Question Project 1: Non-Equilibrium Separations in The Real World
Non-equilibrium separation processes are constantly being developed and optimized for use in a
range of industries including (but not limited to): petroleum, food production, pharmaceuticals,
defense, green energy, and consumer products. This research is conducted both in industrial
R&D departments and in academic research laboratories. For this project, you need to select an
article from the literature that presents on a topic we have covered in the "non-equilibrium
separations" portion of the class (batch distillation, drying, crystallization, membrane
separations, adsorption, ion exchange, chromatography). You will then use this article to develop
a homework-style problem with significant background and detailed solutions.
Tasks:
1. Select an article
2. Background/Introduction: Please summarize the process described in the paper, both the
details of the paper and the theory that underlies the separation process. This will likely
require research beyond your chosen article. Remember to cite all sources and include a
bibliography of the literature you reference. In particular, please discuss:
a. The relevant background information for understanding the paper and problem
statement including the theory discussed in class
b. Why this separation process is being studied. What are its advantages and
disadvantages and how can it be/was it improved?
c. The driving force at play during this separation
d. The factors that were or could be optimized and how they would affect the
separation (purity, rate, time required)/n APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2000, p. 2914-2920
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 66, No. 7
Influence of Salts on Virus Adsorption to Microporous Filters+
JERZY LUKASIK,* TROY M. SCOTT, DIANE ANDRYSHAK, AND SAMUEL R. FARRAH
Department of Microbiology and Cell Science, University of Florida,
Gainesville, Florida 32611-0700
Received 15 March 1999/Accepted 11 April 2000
We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl2, and AICI) on
the adsorption of several viruses (MS2, PRD-1, X174, and poliovirus 1) to microporous filters at different pH
values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose),
and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been
used in virus removal and recovery procedures. The direct effects of added salts were considered to be the ef-
fects associated with the presence of the soluble salts. The indirect effects of the added salts were considered
to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb
viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study
promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption,
depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the
addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the
enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral ad-
sorption observed when aluminum chloride was added to water was related to the decrease in the pH of the
water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7
following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs
by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus
adsorption that we observed was interference with adsorption to microporous filters. Under conditions under
which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Milli-
pore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence
of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).
The effects of salts on virus adsorption to microporous filters
have been studied for many years and have been discussed in
several reviews (4, 13, 16, 17, 28). In previous studies, the
researchers concluded that addition of aluminum ions (or mag-
nesium ions) enhanced viral adsorption to microporous filters,
and from these results it was concluded that the presence of
salts is necessary for optimum viral adsorption to the filters
(4, 13, 16, 17, 27, 28). However, we found two problems with
the earlier studies. First, relatively few viruses and few micro-
porous filters were studied. Much of the information was in-
formation concerning the adsorption of poliovirus to nitro-
cellulose filters (Millipore filters). However, due to recent
developments in filter technology, new filters that have sub-
stantially different properties than the filters previously studied
are currently being used. Therefore, it is necessary to evaluate
the effects of salts on these filters.
Second, previous studies on the effects of salts on virus
adsorption did not distinguish between direct and indirect ef-
fects of the salts that influenced viral adsorption. The direct
effects that have been proposed include formation of salt
bridges between the viruses and the filters (16) and alteration
of the charge of a filter (14). The indirect effects include (i) a
decrease in the pH due to addition of aluminum salts to puri-
fied water (28); (ii) the formation of flocs that adsorb viruses
and are then physically trapped by the filters (9, 28); and (iii)
* Corresponding author. Mailing address: Department of Microbi-
ology and Cell Science, University of Florida, Gainesville, FL 32611-
0700. Phone: (352) 392-1885. Fax: (352) 392-5922. E-mail: george
@micro.ifas.ufl.edu.
+ Paper number R-07534 from the Florida Agricultural Experiment
Station, Gainesville.
2914
the reaction between aluminum ions and humic materials that
interfere with virus adsorption (10, 25).
In order to better understand the forces involved in viral
adsorption to solids, we investigated the adsorption of four
viruses to four commercially available filters having different
compositions and different physical characteristics. Two of the
filters used in this study (Filterite and 1MDS filters) are cur-
rently used for recovering viruses from water (1, 2). The Fil-
terite filters have a net negative charge at pH values near
neutrality, in contrast to the 1MDS filters, which are positively
charged or have a slight negative charge at similar pH values
(14, 24). Nitrocellulose filters (Millipore HA filters) also have
a net negative charge at pH 7 (14) and have been used in
studies on virus adsorption and to recover viruses from water
(6, 7, 12, 28). Cellulose filters (Whatman filters) were included
as an example of filters that poorly adsorb viruses. These filters
are made from material that is electronegative at pH 7 (26)
and have little ability to adsorb viruses unless they are modified
(11).
Mono- and multivalent salts were used at different pH val-
ues, and compounds that have been shown to disrupt hydro-
phobic interactions were also used. In addition, the effects of
different concentrations of aluminum and the effects of differ-
ent concentrations of magnesium on pH and viral adsorption,
respectively, were studied. The previously described direct and
indirect effects were minimized by controlling flocculation and
pH by filtering and buffering. All virus stocks and salt solutions
were prefiltered through 0.2-μm-pore-size filters prior to each
experiment to reduce the effects of aggregation and floccula-
tion. The pH was monitored and controlled, and only purified
water was used. Under these conditions, magnesium chloride
was found to promote virus adsorption, to interfere with virus
adsorption, and to have little or no effect on virus adsorption,
Downloaded from https://journals.asm.org/journal/aem on 12 February 2024 by 128.138.65.188. VOL. 66, 2000
Percent Adsorption
120
100
80
60
40
20
0
0
-20
0.001
-1 MDS
-HA
0.01
0.1
VIRUS ADSORPTION TO MICROPOROUS FILTERS
PercentAdsorption
100
80
60
60
HA
40
+1MDS
20
2915
Magnesium Chloride Concentration (M)
FIG. 1. Influence of magnesium chloride concentration on adsorption of
poliovirus 1 to 1MDS and Millipore HA filters at pH 7.
depending on the virus and the microporous filter tested. The
most consistent effect of sodium chloride and aluminum chlo-
ride on virus adsorption was to interfere with virus adsorption
to 1MDS filters.
MATERIALS AND METHODS
Filtration of samples. All viral stocks and solutions were prefiltered through a
0.2-μm-pore-size filter (Millipore GS; Millipore Corp., Bedford, Mass.) that had
been prewashed with 20 ml of deionized water before use.
Viruses. The phages used in this study, their isoelectric points (1), and their
hosts are as follows: MS2 (= ATCC 15597-B1), pl 3.9, Escherichia coli C-3000
(= ATCC 15597); X174 (= ATCC 13706-B1), pl 6.6, E. coli ATCC 13607; and
PRD-1, pl 4.2, Salmonella typhimurium ATCC 19585. Numbers of phage PFU
were determined by using the appropriate hosts and a soft-agar overlay (23).
Poliovirus 1 (pl, approximately 4 and 7 [13]) was grown on BGM cells, and the
number of poliovirus PFU was determined by an agar overlay method (22).
Microporous filters. The flowing microporous filters were used: Millipore HA
filters (nitrocellulose; Millipore Corp.); Filterite 0.20-μm-pore-size filters (bound
fiberglass; Filterite Corp., Timonium, Md.); Whatman no. 5 filters (cellulose;
Fischer Scientific, Pittsburgh, Pa.); and 1MDS filters (charge-modified fibers;
AMF Cuno, Meridan, Conn.). The Filterite and 1MDS filters were purchased as
cartridge filters. These filters were broken down, and smaller filters were cut
from the filter material. The Millipore, Filterite, and Whatman filters are neg-
atively charged at pH values near neutrality; the 1MDS filters are positively or
slightly negatively charged at the same pH values (14, 19, 24, 26).
Solutions. Solutions of sodium chloride, magnesium chloride, aluminum chlo-
ride, urea, and Tween 80 were prepared with a buffer solution (0.02 M imid-
azole-0.02 M glycine, unless otherwise indicated). The solutions were adjusted to
the required pH by adding 1 M NaOH or 1 M HCl. Tap water was dechlorinated
by adding 10 mg of sodium thiosulfate per liter. Deionized water (>15 MQ-cm)
was obtained from a Barnstead NANOpure II unit.
Aluminum chloride and magnesium chloride titration experiments. For alu-
minum chloride and magnesium chloride titration experiments (Fig. 1 through
3), aliquots of a 1.00 M aluminum chloride or 1.00 M magnesium chloride
solution were added to a 0.002 M glycine-0.002 M imidazole solution or to
purified water to obtain the desired concentrations. Since the concentrations of
salts used in these experiments were lower than the concentrations used in other
experiments, a lower concentration of buffer was used to reduce interference
with the metallic ions by buffer ions.
In addition, aluminum chloride was added to deionized water, and the pH was
recorded. The pH values of samples of deionized water were then adjusted with
HCl to values that matched the pH values of the solutions described above that
0
0.0001
0.001
0.01
Aluminum Chloride Concentration (M)
0.1
FIG. 3. Influence of aluminum chloride concentration on adsorption of po-
liovirus 1 to 1MDS and Millipore HA filters at pH 3.5.
contained aluminum chloride. Virus adsorption experiments were then con-
ducted by using samples containing aluminum chloride and samples containing
HCl at the same pH value (Fig. 4).
The pH values of all solutions were measured at the beginning and at the end
of each experiment.
Experimental procedure. Most experiments were conducted at pH 7.0 or 3.5.
The lower pH (pH 3.5) was selected since it has been used in several other
studies and is the recommended pH for recovering viruses in water when Filterite
filters are used (2, 3, 18, 27). Also, the aluminum salts used (at the concentrations
used) are soluble at pH 3.5. Aluminum salts were not used at pH 7 (Table 1 and
2) since they are relatively insoluble and form flocs that can adsorb viruses (8, 9).
Viruses were added to solutions to obtain an initial titer of approximately 105
PFU/ml. The viruses in the solutions were assayed following dilution in 1%
tryptic soy broth (Difco) for phages or in minimal essential medium supple-
mented with 2% fetal calf serum for poliovirus. In each case the dilution was
sufficient to raise the pH of the sample to approximately 7. To prevent floccu-
lation when experiments were performed with aluminum chloride, an initial 1/10
dilution with deionized water was prepared, and then a second dilution with the
dilution media described above was prepared. The filters used were 25-mm
circles or filters cut into 25-mm circles and placed into stainless steel filter
holders. One layer of Millipore HA filter material, two layers of Filterite filter
material, two layers of Whatman no. 5 filter material, or three layers of 1MDS
filter material were placed in each holder for the experiment. Each filter prep-
aration was first rinsed with 60 ml of deionized water. Then 25 ml of the salt
solution containing viruses was passed through each filter by using a mechanical
syringe infusion pump (Harvard Apparatus Co., Millis, Mass.) at a flow rate 1.5
ml/s. The filter effluents were assayed for viruses, and the percentage removed
was determined. A portion of each sample that had not been passed through any
filter was assayed at the beginning and at the end of each experiment to deter-
mine if the solutions inactivated the viruses. The numbers of viruses in the
unfiltered and filtered samples were used to determine the percentages removed
by the filters. Each experiment was performed in triplicate. In addition, each
experiment was performed at least twice. Therefore, each value reported below
represents a mean based on at least six determinations. The error associated with
the values reported was less than √n, where n is the number of the PFU counted.
A statistical analysis of the data obtained (standard deviations, slopes, correla-
tion, and general t test probabilities) was performed by using PSI-Plot software
(Poly Software International, Salt Lake City, Utah).
Aluminum Chloride (M)
1.1E-7 6.3E-7 3.14E-6 4.9E-5 1.2E-5 1.9E-4 7.8E-4 3.1E-3 1.3E-2 0.05
Percent Adsorption
60
282222°
120
100
80
40
20
0
0
1 MDS
--HA
Percent Adsorption
120
100
60
80
60
40
40
20
0
Buffer
Aluminum chloride.
0.001
0.01
Magnesium Chloride Concentration (M)
0.1
FIG. 2. Influence of magnesium chloride concentration on adsorption of
poliovirus 1 to 1MDS and Millipore HA filters at pH 3.5.
7
6 5.2 4.9 4.65 4.42 4.18 3.96 3.72 3.43
pH
FIG. 4. Influence of aluminum chloride concentration and the corresponding
pH of a buffer solution on adsorption of poliovirus 1 to Millipore HA filters. The
pH values used corresponded to the pH values resulting from dissolution of
aluminum chloride at the concentrations used.
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LUKASIK ET AL.
TABLE 1. Effects of salts on virus adsorption to
microporous filters at pH 7ª
% Virus adsorption
Buffer containing
0.1 M MgCl2
Filter
Virus
Buffer containing
Buffer
0.1 M NaCl
Millipore HA
MS2
8 Ab
18 B
99 C
PRD-1
6 A
8 A
59 B
OX174
4 A
40 B
57 C
Poliovirus 1
3 A
98 B
Mean
5
41
99 B
79
Filterite
MS2
6 A
21 B
PRD-1
11 A
9 A
OX174
3 A
3 A
23 B
10 A
5 A
Poliovirus 1
10 A
9 A
99 B
Mean
7
11
34
Whatman
MS2
18 A
12 A
19 A
PRD-1
7 A
3 A
6 A
OX174
5 A
9 A
38 B
Poliovirus 1
8 A
6 A
Mean
10
8
17
1MDS
MS2
96 A
10 B
PRD-1
97 A
13 B
13 B
OX174
29 A
12 B
Poliovirus 1
Mean
79 A
7 B
75
11
18 C
9 B
12
5 A
7 B
a We added approximately 105 PFU of virus to 25-ml portions of buffer, buffer
containing 0.1 M NaCl, and buffer containing 0.1 M MgCl2, and each type of
solution was passed through the different types of filters at a rate of 1.5 ml/s. The
numbers of viruses in the influent and effluent were measured in order to
determine the percentage of adsorption in each case.
b Values on the same line followed by the same letter are not significantly
different at P = 0.05.
APPL. ENVIRON. MICROBIOL.
the adsorption of poliovirus to filters is shown in Fig. 1 and 2.
At pH 7, increasing the concentration of magnesium chloride
increased poliovirus 1 adsorption to Millipore HA filters but
decreased poliovirus 1 adsorption to 1MDS filters (Fig. 1). At
pH 3.5, increasing the concentration of magnesium chloride
(Fig. 2) did not affect adsorption of poliovirus 1 to Millipore HA
filters but decreased poliovirus 1 adsorption to 1MDS filters.
The effects of different concentrations of aluminum chloride
on the adsorption of poliovirus to Millipore and 1MDS filters
are shown in Fig. 3. Increasing the concentration of aluminum
chloride had little effect on poliovirus adsorption to Millipore
filters but interfered with adsorption of this virus to 1MDS
filters.
The Millipore and 1MDS filters differed in hydrophobic-
ity. The contact angle for chloroform on Millipore filters was
144.2 ± 3.2°, and the contact angle for chloroform on 1MDS
filters was 151.8 ± 1.6°, which showed that the Millipore filters
were more hydrophobic.
At pH 7 and in absence of salt ions, we observed little
adsorption of poliovirus 1 in deionized water to Millipore HA
filters (Fig. 4). As the concentration of aluminum chloride was
increased, the adsorption of poliovirus increased. The pH val-
ues of the solutions also decreased as aluminum chloride was
added. When the pH values of samples of deionized water
were decreased by adding HCl, a similar trend in virus adsorp-
tion was observed. The decrease in the pH of the solution that
was caused by the addition of aluminum chloride was sufficient
to explain the observed increase in virus adsorption associated
with the addition of aluminum chloride.
Adding urea in the absence of salt ions did not have a sig-
Measurement of aluminum concentrations. Solutions were analyzed to deter-
mine total aluminum contents before and after filtration by workers at the
Analytical Research Laboratory of the University of Florida.
Contact angle measurements. Contact angles for chloroform on filters were
measured as previously described (21).
TABLE 2. Effects of 0.1 M MgCl3 and 4 M urea on virus
adsorption to microporous filters at pH 7"
% Adsorption in the presence of:
Buffer containing
4 M urea plus
0.1 M MgCl2
RESULTS
The effects of solutions of salts at pH 7 on the adsorption of
the viruses studied to the filters depended on the filter type and
the salt added (Table 1). In general, addition of salts increased
the adsorption of viruses to Millipore filters and interfered
with the adsorption to 1MDS filters. Except for X174, the
salts did not increase viral adsorption to Whatman filters. Mag-
nesium chloride greatly increased the adsorption of poliovirus
to Filterite filters; the salts had little or no effect on adsorption
of the phages tested to these filters.
The effects of buffer and buffer containing a salt (sodium
chloride, magnesium chloride, or aluminum chloride) at a con-
centration of 0.1 M on adsorption of viruses at pH 3.5 are
shown in Table 3. None of the salts affected viral adsorption to
Millipore or Whatman filters. More than 95% of the viruses
tested adsorbed to Millipore filters, and less than 10% ad-
sorbed to Whatman filters under all of the conditions tested;
however, the salts interfered with viral adsorption to 1MDS
and Filterite filters. The degree of interference depended on
the virus and the salt used. Aluminum chloride interfered with
adsorption of viruses to 1MDS filters more than magnesium
chloride or sodium chloride interfered with such adsorption.
Only aluminum chloride interfered significantly with virus ad-
sorption to Filterite filters.
The effect of the concentration of magnesium chloride on
Filter
Virus
Buffer
Buffer containing
4 M urea
Millipore HA
MS2
PRD-1
8 Ab
6 A
1 B
6 A
3 A
3 A
«Χ174
4 A
4 A
2 A
Poliovirus 1
3 A
7 A
2 A
Mean
5
5
2
Filtrite
MS2
6 A
8 A
3
B
PRD-1
11 A
6 A
2
B
OX174
3 A
3 A
1 A
Poliovirus 1
10 A
11 A
10 A
Mean
8
7
4
Whatman
MS2
18 A
4 B
5
B
PRD-1
OX174
7 A
4 A
4
A
5 A
5 A
7 A
Poliovirus 1
8 A
4 A
7 A
Mean
10
4
6
1MDS
MS2
96 A
96 A
6 B
PRD-1
97 A
90 A
11 B
ΦΧ174
29 A
21 A
10
B
Poliovirus 1
79 A
68 A
8
B
Mean
75
69
9
a We added approximately 105 PFU of virus to 25-ml portions of buffer, buffer
containing 4 M urea, and buffer containing 4 M urea plus 0.1 M MgCl2, and each
type of solution was passed through the different types of filters at a rate of 1.5
ml/s. The numbers of viruses in the influent and effluent were measured in order
to determine the percentage of adsorption in each case.
b Values on the same line followed by the same letter are not significantly
different at P = 0.05.
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VIRUS ADSORPTION TO MICROPOROUS FILTERS
2917
TABLE 3. Effects of salts on virus adsorption to microporous filters at pH 3.5ª
% Virus adsorption
Filter
Virus
Buffer containing
Buffer
0.1 M NaCl
Buffer containing
0.1 M MgCl2
Buffer containing
0.1 M AlCl3
Millipore HA
MS2
>99 Ab
>99 A
>99 A
>99 A
OX174
98 A
>99 A
>99 A
>99 A
Poliovirus 1
>99 A
>99 A
>99 A
>99 A
Mean
>99
>99
>99
>99
Filtrite
MS2
>99 A
98 AB
>99 A
96 B
OX174
95 A
97 A
98 A
23 B
Poliovirus 1
99 A
>99 A
97 A
10 B
Mean
98
98
98
43
Whatman
MS2
3 A
4 A
3 A
4 A
OX174
6 A
2 A
2 A
4 A
Poliovirus 1
9 A
7 A
5 A
7 A
Mean
6
4
3
5
1MDS
MS2
95 A
60 B
44 C
5 D
OX174
85 A
75 B
73 B
10 C
Poliovirus 1
Mean
99 A
95 A
93 A
10 B
93
76
70
14
a We added approximately 105 PFU of virus to 25-ml portions of buffer, buffer containing 0.1 M NaCl, buffer containing 0.1 M MgCl2, and buffer containing 0.1 M
AlCl3, and each type of solution was passed through the different types of filters at a rate of 1.5 ml/s. The numbers of viruses in the influent and effluent were measured
in order to determine the percentage of adsorption in each case.
b Values on the same line followed by the same letter are not significantly different at P = 0.05.
nificant effect on viral adsorption to the filters at pH 7 (Table
2). However, viral adsorption was greatly reduced in the pres-
ence of both 4 M urea and a salt at a concentration of 0.1 M.
We observed a similar effect at pH 3.5; urea alone had little
effect on virus adsorption (Table 4). Adding 0.1 M magnesium
chloride or 0.1 M aluminum chloride to solutions of urea re-
duced the adsorption of both MS2 and poliovirus 1 to the fil-
ters tested. Tween 80 at a concentration of 0.1% had the same
effect on viral adsorption as 4 M urea had (data not shown).
The effects of prefiltering aluminum chloride solutions made
with tap water before viruses were added are shown in Table 5.
When a 0.0001 M aluminum chloride solution was prepared
with tap water at pH 7, viruses added to the solution were
removed by a Millipore HA filter. If the aluminum chloride
solution was first passed through a 0.2-μm-pore-size Millipore
GS filter before the viruses were added, then no significant
viral adsorption to Millipore HA filters was observed. The pre-
filtering step decreased the concentration of aluminum chlo-
TABLE 4. Effects of 0.1 M AlCl3 or 0.1 M MgCl 3 and 4 M urea on virus adsorption to microporous filters at pH 3.5ª
% Adsorption in the presence of:
Filter
Virus
Buffer
Buffer containing
4 M urea
Buffer containing 4 M urea
plus 0.1 M MgCl2
Buffer containing 4 M urea
plus 0.1 M AlCl3
Millipore HA
MS2
>99 Ab
+X174
Poliovirus 1
Mean
98 A
>99 A
98 A
95 A
15 B
13 B
9 B
7 B
>99 A
12 B
15
B
99
Filtrite
MS2
>99 A
ΦΧ174
95 A
Poliovirus 1
99 A
Mean
98
12 5225
97
12
12
95 B
14 B
11
B
92 B
7 B
8
B
99 A
13 B
15
B
95
11
11
Whatman
MS2
3 A
9 A
11 A
7 A
ΦΧ174
6 A
4 A
3 A
6 A
Poliovirus 1
9 A
7 A
8 A
4
A
Mean
6
7
7
6
1MDS
MS2
95 A
95 A
10 B
8
B
ΦΧ174
Poliovirus 1
Mean
85 A
69 A
48 B
30 C
99 A
93
99 A
88
50 B
36
10 C
16
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a We added approximately 105 PFU of virus to 25-ml portions of buffer, buffer containing 4 M urea, buffer containing 4 M urea plus 0.1 M MgCl2, and buffer
containing 4 M urea plus 0.1 M AlCl3, and each type of solution was passed through the different types of filters at a rate of 1.5 ml/s. The numbers of viruses in the
influent and effluent were measured in order to determine the percentage of adsorption in each case.
b Values on the same line followed by the same letter are not significantly different at P = 0.05. 2918
LUKASIK ET AL.
TABLE 5. Effect of prefiltering the aluminum chloride solutions
used in viral adsorption experiments on viral adsorption
to Millipore HA filters at pH 7ª
Virus
MS2
PRD-1
ΦΧ174
Poliovirus 1
Not prefiltered
>99 Ab
98 A
95 A
>99 A
98
APPL. ENVIRON. MICROBIOL.
TABLE 7. Effect of salt type on virus adsorption to
1MDS filters at pH 3.5"
% Viral adsorption
% Viral adsorption
Virus
Buffer'
Prefiltered
Buffer containing Buffer containing Buffer containing
0.1 M AlCl3
0.2 M MgCl2
0.6 M NaCI
13 B
MS2
95 Ac
5 B
10 C
13 C
8 B
«Χ174
85 A
10 B
24 C
31 C
14 B
Poliovirus 1
Mean
99 A
10 B
36 C
20 C
93
8
23
21
16 B
13
Mean
a The concentration of aluminum chloride in 100 ml of dechlorinated water
containing 105 PFU of virus per ml was adjusted to 0.0001 M. Fifty milliliters was
passed through a Millipore HA filter at a rate of 1.5 ml/s. The remaining 50 ml
was first passed through a glass fiber prefilter (Millipore GS). This solution was
then passed through a Millipore HA filter. In each experiment the numbers of
viruses in the influent and effluent were measured, and the corresponding per-
centage of adsorption was determined.
b Values on the same line followed by different letters are significantly different
at P = 0.05.
ride from 0.0001 M to less than the detectable level (<0.00003
M). In contrast, no change in the aluminum chloride concen-
tration was observed following filtration of 0.0001 M aluminum
chloride solutions through the same type of filter at pH 3.5
(where aluminum chloride is more soluble).
In order to separate the effects of salt type from the effects
of ionic strength, we performed experiments in the presence of
added salt at a constant ionic strength. As shown in Table 6,
magnesium chloride solutions were better at promoting ad-
sorption of MS2 and PRD-1 than sodium chloride solutions at
the same ionic strength were. Sodium chloride and magnesium
chloride both promoted adsorption of X174 to nitrocellulose
filters.
Increasing the ionic strength of solutions of magnesium
chloride and sodium chloride to the ionic strength of alumi-
num chloride decreased the adsorption of viruses to 1MDS
filters (Table 7). At a constant ionic strength of 0.6, all of the
salts significantly reduced the virus adsorption that was ob-
served in solutions containing buffer alone. However, the great-
est interference with adsorption was observed with solutions of
aluminum chloride.
DISCUSSION
Based on early studies on the influence of salts on virus
adsorption, it was suggested that salts promote virus adsorp-
tion to microporous filters by promoting electrostatic interac-
TABLE 6. Effect of salt type on virus adsorption to
Millipore HA filters at pH 7
0.1 M MgCl2
% Viral adsorption
Virus
Bufferb
Buffer containing
Buffer containing
0.3 M NaCl
8 A
99 B
PRD-1
6 A
59 B
Χ174
4 A
57 B
Poliovirus 1
Mean
3 A
5
99 B
79
61 C
3 C
42 B
>99 B
51
MS2
a Experiments were performed as described in Table 1, footnote a, except that
the ionic strength of the salts was maintained at 0.3.
b Values from Table 1.
Values on the same line followed by the same letter are not significantly
different at P = 0.05.
Experiments were performed as described in Table 1, footnote a, except that
the ionic strength of the salts was maintained at 0.6.
b Values from Table 1.
© Values on the same line followed by the same letter are not significantly
different at P = 0.05.
tions between the viruses and the filters. The possible mecha-
nisms that were suggested to explain the observations made
were salt bridging and charge neutralization of the filters. It
was also suggested that the ability of salts to promote virus
adsorption was related to the valence of the cation involved.
Trivalent cations (Al³+) were better than divalent cations
(Mg2+), which were better than monovalent cations (Na+), at
promoting virus adsorption when the cations were used at the
same concentration (4, 13, 16, 17, 27, 28).
In examining the early studies on the effects of salts on virus
adsorption, we found three main problems. These were (i) the
role of salts in promoting hydrophobic interactions was not
always considered; (ii) the indirect effects of adding salts on the
pH values of solutions and flocculation of the salts were not
always determined; and (iii) few viruses and few filter types
were studied. Below we discuss these problems in relation to
our study.
Hydrophobic interactions. Previous studies on virus adsorp-
tion to microporous filters led to the suggestion that metal
chelators could interfere with virus adsorption promoted by
salts (14, 16, 17). Later studies showed that chelators, such as
the citrate ion, did not elute viruses adsorbed to Millipore HA
filters (7). In fact, chelators have been found to promote, ra-
ther than interfere with, virus adsorption to microporous filters
(6). The study of Farrah (6) and other studies showed that in-
creased virus adsorption to certain microporous filters in the
presence of cations and anions was influenced by hydrophobic
interactions (6, 12, 21). Therefore, the adsorption of MS2 and
poliovirus 1 in the presence of salts to filters was best explained
by the presence of hydrophobic interactions between the filters
and the viruses. The effect of added magnesium chloride on
virus adsorption was to strengthen hydrophobic interactions
rather than electrostatic interactions (6).
In this study, urea had little effect on virus adsorption. This
compound did not interfere with or promote adsorption of
viruses to the filters tested at pH 7.0 or 3.5. In contrast, it
greatly reduced virus adsorption when salts were present. The
neutral detergent Tween 80 had an effect similar to that of
urea, even though it was used at much lower concentrations.
As discussed above, the salts probably promoted hydrophobic
interactions and interfered with electrostatic interactions be-
tween the viruses and the filters (5, 12, 21).
Indirect effects. Adding salts can change the pH of a solution
or cause the formation of flocs, or salts can interact with or-
ganic material present in the solution. This is especially true
for aluminum salts and makes determining the direct role of
aluminum ions on virus adsorption difficult. As shown in Fig. 4,
the effect of increasing the aluminum chloride concentration
on virus adsorption reported by Wallis et al. (28) can be ex-
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