SPE Method Development Solid Phase Extraction

Summary: Ideal for extraction, concentration and cleanup of biological samples. This 300Å silica-based media has the ... Incorporating the highest gra...

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SPE Method Development SPE method development typically contains four steps:

Solid Phase Extraction Essentials

Solvent Polarity Chart

Step 1: Condition Less Polar (Stronger Reversed-Phase Elution)

The conditioning step is composed of two substeps; the first activates the sorbent ligands, the second equilibrates the sorbent bed.

Hexane

Step 2: Load In the load step, sample is applied to the SPE device. Matrix and flow rate are optimized to quantitatively retain target analytes.

Isooctane Cyclohexane Carbon tetrachloride Toluene

Step 3: Wash In the wash step, choose a solvent that elutes impurities but retains target analytes. Often the second conditioning solvent is a suitable wash solvent.

Chloroform Methylene chloride Tetrahydrofuran

Step 4: Elute

Ethyl ether

The elution step ideally removes all target analytes with this minimal solvent to maximize sensitivity. Sometimes requires a combination of solvents to break both the primary and secondary interactions.

Acetone

General Method Development Procedures Step 1—Condition ~ 4 bed volumes

Reversed-Phase Extraction Procedure Mechanism: Bind moderately polar to non-polar compounds from a polar sample matrix. Normal Phase Extraction Procedure Mechanism: Bind polar compounds from a non-polar sample matrix. Ion-Exchange Extraction Procedure Mechanism: Bind charged (negative/anionic or positive/cationic) compounds.

tech tips To calculate sorbent bed volume, use 150µL for every 100mg of sorbent.

Step 2—Load

Methanol followed by water

Step 3—Wash Step 4—Elute ~ 6 bed volumes ~ 3 bed volumes

Process sample at a Water or flow rate of 1– 5mL/min water:methanol (95:5) IPA followed Process sample at a Hexane or by hexane flow rate of 1– 5mL/min hexane:IPA (98:2) Methanol:water Apply slowly: less than Methanol:low (50:50) followed by or equal to 1mL/min ion ionic strength low ionic strength exchange kinetics are (0.1M) buffer (0.1M) buffer slower than reversed- (10:90) or normal phase

Retention capacity describes the total amount that an SPE sorbent will bind. This includes all compounds retained—analytes of interest as well as the contaminants.

Methanol or acetonitrile. May need to add strong acid or base to organic solvent to break secondary interactions. IPA, ethyl acetate, acetone, or hexane:IPA (50:50) High ionic strength (0.5M –1.0M) buffer or modify pH such that the analyte is uncharged. May need to add organic to break hydrophobic interactions.

The minimum elution volume re ommended recommended in the bed size in chart above will offer best sensitivity, required but more solvent may be required depending on the application

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SPE Introduction

Working with Experts in Media Production Using a consistent and pure silica base that employs tightly controlled bonding techniques, insures predictable analyte-sorbent interactions, critical in ensuring a bonded phase with high and reproducible recoveries. Highest Quality Control Every part of our manufacturing process is carefully monitored. From managing our raw materials to stringent quality controls in the final product, we perform multiple quality tests, and provide a comprehensive quality assurance certificate.

50µm

60Å

100% water wettable

Standard C18 High-Flow C18

Silica Silica

6.0% 8.0%

Yes Yes

50µm 100µm

60Å 60Å

Low carbon load C18 Large particle

High-Capacity C18 Large Pore C18

Silica Silica

17.0% 14.0%

Yes Yes

50µm 50µm

Hydrophilic/hydrophobic retention. Phase remains active even when completely dry. Can omit preconditioning step. General purpose phase. Less flow resistance for faster flow rates of large volume sample. Maximum capacity phase. Ideal for compounds >1500MW.

Octyl (C8)

Silica

4.5%

Yes

50µm

Ethyl (C2)

Silica

5.5%

Yes

50µm

Phenyl (PH)

Silica

3.8%

Yes

50µm

60Å High carbon load 150Å Larger than average pore size 60Å Less hydrophobic than C18 Less retention of highly hydrophobic compounds. Use when C18 is too retentive. 60Å Short chain functional group Less retention of highly hydrophobic c is less hydrophobic than C8 compounds. Use when C8 is too retentive. 60Å Aromatic structure Highly selective for aromatic compounds.

5.0% No

Cyanopropyl (CN) C anoprop l (CN) Sili a Diol (2OH)

Silica 6.0% Silica

6.0% Ye 4.0%

Florisil® (FL)

Sili a

1.5 mL 4 mL

MaxiClean™ SPE Columns

25 mL

Base

Average % EndParticle Pore Size Carbon capped Size

Features

Benefits

DVB

100% DVB





40µm



100% DVB

Carbograph

Graphitized — Carbon



38 – 125µm



Graphitized Carbon

Greater capacity than C18 for general SPE. Also free vinyl surface groups make a suitable solid-phase synthesis support. Retains polar organics in aqueous matrices. Ideally suited for acid, base-neutral extraction of pesticides and herbicides. Ideal for drugs of abuse.

Silica-based mixed mode C8/cation exchange Silica-based mixed mode Ideal for drugs of abuse. C8/anion exchange Polymer-based mixed mode pH stable with no conditioning C8/cation exchange required. Extract acidic, neutral and basic drugs of abuse from single column.

Drug-Clean SB-C

Silica





50µm

60Å

Drug-Clean SB-A

Silica





50µm

60Å

Drug-Clean PB

Polymer





30µm



x

x

x

x

x

x

Maxi-Clean™

x

x

x

Maxi-Clean

Extract-Clean™

Ultra-Clean™

x

Extract-Clean

x

x

x

x

x x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x x x

Retains

Applications

Maxi-Clean™

SCX

Particle Functional Exchange Counter Ion Size Group Exchange Capacity Particle Functional

Cations, (+) charged compounds

Remove/concentrate basic compounds.

x

x

x

Anions, (–) charged compounds

Remove/concentrate acidic compounds.

x

x

x

Maxi-Maxi-Clean™

600 mg Bed

Base

SCX StyreneHydrogen 50µm 2.0meq/mL Si e Group Benzene Capacity Base Counter Ion Sulfonic Styrene- DVB Hydrogen 50µm Acid DVB SAX Styrene- Acetate 50µm Tetramethyl 1.0meq/mL DVB Ammonium Ion Chromatography Sorbent Specifications

Frit Material 20µm Polyethylene 10µm PTFE Glass Fiber Filter Paper with Polyethylene Mesh Support 20µm Polyethylene

Molecular Particle Exclusion Exchange Capacity Limit Counter Ion Size

Packing

Base

IC-OH

StyreneDVB

Hydroxide

IC-H

StyreneDVB

IC-Ag

StyreneDVB StyreneDVB StyreneDVB

IC

Polypropylene

x

x x

Extract-Clean™ -Clean™

Packing

300 mg Bed

Device Specifications

Maxi-Clean™ Cartridges

Packing

900 mg Bed

Housing Polypropylene Treated Polypropylene Polypropylene

x x

x

General Ion-Exchange Sorbent Specifications

• Female luer top and male luer bottom • Process single cartridge by syringe or multiple cartridges by vacuum • Stack for multi-step extractions

Vydac® Columns

Benefits Benefits Most common polar phase.

50µm 60Å Highl polar urfa e Mo t ommon polar pha e. No 50µm 60Å Polar phase with slight anion Ideal for carbohydrates or generally properties withfor analyses 50µm 60Å Polarexchange pha e with light anion Ideal arbohcontaining drate orhydroxyl generall functional e hange propertie with anal egroups. ontaining h dro l Yes 50µm 60Å Unique selectivity Can be used in normal-phase or fun tional group . reversed-phase modes. 50µm 60Å Unique ele ti it Can be u ed in normal-pha e or No 50µm 60Å Polar surface with minor re Wets easily and offers .more er ed-pha e mode reproducibility. 50µm 60Å Polarhydrophobic urfa e withretention minor Wet ea il and offer more — 75 – 60Å Highly polar surface Referenced in many EPA methods. h drophobi retention reprodu ibilit . Ideally suited for pesticides and . 75 – 150µm 60Å Highl polar urfa e Referen ed in man EPA method metals. 150µm Ideall uited for pe ti ide and 75 – — 60Å Specifically tested for Ensures most inert batches suitable metal . 150µm chlorinated pesticides for highly active compounds. 75 – 60Å Spe ifi all te ted for En ure mo t inert bat he uitable —150µm130µm 100Å Aluminapewashed for acidic . hlorinated ti ide with acid forIncrease highl acapacity ti e ompound surface compounds. 100Å Alumina wa hed with a id In rea e apa it for a idi — 130µm 100Å Alumina washed with base Increase capacity for basic surface compounds. — 130µm 100Å Alumina washed with neutral Interacts with highly aromatic surface compounds and neutral hydroxyls.

Drug-

SPE Cartridges

Device Extract-Clean™ Columns Ultra-Clean™ Columns

x

Maxi-Clean™

Silica 5.0%

4.0% No Magnesium — Silicate ® Flori il (FL) Magne ium Sili ate Florisil® PR (FL-PR) Magnesium — Silicate ® Flori il PR (FL-PR) Magne ium Alumina Acidic (AL-A) Aluminum — Oxide Alumina Basic (AL-B) Aluminum — Oxide Alumina Neutral (AL-N) Aluminum — Oxide Specialty Packings Specifications

75 mL

x

Ultra-Clean

Base

Sili a Aminopropyl (NH2) Aminoprop l (NH2) Sili a

Diol (2OH)

15 mL

x x

Extract-Clean™

Silica (SI)

Average Particle Pore End% Average Features Carbon Base % ParticleSizePore Size End- capped capped —Si e 50µm Si e 60Å Features SilicaCarbon — Highly polar surface

Sili a (SI)

8 mL

x

Maxi-Clean™

Yes

Ultra-Clean™

Packing

Ultra-Clean™ Columns

Format: SPE Columns Sizes: 1, 3mL (volume above the packing) Summary: Ideal for extraction, concentration and cleanup of biological samples. This 300Å silica-based media has the same properties as the industry-leading Vydac® TP HPLC packing. Offered in C18 and C4, use for a variety of protein and peptide applications

11.0%

Packing

Format: SPE Cartridges Sizes: 300, 600, 900mg (media amount, not device volume) Summary: The Maxi-Clean™ line is offered in many of the same media as the Extract-Clean™ line, but slightly paired down, with over 20 chemistries available. This lure hub cartridge device is not as prevalent in the SPE industry, and while manual processing is most common, this format offers a number of other interesting processing options, including multimedia extractions.

Vydac® Columns

Benefits

Silica

Normal-Phases (Polar) Sorbent Specifications

Maxi-Clean™ Cartridges

Format: SPE Columns Sizes: 4, 8mL (the entire tube volume) Summary: Choose this ultra-low extractable version for very sensitive applications. Nine selected media are packed into highly inert fluorinated polypropylene tubes with PTFE frits. Less expensive than glass extraction devices, this durable format offers comparable inertness without the added concern of being easily broken.

Features

Prevail™ C18

Ultra-Clean™ Clean

Format: SPE Columns Sizes: 1.5, 4, 8, 15, 25, 75mL (the entire tube volume) Summary: In production for over 25 years, with proven consistency, this is our most comprehensive SPE product line. It includes 30 media types in over 10 different bed weights. And with a complete offering of reversed normal, and specialty medias exhibiting unique retention properties, you are sure to find the packing that delivers a cleaner, more concentrated sample

• Open top tubes with male luer bottom • Process multiple samples with vacuum manifold or automated SPE instruments • Process individual samples manually with use of adapter and syringe

Average % Particle Pore EndCarbon capped Size Size

IC-Ba IC-Na

Retains

1000 Daltons

1.0meq/mL Anions

Hydronium 50µm

1000 Daltons

2.0meq/mL

Silver

50µm

Barium

50µm

Sodium

50µm

1000 Daltons 1000 Daltons 1000 Daltons

Sodium

50µm

IC-Chelate

StyreneDVB

IC-RP

Polystyrene —

50µm

1000 Daltons

550µm —

2.0meq/mL 2.0meq/mL 2.0meq/mL

0.4meq/mL



Applications

Exchanges anions for hydroxide. May be used to remove or concentrate anions from sample and to increase pH of acidic samples. Removes cations that form insoluble hydroxide salts. Cations Exchanges cations for H+. May be used to remove or concentrate cations from sample and to reduce pH of basic samples. Chloride Iodide Bromide Removes excess halides through formation of Ag-halide salts. Sulfate Removes excess sulfate through formation of BaSO. Cations Exchanges cations for Na+. May be used to remove or retain cations from sample without changing the pH of the sample. Polyvalent metal ions Exchanges transition metals and divalent cations for Na+. May be used to remove or retain divalent cations and transition metals from sample. Hydrophobic Removes surfactants, organic acids, and components other organic substances. Inorganic ions pass through.

Extract-Clean™

Extract-Clean™ Columns

SPE Columns

Base

UltraUltra-Clean™

TM

Extract-Clean

Packing

Ultra-Clean™

Incorporating the highest grade of silica in the industry with over a quarter of a century’s experience in making SPEs. S*Pure brings to you a highly comprehensive range of silica-based SPE products. This includes MaxiClean™, Ultra-Clean™, Extract-Clean™, Vydac®; brands synonymous with quality, reproducibility and highest recoveries.

Extract-Clean™

Reversed-Phases (Non-Polar) Sorbent Specifications

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x