PH calibration buffers

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.

pH calibration: temperature dependence

pH electrodes have to be calibrated at the temperature intended for use and the correct pH values of the pH calibration buffer has to be used for the calibration. These values are sometimes difficult to obtain. Sigma-Aldrich supplied us with temperature dependent pH values for the following products:

T	B5020 (pH4)	B4770 (pH 7)	B4985 (pH 10)
°C	pH	pH	pH
0	4.01	7.12	10.31
5	4.01	7.09	10.23
10	4.00	7.06	10.17
20	4.00	7.02	10.05
25	4.01	7.00	10.00
30	4.01	6.99	9.95
35	4.02	6.98	9.91
40	4.03	6.98	9.87
45	4.04	6.97
50	4.06	6.97	9.81
55	4.08

(a) There seems to be a contradiction between the value stated for the pH 4 buffer at 25 °C in this table and on the product itself ("pH 4.00 +-1").

(b) This information is as supplied to Oroboros and with no responsibility by Sigma Aldrich or Oroboros for the correctness of this information.

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).