Bio-Rad SmartSpec Plus Spectrophotometer - Manual (Page 22)

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 SmartSpec Plus 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 Plus 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 Plus.
D. Kinetic. SmartSpec Plus 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 Plus 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 Plus 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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