MAKING GOOD'S BUFFERS GOOD FOR FREEZING: THE ACIDITY
CHANGES AND THEIR ELIMINATION VIA MIXING WITH SODIUM
PHOSPHATE [1]
1
HEPES and Na‐P Mixture,
pH=7.5
Cresol Red (CR) Bromocresol Purple (BCP)
Lukáš Veselý 1, Behera Susrisweta 1, Dominik Heger *
1Department of Chemistry , Masaryk University, Kamenice 5, 625 00, Brno Czech Republic
e‐mail:502102@muni.cz,*hegerd@chemi.muni.cz
MOPS and Na‐P Mixture 
pH=7.5
MES and Na‐P mixture 
pH=5.5
𝑯 𝟐 𝒑𝑲 𝒂,𝑰𝒏𝒅 𝒍𝒐𝒈
𝒄 𝑰𝒏𝒅 𝒙
𝒄 𝑯𝑰𝒏𝒅 𝒙 𝟏Molecular Probes for H0
Conclusion
How we measure the pH Change?
1 2 3
1 2
Introduction: Freezing of (bio)chemicals often leads to deviations from the optimum pH which cause compound’s degradation, e.g., protein
aggregation[2] .So, we would like to present a straightforward and efficient approaches to reduce the freezing-induced acidity changes by
blending two different buffers which are commonly used for biological samples . This approach can be highly helpful in both the pharmaceutical
domain and those branches of science where freezing is the preferred method for long-term preservation.
References
[1] Lukáš Veselý, (2021)” Making good's buffers good for freezing: The
acidity changes and their elimination via mixing with sodium phosphate”
Int J Pharm 593, 120128
[2] Zbacnik, T. J., R. E. Holcomb, D. S. Katayama, B. M. Murphy, R. W.
Payne, R. C. Coccaro, G. J. Evans, J. E. Matsuura, C. S. Henry and M. C.
Manning (2017). "Role of Buffers in Protein Formulations." Journal of
Pharmaceutical Sciences 106(3): 713-733.
[3] Thorat, A. A. and R. Suryanarayanan (2019). "Characterization of
Phosphate Buffered Saline (PBS) in Frozen State and after Freeze-Drying."
Pharm Res 36(7): 98.
B. Freezing–induced acidity changes in Good’s Buffers[1]
A. Freeze concentrate solution
350 400 450 500 550 600 650 700
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Absorbance
350 400 450 500 550 600 650 700
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Absorbance
C. What happens when we Mix Good’s Buffers with Sodium Phosphate Buffer ?
4
5
6
7
8
9
10
×
×
? ??
MESMOPS
Good's Buffer
HEPES
×
pH
0
20
40
60
80
180
200
?
×
× ×
?
×
?
×
?
?
MESMOPS
c/mmolL-1
Good's Buffer
HEPES
?
×
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
pH
H2
HEPES
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
6.5 7.0 7.5 8.0 8.5 9.0
7.0
7.5
8.0
8.5
9.0
9.5
10.0
pH
H2
MOPS
7.0
7.5
8.0
8.5
9.0
9.5
10.0
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
7.0
7.5
8.0
8.5
9.0
9.5
pH
H2
MES
7.0
7.5
8.0
8.5
9.0
9.5
1
pKa=7.45
2
pKa=7.20
3
pKa=6.10 400 500 600 700
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
 nm
6.0
6.5
7.0
7.5
8.0
8.5
9.0
A
2.7 2.4 2.1 1.8 1.5
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
A
~ / (104cm-1)
a
400 500 600 700
0.00
0.05
0.10
0.15
0.20
0.25
nm
6.0
6.5
7.0
7.5
8.0
8.5
9.0
b
2.7 2.4 2.1 1.8 1.5
0.00
0.05
0.10
0.15
0.20
0.25
~ / (104 cm-1) A
A
0 5 10 15 20 25 30
-7
-6
-5
-4
-3
-2
-1
0
1
H2
c(HEPES)
/mmol L-1
(H2pH)
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30
-7
-6
-5
-4
-3
-2
-1
0
1
c(MOPS)
/mmol L-1
(H2pH)
H2
1
2
3
4
5
6
7
8
1 10 100
-2.0
-1.6
-1.2
-0.8
-0.4
0.0
0.4
0.8
1.2
C(MES)/(mmol.L-1
H2
4.0
4.5
5.0
5.5
6.0
6.5
7.0
(H2
pH)
UV‐Vis Spectra of 
HEPES Buffer 
Room Temperature(RT)
Fast Frozen (FF)