[ Care and Use ManUal ] seP-PAK CArtrIdGes And PLAtes

[ Care and Use Manual ]
Sep-Pak Cartridges and plates
Contents
Waters Sep-Pak® cartridges and 96-well plates are convenient,
reproducible, disposable solid-phase extraction (SPE) devices
I. introduction
for sample preparation. Sep-Pak cartridges and plates are
manufactured in a Waters ISO 13485 and ISO 9002-registered
II. USING SEP-PAK CARTIDGES AND PLATES
facility in compliance with cGMP guidelines for the U.S. Food and
Drug Administration for Class 1 Medical Devices.
III. STRATEGIES FOR SOLID-PHASE EXTRACTION
a. Retention-Cleanup-Elution Strategy
b. Pass-Through Cleanup Strategy
IV. Steps of aN SPE Procedure
V. STORAGE AND DISPOSAL OF USED CARTRIDGES
Vi. ADDITIONAL INFORMATION
ViI. LITERATURE REFERENCES
Sep-Pak Cartridges and Plates
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[ Care and Use Manual ]
i. Introduction
Prepare sample: The sample can be applied to the Sep-Pak Cartridge in
either liquid or a gas phase. If the sample is a solid, it must be dissolved or
Sep-Pak devices are available in many styles to accommodate
extracted prior to loading.
manual and automated solid-phase extraction. Devices are available
Condition/Equilibrate Cartridge: A conditioning step is required for
in convenient Plus, Light, Classic, Vac, Vac RC, and 96-well plate
reversed-phase sorbents (e.g., C18). Use a strong solvent to wet the station-
formats.
ary phase. Conditioning is followed by an equilibration step using a weak
Available Sorbent Chemistries:
solvent.
Reversed Phase: C18, tC18, C 8, tC2
Load Sample: The sample is loaded onto the cartridge.
Normal Phase: Silica, Alumina A, Alumina B, Alumina N, Florisil®
Wash: The wash step removed interferences while retaining the analyte. If
Ion Exchange and Mixed Mode: Aminopropyl, PSA, Accell™ Plus
weakly retained interferences are present in the sample matrix, wash with an
QMA, Accell Plus CM, Cyanopropropyl, Diol
appropriate weak solvent.
Speciality Products: Porapak® RDX, DNPH-Silica, XPoSure™, Ozone
Elute: Use an appropriate stronger solvent to elute the analyte while retain-
Scrubber, Dry, PS2, AC2, Carbon Black/Aminopropyl
ing more strongly bound interferences.
II. USING SEP-PAK CARTRIDGES AND 96-WELL PLATES
III. STRATEGIES FOR SOLID-PHASE EXTRACTION
Below is a five step guideline for using Sep-Pak cartridges. Each separation
Purification: Use as a “chemical filter” to retain the component of interest
while interferences elute, or to retain the interferences while the analyte
is different and not all steps may be required for your application. Typical
strong and weak solvents used in the sample preparation steps are listed in
passes through unretained.
Table 1 below.
Trace Enrichment or Concentration: Use to concentrate an analyte from
Table 1: Typical strong and weak solvents used in the
a dilute solution. This is done when the analyte concentration is below the
detection limit of the analytical method.
sample preparation.
Separation Mode
Reversed Phase
Typical Weak
Solvents
Water or bufer.
May contain low
concentrations of organics,
such as methanol.
Acetonitrile,
methanol, or
aqueous/organic
mixture
Normal Phase
Hexane, toluene
Methylene chloride,
methanol
Weak Cation
Exchange
Low ionic strength
buffer, pH >4
High ionic strength
buffer, or pH <2
Strong Anion
Exchange
Low ionic strength
buffer, pH <8
High ionic strength
buffer, or pH >10
Sep-Pak Cartridges and Plates
Fractionation: Use with a step gradient of increasing solvent strength to
Typical Strong
Solvents
selectivity elute and isolate analytes based on differences in polarity.
Solvent Exchange: Use to adsorb the analyte and elute with a
desirable solvent. This is helpful of the sample is dissolved in a solvent
that is incompatible with the analytical method.
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[ Care and Use Manual ]
a. Retention-Cleanup-Elution Strategy
IV. Steps of aN SPE Procedure
As the sample is loaded onto the cartridge, the analytes of interest are
retained by the sorbent. If needed, an optimized series of washes are
The following section describes the steps involved in a complete solid-phase
used to remove matrix interference from the cartridge. A strong solvent
extraction procedure:
is used to elute the analytes from the cartridge. Sample enrichment
1. Pretreatment of Sample
results when the final elution volume is smaller than the load volume.
Solid samples (soil, tissue, etc.)
Load Sample
(Black)
Step
Elute 1
Step
Elute 2
Step
Elute 3
• Shake, sonicate or use soxhlet extraction.
-
-
Stationary
Phase
Particles
NOTE: Different
strength solvents
can be used to
separate the dyes.
extract sample with polar organic solvent (methanol, acetonitrile) for polar analytes.
extract sample with organic solvent and drying agent
(dichloromethane, acetone) for non-polar analytes and
multiresidue extraction.
Non Aqueous Liquid
• If the sample is soluble in water, dilute it with water for
reverse-phase SPE.
• If the sample is soluble in hexane, dilute it with or exchange to One cartridge can separate all three dyes
hexane for SPE.
Figure 1: Retention-Cleanup-Elution
Wastewater
b. Pass-Through Cleanup Strategy
• Filter or centrifuge as necessary.
Pass-through cleanup methods optimize matrix retention while the
2. Condition Step
analytes of interest pass-through the cartridge unretained. No sample
For reversed-phase sorbents, preconditioning of the sorbent with an
enrichment occurs during the solid-phase extraction (SPE) step.
organic solvent, such as methanol, acetonitrile, isopropanol, or tetrahy-
1. Sample is passed through sorbent and collected
drofuran, is usually necessary to obtain reproducible results. Without
this step, a highly aqueous solvent cannot penetrate the hydrophobic
• No sample enrichment
surface and wet the sorbent. Thus, only a small fraction of the sorbent
2. Matrix interferences are retained on sorbent
surface area would be available for interaction with the analyte. For the
same reason, it is important not to let silica-based SPE cartridges dry out
between the solvation step and the addition of the sample. A complete
preconditioning of a reversed-phase cartridge includes the solvation step
and an equilibration with a low-strength solvent such as water or buffer.
Figure 2: Pass-Through
Sep-Pak Cartridges and Plates
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[ Care and Use Manual ]
3. Load Step
5. Elution Step
When the analytes of interest are not retained by the sorbent, this is
Once the interferences are washed off the cartridge, a strong
called analyte breakthrough. For some methods, such as pass-through
solvent is introduced to elute the analytes of interest. The volume
cleanup, analyte breakthrough is desirable and is maximized for
and flow rate of the eluting solvents should be precisely controlled as
those specific methods. However, in all other cases, analyte break-
in the load step to ensure reproducible results.
through is unwanted and contributes to poor recovery and method
Table 2. Guidelines on the various types of separation mechanisms
reproducibility. Breakthrough occurs when:
Reversed Phase
• There is too high an organic concentration in the load solution for very polar analytes. Dilute sample at least 1:1 with water or buffer prior to loading.
• The analytes are bound to proteins, they may pass through the sorbent. Ensure that analytes are not bound to proteins by acidifying or basifying the sample.
Normal Phase
Analyte
Low to
moderate
polarity/
hydrophobic
Moderate to
highly polar/
uncharged
Charged
or ionized
Matrix
Aqueous
Non-polar
organic solvent
Aqueous/low
ionic strength
Condition/
Therefore, it is important to choose the correct sorbent 1. Solvate polar Non-polar
organic
Equilibrate organic
2. Water
mass (see Table 1).
Wash
Aqueous/buffer
enough contact time between the analytes and the sorbent. Elution
Look at the drops and adjust the vacuum so that you see Steps
discrete droplets, not a stream of liquid.
Increase polar Increase
organic content moderate to
in steps
high polarity
organic content
in steps
• Sorbent is overloaded by the matrix component.
Non-polar
• The flow rate of the load step is too fast. There is not Table 1. Choice of cartridges based on sample size
Sample Size
Ion Exchange
Low ionic
strength buffer
Low ionic
strength buffer
Stronger
buffers–ionic
strength or pH
to neutralize
the charge
Cartridge
1 - 10 mL
1 cc/30 mg or 3 cc/60 mg
10 - 100 mL
3 cc/60 mg or 6 cc/200 mg
100 - 500 mL
6 cc/200 mg or 6 cc/500 mg (LP*)
Cartridges stored in their original sealed pouch remain stable for long
500 - 1000 mL
6 cc/500 mg (LP) or 12 cc/1 g (LP*)
periods. To store unused cartridges in opened pouches, squeeze the air out
V. STORAGE AND DISPOSAL OF USED CARTRIDGES
of the pouch, fold over the open end of the pouch twice, seal with tape, and
* LP=large particules (60 µm)
store in a desiccator.
4. Wash Step
Dispose of used cartridges safely in accordance with applicable government
The wash steps are designed to remove unwanted matrix components
or local regulations.
that remain from the loading step. The ideal wash solvent removes
only the matrix while keeping the analytes bound to the sorbent. For
complex samples this is impossible, so the wash steps are optimized
using pH, solvent strength, and solvent polarity to remove as much
matrix as possible while maintaining acceptable analyte recovery.
Sep-Pak Cartridges and Plates
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[ Care and Use Manual ]
Table 4: Accessories and Spare Parts for the Waters Extraction Manifolds
VI. ADDITIONAL INFORMATION
Description
Sep-Pak accessories are available to help with your solid-phase extraction.
Qty.
Part No.
Ordering information for these products is given below.
Needle valves(required when using
silica-based SPE cartridges)
20/pk
WAT200685
For more detailed information, consult the Chromatography Columns
Needle
20/pk
WAT200691
and Supplies Catalog by visiting www.waters.com (literature code:
Cover, 20 position without gauge assembly
1
WAT200686
720002419EN), or contact your Waters representative.
Gauge assembly, Vacuum
1
WAT200687
If you require technical assistance, or have any questions regarding sample
Reservoir, glass with outlet valve
1
WAT200688
preparation, visit www.waters.com or call us at 1-800-252-4752.
Outlet valve kit
1
WAT200689
Table 2: Sep-Pak Cartridge Accessories
Gasket for cover
1
WAT200690
Ejector tool
1
WAT058839
Luer plugs
25/pk
WAT058851
Rubber ball ring (for vacuum gauge assembly)
1
WAT058840
Reversible vial rack for 1 mL or 4 mL
Autosampler vials
1
WAT058871
13 x 75 mm test tube rack
1
WAT200678
13 x 100 mm test tube rack
1
WAT200679
16 x 75 mm test tube rack
1
WAT200680
Description
Qty.
Part No.
30 cc Reservoir
48/pk
WAT011390
60 cc Reservoir
12/pk
WAT024659
Reservoir adapters for 1, 3 and 6 cc VAC
12/pk
WAT054260
Reservoir adapters for 12, 20 and 35 cc VAC
10/pk
WAT048160
Male-male adapter
100/pk
WAT024310
Male luer plugs
100/pk
WAT044395
Female luer plugs
100/pk
WAT044385
1
WAT011400
Sep-Pak connections kit
Table 3: Extraction Manifolds (Includes 20 needle tips, 25 plugs and
ejector tool)
Description
Part No.
20 position extraction manifold without rack
WAT200677
20 position extraction manifold with
13 x 75 mm test tube rack
WAT200606
20 position extraction manifold with
13 x 100 mm test tube
WAT200607
20 position extraction manifold with
16 x 75 mm test tube rack
WAT200608
20 position extraction manifold with
16 x 100 mm test tube
WAT200609
Sep-Pak Cartridges and Plates
1
WAT200681
Reservoir, 30 cc (for Sep-Pak Plus, Light,
Vac & Classic cartridges)
48/pk
WAT011390
Reservoir, 60 cc
(for Sep-Pak Plus, Light & Vac cartridges)
12/pk
WAT024659
Adapter, male-male Luer
(for Sep-Pak Classic cartridges)
100/pk
WAT024310
Adapter (to attach reservoir to 1, 3 & 6 cc
Sep-Pak Vac cartridges)
12/pk
WAT054260
Adapter (to attach reservoir to 12, 20 & 35 cc
Sep-Pak Vac cartridges)
10/pk
WAT048160
Vacuum pump (110 V, 60 Hz)
1
WAT085114
Vacuum pump (220 V, 50 Hz)
1
WAT085115
16 x 100 mm test tube rack
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[ Care and Use Manual ]
VII. Related Literature
Document
A Sample Preparation Primer and Guide to
Solid-Phase Extraction Methods Development
Sales Offices:
Austria and European Export
(Central South Eastern Europe,
CIS and Middle East) 431 877 18 07
Literature Reference
WA20300
Sample Preparation Solutions Brochure
20000848EN
Australia 2 9933 1777
96-Well Collection Plate Options for the Waters
Extraction Plate Manifold
720001263EN
Belgium 32 2 726 1000
720000860EN
Brazil 55 11 5094 3788
Waters Sep-Pak Sample Extraction Products Brochure
Waters 96 and 384-Well Collection Plate
Specifications
WA41941
Canada 800 252 4752
Sorbent Selection Guide for SPE Wall Chart
720002007EN
China 8621 6495 6999
Environmental Analysis Catalog
720002639EN
CIS/Russia +7 495 3367000
Environmental Chromatography Methods Guide
720002543EN
Food Safety Application Notebook
720002565EN
Czech Republic 42 02 617 11384
Denmark 45 46 59 8080
Finland +358 9 5659 6288
France (33) 1 30 48 72 00
Germany 49 6196 400600
Hong Kong 852 29 64 1800
Hungary 36 1 350 5086
India and India Subcontinent
91 80 2 837 1900
Ireland 353 1 448 1500
Italy 39 02 274 211
Japan (81) 3 3471 7191
Korea (82) 2 820 2700
Mexico 5255 5200 1860
The Netherlands +31 (0)76-50 87 200
Norway 47 63 84 60 50
Poland (48) 22 833 4400
Puerto Rico 787 747 8445
Singapore 65 6273 1221
Spain 34 93 600 93 00
Sweden 46 8 555 11500
Switzerland 41 62 889 2030
Taiwan 886 2 2543 1898
United Kingdom 44 208 238 6100
©2008 Waters Corporation, Waters. The Science of What’s
Possible, Sep-Pak, Porapak, Accell, and XPoSure are trademarks
of Waters Corporation. Florisil is a trademark of U.S. Silica.SepPak Cartridges are covered by U.S. Patents # 4,211,658 and/or
# 4,250,035 and several foreign patents.
June 2008 WAT011188 Rev 8 KK-PDF
Sep-Pak Cartridges and Plates
6
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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