The International Mussel Watch 



4.4.1. Alumina clean-up 



Activated alumina, activated at 800°C and 5% deactivated is slurried into a precleaned 

 Pastuer pipette (0.5 cm i. d. with a glass wool plug) to a height of 4 cm and kept under n-hexane. 

 The tissue extract which has been concentrated to below 1 ml in a Kuderna-Danish concentrator or 

 Rotavap is transferred to the column and eluted with n-hexane (10 ml). The eluate received in a 

 Kuderna-Danish concentrator vial is reduced immediately in volume to below 1 ml and reduced 

 further under a a mild stream of nitrogen to about 100 pi ready for injection unto the HPLC 

 column. 



Caution: temperature must be controlled. As an example, concentrate from 1 ml to 100 pi at 

 <10°C in 20 minutes. 



4.4.2. HPLC separation 



The details of the HPLC procedures are described in Petrick, et al, (1988) and are 

 summarized here. The equipment consists of an isocratic HPLC system equipped with a 

 Rheodyne (or equivalent) injector with 200 pi loop capacity, a guard column with a back flush 

 valve and a 20 cm x 0.4 cm, normal phase, 5 um Nucleosil (or equivalent) column. The total 

 volume of extract is transferred with washings. The total volume is kept as small as possible. The 

 organochlorine compounds of interest are eluted with 1 1 ml of n-pentane at a flow rate of 0.5 ml 

 per minute, 4 ml of 20% dichloromethane in n-pentane and finally 10 ml 100% dichloromethane 

 followed by a 5 minute backflush and a 5 minute equilibration with the first eluant. 



The following classes of compounds can be separated consecutively (Petrick, et al., 1988): 



Fraction: ml 



1. n-alkanes and n-alkenes 0.5- 2.0 



2. HCB, PCB's and alkylbenzenes 2.0- 4.5 



3. PAH's and toxaphene 4.5-11.0 



4. pesticides, and toxaphene 1 1.0 - 15.0 



5. acids, etc. (polar compounds) 15.0 - 25.0 



90 



