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Difference between revisions of "Dithionite"

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{{MitoPedia
{{MitoPedia
|abbr=Dith
|abbr=Dit
|description=Zero oxygen solution powder, Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub>, used for [[MiPNet19.18D O2k-Series G and DatLab 6: Calibration |calibration of oxygen sensors]] at zero oxygen, or for stepwise reduction of oxygen concentration in [[MiPNet14.06 Instrumental O2 background |instrumental O<sub>2</sub> background tests]].
|description=The sodium salt of '''Dithionite''' Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub> (Dit) is the 'zero oxygen solution powder' used for [[Oxygen calibration - DatLab |calibration of oxygen sensors]] at [[Zero calibration | zero oxygen concentration]], or for stepwise reduction of oxygen [[concentration]]s in [[MiPNet14.06 Instrumental O2 background |instrumental O<sub>2</sub> background tests]]. It is not recommended to use dithionite in experiments with biological samples or several multisensor approaches, for these see [[Setting the oxygen concentration]].
|info=[[MiPNet06.03 POS-calibration-SOP]], [[MiPNet14.06 Instrumental O2 background]]
|info=[[MiPNet06.03 POS-calibration-SOP]], [[MiPNet14.06 Instrumental O2 background]]
}}
}}
{{MitoPedia concepts}}
{{MitoPedia methods
|mitopedia method=Respirometry
}}
{{MitoPedia O2k and high-resolution respirometry}}
{{MitoPedia topics}}
__TOC__
__TOC__
{{Template:Technical support integrated}}
 
== Preparation ==
== Application in [[HRR]] ==
::: '''Preparation of 30 mM dithionite solution for instrumental oxygen background test''' (dissolved in Phosphate buffer, 50 mM, pH 8)
{{Chemical_description
|abbr=Dit - (The abbreviation 'Dith' has been used previously and is stepwise replaced by Dit.)
|trivial name=Dithionite
|complete name=Sodium hydrosulfite
|chem formula=Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub>
|molar mass=174.11
|vendor=Sigma-Aldrich
|product number=71699
|store at=4 °C
|sensitivity=
|cas=7775-14-6
|h statements= H251, H302, H319
|h info=self-heating; may cause fire, harmful if swallowed, causes serious eye irritation
}}<!--::: '''Dit: Dithionite''' (sodium hydrosulfite, Na<sub>2</sub>S<sub>2</sub>O<sub>4); Sigma P 71699, 250 g, store at 4 °C; ''M'' = 174.11 g·mol<sup>-1</sup>-->
 
:::: '''Note''': Dithionite is oxidized when exposed to air. We recommend to reduce contact to air during preparation in a closed tube. The effective concentration of a solution of reduced dithionite is lower than the nominal concentration (30 mM or 10 mM) after dithionite has been partially oxidized in the powder or in aqueous solution.
::::* To calculate the injection volume of Na-dithionite solution to reduce O<sub>2</sub> concentration to specific desired values, see Section 3.2 in [[MiPNet14.06 Instrumental O2 background]].
 
:::: '''Preparation of 30 mM dithionite solution for instrumental oxygen background test''' (dissolved in phosphate buffer, 50 mM, pH 8)


::::# Weigh 0.051 g dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
::::# Weigh 0.051 g dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
::::# Adjust final volume to 10 mL with Phosphate buffer and keep closed to avoid decomposing.
::::# Adjust final volume to 10 mL with Phosphate buffer and keep closed to avoid decomposing.


::: '''Preparation of 10 mM dithionite solution for instrumental oxygen background test''' (dissolved in Phosphate buffer, 50 mM, pH 8)
:::: '''Preparation of 10 mM dithionite solution for instrumental oxygen background test''' (dissolved in Phosphate buffer, 50 mM, pH 8)


::::# Weigh 0.017 g dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
::::# Weigh 0.017 g dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
::::# Adjust final volume to 10 mL with Phosphate buffer and keep closed to avoid decomposing.
::::# Adjust final volume to 10 mL with Phosphate buffer and keep closed to avoid decomposing.


:::: Note: Experiments performed with 10 and 30 mM dithionite solution showed identical results. Sodium dithionite is oxidized when exposed to air. We recommend to reduce contact to air during preparation and usage (closed tube). We advise to use a concentration of 30 mM for performing the instrumental oxygen background test. For new dithionite, 10 mM dithionite solution may be used.   
:::: Experiments performed with 10 and 30 mM dithionite solution showed identical results. We advise to use a concentration of 30 mM for performing the instrumental oxygen background test. For new dithionite, 10 mM dithionite solution may be used.   


::: '''Preparation of 2.5 mM dithionite solution for instrumental oxygen background test with the [[O2k-sV-Module]] (0.5 mL)''' (dissolved in Phosphate buffer, 50 mM, pH 8)
:::: '''Preparation of 2.5 mM dithionite solution for instrumental oxygen background test with the [[O2k-sV-Module]] (0.5 mL)''' (dissolved in phosphate buffer, 50 mM, pH 8)


::::# Weigh 0.00425 g dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
::::# Weigh 0.00425 g sodium dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
::::# Adjust final volume to 10 mL with Phosphate buffer and keep closed to avoid decomposing.
::::# Adjust final volume to 10 mL with phosphate buffer and keep closed to avoid decomposing.


:::: Note: Experiments performed with 2.5 mM dithionite solution are recommended for the [[O2k-sV-Module]] only. Sodium dithionite is oxidized when exposed to air. We recommend to reduce contact to air during preparation and usage (closed tube).   
:::: Experiments performed with 2.5 mM dithionite solution are recommended for the [[O2k-sV-Module]] only.   




::::* From [[MiPNet06.03 POS-calibration-SOP|MiPNet06.03 POS-calibration-SOP (Version 14)]]: Prepare "zero solution" by dissolving c. 20 mg sodium hydrosulfite ([[OroboPOS-Service Kit]]) or two tips of a spatula in 0.5 mL water. Mix in a small vial with minimum gas space. Use fresh. Dithionite may not work after prolonged storage.
::::* From [[MiPNet06.03 POS-calibration-SOP|MiPNet06.03 POS-calibration-SOP (Version 18)]]: Prepare "zero solution" by dissolving c. 20 mg sodium hydrosulfite ([[OroboPOS-Service Kit]]) or two tips of a spatula in 0.5 mL water. Mix in a small vial with minimum gas space. Use fresh. Dithionite may not work after prolonged storage.


  '''Further information »[[Talk:Dithionite]]'''
  '''Further information »[[Talk:Dithionite]]'''
{{Template:Technical support integrated}}
== Troubleshooting ==
== Troubleshooting ==


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|}


Please note that:
:::: Please note that: On your protocol the event P2 has been missed (if you see, the default P3 and your P2 are the same steps);
On your protocol the event P2 has been missed (if you see, the default P3 and your P2 are the same steps);
A premise for P1 (please see on your DLD file under 'Feedback control' and after loading as protocol 'BG_Feedback-highO2_400-200') is to start when O2 concentration [µM]  > 370 and to stop when O2 concentration [µM] < 360.
A premise for P1 (please see on your DLD file under 'Feedback control' and after loading as protocol 'BG_Feedback-highO2_400-200') is to start when O2 concentration [µM]  > 370 and to stop when O2 concentration [µM] < 360.


However:
:::: However: Immediately after your P1 titration, you already have a O<sub>2</sub> concentration [µM] = 322 and therefore your titration P1 goes on until the requirements for the next possible P titration are met;
immediately after your P1 titration, you already have a O<sub>2</sub> concentration [µM] = 322 and therefore your titration P1 goes on until the requirements for the next possible P titration are met;
The next possible titration requirements corresponds to P3 (in your DLD recognized as P2) (start when O<sub>2</sub> concentration [µM]  > 270 and to stop when O2 concentration [µM] < 260);
The next possible titration requirements corresponds to P3 (in your DLD recognized as P2) (start when O<sub>2</sub> concentration [µM]  > 270 and to stop when O2 concentration [µM] < 260);
Finally the P4 titration takes place (in your DLD recognized as P3) which corresponds to a 100 uL titration just before the event 'TIP end'.
Finally the P4 titration takes place (in your DLD recognized as P3) which corresponds to a 100 uL titration just before the event 'TIP end'.


Conclusion:
::::* Conclusion: Since we observe that your O<sub>2</sub> concentration decreases too fast at each Dit titration point, we can be sure that the Dit solution stock has an high concentration. Our recommendation for Dit concentration is between 10 and 30 mM. Due to the oxidation process that occurs when Dit is open for long time, we recommend to use a concentration of 30 mM for performing the instrumental oxygen background test, however in your case I advise you to use a lower concentration of your Dit solution.
Since we observe that your O<sub>2</sub> concentration decreases too fast at each Dith titration point, we can be sure that the Dith solution stock has an high concentration. Our recommendation for Dith concentration is between 10 and 30 mM. Due to the oxidation process that occurs when Dith is open for long time, we recommend to use a concentration of 30 mM for performing the instrumental oxygen background test, however in your case I advise you to use a lower concentration of your Dith solution.
 
=== During instrumental oxygen background test, the step representative of O<sub>2</sub> consumption is too small after Dith titrations===


* '''Customer ID''': CH Lausanne Kayser B
=== During instrumental oxygen background test, the step representative of O<sub>2</sub> consumption is too small after Dit titrations ===


'''Question:'''
:::: '''Customer ID''': CH Lausanne Kayser B


I ran a zero oxygen calibration after the instrumental background protocol and the O<sub>2</sub> neg slope for O<sub>2</sub> concentrations of 60 and 30 uM is positive and greater compared with the O<sub>2</sub> neg slope at O<sub>2</sub> concentrations of 180 and 120 uM.  The data is attached (2019-09-11).
:::: '''Question:''' I ran a zero oxygen calibration after the instrumental background protocol and the O<sub>2</sub> neg slope for O<sub>2</sub> concentrations of 60 and 30 uM is positive and greater compared with the O<sub>2</sub> neg slope at O<sub>2</sub> concentrations of 180 and 120 uM.  The data is attached (2019-09-11).


[[File:Dith oxidized 2019-09-11 P1-05.DLD.png|center|600px]]
[[File:Dith oxidized 2019-09-11 P1-05.DLD.png|center|600px]]


'''Answer:'''
:::: '''Answer:''' Stepwise analysis of the attached data shows the following:
Stepwise analysis of the attached data shows the following:


<u>Quality control (QC) 1</u>: Your POS operates perfectly: during air calibration the O2 slope neg. approaches zero (see section 2.1 of https://wiki.oroboros.at/images/7/77/MiPNet06.03_POS-Calibration-SOP.pdf);
::::* <u>Quality control (QC) 1</u>: Your POS operates perfectly: during air calibration the O2 slope neg. approaches zero (see section 2.1 of https://wiki.oroboros.at/images/7/77/MiPNet06.03_POS-Calibration-SOP.pdf);


<u>Quality control (QC) 2</u>:  In the closed chamber, the O<sub>2</sub> slope neg. is <4 pmol s<sup>-1</sup> mL<sup>-1</sup> which excludes any biological or chemical contamination from the MiR05 buffer. https://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf;
::::* <u>Quality control (QC) 2</u>:  In the closed chamber, the O<sub>2</sub> slope neg. is <4 pmol s<sup>-1</sup> mL<sup>-1</sup> which excludes any biological or chemical contamination from the MiR05 buffer. https://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf;
* From your dithionite titrations we can observed that:
::::* From your dithionite titrations we can observed that:
::::# After titrations of 2.5 µL of Dit, the step representative of O<sub>2</sub> consumption is too small – '''indicative of a low effective potency of the solution of Dit''';
::::# At increasing concentrations of Dit towards stepwise lower O<sub>2</sub> concentrations, we observe that the O<sub>2</sub> slope negative does not decrease in a titration dependent manner. Therefore, O<sub>2</sub> is being consumed by the solution  even after the initial fast decline of oxygen. Since we excluded other potential problems, the irregular background traces are due to the Dit solution.(see representative trace on page 5 of https://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf)
:::: Taken together, your dithionite is highly oxidised and a continuing oxidation reaction explains the results.


# After titrations of 2.5 µL of Dith, the step representative of O<sub>2</sub> consumption is too small – '''indicative of a low effective potency of the solution of Dith''';
:::: '''Solution:''' Assuming you prepared the Dit according to our SOP (always fresh, http://bioblast.at/index.php/Dithionite), I advise you to purchase a new Dit powder (your Dit may be old and/or has oxidized).
# At increasing concentrations of Dith towards stepwise lower O<sub>2</sub> concentrations, we observe that the O<sub>2</sub> slope negative does not decrease in a titration dependent manner. Therefore, O<sub>2</sub> is being consumed by the solution  even after the initial fast decline of oxygen. Since we excluded other potential problems, the irregular background traces are due to the Dith solution.(see representative trace on page 5 of https://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf)
Taken together, your dithionite is highly oxidised and a continuing oxidation reaction explains the results.


'''Solution:'''
{{Template:OSC Oxygen signal1}}
Assuming you prepared the Dith according to our SOP (always fresh, http://bioblast.at/index.php/Dithionite), I advise you to purchase a new Dith powder (your Dith may be old and/or has oxidized).


{{Template:OSC Oxygen signal1}}
{{MitoPedia methods
|mitopedia method=Respirometry
}}
{{MitoPedia O2k and high-resolution respirometry
|mitopedia O2k and high-resolution respirometry=O2k-Open Support
}}
{{MitoPedia topics
|mitopedia topic=Substrate and metabolite
}}

Latest revision as of 06:33, 13 October 2023


high-resolution terminology - matching measurements at high-resolution


Dithionite

Description

The sodium salt of Dithionite Na2S2O4 (Dit) is the 'zero oxygen solution powder' used for calibration of oxygen sensors at zero oxygen concentration, or for stepwise reduction of oxygen concentrations in instrumental O2 background tests. It is not recommended to use dithionite in experiments with biological samples or several multisensor approaches, for these see Setting the oxygen concentration.

Abbreviation: Dit

Reference: MiPNet06.03 POS-calibration-SOP, MiPNet14.06 Instrumental O2 background

Application in HRR

Dit - (The abbreviation 'Dith' has been used previously and is stepwise replaced by Dit.): Dithionite (Sodium hydrosulfite; Na2S2O4), Sigma-Aldrich: 71699, store at 4 °C, CAS: 7775-14-6, M = 174.11 g·mol-1
Hazard statements: H251, H302, H319; self-heating; may cause fire, harmful if swallowed, causes serious eye irritation


Note: Dithionite is oxidized when exposed to air. We recommend to reduce contact to air during preparation in a closed tube. The effective concentration of a solution of reduced dithionite is lower than the nominal concentration (30 mM or 10 mM) after dithionite has been partially oxidized in the powder or in aqueous solution.
Preparation of 30 mM dithionite solution for instrumental oxygen background test (dissolved in phosphate buffer, 50 mM, pH 8)
  1. Weigh 0.051 g dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
  2. Adjust final volume to 10 mL with Phosphate buffer and keep closed to avoid decomposing.
Preparation of 10 mM dithionite solution for instrumental oxygen background test (dissolved in Phosphate buffer, 50 mM, pH 8)
  1. Weigh 0.017 g dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
  2. Adjust final volume to 10 mL with Phosphate buffer and keep closed to avoid decomposing.
Experiments performed with 10 and 30 mM dithionite solution showed identical results. We advise to use a concentration of 30 mM for performing the instrumental oxygen background test. For new dithionite, 10 mM dithionite solution may be used.
Preparation of 2.5 mM dithionite solution for instrumental oxygen background test with the O2k-sV-Module (0.5 mL) (dissolved in phosphate buffer, 50 mM, pH 8)
  1. Weigh 0.00425 g sodium dithionite, sodium hydrosulfite, and transfer to 10 mL volumetric glass flask.
  2. Adjust final volume to 10 mL with phosphate buffer and keep closed to avoid decomposing.
Experiments performed with 2.5 mM dithionite solution are recommended for the O2k-sV-Module only.


Further information »Talk:Dithionite

Template NextGen-O2k.jpg


MitoPedia O2k and high-resolution respirometry: O2k-Open Support 



Troubleshooting

Zero oxygen concentration reached but 'Instrumental O2 background_TIP2k.DLP' is not finished

Question:

We have always had the same issue with our TIP2k running the background calibration that it has always reached ‘TIP2 end’ with the oxygen concentration reaching zero before the event P004 was activated meaning we are not able to get mark J°4h. The data is attached (2019-11-04).

2019-11-04 P2-01 Instrumental Background.PNG

Answer:

Stepwise analysis of the attached data shows the following:

Observations:

Default parameters high O2 background TIP2K (.DLD)
Volume (nL) Flow (nL/s)
P1 100000 250
P2 100000 250
P3 50000 125
P4 100000 50000
Your high O2 background TIP2K (.DLD)
P1 100000 250
P2 50000 125
P3 100000 50000
P4 x x
Please note that: On your protocol the event P2 has been missed (if you see, the default P3 and your P2 are the same steps);

A premise for P1 (please see on your DLD file under 'Feedback control' and after loading as protocol 'BG_Feedback-highO2_400-200') is to start when O2 concentration [µM] > 370 and to stop when O2 concentration [µM] < 360.

However: Immediately after your P1 titration, you already have a O2 concentration [µM] = 322 and therefore your titration P1 goes on until the requirements for the next possible P titration are met;

The next possible titration requirements corresponds to P3 (in your DLD recognized as P2) (start when O2 concentration [µM] > 270 and to stop when O2 concentration [µM] < 260); Finally the P4 titration takes place (in your DLD recognized as P3) which corresponds to a 100 uL titration just before the event 'TIP end'.

  • Conclusion: Since we observe that your O2 concentration decreases too fast at each Dit titration point, we can be sure that the Dit solution stock has an high concentration. Our recommendation for Dit concentration is between 10 and 30 mM. Due to the oxidation process that occurs when Dit is open for long time, we recommend to use a concentration of 30 mM for performing the instrumental oxygen background test, however in your case I advise you to use a lower concentration of your Dit solution.

During instrumental oxygen background test, the step representative of O2 consumption is too small after Dit titrations

Customer ID: CH Lausanne Kayser B
Question: I ran a zero oxygen calibration after the instrumental background protocol and the O2 neg slope for O2 concentrations of 60 and 30 uM is positive and greater compared with the O2 neg slope at O2 concentrations of 180 and 120 uM. The data is attached (2019-09-11).
Dith oxidized 2019-09-11 P1-05.DLD.png
Answer: Stepwise analysis of the attached data shows the following:
  1. After titrations of 2.5 µL of Dit, the step representative of O2 consumption is too small – indicative of a low effective potency of the solution of Dit;
  2. At increasing concentrations of Dit towards stepwise lower O2 concentrations, we observe that the O2 slope negative does not decrease in a titration dependent manner. Therefore, O2 is being consumed by the solution even after the initial fast decline of oxygen. Since we excluded other potential problems, the irregular background traces are due to the Dit solution.(see representative trace on page 5 of https://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf)
Taken together, your dithionite is highly oxidised and a continuing oxidation reaction explains the results.
Solution: Assuming you prepared the Dit according to our SOP (always fresh, http://bioblast.at/index.php/Dithionite), I advise you to purchase a new Dit powder (your Dit may be old and/or has oxidized).

Anonymous user

Question:

Q1: The Oxygen Concentration couldn't go down while I tried zero oxygen calibration. No matter how much solution I added, the oxygen concentration still remain the same. I reassembled, but it still keeps the same. The data is attached.

No response to dithionite.PNG


Q2: The chamber B's oxygen slope is not stable. This is a new problem I have never seen. Please see the attachment.(2019-04-26)

Signal stability.PNG


Q3. The maximum Oxygen concentration of another Oroboros O2k is only about 140 μM. I don't how to increase the starting point of oxygen concentration.


Answer:

A1: I suspect that the dithionite solution was not correctly prepared or not fresh. Dithionite loses activity quite fast, so you need to make it freshly. Please check this site: http://bioblast.at/index.php/Dithionite

A2: While there is a difference between chambers A and B, the stability of the signal is still within the acceptable range of noise given by the specifications of the instrument, see: Oxygen_sensor_test.

A3: You may simply need to calibrate, please see here: http://wiki.oroboros.at/index.php/O2_calibration_-_DatLab


MitoPedia methods: Respirometry 


MitoPedia O2k and high-resolution respirometry: O2k-Open Support 


MitoPedia topics: Substrate and metabolite