Most of the chemicals for SUIT protocol titrations are prepared by weighing the substance on the balance, transferring to a volumetric glass flask and adding solvent until the intended volume is reached. However, for practical reasons some of the chemical compounds are prepared by just adding the solvent instead of adjusting it's volume. For example, this approach is useful if the substance is very toxic. Then an arbitratry amount is taken, its mass determined on the balance without trying to reach a specific value and the necessary amount of solvent is added. Adding the solvent instead of adjusting its volume is also useful if small amounts are needed (e.g. 1 mL) or if the compound has to be prepared directly before using it like Pyruvate. In these cases the volume contributed by the solute was tested.
Reference: MiPNet09.12 O2k-Titrations
The volume that the solute contributes to the stock solution depends on the concentration and to some extent to the molar mass. The compound with the lowest concentration that was tested was TMPD, 200 mM. The dry substance contributed only about 3 % to the volume of the stock solution. Therefore further tests were focused on stock solution of a concentration about 1 M and higher
KCN 500 mM: Because of its toxicity KCN was not tested yet. However, the low concentration of the stock solution together with the low molar mass of KCN lead to the assumption, that dry KCN will contribute only some percent to the volume of the stock solution.
Determining the volume of the solute
Tested with pipettes
Strategy: P, Gp and TMPD were directly weighted in 2 mL Eppendorf tubes. Azide turned out to be too electrostatic to be prepared in an Eppendorf tube and was therefore prepared in a small glass vial. Then the amount H2O of the uncorrected preparation instructions was added with one pipette (between 1 and 2 mL). After dissolving the compounds, the volume added was removed with the same pipette to avoid errors between pipettes. A smaller pipette was used to pick up the remaining volume. The volume of the pipette was first set to a higher volume then the volume left in the tube. After picking the liquid up (together with some air in the end), the pipette volume was turned down until the liquid filled the pipette tip without a air bubble at the bottom.
Advantage: This strategy avoids errors between different volume measuring instruments. Working in Eppendorf tubes has the advantage, that all the liquid collects very well in the lowest point of the tubes where it is easy to pick it up.
- Pyruvate: for 200 µL of 2 M stock solution, weigh 44 mg and add 180 µL H2O
- Glycerophosphate: for 1 mL of 1 M stock solutin, weigh 324.13 mg and add 845 µL H2O
- TMPD: for 1 mL of 0.2 M stock solution, weigh 47.4 mg and add 970 µL H2O
- Azide: for 1 mL of 4 M stock solution, weigh 260 mg and add 900 µL H2O
Tested with volumetric glasses
Strategy: The powder (only tried with Glucose) was transferred to a volumetric glass, H2O was added and the volume recorded with the aid of pipettes until the line of the volumetric glass is reached.
Graduated cylinders as well as a volumetric glass flask were used, because Glucose is cheap enough to prepare larger amounts without needing them. The size of the glasses allows for thorough stirring, which is necessary to solve Glucose. Disadvantage: Glucose is very sticky, the bottlenecks are narrow, funnels have to be used and a small amount of Glucose as well as H2O will remain on spatels and funnels. Still, the results of three experiments (3 mL and 4 mL in graduated cylinders, 5 mL in volumetric glass flask) were in very good agreement to each other.
Disadvantage: A comparison between pipette volumina and volumetric glass flasks (5 mL) showed, that there are deviations between volumes determined by pipettes and the volumetric glass flasks. This is not surprising taking into account that the error of a volumetric glass flask is +/- 0.04 mL, which is only about 1 % of a 5 mL-glass. Moreover, H2O was added in several steps, which could lead to accumulation of pipette errors. Since we are interested in measuring the volume contributed by the solute (which contributes e.g. 10 %, i.e. 50 µL in 5 mL) an deviation of e.g. 40 µL between pipettes and volumetric glasses is too large.
- Glucose: for 1 mL of 2 M stock solution, weigh 360.32 mg and add 775 µL H2O
Testing unadjusted concentrations
The concentrations prepared until 2021-02 were lower than indicated in the preparation instructions, because the volume of the solute was neglected. In a SUIT experiment Pyruvate and Azide were tested, their concentrations were between 10 and 15 % lower than indicated. For Pyruvate and Azide it could be shown that the concentrations prepared so far are sufficient to reach kinetically saturated conditions in SUIT experiments.
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