Automation of Sample Preparation

for Trace Analysis

Haibin Wan

Purpose of Sample Preparation

Remove interference for instrumental analysis

Enrich analytes

Convert analytes to a form suitable for instrumental analysis

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

Procedures in Sample Preparation

Extraction

Cleanup using L/L partitionand/or

Column chromatography

Concentration Solvent change Concentration

Approaches for Automation

Direct automation of manual procedures

Integration of sample preparation with

instrumental analysisNew techniques

1. Column switching

2. SPE-LC

3. LC-GC

Aanlysis of Organophosphorus Pesticides in Tea

Grind10 g tea, soak with 80 mL acetone overnight

Filter out the extract solvent under vacuum with extra wash using 50 mL acetone, evaporate to 10 mL

Cleanup 1 mL concentrated extract (=1 gram tea) using 500 mg graphitized carbon black column with elution of

25 mL acetonitrile + toluene (3:1)

Evaporate the eluate to 2 mL for GC-FPD

SPE-03 Working PrincipleIntroduced for automation of column cleanup and SPE

Sample volume 1-4000 mL

Process 8 samples in parallel mode with 8 pumps

Heated evaporation while fraction collection is going on

SPE-03 Working Principle

SPE-03 Operation

Available actions

1. Add samp2-6. Elute 1-57. Blow air8-9. Collect 1&2 10-11. Rinse 1&212-13. Clean 1&214. Blow N215. Pause16. Con F217-18. Evap F1&219. Heat

SPE-03 Modification for More Functions

Place sample extract or sample here

Approach 1: Extracts evaporation+ cleanup + final evaporation

Approach 2: Sample extraction + extracts evaporate + cleanup + final evaporation

Modification of Procedures to Fit Automation

10 g tea + 80 mL acetone, soak overnight.

Filter out with 50 mL acetone wash,

reduce to 10 mL

Clean up 1 mL extract (=1 g tea) using 500 mg GCB column with elution of 25 mL acetonitrile + toluene.

Reduce fraction to 2 mL

Original procedure

10 g tea + 100 mL acetone, soak overnight.

Take 5 mL extracts (=0.5 g tea) for online evaporation to 2 mL

Cleanup using 250 mg GCB column with elution of 12 mL aceton +CH2Cl2(1:1)

Reduce fraction to 0.5 mL

Modified for approach 1

Selection of Evaporation Parameters

For sample extracts (acetone)

Temperature, 60 oC; preheating time, 5 minutes; nitrogen flow rate, 2.8 L/min (350 mL/min for each channel).

12 minutes to evaporate dry 5 mL acetone.

For fraction while elution is going on (acetone + dichloromethane 1:1)

Temperature, 60 oC; preheating time, 5 minutes; nitrogen flow rate, 2.8 L/min; elution flow rate; 0.3 mL/min; wait time, 1 minute.

The level of liquid in the collection vial can be maintained at 0.1-0.5 mL

Loss of Methyl Parathion (MP) during Evaporation

Solvents of good volatility should be used, such as acetone, ethyl acetate, hexane, dichloromethane.

When the heating temperature is high, complete dry must be avoided by carefully selecting parameters or use of high boiling solvent (DMF, DMSO etc.)

Treatment 1 Treatment 2 Treatment 310 ug MP in 5 mL acetone. Evaporate to dry at 60 oC with 2.8 L/min N2. Treat extra 3 minutes.

10 ug MP in 5 mL acetonitrile + toluene. Evaporate to dry at 85 oC with 2.8 L/min N2. Treat extra 1-3 minutes.

10 ug MP in 0.5 mL methanol. left in a open 20-mL vial for 20 hours.

Cleanup effect of two elution solvents

5 mL tea extract evaporated to dry and dissolve with 0.5 mL methanol;5 mL tea extract cleanup with acetonitrile + toluene (3:1) as elution solvent;5 mL tea extract cleanup with aceton + dichloromethane (1:1) as elution solvent.

Methyl parathion

Caffeine

HPLC: Model, Agilent 1100 with quaternary pump and VWD; Mobile phase A, water; Mobile phase B, methanol; gradient profile, increase B from 30% to 95% over 6 min, keep for 2.5 min, then return to 30%; column, PCT-C18 250 X 4.6 mm, 5 um; detection wavelength, 276 nm; flow rate 1.5 mL/min; injection volume, 20 uL.

Recovery Test for Approach 1Sample: 5 mL tea extracts (acetone) is spiked with 5 ug methyl parathion (=10 ppm) and processed as below

Evaporate sample to dry at 60 oC for 12 minutes under N2 (350mL/min)

Dissolve in 2 mL elution solvent and transfer to GCB column. Elute another 8 mL. Collect all eluate.

Evaporate fraction to dry at 60 oC for 23 minutes under N2. Dissolve in 0.5 mL methanol for HPLC analysis.

Corresponding method for SPE-03:

No. Acts Flow rate Vol. 5 Add sam 10 0.11 Heat 60(oC) 5.0 (min) 6 Collect 1 10 3.02 Elute 1 10 10.0 7 Elute 1 10 0.03 Evap F1 10 12 8 Collect 1 10 8.04 Clean 1 10 2.0 9 Evap F1 10 23.0

Recovery Test for Approach 1

Sample 1 Sample 2 Sample 2 Sample 497.4% 91.5% 97.3% 98.0%

Fraction evaporate to just dry

Fraction evaporate extra 3 minutes

Fraction evaporate to just dry

Fraction evaporate extra 2 minutes

By further optimization on parameters and improvement of the heating homogeneity, over evaporation can be avoided.

Automation of Sample Extraction

Use heated solvent to accelerate the extraction Evaporate solvent without filtering out the solid sample

1 g tea +10 mL acetone, heat for 10 minutes.

Evaporate away acetone, analytes stay on surface of sample solid.

Add 3 mL acetone, heat for 2 min, transfer all liquid to column.

Assumption on Behavior of Analytes in Proposed Approach

Extraction solvent is added to sample

Aanlytes diffuse into solvent with assistance of heat

Majority analytes stay on surface of sample after solvent is removed

Solvent is added to sample for loading to column. Analytes distribute into solvents with help of air bubbling and heat.

Assumption on Behavior of Analytes in Proposed Approach (Proof)

Left. Tea soaked in ethanol for 1 hr with occasional shaking.

Right. Same treatment as above plus heat at 80 oC.

Left. Tea soaked with hot water for 1 hr with occasional shaking.

Right. Tea soaked with hot ethanol, evaporated to dry and soak again with hot water.

Recovery Test for Approach 2Sample: 1 g tea and 10 mL acetone is placed in 20-mL vials. 2 ug methyl parathion (=2 ppm) is spiked. Samples are processed as below:

Heat sample at 60oC for 5 min, Evaporate sample to dry at 60 oC for 23 minutes under N2 (2.8 L/min)

Add 3 mL elution solvent to sample, purge with air and heat for 2 min. Transfer all liquid to GCB column. Collect fraction and do evaporation.

Elute another 8 mL. Collect fraction and evaporate at same time. Adjust to 0.5 mL with methanol.

Corresponding method for SPE-03:

No. Acts Flow rate Vol. 5 Add sam 10 0.11 Heat 60(oC) 5.0 (min) 6 Con F2 10 4.02 Evap F1 10 23.0 (min) 7 Elute 1 10 0.03 Clean 1 10 3.0 8 Con F2 10 8.04 Heat 60 2.0(min)

Recovery Test for Approach 2

Sample 1 Sample 264% 50%

The low recovery could be due to incomplete transfer of sample to the column. After sample loading, there is still some liquid left in the vial. More optimization in sample loading step is necessary.

1 g tea +10 mL acetone, heat for 10 minutes.

Evaporate away acetone, analytes stay on surface of sample solid.

Add 3 mL acetone, heat for 5 min, transfer 1.5+mL liquid to column.

Questions?

Acknowledgement: This project is financially supported by the Industrial Research Assistance Program from National Research Council Canada.