Brief Introduction For The Development of High-Performance Liquid Chromatography (HPLC) Methods

High-Performance Liquid Chromatography (HPLC) uses liquid as the mobile phase and a high-pressure infusion system to pump a single solvent with different polarities or a mixture of solvents and buffers of different proportions into a column with a stationary phase. After the different components are separated in the column, they enter the detector for detection, thus realizing the analysis of the specimen.

The general principle of method development in HPLC:

  • Find the peak of the target compound
  • Adjust the peak shape
  • Refine the method

1. Find the peak of the target compound

  • HPLC column
  • Wavelength
  • Mobile phase
  • Temperature
  • Flow rate
  • Injection volume
Rosmarinic Acid Purification

Choose a proper HPLC column

The molecular weight, structural formula, and solubility of the sample in solvents (water, methanol, acetonitrile, tetrahydrofuran, n-hexane, isopropanol, etc.).

Use the molecular weight of the sample to choose the pore size of the packing material

The molecular weight of the sample determines the selection of the pore size of the HPLC column. Usually, the pore diameter needs to be more than 3 times the molecular diameter to not affect the analysis. This is because the better retention of the sample molecules on the column is due to the access to the micro-pores of the packing material and the interaction with the long C18 chains bonded to the surface.

HPLC columns with a pore size of 120 Å are usually suitable for molecules with molecular weights <10,000.

If the molecular weight is too large, the separation will be poor on a 120 Å pore size column.

Molecular weights greater than 100,000 less than 200,000 are analyzed on columns with 300 Å pore size.

Samples with molecular weights greater than 200,000 require a gel chromatography column.

Use the structural formula of the sample to select the phase of the column

The structural formula plays a very important role in predicting the polar size of molecules and in the subsequent adjustment of peak shapes.

-COOH, -NH2, -NHR, -NR2, and -OH are polar groups.

Benzene ring, hexane ring, -CH=, -CH2-CH3 are non-polar groups.

Make a general judgment of their polarity based on experience and estimate how the retention performance might be on C18 (the most familiar column). Combined with the solubility status of the target compound in several commonly used solvents, a rough judgment is made as to whether a normal-phase or reversed-phase column will be used for method development.

Selection of particle size and column size

Unless otherwise specified, particle size 5 μm, 4.6 × 150 mm or 4.6 × 250 mm columns are preferred.

Selection of HPLC Column

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.

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.


1. use pure methanol as the mobile phase to see if the compound is eluted down and if so what the peak height is and collect these data.

2. Decrease the proportion of methanol in the mobile phase. Methanol : Water = 100 : 0, 90 : 10, 80 : 20, 70 : 30, ..., 0 : 100. The change of peaks in the spectrum as the mobile phase goes from the initial 100% methanol to 100% water, the change in peak height, and where the extra peaks are located.


(1) It did not elute down under pure methanol conditions, which means that the compound is very weakly polarized and has a very strong retention capacity, and should be considered for a normal phase system, such as a silica gel column.

(2) The peak was difficult to come out under pure water conditions, but eluted down quickly in pure methanol. This peak can be found by adjusting the ratio of the mobile phase, and combined with the data of the previous series of mobile phase ratio 10% change, choose the appropriate mobile phase.

(3) Under pure water conditions, the peak still appears quickly, which indicates the strong polarity of the compound. Combined with the structural formula of the molecule to determine, it is possible which groups caused the sample to peak so quickly. Consider adding buffer salts, adjusting pH, or even ion-pairing reagents if needed.

2. Adjustment for the peak shape

Adjust the pH of the acid, base and buffer salt in the mobile phase.

pH in buffer

The pH of the mobile phase is one of the most effective variables in changing the selectivity of HPLC. In many cases, a change in the pH of the mobile phase causes a change in the retention factor k of the analyte, in addition to having a significant effect on peak broadening and tailing.
For the correction of mobile phase pH in the presence of organic solvents, two steps are performed: 1. Calculate the effect of organic solvent addition on the pH of the buffer and on the pKa of the ionizable analyte, respectively; 2. Sum the effects of both.

If both the buffer and the analyte are acidic or basic, the effects of the addition of the organic phase on pH and pKa will largely cancel out, and the total correction factor will be about 0; in this case, the pH of the configured buffer can be determined directly from the pKa of the analyte.

If the pH of the buffer and the analyte do not match, i.e., one is acidic and the other is basic, the effect of adding the organic phase will be very significant. In the range of 0-60% for the organic phase, the total correction factor is 0.4 units for every 10% change in the ratio for acetonitrile, i.e. +0.4 units for acidic analytes and -0.4 units for bases; for methanol, the correction factor is 0.2 units for every 10% change in the ratio, i.e. +0.2 units for acidic analytes and -0.4 units for bases. units for acidic analytes and -0.2 units for alkalis.

3. Refinement of the method

Solve problems in sample testing, such as retention time, separation of target compounds from impurities, etc.

GALAK ChromatographyAuthor
Tian Jing

Manager & Engineer in GALAK Chromatography. Master of Chemical Engineering.
During my college study, I found liquid chromatography to be a profound subject. I know the painful struggle a novice needs to go through to get started. I share this article to help you solve your problems quickly.

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