Automation of Sample Preparation for Trace Analysis Haibin Wan Promo. Chrom Technologies www. promochrom. com
Purpose of sample preparation 1. Remove interference for instrumental analysis 2. Enrich analytes 3. Convert analytes to a form suitable for instrumental analysis Promo. Chrom 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 Promo. Chrom Technologies www. promochrom. com
Procedures in sample preparation Extraction Cleanup using L/L partition and/or Column chromatography Concentration Solvent change Promo. Chrom Technologies www. promochrom. com Concentration
Approaches for automation New techniques Direct automation of procedures Integration of sample preparation with instrumental analysis 1. Column switching 2. SPE-LC 3. LC-GC Promo. Chrom Technologies www. promochrom. com
Direct automation of column cleanup SPE-03 SPE-01 Designed for pesticides and drug residues in food samples Up to 5 elution solvents Collect 2 fractions for each sample 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. Promo. Chrom Technologies www. promochrom. com
Direct automation of column cleanup Working principle of SPE-01 waste Advantages over manual column cleanup and SPE: 1. Faster and well controlled flow rate 2. Enable more complex elution and collection steps 3. Release chemist from tedious work Promo. Chrom 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 Promo. Chrom Technologies www. promochrom. com
Application of column switching High pressure valve Low pressure valve • Can take hundreds of bars • Can take less than 3 Bars • Can be used between pump and columns • Can only be used before pump or after detector Low pressure zone High pressure zone Promo. Chrom Technologies www. promochrom. com Low pressure zone
Application of column switching Multi position stream selection valve Two-position switching valve Rotor Stator Promo. Chrom Technologies www. promochrom. com
Typical application of column switching General Considerations 1. The pre-column should be different from the analytical column. 2. The pre column can tolerant repeated use. 3. 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/C 18; HLB/C 18; RAM/C 18 RAM=Restricted access material 6 -10 nm pore coated with C 4, C 8 or C 18. Exclude MW >15 K Promo. Chrom Technologies www. promochrom. com
Advanced application of column switching RAM A continuous 2 -D LC for direct analysis of proteins with MW < 20 k. Da in biological samples. Resolve 1, 000 peaks of peptides within 96 minutes. SCX C 18 As a complementary technique for 2 D gel electrophoresis for small proteins. Fraction collection for MALDI -TOF MS analysis 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 Promo. Chrom Technologies www. promochrom. com
LC-04 SP Valve system - Focus on ease of use and flexibility Promo. Chrom Technologies www. promochrom. com
Online SPE based on column switching LC pump Detector Waste Syringe pump Components from online SPE Ports 5 -14 for samples Ports 1 -4 for solvents 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. Promo. Chrom Technologies www. promochrom. com
Direct Analysis of PAH in Tap Water at ppt level Phenanthrene in tap water at 50 ppt; sample volume, 40 m. L. Estimated quantitation limit 5 ppt Phenanthrene in tap water at 5 ppb; sample volume, 40 m. L. Promo. Chrom Technologies www. promochrom. com
Integration of SPE with Instrumental analysis 1. One SPE column per sample 2. Between column derivatization with controlled temperature 3. Lower cost than other online SPE Promo. Chrom Technologies www. promochrom. com
Working principle of online SPE Normal procedures: Syringe pump 1. Condition SPE column using 1 -2 solvents heater 2. Load sample to SPE column 3. Elute SPE column to remove interference 4. Collect fraction from SPE column 5. Mix the fraction with derivatization reagents and send to the heater (if necessary) 6. Transfer fraction or derivatized fraction to HPLC for analysis Promo. Chrom Technologies www. promochrom. com
Application example using online SPE + Estrone 60 o. C/p. H 10 Dansyl chloride Analysis of hormone in milk powder: 1. Extract estrone from milk powder using acetonitrile 2. Load extracts to SAX SPE column 3. Derivatize the collected fraction 4. Inject into HPLC 5. HPLC analysis using a C 18 column for separation and UV detection at 360 nm. Promo. Chrom Technologies www. promochrom. com
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