Types Of IEC Buffers- Welch Materials, Inc.

It is very important to maintain a constant pH during ion exchange. As discussed earlier, changes in pH can cause changes in the amount and distribution of protein charges, which directly affects whether the protein can bind to the exchanger and the binding capacity. strength. Therefore, buffers must be used for the mobile phase in ion exchange chromatography.

There are many types of buffers, and the substances that can act as buffers can be divided into two categories: the first category is a system composed of weak acids (or weak bases) and corresponding salts. The second category is zwitterionic compounds. For the first type of buffer substances, during ion exchange, if the charge of the buffer ion is opposite to the functional group on the ion exchanger, it will participate in the ion exchange process and may affect the local pH, so it should be used as much as possible. Buffer ions with the same charge as the functional group, that is, when using an anion exchanger, choose a buffer ion with a positive charge, and when using a cation exchanger, choose a buffer ion with a negative charge.

Of course, this is not absolute. For example, phosphate buffer is often used in the anion exchange process, but in this case, special attention should be paid to fully equilibrating before loading the sample to ensure that the pH and ionic strength of the chromatographic system are consistent with the initial value. Buffers are the same. The second type of buffer substances can be used in both anion and cation exchange. Commonly used buffers for cation exchange chromatography and anion exchange chromatography are listed in Tables 1 and 2, respectively.

Table 1 Commonly used buffers for cation exchange chromatography

Buffer substance	pKa	Recommended concentration/(mmol/L)
Maleic acid	2.00	20
Malonate	2.88	20
Citric acid	3.13	20
Lactic acid	3.81	50
Formic acid	3.75	50
Succinic acid	4.21	50
Acetic acid	4.76	50
Malonate	5.68	50
Phosphate	7.20	50
Hepes	7.55	50
Bicine	8.35	50

Although many impurity proteins can be removed during the ion exchange process to achieve purification effect, the elution peak of the target protein must contain a large amount of buffer substances and salt components, and the introduction of these components is also an impurity for the target protein itself. Especially when the elution peaks need to be freeze-dried after chromatography to obtain pure protein samples, most of the powders after freeze-drying are buffer substances and salts. If desalting or dialysis is performed before lyophilization, although these impurities can be basically removed, the recovery rate of protein activity may also be reduced. At this time, priority should be given to using volatile buffer substances, so that this part of impurities can be removed during the lyophilization stage. Common volatile buffer substances are listed in Table 3.

Table 2 Commonly used buffers for anion exchange chromatography

Buffer substance	pKa	Recommended concentration/(mmol/L)
N-Methylpiperazine	4.75	20
Piperazine	5.68	20
L-Histidine	5.96	20
Bis-Tris	6.46	20
Bis-Tris propane	6.80	20
Triethanolamine	7.76	20
Tris	8.06	20
N-Methyldiethanolamine	8.52	20
Diethanolamine	8.88	20（pH=8.4）50（pH=8.8）
1,3-Diaminopropane	8.64	20
Ethanolamine	9.50	20
Piperazine	9.73	20
1,3-Diaminopropane	10.47	20
Piperazine	11.12	20
Phosphate	12.33	20

Table 3 Volatile buffer systems

Buffer system (base/acid)	pKa
Formic acid/ammonia	3.75
Pyridine/formic acid	3.75, 5.25
Trimethylamine/formic acid	3.75, 9.25
Pyridine/acetic acid	4.76, 5.25
Trimethylamine/HCl	9.26
N-Ethylmorpholine/acetic acid	4.76, 7.8
Ammonia/formic acid	3.75, 9.25
Ammonia/acetic acid	4.76, 9.25
Trimethylamine/carbonic acid	6.50, 9.25
Ammonium bicarbonate/HCl	6.50, 9.26
Ethanolamine/HCl	9.5