Prolonged exposure to ultraviolet radiation (UVR) from the sun has negative effects on DNA and can also damage amino acids and proteins. To protect themselves against UVR, marine organisms possess UV-absorbing compounds called mycosporine-like amino acids (MAAs). Currently, there are no commercial standards of MAAs available. However, it is possible to isolate MAAs from methanol extractions of marine organisms using high-performance liquid chromatography (HPLC). Over the course of several months, HPLC was successfully used to isolate MAAs from the Rhodophyta (red algae) species of Iridaea cordata, Gigartina papillata, Mastocarpus papillatus, and Mazzaella splendens. In addition, co-chromatography was used to identify common compounds between different red algae. Although inadequate storage methods lead to MAA breakdown following isolation, the work spent making the HPLC operational and calculating the collection windows will make future MAA isolation a simple matter of repeating procedures. Future implications of this research include scaling up MAA isolation through preparative HPLC in addition to analysis by mass spectrometry.

2. I. Purpose
II. Background Information
III. Methods
IV. Results
V. Conclusion
VI. Future Considerations
VII. Acknowledgements

3. There are no commercial standards of
mycosporine-like amino acids (MAAs) available.
 Isolate MAAs to be used for standards
 Become proficient with HPLC system and
software
 Document methods for future student research

4.  Ultraviolet radiation (UVR)
 Mycosporine-like amino acids (MAAs)
 Reversed-phase high performance liquid chromatography
(HPLC)

5.  A portion of the electromagnetic spectrum
between visible light and x-rays
http://missionscience.nasa.gov/ems/10_ultravioletwaves.html

6. Karentz 2001
adeletang8.edu.glogster.com

7. http://earthobservatory.nasa.gov/Features/UVB/

8.  UV-absorbing compounds that filter radiation in
the 309-360 nm range
Karentz 2001

9. O NH
OCH3 OCH3
mycosporine-glycine palythine
max = 310 nm max = 320 n
HO OH NH HO OH NH
CO2 H CO2 H
CO2 H CO2 H
N N
OH OH porphyra-33
shinorine OCH
max = 334 n
OCH3 3
max = 334 nm
HO OH NH HO OH NH
CO2 H CO2 H
Karentz 2001

10. Karentz 2001

11.  Chromatography = the separation of mixtures
 Liquid = liquid mobile phase
 High-performance = small packing materials and
high pressure
 Reversed-phase = non-polar stationary phase

12. http://commons.wikimedia.org/wiki/File:HPLC_apparatus.svg

13.  Conditions to consider:
› Composition of mobile phase (running solvent)
› Composition of stationary phase (packing material of
column)
› Flow rate
› Injection volume
› Run time

14. 1. Sample collection and storage performed by
Professor Karentz
2. Extraction with methanol
3. Separation of MAAs by HPLC
4. Isolation of MAAs by fraction collecting
5. Run samples in scanning spectrophotometer
6. Co-chromatography to identify common MAAs

15. 1. Sample collection and storage performed by Professor Karentz

16.  Rhodophyta (red algae)
› Anthopleura sola
› Erythrophyllum delesserioides
› Gigartina papillata
› Hymenena flabelligera
› Iridaea cordata
› Mastocarpus papillatus
› Mazzaella splendens
› Plocamium cartilagineum
› Smithora naiadum
 Other
› Euphausia superba (krill)
› Limacina helicina (pteropod)
› Orchomene plebes (amphipod)
› Prasiola crispa (green algae)

17. 2. Extraction with methanol

18. 3. Separation of MAAs by HPLC

19. 4. Isolation of MAAs by fraction collecting

20. 5. Run samples in scanning spectrophotometer

21. 6. Co-chromatography to identify common MAAs

22.  Species selected for MAA isolation
› Gigartina papillata
› Iridaea cordata
› Mastocarpus papillatus
› Mazaella splendens
 Co-chromatography of Iridaea cordata and
Gigartina papillata

23. Species Peak Landmark Delay Collection
Retention Time Amount
Iridaea 3 minutes, 30 seconds 25 drops
cordata 22 seconds
Gigartina 3 minutes, 25 seconds 25 drops
papillata 17 seconds
Mastocarpus 3 minutes, 1 minute, 30 drops
papillatus 18 seconds 15 seconds
Mazzaella 3 minutes, 50 seconds 15 drops
splendens 30 seconds

25. Tube 1 Tube 2
Tube 3 Tube 4
Mazaella splendens

28.  MAA isolation was successful
 Co-chromatography confirmed identical MAAs
 Reproducible for future student research

29.  Preparative HPLC  Mass spectrometry
http://www.mac-mod.com/ace_catalog/ac-prep.html
http://www.chemguide.co.uk/analysis/masspec/

30.  Professor Deneb Karentz
 Professor Patricia Schulz
 Professor Megan Bolitho
 David and Bela Villongco