Temperature Control & Stability
Simple Test using NaCl to demonstrate linearity of
apparatus response to changes in [NaCl]
The graph above refers to the circuit which used the long-tailed pair differential amplifier. The chart below shows the equivalent result using the 741 opamp amplifier
The graph below shows the original data used to construct the chart above. Note that the sensitivity of the 741 circuit is approximately half that of the long-tailed pair. However as mentioned elesewhere the noise is considerable better owing to common mode noise reduction in the input square wave.
It is obvious that 10% of this addition i.e. 100 micromolar can easily be measured and 1% i.e. 10 micromolar is possible with attention to detail.
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The volume given above
is incorrect it should
read 0.0067 ml
The graph below shows the same 1 mM additions to 1% NaCl. This is much more difficult to measure against a strong ionic background. The measurement is also less sensitive
The cell constants were measured as 1.27 & 1.34 cm-1 for the two cells
The calculated value is 1.2, taking fringe fields into account
Impedance of the electrode assemblies
For optimum sensitivity of a Wheatstone bridge the four resistances should be approximately the same at the start of an experiment. For an AC bridge the reactances (or impedances) should be similar.
The reactance of the buffer used in enzyme experiments is ca. 500 ohms for the 28 kHz square wave used in the bridge. [This increases during a 36 microsecond period of the half wave if polarisation occurs]. The reactances of the two cells are 670 & 633 ohms for a 0.1% NaCl solution that was used to calibrate & test the system.
This is the reason that a 1 k ohm potentiometer was chosen to balance the bridge, since each half gives a resistance of 500 ohms at balance before the start of an experiment & this leads to optimum sensitivity of the bridge. It is advisable to change the resistance of the potentiometer if a very different buffer is used to give as good a match as is practicable. However tests have shown that reasonable results can be obtained even if the buffer strength is reduced 10 – fold, while the potentiometer is unchanged (although clearly 10 k ohm pot would be better). For best results at lowest concentrations change the pot!
More generally a change of 1% in the overall ionic strength can be measured when the pot is approximately (+/- 50%) matched to the impedance of the reaction solution. If the pot rsistance differs by more than 10-fold from the solution impedance the best that can be measured is a 10% change. This is a general rule that is valuable in planning experiments.
The Table below shows how the sensitivity of the apparatus changes with the salt concentration. The Log-Log plot below the Table shows that the sensitivity is nearly inversely proportional to the salt concentration. This empahsises how important it is to choose an appropriate buffer concentration for your enzyme assays. You do not want the pH to change during the assay but also you want to minimise the salt concentration, i.e. the buffer concentration. The buffer concentration should be a minimum of 10 times the change in ions measured as a result of the enzyme reaction in the assay.
MAKE SURE THAT ADDITION OF THE ENZYME SOLUTION DOES NOT CHANGE THE pH - DIALYSE AGAINST REACTION BUFFER IF NECESSARY TO AVOID THIS.
1 micromolar additions to 0.0001% NaCl [16.7 micromolar].
This trace shows how good the sensitivity is at low background ionic concentrations
Some general Rules
Tests with Sodium Chloride
Test with urease & Urea
In the second of the references quoted in the ref section it is reported that the hydrolysis of 2 nmoles of urea could be detected using the precision apparatus used for these experiments. I thus set out to try & demonstrate that the apparatus used here could detect a 1 nM change. In the event I was stymied by a high salt concentration (400 mM!) in the enzyme stock solution (100 mg/ml crude jack bean powder), which I was unable to remove as I do not have enough material for dialysis. The reaction solution (after dilution of 0.01 ml enzyme to 1 ml) was 4 mM in salt, too high to measure a 1 nmole change.
I thus tried to detect hydrolysis of 10 nmoles of urea, with success. The graph below shows two 10 nmole additions.
The graphs below shows the temperature resilience that comes from using two active electrochemical cells in the bridge rather than just one. The left hand side graph shows the response to a 1 degree rise in temperature within the box at balance with 0.1% NaCl in both the cells. There is little change because the effects in each cell cancel each other.
The right hand side graph above shows the response to a 1 degree temperature rise when the instrument is away from balance. 5 mM NaCl was added to the sample cell, giving a voltage of ca. 1V. Note that the voltage change is now larger as would be expected.