binding of rhodamine .   One such series of compound were free fatty acids of the types found in acylsugars .    Glycerols esterified to fatty acids from C4 to C10, the range of chain lengths found in acylsugars of L . pennellii were also used as test compounds .   The tests of rhodamine B binding to free fatty acids indicates that this test could be strongly biased depending on fatty acid chain length (Table 1) . The assay’s response is very strong in fatty acids of C7 and greater, but weak to non - existent in fatty acids shorter than C7 .   The Rhodamine B assay was similarly affected by fatty acid chain length in tests on diacylglycerols; however, diacylglycerols with shorter fatty acids appeared to have greater affinity for Rhodamine B than free fatty acids of the same length (data not shown) .    Since the relative levels of the different fatty acids present in acylsugars varies with genotype, the bias for fatty acid chain length could result in false negatives or in errors in estimation of relative acylsugar levels among genotypes when the Rhodamine B assay is used .   Table 1 . Average absorbence values from Rhodamine binding to increasing amounts of different fatty acids . Concentration . 003M . 045M . 067M . 1M . 15M . 224M . 335M . 5M . 045M propionic acid (C3) 0 . 01 0 . 01 0 . 01 0 . 01 0 . 00 0 . 01 0 . 00 0 . 01 0 . 01 isobutyric acid (C4) 0 . 01 0 . 01 0 . 01 0 . 02 0 . 01 0 . 03 0 . 03 0 . 05 0 . 01 2 - methylbutyric acid (C4) 0 . 01 0 . 01 0 . 02 0 . 02 0 . 03 0 . 06 0 . 11 0 . 23 0 . 01 n - valeric acid (C5) 0 . 01 0 . 01 0 . 01 0 . 02 0 . 03 0 . 06 0 . 26 0 . 53 0 . 01 3 - methyl - n - valeric acid (C6) 0 . 01 0 . 01 0 . 02 0 . 04 0 . 10 0 . 21 0 . 23 0 . 79 0 . 01 heptanoic acid (C7) 0 . 21 0 . 44 1 . 00 1 . 71 2 . 00 2 . 00 2 . 00 2 . 00 0 . 44 octanoic acid (C8) >2 . 00, >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 8 - methylnonanoic acid (C10) >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 decanoic acid (C10) >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 >2 . 00 Since these results suggested that chain length biases were the basis for the discrepancies between the results of the Nelson’s and Rhodamine B - based tests, gas chromatograph analysis was performed of the