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Abstract: Fasching M, Gradl L, Gradl P, Gnaiger E (2016) Titration-Injection microPump. TIP2k user manual. Mitochondr Physiol Network 12.10(08):1-29. - Versions
The TIP2k provides unique advantages for applications which require highest accuracy of titration volumes and automatic control of complex titration-injection protocols. The TIP2k was specially developed for the O2k. The main modes of operation are automatic multi-step injections, continuous injections for steady-state respirometry, (both operated form the direct control mode), and feedback controlled operation.
Direct Control Mode: Operation mode for delivering a defined volume in a short pulse. A number of cycles, N, can be defined, and the duration of the interval from injection to injection determined, repeating the process N times. Several such steps can be programmed to run in sequence to give an entire TIP2k program.
Feedback Control Mode: Operation mode to reach and maintain a certain parameter (e.g. oxygen concentration) by automatic TIP injections.
• Keywords: HRR, TIP2k• O2k-Network Lab: AT_Innsbruck_Oroboros
This information is part of O2k-technical support and open innovation.
- To prevent washing out effects when the needle is inserted into the chamber the inner diameter of the TIP needle has to be very small. This means that the needle is easily blocked by small particles either precipitated from the solution or introduced with the solution.
- While a TIP syringe has to be considered a consumable, it is possible to lengthen its lifetime by carefully following the procedures suggested in the TIP-2k Manual.
- The main way to prevent clogging of needles is to follow rigorously the cleaning procedure after each usage.
- Additionally, it has to be taken care that no particles are suspended in the used solutions.
- For not very well soluble substances, filtering of the solution may be necessary. Small particles can also be introduced by laboratory activities, e.g. from paper towels or from powdered gloves. Only use underpowered gloves when working with TIP syringes!
- Examples for proper TIP use and cleaning protocols
- Instrumental O2 background (dithionite in phosphate buffer or MiR05)
- Clean (fill and empty) the syringe with water.
- Fill the syringe with dithionite solution for washing, discard immediately.
- Fill the syringe with dithionite solution (in buffer) for use during the assay.
- After the assay empty the syringe and immediately wash the outside of the needle with water (because there is medium sticking to the outside).
- Wash (fill and empty cycles) the syringe three times with water.
- Store the syringe (will be partially filled with water) or after cleaning with ethanol (then it will be partially filled with ethanol.
- CCCP titration (CCCP dissolved in ethanol)
- Clean (fill and empty cycle) the syringe with pure ethanol.
- Clean with CCCP solution (in this case more important than in example 1 because you want to have a precise CCCP concentration).
- Fill with CCCP stock solution and start the experiment.
- After the assay empty the syringe and immediately wash the outside of the needle with water (because there is medium sticking to the outside)
- Clean (fill and empty cycles) the syringe three times with ethanol.
- Store the syringe (will be partially filled with ethanol) or wash it with water and then store (then it will be partially filled with water).
TIP2k syringe blocked
- The TIP syringe is harder to operate than usually.
- Start immediately with extensive cleaning procedures. Once a needle is totally blocked it is usually impossible (with exceptions) to clean it.
- Suitable solvents for extensive cleaning procedures depend on the substances used.
- Chloroform was successful for some fatty acids.
- An ultrasonic bath may be helpful in cleaning partially blocked syringes.
Programming the TIP2k for "BG_Feedback"
- In the TIP2k window the modus can be changed by clicking on either direct control or feedback control. In the feedback control several lines can be inserted, all of which take effect during the currently selected line of the main program. In the figure the implementation of program line 1 of the example set up is shown.
- In the main window (Program line), blue highlighting shows that the first program line is selected. The first line in the feedback control window (upper right corner) sets the "start injection" value to 120 mM. The injection is only started when this condition is valid for two consecutive data points (DataN = 2). The interval is set to 200 seconds, which equals the maximum injection time when the maximum volume is set to 100 µl. These parameters are not important for the discussed purpose; however the interval has to be set to at least the (maximum) injection time calculated at the left side. Once an injection is started the program checks the following lines for "stop" instructions. If just one of these conditions is met, the injection will stop. In our example a stop condition is set at 100 µM.
TIP2k Volume + Time
- Problem: If the user programs an injection by setting volume and time (button "Vol+Time") the required flow is calculated by DatLab. The result is rounded to a precision that can be handled by the TIP2k. For long, slow injections this may result in significantly more or less time being necessary to inject the desired volume than the time entered by the user. If this injection is immediately or with a short delay followed by another injection this may lead to a conflict between the not finished first and the already starting second injection. Using DatLab 4 in this situation will crash the TIP program (error "TIP already running"). In DatLab 6 the program will be executed with the start of the second injection delayed. However the TIP2k status line will behave strangely in the overlapping time and events may be set at the wrong time.
- Suggestions: Use the rounded flow as a starting point to set you own Flow + Volume or Flow + Time values that fit your experimental design.
- Alternative, not tested for all DatLab versions: After entering the desired volume and time, press either the button "Vol+Flow" or "Flow+Time". DatLab will use the rounded flow to calculate a new time, or a new volume, respectively.
List of publications: TIP2k
Labels: MiParea: Instruments;methods
HRR: Oxygraph-2k, TIP2k, O2k-Manual
DatLab, DL6, DL6a7