Automation of Sample Preparation for Trace Analysis Haibin Wan PromoChrom Technologies www.promochrom.com

Purpose of sample preparation Remove interference for instrumental analysis Enrich analytes Convert analytes to a form suitable for instrumental analysis PromoChrom Technologies www.promochrom.com

PromoChrom Technologies www.promochrom.com Type of interference a. Affect instrument functioning GC: Anything that has low volatility and poor thermal stability, LC: large molecules, particles b. Affect detection Chemicals with similar retention time as the analytes and detection response PromoChrom Technologies www.promochrom.com

Procedures in sample preparation Extraction Cleanup using L/L partition and/or Column chromatography Concentration Solvent change Concentration PromoChrom Technologies www.promochrom.com

Approaches for automation New techniques Direct automation of procedures Integration of sample preparation with instrumental analysis Column switching SPE-LC LC-GC PromoChrom Technologies www.promochrom.com

Direct automation of column cleanup SPE-01 Designed for pesticides and drug residues in food samples Up to 5 elution solvents Collect 2 fractions for each sample SPE-03 Designed for extraction of large volume water samples The major difference with SPE-01 is in number of pumps. SPE-03 has four pumps and SPE-01 has only one. PromoChrom Technologies www.promochrom.com

Direct automation of column cleanup Working principle of SPE-01 waste Advantages over manual column cleanup and SPE: Faster and well controlled flow rate Enable more complex elution and collection steps Release chemist from tedious work PromoChrom Technologies www.promochrom.com

Computer free and easy operation Step 1, edit method (only necessary for new method) Step 2, pick samples and method Step 3, press start button PromoChrom Technologies www.promochrom.com

Application of column switching High pressure valve Low pressure valve Can take less than 3 Bars Can only be used before pump or after detector Can take hundreds of bars Can be used between pump and columns Low pressure zone High pressure zone Low pressure zone PromoChrom Technologies www.promochrom.com

Application of column switching Two-position switching valve Multi position stream selection valve Rotor Stator PromoChrom Technologies www.promochrom.com

Typical application of column switching General Considerations The pre-column should be different from the analytical column. The pre column can tolerant repeated use. When analytes are transferred from pre-column to the analytical column, the solvent must be compatible to the two columns, and there should not be significant peak dispersion. Example: Ion exchange/C18; HLB/C18; RAM/C18 RAM=Restricted access material 6-10 nm pore coated with C4, C8 or C18. Exclude MW >15K PromoChrom Technologies www.promochrom.com

Advanced application of column switching Fraction collection for MALDI -TOF MS analysis SCX RAM A continuous 2-D LC for direct analysis of proteins with MW < 20 kDa in biological samples. Resolve 1,000 peaks of peptides within 96 minutes. As a complementary technique for 2D gel electrophoresis for small proteins. Reference: An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation Knut Wagner et. al..Anal. Chem. 2002, 74, 809-820 PromoChrom Technologies www.promochrom.com

LC-04SP Valve system - Focus on ease of use and flexibility PromoChrom Technologies www.promochrom.com

Online SPE based on column switching Waste Ports 1-4 for solvents LC pump Syringe pump Ports 5-14 for samples Components from online SPE Detector The online SPE looks after sample injection and elution of SPE column. The HPLC does not need a secondary pump and sampler. This is the online SPE system with the lowest cost. PromoChrom Technologies www.promochrom.com

Direct Analysis of PAH in Tap Water at ppt level Phenanthrene in tap water at 50 ppt; sample volume, 40 mL. Estimated quantitation limit 5 ppt Phenanthrene in tap water at 5 ppb; sample volume, 40 mL. PromoChrom Technologies www.promochrom.com

Integration of SPE with Instrumental analysis One SPE column per sample Between column derivatization with controlled temperature Lower cost than other online SPE PromoChrom Technologies www.promochrom.com

Working principle of online SPE heater Syringe pump Normal procedures: Condition SPE column using 1-2 solvents Load sample to SPE column Elute SPE column to remove interference Collect fraction from SPE column Mix the fraction with derivatization reagents and send to the heater (if necessary) Transfer fraction or derivatized fraction to HPLC for analysis PromoChrom Technologies www.promochrom.com

Application example using online SPE + 60 oC/pH10 Estrone Dansyl chloride Analysis of hormone in milk powder: Extract estrone from milk powder using acetonitrile Load extracts to SAX SPE column Derivatize the collected fraction Inject into HPLC HPLC analysis using a C18 column for separation and UV detection at 360 nm. PromoChrom Technologies www.promochrom.com

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