Selection of mobile phase

The selection of the mobile phase needs to be determined according to the detection method. In this paper, the UV detector is used as an example.
The mobile phase of the liquid phase is most commonly used in two systems, one is the system of methanol and water, and one is the system of acetonitrile and water. These two mobile phase systems are not very different, the main difference is，

• Methanol is cheaper than acetonitrile.
• The elution capacity of acetonitrile is stronger than that of methanol.
• The absorption wavelength of methanol on the UV detector is around 210 nm. The absorption cut-off wavelength of acetonitrile is around 190 nm. If the wavelength is below 210 nm, then acetonitrile and water should be selected as the mobile phase system. If the wavelength is above 210 nm, methanol is preferred.

Other influencing factors include mobile phase mixing, mobile phase degassing, pH.

• The mixing of the mobile phase is based on the requests of different laboratories and testing methods. Pay attention to the mix techniques, if you need methanol solvent 50/50 v/v. Measure each volume, then mix them together.
• The degassing of the mobile phase is common to use in HPLC systems. If you don't have one, use helium gas or other gas.  Because bubbles will affect the pump and detector.
• pH will affect the selectivity, peak shape and retention, especially for polar compounds.

Buffer

The buffer solution is usually a solution composed of "weak acid and its conjugate base" or "weak base and its conjugate acid" buffer pair, which can slow down the change of pH when adding a certain amount of other substances. In order to ensure that the buffer solution has a strong enough buffering capacity, when preparing the buffer solution, in order to make the concentration ratio of the conjugate acid-base pair close to 1, a suitable buffer pair should be selected according to the pH range to be maintained, so that the PKa of the weak acid in it is equal to or close to the required pH.

The acids commonly used as buffer solutions are buffered by the combination of weak acids and their conjugate acid salts. The common buffer systems are.

• Weak acid and its salt (HAc - NaAc)
• Weak base and its salt (NH3·H2O --- NH4Cl)
• Aqueous solutions of acid salts of multiple weak acids and their corresponding secondary salts (NaH2PO4 --- Na2HPO4) are composed.

Selection of flow rate

The flow rate corresponding to the selected column type, 1 mL/min is selected for the 4.6 mm inner diameter column.

Selection of detector and wavelength

The most commonly used detector is the UV detector. If we do not know whether the sample has UV absorption, we first use the standard to prepare a suitable solution for UV scanning and collect data on the maximum absorption wavelength of the target compound to determine the wavelength. Or in the condition that there is a DAD detector into a few shots of standard solution, through the three-dimensional spectrum of the DAD can obtain information about the maximum absorption wavelength of the compound.
If UV absorption is not available, a suitable detector is required. Because the detector is our tool to monitor the peak of the compound, it is our "eye" to see the compound, and it is also the basis of our method development, so it is very important to choose carefully.

Selection of temperature

If the sample does not have a particular need for temperature (at high temperatures, the sample is unstable), try to use room temperature at the beginning of the method development. Increasing the temperature is done to improve the separation of the target compound from other impurities, or to reduce the column pressure, etc. These are taken into account when further optimizing the chromatographic conditions.