WPI APOLLO 4000, Instruction Manual

Page: 34 / 74

APOLLO 4000
NITRIC OXIDE DETECTION
WORLD PRECISION INSTRUMENTS C-10
will reach a plateau and the second aliquot of SNAP can then be added. Successive
additions of the remaining aliquots of SNAP can be made in a similar way.
A calibration curve can be constructed by plotting the signal output (pA) vs
concentration (nM) of SNAP. Each addition of SNAP corresponds to equivalent NO
concentration. The response should be very linear from 10 to 1000 nM.
The sensitivity of the NO probe can be established from the gradient of the response
curve. The sensitivity of the ISO-NOP sensor is about 1 pA/nM. Once the slope of the
probe has been determined the value can be entered into the APOLLO4000
software program (see previous section) if the user wishes the observe data in
concentration mode ( i.e., nM,
µ
M).
Note: Remember that most NO probes are sensitive to temperature changes. It is
therefore recommended that the calibration of a NO sensor is performed at the
experimental temperature.
Method 2: Calibration by decomposition of SNAP using
Cu(II) as a catalyst
This method of calibration relies on the use of Cu(II) for the partial but quantifiable
conversion of SNAP to NO. This procedure can be used as an alternative to the
previous method in which Cu (I) is the active catalyst for the conversion of SNAP to
NO. In this procedure Cu(II) is substituted as a catalyst for ease-of-handling.
NOTE: Experimentally it has been shown that Cu(II) is less efficient as a catalyst in
the conversion of SNAP to NO ( e.g., conversion ratio is reduced to approximately
60%). The accuracy of the calibration may also be reduced (see discussion).
S-Nitriso-N-acetyl-D,L-penicillamine (SNAP) is a stable NO containing compound
that can be used for quantitative generation of NO in solution. SNAP decomposes
to NO and a disulfide byproduct when dissolved in water. However, the rate of
decomposition is very slow. The kinetics of decomposition for this reagent is a
function of several parameters including pH, presence of a catalyst, temperature
and light.
In the procedure described here, SNAP is used in combination with a catalyst,
copper (II) sulfate (CuSO
4
) or cupric (II) chloride (CuCl
2
), to generate a known
quantity of NO in solution. Note that this protocol does not investigate the effects of