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

[ Care and Use ManUal ]

Sep-Pak Cartridges and Plates 1

Contents

I. IntroduCtIon

II. usInG seP-PAK CArtIdGes And PLAtes

III. strAteGIes For soLId-PHAse eXtrACtIona. 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

Waters Sep-Pak® cartridges and 96-well plates are convenient,

reproducible, disposable solid-phase extraction (SPE) devices

for sample preparation. Sep-Pak cartridges and plates are

manufactured in a Waters ISO 13485 and ISO 9002-registered

facility in compliance with cGMP guidelines for the U.S. Food and

Drug Administration for Class 1 Medical Devices.



I. IntroduCtIon

Sep-Pak devices are available in many styles to accommodate

manual and automated solid-phase extraction. Devices are available

in convenient Plus, Light, Classic, Vac, Vac RC, and 96-well plate

formats.

Available Sorbent Chemistries:

Reversed Phase: C18, tC18, C8, tC2

Normal Phase: Silica, Alumina A, Alumina B, Alumina N, Florisil®

Ion Exchange and Mixed Mode: Aminopropyl, PSA, Accell™ Plus

QMA, Accell Plus CM, Cyanopropropyl, Diol

Speciality Products: Porapak® RDX, DNPH-Silica, XPoSure™, Ozone

Scrubber, Dry, PS2, AC2, Carbon Black/Aminopropyl

II. usInG seP-PAK CArtrIdGes And 96-WeLL PLAtes

Below is a five step guideline for using Sep-Pak cartridges. Each separation

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

Table 1 below.

Table 1: Typical strong and weak solvents used in the

sample preparation.

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

extracted prior to loading.

Condition/Equilibrate Cartridge: A conditioning step is required for

reversed-phase sorbents (e.g., C18). Use a strong solvent to wet the station-

ary phase. Conditioning is followed by an equilibration step using a weak

solvent.

Load Sample: The sample is loaded onto the cartridge.

Wash: The wash step removed interferences while retaining the analyte. If

weakly retained interferences are present in the sample matrix, wash with an

appropriate weak solvent.

Elute: Use an appropriate stronger solvent to elute the analyte while retain-

ing more strongly bound interferences.

III. strAteGIes For soLId-PHAse eXtrACtIon

Purification: Use as a “chemical filter” to retain the component of interest

while interferences elute, or to retain the interferences while the analyte

passes through unretained.

Trace Enrichment or Concentration: Use to concentrate an analyte from

a dilute solution. This is done when the analyte concentration is below the

detection limit of the analytical method.

Fractionation: Use with a step gradient of increasing solvent strength to

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.

Separation Mode Typical Weak Solvents

Typical Strong Solvents

Reversed Phase 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



IV. stePs oF An sPe ProCedure

The following section describes the steps involved in a complete solid-phase

extraction procedure:

1. Pretreatment of Sample

Solid samples (soil, tissue, etc.)

• Shake, sonicate or use soxhlet extraction.

- 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

hexane for SPE.

Wastewater• Filter or centrifuge as necessary.

2. Condition StepFor reversed-phase sorbents, preconditioning of the sorbent with an

organic solvent, such as methanol, acetonitrile, isopropanol, or tetrahy-

drofuran, is usually necessary to obtain reproducible results. Without

this step, a highly aqueous solvent cannot penetrate the hydrophobic

surface and wet the sorbent. Thus, only a small fraction of the 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.

a. Retention-Cleanup-Elution StrategyAs the sample is loaded onto the cartridge, the analytes of interest are

retained by the sorbent. If needed, an optimized series of washes are

used to remove matrix interference from the cartridge. A strong solvent

is used to elute the analytes from the cartridge. Sample enrichment

results when the final elution volume is smaller than the load volume.

Figure 1: Retention-Cleanup-Elution

b. Pass-Through Cleanup StrategyPass-through cleanup methods optimize matrix retention while the

analytes of interest pass-through the cartridge unretained. No sample

enrichment occurs during the solid-phase extraction (SPE) step.

1. Sample is passed through sorbent and collected

• No sample enrichment

2. Matrix interferences are retained on sorbent

Figure 2: Pass-Through

Load Sample(Black)

StepElute 1

StepElute 2

One cartridge can separate all three dyes

StepElute 3

NOTE: Different strength solvents can be used to separate the dyes.

Stationary Phase

Particles



5. Elution StepOnce the interferences are washed off the cartridge, a strong

solvent is introduced to elute the analytes of interest. The volume

and flow rate of the eluting solvents should be precisely controlled as

in the load step to ensure reproducible results.

Table 2. Guidelines on the various types of separation mechanisms

V. storAGe And dIsPosAL oF used CArtrIdGes

Cartridges stored in their original sealed pouch remain stable for long

periods. To store unused cartridges in opened pouches, squeeze the air out

of the pouch, fold over the open end of the pouch twice, seal with tape, and

store in a desiccator.

Dispose of used cartridges safely in accordance with applicable government

or local regulations.

3. Load StepWhen the analytes of interest are not retained by the sorbent, this is

called analyte breakthrough. For some methods, such as pass-through

cleanup, analyte breakthrough is desirable and is maximized for

those specific methods. However, in all other cases, analyte break-

through is unwanted and contributes to poor recovery and method

reproducibility. Breakthrough occurs when:

• 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.

• Sorbent is overloaded by the matrix component.

Therefore, it is important to choose the correct sorbent

mass (see Table 1).

• The flow rate of the load step is too fast. There is not

enough contact time between the analytes and the sorbent.

Look at the drops and adjust the vacuum so that you see

discrete droplets, not a stream of liquid.

Table 1. Choice of cartridges based on sample size

* LP=large particules (60 µm)

4. Wash StepThe wash steps are designed to remove unwanted matrix components

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.

Sample Size 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*)

500 - 1000 mL 6 cc/500 mg (LP) or 12 cc/1 g (LP*)

Reversed Phase Normal Phase Ion Exchange

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/

Equilibrate

1. Solvate polar organic 2. Water

Non-polar organic

Low ionic strength buffer

Wash Aqueous/buffer Non-polar Low ionic strength buffer

Elution

Steps

Increase polar organic content in steps

Increase moderate to high polarity organic content in steps

Stronger buffers–ionic strength or pH to neutralize the charge



Table 4: Accessories and Spare Parts for the Waters Extraction Manifolds

Description Qty. Part No.

Needle valves(required when using silica-based SPE cartridges)

20/pk WAT200685

Needle 20/pk WAT200691

Cover, 20 position without gauge assembly 1 WAT200686

Gauge assembly, Vacuum 1 WAT200687

Reservoir, glass with outlet valve 1 WAT200688

Outlet valve kit 1 WAT200689

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




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

VI. AddItIonAL InForMAtIon

Sep-Pak accessories are available to help with your solid-phase extraction.

Ordering information for these products is given below.

For more detailed information, consult the Chromatography Columns

and Supplies Catalog by visiting www.waters.com (literature code:

720002419EN), or contact your Waters representative.

If you require technical assistance, or have any questions regarding sample

preparation, visit www.waters.com or call us at 1-800-252-4752.

Table 2: Sep-Pak Cartridge Accessories

Table 3: Extraction Manifolds (Includes 20 needle tips, 25 plugs and

ejector tool)

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

Sep-Pak connections kit 1 WAT011400

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



VII. reLAted LIterAture

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

©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.Sep-Pak 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

Sales Offices:

Austria and European Export

(Central South Eastern Europe,

CIS and Middle East) 431 877 18 07

Australia 2 9933 1777

Belgium 32 2 726 1000

Brazil 55 11 5094 3788

Canada 800 252 4752

China 8621 6495 6999

CIS/Russia +7 495 3367000

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

Document Literature Reference

A Sample Preparation Primer and Guide to Solid-Phase Extraction Methods Development

WA20300

Sample Preparation Solutions Brochure 20000848EN

96-Well Collection Plate Options for the Waters Extraction Plate Manifold

720001263EN

Waters Sep-Pak Sample Extraction Products Brochure 720000860EN

Waters 96 and 384-Well Collection Plate Specifications

WA41941

Sorbent Selection Guide for SPE Wall Chart 720002007EN

Environmental Analysis Catalog 720002639EN

Environmental Chromatography Methods Guide 720002543EN

Food Safety Application Notebook 720002565EN

Documents

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