muhero at globalnet.co.uk
Wed Apr 1 16:26:58 EST 1998
Is the optical path length through the sample the same? If it is less you
will need a higher concentration of the pigment to achieve the same
absorbance. Typically you'll be using a 1cm path length on a bench
spectrophotometer; in a microtitre tray the most you'll get is about half
that. That probably also accounts for the flattening of the curve, although
without knowing the details of the setup I can't be sure. You can
investigate this very simply by taking a standard solution of dye and
preparing a dilution series - then read them in both systems. That will show
you the different responses for the same signal.
Gary Thomson wrote in message <35185338.A2112D4E at dsto.defence.gov.au>...
>We are currently working up bioassay methods for folic acid and thiamin
>for analysing blood samples. We have achieved very good standard curves
>when the assays are carried out in test tubes. Unfortunately we lack the
>automated equipment to read the absorbance values for the tubes, so they
>have to be individually read. We are now trying to decrease the time
>taken to read the assays by using microtitre trays. We have kept the
>same proportions of sample:assay medium, but we are now finding that the
>incubation time for thiamin has to be extended and that the standard
>curves are flatter (i.e. less sensitive). We are using polysyrene tissue
>culture treated plates with lids.
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