Dr. K. van Lenning Institute of Marine Science ICM-CSIC, Barcelona

1. Losses of chlorophylls and carotenoids in aqueous acetone and methanol extracts prepared for RP-HPLC analysis of pigments Dr. K. van Lenning Institute of Marine Science ICM-CSIC, Barcelona published in Chromatographia - April 2001 M. Latasa, K. van Lenning, J. L. Garrido, R. Scharek, M. Estrada, F. Rodríguez and M. Zapata

2. 1: Sample collection at sea (volume usually limited) 2: Filtration on board (25 or 47 mm GF/F) 3: Storage until extraction (liquid N2 or fridge) 4: Extraction (acetone or methanol) 5: Pigment analyses (RP-HPLC procedures) 4: Extraction (is methanol really the best option?) 2

3. May cause peak distortion Large injection volumes wanted.... Minimum solvent volume wanted to achieve high concentrations - determined by filter size May cause peak distortion 4: Extraction (is methanol really the best option?) 3

4. Peak distortion; solutions and consequences Related to differences between extraction solvent and initial mobile phase composition; strength, viscosity, ... ? We do not know Two solutions 1: Reduce injection volume and/or 2: Dilute sample with water Dilute sample with water: risky! 4

5. “Critical dilution rate” expected (polar- and nonpolar pigments) Pigments dissolve in organic solvents, but not in water Higher dilution rates: larger injection volumes in HPLC (frequently 1:0.3 - 1:1) Solvent- and time dependant losses of individual compounds 1: Maximum sample dilution permitted 2: Best solvent for storage and injection Dilute sample with water 5

6. Experiment consisted of 1 main and 2 additional parts: 1 Influence of dilution rates on pigment losses in acetone and methanol extracts. a: Extract Emiliania huxleyi and Dunaliella tertiolecta in 100% organic solvent and inject small volume. b: Dilute extracts (to 90%, 80%, ....) and inject directly in HPLC. c: Keep diluted extracts in refrigerated autosampler (4ºC) and reinject entire range after 24 and 48 hours. d: Establish critical dilution rate and maximum storage time in autosampler. Experiments with E. Hux and D. tertiolecta in acetone or methanol were performed individually under subdued light conditions 6

7. 2 Check if the observed anomalies in first injection of diluted samples were induced by losses in the 100% extracts prior to water addition. a: Prepare new extracts of E. huxleyi and D. tertiolecta (first 100% acetone, than methanol). b: Inject fresh extracts directly in HPLC. c: Fill 23 additional vials, keep them in autosampler at 4ºC, and inject as time series. 7

8. 3 Check if observed anomalies of diluted samples were related to chromatographic problems. a: Prepare new extracts of E. huxleyi and D. tertiolecta (first 100% acetone, than methanol). b: Measure absorption of red (mainly Chls a and b) and blue light (all pigments) using a spectrophotometer. c: Dilute to same range employed for HPLC experiment and measure red and blue absorption directly. d: Keep samples at 4ºC, and repeat readings after 30, 75, 180, 360 min (6 h), 24 h and 48 h. This quick approach confirmed losses observed by HPLC and results will not be presented. 8

9. Time series Emiliania huxleyi extracted in 100% acetone Chlorophyll a Carotenes Total Chl c3 19’Hex-fucoxanthin Chlorophyll c2 Differences time zero (%) Time (hours) - symbols indicate injections Deviations  5% were attributed to equipment and sample treatment 9

10. Emiliania huxleyi extracted in acetone, diluted with water First injection: t = 0 = Differences larger than 5% = Dilution rate with maximum differences 123 10

11. Second injection: t = 24 hours = Differences larger than 5% = Dilution rate with maximum differences 123 Emiliania huxleyi extracted in acetone, diluted with water 11

12. Third injection: t = 48 hours = Differences larger than 5% = Dilution rate with maximum differences 123 Emiliania huxleyi extracted in acetone, diluted with water 12

13. Time series Dunaliella tertiolecta extracted in 100% acetone Chlorophyll a Carotenes Chlorophyll b Violaxanthin Lutein Differences time zero (%) Time (hours) 13

14. Dunaliella tertiolecta extracted in acetone, diluted with water First injection: t = 0 hours = Differences larger than 5% = Dilution rate with maximum differences 123 14

15. Second injection: t = 24 hours = Differences larger than 5% = Dilution rate with maximum differences 123 Dunaliella tertiolecta extracted in acetone, diluted with water 15

16. Third injection: t = 48 hours = Differences larger than 5% = Dilution rate with maximum differences 123 Dunaliella tertiolecta extracted in acetone, diluted with water 16

17. Time series Dunaliella tertiolecta extracted in 100% methanol Chlorophyll a Carotenes Chlorophyll b Violaxanthin Lutein Differences time zero (%) Time (hours) 17

18. Dunaliella tertiolecta extracted in methanol, diluted with water First injection: t = 0 hours = Differences larger than 5% = Dilution rate with maximum differences 123 18

19. Second injection: t = 24 hours = Differences larger than 5% = Dilution rate with maximum differences 123 Dunaliella tertiolecta extracted in methanol, diluted with water 19

20. Third injection: t = 48 hours = Differences larger than 5% = Dilution rate with maximum differences 123 Dunaliella tertiolecta extracted in methanol, diluted with water 20

21. Time series Emiliania huxleyi extracted in 100% methanol Chlorophyll a Carotenes Total Chl c3 19’Hex-fucoxanthin Chlorophyll c2 Differences time zero (%) Time (hours) 21

22. Emiliania huxleyi extracted in methanol, diluted with water First injection: t = 0 = Differences larger than 5% = Dilution rate with maximum differences 123 22

23. Second injection: t = 24 = Differences larger than 5% = Dilution rate with maximum differences 123 Emiliania huxleyi extracted in methanol, diluted with water 23

24. Third injection: t = 48 = Differences larger than 5% = Dilution rate with maximum differences 123 Emiliania huxleyi extracted in methanol, diluted with water 24

25. Conclusions: 1: Concentrations of pigments only remain stable for at least 24 h in 80% and 90% acetone. This obviously excludes the use of methanol when *normal autoinjectors are involved. 2: The acetone range can be extended to 70% and 100% when injecting fresh extracts immediately after dilution step. Such procedures also allow the use of methanol (90 - 100% range). 3: Extended ranges are valid for up to 24 h when concentrations of ,- and ,-carotenes are not considered to be important. 4: Amount of pigment losses due to degradation or precipitation may vary between experiments, but tendencies always hold true and can be observed directly or within a few hours. *no automatic sample dilution prior to injection possible

26. Recommendations: 1: The established limits of the “safe” dilution rates are critical and seawater volumes retained by the filters are thus very important. Individuals should check their own limits for which they may (initially) employ a spectrophotometric approach. 2: An HPLC setup should preferably include a refrigerated auto- injector capable of sample dilution prior to injection. 3: In this case it is safe to use a final concentration of 80 - 90% acetone for sample storage at 4ºC (reliable up to at least 48 h), diluted to 70% before injection (maximum injection volume in HPLC without risk of pigment losses at t=0).