Operation of the fully computer-controlled
Olis^® Two-Syringe Titrator

Compatible for use with all cuvette styles and pathlengths, and precise to submicroliter volumes

The stratagem behind the operation of the Olis Two-Syringe titrator is that the sample remains in its syringe until a scan is to be made, and then it is pushed into the measuring cuvette with or without the addition of an aliquot of titrant. After the scan is taken, the sample and titrant in the measuring cuvette are withdrawn into the sample syringe, to await the next scan.

The volume of sample and titrant to push into the cuvette, the number of mix cycles¹, and the incubation time are scripted in a simple ASCII file written by the technician and executed by the computer and titrator.

With the two syringe Olis titrator, "sample" and "titrant" could be protein and protein, protein and denaturant, DNA and protein, or any other pairing.

There is a great deal of flexibility in this computer controlled titration accessory. Volumes added can be 0 or 100% of the volume of the syringe, in 0.0001th of the syringe volume increments. The two syringes in the titrator can be the same or different volumes; volumes can be 0.25, 0.5, 1.0, 2.5, 5, or 10 mL.

Stirring within the cuvette is not done, so cuvettes of any pathlength and geometry can be used. The cuvette can be cylindrical or rectangular, long pathlength or short, as the measurement requires.

Any number of scans can be made of each sample preparation, so one can test for temporal stability, too.

Volumes used in the following example description are for descriptive purpose only.

Consider four additions of titrant, of 0, 10, 10 and 30 µl. Let us start with a cuvette requiring 1,000 µl. Let us use sample syringe, "S," of 2.5 mL and a titrant syringe, "T," of 1 mL. Let us fill S with 1 mL of protein and T with 0.1 mL of titrant. (Recall, these solutions could be protein (S) and denaturant (T), protein at pH 1 (S) and protein at pH 7 (T), or any other pairing of S and T.)

1. S pushes 1000 µl into the empty cuvette, T pushes 0 µl. This first scan is recorded as pure sample (perhaps to use as baseline).
2. S withdraws the 1000 µl from the cuvette into itself.
3. S pushes 990 µl (10 µl remain) into the cuvette while T simultaneously pushes 10 µl into the cuvette, so that a total volume in the cuvette is 1000 µl. Mix cycle is initiated and stopped. Second scan is recorded.
4. S withdraws the 1000 µl from the cuvette; now S contains 1010 µl, 10 µl of which are titrant.
5. S pushes 990 µl (20 µl remain) into the cuvette while T simultaneously pushes 10 µl into the cuvette. Mix cycle is initiated and stopped. Third scan is recorded.
6. S withdraws the 1000 µl from the cuvette, now S contains 1020 µl, 20 µl of which are titrant.
7. S pushes 970 µl (50 µl remain) while T simultaneously pushes 30 µl. Fourth scan is recorded.

The user can specify the number of mixing cycles for each individual titration. A "mix cycle" consists of pushing and withdrawing the plunger of the sample syringe (filling and emptying the cuvette) after the titrant was added but before the measurement is taken.

The user determines and/or tells the Olis software:

1. size of sample syringe
2. size of titrator syringe
3. volume of measuring cuvette
4. sample volume [amount of sample to be added to sample syringe]
5. titrant volume [amount of titrant to be added to titrant syringe]
6. He then tells the software (1) how much titrant to add to sample, (2) how many mixes to make, and (3) how long the incubation time should be before taking the scan.

Referring to (3) above, "volume of measuring cuvette," the software must know how much solution is required to fill the measuring cuvette². With cylindrical cuvettes, used principly in CD spectrophotometers, this is the volume to fill the cuvette. With rectangular cuvettes, this is the volume necessary to have solution in the path of the measuring beam³. (One must also account for the volume of fluid to fill the tubings.)

Microvolume cuvettes are also practical, as long as the volume of titrant to add per titration is less than or equal to the volume of the cuvette. If the volume of titrant added is equal to the volume of the cuvette, then the titrant alone fills the cuvette, is drawn into the sample syringe, the mix is achieved, and the resulting mixture is put into the cuvette.

Referring to (4) and (5) above, "sample volume" and "titrant volume," notice that the titrator fills itself with the volume specified. The technician does not directly fill the syringes from a pipette, but fits a pipette, syringe, or other reservoir container to the titrator and the titrator draws in exactly the right volume. The available precision from the titrator makes this the right method of filling. The unit calibrates itself for each syringe. While it is arguable that the titrant syringe can have any volume higher than will be used in the experiment, we think it only makes sense to treat the two syringes in the same manner, so the titrant syringe is filled in the same manner as the sample syringe.

The Olis software also will verify that the volume proposed is equivalent to what the measuring cuvette can hold, otherwise prompting you to change volumes in the script or change to a different volume cuvette, and that the completed experiment will not result in more volume in the sample syringe than the sample syringe can hold, otherwise prompting you to change volumes or change to a larger syringe.

With considerable effort, one can do precise titration studies by hand. Or, one can let the computerized titrator do the technician's work over and over again, perfectly.

Download the default script for using the Olis^® Two-Syringe Titrator