ENVIRONMENTAL ANALYSIS I. Solid Phase Extraction step-by-step method development

Goal of Six-Step Mode Isolation Purification Trace enrichment

Six-Step Method Development Compile known parameters Tech Support Form Follow step-by-step guide Fill in method development form

Technical Support Form

Pertinent Data The analyte(s) The matrix The analytical method Existing method(s)

The Analyte Structure pKa(s) Concentration Sample volume Solubility Stability

The Matrix Type of matrix pH and ionic strength Interferences

The Analytical Method Final technique Compatible solvents HPLC conditions

Existing Method Liquid-liquid method Current SPE method details Data from current method

Method Development Example Pollution Unlimited 1224 Sunnyside Drive Elution, Alaska (907) 663-3217 573 (907) 663-3218 Dr. Anna Light

The Analyte

The Analyte (cont.)

The Matrix

The Analytical Method

Other Information

Following the Guide

The “Form” Sample name _________________ Analyte of Interest ________________ Sample size ____________________ Sorbent Bed Mass _______ Cartridge volume_____ Sorbent phases to try 1) _____ 2) _____ 3) _____ 4) _____ 1. Sample pretreatment ___________________________ 2. Condition column with ____ mL of _________ at ___ mL/min 3. Equilibrate column with ____ mL of _________ at ___ mL/min. 4. Load sample at ______ mL per minute. 5. Remove interference with ____ mL of _________ at ___ L/min. Drying step _____ minutes 6. Elute analyte with ____ mL of _________ at ___ mL/min. Remember to include soak step if possible!

Filling in the Form

Following the Guide

Sample Size Determination

Filling in the Form

0.3 to 0.6 milli-equivalents per gram Sorbent Capacity Hydrophobic phases: 1- 5% of Sorbent Mass Ion Exchangers 0.3 to 0.6 milli-equivalents per gram

Following the Guide

Select a Mechanism

Select a Mechanism

Select a Mechanism

Select a Mechanism

Following the Guide

Selecting a Sorbent

Filling in the Form

Following the Guide

A Reminder...

Sample Pretreatment

Filling in the Form

Following the Guide

Conditioning and Equilibration

Conditioning and Equilibration

Filling in the Form

Following the Guide

Sample Loading Rate

Filling in the Form

Following the Guide

Removing Interferences

Removing Interferences

Filling in the Form

Following the Guide

Drying Time

Filling in the Form

Following the Guide

Selecting an Elution Solvent

Elution Solvent Volume

The Completed Form

What if Low Recovery? Losses during rinse step Analyte breakthrough Poor elution

Losses During Rinse Step Check rinse for presence of analyte

Analyte Breakthrough Stack Columns Elute Separately

Analyte Breakthrough Check pH and ionic strength at each step of extraction procedure Select phase offering greater interaction

Impact of pH Analyte loaded at pH=2 Analyte loaded at pH=7 The analyte loaded at pH=2 is well retained on the column. The analyte loaded at pH=7 bleeds through the column. At a pH=2, the surface of the silica is neutral, since the silanols are protonated (SiOH). At pH=7, the surface is negatively charged, since the silanols are ionized (SiO-). The very water soluble, negatively charged analyte is much more likely to be retained on a surface that is neutral than one that is negatively charged.

Selection of Bonded Phase Analyte poorly retained on C2 Analyte well retained on C18(EC) Analyte well retained on C8

Poor Elution Select stronger elution solvent pH, ionic strength, % organics Select less retentive sorbent Include a soak step

Solvent Strength Elution with 5% methanol in water

Less Retentive Sorbent Analyte completely eluted from C8 Traces of analyte remain on C18(EC)

Soak Step The analyte is completely eluted from the column which was soaked for one minute with the elution solvent. Some analyte remains on the column that was not soaked in the elution solvent.