EPA/600/2-90/020
TRANSPORT OF MACROMOLECULES AND HUMATE COLLOIDS
THROUGH A SAND AND A CLAY AMENDED SAND LABORATORY COLUMN
by
Candida Cook West
Processes and Systems Research Division
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ADA, OKLAHOMA 74820
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA/600/2-90/020
3. RECIPIENT'S ACCESSION NO.
PB90 219205ftS
4. TITLE AND SUBTITLE
TRANSPORT OF MACROMOLECULES AND HUMATE COLLOIDS THROUGH
A SAND AND A CLAY AMENDED SAND LABORATORY COLUMN
5. REPORT DATE
>. PERFC
April 1990
ORMING ORGANIZATION CODE
7. AUTHOR(S)
Candida Cook West
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Processes and Systems Research Division
Robert S. Kerr Environmental Research Laboratory
P.O. Box 1198
Ada, OK 74820
10. PROGRAM ELEMENT NO.
ABFC1A
11. CONTRACT/GRANT NO.
In-house
12. SPONSORING AGENCY NAME AND ADDRESS
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
P.O. Box 1198
Ada, OK 74820
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
EPA/600/I5
15. SUPPLEMENTARY NOTES
Project Officer: Candida Cook West,
FTS: 743-2257
16. ABSTRACT
Laboratory experiments were conducted to determine if macromolecules or humate
colloids would transport through sand columns and if they would exhibit any variations
in their relative velocity based upon their molecular volumes and the pore size
distribution of the column packing. PolyCethylene oxide) standards ranging in molecular
weights.from 50,400 to 900,000 were investigated. Humate colloids were prepared from a
.humate muck as their calcium and sodium salts. Columns were packed with a fine-grained
sand of uniform pore size (=? 20 um in diameter) and the same sand amended with 8% clay
(4% each kaolinite and illite) resulting in a pore size distribution in which ^=10% of
the pores had diameters less than 2 ym. The polyfehtylene oxides) and calcium and
sodium humate colloids were transported virtually conservatively through the Oil Creek
sand, with no evidence of size exclusion phenomena. Calcium humate was retarded in the
amended sand due to complexation with the clay fraction but moved through the column
with 77% recovery of the humate mass. The mobilization of clays was observed as a temp-
orary increase in column effluent turbidity and a significant shift in the particle
size distribution of tha effluent (150 to 450 nn).
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COSATi Field,Group
LIBRARY
RSKERL
U S ENVIRONMENTAL PROTECTIOII AGENCY
P.O. BOX 1198
919 KERR RESEARCH DRI\fE
ADA. OKLAHOMA 7482C
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS iTIns Ktporri
UNCLASSIFIED
21. NO. OF PAGES
51
20. SECURITY CLASS (This pu?i"
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (R«v. 4-77) PREVIOUS EDITION is OBSOLETE
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DISCLAIMER
The information in this document has been funded wholly by the United States
Environmental Protection Agency. It has been subjected to the Agency's peer and
administrative review and has been approved for publication as an EPA document.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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FOREWORD
EPA is charged by Congress to protect the Nation's land, air and water systems. Under a
mandate of national environmental laws focused on air and water quality, solid waste
management and the control of toxic substances, pesticides, noise and radiation, the Agency
strives to formulate and implement actions which lead to a compatible balance between human
activities and the ability of natural systems to support and nurture life.
The Robert S. Kerr Environmental Research Laboratory is the Agency's center of
expertise for investigation of the soil and subsurface environment. Personnel at the Laboratory
are responsible for management of research programs to: (a) determine the fate, transport and
transformation rates of pollutants in the soil, the unsaturated and the saturated zones of the
subsurface environment; (b) define the processes to be used in characterizing the soil and
subsurface environment as a receptor of pollutants; (c) develop techniques for predicting the
effect of pollutants on ground water, soil, and indigenous organisms; and (d) define and
demonstrate the applicability and limitations of using natural processes, indigenous to the soil
and subsurface environment, for the protection of this resource.
This research was conducted for the Office of Ground Water Protection as part of the
objectives to protect human health and the environment from ground water contamination. This
project report is intended to support this effort by supplementing basic understanding of the
mechanisms by, and extent to, which macromolecules and colloids transport in the subsurface.
Clinton W. Hall
Director
Robert S. Kerr Environmental
Research Laboratory
in
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ABSTRACT
Laboratory experiments were conducted to determine if macromolecules, fluorescent
microspheres or humate colloids would transport through sand columns and if they would exhibit
any variations in their relative velocity based upon their molecular volumes and the pore size
distribution of the column packing. Blue dextran, molecular weight 2,000,000, poly(ethylene
oxide) standards ranging in molecular weights from 50,400 to 900,000 and 0.05 and 0.51 um
carboxylated and 0.13 and 0.487 (im neutral polystyrene latex particles were investigated.
Humate colloids were prepared from a humate muck as their calcium and sodium salts. Columns
were packed with fine-grained Oil Creek sand (OCS) of uniform pore size (= 20 um in diameter)
and the same sand amended with 4% each kaolinite and illite resulting in a pore size distribution
in which = 10% of the pores had diameters less than 2 um. Blue dextran transported
conservatively through the OCS with the average linear velocity of the pore water. Early
breakthrough was observed in the amended OCS (0.296 pore volumes before the tritiated water),
but only after the column was pre-saturated with blue dextran. The exclusion of the blue dextran
could not be explained by either steric exclusion or hydrodynamic velocity gradients. It is
possible, however, that diffusional non-equilibrium may be a contributor. The poly(ethylene
oxides) and calcium and sodium humate colloids were transported virtually conservatively
through the OCS, with no evidence of size exclusion phenomena. Calcium humate was retarded
due to complexation with the clay fraction of the amended sand but moved through the column
with 77% recovery of the humate mass. The mobilization of clays was observed as a temporary
increase in column effluent turbidity and a significant shift in the particle size distribution of the
effluent (150 to 450 nm).
The data presented here neither supports nor refutes the application of chromatographic
principles to particle transport in soil columns. The behavior of the blue dextran in the amended
OCS may be unique. The relative velocities of macromolecules and colloids investigated were
not described using a simplistic model discussed in this study. Subsequent studies may need to
include diffusional non-equilibrium conditions and solid surface to particle interactions.
This report covers a period from February, 1988 to February, 1990 and work was completed as
of April, 1990.
IV
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CONTENTS
Disclaimer ii
Foreword iii
Abstract iv
Abbreviations and Symbols vi
Acknowledgements vii
I. Introduction I
n. Background 1
IE. Theoretical Development 5
IV. Conclusions 7
V. Materials and Methods
A. Polymers and Macromolecules 8
B. Columns 9
C. Column Packing 10
D. Analytical Methods 10
VI. Results and Discussion
A. Pore Size Frequency Distributions 12
B. Particle Size Distributions 12
C. Column Breakthrough and
Batch Sorption Studies 13
1. Blue Dextran 13
2. Poly(ethylene oxides) 14
3. Humate Colloids 14
4. Fluorescent Microspheres 15
Figures 16
Tables 38
References 40
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ABBREVIATIONS AND SYMBOLS
V = Darcy velocity
v = interstitial velocity of water
w
= V /9 where 0 = total saturated volume
v = interstitial velocity of a particle of radius, r
p,w
v = excluded interstitial velocity of a particle
= V_/6 where 9 = total available saturated volume
D e e
DCS = Oil Creek sand
AOCS = Clay amended Oil Creek sand
VI
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ACKNOWLEDGEMENTS
I acknowledge the participation of Dr. Narong Chamkasem, and Ms. Lynda Pennington
of NSI (RSKERL) for their assistance in the analysis of poly(ethylene oxides) and organic
carbon. I also thank Dr. Jong Soo Cho for his contributions to the theoretical development of
size contributions to particle velocity.
vu
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I. INTRODUCTION
Evaluating contaminant fate in any environment necessitates determining the compartments into
which the contaminant will distribute. In saturated subsurface environments the partitioning of a
contaminant between the mobile aqueous phase and the immobile aquifer solid matrix is often
the controlling distribution. The association of a contaminant with immobile solids results in a
reduction in the dissolved mobile concentration of the contaminant, sometimes by orders of
magnitude. However, it has been suggested that mobile entities such as suspended organic and
inorganic colloids and macromolecules may increase the "apparent" solubility of some
contaminants (Abdul et al. 1990; Penrose et al., 1990; Puls and Barcelona, 1989; Enfield and
Bengtsson, 1988; Chiou et at., 1987). If this is the case, then predictions of contaminant
transport based upon a two-phase system may seriously underestimate observed aqueous phase
concentrations of contaminant in laboratory and field studies. The influence of these entities
would need to be addressed by modeling systems as three phases consisting of immobile sorbed,
dissolved and mobile sorbed phases.
There is a good deal of research activity in the areas of colloid origination, chemistry, stability
and mobility. Recently, colloidal entities such as macromolecules and viruses have been
observed to be capable of eluting prior to conservative solutes in column and field studies. The
intent of this study was to observe the transport of colloid-sized entities and to examine the
validity of two proposed mechanisms by which this phenomenon may occur in the subsurface.
II. BACKGROUND
Definition of Colloidal Phases
Macromolecules, sometimes referred to as molecular colloids, are colloidal by virtue of their
size, interactions with other molecules and colloidal phenomena such as swelling, precipitation,
sorption, flocculation and micelle formation (Breger, 1970). Macromolecules can be loosely
defined as those molecules whose molecular weight exceeds 10,000 and may range in diameter
from one to several hundred nanometers.
The definition of a colloid is unclear because it is not strictly solute or solid. Colloid-associated
contaminants cannot be considered "truly" dissolved. They cannot be considered part of the
immobile solid matrix because they do not settle out and are carried with the mobile phase.
Strictly speaking, a colloidal system is comprised of a dispersing medium and a suspended
dispersed phase (colloid). Colloidal systems are solid/liquid, liquid/liquid or gas/liquid. In the
case of solid/liquid systems, the colloid is a hydrated solid held in suspension and treated as a
solute. One recommended definition of colloids is those species or particles subject to Brownian
motion with a size between 0.001 - 10 urn (Buddemeier and Hunt, 1988; Stumm, 1977). Another
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useful, but rarely used, definition of a colloid that addresses long-chained linear and highly
asymmetrical colloids is that of a particle containing approximately one thousand to one billion
atoms (Jirgensons, 1958). An empirical definition of colloids recognized in this research includes
those phases that cannot be separated from solution by conventional means such as gravity
settling, filtration (0.45 ^im) or standard centrifugation.
Size is not the only consideration to be made when classifying a particle or macromolecule as a
colloid. Defined size limitations are made on the assumption of spherical shape; but for other
tertiary shapes, charge density is a determining factor. The higher the specific surface charge,
the larger the particle can be and still exhibit colloidal behavior. Extremely high surface area to
volume ratio is essential to colloidal stability in most cases.
Colloids in the Subsurface Environment
Colloidal systems of environmental interest are primarily solid/liquid (clay, bacteria, viruses,
precipitates) and liquid/liquid (micelles and macromolecules of molecular weight > 10,000). A
gas/liquid colloidal system may exist in some cases of vapor transport in unsaturated systems.
Colloids may be formed in situ by condensation or homogeneous nucleation of particles from
dissolved species when a mineral phase is supersaturated or release of particles from bulk
material into a suspension of particulate solids (Buddemeier and Hunt, 1988; Apps et al, 1982).
The latter mechanism is likely largely responsible for considerable artifacts in compiling
experimental partition coefficients and desorption kinetics. Colloids may be introduced into a
system by virtue of being a component in original source of pollution. A scenario exemplifying
this would be wood pulp wastes or the wasting of polymeric materials. Water-soluble polymer
additives used in oil well drilling fluids form hydrocolloids that may be mobile. That is, they
hydrate, swell and disperse in water rather than dissolve (Lauzon, 1982). The chemistry,
formation and environmental significance of colloids in the subsurface environment has been
reviewed extensively by McCarthy and Zachara (1989).
Examples of typical colloids and colloid-sized particles that may be mobile in the subsurface are
metal oxides and hydroxides, clay particles, viruses and bacteria, and indigenous organic
macromolecules. Less typical would be synthetic macromolecules such as the high molecular
weight polymers that are in prevalent use today. Mobilization of clay fines or organic carbon
when destabilized by low ionic strength eluents or changes in counterion is a common experience
when conditioning a laboratory soil column. Colloids are typically generated in response to
sudden changes in geochemistry. Changes in pH, pE and ionic strength are common as wastes or
meteoric water percolate through a fonnation. There is substantial evidence that colloids can
move with the average linear ground water velocity and can significantly impact the mobility,
and sometimes the toxicity, of contaminants that otherwise would be considered too highly
retained by the solid matrix to be of concern. An excellent literature review and description of
mechanisms of particle release and capture is presented by McDowell-Boyer etal. (1986).
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Gschwend and Reynolds (1987), using SEM and SEM-EDAX analysis, found monodisperse (100
nm) colloidal materials consisting primarily of iron and phosphorus in ground water samples
collected 300 meters downgradient and 11 meters below the water surface of a secondary-sewage
infiltration site on Cape Cod, Masschusetts. These colloids apparently were formed in situ in
response to oxygen depletion and were speculated to be mobile. Organic macromolecules in the
influent (6 ± 3 nm diameter) appeared not to be transported independently in the system.
However, it was hypothesized that they may have coated the positively charged surfaces of the
ferrous phosphate particles, thus preventing them from coagulating or filtering out of the system.
Under pristine conditions, there can be movement of colloids induced by rain events. For
instance, Murphy et al. (1989) determined by isotopic composition that the high molecular weight
fractions of indigenous dissolved organic carbon (DOC) in samples taken from the Milk River
aquifer in Alberta, Canada were mobile and originated in the interstitial waters of the soil zone in
the area of recharge. Organic and inorganic colloids ranging in sizes from 0.04 to 1 u.m have
been found to be mobile in a subsurface fracture in granitic rock (Degueldre et a I., 1989). The
nature of the colloids was highly complex, with the inorganic colloids being comprised of Si, Ca,
Mg, Sr, Ba, Fe and S. A threshold level of 5 mg/1 has been suggested by Leenheer et al. (1974)
to differentiate between indigenous and introduced organic carbon. This was established by
analyzing base levels of naturally occurring DOC from 100 sites in 27 states.
Implications of Colloid Formation
Regardless of the origin, conditions which lead to the destabilization of the solid matrix to form
colloids or precipitation of sparingly soluble molecules from the aqueous phase may occur until
the plume is diluted such that its geochemistry readjusts to the environment. Increases in ground
water turbidity in response to artificial low salinity ground water recharge has been observed at
many field sites, some of which were studied as early as 1969 (Nightingale and Bianchi, 1977).
The size of the particles that were transported indicate that the probability for filtration and
interaction of particles may be a function of whether the particles were introduced from outside
the system or generated from the system.
The literature suggests that colloid function has added implications with respect to contaminant
transport. There have been numerous publications concerning the partitioning and reactions of
hydrophobic organic compounds to dissolved humic materials and micelles greatly increasing the
solute aqueous solubility (Kan and Tomson, 1990; Kile and Chiou, 1989; Valsaraj and
Thibodeaux, 1989 and Schnitzer et al., 1988). It is not known if the humates responsible for
increases in apparent solute solubilities exist as macromolecules of discrete units or aggregated
units operating essentially as micelles (Wershaw, 1986). Buddemeier and Hunt (1988)
hypothesized that colloid-bound nuclides exhibit reduced mobility as compared to free nuclides.
This may be due to increased interactions with the environment as the bound species. Penrose et
al. (1990) found that plutonium and americium species were mobilized by colloidal material
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between 25 and 450 nm in size and transported to monitoring wells 3390 meters downgradient
from the source.
There are practical implications to interactions between colloids and contaminants. One
implication concerns well sampling. It has been argued that the standard method for
differentiating between free and bound metals by 0.45 um filtration may be inappropriate (Puls
and Barcelona, 1989). The presence of metals associated with colloids less than 0.45 um would
result in erroneously high dissolved concentrations. The argument may apply to organic
contaminants as well. Another implication relates to the use of colloids for remediation. The
possibility of using humate colloids to facilitate the removal of contaminants from soil has been
suggested in a recent study where a solution of 29 mg/L humic acid more effectively removed
non-polar organic compounds than did water (Abdul et al., 1990). Further studies on the
interaction between previously sorbed contaminants and humic materials is warranted. There
would be significant advantages using natural humate colloids for remediation since they are
cheaper and more environmentally acceptable than commercially prepared surfactants.
There has been evidence to suggest that the toxicity of contaminants may be greatly reduced
when associated with colloids or organic macromolecules. Black and McCarthy (1988) found
that when benzo(a)pyrene and 2,2',5,5'-tetrachlorobiphenyl were bound to dissolved Aldrich
humic acid, they did not diffuse across the gill membrane of rainbow trout. It has also been
observed that when gold is bound to commerically prepared (Aldrich) humic acid it is
unavailable for soil cycling (Jones and Peterson, 1989). However, it may not be appropriate to
extrapolate the extent of sorption of hydrophobic compounds to commercial humic acid to real
world situations (Chiou et al. 1987).
Suspended colloids are subject to physical and chemical mechanisms that can limit their transport
in porous media. Two physical mechanisms leading to removal of colloids are surface and
straining filtration and are a function of the particle/pore diameter ratio. In the case of surface
filtration, particle accumulation leads to significant reduction in formation permeability.
Physical-chemical filtration is also a function of particle and media surface charges.
Mobilization of quartz fines during oil production by fluid injection has been found to be
dependent on both ionic strength and the pH of injection fluid and virtually independent of fines
size (Cerda, 1988). Physical-chemical interactions of stable particles to media surfaces often are
not considered to result in permeability reduction because there is only monolayer coverage.
Destabilized colloids can, however, create a situation where particle-particle interactions are
sufficient such that the thickness of deposition may cause a reduction in permeability.
In an investigation conducted by Smith et al. (1985), it was shown that when an intact soil core
was sieved and repacked, E. coli transport was reduced from 22-79% to 0.2-7% penetration. All
other conditions being held equal, this indicated that the pathway was by way of secondary
permeability rather than intergranular pore spaces. However, Bales et al. (1989) demonstrated
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that bacteriophage are conservatively transported in a homogeneously packed sandy soil column.
It was also observed that the phage broke through prior to tritiated water and were excluded
from 35 to 45% of the void volume. Enfield and Bengtsson (1988) had previously observed that
blue dextran (molecular weight two million) broke through a laboratory column prior to tritiated
water and that the relative position of the breakthrough appeared to be dependent on the pore size
distribution of a homogeneously packed column. It was hypothesized that this phenomena was a
result of the particle/pore diameter ratios as in size exclusion chromatography.
The purpose of this study was first to gather more evidence for organic colloid mobility, both
synthetic and natural, and secondly, to examine the validity of proposed mechanisms for early
breakthrough observations.
The interstitial velocity of ground water (v ) is defined as the Darcy velocity corrected for
porosity. By analogy, the intersitital velocity of a mobile particle (v ) may be normalized to the
porosity it is able to travel.
III. THEORETICAL DEVELOPMENT
For the most part, discussions of colloid and macromolecule transport have been confined to
observations influenced by physical and chemical interactions that would retard their transport,
relative to conservative species. As discussed in the previous section, there may be mechanisms
by which macromolecules and colloids can elute prior to conservative species as has been
observed in laboratory and field studies. Two mechanisms which have been proposed to account
for this behavior are discussed.
The first mechanism is that of particle exclusion from pores. Concepts from steric (size)
exclusion chromatography (SEC) have been examined with respect to applicability to classic
dispersion/advection theory (Peyton et al., 1985; Peyton et al., 1986). A particle may be
excluded from dead end pores and pores smaller than some critical diameter based upon the ratio
of the particle diameter to pore diameter and flowrates (dynamic porosity). The exclusion from
these pores is a function both of molecular volume and fluid velocity, that is, diffusional
equilibrium into a pore is a function of the velocity of the fluid as it moves past the pore. This
can result in an "effective" porosity for that particle (9 ) that may be both specific for the particle
(i.e species) involved and less than the total porosity (9 ). A solute diffusing through less than
the total porosity would elute from a soil column prior to a smaller chemical specie like water (v
= V/9>v=V/9). P
D e w D t
The second mechanism by which the elution velocity of a particle may be a function of its size
can be described by analogy to hydrodynamic velocity gradients in capillary flow.
Hydrodynamic chromatography (HOC) is an analytical technique used to separate particles based
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on their size and rate of transport through a bed packed with solid, non-porous particles. The use
of HOC was first described by Small (1974). According to this theory, the rate of transport of
conservative colloidal particles depends on the size of the colloid and the inter-particle volume of
the column packing. Larger particles move faster than the smaller ones, which in turn move
faster than the molecules of the elution fluid (Stoisits, et al. 1976). There exists across the flow
channel a velocity gradient such that the velocity of the fluid is maximum at the center and
diminishes toward the wall. The mean velocity of a particle is a function of the velocity gradient
to which it is subjected, based upon the particle radius. A small molecule, such as water, can
access the entire gradient; and hence, its average interstitial velocity is 1/2 v .A large particle
will be subject to higher velocities than a relatively smaller particle. The rate of transport
through a column bed is expressed by the quantity R , the average moving rate of a particle
through the bed relative to the average flow rate of the mobile phase (v / v ). R is analogous to
p,w w f
the relative velocity of a contaminant in groundwater (i.e. velocity of tne contaminant/velocity of
the water). Stoisits et al. (1976) first mathematically verified hydrodynamic chromatography
flow based on a model by DiMarzio and Guttman (1969, 1970) derived for rigid and flexible
polymer molecules flowing in Poiseuille flow through a capillary tube. The velocity at a point r,
v(r), from the center of the capillary in the radius of the tube is given by the equation:
v(r) = [(Apr02)/(4r,i)][l-(r/ron
where
Ap = pressure drop across the capillary
r0 = radius of the cylinder
T| = dynamic viscosity of the fluid
i = total length of the cylinder
and the maximum velocity at r = 0 is
v = (Apr02)/(4r|i) = 2v
max w
Since a particle of radius r will be subject only to the portion of the velocity gradient > r from
the wall, the average velocity of the particle (v ) can then be calculated as:
p,w
v = v [1 -l/2(l-a)2]
p,w max
where
a = radius of the particle/radius of the pore (r /r „)
P
This then can be rewritten as:
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v = v [1 + a(2-a)]
p,w w
Based on the theories of size exclusion and hydrodynamic velocity in a system of conservative
solutes, those particles not subject to physical straining should transport according to size and
fluid velocities and the two mechanisms contributing to particle velocity would be operational.
Assuming that the effective porosity is the total pore volume available to an excluded
macromolecule and that corrections for the reduced fluid volume in which it travels is
insignificant, then the elution velocity of an excluded particle (v ) can be approximated:
p,e
v = V/9
p,e d e
where
VJ = v * 6
d w t
Since the average linear velocity of the excluded particle can be calculated by its hydrodynamic
velocity as defined above (v = v ) we now have:
w p,w
v /v = (9 /9 ) [1 +
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The calcium humate colloid was retarded due to irreversible clay-humate interactions.
Subsequent flow of calcium humate through the column showed significantly less retardation of
the humate. Based on the theories of relative velocity presented here, it would be expected to see
early breakthrough of the humate colloids. However, driving the system to saturation would
have caused the same doubtful situation as in the case of blue dextran.
An additional observation made in these studies was that the behavior of the tritiated water was
erratic as evidenced by data-scatter, breakthrough curve asymmetry and excessive tailing. This
may have been due to the equilibrium of water molecules from the bulk phase to the hydrated
colloid. If this is the case, then tritiated water may not be an appropriate conservative tracer for
the transport studies.
In many ways, the neutral or charged microspheres would have been ideal models for these
studies as they can be purchased in a wide range of particle sizes with small standard deviations
and specified surface charge. It was observed in these studies, however, that considerable
cleaning of the microspheres is necessary to remove interfering contamination of the charge
characteristics by surfactants used in the manufacture of the beads. The humate colloids do
however present an interesting possibility for further study. Their size can be controlled by
variation in pH, ionic strength and counterion, and they are more relevant to subsurface transport
studies.
The observation of column transport of the colloids and macromolecules present additional
evidence for colloidal mobility. The ability to predict the rate at which these panicles transport
in media other than non-interactive is highly complex and may ultimately be of minor
significance except under very specific circumstances.
V. MATERIALS AND METHODS
A. Polymers and Macromolecules
Blue dextran is a polysaccharide (a 1-6 polyglucose) produced by Leuconostoc
mesenteroides (B512) and dyed with Cibacron blue . It can be purchased in several
different average molecular weights and is used primarily for calibrating gel filtration
columns. The blue dextran was purchased from Sigma Chemical Company in 95%
purity and two million average molecular weight. The blue dextran concentration in
column experiments was 100 mg/L in 0.01 M CaCl 2. Experiments were conducted in a
controlled temperature room at 12°C. Feed solutions were poisoned with 0.1% by
weight NaN to prevent bacterial growth.
Poly(ethylene oxide) polymers have the monomeric formula (-CH CH O-). Standards
were obtained from Alltech Assoc. (Deerfield, IL) as a kit containing 0.2 g each of eight
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high purity polymers ranging in average molecular weight from 14,200 to 900,000.
Poly(ethylene oxides) were prepared in 0.01 M CaCl 2 and 0.1% by weight NaN,.
Experimental concentrations of the poly(ethylene oxides) ranged from 961 to 1261 ppm
and are given for each experiment in the experimental section.
Humate colloids were prepared from a humate muck that is a produced as a co-product of
titanium dioxide mining in Northern Florida. Prior to preparation of colloids for column
studies, a series of humate solutions was prepared for the purpose of determining the
range of colloidal sizes that could be produced varying solution pH and cation
concentration. Two sets of humate solutions were made by adding 1.0 g of humate muck
to 1 liter of deionized water and 1 liter of 0.01 M CaCl 2. The solutions were weakly
buffered to pH 6, 7 and 8 using sulfonic acid buffers (MES, BES and bicine, Sigma
Chemical Co., St. Louis, MO).
The humates prepared for experimental purposes contained no buffers as further work
showed them to be stable. Additionally, higher concentrations were prepared for
analytical reasons. Sodium humate was prepared by weighing 100 grams of humate
muck (25% solids by weight as determined by oven drying at 100° C for 24 hours) into
0.01 M NaCIO and bringing the solution up to a total volume of one liter. The initial pH
was 3.92. The solution pH was then adjusted to match that of the background NaCIO
solution, pH 5.76, using 1.0 M NaOH. After stirring overnight, the humate solution was
allowed to settle for 24 hours. The supernatant was collected by vacuum suctioning,
then centrifuged at 3,000 rpm for one hour and filtered in small aliquots through 0.45um
Millipore filters. The calcium humate was prepared in the same manner, except the
diluting solution was 0.01 M CaSO . The initial pH was 4.04 and was adjusted to 6.41.
Yellow-green fluoresbrite, carboxylated (0.05 and 0.51 urn) and neutral (0.13 and 0.487
fim) polystyrene latex microspheres were purchased from Polysciences, Inc.
(Warrington, PA).
B. Columns
Two column types were used in this study. The first consisted of beaded glass pipe (Ace
Glassware, Inc., Vineland, NJ) and end caps fitted with threaded glass connectors. The
column dimensions were 15.2 cm in length and 5 cm inside diameter. The endcaps were
connected to the pipe by coupling-bolts with Teflon liners. The columns were dry-
packed to minimize introduction of secondary channels. All blue dextran breakthrough
curves were conducted through these columns. Flow in all experiments was from bottom
up to ensure saturation.
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The second column type was specially fabricated of 316 stainless steel, 1 cm in length
and 5.0 cm internal diameter. The columns were fitted with removable stainless steel
porous endplates, 0.078 cm thickness and 40 nm pore size (Mott Company, Farmington,
CT) and Viton o-rings. These were used for all poly(ethylene oxide) and humate studies,
primarily to minimize the quantity and cost of the polymers necessary for the
experiments.
Solutions were pumped into the columns by a Technicon auto analyzer proportioning
pump using tygon pump tubes. The columns were plumbed to the feed solutions and
pump via teflon tubing. Flowrates were 9.6 ml/hour (0.474 cm/hr linear velocity) for all
column experiments. Samples were collected into pre-weighed vials by a model 3100
Cygnet fraction collector (ISCO, Inc., Lincoln, KB). After collection, vials were re-
weighed for flowrate and volume verification. Appropriate sampling aliquots were taken
from the vials for analyses.
C. Column Packing
«t
The column packing material selected for this study is a fine-grained sand referred to as
OCS (OCS) mined commercially in Oklahoma. It is comprised of 99.6% sand and 0.4%
silt. The total organic carbon content of the sand is 0.0045%. Where noted, the sand
was amended with 4% each kaolinite and illite clay standard (8% total clay) obtained
from the Clay Minerals Society for the purpose of adjusting the pore size distribution.
D. Analytical Methods
Blue dextran concentrations were analyzed on a Shimadzu DV160 scanning
spectrophotometer at 630 nm using proper quality assurance procedures. The
poly(ethylene oxides) were analyzed using HPLC gel permeation chromatography. The
system was comprised of a Waters model 590 high pressure pump, model 712B auto
sampler, model 401 refractive index detector, a column heater module to maintain
constant column temperature and a Nelson Analytical microcomputer-base integrator.
Ultrahydrogel GPC columns (250, 500 and 1000) were purchased from Waters. The
limit of detection for the poly(ethylene oxides) was * 100 ppm. High feed solution
concentrations were used in column studies (961-1261 ppm) so that breakthrough could
be discerned at * 10% relative concentration and quality control objectives could be met.
Humate concentrations were expressed as mg/L organic carbon determined using low
temperature ultraviolet/potassium persulfate oxidation after acidification with
concentrated phosphoric acid.
10
-------
The total organic carbon in column packings was determined by digestion in 10%
perchloric acid with subsequent solids analysis by Leco WR-110 and filtrate analysis on
a Dohrmann DC-80 using low temperature ultraviolet/potassium persulfate oxidation.
Fatty acid determinations were performed on column effluents as a qualitative indicator
of presence or absence of microbial growth in blue dextran and poly(ethylene oxide)
column experiments. Column effluents were analyzed for C to C fatty acids by acidic
ethyl ether extraction and subsequent analysis by GC/FID using DB-5 capillary columns.
The limit of detection was approximately 1 ppm. Standards were purchased from
Supelco (volatile acid standard mix).
Particle size distributions were determined on a Malvern Autosizer 2C. Microsphere
analysis was by O.K. Turner fluorometer using appropriate primary and secondary
filters. Excitation and emission wavelengths are 458 and 540 nm, respectively. The
lower detection limit was 1 ppb.
Microspheres were dialyzed against distilled, reverse-osmosis water using Spectra/Por
dialysis membrane, 1,000 molecular weight cut-off (Spectrum Medical Industries, Los
Angeles, CA). Diafiltration was performed using hollow fiber filters with .01 um
opening and 500 |j.m diameter (H1P100-20 filter, Amicon Corporation, Danvers, MA).
Tritium concentrations were analyzed on a LKB model 1219 Rackbeta liquid scintillation
counter with automatic quench correction. Water soluble Ready Safe scintillation
cocktail (Beckman) was used in all experiments. Sample to cocktail ratios varied from
0.1:6 to 1.0:6 ml, respectively.
Soil moisture characteristic curves were performed on both the OCS and amended OCS
using standard Tempe cells in which a drainage curve is established by pairing
volumetric water content with matric pressure head (American Society for Testing and
Materials, F316). From these curves, assuming spherical particles, pore size
distributions were calculated using the equation:
27irycos0 = r2 rc(hpg)
where
r = radius of the pore or capillary
y = surface tension of water
0 = contact angle (cos 0 = 1 for a wetted sample)
hpg = hydrostatic pressure
11
-------
V. RESULTS AND DISCUSSION
A. Pore Size Frequency Distributions of Column Packings
The interparticle pore size frequency distribution for OCS is very uniform, with =74% of
the pore radii between 13 to 8 (Am and less than 1% £ 1.0 \im (figure 1). When the sand
was amended with 6% clay (equal weight kaolinite and illite), =10% of the pores were
less than 1.0 |xm (figure 2), which could be expected to provide a significant proportion
of pore openings from which a large colloid would be excluded if size exclusion is
functional.
B. Particle Size Distributions
The particle size distributions for the study polymers are given in table 1. The blue
dextran and poly(ethylene oxides) could only be estimated due to their non-spherical
shapes. Nevertheless, the range of particle diameters (= 100 to 200 nm) can be used to
estimate if there would be any significant effect of molecular volume on breakthrough
velocity in either of the column packings used based on the theories described above.
The cumulative particle size distribution (figure 3) for the humate suspensions made at
varying pH and counterion had trends in size that are in keeping with observations made
by Ghosh and Schnitzer (1980) using surface pressure and viscosity measurements. It
appears that the ionic strength of the buffer was sufficient to keep the overall particle
sizes small. As a general rule, humate solubility increases with pH due to increasing
negative charge; and conformation changes from coiled to linear as the distance between
like charges is maximized. Addition of a divalent cation like calcium aggregated
humate, which, as in this case, increases the size of the particles. Additionally, the
numbers of particles were greater for all the humates in deionized water than those
prepared in the calcium solution (data not shown), again indicating humate aggregation.
The humate suspension in buffered deionized water had sufficient ionic strength to
minimize the effect of pH. Based on this behavior, it was decided not to buffer humate
solutions.
The colloids prepared for column studies were examined for particle size distribution,
stability of size distribution from one batch preparation to another and over time, and
maintainence of distribution after transport through columns. The latter will be
discussed with the column results. The calcium humate had a particle size distribution
predominantly between 350 to 525 nm and a small contribution in the 200 to 250 nm
range. The sodium humate had a significant particle population in the same size
distribution as the calcium humate, but was bimodal with a significantly higher
population in the range of 200 to 250 nm (figure 4). The particle size distributions of
12
-------
both humates were stable over a period of five days (figure 5 and 6). The reproducibility
of the sodium humate from one batch preparation to the next was good (figure 7),
reaffirming that the bimodal distribution was not an artifact.
C. Column Breakthrough and Batch Sorption Studies
Experimental concentrations, recoveries and experimental R 's for column breakthrough
results for the study macromolecules are listed in table 2 for reference.
1. Blue Dextran
Blue dextran transported through the OCS conservatively (98% recovery) and moved
at the average interstitial velocity of the bulk fluid (figure 8). Attempts to conduct
column studies through the clay amended OCS were unsuccessful due to significant
sorptive losses of the blue dextran to the column packing. Batch sorption isotherms
were conducted for blue dextran at pH 4.0 and 8.0 to determine if there was any pH
dependency. It appeared that the sorption of blue dextran to the clay amended OCS
was independent of pH and highly non-linear (log Kd = 4.2; 1/n = 0.24 at pH 8.0,
0.28 at pH 4.0) (figure 9). Batch sorption experiments were then conducted for blue
dextran and its components, dextran and cibacron blue. This was done to verify that
there was no separation of the chromophore from the dextran. In some
manufacturing processes, dyes are only hydrophobically "bound" as opposed to
- forming covalent bonds. The batch sorption experiments were also run to determine
if the dye alone was responsible for the sorption. Sorption for both the dextran and
cibacron blue were fairly linear (1/n > 0.9) with log Kd's of 1.6 and .996,
respectively. Desorption for both components over 72 hours was minimal (figures
10 and 11). This indicated that both compounds contributed to the sorption of the
blue dextran to the clay fraction of the column packing. This was not unexpected as
cibacron blue is subject to hydrophobic and ionic interactions of sulfonated and
amino groups (personal communication, Sigma Chemical Company) and
clay/polysaccharide complexes have been studied (Clapp and Emerson, 1972).
However, the column elution behavior of the blue dextran was more complex than
that which may have been predicted from linear additive sorption of the two
components. Blue dextran was pumped through the amended OCS column until
saturated (the effluent and influent concentrations were approximately equal). The
column was flushed with background solution until the effluent blue dextran
concentration became relatively constant at =18 ppm. A pulse of blue dextran in
tritiated water was then pumped into the column and the effluent monitored. The
blue dextran C/Co = 0.5 eluted from the column approximately 0.296 pore volumes
prior to the tritiated water tracer (figure 12).
13
-------
The relative velocity (R ) based on hydrodynamic flow is calculated to range
between 1.005 to 1.044 for blue dextran in the OCS assuming the macromolecular
diameter is 0.076 \im and the pore radius distribution limits are = 3 to 35 urn. If it
could be assumed that in the clay amended column blue dextran was completely
excluded from those pores < 1 u.m in radius, the effective porosity would be reduced
10%, thus increasing the relative velocity to 1.117 to 1.160. The high relative
velocity of the blue dextran (1.296) cannot be explained on the basis of
hydrodynamic velocity and size exclusion. Its elution behavior may also have been
influenced by surface repulsive forces such as in anion exclusion or by diffusional
non-equilibrium effects.
2. Poly(ethylene oxides)
Except for 900,000 molecular weight fraction, all the poly(ethylene oxides) broke
through the OCS after the tritiated water (R < 1.0) (figures 13 to 15). Recoveries of
the 50.7K, 83.8K and 770K polymers were relatively high (84-98%) although some
tailing was observed. The 900,000 molecular weight poly(ethylene oxide) eluted
prior to tritium (R = 1.116). It is difficult to determine if this is real or an artifact,
perhaps a reflection of inaccurate analysis as evidenced by the high recovery
(109%). No polymer was detected in the effluent when transport studies of two of
the polymers through an amended OCS column was conducted. Batch sorption tests
(figure 16) showed that due to sorption excessive quantities of the expensive
polymers would need to be purchased to conduct further studies. For this reason the
column elution studies were not pursued.
3. Humate Colloids
Both the sodium and calcium humates transported conservatively (== 100% recovery)
through the OCS (figures 17 and 18). The particle size distribution for the sodium
humate effluent was virtually identical to that in the influent (figure 19). However, a
percentage of the particles in the calcium humate effluent shifted to significantly
smaller particle diameters (figure 20), most probably due to calcium ion loss from
the humate complex by the low cation exchange capacity of the sand causing
disaggregation of the humate particles. The calcium humate was retarded in the
amended OCS (figure 21) in an apparently irreversible fashion. Approximately 77%
of the humate mass was recovered. The effluent temporarily became turbid and the
particle size distribution (figure 22) showed a marked change from a primary particle
size of 150 nm to = 450 nm indicating that the elution species was probably a
clay/humate complex. There was considerable scatter in the tritium data for this
breakthrough curve which was probably due to interference by the clay particles.
When calcium humate was pulsed a second time it was observed that the humate
14
-------
broke through with less retardation, due to saturation of some fraction of the
immobile clay sites.
4. Fluorescent Microspheres
Several attempts to conduct column breakthrough curves through OCS were
unsuccessful due to interactions of the microspheres with the glass and sand.
Electrophoresis qualitatively indicated that the net surface charge of the
microspheres at fixed pH was not that provided by the manufacturer. Microscopic
examination of the microspheres revealed the presence of bacteria and molds,
apparently growing on the surfactant residuals left from the manufacturing process.
Dialysis of uncontaminated the 0.05 u.m carboxylated microspheres did not
adequately reduce interactions between the microspheres and sand, although there
was some charge improvement. A final attempt to clean the microspheres by
diafiltration was unsuccessful. The beads sorbed to the filter and reversing flow was
unsuccessful in removing them. The ionic strength of all solutions were below the
manufacturer's stated critical coagulation concentrations. Additionally, microscopic
examinations confirmed that the beads were monodisperse. Since no reasonable
explanation for this could be made by either supplier (Polysciences or Amicon), no
further attempts were made to use the microspheres as colloid surrogates.
15
-------
0.25
0.20+
Gravimetric 0.15
water
content
(g/g) 0.10
0.05--
0.00
-600 -400 -200 0 200
Matric suction (cm water)
Volume
fraction
1.0 10.0 100.0 1000.0
Pore radius (microns)
Figure 1. Soil moisture characteristic curves
(replicates = 2) for Oil Creek sand (upper)
and resultant pore size distribution
(lower).
16
-------
0.25
0.20--
Gravimetric 0.15
water
content
(g/g) 0.10
0.05--
0.00
-600 -400 -200 0 200
Matric suction (cm water)
Volume
fraction
1.0 10.0 100.0 1000.0
Pore radius (microns)
Figure 2. Soil moisture characteristic curves
(replicates = 3) for amended Oil Creek sand
(upper) and resultant pore size distribution
(lower).
17
-------
100
number
200 400 600 800
Particle size (nm)
1000
Figure 3. Cumulative particle size distribution of
filtered humate muck (100 mg/1). Open
symbols, distilled water; solid symbols,
0.001 M calcium chloride.
18
-------
% by
number
200 400 600 800
Particle size (nm)
1000
Figure 4. Particle size distribution for humate
prepared in 0.01 M calcium sulfate (solid
line) and 0.01 M sodium perchlorate (dashed
line).
19
-------
% by 20+
number
200 400 600 800
Particle size (nm)
1000
Figure 5. Particle size distribution stability of
calcium humate. Time = 0 (solid line;
distribution mean = 428 nm) and time = 5
days (dashed line; distribution mean = 407
nm) .
20
-------
% by
number
100 200 300 400 500 600 700 800
Particle size (nm)
Figure 6. Particle size stability of sodium humate
Time = 0 (solid line; distribution mean =
314 nm) and time = 5 days (dashed line;
distribution mean = 265 nm).
21
-------
30
25--
20--
% by 15+
number
10--
100 200 300 400 500 600 700
Particle size (nm)
Figure 7. Reproducibility of particle size distribution
of sodium humate. Batch #1, double line;
batch |2, single line.
22
-------
Relative
Concentration 0.5--
Figure 8. Blue dextran (solid circles) transport
through Oil Creek sand. Tritiated water,
open circles.
23
-------
LoglO 4<8_.
soil
cone.
(ug/g)
4.2
0123
LoglO aqueous cone, (mg/1
Figure 9. Freundlich sorption isotherm (log S =l/n
(log C) + log Kp) for blue dextran to
amended Oil Creek sand. At pH 4.0 (solid
squares) 1/n = 0.28; log Kp = 4.2. At pH
8.0 (open squares) 1/n = 0.24; log Kp = 4.2,
Samples were run at three concentrations at
both pH's.
24
-------
Soil
concentration 300--
(ug/g)
250--
200--
150--
100
0 20 40 60
Aqueous concentration (mg/1)
Figure 10.
Sorption/desorption isotherm for cibacron
blue to amended Oil Creek sand. Sorption
data at 72 hours (solid circles; R-squared =
0.999); desorption data at 1,4,24,48 and 72
hours (open circles; average of triplicate
samples).
25
-------
750
Soil
concentration 450--
(ug/g)
400--
20 40 60 80 100
Aqueous concentration (mg/1)
Figure 11.
Sorption/desorption isotherm for dextran to
amended Oil Creek sand. Sorption data at
72 hours (solid circles; R-squared =
0.999); desorption data at 3,6,24,48 and 72
hours (open circles; average of triplicate
samples).
26
-------
0.6
Relative
concentration 0.5
0.0
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
Pore volumes
Figure 12.
Blue dextran (solid circles) transport
through amended Oil Creek sand after pre-
saturation with blue dextran. Tritiated
water, open circles.
27
-------
Relative
concentration
1.
0.8--
0.6--
0.4--
0.2--
o
O
0
V
^
Relative
concentration
Figure 13.
770K poly(ethylene oxide) transport through
Oil Creek sand (closed circles, top
figure). Tritiated water data (open
circles) was fitted using a non-linear
least squares fitting procedure (double
line, bottom figure). Porosity 0.32;
dispersivity 0.16 cm, Darcy velocity 0.47
cm/hr.
28
-------
Relative 0.6
concentration
Relative 0.6--
concentration
0.2--
0.0
2468
Pore volumes
Figure 14. 900K and 83.8K poly(ethylene oxide)
transport through Oil Creek sand (closed
triangles and closed circles, respectively)
Tritiated water data (open circles) was
fitted using a non-linear least squares
fitting procedure (double line, bottom
figure). Porosity 0.32; dispersivity 0.27
cm; Darcy velocity 0.47 cm/hr.
29
-------
1.0--
0.8--
Relative 0.6--
concentration
1.0--
0.8--
Relative 0.6--
concentration
Pore volumes
Figure 15.
50.4K poly(ethylene oxide) transport
through Oil Creek sand (closed circles,
top figure). Tritiated water data
(open circles) fitted using a non-linear
least squares procedure (double line,
bottom figure). Porosity 0.32; dispers-
ivity 0.21 cm; Darcy velocity 0.47 cm/hr,
30
-------
Log sorbed
cone (ug/g) -
o . ^ j-
3.20-
3.15-
3.10-
3.05-
3.00-
2.95-
2.90-
•5 01;
i i i
•
_
*
/
/ 7 -
/
/
/
/
/
/ V
// i i i •
2.1 2.2 2.3 2.4 2.5
Log aqueous cone. (mg/L)
Figure 16.
Freundlich sorption isotherm for
770K (diamonds; 1/n = 0.67; log Kp = 1.41)
and 160K (circles; 1/n = 1.26; log Kp =
0.13) poly(ethylene oxides) to clay amended
Oil Creek sand. All points are an average
of triplicate samples.
31
-------
Relative
concentration
Figure 17.
Calcium humate transport (closed circles)
through Oil Creek sand. Tritiated water
data (open circles) fitted by a non-linear
least squares procedure (double line).
Porosity 0.32; dispersivity 0.09 cm; Darcy
velocity 0.47 cm/hr.
32
-------
Relative
concentration
10
Pore volumes
Figure 18.
Sodium humate transport (closed circles)
through Oil Creek sand. Tritiated water
data (open circles) fitted by a non-linear
least squares procedure (line). Porosity
0.32; dispersivity 1.2 cm; Darcy velocity
0.47 cm/hr.
33
-------
number
200 400 600 800
Particle size (nm)
Figure 19.
Particle size distribution of influent (solid
line) and effluent (dashed line) of sodium
humate transported through Oil Creek sand
column.
34
-------
% by 20+
number
200 400 600
Particle size (nm)
800
Figure 20.
Particle size distribution of influent
(single line) and effluent (double line,
1.0 pore volumes; dotted line, 1.9 pore
volumes) of calcium humate transported
through Oil Creek sand.
35
-------
Relative
concentration
Figure 21.
Calcium humate transport (closed circles)
through amended Oil Creek sand. Tritiated
water data (open circles) fitted by a non-
linear least squares procedure (line).
Porosity 0.32; dispersivity 0.26 cm; Darcy
velocity 0.47 cm/hr.
36
-------
number
200 400 600
Particle size (nm)
800
Figure 22.
Particle size distribution of influent
(solid line) and effluent (1.13, 1.73
and 2.27 pore volumes, double, dotted and
dashed lines, respectively) of calcium
humate transport through amended Oil Creek
sand column.
37
-------
Primary particle size
Macromolecule Cone, (ppm) frequency distribution (rim)
Blue dextran 100 76
Poly(ethylene oxides)
50,700 MW 1261 ND2
83,800 MW 1119 190
770,000 MW 1044 140
900,000 MW 961 201
Humates
NaClO4/humate 327 200-250,350-525
CaSO4/humate 285 350-525
Molecular Weight
Not Determined
Table 1. Particle size frequency distribution for study macromolecules.
38
-------
Macromolecule
Cone (ppm) Column Packing % Recovery
Observed Relative
Velocity (Rf)'
Blue dextran
100
100
OCS2
AOCS3
98
77
1.00
1.30
Poly(ethylene oxides)
50,700 MW4 1261
83,800 MW 119
770,000 MW 1044
900,000 MW 961
OCS
OCS
OCS
OCS
95
98
84
109
0.86
0.98
0.96
1.12
Calcium humate
285
320
OCS
AOCS
107
100
1.01
Sodium humate
325
OCS
96
0.98
1 R =(v /v )
1 Oil Creik sand
3 amended Oil Creek sand
4 molecular weight
Table 2. Concentrations, recoveries and relative velocities of study macromolecules.
39
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