1. Automation of Sample Preparation for Trace Analysis Haibin Wan
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2. Purpose of sample preparation
Remove interference for instrumental analysis
Enrich analytes
Convert analytes to a form suitable for instrumental analysis
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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
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4. Procedures in sample preparation
Extraction
Cleanup using L/L partition and/or Column chromatography
Concentration
Solvent change
Concentration
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5. Approaches for automation
New techniques
Direct automation of procedures
Integration of sample preparation with instrumental analysis
Column switching
SPE-LC
LC-GC
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6. 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.
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7. 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
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8. 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
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9. 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
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10. Application of column switching
Two-position switching valve
Multi position stream selection valve
Rotor
Stator
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11. 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
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12. 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,
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13. LC-04SP Valve system - Focus on ease of use and flexibility
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14. 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.
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15. 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.
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16. Integration of SPE with Instrumental analysis
One SPE column per sample
Between column derivatization with controlled temperature
Lower cost than other online SPE
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17. 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
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18. 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.
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Thank You
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