Solid phase extraction (SPE) of hydrophobic components from a model white
water.
John Mosbye1, Berit Harstad2 and Anne Fiksdahl2
1
PFI, Trondheim, Norway.
2
Norwegian University of Science and Technology, Trondheim, Norway.
Keywords: Solid phase extraction, Fatty acids, Resin acids, Sterols, Extractives, White water,
TMP.
SUMMARY: A solid phase extraction (SPE) procedure for the analysis of fatty acids, resin
acids and sterols in white water from mechanical pulp and paper industry has been developed.
Quantification was based on gas chromatography of the silylated compounds. Comparison
between the new SPE isolation method and the established liquid-liquid extraction procedure
show that the SPE procedure represents an improvement and that the SPE procedure can
replace the established method.
ADRESSES OF THE AUTHORS: Anne Fiksdahl, Berit Harstad, Organic Chemistry
Laboratories, Department of Chemistry, Sem Sælands vei 8, Norwegian University of Science
and Technology, NTNU, N-7491 Trondheim, Norway. John Mosbye, Norwegian Pulp and
Paper Research Institute, PFI, Høgskoleringen 6b, PFI, N-7491 Trondheim, Norway.
Wood extractives often cause problems in pulping and paper making because of pitch deposits
in the process system and reduced quality of the paper product. There are also additional
environmental problems such as toxic effects on marine life. Nordic mechanical pulping
production is mostly based on the Norway spruce (Picea abies), and some of the major groups
of extractives in white water from the TMP process are fatty acids, resin acids and sterols
(Hoel et al. 1996; Ekman, Holmbom 1989), see Scheme 1. The total concentration of
extractives in the process varies over a wide range (10–1000 ppm) depending on the degree of
closure, chemical additives and the stage in the process. They exist as dissolved materials, in
colloids and as colloidal particles associated with the surface of fines, fibres and fillers
(Magnus et al. 1999; Luuokko, Paulapuro 1998; Rundlöf 1996). Quantification of these
hydrophobic compounds has so far been based on a laborious and solvent consuming MTBE
liquid-liquid extraction procedure (Hoel et al. 1996; Örsa, Holmbom 1994) followed by GLC
analysis (Örsa, Holmbom 1994; Ekman, Holmbom 1989; Zinkel et al. 1968). Solid phase
extraction (SPE) has recently become an important tool for the concentration and isolation of
trace amounts of hydrophobic components in water (Thurman, Mills 1998), and the method
has been applied for sample preparation in the analysis of wood extractives from kraft pulping
(Dethlefs, Stan 1996; Chen et al. 1995; Chen et al. 1994; Backa et al. 1989). However, white
water from the production of wood containing paper is a different matrix and other challenges
arise. Some results has been reported for an SPE/GLC study of white water, however not
including a comparison with solvent extraction (Sweeney 1988).
Our aim was to develop a rapid preparation method for the three important groups of
hydrophobic extractives in white water based on SPE to replace the established liquid-liquid
extraction method. The SPE method was compared with the liquid-liquid extraction method in
1
terms of reproducibility, recovery data and amount of extracted material as measured by
GLC-FID.
Materials and methods
Preparation of model white water
Model white water was produced in the laboratory (Örså, Holmbom 1994; Thornton et al.
1991) due to the detoriation of industrial white water during storage. Model white water was
made by adding distilled water (4.89 l) to a frozen TMP portion (110 g, 45 % dry material)
taken directly from the refiners at a Norwegian integrated newspaper mill, giving a
suspension of about 1 %. The suspension was stirred for three hours at 60 °C and cooled.
Instead of centrifugation (Örså, Holmbom 1994), the sample was filtered (Britt Dynamic
Drainage Jar/BDDJ, pore size 76 m) and stored in bottles at 5°C. This suspension consisting
of dissolved and colloidal material and fines was used as model white water.
Standard solutions
Recovery standard: Calculations of % recovery for respective fatty acids, resin acids and
sterols were based on one reference substance for each group; an acetone solution with exact
concentrations of heptadecanoic acid (Fluka), o-methylpodocarpic acid (Helix Biotech Corp.)
and cholesterol (Sigma) (about 30 mg of each in 100 ml acetone) was used. This solution (1
ml) was added to each sample.
Injection standard: An exact concentration (0.27 mg/ml) of heptadecane (Sigma) in
cyclohexane was used.
Calibration standards: Calibration curves were based on five standard solutions with the
respective approximate concentrations 0.0015, 0.015, 0.075, 0.15 and 0.3 mg/ml of each of
the following fatty acids, resin acids and sterols: heptadecanoic acid (Fluka), linoleic acid
(Sigma), pimaric acid, sandaracopimaric acid, isopimaric acid, palustric acid, omethylpodocarpic acid, dehydroabietic acid, abietic acid, neoabietic acid (Helix Biotech
Corp.), cholesterol, ß-sitosterol, stigmasterol (Sigma).
Calibration curves for each component were established for each series of GLC analysis of
extractives based on the calibration standards and the injection standard.
Sample preparation prior to SPE
The model white water bottle was shaken for 1 minute and three parallel samples (á 20 ml)
were transferred to 100 ml sample bottles. The recovery standard (1 ml) and acetone (20 ml)
were added and the solutions were vigorously stirred for 30 minutes before filtration using
Whatman Glass Microfibre filters GF/A (approximate pore diameter 1.6 m). The filters and
the sample bottles were separately washed with acetone/distilled water (5 ml, 1:1) which was
added to the filtered samples.
Solid Phase Extraction.
Disposable syringe columns packed with sorbent (1 g) (IST, International Sorbent
Technology, Ltd.) and a vacuum manifold system were used. Different sorbents were tested;
C18 (EC, end-capped), C18 (not end-capped) and C8 (EC, end-capped). Based on % recovery
and reproducibility, the C18 (EC) column was used in the following work. The column was
solvated with methanol (10 ml) and washed with distilled water (10 ml) before the sample
(about 50 ml) was applied to the column and eluted at a flowrate of about 5 ml/min. The
2
sample bottle was washed with acetone/water (1:1, 5 ml) which was added to the column. The
sample matrix was removed by a flow rate of maximum 5 ml/min. Before elution of the
extractives the column was dried by suction for 30 min. Methanol (5 ml) was added and
eluted after 2 min. The elution was completed by methanol (10 ml) followed by
dichloromethane (15 ml). The sample was transferred to a smaller test tube. The solvents were
removed by flushing N2, and the injection standard (1 ml) was added to the dried sample. This
sample could be stored in a freezer before silylation.
Liquid-liquid extraction.
The results obtained by SPE were compared with corresponding results using the established
MTBE extraction method described elsewhere (Holmbom, Örså 1993). Four parallel
extractions were carried out at pH 3.5 and pH 9.0 (±0.1).
Silylation.
The extracts were silylated with HMDS : TMCS (Tri-Sil®, Pierce, 50 l) and analysed by
GLC-FID (Örså, Holmbom 1994; Ekman, Holmbom 1989).
GLC-FID.
Quantitative GLC analyses were carried out on a Hewlett Packard HP 6890 Series System;
injector (300°C), split 1:10, helium, detector temp. = 320°C, column: HP-5 (30 m x 0.32 mm;
0.25 m), temperature program: 100°C/1 min. - 5°C/min. – 300°C/5 min.
Calculation of the concentration of each component was based on integration area
component:injection standard ratio, calibration curves for the component and the % recovery.
Results are given in Table 2 and Figure 1 - 3.
Results and discussion
Our work focused on the quantification of the free fatty acids, resin acids and sterols. For the
quantification of the total amount of fatty acids and sterols, including the esterified
compounds, the procedure should include a hydrolysis step. Another group of important
extractives is the lignans. The semi-polar lignans can be extracted from water with MTBE or
Diethyl ether (Örså, Holmbom 1994), and was therefore expected to be retarded by the
unpolar stationary phase. An examination of the water phase eluted from the column showed
that the lignans wasn’t retarded. The presence of eight of the most important lignans could be
identified by GC-MS of the MTBE extracted eluted water phase. The reason for this could be
the addition of aceton to the matrix. By adding aceton the polarity of the model white water
could be changed and thus making the lignans more soluble. If the acetone is absent it is
possible that the lignans will be retarded on the column, but this has not been checked.
Since white water is a complex matrix which also contains particles such as fines and
colloidal extractives in addition to dissolved material, the sample had to be filtered before
SPE. Even after filtering the column plugged. However, we observed that this problem was
reduced by the addition of acetone to the sample (1:1). Subsequent stirring for 30 minutes is
recommended to avoid the loss of extractives at the filter. To confirm that no extractives had
been lost at the filter or in the water phase, the filter was Soxhlet extracted with MTBE, the
filtered water phase was MTBE extracted and both extracts were analysed by GLC-FID.
The extractives, which are dissolved or as colloids, are either neutral or acidic. When
the pH is around 5, some of the acidic groups will be dissosiated and thus making the surface
of the colloids negatively charged. This negative charge will electrostatically stabilize the
colloids by creating a layer of counterions, stretching from the surface of the colloid and into
3
the model water phase. In pure systems this charge will be the only way to stabilize the
colloids, and thus making them susceptible for destabilization by adding simple electrolytes
(Sundberg et al. 1994). In prosess waters the colloids will also be stabilized sterically by
dissolved carbohydrates (Sundberg et al. 1996), and possibly also by lignosaccharides
(Alvarado Jacobs 1995). It is possible that the addition of aceton prior to SPE increased the
amount of dissolved hydrophobic colloidal material in the water phase and thereby redused
the plugging of the column. Alternatively it is possible that the aceton reduces the amount of
carbohydrates or lignosaccharides on the surface of the colloids so that the hydrophobic
material are more easily detatched from the surface of fines and other particles and adsorbed
on the sorbent in the SPE columns.
Several reversed phase sorbents were tested: C18 (EC, end-capped), C18 (not EC)
and C8 (not EC). The % recovery of three recovery standards that were representative for the
fatty acids, the resin acids and the sterols respectively, was calculated and the concentration of
extractives in white water samples was determined. The best results regarding % recovery and
reproducibility (standard deviation) were obtained using the C18 (EC) sorbent. This sorbent
was concluded to be best suited for the actual extraction and was applied in the further work.
The effect of pH on recovery and yields has been discussed in previous works by
several authors regarding both the SPE and the liquid-liquid extraction of extractives
(Dethlefs, Stan 1996; Hoel et al. 1996; Peng, Roberts 1996; Örså, Holmbom 1994; Portugal et
al. 1992; Backa et al. 1989; Voss, Rapsomatiotis 1985). No simple conclusion has been given
about whether pH adjustment gives improved results. At low pH a stronger retention on the
sorbent and a better recovery of the fatty acids and the resin acids could be expected because
of the fully protonated carboxylic acid functions. On the other hand the amount of extractives
in colloidal form is expected to be reduced at high pH caused by the more water-soluble
nature of the ionic groups. A higher solubility in the matrix would make these extractives
more available for retention at the sorbent. However, the major fraction of the lipophilic
extractives still exists as colloids. Diverging results regarding the effect of pH on recovery
and yields has been reported. Our SPE results based on original samples and pH adjusted
samples (pH 3 and 12) showed that the % recovery was higher and the reproducibility
(standard deviation of totals) was better using the original pH which is about 5, see Table 1.
At pH 3 and 12 the recovery of the three recovery standards was lower (2-76 %) than at pH 5
(73 – 94 %). The standard deviation of totals was respectively ±56 % (pH 3) and ±62 % (pH
12) based on five parallels of white water samples, while the respective standard deviation of
totals was ± 5 % without adjusting the pH. This is comparable or better than the respective
data from the established MTBE liquid-liquid extraction method (69 – 99 % recovery, ±15 %
standard deviation of totals). We conclude that the pH should not be adjusted and furthermore
that the SPE method gives more reproducible results than the liquid-liquid extraction method.
Table 1. Recovery and reproducibility obtained by different isolation methods and pH.
Method
SPE
Liquid-liquid
(MTBE) extraction
pH of sample
Recovery of
3 recovery standards
73 – 94 %
50 – 70 %
2 – 76 %
69 - 99 %
original pH (5)
pH 3
pH 12
pH 3.5 and 9
4
Standard deviation
of totals
±5%
± 56 %
± 62 %
± 15 %
A comparison between results obtained by the established MTBE liquid-liquid
extraction procedure and the SPE method was made, see Table 2 and Figures 1 - 3. Two fatty
acids; palmitic and stearic acids, and two resin acids; pimaric and levopimaric acids, could not
be isolated and detected in all parallel samples neither by SPE or by liquid-liquid extraction.
Because of this, their standard deviation (>100 %) is not given in Table 2 and Figures 1 and 2.
In general our results showed that the concentrations of the single fatty acids (Fig. 1)
and resin acids (Fig. 2) were higher, while the sterol β-sitosterol (Fig. 3) were lower using the
SPE method than using the MTBE procedure. The β-sitosterol was chosen to be
representative for the total extracted amount of sterols and it was quantified as done elsewhere
(Magnus et al., 1999). β-Sitosterol is the major sterol in white water samples (Ekman,
Holmbom 1989). In conclusion, as can be seen from Figure 3, the yield of the three groups of
extractives in total is slightly higher and the reproducibility is better for the SPE method. By
using the SPE and the MTBE extraction methods the total concentration of extractives was
respectively 17.1 ± 0.8 mg/l (± 4.8 %) and 15.3 ± 2.3 mg/l (± 15.1 %).
An added benefit which was as important as the results above is that only half the time
was needed for an analysis and that the solvent consumption was reduced by approximately
80 % by replacing the MTBE extraction procedure with the SPE method.
Conclusions
In conclusion, the developed SPE procedure can replace the established MTBE liquid-liquid
extraction procedure for the preparation of fatty acids, resin acids and sterols in the analysis of
extractives in white water. The yields in total are slightly increased, the % recovery was found
to be comparable with the MTBE method and the reproducibility of the results was improved
using the SPE method. Moreover, the time required and the solvent consumption for each
analysis were both reduced.
Literature
Alvarado Jacobs F (1995): Extractives in process waters from newsprint papermaking
(TMP). Thesis for the doctoral degree, KTH, Stockholm, Sweden.
Backa, S., Brolin, A. and Nilvebrant, N.-O. (1989): Analyzing wood extractives from
process streams by using alkaline reversed-phase extraction followed by in situ ion pairing,
Tappi J. 72(8), 139.
Chen, T., Breuil, C. and Carriere, S. (1994): Solid-phase extraction can rapidly separate lipid
classes from acetone extracts of wood and pulp, Tappi J. 77(3), 235.
Chen, T., Wang, Z., Zhou, Y., Breuil, C., Aschim, O. K., Yee, E. and Nadeau, L. (1995):
Using solid-phase extraction to assess why aspen cause more pitch problems than softwood in
kraft pulping, Tappi J. 78(10), 143.
Dethlefs, F. and Stan, H.-J. (1996): Determination of resin acids in pulp mill EOP bleaching
process effluent, Fresenius J. Anal. Chem. 356, 403.
Ekman, R. and Holmbom, B. (1989): Analysis by gas chromatography of the wood
extractives in pulp and water samples from mechanical pulping of spruce, Nord. Pulp Pap.
Res. J. 1,16.
Hoel, H., Løken, T. and Hemming, J. (1996): Kritiske parametre ved kvantitativ
bestemmelse av harpikssyrer i TMP-avløp ved væske-væske-ekstraksjon, NordPap DP 4/7
Scan Forsk report 671.
5
Holmbom, B. and Örså, F. (1993): Methods for analysis of dissolved and colloidal wood
components in papermaking process waters and effluents, 7th Intern. Symp. Wood Pulping
Chem., Beijing, Proc. Vol 2, 810.
Luuokko, K. and Paulapuro, H. (1998): Development of fines quality in the TMP process,
84th Annual meeting, Technical section CPPA, B23-B28, Montreal Canada.
Magnus, E., Hoel, H. and Carlberg, G. E. (1999): TMP wastewater treatment including
biological high-efficiency compact reactor, Part 2: Toxicity reduction and removal of
extractives, accepted for publication in Nord. Pulp Pap. Res. J.
Peng, G. M. and Roberts, J. C. (1996): Resin acid formation during thermomechanical
pulping, Intern. Environm. Conf., Proc., 1.
Portugal, I., Vital, J. and Lobo, L. S. (1992): Resin acids isomerization, a kinetic study,
Chem. Eng. Sci. 47, 2671.
Rundlöf, M. (1996): Quality of fines of mechanical pulp, Thesis for the degree of Licentiate
of Engineering, KTH Sweden.
Sundberg, K., Thornton, J., Ekman, R. and Holmbom, B. (1994): Interactions between
simple electrolytes and dissolved and colloidal substances in machanical pulp, Nord. Pulp
Pap. Res. J. 9(2), 125-128
Sundberg, K., Thornton, J., Holmbom, B. and Ekman, (1996): Effects of wood
polysaccharides on the stability of colloidal wood resin, J. Pulp Pap. Sci. 22(7), J226-J230
Sweeney, K. M. (1988): Solid phase extraction techniques in the pulp and paper industry,
Tappi J. 71(1), 137.
Thornton, J., Eckerman, C. and Ekman, R. (1991): Effects of peroxide bleaching of spruce
TMP on dissolved and colloidal organic substances, 6th Intern. Symp. Wood Pulping Chem..
Proc. Vol. 1, 571.
Thurman, E. M. and Mills, M. S. (1998): “Solid-Phase Extraction. Principles and Practice”.
Whiley, New York.
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for the gas chromatographic analysis of resin and fatty acids in pulp mill effluents, J. Chrom.
346, 205.
Zinkel, D. F., Lathrop, M. B. and Zank, L. Z. (1968): Preparation and gas chromatography
of the trimethylsilyl derivatives of resin acids and the corresponding alcohols, J. Gas Chrom.
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6
Table 2. Concentrations of extractives in white water (mg/l) obtained by SPE and the liquidliquid extraction method.
Compound
SPE
St.dev. (%)
Palmitic acid*
Pinolenic acid
Linoleic acid
Oleic acid
Stearic acid*
FATTY ACIDS IN
TOTAL
1.2893
1.5368
2.5788
2.0408
0.2563
0.1132 (7.4)
0.2045 (7.9)
0.1294 (6.3)
7.7020
0.4471 (5.8)
Pimaric acid*
Sandaracopimaric acid
Isopimaric acid
Palustric acid
Levopimaric acid*
Dehydroabietic acid
Abietic acid
Neoabietic acid
RESIN ACIDS IN
TOTAL
0.1150
0.3586
1.0280
1.4252
0.7246
1.5889
1.9989
1.2103
Liquid-liquid
extraction
0.0000
1.6232
2.0484
1.7908
0.0000
5.4624
St.dev. (%)
0.2305 (14.2)
0.2326 (11.4)
0.1897 (10.6)
0.6528 (12.0)
0.0253 (1.6)
0.0774 (1.9)
0.0413 (3.4)
0.1972
0.3411
1.0106
1.3042
0.5501
1.5054
1.9969
1.3692
8.4495
0.2814 (3.3)
8.2747
1.3846 (16.7)
STEROLS, ß-Sitosterol
0.9830
0.0990 (10.1)
1.6036
0.2727 (17.0)
Fatty acids in total
Resin acids in total
Sterols
TOTAL
CONCENTRATION
7.7020
8.4495
0.9830
0.4471 (5.8)
0.2814 (3.3)
0.0990 (10.1)
5.4624
8.2747
1.6036
0.6528 (12.0)
1.3846 (16.7)
0.2727 (17.0)
17.1345
0.8275 (4.8)
15.3407
2.3101 (15.1)
*
0.0498 (13.9)
0.0198 (1.9)
0.0678 (4.8)
0.2700 (79.2)
0.1458 (14.4)
0.1748 (13.4)
0.2484 (16.5)
0.3143 (15.7)
0.2313 (16.9)
Standard deviation is not indicated since these compounds could not be detected at all or only in one or
two parallel samples.
7
Scheme 1. Wood extractives.
Fatty acids:
COOH
Oleic acid
COOH
Linoleic acid
COOH
Pinolenic acid
Sterol:
R
H3C
CH3
CH3
H
H
R = CH2CH3 ß-sitosterol
H
HO
Resin acids:
CH3
H3C
COOH
Abietic acid
H3C
COOH
COOH
Dehydroabietic acid
CH3
H3C
H3C
COOH
Levopimaric acid
CH3
CH3
COOH
Pimaric acid
H3C
H3C
COOH
Sandaracopimaric acid
8
COOH
Neoabietic acid
CH3
Palustric acid
H3C
CH3
CH3
CH3
H3C
COOH
Isopimaric acid
Figure 1. Concentration of fatty acids (mg/l) obtained by SPE and the liquid-liquid extraction
method.
9
8
7
SPE
6
Liquid-liquid extraction
mg/l
5
4
3
2
1
0
Palmitic Pinolenic Linoleic
acid*
acid
acid
Oleic
acid
Stearic
FATTY
acid* ACIDS IN
TOTAL
Figure 2. . Concentration of resin acids obtained by SPE and the liquid-liquid extraction
method.
12
10
SPE
6
liquid-liquid
extraction
4
2
L
0
Pi
Sa
m
nd
ar
ic
ar
ac
ac
id
op
*
im
ar
ic
ac
Is
id
op
im
ar
ic
ac
Pa
id
lu
st
ri c
Le
ac
vo
id
pi
m
ar
D
ic
eh
ac
yd
id
ro
*
ab
ie
t ic
ac
id
Ab
ie
ti c
ac
N
eo
id
R
ab
ES
ie
IN
ti c
AC
ac
id
ID
S
IN
TO
TA
mg/l
8
9
Figure 3. Concentration of extractives in total (mg/l) obtained by SPE and the liquid-liquid
extraction method.
20
18
16
mg/l
14
12
SPE
10
8
Liquid-liquid
extraction
6
4
2
0
Fatty acids in total
Resin acids in
total
Sterols, ßSitosterol
10
EXTRACTIVES,
TOTAL
CONCENTRATION