SPE Method Development Guide Sample
96-Well Plate Aqueous Samples
Organic Sample
SPE Cartridges
Cationic
Polar
Anionic
Moderate Polar
CN Weak Cationic Strong Cationic Strong Anionic Weak Anionic Polymeric
PCX
PWCX
PWAX
PAX
Silica-based
SCX
COOH
DEA
SAX
SPE Product Cross Reference Table Agela C18 (End Capped ) C18 (Without End Capping) C8 Cyano Group Amino Group Propyl Ethylene Diamine Quaternary Ammonium Salt (Strong Anion-exchange Cartridge) Carboxyl Group (Weak Cation-exchange Cartridge) Propylsulfonic Acid Benzene Sulfonic Acid (Strong Cation-exchange Cartridge) Silica Gel
Waters
—
—
Sep-pak C8 Sep-pak CN Sep-pak NH2 — Accell Plus QMA
ENVI-8 LC-CN LC- NH2 — LC- SAX
AccuBOND C18 Bond Elut C18-OH AccuBOND C8 Bond Elut C8 AccuBOND CN Bond Elut CN AccuBOND NH2 Bond Elut NH2 — Bond Elut PSA AccuBOND Bond Elut SAX SAX
Accell Plus CM
LC- WCX
Cleanert PRS — Cleanert SCX —
Polar Polymer Cartridge
Cleanert PEP Oasis HLB
Acidic Alumina Basic Alumina Mixed Graphitized Carbon and Amino Group Cartridge Cartridge Specialized for Sulfanilamides DNPH-Silica Cartridge (Specialized for Pretreatment of Aldehydes and Ketones in Air)
Cleanert PAX Oasis MAX Cleanert PCX Oasis MCX Cleanert HXN —
— LC- SCX
LC- Silica LC-Diol ENVIChrom P — — — —
— AccuBOND SCX AccuBOND Silica AccuBOND Diol AccuBOND ENV PS-DVB — — — —
Neutral
PSA
Acidic
Basic
Amphoteric
PEP
PEP-2
NH2
MAS-A
MAS-A
MAS-B
C18 C8-N C8
MAS-WA
Causes
1. Low Recovery
Organic：POLAR
Aqueous
Your analyte is ionisable
(When interferences are mostly neutral) adjust pH to ionise analyte. If your analyte is:
Your analyte is non-ionisable: NON-POLAR
(When interferences are mostly ionic) adjust pH to neutralise analyte. If your analyte is:
Load standard solution in the sample solvents without matrix, collect the elutes from Step 2,3,4 respectively
Bond Elut PRS Bond Elut SCX
Bond Elut Silica Bond Elut Diol —
Basic: adjust pH 2 units
Bond Elut® Plexa — — —
Acidic: adjust pH 2 units>pKa ANION EXCHANGE
Basic: adjust pH 2 units>pKa NON-POLAR
Acidic: adjust pH 2 units
Your matrix is of high ionic strength(e.g.biological fluid) and interferences are neutral and ionised
SiOH
The analytes were not retained on the column
NH2
Al-N
CN
SiOH
Solution Choose another Sorbent
Incorrect activation process
Follow the standard method development procedure
Sample solvent too strong
Properties
Adjust pH or polarity of the eluent
Normal Phase
Cleanert SUL-5 Cleanert DNPH-Silica
Sep-pak Florisil — Sep-pak Alumina N Sep-pak Alumina A Sep-pak Alumina B Sep-pak Carb/ NH2 —
Sep-pak DNPH-Silica
—
Bond Elut FL
ENVI Carb
—
—
LCAlumina N LCAlumina A LCAlumina B ENVI Carb/ NH2
AccuBOND Alumina N AccuBOND Alumina A AccuBOND Alumina B —
Bond Elut Alumina N Bond Elut Alumina A Bond Elut Alumina B —
—
—
—
—
—
—
Bonna-Agela Technologies
Your analyte is basic: MIXED-MODE NON-POLAR / CATION EXCHANGE
Your analyte is acidic: MIXED-MODE NON-POLAR / ANION EXCHANGE
Selection of Column Size, Sample Loading and Elution Volume Specification 50 mg/1mL 100 mg/1mL 200 mg/3mL 500 mg/6mL 1 g/6mL
Quantity of Loading Sample 2.5 mg 5 mg 10 mg 25 mg 50 mg
The Minimum Volume of Elution
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125 μL 250 μL 500 μL 1.2 mL 2.4 mL
PEP series
Florisil Typical Sorbents
Ion Exchange Phase or Mix-phase
C18
Amine（NH2）
C18-N
Cyano（CN）
C8
PAX SAX PWAX
PCX SCX PWCX
Diol Sorbent Polarity
High
Low
High
Matrix Properties
Organic
Organic or Aqueous
Organic or Aqueous
Slightly to Eluent was not enough
Take more eluent
Incorrect SPE
Use a different sorbent
Strong binding of the analytes with matrix
SPE was dry during activation (Only for C18, C18-N, C8)
Analyte Properties
Retention
Adjust pH or adjust solvents to break the binding Step 1：Activation
2. High Deviation
SPE cartridge was over loading
LC Florisil
Reversed-Phase
Silica
Reduce amount of the strong solvent
Incorrect eluent
Sorbent Phase Type
Re-activated the cartridge or change another SPE cartridge Reduce the sample amount or increase the sorbent amount
Moderately Polar
polar
Sample solvent or
Water-miscible
Water-miscible organic solvents
other organic
organic solvents
followed by aqueous solution with
solvents
followed by Water
pH adjusted
Step 2：Sample or with dilution in
Dry the column when change different solution
The solution used in four steps was not miscible to each other
low polarity
Change the polarity and pH of the loading solution
Load the sample or with dilution in high polarity solvents
The sample was sticky
Dilute the sample
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polarity solvents with pH adjusted
of aqueous solution
solvents followed by aqueous
low-polar solvents or buffer with a small
solvents with pH adjustment to
Washing with
maintain analytes ionized
Eluting with mixture of Step 4：Elution
non-polar and
Eluting with non-polar or polar organic solvents
3. Clogging Centrifuge or filtration before SPE
Load sample or with dilution in high
Washing with Polar organic
polar solvents
The sample contains colloidal particles
Ionic Strength
Washing with mixture
amount polar solvent
Change the polarity of the washing solution
The washing solution was too strong
The loading solution was too strong
Step 3：Washing
Basic
Non-Polar
Reduce the flow rate below 5 mL/min
Flow rate was too high
Acidic
Non-Polar
Load the sample
Loading
Non-Polar or Polar
Polar
solvents Cleanert Florisil Cleanert PestiCarb Cleanert Alumina N Cleanert Alumina A Cleanert Alumina B Cleanert PestiCarb/NH2
Nonpolar
Standard Method Development Procedure
Incorrect SPE
The analytes were not eluted from column
Polar
Diol
Trouble Shooting
Bond Elut C18
Bond Elut CBA
Suitable for moderately polar and polar substance especially DMPK sample, equivalent to SLE.
Comptemetary of PEP
NH2
Moderate Polar
SLE
PEP-H
More Polar Hydrophilic Compoards
Your sample matrix is:
Cleanert C18-N Cleanert C8 Cleanert CN Cleanert NH2 Cleanert PSA Cleanert SAX
—
OH
Varian
—
Diol Polystyrene/Divinyl-benzene
Neutral Alumina
Aglient
Sep-pak C18 ENVI-18
Cleanert COOH
SiOH
Nonpolar
Selection Guide of Sorbent Retention Mechanism
Cleanert C18
Cleanert Silica Sep-pak Silica Cleanert Diol Sep-pak Diol Cleanert PS —
Mixed Anion-exchange Cartridge Mixed Cation-exchange Cartridge Cartridge Specialized for Sulfonylureas Magnesium Silicate (Florisil) Graphitized Carbon
Supelco
Analytes
Non-ionic Analytes
Ionic Analytes
Bonna-Agela China
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[email protected]
Eluting with polar solvents with pH adjustment
