This software is designed for carrying out online titrations using one or more Microlab titrators. It is designed to run as a slave to a master data acquisition program (e.g., a commercial or custom instrument that can perform repeat scans using a macro). Many commercial instruments and programs these days don't have the option of a having a TTL trigger to initiate a scan so this program will synchronize off of a file creation by the master program. The sequence of events would work like this:
- the titration program has been started and is waiting for a synchronization signal (the file creation by the master program)
- the master program performs a scan and saves a file.
- after the file is saved the titration program withdraws the appropriate amount from the cuvet ("Vol. to Add") using the right syringe and dispenses the same amount from the left syringe, which contains the titrant. The volume of the syringe is kept constant during the titration.
A mechanism to stir the sample during titrant addition is needed.
Note that it is assumed that the titration starts with the concentration of ligand specified by the variable "Conc of Ligand".
If multiple titrators are hooked up, the program will alternate between the two titrators, i.e. the first synchronization signal will withdraw/inject from titrator #1 and the second synchronization signal will withdraw/inject from titrator #2 and so on. There is a limit of 16 simultaneous titrators and they all have to follow the same titration schedule.
The link to download the software is at the bottom of the page.
The "Sync Pattern" field is designed for when you have multiple samples (e.g., 3 in the screenshot below) but do not want the titrator (or titrators) to inject after each scan. A "0" (zero) in the field means don't inject when a file is created (i.e. when a scan is done acquiring) and a "1" means to inject when a file is created. For example, to inject after each acquisition you would enter:
if you have 3 samples.
If you only wanted to inject after the first two samples but not the third (e.g. if the third sample was a control not hooked up to a titrator) you would enter:
You can also account for performing data acquisition at multiple wavelengths by injecting only after the data is acquired for the second wavelength. For the case above (2 protein samples and 1 NATA sample) this would be:
The same principle would be used if you wanted to split acquisition into multiple cycles, i.e. do 2 repeats and inject only after the second repeat of the protein samples).
It may seem confusing but it gives the user complete control, which is what we want :)
Doing a titration this way is very efficient in terms of sample consumption but does have some drawbacks.
Systematic error: The most serious of these is its susceptibility to systematic error. If a valve is very slightly leaky (especially the one doing the withdrawing with viscous samples like >4M urea) you will get beautiful looking titrations that will be physically meaningless. The way to check for this is to make sure your volume at the end of your titration is exactly the same as your starting volume and that the index of refraction of your sample at the end of the titration is what you expected. If these two do not match up, I strongly recommend checking and fixing all of the plumbing and repeating the experiment.
Equilibration time: If you're performing a titration on a protein sample the equilibration time is an important parameter to consider. The time required for a protein to establish an equilibrium between two states could take hundreds to thousands of seconds near the midpoint of a transition. We typically wait for 3x the time constant (assuming a high density of titration points) which places an upper limit of ~100 to 200 s as the longest time constant for which we will attempt a protein denaturation titration. Using too short of an equilibration time will produce a beautiful looking titration that is meaningless :) An autosampler is more efficient with respect to time for very slow folding/unfolding proteins.
An alternative to doing the titration this way is to use an autosampler and a flow cuvette to place inside the spectrometer. We've recently adopted this approach and it's less susceptible to systematic errors. You can build your own autosampler with off the shelf parts for half the cost of a commercial one. The link is here (coming soon).