RAPIDLY MONITORING BIODIESEL PRODUCTION ... DOWNLOAD A COPY OF THIS POSTER, VISIT ©2009 Waters...
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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2009 Waters Corporation | COMPANY CONFIDENTIAL RAPIDLY MONITORING BIODIESEL PRODUCTION PROCESSES USING UPLC Peter J. Lee, Gerard C. Bondoux*, and Alice J. Di Gioia Waters Corporation, Milford, MA 01757 USA AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m n m n TAG of soybean oil UV ELS AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m n m n TAG of soybean oil UV AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m n m n TAG of soybean oil AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m n m n AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m n m n AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m n m AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m n AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a AU 0.00 0.40 0.80 LSU 0.00 800.00 1600.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 a b b c c d d e e f f g g h h i i j j k k l l m n m n TAG of soybean oil UV ELS ELS LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l TAG ELS DAG Expanded ELS LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l TAG ELS DAG ELS LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l TAG ELS ELS LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l TAG ELS LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l TAG LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l TAG LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.00 200.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 LSU 0.0 1000.0 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 a c b d e f g h i l TAG ELS DAG Expanded AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG DAG UV UV b h g AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG DAG UV UV b h AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG DAG UV UV b AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG DAG UV UV AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG DAG UV AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG DAG AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 1.50 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 AU 0.00 0.06 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Expanded b c e g i l TAG DAG UV UV b h g OCOR' OCOR" OCOR''' OCOR" OCOR''' OH OCOR" OCOR''' OH OCOR''' OH OH OCOR''' OH OH OH OH OH Triacylglycerol (TAG) + ROH catalyst + ROH catalyst + ROH catalyst Step 1 Diacylglycerol (DAG) Step 2 DAG + R'COOMe Fatty Acid Methyl Ester Monoacylglycerol (MAG) + R''COOMe (FAME) Step 3 MAG Glycerol + R'''COOMe (FAME) OH OH OH + H 2 O catalyst Glycerol Hydrolysis of Esters TAG + R'COOH R''COOH R'''COOH FFA (FAME) Transesterification reactions DAG, MAG FAME + MeOH ROH = MeOH Note: EtOH and butanol are also reported for transesterification EXPERIMENTAL Biodiesel was synthesized using the kitchen biodiesel method with soybean oil, MeOH and NaOH. 3 Crude biodiesel was diluted to 12 mg/mL with 2-propanol (IPA) and the solution was filtered with a 0.45μm PVDF syringe filter for UPLC analysis. Standards (Table 1) were dissolved in IPA to make 0.5mg/mL solution for the analysis. UPLC™ System and Operation Conditions 1,2 System: ACQUITY UPLC®/ACQUITY PDA/ELSD/Empower™ 2 Detection: PDA 195 to 300 nm; Weak Wash: IPA (600 μL) Column Temp: 30 °C; Injection: 2 μL (full loop) Column: ACQUITY UPLC™ BEH C18 2.1x 150 mm Mobile Phase A: CH3CN; Mobile Phase B: IPA (Fisher, Optima) Gradient Table INTRODUCTION Biodiesel is gaining significant interest as the replacement for petroleum-based diesel fuel since it is renewable and cleaner-burning. Besides these environmental factors, the increase in petroleum price and the government mandates have all contributed to its popularity. Biodiesel is fatty acid methyl ester (FAME) made from vegetable oil or animal fat, by transesterification with methanol in the presence of a catalyst. To maximize the yield of FAME and reduce non-volatile contaminants including unreacted feedstock (TAG), reaction intermediates (MAG and DAG), unwanted hydrolysis contaminants FFA, and glycerol (Scheme 1), the status of reaction and refining processes need to be monitored. Biodiesel samples can be analyzed by GC; however, the non- volatile contaminants (FA, TAG, MAG, and DAG) require derivatization prior to GC analysis, multiple injections with different GC experimental conditions are needed to characterize FAME and impurities, which are laborious. Analyzing biodiesel by conventional HPLC methods requires 30 to 80 minute run times and use halogenated solvents that are known carcinogens and restricted in laboratories. This poster illustrates the application of a UPLC system equipped with photodiode array and evaporative light scattering detectors to analyze biodiesel samples without derivatization and halogenated solvents. Biodiesel related standards, biodiesel, and non-volatile contaminants were separated in 12 minutes. 1,2 The method can be used to rapidly monitor biodiesel production to increase productivity. Compared with conventional HPLC methods, the UPLC solution shortens analysis time and reduces solvent usage, leading to cost benefits and aligning with the principles of green chemistry. Scheme 1. Transesterification and hydrolysis reactions of esters involved in biodiesel production Table 1. Standards of FFA, FAME, MAG, DAG and TAG Figure 1. ELS and UV (210 nm) chromatograms of a solution containing soybean oil (0.7mg/mL) and standards of FFA, FAME, MAG, DAG and TAG (0.5mg/mL each, see table 1 for peak labeling). Figure 2. ELS chromatogram of homemade biodiesel (12mg/mL) made from soybean oil. Note: The lower chromatogram represents a baseline expansion. Figure 3. UV (210 nm) chromatogram of homemade biodiesel (12mg/mL) made from soybean oil. Note: The lower chromatogram represents a baseline expansion. CONCLUSION ● Biodiesel components, FAME and non-volatile contaminants including TAG, reaction intermediates (MAG and DAG), hydrolysis and reaction byproducts (FFA, and glycerol) are separated in 12 minutes. ● UPLC enables rapid monitoring of production processes that can leads to the benefits of higher yield, lower contamination and higher quality products. ● By incorporating UV with the evaporative light scattering detection, more information per chromatographic run can be obtained, thus dramatically increasing productivity. ● The separation is several times faster than conventional HPLC, minimizes solvent usage and eliminates the use of toxic halogenated solvents, resulting in cost benefits and alignment with the principles of green chemistry. REFERENCE 1. Lee, P. J. and Di Gioia, A. J. (2007) ACQUITY UPLC/ELSD/UV one methodology for FFA, FAME & TAG analysis, Waters application note 720002155en. 2. Lee, P. J. et al. (2009) Automating calculations for rapid seed oil quality control and Authenticity. Waters Corporation, application note 720002973en. 3. http://kitchen-biodiesel.com/ UPLC SEPARATION OF BIODIESEL & NON-VOLATILE CONTAMINANTS Time (min) mL/min %B Curve 0 0.17 11 6 7 0.17 37.5 6 7.01 0.17 90 11 12 0.17 90 11 8001-22-7 Soybean oil 19 n 122-32-7 Glyceryl trioleate 18 m 537-40-6 1,2,3-trilinoleoylglycerol 17 l 15818-46-9 1,3-dilinoleoyl-rac-glycerol 16 k 1120-28-1 Methyl arachidate 15 j 506-30-9 Arachidic acid 14 i 112-61-8 Methyl stearate 13 h 57-11-4 Stearic acid 12 g 112-39-0 Methyl palmitate 11 g 112-62-9 Methyl oleate 10 f 57-10-3 Palmitic acid 9 f 112-80-1 Oleic acid 8 e 112-63-0 Methyl linoleate 7 d 111-03-5 1-Oleoyl-rac-glycerol 6 d 60-33-3 Linoleic acid 5 c 301-00-8 Methyl linolenate 4 b 2277-28-3 1-Linoleoyl-rac-glycerol 3 b 463-40-1 Linolenic acid 2 a 56-81-5 Glycerol 1 Peak Label CAS No. Name ID 8001-22-7 Soybean oil 19 n 122-32-7 Glyceryl trioleate 18 m 537-40-6 1,2,3-trilinoleoylglycerol 17 l 15818-46-9 1,3-dilinoleoyl-rac-glycerol 16 k 1120-28-1 Methyl arachidate 15 j 506-30-9 Arachidic acid 14 i 112-61-8 Methyl stearate 13 h 57-11-4 Stearic acid 12 g 112-39-0 Methyl palmitate 11 g 112-62-9 Methyl oleate 10 f 57-10-3 Palmitic acid 9 f 112-80-1 Oleic acid 8 e 112-63-0 Methyl linoleate 7 d 111-03-5 1-Oleoyl-rac-glycerol 6 d 60-33-3 Linoleic acid 5 c 301-00-8 Methyl linolenate 4 b 2277-28-3 1-Linoleoyl-rac-glycerol 3 b 463-40-1 Linolenic acid 2 a 56-81-5 Glycerol 1 Peak Label CAS No. Name ID
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RAPIDLY MONITORING BIODIESEL PRODUCTION PROCESSES USING UPLC
Peter J. Lee, Gerard C. Bondoux*, and Alice J. Di Gioia Waters Corporation, Milford, MA 01757 USA
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LSU
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LSU
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DAG
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UV
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1.50
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0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
TAG
DAG
UV
UV
b
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
TAG
DAG
UV
UV
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
TAG
DAG
UV
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
TAG
DAG
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
TAG
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
TAG
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
1.50
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.06
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Expanded
b
c
e
g i
l
TAG
DAG
UV
UV
b
h
g
OCOR'
OCOR"
OCOR'''
OCOR"
OCOR'''
OH
OCOR"
OCOR'''
OH
OCOR'''
OH
OH
OCOR'''
OH
OH
OH
OH
OH
Triacylglycerol (TAG)
+ ROHcatalyst
+ ROHcatalyst
+ ROHcatalyst
Step 1
Diacylglycerol (DAG)
Step 2
DAG
+ R'COOMe
Fatty Acid Methyl Ester
Monoacylglycerol (MAG)
+ R''COOMe (FAME)
Step 3
MAG Glycerol
+ R'''COOMe (FAME)
OH
OH
OH
+ H 2Ocatalyst
Glycerol
Hydrolysis of Esters
TAG
+
R'COOH
R''COOH
R'''COOH
FFA
(FAME)
Transesterification reactions
DAG, MAGFAME
+ MeOH
ROH = MeOHNote: EtOH and butanol are also reported for transesterification
EXPERIMENTAL
Biodiesel was synthesized using the kitchen biodiesel method with soybean oil, MeOH and NaOH.3 Crude biodiesel was diluted to 12 mg/mL with 2-propanol (IPA) and the solution was filtered with a 0.45µm PVDF syringe filter for UPLC analysis. Standards (Table 1) were dissolved in IPA to make 0.5mg/mL solution for the analysis. UPLC™ System and Operation Conditions1,2 System: ACQUITY UPLC®/ACQUITY PDA/ELSD/Empower™ 2 Detection: PDA 195 to 300 nm; Weak Wash: IPA (600 µL) Column Temp: 30 °C; Injection: 2 µL (full loop) Column: ACQUITY UPLC™ BEH C18 2.1x 150 mm Mobile Phase A: CH3CN; Mobile Phase B: IPA (Fisher, Optima) Gradient Table
INTRODUCTION Biodiesel is gaining significant interest as the replacement for petroleum-based diesel fuel since it is renewable and cleaner-burning. Besides these environmental factors, the increase in petroleum price and the government mandates have all contributed to its popularity. Biodiesel is fatty acid methyl ester (FAME) made from vegetable oil or animal fat, by transesterification with methanol in the presence of a catalyst. To maximize the yield of FAME and reduce non-volatile contaminants including unreacted feedstock (TAG), reaction intermediates (MAG and DAG), unwanted hydrolysis contaminants FFA, and glycerol (Scheme 1), the status of reaction and refining processes need to be monitored. Biodiesel samples can be analyzed by GC; however, the non-volatile contaminants (FA, TAG, MAG, and DAG) require derivatization prior to GC analysis, multiple injections with different GC experimental conditions are needed to characterize FAME and impurities, which are laborious. Analyzing biodiesel by conventional HPLC methods requires 30 to 80 minute run times and use halogenated solvents that are known carcinogens and restricted in laboratories. This poster illustrates the application of a UPLC system equipped with photodiode array and evaporative light scattering detectors to analyze biodiesel samples without derivatization and halogenated solvents. Biodiesel related standards, biodiesel, and non-volatile contaminants were separated in 12 minutes.1,2 The method can be used to rapidly monitor biodiesel production to increase productivity. Compared with conventional HPLC methods, the UPLC solution shortens analysis time and reduces solvent usage, leading to cost benefits and aligning with the principles of green chemistry.
Scheme 1. Transesterification and hydrolysis reactions of esters involved in biodiesel production
Table 1. Standards of FFA, FAME, MAG, DAG and TAG
Figure 1. ELS and UV (210 nm) chromatograms of a solution containing soybean oil (0.7mg/mL) and standards of FFA, FAME, MAG, DAG and TAG (0.5mg/mL each, see table 1 for peak labeling).
Figure 2. ELS chromatogram of homemade biodiesel (12mg/mL) made from soybean oil. Note: The lower chromatogram represents a baseline expansion.
Figure 3. UV (210 nm) chromatogram of homemade biodiesel (12mg/mL) made from soybean oil. Note: The lower chromatogram represents a baseline expansion.
CONCLUSION ● Biodiesel components, FAME and non-volatile contaminants including TAG, reaction intermediates (MAG and DAG), hydrolysis and reaction byproducts (FFA, and glycerol) are separated in 12 minutes. ● UPLC enables rapid monitoring of production processes that can leads to the benefits of higher yield, lower contamination and higher quality products. ● By incorporating UV with the evaporative light scattering detection, more information per chromatographic run can be obtained, thus dramatically increasing productivity. ● The separation is several times faster than conventional HPLC, minimizes solvent usage and eliminates the use of toxic halogenated solvents, resulting in cost benefits and alignment with the principles of green chemistry.
REFERENCE 1. Lee, P. J. and Di Gioia, A. J. (2007) ACQUITY UPLC/ELSD/UV one methodology for FFA, FAME & TAG analysis, Waters application note 720002155en. 2. Lee, P. J. et al. (2009) Automating calculations for rapid seed oil quality control and Authenticity. Waters Corporation, application note 720002973en. 3. http://kitchen-biodiesel.com/
UPLC SEPARATION OF BIODIESEL & NON-VOLATILE CONTAMINANTS
Time (min) mL/min %B Curve
0 0.17 11 6
7 0.17 37.5 6
7.01 0.17 90 11
12 0.17 90 11
8001-22-7Soybean oil19
n122-32-7Glyceryl trioleate18
m537-40-61,2,3-trilinoleoylglycerol17
l15818-46-91,3-dilinoleoyl-rac-glycerol16
k1120-28-1Methyl arachidate15
j506-30-9Arachidic acid14
i112-61-8Methyl stearate13
h57-11-4Stearic acid12
g112-39-0Methyl palmitate11
g112-62-9Methyl oleate10
f57-10-3Palmitic acid9
f112-80-1Oleic acid8
e112-63-0Methyl linoleate7
d111-03-51-Oleoyl-rac-glycerol6
d60-33-3Linoleic acid5
c301-00-8Methyl linolenate4
b2277-28-31-Linoleoyl-rac-glycerol3
b463-40-1Linolenic acid 2
a56-81-5Glycerol 1
Peak Label
CAS No.NameID
8001-22-7Soybean oil19
n122-32-7Glyceryl trioleate18
m537-40-61,2,3-trilinoleoylglycerol17
l15818-46-91,3-dilinoleoyl-rac-glycerol16
k1120-28-1Methyl arachidate15
j506-30-9Arachidic acid14
i112-61-8Methyl stearate13
h57-11-4Stearic acid12
g112-39-0Methyl palmitate11
g112-62-9Methyl oleate10
f57-10-3Palmitic acid9
f112-80-1Oleic acid8
e112-63-0Methyl linoleate7
d111-03-51-Oleoyl-rac-glycerol6
d60-33-3Linoleic acid5
c301-00-8Methyl linolenate4
b2277-28-31-Linoleoyl-rac-glycerol3
b463-40-1Linolenic acid 2
a56-81-5Glycerol 1
Peak Label
CAS No.NameID
