Difference between revisions of "PH calibration buffers"

Revision as of 10:46, 8 September 2018

high-resolution terminology - matching measurements at high-resolution

PH calibration buffers

Description

pH calibration buffers are prepared to obtain two or more defined pH values for calibration of pH electrodes and pH indicator dyes.

MitoPedia methods: Respirometry, Fluorometry

MitoPedia topics: Media for respirometry

Modified hanks buffer

Substance	Formula	Concentration [mM]
Calcium chloride dihydrate	CaCl₂ * 2 H₂O	0.42
Imidazole	C₃H₄N₂	2.0
Potassium chloride	KCl	2.4
Magnesium chloride hexahydrate	MgCl₂*6 H₂O	0.49
Sodium chloride	NaCl	137

pH adjusted to 7.1 at 30 °C.
pH adjusted with KOH or HCl to create a buffer series: (pH values at 25 °C)

A (pH 3.3),
B (pH 6.1)
C (pH 6.6)
D (pH 6.9)
E (pH 7.3)
F (pH 7.5)
G (pH 11.1) pH 10.1 at 37° did not represent the basic endpoint for carboxy-SNARF-1.

Calibration

Basic endpoint (for SNARF): fluorophore completely deprotonated.
Acidic endpoint (for SNARF): fluorophore completly protonated.

Media with low buffering capacity

Media with low pH buffering capacity are required for measurement of proton flux in intact cells. Commercially available variants of RPMI1640 that contain neither hydrogen carbonate nor HEPES may constitute a starting point, see e.g. Sigma Product number 6504 (not tested by Oroboros Instruments, due to the inclusion of phenol red this medium may (or may not) be problematic for fluorescence).

@@ Line 2: / Line 2: @@
 |description='''pH calibration buffers''' are prepared to obtain two or more defined pH values for calibration of pH electrodes and pH indicator dyes.
 }}
-{{MitoPedia concepts}}
 {{MitoPedia methods
 |mitopedia method=Respirometry, Fluorometry
 }}
-{{MitoPedia O2k and high-resolution respirometry}}
 {{MitoPedia topics
 |mitopedia topic=Medium
 }}
-== Hanks based calibration buffers ==
+== Modified hanks buffer ==
-::: '''Modified hanks buffer'''
+{| class="wikitable" border="1"
-::::* CaCl2 * 2 H2O  0.42 mM, imidazole 2.0 mM, potassium chloride 2.4 mM, magnesium chloride hexahydrate 0.49 mM, sodium chloride 137 mM, pH adjusted to 7.1 at 30°C; see also: [http://wiki.oroboros.at/index.php/Talk:O2k-pH_ISE-Module#Media_with_low_buffering_capacity]
+|-
+! Substance
+! Formula
+! Concentration [mM]
+|-
+| Calcium chloride dihydrate
+| CaCl<sub>2</sub> * 2 H<sub>2</sub>O
+| 0.42
+|-
+| Imidazole
+| C<sub>3</sub>H<sub>4</sub>N<sub>2</sub>
+| 2.0
+|-
+| Potassium chloride
+| KCl
+| 2.4
+|-
+| Magnesium chloride hexahydrate
+| MgCl<sub>2</sub>*6 H<sub>2</sub>O
+| 0.49
+|-
+| Sodium chloride
+| NaCl
+| 137
+|}
+::::* pH adjusted to 7.1 at 30 °C.
+::::* pH adjusted with KOH or HCl to create a buffer series: (pH values at 25 °C)
+::::# A (pH 3.3),
+::::# B (pH 6.1)
+::::# C (pH 6.6)
+::::# D (pH 6.9)
+::::# E (pH 7.3)
+::::# F (pH 7.5)
+::::# G (pH 11.1) pH 10.1 at 37° did not represent the basic endpoint for carboxy-SNARF-1.
-ph adjusted with KOH, HCl to create a buffer series: (pH values at 25 °C): A(3.3), B(6.1), C(6.9 initial measurement showed 6.4), D (6.9), E(7.3), F(7.5), G(10.1).  for experiments after MF613 buffer C was further adjusted to 6.6 and G was adjusted to 11.1 ( MF613 showed that buffer G at 37° was not yet representing the basic endpoint for carboxy-SNARF-1)
+:::'''Calibration'''
+::::* Basic endpoint (for SNARF): fluorophore completely deprotonated.
+::::* Acidic endpoint (for SNARF): fluorophore completly protonated.
+== Media with low buffering capacity ==
+:::: Media with low pH buffering capacity are required for measurement of proton flux in intact cells. Commercially available variants of RPMI1640 that contain neither hydrogen carbonate nor HEPES may constitute a starting point, see e.g. Sigma Product number 6504  (not tested by Oroboros Instruments, due to the inclusion of phenol red this medium may (or may not) be problematic for fluorescence).