Limitations of the methods. The linear regression, when fit properly, generally
produces reliable results at the extremes of the range of the standard curve. Results
calculated from mean absorbance data outside the range of the standard curve may
be unreliable. (For the purposes of this discussion, the range of the standard curve is
defined as the absorbance range between the highest and lowest mean absorbances
of the standards used to form the standard curve.) Calculated results that are
determined from mean absorbances outside the range of the standard curve are
extrapolations, and should be treated as such when determining the final results of
your analysis.
The firmware will not be able to give a unique concentration for a given absorbance
if the calibration data are not monotonic (i.e., two standards of differing concentration
have the same absorbance reading).
C.
Scan. SmartSpec 3000 can read any subset of the interval between 200 nm and 800 nm
and then print the scan; the interval can be further subdivided into as many as four
regions, and for each region the local minimum and maximum are identified. You
may select a background wavelength and its absorbance will be subtracted from the
absorbance at all other wavelengths in the interval of the scan. There are two
scanning speeds: fast and slow. If you choose a slow scan, you will get the best
data, but will take several minutes to complete, depending on the sample and the
range being read. You may not specify more than one repetition for slow
scanning. Scan data may be downloaded to an external computer via the serial port,
or may be printed by SmartSpec 3000.
D.
Kinetic. SmartSpec 3000 can collect absorbance readings at periodic intervals for
a specified length of time so that you can collect kinetic data. You may specify a
wavelength for background subtraction; background absorbances are collected at the
same interval and are subtracted from the reading wavelength. You may then print
out the data and/or download the data to another program for analysis. Permissible
reading intervals range from 2 seconds to 999 seconds and data may be collected
for as long as 9999 seconds.
E.
OD600. This function is useful for monitoring growth of bacterial cultures.
SmartSpec 3000 can also convert the absorbance reading into a concentration with
units of cells/ml if provided with a conversion factor. The conversion factor is
multiplied by the absorbance reading and the dilution factor to estimate the
concentration of cells in cells per ml. For example, if for certain E. coli an OD of 1.0
indicates a concentration of 5x10
8
cells/ml, then the conversion factor would be
5x10
8
. On the SmartSpec 3000 5x10
8
is entered in exponential notation using the
exp key (press 5, exp, 8 and Enter) and displayed as 5 e8. On the display, e refers
to the base 10 and not to the base of the natural logarithm.
F.
λλ
. The wavelength function allows you to choose up to three wavelengths for
measurement and any other wavelength for optional background subtraction. It is a
rapid and simple way to collect absorbance data without constructing standard curves
or otherwise calculating concentrations.
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