United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park, NC 27711
Research and Development
EPA-600/S1-84-026 Jan. 1985
Project Summary
Sorption Properties of Model
Compounds on Cis Adsorbents
Harold F. Walton
The bonded silica adsorbent, Bonda-
pak-Cis® (Waters Associates) has been
evaluated for removing organic matter
from secondary sewage effluents and
from solutions of pure organic com-
pounds. The adsorbent is hydrophobic
and will not function effectively in
water unless it is first wet with methanol
or another organic solvent; therefore,
its behavior in water may be erratic. In
water-methanol mixtures on the other
hand it behaves reproducibly and gives
linear absorption isotherms. Advantages
of Bondapak-CiB®, compared with
microparticulate adsorbents, include
greater chemical robustness and toler-
ance to regeneration, lower flow resist-
ance and lower cost. It has been used to
remove organic compounds from water
and as a support in low-resolution
chromatographic analysis.
Part of this effort was to develop
liquid-chromatographic methods of
analysis for organic constituents of
wastewater. For this purpose, micro-
particulate adsorbents were used along
with a Sep-Pak® precolumn for rapid
trace enrichment. Identification of the
many chromatographic peaks was
difficult, but the chromatograms were
reproducible and permit different ef-
fluents to be compared and tertiary
treatment processes to be evaluated.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Research Triangle Park.
NC, to announce key findings of the re-
search project that is fully documented
in a separate report of the same title (see
Project Report ordering information at
back).
Introduction
The research described herein is a
continuation of that reported in March
1979: EPA Report No. 600/1-79-014;
"Chemistry and Cytotoxicity of Renovated
Wastewater." That report described a
method of analysis of filtered secondary
sewage-effluents using selective adsorp-
tion and desorption of the less polar
organic constituents and yielded a
number of fractions of successively
decreasing polarity. These fractions were
tested for toxicity' by a micro-scale
cellular technique; some tests of muta-
genic action were made, and attempts
were made to resolve the fractions into
individual, identifiable chemical com-
pounds.
The primary purpose of the continued
investigation, described in the final
report, was to study in detail the
adsorptive properties of Bondapak-Cis®.
A secondary purpose was to determine
how this material, and others like it, could
be applied to the chromatographic
analysis of the organic compounds in
treated wastewater. The Bondapak-da®
adsorbent was chosen because, when
packed into a column, it retained an ap-
preciable proportion of the organic
material, about one-half of the total or-
ganic carbon, and released nearly all of it
when water-methanol mixtures were
passed through the column.
Bondapak-Cia® is a bonded-phase
adsorbent designed for large-scale or
preparative-scale liquid chromatography.
It consists of irregular particles of
superficially porous silica, size range 37-
75 micrometers, to whose surface is
chemically bonded a hydrocarbon layer,
equivalent to a coating of paraffin one-
molecule thick. The molecular fragments
bound to silica are octadecyl groups,
Ci8H37. For high-performance liquid
chromatography (HPLC) very small and
uniform particles, diameter 10 micro-
meters or less, are used, and they are
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"totally porous," that is, they are
penetrated by internal channels and have
a large effective surface area. Ideally,
every silicon atom on this surface carries
a carbon chain, if not CisHs?, then CH3
groups used for "capping." The idea is to
prevent free Si-OH groups that would
otherwise exist at the surface of silica. It
is impossible to avoid Si-OH groups
entirely, however, and a part of the
surface of Bondapak-d8® may be con-
sidered to consist of Si-OH groups. They
change the adsorptive properties, making
the particles more prone to adsorb polar
compounds, and even ionic compounds.
Chemically, the coarse Bondapak-Ci8®
closely resembles fine-particle bonded-
phase adsorbents such as Micro Bonda-
pak-Cia® (Waters Associates), Partisil
ODS® (Whatman), and Zorbax ODS®
(Dupont)*.
Experimental Procedures
Model Solutes
Breakthrough and
Elution Studies
Two kinds of tests were made: 1) break-
through, in which solutions of the organic
compounds were pumped through the
column until the compounds "broke
through" the column and emerged at the
same concentration level as they went in,
and 2) elution, in which small portions
(0.1 ml or less) of solution were injected
into a flowing stream of solvent, and the
compounds were detected as a peak in
ultraviolet absorbance when they emerged.
"Elution" is the normal mode used in
chromatographic analysis, but "break-
through" and its reverse, "stripping,"
provides a better evaluation of the be-
havior of the adsorbent in retaining trace
organic compounds from water. Further,
"breakthrough" permits study of the ef-
fect of concentration and column loading.
Benzophenone was tested utilizing a
Bondapak-CiŁ(® column (0.45 cm ID x 10-
15 cm long). Methanol-water mixtures
were pumped through them under
pressure, using a Waters pump, model
6000-A or 45; an ultraviolet absorbance
detector operated at 254 nm was con-
nected to the column exit. Methanol-
water mixtures were used, rather than
pure water, because benzophenone is
almost insoluble in water; moreover,
retention times in pure water are ex-
tremely large. Curves for breakthrough,
stripping (desorption) and elution of
benzophenone in 50% methanol are
shown in Figure 1.
Caffeine was also chosen for detailed
study because, as a common contami-
nant in wastewater, it is sufficiently
hydrophobic to be adsorbed by Bondapak-
Cis®, yet is freely soluble in water.
Adsorption-desorption curves (Figure 2)
were obtained for caffeine at 10 ppm, in
simulated city water (distilled water
containing anhydrous CaSO.», 120 ppm;
NaHCO3, 70 ppm; and CaCI2 • 2H;>0, 47
ppm); on a Bondapak column (0.45 cm ID
x 5.5 cm long) that had been previously
conditioned with methanol.
Other compounds selected for evalua-
tion were dibutylphthalate, dioctyl-
phthalate, 2,4'-diachlorobiphenyl, an-
thraquinone, pyrene, atrazine, 4,4'-
dichlorobiphenyl, 2,4-dichlorophenol,
furfural, and pentachlorophenol. The
majority of these compounds were
selected from a list of Consensus
Voluntary Reference Compounds (Keith
et a/., Environ. Sci. Tech., 13, 1469,
1979). These substances were passed
separately through a column of Bondapak-
Cis® (0.45 cm ID x 10.5 cm long) and
elution volumes, (Vei) measured. Most of
these substances are sparingly soluble in
water and are strongly retained; retention
volumes were therefore measured in
water-methanol mixtures. Each mea-
B s
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Abs.
100 mL
Figure 2. Adsorption and desorption of caffeine on Bondapak-C^ from water. These curves
are copied from strip-chart records. Caffeine concentration was 10 mg/L; flow rates,
curve (a). 2.5 mL/min, 0.275 cm/sec; (b), 5.0 mL/min, 0.55 cm/sec; fcj, 7.5
mL/min, 0.825 cm/sec. Column dimensions, 0.45 cm x 5.5 cm. Absorbance
measured at 280 nm, 1.0 absorbance units full scale. Note the small dependence on
flow rate and the unsymmetrical shape, a nonlinear adsorbtion isotherm is
indicated.
tubing with appropriate end fittings.
Columns were packed dry with Bondapak-
Cis®, using a vibrator. Dry packing is
recommended by the manufacturer. Dry
packing is easier than slurry packing
(which was also used) and gives equal
performance. Pure methanol was pumped
through the packed columns to displace
air and render the packing hydrophilic.
Columns of different lengths and diame-
ters were used; the widest were 1.0 cm
ID; most were 0.45 cm ID. The wall
thickness was 1 mm; end fittings were of
the standard "zero dead volume" type
with stainless steel frits. Back pressures
were of the order of 100-500 psi and
increased with use.
Small-Scale Trace Enrichment
These experiments were conducted
using Sep-Pak® (small disposable cart-
ridge packed with a micro particulate
bonded adsorbent — Waters Associates)
for preconcentration. The procedure was
as follows:
a) Sample preparation. Filter a quantity
(50-200 ml) of secondary sewage effluent
through Whatman glass-fiber filter
paper, grade GF/F, adjusting the pH with
nitric acid where desired. Next, condition
the Cie Sep-Pak® by passing 2 ml
methylene chloride followed by 5 ml of
methanol. Attach the Sep-Pak® to a Luer
tip syringe and drive the filtered waste-
water through the Sep-Pak® at 5 ml/min
using a Sage pumping assembly or
equivalent. Now displace as much water
as possible by passing 50 ml of air
downwards thru the column. Evaporate
the methylene chloride just to dryness
using a stream of nitrogen. Dissolve the
residue in 0.1-0.15 ml methanol and add
1 ml of water.
b) Sample introduction-liquid chromat-
ograph. A liquid chromatography as-
sembly consisting of two Water Model
6000A pumps (Waters Associates), a
Model 660 solvent programmer (Waters
Associates), a U6K injector (Waters
Associates), a Valco six-port valve, a
Whatman PXS-ODS analytical column, a
Brownlee precolumn, a Schoeffel vari-
able-wavelength ultraviolet detector and
a Schoeffel fluorescence detector with a
Linear Instruments two-pen recorder
was used. The valving system allows one
to inject a fairly large sample, up to 2 ml,
which is the volume of the storage loop of
the U6K injector, and collect the dissolved
compounds on the Brownlee precolumn;
then, by passing a higher concentration
of methanol and reversing the solvent
flow, the various dissolved compounds
are focused so that they enter the
analytical column together in a small
volume.
Results and Discussion
Adsorptive Properties of
Bondapak-C^a
The Adsorbent
Regarding chemical selectivity, the
partition of pure organic compounds
between the adsorbent and a solvent
(water or methanol-water mixture) and
the order in which a series of compounds
is adsorbed and released are the same for
the 37-75 micron Bondapak-de® as the
fine particle (< 10 micron) Cis bonded
phases used in HPLC. The main advant-
ages of Bondapak-Cia® for wastewater
analysis and the preparation of fractions
for toxicity testing are that it is more
robust and less prone to fouling by humic
substances, and back pressures are
much less than those observed with 10-
micron particles, and the material is
considerably less expensive.
When using Bondapak-Cis® to adsorb
organic compounds from water, it must
be recognized that it is hydrophobic and
not wetted by water. It must first be
thoroughly wetted with at least 50%
methanol or another water-miscible
solvent; then, when the greater part of
this solvent has been washed out, the
absorbent can be used to take up organic
compounds from water. Its behavior
towards purely aqueous solutions depends
strongly on the small amounts of methanol
sticking to it, and inevitable the behavior
is unreproducible, making it impractical
to try to characterize it by exact physical
tests. In mixed water-methanol solvents,
however, its behavior is reproducible and
similar to that of microparticulate -Cis
bonded packings, except for the lower
plate numbers.
Model Solutes
Experimental values of Vads, volume of
desorption (Vdes), and Vei for benzophe-
none; listed in Table 1, indicate that the
adsorption of benzophenone on Bonda-
pak-Cie follows a linear isotherm. As-
suming the isotherm to be linear, even at
lowest concentrations, one can predict
that the breakthrough volume, Vads, is the
same at the very low concentrations
prevailing in treated sewage effluents
and in polluted water as it is using the
concentrations in this work.
Figure 2 shows the adsorption-desorp-
tion curves for caffeine. A marked
contrast exists between these curves and
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I
.O
I
Qi
O
-J
1.5
1.0
0.5
Benzophenone
2.5
2.0
1.5
1.0
W
20 30 40
Volume Percent Methanol
50
Figure 3.
Adsorption (breakthrough) of furfural and bemophenone on Bondapak-Cia versus
volume fraction of methanol. Furfural column was 0.45 cm x 10.5 cm, void volume
0.8 mL; bemophenone column was 0.32 cm x 11.6 cm, void volume 0.6 mi-
Adsorption volumes are in mL, corrected for the void volume.
those shown in Figure 1 for benzophenone.
In Figure 2, there is a rapid concentration
rise during breakthrough and a slower
concentration fall during stripping. This
behavior is due to a nonlinear adsorption
isotherm. When nonlinear adsorption
occurs, the adsorption becomes less
strong as the concentration rises; this is a
common phenomenon in chromatography.
Measurements of Vads at different con-
centrations confirms this interpretation;
as shown in Table 2.
Breakthrough volumes were measured
for furfural at methanol concentrations
ranging from 50% to 20% and in pure
water. Figure 3 shows the relationship.
At low methanol concentrations, the
linear relationship breaks down. Adsorp-
tion on hydrophobic bonded-phase adsor-
bents is stronger in pure water and at very
low methanol concentrations than the
straightline plots predict. It has been
suggested that in solvents rich in
methanol, the long bonded hydrocarbon
chains stretch out into the solvent and
move freely about their anchor points on
the silica surface, but irr pure water they
stick to each other to form a compact,
hydrophobic, matted layer.
Data for the other model solutes (Figure
4) suggests a linear isotherm, however,
the limited number of points makes it
impossible to determine if a linear
relationship holds at low methanol
concentrations or whether an effect
similar to that observed for furfural
occurs.
Wastewater Studies
Large Scale Trace Enrichment
with Gradient Elution
FigureB shows the results of tests with
two Bondapak-da® columns. The erratic
rise and fall of the dissolved TOC was
mainly attributed to the inability to
maintain a consistent flow rate. The
effect of pH is clearly seen. Sorption was
more effective at lower pH as expected
since undissociated acids (mainly humic
acids) are more strongly retained than the
acid anions.
With the larger column, the TOC falls
as more liquid is passed. At the very
beginning of a run, the TOC is abnormally
high due to methanol that is washed out
of the adsorbent, but this effect only lasts
for 100 mL. at most. The gradual fall in
TOC seen in Figure 5 might be due to
another effect. Perhaps the humic acid
adsorbed on the Bondapak-Cia® enhances
the adsorption of other organic com-
pounds. With the small column, however,
the TOC rises slowly, that is, sorption
becomes less efficient as time goes on.
This is a more understandable effect, for
the column must eventually become
saturated with adsorbed material. Curve
(d), Figure 5, shows that the column had
adsorbed 6 mg of organic carbon during
the run. The dry weight of Bondapak-Cia®
in this column was 0.7 gram.
Small-Scale Trace Enrichment
A serious difficulty in analyzing the
potentially toxic trace organic compounds
in treated sewage isolated by the above
procedure is the presence of humic
material. Humic material is not a single
chemical substance but an ill-defined
high molecular weight polyelectrolyte
with a variety of functional groups. By
utilizing a" Ci8 Sep-Pak® that has been
conditioned with methanol, the humic
material is held back as a thin brown ring
at the entrance to the Sep-Pak®. Elution
with methylene chloride leaves nearly all
the humic material behind. Despite this
separation, the identification of individual
peaks is nearly impossible because of
their number and peak overlap. However,
portions of the same filtered waste water
passed through different Sep-Paks® and
analyzed separately produced essentially
identical chromatograms. Figure 6 shows
the fluorescence record for a Denver and
a Boulder wastewater. Note the prominent
peaks in the Boulder sample.
Conclusions
Bondapak-Cia® adsorbs relatively
hydrophobic compounds, including caf-
feine, phenols and chlorophenols, most
pesticides, plasticizers and polycyclic
aromatic hydrocarbons.
The main advantages of Bondapak-Cia
(37-75 microns) over microparticulate
Bondapak-Cia (less than 10 microns) for
wastewater analysis and the preparation
of fractions for toxicity testing are: its
lower cost and the fact that it is more
robust and less prone to fouling by the
humic substances.
Adsorption and retention volumes
were determined for a variety of model
solutes. These values were not affected
appreciably by levels of salts and humic
substances normally encountered in
drinking water.
Preliminary data are given on a method
of HPLC that uses a Sep-Pak® to adsorb
and separate the less polar organic
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Figure 4.
30 40 50 60
Volume Percent Methanol
Plots of capacity factors versus methanol concentration Bondapak-C^. Column
(0.45 cm ID x 10.5 cm long); void volume 0.6 mL; to find the adsorption volumes in
ml, add (log 0.6 = -0.221 to ordinates.
compounds from water and humic
material.
Recommendations
Two practical purposes remain to be
fully realized: one is the separation of the
dissolved organic material in wastewater
and treated sewage into well-character-
ized fractions, with a view to toxicity
testing, and the other is the chemical
analysis and recognition of specific
organic compounds, including nonvolatile
compounds, by high-resolution liquid
chromatography. This work advanced
significantly toward this goal through
Sep-Pak® trace enrichment and high
resolution liquid chromatography, as
described in the latter part of the final
report. These efforts should be continued,
with emphasis on selective methods of
chromatographic detection, particularly
electrochemical, which distinguish oxidi-
zable compounds like phenols and
benzidines. Electrochemical techniques
are now possible that can be used with
gradient elution, which the older tech-
niques could not.
A few tests were made with other
adsorbents, notably anion-exchange
resins, Duolit* A-7 and Bio-Rad MP-1.
Those resins removed organic compounds
from water that were not removed by
Bondapak-Cis®. Stripping each sorbent
in turn using pH and solvent gradients
would permit further selective fractiona-
tion. Additional work is needed in this
area.
'able 1. Adsorption and Desorption Volumes of Benzophenone on Bondapak-C\a from Solu-
tions in 50% Methanol (V/V)
Benzophenone
concentration, ppm:
0.5
2.5
5.0
6.5
c/ow rate, mL/min:
/ads (mL):
/des* (mL):
1.0 0.5
9.6 9.9
9.6 9.9
0.5
9.9. 9.9
10.2. 9.0
9.9, 9.9
9.8, 9.8
0.5
9.9
9.8
0.5
9.9, 9.8
9.8
9.8, 9.8
9.8
NOTES: (a)column dimensions, 0.45 cm x 10.5 cm.
(b) theoretical-plate numbers, from adsorption and desorption profiles, 115(1 mL/min),
165 (0.5 mL/min).
(c)elution volumes, Vei, for injections of benzophenone varying between 0.2 and 5.0
micrograms. were 10.0 ± 0.05 mL (mean of 14 measurements).
*Vaes - stripping or desorption volume.
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Table2. Adsorption Volumes of Caffeine on Bondapak-Cie from Water
Caffeine concentration, ppm:
Adsorption volume, mL,
at flow rates:
2.5 mL/min
5.0 mL/min
9.9 mL/min
70
65
63
2.5
71
62
56
10
48
46
42
Column (0.45 cm ID x 10 cm long), freshly packed.
I
10
I
13
(a)
\
1000
2000
10
(d)
Figure 6. Fluorescence Chromatograms
of two secondary sewage efflu-
ents; (a) Boulder, (b) Denver.
Each injection corresponds to 10
mL of secondary effluent. Sol-
vent gradient went from 25% to
75% methanol in 40 minutes.
500
1000
mL Effluent
Figure 5. Dissolved organic carbon in effluent from Bondapak- Ci e- Curves (a) and(c), column 1
cm x 1O cm; influent, 19.5 ppm carbon; (a), pH 7.2; (c), pH 3.5. Curve (b). column
1 cm x10 cm; influent, 17 ppm carbon; pH 7.5. Curve (b) was taken a month after (a)
andfc) but with the same packed column. Flow rate was about 5 mL/min but could
not be controlled accurately. Points (d), column 0.45 cm x 10 cm; influent, 14 ppm
carbon, pH 7.5; flow rate, 1.5 mL/min.
&U. S. GOVERNMENT PRINTING OFFICE: 1985/559-111/10765
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Harold F. Walton is with the University of Colorado, Boulder. CO 8030 J.
H. P. Ringhand is the EPA Project Officer (see below).
The complete report, entitled "Sorption Properties of Model Compounds on Ci8
Adsorbents, "(Order No. PB 85-125 839; Cost: $8.50, subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
-------
Harold F. Walton is with the University of Colorado, Boulder, CO 80301.
H. P. Ringhand is the EPA Project Officer (see below).
The complete report, entitled "Sorption Properties of Model Compounds on Ci$
Adsorbents, "(Order No. PB 85-125 839; Cost: $8.50, subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park. NC 27711
-------
HaroldF. Walton is with the University of Colorado. Boulder. CO 80301.
H. P. Ringhand is the EPA Project Officer (see below).
The complete report, entitled "Sorption Properties of Model Compounds on Cia
A dsorbents." (Order No. PB85-125 839; Cost: $8.50, subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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