Effects of buffer pH and temperature on chromatographic peaks

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Effect of pH on Retention Time

In reversed-phase chromatography, with the increase of pH, the retention time of acidic compounds is shortened, while the retention time of basic compounds is prolonged; the buffer solution can buffer the change of pH of the solution. The definition of buffer solution is that a small amount of acid or base can buffer pH, that is to say There is little change in pH when acid or base is added. This ability to maintain pH is called buffer capacity, which is determined by the concentration of the buffer and the pKa of the buffer salt. The buffer salt has the greatest buffer capacity near the pKa.

Table 1 lists the pKa of some commonly used buffer salts in liquid phase. Note that this was measured in water, no organic solvent was added. The pKa changes when organic solvents are added, but generally the pKa of all compounds, including your analyte, will change the same. For common concentrations in liquid phase, the buffer should be maintained at about 1.5 pH units of the pKa of the buffer salt. When the concentration is particularly dilute, such as less than 5mM, the range should be reduced, about +/- 1 pH unit .

borate 1


borate 2


borate 3


Citrate 1


Citrate 2


Citrate 3


2-(N-Morpholino)ethanesulfonic acid (MES)


Trichloroacetic acid




It is recommended to first select buffer salts and pH ranges when developing a method, and then use the selectivity of the solution to fine-tune the separation. This strategy is fast and efficient, and it will keep you from getting bogged down in buffer pH.

The effect of temperature on chromatographic peaks

1) It is recommended to set the temperature at 25-40. The appropriate column temperature can maintain good reproducibility of retention time, improve the column efficiency to a certain extent, and increase the degree of separation.

2) When the column temperature is too low, the viscosity of the mobile phase decreases, and the back pressure of the chromatographic column decreases.

3) The column temperature is too high, and the temperature difference between the detector and the detector is too large, which will deform the peak, accelerate the attack of the ion-pair reagent or buffer salt on the silica matrix, dissolve the silica gel, and shorten the life of the chromatographic column; the column temperature is too low, which requires refrigeration The hardware requirements are too high, which is not conducive to the promotion of the method.

4) The change of temperature will lead to the drift of the retention time. If the instrument automatically injects samples overnight or over the weekend, it will be found that the retention time will drift with the change of the laboratory temperature. Obviously, this problem can be avoided by using the column thermostat.

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