1. Micro Analytical GC System for Tea Aroma in Product Quality Control Fred Lewis, Principal Chemist, Analytical Science Laboratory, Unilever John Crandall, Analytical Specialists – presenter Brian Rohrback – President, Infometrix

2. Copyright January 31, 2007 2 Introduction to the Problem Rigors of achieving process control and consistency Natural products have natural variances “Good” and “Bad” assessments are subjective Historically assessed by human sensory panels Human customers still assess the product “goodness” or… “not good” Human sensory panel and production staff turnover “Personal expert” databases of “good” and “bad” leave with the turnover Production staff may or may not be able to consistently continue Advanced analytical chemistry Used to make assessments objective & instrumental measurement based GC/MS systems quantify flavor and fragrance chemicals Chemometric modeling including “sensory panel” information has been used These techniques work very well in an R&D environment with experts They are too slow and technically unsuitable for real world process control

3. Copyright January 31, 2007 3 Current Analytical Method & Results Column & Injection DB1 60 m X.32 mm I.D. X 1u thickness 1 uL C 6 /MeCl 2 Injector - 250 C Detector – 300 C Temperature Program Initial - 40 C for 2 min. Rate - 4 C/min., 52.5 min. Final - 250 C for 10 min. Total run time - 64.5 min. He carrier gas at 1 mL/min. GC/MS Trace of Standard Run time about 65 MINUTES Method time more than 90 MINUTES Sample turnaround time up to 4 HOURS

4. Copyright January 31, 2007 4 Slow, Complicated & Expensive The GC/MS method Does an excellent job Requires advanced expertise Costs in excess or $100K to implement A faster, simpler, more cost effective solution was sought Solution Process First step Reduce chromatography time to seconds, not minutes Second step Simplify sample collection, preparation and injection process Third step Simplify & automate the data handling Automate the instrumentation

5. Copyright January 31, 2007 5 First Step Solution, Fast GC Faster GC requirements Preserve resolution as required Preserve sensitivity Simplify operation Significantly reduce capital & operating costs

6. Copyright January 31, 2007 6 Analytical Process (initially the same) & microFAST GC System Prepared Standards Selected “standard” aldehydes, ketones & acids are prepared in C 6 /MeCl 2 Bromobenzene used as an internal standard Prepared Samples An aliquot of aqueous sample is measured & extracted Extract solvent is C 6 /MeCl 2 with bromobenzene included GC/MS System Microliters of the extracted liquid are injected Using pre-established response factors & internal standard, the concentration of the “standards” is determined

7. Copyright January 31, 2007 7 microFAST GC Analytical Method Column & Injection DB5 2 m X 0.1mm I.D. X 0.4u thickness 1 uL C 6 /MeCl 2 Injector - 150 C Detector - 225 C Temperature Program Initial - 35 C for 6 sec. Rate - 3 C/sec., 58 sec. Final - 250 C for 2 sec. Total run time - 80 sec. Pressure Program Initial – 8 psi for 10 sec. Rate – 0.3 psi/sec., 57 sec. Final - 25 psi for 13 sec. Total run time - 80 sec. H 2 carrier gas, variable flow

8. Copyright January 31, 2007 8 Results Exp. GC/MS Trace GC Trace of Standard Run time about 80 Seconds Method time about 30 MINUTES Sample turnaround time up to 3 HOURS

9. Copyright January 31, 2007 9 Direct Comparison Essentially, the chromatograms are the same except Isomer pair at the beginning Two other pairs “shoulder” Analytical times 65 minutes 65 minutes 80 seconds

10. Copyright January 31, 2007 10 Results Standard components 2 pairs measured as a “a pair” Are identified by the method Quantify as necessary with adequate Resolution Sensitivity

11. Copyright January 31, 2007 11 Second Step Solution, Fast Sampling System Faster sampling requirements Preserve important sample components Take care to prevent sampling “bias” Preserve sensitivity Simplify operation (eliminate L/L extraction) Significantly reduce capital & operating costs

12. Copyright January 31, 2007 12 Analytical Process Changes & Fast Sampling System Prepared Standards Selected “standard” aldehydes, ketones & acids are prepared in C 6 /MeCl 2 Bromobenzene used as an internal standard Prepared Samples An aliquot of aqueous sample is measured & put in a vial SP/ME fiber is exposed to heated sample headspace gas SP/ME fiber is “desorbed” in the microFAST GC inlet microFAST GC System Desorbed fiber components are trapped, then desorbed into GC columns Using pre-established response factors & internal standard, the concentration of the standard & samples are determined

13. Copyright January 31, 2007 13 Initial Results a SP/ME run on 4 marker aldehydes in Neobee

14. Copyright January 31, 2007 14 Initial Results SP/ME of Tea Aroma overlaid with the previous 4 aldehydes run

15. Copyright January 31, 2007 15 Results & More Questions Tea Aroma analysis is possible in 5 minutes No lab chemicals or extraction skills are necessary Production could use system “near line” To speed up sample transportation & result generation Simplify production quality decision process Turn around could be reduced From up to 4 hours 15 minutes To less than 15 minutes What about sample bias? What about data processing? What about automation?

16. Copyright January 31, 2007 16 Sampling Bias from SP/ME Fiber to sample exposure time 1 minute is inadequate 15 minutes is excellent but too long 5 minutes is adequate Sample temperature set at 60 C 1 minute 5 minutes 15 minutes Overlaid

17. Copyright January 31, 2007 17 Data Processing: PCA Scores Plot Examples Principal Component Analysis Simple example demonstrates distinct data clusters Models will include microFAST GC data Sensory panel information Production parameters And be automated for production consistency & control

18. Copyright January 31, 2007 18 Third Step Solution, Automation Prospects Laboratory hardware and methods testing SP/ME holder Auto sampler Synchronization of devices Laboratory software and data processing Chemometric modeling System automation, data presentation, reporting and archiving

19. Copyright January 31, 2007 19 What’s Next? Moving on towards Standardized product profiling Monitoring product changes during storage Objectively determining if and when our processes have changed Supporting human sensory panels in mapping product attributes Here are examples Concept is proven Fast GC provides sufficient Data Simplicity Speed With system costs under $50,000 Chemometrics provides Total system automation Data processing and Sophisticated results in the hands of normal process operators.

20. Copyright January 31, 2007 20 SP/ME Product Profiles Peanut Butter Spaghetti Sauce Olive Oil

21. Copyright January 31, 2007 21 Reconstructed SP/ME Product Profiles

22. Copyright January 31, 2007 22 Reconstructed SP/ME Product Profiles – Expanded Scale

23. Copyright January 31, 2007 23 SP/ME Product Profiles - PCA Peanut Butter 1,2 &3 Olive Oil 1, 2 & 3 Spaghetti Sauce 1, 2 & 3

24. Copyright January 31, 2007 24 Re-analysis to Separate Peanut Butter and Olive Oil Peanut Butter 1 Peanut Butter 2 Peanut Butter 3 Olive Oil 1 Olive Oil 2 Olive Oil 3

25. Copyright January 31, 2007 25 Latest SP/ME Tea Aroma Overlaid SP/ME Standard Standard Tea Aroma

26. Questions?

27. Copyright January 31, 2007 27 Expanded GC/MS Trace Back

28. Copyright January 31, 2007 28 Optimum Fiber Exposure ( 5 Minutes) Back

29. Copyright January 31, 2007 29 Overlaid Results Back 1 minute 5 minutes 15 minutes 5 minute exposure Low and high boilers sufficiently captured Pattern is “balanced” across the boiling range Sufficient for future model building

30. Copyright January 31, 2007 30 Olive Oil

31. Copyright January 31, 2007 31 Peanut Butter

32. Copyright January 31, 2007 32 Spaghetti Sauce