1) Use a pH buffer along with a chelator to avoid unpredictable changes in pH and therefore metal ion values. All of the equations are affected by changes in pH.
2) Being within the buffering range of the chelator is preferable.
· You loose accuracy below the chelator buffering range, not from measuring error, but from not knowing accurately the contamination levels of metal ions already present.
· Being above the buffering range is to be avoided. You lose both accuracy and buffering capacity and are VERY likely to have a different metal concentration than you expected.
3) Buffering a metal ion can never result in the metal ion being reduced to zero. It may be very low, but it will never be zero.
4) A chelator with a lower Kd can remove metal from a chelator with a higher Kd. [this relationship reverses when talking about the log form, pKd, a higher pKd can remove metal from a lower pKd].
example: EGTA can remove calcium from ATP.
Some cellular proteins have lower Kds than some chelators and therefore can remove calcium from the chelator.
Know what you are working with!
5) NO program is as good as a calibrated metal electrode. The equations cannot take into account EVERY consideration. Programs don't know how pure reagents are or how accurately materials were weighed out or pH measured.
6) Always report what program and version you used in your papers so others can follow along. There is still much debate as to which constants are the best. Different chelator programs often use different constants. This is very confusing, but is the current state of these software offerings.
MAXCHELATOR is an experimental program using constants and understandings developed by many people. Use the information generated by this and all chelator programs at your own risk. See #5 above. [legal disclaimer]