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== Compounds tested ==
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
:::* [[Pyruvate]] 2 M
:::* [[Glycerophosphate]] 1 M
:::* [[Glucose]] 2 M
:::* [[Azide]] 4 M
:::* [[TMPD]] 200 mM
[[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 H<sub>2</sub>O 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 H<sub>2</sub>O
:::* [[Glycerophosphate]]: for 1 mL of 1 M stock solutin, weigh 324.13 mg and add '''845''' µL H<sub>2</sub>O
:::* [[TMPD]]: for 1 mL of 0.2 M stock solution, weigh 47.4 mg and add '''970''' µL H<sub>2</sub>O
:::* [[Azide]]: for 1 mL of 4 M stock solution, weigh 260 mg and add '''900''' µL H<sub>2</sub>O
=== Tested with volumetric glasses ===
'''Strategy:''' The powder (only tried with Glucose) was transferred to a volumetric glass, H<sub>2</sub>O 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 H<sub>2</sub>O 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, H<sub>2</sub>O 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''' H<sub>2</sub>O
== 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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