them high-perfor-
mance liquid chromatography (HPLC) is one of the most pow-
erful analytical tools for this purpose (2125).
Both normal-phase (2628) and reversed-phase (2935) HPLC
conditions have been widely used. However, many of these
methods include gradient elution (3639), flow rate (34,36),
wavelength time-programmation (36,40), a switching device
between coupled columns (41,42), and the use of two different
detectors in series (43,44). All of these approaches are time-con-
suming because of their long-equilibration period between each
run and troublesome because of the hyphenated systems needed.
Indeed, the main difficulty for the simultaneous determination
of liposoluble vitamins and carotenoids results from their dif-
ferent spectral characteristics (absorption maxima vary in the
Abstract
An Isocratic Liquid Chromatographic Method with
Diode-Array Detection for the Simultaneous
Determination of -Tocopherol, Retinol, and
Five Carotenoids in Human Serum
Sonia Gueguen
1
, Bernard Herbeth
1
, Grard Siest
1
, and Pierre Leroy
2
1
Inserm U525, Centre de Mdecine Prventive, 2 rue du Doyen Jacques Parisot, 54500 Vandoeuvre-ls-Nancy, France and
2
Thiols and
Cellular Functions, Facult de Pharmacie, Universit Henri Poincar Nancy 1, 30 rue Lionnois, 54000 Nancy, France
Reproduction (photocopying) of editorial content of this journal is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, February 2002
* Author to whom correspondence should be addressed: email pierre.leroy@pharma.uhp-nancy.fr.
Journal of Chromatographic Science, Vol. 40, February 2002
70
range of 292 to 450 nm). This problem has been solved by using
multichannel UVvis spectrophotometric detectors (31,37,40,
4547). More recently, a technique combining both isocratic elu-
tion in reversed-phase mode and diode-array detection was
reported, providing selectivity between the three classes of
liposoluble vitamins and thus a convenient way for their simul-
taneous measurements (32).
For all these methods, the preanalytical treatments, especially
the extraction procedure relying upon either liquidliquid
(2628,3035,39,43,47,48) or solidliquid (38,49,50) partition,
are critical steps to obtain reliable data.
This study deals with some improvements of a previously
reported method (32); the full validation of the optimized assay;
and its use to quantitate retinol, -tocopherol, luteinzeaxan-
thin, -cryptoxanthin, lycopene, and - and -carotene in
healthy subjects.
Experimental
Chemicals, reagents, and standards
All solvents and reagents used were of analytical- or HPLC-
grade. Ultrapure water was prepared using a Milli-Q system
(Millipore Milford, MA). Tert-butylated hydroxytoluene (BHT)
was purchased from Sigma-Aldrich (St. Quentin Fallavier,
France).
All-trans retinol (henceforth simply referred to as retinol),
retinol acetate, -tocopherol, -tocopherol acetate, and
-carotene standards were obtained from Fluka (Buchs,
Switzerland). Zeaxanthin and -cryptoxanthin were a generous
gift from Hoffman-Laroche (Basle, Switzerland). Lycopene and
echinenone were purchased from CaroteNature (Lupsingen,
Switzerland). Stock solutions of retinol, -tocopherol, and their
corresponding internal standards (acetate form) were prepared
in ethanol (EtOH) added with 0.01% (w/v) BHT. Carotenoids
were prepared in tetrahydrofuran (THF) added with 0.01% BHT.
Stock solutions were protected from light in ambered glass bot-
tles, titrated by spectrophotometry using their specific
absorbance (Table I), and stored under nitrogen at 80C for up
to 2 mo. The concentrations of stock solutions were 0.250.5
mg/mL for retinol and retinol acetate, 34 mg/mL for -toco-
pherol and -tocopherol acetate, and 0.10.2 mg/mL for
carotenoids.
Daily working solutions for calibration curves were prepared
by diluting stock solutions in EtOH containing 0.01% BHT. The
ranges of tested concentrations are indicated in Table II. An
internal standard mixture containing retinol acetate, -toco-
pherol acetate, and echinenone was also prepared
daily following a similar procedure (combining
100 L of each stock solution of internal standard
and diluting the volume to 20 mL with
EtOH0.01% BHT). All the operations were per-
formed by handling solutions in darkness and ice.
The standards of -carotene and zeaxanthin
were used to quantitate -carotene and both
lutein and zeaxanthin, respectively.
Blood collection and storage conditions
Blood was collected at the antecubital vein of
168 healthy control subjects from ages 9 to 55
years old (informed consent was obtained, and
the research protocol was in agreement with the
Helsinki Declaration) in a reclined position in dry
tubes (Vacutainer Tube, Becton Dickinson,
Grenoble, France). Blood samples were cen-
Table I. Characteristics of Standards Used
Maximum
Molecular weight wavelength
Compounds (g/mol) (nm) A
1%
1 cm
* (mol
1
/L/cm
1
)
Retinol 286.5 325 1835 (32,61) 52573
Retinol acetate 328.5 326 1550 (32,61) 50912
-Tocopherol 430.7 292 75.8 (45) 3265
-Tocopherol acetate 472.8 290 40 (32) 1891
Echinenone 550.9 458 2244 123622
(Hoffmann-Laroche data source)
Luteinzeaxanthin 568.9 452 2765/2416 (45) 157301/137446
-Cryptoxanthin 552.9 452 2486 (45) 137451
Lycopene 536.9 472 3450 (32,61) 185231
-Carotene 536.9 450 2620 (35) 140667
* In EtOH as the solvent. Data references appear in the parentheses.
Table II. Equations of Calibration Curves and Values of LODs and LOQs*
Equations of calibration curves
Concentration Slope
Intercept Correlation LOD LOQ
range (mol/L) (SD
, n = 5) (SD, n = 5) coefficient
(mol/L) (mol/L)
Retinol 0.457.50 0.16 (0.015) 0.021 (0.016) 0.998 0.45 0.66
-Tocopherol 4.8080.0 0.01 (0.000) 0.027 (0.008) 0.996 2.64 5.36
Luteinzeaxanthin 0.101.90 0.35 (0.034) 0.024 (0.006) 0.997 0.06 0.11
-Cryptoxanthin 0.091.50 0.34 (0.031) 0.022 (0.019) 0.996 0.03 0.09
Lycopene 0.121.90 0.24 (0.018) 0.018 (0.020) 0.997 0.03 0.08
-Carotene 0.132.00 0.35 (0.019) 0.014 (0.006) 0.997 0.03 0.06
* Each calibration curve included six points, and each point was assayed in five replicates.
Calculated by internal standardization: (standard peak area/internal standard peak area)/standard concentration.
(mol/L) %RSD
Retinol 1.90 (0.06) 3.3 2.1 (0.09) 4.4
-Tocopherol 34.9 (1.31) 3.8 29.3 (1.1) 3.8
Luteinzeaxanthin 0.65 (0.02) 3.8 0.51 (0.02) 4.5
-Cryptoxanthin 0.13 (0.01) 7.8 0.10 (0.01) 13.7
Lycopene 0.53 (0.05) 9.5 0.28 (0.04) 12.5
-Carotene 0.18 (0.02) 8.8 0.14 (0.02) 12.1
-Carotene 0.57 (0.04) 6.7 0.52 (0.05) 9.1
* Mean (standard deviation), n = 20.
0.43 (0.24)
0.49 (0.23)
0.52 (0.25)
0.26 (0.11)
0.71 (0.30)
0.24 (0.21)
0.36 (0.140.74)
Zeaxanthin
-Cryptoxanthin 0.13 (0.08) 0.13 (0.11) 0.19 (0.14) 0.17 (0.12) 0.55 (0.11) 0.35 (0.27) 0.60 (0.47) 0.22 (0.050.52)
Lycopene 0.33 (0.16) 0.28 (0.16) 0.31 (0.16) 0.32 (0.22) 0.37 (0.18) 0.56 (0.43) 0.42 (0.24) 0.40 (0.110.80)
-Carotene 0.08 (0.06) 0.10 (0.13) 0.13 (0.11) 0.14 (0.14) 0.07 (0.04) 0.36 (0.26) 0.07 (0.05) 0.08 (0.020.22)
-Carotene 0.49 (0.43) 0.42 (0.29) 0.60 (0.37) 0.64 (0.72) 0.38 (0.20) 0.81 (0.45) 0.37 (0.23) 0.34 (0.070.88)
* Means (standard deviation).
Concentrations in serum for men and women ranging from ages 19 to 62 years old, n = 111.
Concentrations in serum for women ranging from ages 35 to 50 years old, n = 96.
Concentrations in serum for women ranging from ages 25 to 59 years old, n = 54.
** Concentrations in serum for men and women ranging from ages 4 to 93 years old, n = 3480.
100 21.9
2,4,6-TNT 101 14.5
RDX 125 17.3
HMX 118 14.3
2-Nitrotoluene 77 16.9
3-Nitrotoluene 94 5.0
4-Nitrotoluene 95 19.3
Nitrobenzene 85 15.6
1,3-Dinitrobenzene 93 17.7
1,3,5-Trinitrobenzene 94 16.5
4-Amino-2,6-dinitrotoluene 102 15.5
2-Amino-4,6-dinitrotoluene 109 14.3
Tetryl 100 18.3
Nitroglycerin 125 17.8
Diphenylamine 91 8.3
Di-n-butylphthalate 113 8.4
Dioctylphthalate 109 8.2
* 2.62.7 m
3
volume sampled. Seven spikes at 15 g (energetics) or 75 g (propellants).
87 6.2
2,4,6-TNT 91 6.9
RDX 101 5.2
HMX 107 17.7
2-Nitrotoluene 99 13.9
3-Nitrotoluene 103 18.8
4-Nitrotoluene 114 17.2
Nitrobenzene 89 9.1
1,3-Dinitrobenzene 87 7.4
1,3,5-Trinitrobenzene 85 7.9
4-Amino-2,6-dinitrotoluene 93 6.6
2-Amino-4,6-dinitrotoluene 103 5.1
Tetryl 96 11.2
Nitroglycerin 104 16.0
Diphenylamine 88 3.9
Di-n-butylphthalate 100 3.5
Dioctylphthalate 106 3.3
* 130 m
3
volume sampled. Seven spikes at 100 g (energetics) or 500 g (propellants).
app
*
p
(k
D
/k
L
) (k
lsD
/k
lsL
) (k
nsD
+ k
sD
/k
nsL
+ k
sL
)
Dansyl valine 1.26 1.47 1.14
Dansyl serine 1.29 1.48 1.09
Dansyl leucine 1.18 1.26 1.11
Dansyl phenylalanine 1.18 1.28 1.08
* The apparent enantioselectivity.
The enantioselectivity resulting from the compound binding to the aglycone pocket.
and a Powdered
Enteral Formula
Displaced hydrolysate
Sample Discarded Collected
Powdered milk B
Elution with 3 mL 3M HCl 120.83 6.00 124.05 2.34
Elution with 5 mL 3M HCl 131.94 2.87 132.86 2.55
Powdered enteral formula B
Elution with 3 mL 3M HCl 128.25 4.56 133.12 4.41
Elution with 5 mL 3M HCl 139.49 4.99 143.53 6.91
* Milligrams per 100 g of product. Values are the means of four replications standard deviation.
Determination coefficient.
Table IV. Percentage Furosine Recovery for a Powdered
Enteral Formula and a Powdered Milk
Furosine (g/mL)
Recovery
Sample Initial Added Recovered (%)
Enteral formula A 3.39 0.95 4.30 99.08
1.42 4.78 99.38
1.89 5.25 99.43
2.37 5.72 99.31
Mean value 99.30 0.15
Powdered milk A 2.10 0.95 3.02 99.02
1.89 3.94 98.75
2.84 4.89 98.99
3.79 5.84 99.15
Mean value 98.98 0.17
Table V. Effect of HCl Concentration and Ratio During
Hydrolysis on Furosine* Formation in a Powdered
Enteral Formula and a Powdered Milk
Hydrolysis conditions Enteral formula A
Powdered milk A
1 mg protein to 1 mL acid
10M HCl 546.22 4.39 (100) 322.95 0.95 (100)
8M HCl 460.45 2.41 (84.3) 289.37 4.55 (89.6)
6M HCl 332.66 1.24 (60.9) 195.63 0.43 (60.6)
5 mg protein to 1 mL acid
10M HCl 493.59 4.40 (100) 306.39 0.98 (100)
8M HCl 428.14 5.56 (86.7) 268.89 5.53 (87.8)
6M HCl 320.26 2.56 (64.9) 161.66 1.47 (52.8)
* Milligrams per 100 g protein. Values are the means of three replications
standard deviation.
) correlations (g/mL)
Paracetamol 0.13 0.9999 25120
Phenylephrine HCl 1.95 0.9999 0.310
Chlorpheniramine maleate 1.36 0.9999 0.23
* n = 10.
, Cl
, Br
, NO
3
, SO
4
2
, and
PO
4
3
in serum samples. The factors studied are various sample
deproteinization procedures, eluent composition, and flow rates.
Deproteinization using either acetonitrileNaOH or ultrafiltration
can be used in order to obtain a significant protein removal before
IC analysis; however, the former is recommended because it is less
time-consuming and cheaper. Better resolution is obtained when
a sodium hydroxide solution is used as the eluent. There is no
influence of the samples deproteinization procedures on the
chromatographic resolution.
Introduction
The ability to determine the concentration of physiologically
and pathologically related anions with a single sample treatment
and technique is of great importance. Ion chromatography has
become one of the most powerful tools for the quantitative anal-
ysis of anions in a wide variety of matrices. One of the problems
associated with serum anion determination by this technique is
the lack of similar sensitivity for all the anions to be determined
simultaneously. Of all the inorganic anions present in human
serum, chloride, bicarbonate, and phosphate are the most rou-
tinely determined. However, the demand for sulfate, bromide,
nitrate, and fluoride serum analysis is increasing because of the
clinical information derived from the levels in serum. The impor-
tance in human physiology of the inorganic anions sulfate (1),
bromide (24), nitrate (5,6), and chloride (7) have been described.
The biochemical analysis of metabolites in whole blood and
serum requires initial deproteinization to remove hemoglobin
and other proteins that may interfere with the assays. Different
agents have been used. One of them is perchloric acid (8,9); how-
ever, it has the disadvantage that percholorate inhibits some
enzyme reactions considerably and it has to be removed by pre-
cipitation with potassium hydroxide. A simple deproteinization
procedure has been reported by Khan et al. (10) involving the use
of sulfosalycylic acid (SSA). However, the SSA agent is often con-
taminated with sulfate. Tricholoroacetic acid has been reported
(11) as a time-consuming procedure and interferes with the
anion elution profile. The precipitation method based on acetoni-
trile (ACN) (12) has not been successful because proteins are not
quantitatively precipitated by this method, which limits the life-
time of the separator column to a maximum of two months.
Centrifugation or ultracentrifugation has been reported (11) as
an efficient method for the deproteinization of serum samples.
A number of studies have reported the determination of some
inorganic anions in serum by ion chromatography. These have
included the simultaneous determination of inorganic phos-
phate, bromide, nitrate, and sulfate in human serum (1) and the
determination of thiocyanate (13), bromide (14), and sulfate (15).
In measuring systems in which the signal is linearly related to the
component of interest, matrix components, and instrumental
parameters, factorial analysis is a convenient tool to apply.
Factorial approaches to experimental designs contrast with sim-
plex approaches in that several experiments can be performed
simultaneously and are used to calculate the main effects and the
interaction effects of several factors. Full factorial designs at two
levels of variation for the input factors are often used in analytical
chemistry (16,17).
This research deals with the optimization of a method for the
simultaneous analysis of chloride, fluoride, nitrate, bromide, sul-
fate, and phosphate in human serum by isocratic ion chromatog-
raphy. A factorial design at two levels was applied in order to
estimate the magnitude of the main effects and various two-factor
interactions under the experimental conditions of interest. It fur-
ther allowed for a better interpretation of the results obtained.
The evaluation of parameter significance is a very important step
in the optimization procedure. The selection criterion for
choosing the factors involved in this design was dictated by
Abstract
Evaluation of Select Variables in the Ion
Chromatographic Determination of F
, Cl
, Br
,
NO
3
, SO
4
2
, and PO
4
3
in Serum Samples
Z. Benzo
1,
*, A. Escalona
3
, J. Salas
1
, C. Gmez
1
, M. Quintal
1
, E. Marcano
1
, F. Ruiz
1
, A. Garaboto
1
, and F. Bartoli
2
1
Centro de Qumica and
2
Dpto. de Biologia estructural, Instituto Venezolano de Investigaciones Cientficas, IVIC, Apdo. Postal 21827,
Caracas 1020-A, Venezuela and
3
Centro de Qumica Analtica, Facultad de Ciencias, Universidad Central de Venezuela, Apdo. Postal 47102,
Caracas 1041-A, Venezuela
Reproduction (photocopying) of editorial content of this journal is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, February 2002
* Author to whom correspondence should be addressed: email zbenzo@ivic.ve.
Journal of Chromatographic Science, Vol. 40, February 2002
102
variables that may influence the resolution such as deproteiniza-
tion treatment, eluent composition, and flow rate.
Experimental
Apparatus and reagents
The ion chromatographic equipment used was a Dionex
(Sunnyvale, CA) DX 500 system with a 100-L injection loop, an
Ion Pac AS11 analytical column (4 250 mm), and an Ion Pac
AG11 Guard column (4 50 mm) (Dionex). The column temper-
ature was 25C. The apparatus was equipped with an ion fiber
suppressor ASRS-11-4 mm (Dionex Anion self regenerating) that
was continuously regenerated by water and a conductimetric
detector (Dionex) PeakNet Chromatography Workstation.
All chemicals were of analytical-reagent grade. Sodium
hydroxide was obtained from Merck (Darmstadt, Germany), and
Na
2
CO
3
, NaHCO
3
, NaCl, NaNO
3,
NaF, Na
2
SO
4
, Na
3
PO
4
, and KBr
(> 99.9% pure) were from Aldrich (St. Louis, MO). Milli-Q deion-
ized water was used throughout (Milli-Q water purification
system, Millipore, MA) as well as HPLC-grade ACN (EM Science,
NJ). Ultrafree-Cl low binding cellulose 10,000 nominal molecular
weight limit filters were used (Millipore) as well as 0.45- and 0.47-
m nylon filters (Alltech, Deerfield, IL).
A protein assay was performed with the Coomassie Plus Protein
assay reagent kit (Pierce, Rockford, IL).
Serum samples
A pooled serum sample was obtained from the Medical Center
at our research institute. Each deproteinization method was per-
formed on an aliquot of this pooled sample.
Sample treatment
A preliminary study was carried out in order to assess the effi-
ciency of protein removal by different methods. For this reason,
three deproteinization treatments that have been reported in the
literature were tested: ACN (18,19), ACNNaOH (20), and ultrafil-
tration (19). Protein quantitation was carried out on an aliquot of
the same serum sample after each deproteinization treatment,
measuring the absorbance at 595 nm.
The ACN treatment consisted of mixing 0.2 mL of serum
sample with an equal volume of ACN, then centrifuging at 2000
g for 5 min.
For the ACNNaOH treatment, 500 L of a serum sample,
50 L of NaOH (2M), and 150 L of deionized water were added
and shaken for a few seconds. Then, 1 mL of ACN was added and
vortex mixed for 10 s. The resulting mixture was centrifuged for
5 min at 755 g. Finally, 1 mL of the supernatant solution was
diluted with 5 mL of deionized water and injected into the chro-
matograph.
For the ultrafiltration, a 1:10 diluted serum sample was filtered
for 30 min and injected.
Results and Discussion
Protein removal
The sample preparation for ion chromatographic analysis of
serum is much more elaborate than for other physiological fluids.
Proteins that are present in high concentration must be removed
before the sample is injected, because they negatively affect the
separation efficiency of the ion-exchange columns.
The principal objective of this study was to find an efficient, fast,
and reliable deproteinization method that yields a solution suit-
able for subsequent ion chromatographic analysis. The three
deproteinization methods described in the Experimental section
were applied, and the resulting percentages of protein removal
were 21.5%, 93.3%, and 98.6% for the ACN, ACNNaOH, and
ultracentrifugation treatments, respectively.
After the efficiency of these methods was verified, it was decided
to choose the ACNNaOH mixture and ultrafiltration depro-
teinization procedures as variables to be considered in the exper-
imental design in order to observe their influence on the
chromatographic resolution.
Optimization
In order to obtain proper information on the significance of the
factors mentioned, a full 2
4
factorial design at two levels was
applied. This type of design involved sixteen experiments. Four
factors were examined in this design: a sodium hydroxide solu-
tion (X
1
), a sodium carbonatesodium bicarbonate mixture (X
2
),
Table I. Experimental Variables Considered in the
Application of the 2
4
Full Factorial
Natural Coded
variable variable Level (+1) Level (1)
NaOH X
1
12mM 6mM
Na
2
CO
3
NaHCO
3
X
2
3mM,2.4mM 0
Flow rate X
3
1.5 mL/min 1.0 mL/min
Deproteinization
procedure X
4
ultrafiltration NaOHACN
Table II. 2
4
Experimental Design*
Resolution
Experiment X
1
X
2
X
3
X
4
criterion
1 1 1 1 1 11.36
2 1 1 1 1 10.62
3 1 1 1 1 12.30
4 1 1 1 1 7.41
5 1 1 1 1 11.03
6 1 1 1 1 10.99
7 1 1 1 1 11.50
8 1 1 1 1 7.14
9 1 1 1 1 10.04
10 1 1 1 1 10.84
11 1 1 1 1 12.69
12 1 1 1 1 7.56
13 1 1 1 1 10.52
14 1 1 1 1 11.07
15 1 1 1 1 11.32
16 1 1 1 1 9.26
* Resolution taken as the response.
Journal of Chromatographic Science, Vol. 40, February 2002
103
the flow rate (X
3
), and a deproteinization procedure (X
4
) (Table I).
Two levels were chosen associated with the 1 and +1 levels of the
corresponding coded variables. These were selected on the basis
of the literature review on this topic, and the deproteinization
procedures were selected according to the results obtained and
described in the previous section.
The selected experimental response was a criteria of peak reso-
lution (R
s
) and the peak area. This criterion of resolution con-
sisted in the addition of the resolution values of all consecutive
peaks plus the number of peaks that appears in the chro-
matogram (21). Table II shows the results.
The significance was evaluated through the application of the
student t-test with a 0.05 significance level. Table III shows these
results. The variables of X
1
at the 1 level (6mM), X
2
at the 1
level, and the interaction of X
1
and X
2
resulted as being significant
at the 95% level. The influence of X
1
revealed that a better reso-
lution was obtained when this sodium hydroxide concentration
was used. The use of the Na
2
CO
3
NaHCO
3
mixture did not influ-
ence the resolution, and this agreed with the result of the signifi-
cance test. The X
1
X
2
interaction revealed that an improvement
on the resolution was obtained when both mixtures were used.
Identification
Figure 1 shows the chromatograms of an aqueous solution run
at an eluent composition of 6 and 12mM NaOH (Figures 1A and
1B) and a serum sample using isocratic conditions (Figures 1C
and 1D). Peak identification was based on retention times.
As it can be seen from these chromatograms, in general, the
aqueous standard and the serum samples exhibited a well-defined
resolution and symmetrical peaks (not broadened) in less than 16
min. Shorter retention times, mainly for the monovalent and
divalent anions, were observed when changing the concentration
of the eluent mixture (12mM). Furthermore, higher signals were
obtained at this condition. Observed in the chromatogram of the
serum sample was the appearance of a second peak (probably
acetate), which was coeluted with fluoride.
Figures 1C and 2 show the effect on the resolution from the
deproteinization treatments. It can be observed that there was not
a significant change between the results obtained. Therefore,
either treatment (deproteinization by using ACNNaOH or by
ultrafiltration) could be used. This result was consistent with that
obtained in the experimental design in which the deproteiniza-
Table III. Calculation of the Effects
Estimated Standard Experimental Probability
Effect value deviation t-value level
b
0
10.42 0.1613 64.58 0.0000
b
1
1.0574 0.1613 6.55 0.0012*
b
2
0.4545 0.1538 2.95 0.0317*
b
3
0.0619 0.1613 0.10 0.7039
b
4
0.1256 0.1613 0.77 0.4714
b
12
1.0655 0.1538 6.92 0.0012*
b
13
0.2638 0.1945 1.35 0.2331
b
14
0.1955 0.1613 1.21 0.2797
b
23
0.0945 0.1538 0.61 0.5659
b
24
0.2514 0.1538 1.63 0.1630
b
34
0.0621 0.1613 0.38 0.7158
* Significant effect.
Figure 1. Chromatograms of an aqueous solution run at two different eluent compositions (A and B) and a serum sample using two different isocratic conditions (C and
D): (A) an aqueous standard of 6mM NaOH eluent and 1-mL/min flow rate; (B) an aqueous standard of 12mM NaOH eluent and 1-mL/min flow rate; (C) a serum
sample of 6mM NaOH eluent, ACNNaOH deproteinization, and 1-mL/min flow rate; and (D) a serum sample of 12mM NaOH eluent, ACNNaOH deproteiniza-
tion, and 1-mL/min flow rate.
Journal of Chromatographic Science, Vol. 40, February 2002
104
tion treatment did not have any statistical significance. However,
we recommend the ACNNaOH mixture because it is less time-
consuming (the sample can be deproteinized in approximately
5 min compared with the 30 min that it takes for the ultrafiltra-
tion procedure).
Even when the flow rate was not significant, according to the
results from the significance test a flow rate of 1.5
mL/min (+1 level, X
3
) is more convenient if anal-
ysis time is considered important (as shown in
Figures 1C and 3).
The significance of the b
12
interaction was veri-
fied by the use of the interaction diagrams (22).
The b
12
interaction at the 1 level revealed that the
resolution was improved when the NaOH (6mM)
and Na
2
CO
3
NaHCO
3
mixtures were used. The
chromatogram resulting from this experimental
condition is shown in Figure 4. However, this con-
dition is not recommended when fluoride has to
be determined, because its peak appeared within
the water dip.
In order to further evaluate the efficiency of the
deproteinization procedures, an evaluation of the
signal sensitivity was carried out for each anion by
taking into account the peak area as a measure-
ment of the response in the experimental design.
The result of this study (not shown) led to the con-
clusion that the treatment procedure does not
have any significant effect on the signal sensitivity.
Because of the complexity of the serum matrix,
the column efficiency was checked in order to see
if any modification (probably occasioned by the
matrix) could have affected it. For this reason, a
control solution containing all the anions consid-
ered in this study was passed through the column
before and after running the serum samples for
their analysis (shown in Figure 5). In order to
evaluate column efficiency, the theoretical plates
were calculated for each anion before and after the
set of experiments involved in this research, and
no significant change was observed. No change in
the signal sensitivity and resolution was obtained.
Therefore, it can be said that the serum matrix did
not alter the columns original characteristics
with it being able to analyze the inorganic anions
in this matrix under the conditions developed in
this work.
Conclusion
It has been shown in this work that the simulta-
neous determination of six important physiolog-
ical anions in human serum is possible using ion
chromatography under isocratic conditions
without altering the columns original conditions
and thus its efficiency. This is an important aspect,
taking into account the complexity of the serum
matrix. Scarce information has been given about
column integrity after this type of analysis.
The application of the experimental design to
Figure 2. Chromatogram of a serum sample with ultrafiltration: 6mM NaOH eluent and 1-mL/min flow
rate.
Figure 3. Chromatogram of a serum sample with ACNNaOH: 6mM NaOH eluent and 1.5-mL/min
flow rate.
Figure 4. Chromatogram of a serum sample with ultrafiltration: 6mM NaOHNa
2
CO
3
NaHCO
3
eluent and 1-mL/min flow rate.
Journal of Chromatographic Science, Vol. 40, February 2002
105
this particular application for evaluating the significance of input
parameters for analytical determinations allows for the obtaining
of important information on the variables that most influence the
determination of the anions in this matrix and on the interactions
between chemical and instrumental parameters.
There was no influence of the samples deproteinization proce-
dures on the chromatographic resolution.
Acknowledgments
The authors gratefully acknowledge the valuable support of the
program BID-CONICIT through the project QF-10 and Prof. Luis
Gmez of the chromatography laboratory at the Analytical
Chemistry Center (UCV) for his valuable assistance.
This research is dedicated to our late and dearest colleague
Santos Melendez who could never fulfill his dream of working
with us in this project.
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Figure 5. Column performance (A) before and (B) after 40 injections of serum sample. Data for A and
B are shown in Tables IV and V, respectively.
Table IV. Data for the Anions in Figure 5A
Anion Retention time (min) Area
Fluoride 1.82 71263
Chloride 2.30 674127
Nitrite 2.52 42713
Bromide 3.48 91697
Nitrate 3.60 294013
Sulfate 4.48 1065847
Table V. Data for the Anions in Figure 5B
Anion Retention time (min) Area
Fluoride 1.82 71282
Chloride 2.27 666512
Nitrite 2.48 37662
Bromide 3.43 107169
Nitrate 3.55 284737
Sulfate 4.25 1074038
Journal of Chromatographic Science, Vol. 40, February 2002
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107
Subcritical water has been recently employed as the mobile phase
to eliminate the use of organic solvents in reversed-phase liquid
chromatography. Although the influence of temperature on
retention in subcritical water chromatography has been reported,
the temperature effect on peak width and column efficiency has not
yet been quantitatively studied. In this work, several polar and
chlorinated compounds are separated using pure subcritical water
on Zorbax RX-C8, PRP-1 (polystyrenedivinylbenzene), Hypersil
ODS, and ZirChrom-polybutadiene columns. Isothermal separations
are performed at temperatures ranging from 60C to 160C. The
retention time and peak width of analytes are reduced with
increasing temperature. However, the column efficiency is either
improved or almost unchanged with the increasing temperature in
the low-temperature range (lower than the 100C to 120C range),
but it is decreased when temperature is further raised in the high-
temperature range (higher than the 100C to 120C range).
Therefore, a maximum in column efficiency is obtained at
temperatures within the 100C to 120C range in most cases.
Introduction
Reversed-phase liquid chromatography (RPLC) is a very
popular separation and analysis technique used today.
Unfortunately, organic solvents are required to achieve separa-
tion in RPLC. An enormous amount of these organic solvents is
consumed every day worldwide. These organic solvents are
expensive in terms of both purchasing and waste disposal. In
addition, they are also potentially harmful to the laboratory envi-
ronment and the operator. Therefore, searching for nontoxic sol-
vents as the mobile phase for RPLC is of great interest.
Ambient water is too polar to serve as an eluent for reversed-
phase separation. Fortunately, the polarity of water decreases
with increasing temperature. Therefore, the solubility of organic
compounds is dramatically increased in water at elevated tem-
peratures (14). For example, the solubility of some pesticides
and polycyclic aromatic hydrocarbons is increased several orders
of magnitude by raising the water temperature from ambient to
200C (13). Thus, liquid chromatographic (LC) separations can
be achieved by using high-temperature (subcritical) water
(511). With two additional components (an oven and a back-
pressure regulator or restrictor), a conventional high-perfor-
mance liquid chromatographic (HPLC)UV system can be easily
modified to a subcritical water separation system. The oven is
used to provide the temperature for subcritical water separation,
and the backpressure regulator prevents water from boiling
when working with temperatures higher than 100C. The UV
detector is placed outside the oven. Depending on the separation
temperature and the flow rate of water used, the temperature of
the water eluent in the UV flow cell varies, but it is lower than the
oven temperature. If a backpressure regulator or a short packed
LC column is used to provide the backpressure, they are nor-
mally connected to the outlet of the UV flow cell. Thus, the flow
cell is under pressure and may be damaged.
Most reports on subcritical water chromatography mainly
focus on testing the feasibility of using subcritical water as the
mobile phase for reversed-phase separation (511). Even though
the effect of water temperature on the retention is mentioned in
some of these reports (511), a quantitative study of the temper-
ature effect on peak width and column efficiency in subcritical
water separation has not yet been reported. It should be pointed
out that the effects of temperature on retention, viscosity, diffu-
sivity, and the number of plates have been well-investigated in
conventional HPLC (1217). However, the temperature range
was generally much narrower and normally went up to 80C. In
addition, organic solvents were involved in the mobile phases of
these studies (1217).
In this work, pure water at elevated temperatures and pres-
sures was used as the eluent to separate several polar analytes
and chlorophenols on four commercial columns, which
included the Zorbax RX-C8, polymeric PRP-1, Hypersil ODS, and
ZirChrom-PBD columns. Separations were performed at tem-
peratures ranging from 60C to 160C in an isothermal manner.
The peak width was monitored and the number of theoretical
plates was calculated to evaluate the temperature effect on
column efficiency.
Abstract
Temperature Effect on Peak Width and Column
Efficiency in Subcritical Water Chromatography
Yu Yang
1,
*, Lori J. Lamm
1
, Ping He
1
, and Toru Kondo
2
1
Department of Chemistry, East Carolina University, Greenville, NC 27858 and
2
Fuji Silysia Chemical Ltd., Kasugai-Shi, Aichi-Ken,
487-0013, Japan
Reproduction (photocopying) of editorial content of this journal is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, February 2002
* Author to whom correspondence should be addressed: email yangy@mail.ecu.edu.
Journal of Chromatographic Science, Vol. 40, February 2002
108
Experimental
Separation columns
A polystyrenedivinylbenzene column (PRP-1, 250- 4.1-mm
i.d.) was purchased from Hamilton Company (Reno, NV). A
Zorbax RX-C8 column (250- 4.6-mm i.d.) was obtained from
DuPont (Wilmington, DE). A Hypersil ODS column (100- 4.6-
mm i.d.) (Keystone, Bellefonte, PA) was used to separate a phenol
mixture. Because the recently developed zirconia-based columns
have shown excellent thermal stability and column efficiency
(1820), a ZirChrom-polybutadiene (PBD) column (100- 2.1-
mm i.d.) (ZirChrom Separation Inc., Anoka, MN) was also
employed in this study. The particle size was 3 m for the
ZirChrom-PBD column and 5 m for the other three columns.
Reagents
All analytes used in this study were obtained from Sigma (St.
Louis, MO). The stock solutions of the solutes were prepared in
methanol (HPLC grade) (Fisher Scientific, Fair Lawn, NJ). The
deionized water (18 M) was prepared in our laboratory using a
Sybron/Barnstead (Boston, MA) system. All mobile phases were
purged using helium gas prior to each use.
Subcritical water separation
A homemade subcritical water chromatographyUV system
was employed in this work. A Hewlett-Packard (Avondale, PA)
gradient pump Series 1050 was used to deliver the mobile phase.
The flow rate was 0.2 mL/min for the ZirChrom-PBD column and
1 mL/min for the other three columns. The outlet of the pump
was connected to a Valco injector fitted with a 2-L sample loop
(purchased from Keystone Scientific). The injector was located
just outside a Fisher Isotemp oven. A piece of stainless steel
tubing (100-cm 0.005-inch i.d.) (Keystone) was connected
between the injector and the separation column as a preheating
coil. Both the preheating coil and the separation column were
placed inside the oven. The preheating coil acted like a high-tem-
perature water reservoir to ensure that the water eluent reached
the desired temperature before entering the separation column.
Because water will be vaporized at 1 atm and temperatures at
100C or higher, backpressure must be applied to the outlet of the
column in order to keep water in the liquid state. There are sev-
eral reasons for avoiding steam in subcritical water chromatog-
raphyUV systems. The water mobile phase may stay in liquid
near the column inlet, thus causing steam to form inside the sep-
aration column near the outlet end if there is not enough back-
pressure applied. Thus, the mobile phase exists as two separate
phases (liquid water and steam) in the separation column. In
addition, the UV signal strongly fluctuates if steam exists in the
system. This means that the UV detector is not stable when steam
passes through the flow cell. In this study, a capillary restrictor
(7-cm 75-m) (Polymicro Technologies Inc., Phoenix, AZ) was
placed outside the oven and between the separation column and
the UV flow cell in order to ensure that the water inside the sepa-
ration column stayed in the liquid state at higher temperatures.
Connection unions (
1
/
16
inch to
1
/
16
inch) (Supelco, Bellefonte,
PA) were used to connect the restrictor. By
1
/
16
-inch stainless steel
tubings, the inlet of union 1 and the outlet of union 2 were con-
nected to the column and UV detector, respectively. The fused-
silica capillary restrictor was connected with both unions using
graphite ferrules (Alltech, Deerfield, IL). We evaluated the influ-
ence of the restrictor dimension on the retention time using
restrictors having 7 to 30 cm in length and 51 to 103 m in inner
diameter. However, there was no significant effect of the restrictor
dimension on the retention time. A restrictor with a length of
7 cm and a 75-m inner diameter was chosen for all of the exper-
iments reported in this work. An LDC variable wavelength
detector (spectro Monitor 3200, Riviera Beach, FL) was used in
this separation system. The UV detector was set at a wavelength of
254 nm for the entire work.
After purging the deionized water with helium, the water was
continuously pumped through the separation column at either
0.2 or 1 mL/min, depending on the columns used. Then, the
oven was turned on and set to a desired temperature. In order to
ensure that separations were carried out at the set temperature,
the first injection was not made until approximately 20 min after
the oven temperature was reached. This allowed the stationary
phase in the packed column and the mobile phase to equilibrate
to the desired temperature. It should be noted that the tempera-
ture of the stationary phase and the mobile phase inside the
column lagged behind the oven temperature by approximately 5
to 20 min, depending on the temperature employed. A Hewlett
Packard 3396 Series II integrator was used as the data-recording
device. The peak width monitored in this work was at half-
height, and the number of theoretical plates (N) was computed
using the following equation:
N = 2(t
R
H/A)
2
Eq. 1
where H and A are the peak height and area, respectively.
Results and Discussion
Zorbax RX-C8 column
The Zorbax RX-C8 column was first used to study the temper-
ature effect on the peak width and column efficiency. The test
solutes in this study included pyridine, benzamide, catechol, and
guaiacol. The temperature used for the separation of these ana-
lytes ranged from 60C to 100C because this column was
proven to be thermally stable at temperatures up to 100C for
several thousand column volumes (18). In case of coelution, the
analytes were injected individually. It is known that the retention
time is decreased with increasing temperature. The same trend
was observed in this study with all four solutes tested. For
example, pyridine was not eluted until approximately 44 min at
60C (as shown in Figure 1A) (t
0
= ~2.4 min). However, the same
analyte was eluted within approximately 16 min at 100C. It
should be noted that the decrease in retention with increasing
temperature was in an almost linear fashion (Figure 1A). Figure
1B demonstrates the temperature effect on the peak width for
the test analytes. Because the viscosity of water decreased dra-
matically when the temperature was raised (as shown in Table I)
(21,22), the diffusivity was greatly enhanced. Thus, narrower
Journal of Chromatographic Science, Vol. 40, February 2002
109
peaks were obtained at elevated temperatures. Similar to the
retention time, the reduction in the peak width with increasing
temperature was not dramatic.
The influence of temperature on column efficiency is demon-
strated in Figure 1C. Based on the type of curves in Figure 1C,
the solutes can be divided into two groups. The first group
includes benzamide and pyridine. The peak efficiency of these
two solutes was significantly improved with increasing tempera-
ture. The number of theoretical plates obtained at 100C was
5384% higher than that at 60C for benzamide and pyridine.
This uptrend temperature effect on efficiency was achieved
because the reduction in retention was slower than the reduc-
tion in peak width when the temperature was raised. This phe-
nomenon can be seen from Figures 1A and 1B. When the
temperature was increased from 60C to 100C, the reduction in
retention time and peak width for benzamide was 49% and 62%,
respectively. However, the plate number of catechol and guaiacol
was almost unchanged when the temperature was raised from
60C to 100C. This means that both the retention time and peak
width of catechol and guaiacol were decreased with similar rates
when the temperature was increased.
PRP-1 column
Because the polymeric PRP-1 column is thermally stable at
temperatures up to 160C based on our previous work (18), the
temperature range was expanded to 160C to evaluate the tem-
perature effect on the column efficiency with a greater tempera-
Table I. Temperature Effect on the Viscocity of Water*
Viscocity (cP)
Temperature (C) At 50 bar At 100 bar
25 0.8898 0.8889
50 0.5479 0.5487
100 0.2836 0.2849
150 0.1832 0.1844
200 0.1345 0.1357
250 0.1061 0.1075
* Obtained from references 21 and 22.
Figure 1. Temperature effect on (A) the retention time, (B) peak width, and
(C) number of theoretical plates for separation on the Zorbax RX column.
Figure 2. Temperature effect on (A) the retention time, (B) peak width, and
(C) number of theoretical plates for separation on the PRP-1 column.
Journal of Chromatographic Science, Vol. 40, February 2002
110
ture range. Therefore, the temperature effect on the peak effi-
ciency of the same or similar solutes (resorcinol, catechol, ben-
zamide, and pyridine) was also investigated by using the PRP-1
column. The separation temperatures ranged from 60C to
160C at an interval of 20C. The analytes were injected individ-
ually in case of coelution at higher temperatures. The retention
time and peak width were also significantly decreased with
increasing temperature as shown in Figure 2 (t
0
= ~2.0 min). The
reduction in retention time and peak width was up to 80% for
these analytes by raising the separation temperature from 60C
to 160C. This means that the analysis was 79 times faster at
160C than that at 60C.
The effect of temperature on peak efficiency is depicted in
Figure 2C. Similar to the separation on the Zorbax column,
uptrend curves of temperature versus column efficiency were
obtained in the temperature range of 60C to 120C. However, the
number of theoretical plates was decreased when the temperature
was further raised to 160C. Thus, the peak efficiency reached a
maximum at temperatures in the 100C to 120C range. This
maximum of peak efficiency shows that the reduction in reten-
tion time was smaller than the reduction in peak width at the low-
temperature range, and the decrease in retention time was
greater than the decrease in peak width at the high-temperature
range. This phenomenon can be clearly seen from Figures 2A and
2B. For example, the retention time of resorcinol was reduced
35% while its peak width experienced a 48% reduction when tem-
perature was raised from 60C to 80C (low-temperature range).
However, the opposite phenomenon was observed at the higher
temperature range. When the temperature was increased from
140C to 160C, the decrease in retention time and peak width for
catechol was 20% and 8%, respectively.
Hypersil ODS column
In order to further explore the effect of temperature on
column efficiency, a mixture of phenol, 2-chlorophenol, and 2,3-
dichlorophenol was separated using a Hypersil ODS column.
Even though the thermal stability of this column is poorer than
the PRP-1 column, we still used a temperature range of 60C to
140C. Because the column was exposed to high temperatures
only for several hours (the most) in this study, the thermal sta-
bility did not get significantly worse within this short period of
time based on our previous study (18). Again, both the retention
time (t
0
= ~1.0 min) and peak width were decreased with
increasing temperature as shown in Figures 3A and 3B. The
number of theoretical plates (equivalent to a 25-cm column) was
slightly increased for chlorophenols but stayed almost
unchanged for phenol when the temperature was increased from
60C to 100C. Further raising the temperature from 100C to
140C caused a significant decrease in column efficiency (as
demonstrated in Figure 3C). This was in agreement with the
results obtained by using the PRP-1 column even though dif-
ferent analytes were used.
ZirChrom-PBD column
Based on references 19 and 20, the ZirChrom-PBD column
was stable at temperatures up to the range of 150C to 200C.
Therefore, the phenol mixture was also separated on the
ZirChrom-PBD column at temperatures ranging from 60C to
140C. Because the inner diameter of the ZirChrom-PBD
column was 2.1 mm, a flow rate of 0.2 mL/min was used for this
column. Similar to separations on the Hypersil ODS column, the
column efficiency was either increased or unchanged when the
temperature was raised from 60C to 100C (as shown in Figure
4C), but the plate number (equivalent to a 25-cm column) was
decreased when the temperature was further increased from
100C to 140C. However, the decrease in efficiency was less sig-
nificant for this zirconia-based column compared with that for
the Hypersil column. As can be seen from Figure 4C, increasing
the temperature from 60C to 140C resulted in either no
decrease or a very little decrease (approximately 15%) in effi-
ciency with the ZirChrom-PBD column but a typical 40%
decrease with the Hypersil ODS column. This means that the
Figure 3. Temperature effect on (A) the retention time, (B) peak width, and
(C) number of theoretical plates (equivalent to a 25-cm column) for separation
on the Hypersil ODS column.
Journal of Chromatographic Science, Vol. 40, February 2002
111
zirconia-based column is more suitable for separations at higher
temperatures. Another benefit associated with the ZirChrom-
PBD column is that the analysis time required by this column
was much shorter than that required by the Hypersil column. As
shown in Figures 3 and 4, separation on the ZirChrom-PBD
column was approximately four times faster than that on the
Hypersil column, but the column efficiency of the ZirChrom-
PBD under the fast analysis conditions was still competitive
compared with that obtained by the Hypersil column.
Mechanism for the temperature effect on column efficiency in
subcritical water chromatography
To the best of our knowledge, this is the first report that quan-
titatively describes the effect of water temperature on column
efficiency over a wide temperature range in subcritical water
chromatography. In many cases, a maximum in column effi-
ciency in subcritical water chromatography was obtained when
the water temperature was varied in this work. We believe that
this maximal efficiency was caused by two factors that are asso-
ciated with water temperature. The first one is the mass transfer
that improves the efficiency, and the second is the longitudinal
diffusion that worsens the column efficiency.
It is well-known that the diffusivity or diffusion coefficient of
the mobile phase (D
m
) is directly proportional to the absolute
temperature and inversely proportional to the viscosity of the
mobile phase. Because the viscosity of water is decreased with
increasing temperature as demonstrated in reference 22 (Table I),
the diffusivity (mass transfer) of water is dramatically increased
and the mass transfer resistance (the C
m
term in the van Deemter
equation) is greatly decreased at elevated temperatures. Thus,
narrower bands and higher column efficiency should be expected
with increasing temperature. This is why the number of theoret-
ical plates was generally increased when the temperature was
raised from 60C to the 100C to 120C range (as illustrated in
Figures 14). Therefore, we believe that mass transfer may domi-
nate the subcritical water separation process at the lower temper-
ature range (lower than the 100C to 120C range).
By increasing the temperature from 100C to 120C, the diffu-
sivity is further increased and even better mass transfer results.
However, the better mass transfer also causes a greater axial
molecular diffusion (longitudinal diffusion, the B term in the van
Deemter equation), which makes the column efficiency become
poorer. Therefore, the higher the temperature, the greater the
longitudinal diffusion (B is directly proportional to D
m
in the van
Deemter equation) and the lower the column efficiency. This is
the reason why the number of plates was decreased when the
temperature was raised from the 100C to 120C range to the
140C to 160C range (Figures 24). Therefore, longitudinal dif-
fusion may be the dominating factor that controls the subcritical
water separation at the higher temperature range. Carr et al.
(20) reported that the column efficiency was decreased for sepa-
rations using organic solventwater mixtures at temperatures of
150C and 200C, although the authors indicated that this might
be caused by the interaction of molecules with the column walls
at higher temperatures (20).
Because increasing the separation temperature causes lower
mass transfer resistance (the C term in the van Deemter equa-
tion decreases) but also greater longitudinal diffusion (the B
term in the van Deemter equation increases), a maximal column
efficiency may be observed. However, if the decrease in the C
term and increase in the B term are similar in the lower temper-
ature range, then they compensate each other. Thus, the effi-
ciency will stay unchanged when the temperature is increased
from low temperature to the 100C to 120C range. This is evi-
denced in Figures 1, 3, and 4. However, at a higher temperature
range the increase in the B term always exceeds the decrease in
the C term. Therefore, the column efficiency was always
decreased when the temperature was further raised. This may
explain why the number of plates was always decreasing with all
of the columns and solutes tested when the temperature was
increased from the 100C to 120C range to the 140C to 160C
range (as shown in Figures 24).
Figure 4. Temperature effect on (A) the retention time, (B) peak width, and
(C) number of theoretical plates (equivalent to a 25-cm column) for separation
on the ZirChrom-PBD column.
Journal of Chromatographic Science, Vol. 40, February 2002
112
Conclusion
The elution of several polar analytes has been achieved by
using pure water and four different types of commercially avail-
able reversed-phase columns at elevated temperatures under
moderate pressure to keep the water in the liquid state. A fused-
silica capillary restrictor was connected between the separation
column and the UV flow cell to provide the backpressure needed
to avoid water from boiling at higher temperatures. For all of the
analytes studied and columns used, the peak width was
decreased with increasing water temperature. For example, the
peak width of benzamide obtained on the PRP-1 column was
reduced by as much as 93% by raising the temperature from
60C to 160C. However, the column efficiency was either
improved or remained unchanged initially but then decreased
with increasing temperature. Thus, a maximum in efficiency was
observed at temperatures in the 100C to 120C range in most
cases.
Acknowledgments
This research was supported by an award from Research
Corporation (CC4607). The authors would also like to thank
ZirChrom Separations Inc. for providing the ZirChrom-PBD
column. Toru Kondo acknowledges Fuji Silysia Chemical Ltd.
and the Department of Chemistry at East Carolina University for
providing the opportunity to conduct this research.
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113
A technique is presented for the specific and sensitive determination
of ethambutol concentrations in plasma, bronchoalveolar lavage
(BAL), and alveolar cells (AC) using a high-pressure liquid
chromatographic (HPLC)tandem mass spectrometric (MSMS)
method. The preparation of samples requires a deproteinization step
with acetonitrile. The retention times for ethambutol, neostigmine
bromide, and propranolol are 2.0, 1.4, and 1.1 min, respectively, with
a total run time of 2.8 min. The detection limits for ethambutol are
0.05 g/mL for plasma and 0.005 g/mL for the BAL supernatants and
AC suspensions. The assay has excellent performance characteristics
and has been used to support a study of the intrapulmonary
pharmacokinetics of ethambutol in human subjects.
Introduction
Ethambutol has a primary role in the treatment of tuberculosis
and is recommended with isoniazid, rifampin, and pyrazinamide
as initial therapy (1). Ethambutol is rapidly absorbed and has a
bioavailability of 7% after oral administration (2,3). Under fasting
conditions, the maximum concentration (mean standard devi-
ation, SD) of the drug in serum is 4.5 1.0 g/mL and the time
to maximum concentration is 2.5 0.9 h (2). The minimum
inhibitory concentration of ethambutol for M. tuberculosis
ranges from 0.5 to 2 g/mL in broth media (4). A microbiological
assay (detection limit of 0.4 g/mL) using M. smegmatis as the
test organism for determining ethambutol in serum has been
reported (5). A gas chromatographic (GC)mass spectrometric
(MS) method has been used for the determination of ethambutol
in tablets (6). A GCliquid chromatographic (LC) assay with
improved performance characteristics (detection limit of 0.1
g/mL in plasma) has been used to study the pharmacokinetics of
ethambutol in humans (3,79) and rabbits (10), and a high-pres-
sure liquid chromatographic (HPLC) method for the determina-
tion of ethambutol in plasma (detection limit of 10 ng/mL) and
urine (detection limit of 10 g/mL) has been described (11).
We report the use of a sensitive HPLCtandem mass spectro-
metric (MSMS) technique to measure ethambutol in human
plasma, bronchoalveolar fluid (BAL) (detection limit of 0.05
g/mL), alveolar cells (AC) (detection limit of 0.005 g/mL), and
plasma (detection limit of 0.05 g/mL). Compared with other
methods, the technique has the advantages of increased sensi-
tivity and a capability to analyze small sample volumes. The speci-
ficity of HPLCMSMS detection greatly minimizes the risk of
interference from other substances. This is especially important
when analyzing specimens from patients such as those with AIDS
who are taking numerous concomitant medications. It currently
is being used to support a phase-one study of the intrapulmonary
pharmacokinetics of ethionamide in normal subjects and sub-
jects with AIDS.
Experimental
Chemicals
All solvents and chemicals were HPLC grade except ammo-
nium acetate, which was certified. A 1.0-mg/mL solution of
ethambutol HCl (Lederle Laboratories, Wayne, NJ) was made in
50% methanol and stored refrigerated. This solution was further
diluted to produce working stock solutions of 0.1, 1.0, and 10
g/mL of ethambutol. Stock solutions of 1.0 mg/mL neostigmine
bromide (Aldrich Chemical Co., Milwaukee, WI) and propranolol
(USP Reference, Rockville, MD) were prepared in 50% methanol.
Neostigmine bromide and propranolol were then diluted to a con-
centration of 0.050 g/mL in acetonitrile and used as the internal
standard for plasma, and propranolol was diluted to 0.300 g/mL
and used as the internal standard for BAL and AC.
Abstract
A High-Pressure Liquid ChromatographicTandem
Mass Spectrometric Method for the Determination of
Ethambutol in Human Plasma, Bronchoalveolar Lavage
Fluid, and Alveolar Cells
John E. Conte, Jr.
1,2,3,
*, Emil Lin
4
, Yeping Zhao
4
, and Elisabeth Zurlinden
1
1
Department of Epidemiology and Biostatistics, Infectious Diseases Research Laboratory,
2
Department of Medicine,
3
Department of
Microbiology and Immunology, and
4
Department of Biopharmaceutical Sciences, University of California, San Francisco, 350 Parnassus
Avenue, Suite 507, San Francisco, CA 94117
Reproduction (photocopying) of editorial content of this journal is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, February 2002
* Author to whom correspondence should be addressed.
Journal of Chromatographic Science, Vol. 40, February 2002
114
Instrumental
Chromatography
The mobile phase (containing 80% acetonitrile, 4mM ammo-
nium acetate, and 0.10% trifluoroacetic acid) was run through a
hypersil silica column (50- 4.6-mm i.d., 5-m particle size) at a
flow rate of 0.8 mL/min using a Shimadzu (Columbia, MD) LC-10
AD pump. Extracts from samples were injected onto the system
with a Waters (Milford, MA) Intelligent Sample Processor 717
Plus. The retention times for ethambutol, neostigmine, and pro-
pranolol were 2.0, 1.4, and 1.1 min, respectively, with a total run
time of 2.8 min.
MS
We used two different MS systems during the development and
validation of this assay to explore different types of MS equipment.
Neostigmine bromide was the internal standard used for the
plasma and BAL that were assayed on the PE Sciex API III
(PerkinElmer, Foster City, CA), whereas propranolol was used as
the internal standard for the assays in plasma, BAL, and ACs per-
formed on the Micromass (Manchester, U.K.) Quattro LC. Peak
detection and area determinations for some plasma and BAL were
Figure 1. Daughter ion spectra and chemical structures of ethambutol using the
Sciex APCI mode.
Figure 2. Daughter ion spectra and chemical structures of neostigmine (the
internal standard) using the Sciex APCI mode.
Figure 3. Daughter ion spectra and chemical structures of ethambutol using the
Micromass Quattro LC electrospray mode.
Figure 4. Daughter ion spectra and chemical structures of propranolol (the
internal standard) using the Micromass Quattro LC electrospray mode.
Journal of Chromatographic Science, Vol. 40, February 2002
115
carried out with a PE Sciex API III.
The MS used the following settings and conditions. The mul-
tiple reaction monitor scanning mode was set at m/z 205116 for
ethambutol and m/z 20971 for neostigmine (Figures 1 and 2).
Atmospheric pressure chemical ionization (APCI)positive ion-
ization was used. The sample inlet used a heated nebulizer at
450C. The discharge current was +3 A. The gas curtain flow was
1.2 L/min (N
2
= 99.999%). The nebulizer pressure was 551.4 kPa.
The collision gas consisted of a 9.99% nitrogen90.01% argon
mixture (set at 250 10
12
molecules/cm
2
). Peak detection for the
ACs and some plasma and BAL specimens was carried out on a
Micromass Quattro LC. For these specimens the reaction channel
was m/z 205.35116.10 for ethambutol and m/z 260.18115.95
for propranolol (Figures 3 and 4). Electrospraypositive ioniza-
tion with a flow rate of 0.2 mL (5-to-1 split ratio of 1.0 mL/min)
to the Micromass system was used. The sample inlet used a heated
nebulizer. The sample cone was set to 25 V for ethambutol and
35 V for propranolol. The energy collision was set to 15.0 eV for
both ethambutol and propranolol. A Macintosh Quadra 800 com-
puter (Apple Computers, Cupertino, CA) was used for peak inte-
gration and analysis.
Sample preparation
Standard curves
Plasma standard curves were prepared by adding appropriate
volumes of ethambutol working stock solutions into 0.2 mL of
blank plasma to yield the concentrations of 0.05, 0.10, 0.20, 0.40,
0.80, 1.2, 1.6, and 2.4 g/mL of ethambutol. The standards for
BAL supernatants were spiked to yield concentrations of 0.005,
0.010, 0.020, 0.040, 0.080, 0.160, 0.320, and 0.640 g/mL of
ethambutol. The AC suspension standards were spiked to yield
concentrations of 0.005, 0.010, 0.020, 0.040, 0.100, 0.400, 0.800,
1.600, and 2.000 g/mL ethambutol. Standard curves were con-
structed by plotting a 1/y weighted least-squares linear regression
of ethambutol to the internal standard peak-area ratios versus the
spiked concentration of ethambutol.
Preparation of plasma standards and samples
In order to ensure consistency of recovery, 200 L of acetoni-
trile containing 0.050 g/mL neostigmine or propranolol as the
internal standard was added to 0.2 mL plasma standards and sam-
ples. After vortexing, an additional 0.2 mL of the internal standard
solution was added. After vortexing and then centrifuging for
5 min at 1800 g, the solvent phase was transferred to a 400-L
microfuge tube, and 2.0 L were injected onto the HPLC system.
Preparation of BAL supernatants and AC pellet standards
and samples
A cell count and differential was performed on the BAL lavage
fluid, then a 30-mL aliquot was centrifuged at 400 g for 5 min
and the supernatant immediately separated from the cells. BAL
supernatant standards and samples were prepared by adding 0.5
mL of the internal standard solution (0.015 g/mL neostigmine
or 0.150 g/mL propranolol) to 0.25 mL of the sample, vortexing,
and then centrifuging for 5 min at 1800 g. The solvent phase
was transferred to a 400-L microfuge tube, and 2.0 L were
injected onto the HPLC system.
ACs were resuspended volumetrically in deionized water and
sonicated for 2 min on a Fisher 550 dismembrator (Fisher
Scientific, Santa Clara, CA) to lyse the cells. A 250-L volume of
the internal standard (0.300 g/mL propranolol) was added to 250
L of an AC cell suspension and vortexed. A 250-L volume of ace-
tonitrile was added and mixed by vortexing. Following centrifu-
gation for 5 min at 1800 g, 2 L of the solvent phase was
injected onto the HPLC system.
Preparation of controls for method validation
Two sets of stock solutions were prepared; one was used for
spiking standards and the other for spiking controls. Measured
amounts of plasma were spiked at 0.15, 0.4, 0.8, and 1.4 g/mL;
aliquoted; and frozen at 70C for stability studies. Aliquots were
Figure 5. Chromatograms of blank plasma: (A) the internal standard and
(B) ethambutol.
Figure 6. Chromatograms of a study subjects plasma obtained 4 h after the fifth
dose of 15 mg/kg ethambutol administered once a day: (A) the internal standard
and (B) ethambutol. The ethambutol concentration was 0.734 g/mL.
A
B
A
B
Journal of Chromatographic Science, Vol. 40, February 2002
116
analyzed in duplicate weekly over a period of six weeks. In order
to assess interday reproducibility, standard curves with controls
spiked at concentrations of 0.1, 0.3, 1.2, and 2.4 g/mL were ana-
lyzed on five different days. Intraday reproducibility was assessed
by analyzing six preparations of each of the four concentrations
on the same day. The validation for BAL supernatants was carried
out in the same time frames as for plasma, with controls spiked at
concentrations of 0.015, 0.04, 0.16, and 0.24 g/mL. The valida-
tion for ACs was performed at concentrations of 0.010, 0.40, and
1.60 g/mL.
Statistics
The statistical analysis was performed using the PROPHET
Computer Resource (12). Linearity (r
2
), precision (coefficient of
variation, CV), recovery (relation of test result to the true concen-
tration) (13), and percentage accuracy (14) were calculated. The
detection limit was defined as the lowest point of the standard
curve. Drug concentrations in epithelial lining fluid (ELF) were
calculated using the urea diffusion method, and AC concentra-
tions were calculated using cell counts in alveolar fluid as we have
previously reported (1517).
Results and Discussion
Linearity, assay precision, recovery, and accuracy assessments
HPLCMSMS chromatograms of ethambutol and the internal
standard in plasma, BAL supernatant, and AC suspension are
shown in Figures 510. The detection limits for ethambutol were
0.05 g/mL for plasma and 0.005 g/mL for the BAL supernatants
Figure 7. Chromatograms of blank BAL supernatant: (A) the internal standard
and (B) ethambutol.
Figure 10. Chromatograms of a study subjects AC suspension obtained 4 h
after the fifth dose of 15 mg/kg ethambutol administered once a day: (A) the
internal standard and (B) ethambutol. The ethambutol concentration was 0.316
g/mL.
Figure 9. Chromatograms of blank AC suspension: (A) the internal standard and
(B) ethambutol.
Figure 8. Chromatograms of a study subjects BAL supernatant obtained 4 h
after the fifth dose of 15 mg/kg ethambutol administered once a day: (A) the
internal standard and (B) ethambutol. The ethambutol concentration was 0.053
g/mL.
A
B
A
B
A
B
A
B
Journal of Chromatographic Science, Vol. 40, February 2002
117
and AC suspensions. The detection limit referred to the lowest
point of the standard curve and was at least five times the noise
level. The mean SD of the r
2
from 24 standard curves (8 in
plasma, 8 in BAL, and 8 in ACs) was 0.9941 0.0060. Results for
the assay precision, recovery, and accuracy assessments in the
plasma, BAL, and AC suspensions are summarized in Tables IIII.
CV
The mean ( SD) CVs and the ranges of the assay for intraday
and interday determinations together for plasma, BAL super-
natants, and ACs were 7.81% 2.02% (ranging from 3.9% to
10.14%), 6.46% 3.69% (ranging from 1.42% to 11.42%), and
12.67% 4.59% (ranging from 6.0% to 20.0%), respectively
(Tables IIII).
The mean ( SD) recoveries and the ranges of the assays for
intraday and interday determinations together in plasma, BAL
supernatants, and ACs were 105.91% 7.73% (ranging from
93.3% to 119.0%), 95.94% 10.43% (ranging from 80.0% to
106.88%), and 105.48% 3.60% (ranging from 100.00% to
110.00%), respectively (Tables IIII). The accuracy ranges for all
of the determinations in plasma, BAL supernatants, and ACs were
6.67% to 19.0%, 20.0% to 6.88%, and 0.0% to 10.0%, respec-
tively (Tables IIII).
Stability
The results of repeated determinations of ethambutol in spiked
plasma, BAL supernatants, and ACs stored at 70C revealed no
significant degradation of the drug. These determinations were
performed over a period of 4 mo for plasma, 7 weeks for BAL
Table II. Assay Precision, Recovery, and Accuracy for
Ethambutol Determination in BAL Supernatant
Measured
Spiked concentration
concentration (mean SD) Recovery* Accuracy
(n = 6)
0.240 0.248 0.004 1.4 103.33 3.33
0.160 0.171 0.004 2.2 106.88 6.88
0.040 0.040 0.002 6.2 100.00 0.00
0.015 0.012 0.001 4.4 80.00 20.0
Interday
(n = 12)
0.240 0.238 0.023 9.9 99.17 0.83
0.160 0.165 0.011 6.4 103.13 3.13
0.040 0.038 0.004 11.4 95.00 5.0
0.015 0.012 0.001 9.8 80.00 20.0
* Measured/spiked 100%.
Plasma spiked at four concentrations and analyzed in duplicate on six different days.
Table III. Assay Precision, Recovery, and Accuracy for
Ethambutol Determination in Alveolar Cells
Measured
Spiked concentration
concentration (mean SD) Recovery* Accuracy
(n = 6)
1.600 1.643 0.099 6.0 102.69 2.69
0.400 0.423 0.053 12.6 105.75 5.75
0.010 0.010 0.001 14.5 100.00 0.00
Interday
(n = 10)
1.600 1.707 0.207 12.1 106.69 6.69
0.400 0.431 0.047 10.8 107.75 7.75
0.010 0.011 0.002 20.0 110.00 10.00
* Measured/spiked 100%.
Plasma spiked at three concentrations and analyzed in duplicate on five different days.
Table I. Assay Precision, Recovery, and Accuracy for
Ethambutol Determination in Plasma
Measured
Spiked concentration
concentration (mean SD) Recovery* Accuracy
(n = 6)
2.4 2.24 0.227 10.1 93.33 6.67
1.2 1.30 0.111 8.6 108.33 8.33
0.3 0.32 0.012 6.1 106.67 6.67
0.1 0.12 0.011 9.2 119.00 19.00
Interday
(n = 10)
2.4 2.35 0.168 7.2 97.92 2.08
1.2 1.26 0.114 9.1 105.00 5.00
0.3 0.33 0.013 3.9 110.00 10.00
0.1 0.107 0.009 8.3 107.00 7.00
* Measured/spiked 100%.
Plasma spiked at four concentrations and analyzed in duplicate on five different days.
Table IV. Ethambutol Concentrations* in Plasma, ELF, and
AC in Five Adult Volunteer Subjects
Subject Subject Subject Subject Subject
Sample #1 #2 #3 #4 #5
Plasma
(2 h after fifth dose
The amount of ELF collected in the BAL fluid was calculated from the urea
concentration in BAL and serum, as previously reported (1517).