In reversed-phase chromatography, the pH of the mobile phase is generally between 2-7. When the analyte can dissociate under reversed-phase conditions, or when the pH of the sample is outside the range of 2-7, a buffer salt is required. Compounds that can dissociate under reversed-phase conditions generally have amino and carboxyl groups, with pKa values between 1-11. Choosing the correct buffer pH can ensure that the dissociated functional groups exist in either an ionic form or a neutral compound form. If the pH of the sample is harmful to the column, then a buffering solution can make it milder or reduce the harm. The pH tolerance range for a conventional silica-based column is 2-8. The common buffered electrolyte salts are classified as follows.
1. Alkaline buffer salts
Common additives include ammonia and triethylamine, both of which can increase the pH of water to a very high level, high enough to dissolve silica gel particles. Therefore, they need to be used with caution. In addition, these two solvents are usually not easily removed through washing and maintenance methods. Especially for triethylamine, even if it is of chromatographic purity, it may be contaminated due to deterioration after a long period of storage.
2. Acidic buffer salts
1. Formic acid: usually used at a concentration below 1%, it can increase the pH of water to around 2.
2. Acetic acid: slightly less acidic than formic acid, usually used at a concentration not exceeding 5%, it can increase the pH of water to around 2.
3. Trifluoroacetic acid: a relatively strong acid, usually used at a concentration not exceeding 0.5%, it can decrease the pH of water to below 2. It has a large ion intensity and a certain ion pair effect, and is often used as an additive when analyzing proteins and peptides. The most fatal problems of these three acids lie in their UV absorption at low wavelengths. It is usually not recommended to use them at wavelengths less than 215nm. In addition, formic acid, acetic acid, and trifluoroacetic acid are volatile and their concentration may change over time.
4. Phosphoric acid: this is a non-volatile acid with good ion intensity. The most important thing is that there is no background absorption in the low-wavelength UV area. However, it also has some disadvantages, such as being incompatible with mass spectrometry methods. In addition, there are some other acids, such as sulfuric acid, hydrochloric acid, or citric acid, but they are rare.
3. Salt buffer salts
Compared with simple acid-base buffers, by adjusting the proportions of the two salts in different buffer salts, a buffer solution with a certain pH can be easily prepared, and buffer salts with buffering capability can control the pH value of the mobile phase.
1. Formate salts: commonly used formate salts in liquid chromatography are ammonium formate. This type of salt has good solubility, is completely compatible with liquid-mass spectrometry methods, and has a relatively small impact on chromatographic columns. However, the disadvantage is that the ion intensity is relatively low. In addition, formate salts are prone to deliquescence, making it difficult to weigh and potentially volatile in the mobile phase. When combined with formic acid, the formic acid-ammonium formate buffer system can achieve buffering capability within a pH range of 3~4.5, just by adjusting the ratio of the two.
2. Acetate salts: common examples are ammonium acetate. The use of Acetate salts is similar to formate salts, and the ion intensity is not high. The acetic acid-ammonium acetate buffer system can control the pH in a range of 4~5.5.
3. Phosphate salts: one of the most commonly used buffer solutions in HPLC under UV detection conditions because it can be used at wavelengths below 220 nm. Phosphate salts have three pKa values. Although they are widely used, attention must be paid to their solubility issues to prevent them from precipitating in HPLC pumps or chromatographic columns under high-concentration conditions.
Sodium and potassium salts of phosphoric acid are commonly used, and the pH control capability of the two is very similar, with only a slight difference in ion intensity. In most cases, they can be used interchangeably. Because phosphoric acid is a polybasic acid, it can form a monohydrogen phosphate and a dihydrogen phosphate, so the use of the phosphate buffer system to adjust the pH is more flexible and can cover a wide range, and can provide high ion intensity. Unfortunately, the phosphate buffer system for mobile phase is not compatible with mass spectrometry.