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INTRODUCTION
Sample preparation is an essential part of any solidliquid extraction. Most food and animal feed samples
require some type of treatment either before or after
extraction. This can include preparing the sample prior
to extraction, adding substances to the ASE cell to retain
unwanted extractables, as well as treating the final extract.
This technical note describes different techniques
that can be used to prepare various food and animal feed
samples, as well as techniques used during and after
extraction to ensure the best recoveries of analytes while
maintaining a clean extract for analysis.
Pretreatment
Wet Food/Animal Feed Samples
100
Recent Recovery vs Mojonnier
Plant Material
Plant material can contain a varying degree of
moisture and often requires treatment prior to extraction.
Lyophilization is an excellent pretreatment method for
plant materials because this process yields a fine, dry
sample that can be easily loaded into an ASE extraction
cell. However, if freeze-drying is not a viable option,
simply grind (or mix) the plant material with ASE Prep
DE using a mortar and pestle. Initially, a ratio of plant
material to DE of 1:1 is a good mixture, but the amount
of DE may need to be increased if the plant material is
very wet. Sufficient DE has been mixed with the sample
if the sample can be easily removed from the mortar
without adhering to the sides of the mortar. If the plant
material is low in moisture, it can be ground in a blender
or mill to produce small particles (<2 mm). Cryo-grinders have also been used successfully.
80
60
40
20
0
Whole
Chopped
Hex: IPA, 125 C, gravimetric analysis
Ground
225690
Accelerated Solvent Extraction (ASE) Sample Preparation Techniques for Food and Animal Feed Samples
Choosing a Filter
It is very important to use a filter in the bottom of
the extraction cell. This will keep fine sample particulates from getting trapped in the frit or from leaving the
cell and possibly causing damage to the static valve.
Cellulose filters (for ASE 200, Dionex P/N 049458, pkg.
of 100 and for ASE 300 and 100, Dionex P/N 056780,
pkg. of 100) are the most common filters used, but if
the extraction solvent chosen is an aqueous solvent and
the temperature is above 100 C, then the cellulose filter
should not be used. Glass-fiber filters (for ASE 200,
Dionex P/N 047017, pkg. of 100, and for ASE 300 and
100, Dionex P/N 056781, pkg. of 100) are available for
these applications. The glass-fiber filters are available in
the same diameter sizes as the cellulose filters. If the
sample contains very fine particulates and there is
evidence of these particulates passing through into the
extraction vial, more than one filter (glass fiber or cellulose) can be used in the extraction cell to help eliminate
this problem. In most cases, 23 filters will be sufficient.
In-Cell Clean Up
In an effort to eliminate post-extraction clean-up
steps, Dionex has studied the addition of various sorbents
to the extraction cell. For many sample types, this approach has proven successful in producing clean extracts
that are ready for direct analysis. For example, lipids
are often co-extracted with fish tissue. Adding alumina
(aluminum oxide, Al2O3, acidic, activated by oven drying
at 350 C for 15 min) to the extraction cell before adding
the sample or sample mixture prevents the extraction of
unwanted lipids. Mixing the sample with C18 resin (1:2)
retains organic contaminants (C18 bonded silica, 3570
m diameter and porosity of 60 from Alltech has been
used, but similar material from any vendor can be used).
When performing in-cell clean up, solvent choice
can impact the retention of unwanted components. For
example, a mixture of hexane/acetone (1:1) is a common
solvent choice for extracting organochlorine pesticides
from animal tissues. However, after extraction, a cleanup step is usually required to remove co-extracted lipids.
If alumina is placed in the outlet (or exit) end of the extraction cell (the tissue sample that has been thoroughly
mixed with ASE Prep DE is placed on top of the alumina), and hexane is used as the extraction solvent, the
lipids will be retained on the alumina and remain in the
extraction cell. On the other hand, if hexane/acetone (1:1)
is used as the extraction solvent, almost no lipid material
Choosing a Solvent
When transferring extraction methods from traditional or manual techniques to ASE, the question, What
solvent should I use? often arises. In most cases, the
same solvent that was used in the previous technique can
be used in the ASE system with excellent results. Remember that the ASE system is compatible with almost
all liquid solvents with the exception of mineral acids
(HCl, H2SO4, and HNO3) and strong caustic
solutions (KOH and NaOH). The use of mineral acids
in the ASE system will etch the stainless steel cells and
tubing, which leads to premature failure of key instrument components. Strong bases destroy the ASE pump
components, leading to pump failure. As stated in the
ASE Users Manual, buffers, weak acids, and weak
bases have all been successfully employed as ASE
solvents. If acidic extraction conditions are required,
weaker acids, such as acetic or phosphoric should be
used, usually in the 110% (v/v) range mixed with
aqueous or polar solvents.
If there is uncertainty about which solvent to use,
note that the polarity of the extraction solvent should be
similar to the polarity of the target analytes. The solvent
should not only be able to extract the analytes but leave
the sample matrix generally intact. For example, when
the goal is to extract an analyte that is moderately polar
using an extremely polar solvent, the polar solvent may
extract not only the target analytes, but much of the
sample matrix as well. This scenario could create an
extract that contains co-extractable compounds that may
require extensive clean-up procedures prior to analyTechnical Note 209
TABLE 1. SUGGESTED ASE SOLVENTS FOR VARIOUS FOOD MATRICES AND ANALYTES
Matrix
Analytes Group
Pesticides
Sterols
Ions: sulfates and nitrates
Antibiotics
Environmental toxins (organic)
Environmental toxins (inorganic)
Environmental toxins (organic)
Environmental toxins (inorganic)
Fats and lipids
Fats and lipids
Acrylamide
Suggested Solvents
DCM/acetone (1:1, v:v), hexane/acetone (1:1, v:v), toluene, ethyl acetate
Water
7% acetic acid in methanol, 0.1% TFA in methanol
Hexane, hexane/isopropanol (3:2, v:v), petroleum ether
DCM/acetone (1:1, v:v), hexane/acetone (1:1, v:v), toluene, ethyl acetate
Methanol/water (1:1, v:v), acetonitrile/water (1:1, v:v),
methanol/water/ H3PO4
Acetone/hexane (1:1, v:v), DCM/acetone (1:1, v:v)
Chloroform/methanol (2:1, v:v), hexane /isopropanol (3:2, v:v)
Water
Water
DCM/acetone (1:1, v:v), hexane/acetone (1:1, v:v), toluene
Methanol, methanol with 1% acetic acid
DCM/acetone (1:1, v:v), hexane/acetone (1:1, v:v)
Water
Hexane, hexane/isopropanol (3:2, v:v), petroleum ether
Hexane, hexane/isopropanol (3:2, v:v), petroleum ether
Water with 10 mM formic acid or acetonitrile (100%)
Accelerated Solvent Extraction (ASE) Sample Preparation Techniques for Food and Animal Feed Samples
5 min
Flush Volume:
60 % of cell volumea
Purge Time:
Static Cycles: 1
Pressure: 1500 psi (only applies to ASE 200
instruments)
Decrease flush volume to 50% if instrument was manufactured in 1998 or later.
Remember the purge time depends on the ASE cell size: 60 s for 11-mL cells
and 100 s for 34-mL cells. Purge time may need to be increased depending on
the size of the ASE extraction cell.
a
b
Post-Extraction Treatment
Sometimes, in spite of pretreatment efforts, some
type of post-extraction treatment is necessary to produce
high quality analytical data. For example, at ASE extraction
temperatures above 100 C, water from wet samples usually
appears in the final extract, even after pretreatment with
ASE Prep DE. If acetone is used as the extraction solvent,
the extracted water will be miscible with the acetone and
may not be visible until the evaporation step (in most cases,
the extract will appear cloudy if water is mixed with acetone). On the other hand, if hexane is used as the extraction
solvent, the extract will contain two distinct phases: (1) a
visible water layer, and (2) a hexane layer. To easily remove
unwanted water from extracts, add sodium sulfate directly
to the collection vial containing the extract (remember it is
important not to use sodium sulfate in the extraction cell).
Shake the collection vial and decant the solvent into a clean
collection vial or other suitable container. Rinse the sodium
sulfate with fresh solvent to ensure that analytes of interest
are not trapped in the sodium sulfate layer.
Example of Method Development of an Animal
Feed Sample
The following is an example of developing an ASE
method for an animal feed sample:
Sample: Pelletized rodent food (dry)
Analyte: Anti-schizophrenic drugs
Step One: Pretreatment of sample
Because methanol was used for the traditional extraction of this sample (wrist shaker) methanol was also
chosen as the extraction solvent for the ASE.
Accelerated Solvent Extraction (ASE) Sample Preparation Techniques for Food and Animal Feed Samples
Suppliers
Fisher Scientific, 2000 Park Lane, Pittsburgh, PA
15275-1126 USA, Tel: 800-766-7000,
www.fishersci.com.
Alltech Associates, Inc., 2051 Waukegan Road,
Deerfield, IL 60015 USA, Tel: 847-948-8600,
www.alltechweb.com.
References
1. Extraction of Drugs from Animal Feeds Using
Accelerated Solvent Extraction (ASE). Application
Note 326; Dionex Corporation, Sunnyvale, CA.
2. Rapid Determination of Organochlorine Pesticides
in Animal Feed Using Accelerated Solvent Extraction
(ASE). Application Note 349; Dionex Corporation,
Sunnyvale, CA.
3. Rapid Determination of Sulfonamide Residues in
Animal Tissue and Infant Food Containing Animal
Products Using Accelerated Solvent Extraction
(ASE). Application Note 353; Dionex Corporation,
Sunnyvale, CA.
6. Extraction of Oil from Oilseeds by Accelerated Solvent Extraction (ASE). Application Note 325; Dionex
Corporation, Sunnyvale, CA.
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