Regarding high-performance liquid chromatography, the preference is for isocratic elution, and simpler mobile phase ratios are considered more effective. If possible, avoiding the use of buffer salts is recommended, and simplicity is key in elution methods. Common chromatographic columns are favored, but there are instances where a single mobile phase ratio or common columns may not suffice for separating numerous impurities. In such cases, it becomes essential to redevelop chromatographic separation methods. Below are some personal insights into analytical method development, hoping to provide experimental inspiration for newcomers.

  • Identifying the Properties of the Target Compound: First, it is essential to understand some properties of the impurities to be separated, such as molecular weight, structural formula (primarily focusing on functional groups), and the solubility of the substance in various organic solvents. The structural formula plays a crucial role in predicting the molecular polarity and determining whether the substance is prone to hydrolysis. Common polar functional groups include -NH₂, -OH, -COOH, while non-polar ones include benzene rings, -CH=, and alkyl groups. Based on experience, one can roughly assess the substance’s polarity, estimate its retention on commonly used chromatographic columns, and, combined with the solubility of impurities in various eluting solvents like methanol, acetonitrile, and hexane, choose an organic mobile phase suitable for elution. This helps determine whether to use a normal-phase or reverse-phase chromatographic column in the preliminary development of the method.
  • Determining Normal-Phase or Reverse-Phase Elution: Once the elution mode is determined, for reverse-phase, I personally recommend starting with 60% acetonitrile or methanol. First, check if the substances you need to separate can be effectively isolated (similar for normal-phase elution). Assuming successful separation, observe the retention time of the last peak. If the time is relatively long, assess the resolution of the preceding peaks to decide whether to increase or decrease the proportion of the organic phase. The commonly used elution separation method is reverse-phase, which further divides into two systems: methanol-water and acetonitrile-water.

The specific choice between the two reverse-phase elution systems depends on the following conditions:

A. Acetonitrile has better elution ability than methanol. Under equal concentrations and sample conditions, the target peak in the acetonitrile-water system elutes faster than in the methanol-water system.

B. Methanol releases heat when dissolved in water, while acetonitrile absorbs heat. In a gradient elution with the same method, due to molecular thermal motion, the mixture of methanol and water exhibits more vigorous molecular thermal motion than acetonitrile and water. This results in a more even mixing of the two mobile phases, reflected in a smoother baseline and fewer gradient peaks.

C. Methanol is generally less expensive than acetonitrile in the market (unless cost is not a significant factor).

D. The molecular weight of methanol is greater than that of acetonitrile. Under the same chromatographic column and the same concentration of the organic phase, the column pressure is lower in the acetonitrile-water system.

E. Methanol’s cutoff absorption wavelength is around 210nm, while acetonitrile’s is around 190nm. When selecting methanol or acetonitrile-water systems at wavelengths far from 210nm, other influencing factors are considered. However, when the detection wavelength is around 210nm, as methanol’s cutoff absorption wavelength is greater than acetonitrile’s, under the same detection conditions, the sample response in the acetonitrile-water system is higher, resulting in a lower detection limit. Hence, it is advisable to prefer the acetonitrile-water system in such cases.

Figure 2-1 System suitability diagram using methanol-water as system
Figure 2-2 System suitability diagram using acetonitrile-water as the system

In the above two figures, Figure 2-2 introduces an additional impurity, namely Peak 5, compared to Figure 2-1. The types and elution positions of the other impurities remain unchanged. In both figures, the main focus is on the baseline and the elution positions of each impurity on the chromatogram.

  • Determining Chromatographic Column, Flow Rate, and Column Temperature:

The most commonly used chromatographic columns in laboratory analysis are typically of 5μm, 0.46×250mm, or 0.46×150mm dimensions. In the early stages of experimentation, without reference data, the preference is to start with the most common 5μm, 0.46×250mm chromatographic column. If, during the separation of multiple impurities with a 5μm, 0.46×250mm column, split peaks or a low theoretical plate number are observed, it suggests that this type of column may not be suitable for effective separation of the substance. In such cases, it’s advisable to switch to a column with higher efficiency, often with smaller particle size and different packing material. The suitable chromatographic column for detection is determined through experimentation based on practical considerations.

The choice of flow rate is generally determined by the specific type of chromatographic column and its maximum pressure tolerance. Commonly used chromatographic columns typically operate at a flow rate of 1.0mL/min, and the impact of flow rate on impurity separation is relatively minor.

For column temperature, room temperature is preferred. An increase in column temperature affects the speed of impurity elution. In theory, higher temperatures result in faster elution of various substances, and the column pressure decreases. However, excessively high temperatures can cause the stationary phase in the chromatographic column to be easily flushed away by the mobile phase, leading to irreversible loss of column efficiency. Therefore, the column temperature should not be too high, generally set between 25-35℃. Some special types of columns may require temperatures at 40℃ or even higher.

  • Gradient Elution Program Selection:

After selecting the appropriate normal or reverse-phase elution mode, determining the chromatographic column, column temperature, and flow rate, if adjustments to the gradient and mobile phase ratio still cannot separate two impurities effectively, gradient elution becomes necessary. Gradient elution involves introducing different organic phase ratios at different times. Compared to traditional isocratic elution, gradient elution offers several advantages:

A. Increased Separation Efficiency: Gradient elution enhances the separation of two substances by introducing varying concentrations of the organic phase at different time intervals. This differential concentration during different time intervals facilitates the elution of impurities at different rates. The general principle is to gradually increase the proportion of the organic phase, accelerating the elution of impurities that are more difficult to wash out.

B. Reduced Analysis Time and Cost: The ability to adjust the concentration of the organic phase allows for shorter analysis times compared to isocratic elution. Moreover, it enables the separation of substances that cannot be effectively separated by isocratic elution in a single run. This capability allows for the analysis of multiple substances using one analytical method, ultimately reducing costs.

The exploration process of a gradient elution program should adhere to the following principles:

Principle 1: Avoid using a single mobile phase In the early stages of gradient elution, novice experimenters typically use a buffer salt as mobile phase A and pure organic solvent as mobile phase B. However, experienced experimenters often incorporate a certain proportion of organic solvent into mobile phase A, not solely relying on a buffer salt. There are two reasons for this approach. Firstly, adding a minimum of 5% methanol or acetonitrile helps prevent the formation of microorganisms in the mobile phase, reducing the need for frequent filtration. Secondly, introducing a certain proportion of organic solvent helps minimize baseline fluctuations during the mixing of mobile phases A and B in the instrument, resulting in a smoother baseline. Whether mobile phase B should consist of pure organic solvent or a specific proportion of organic solvent (such as 80% or 90% methanol or acetonitrile) depends on experimental considerations. I recommend using a certain proportion of organic solvent as mobile phase B. When pure organic solvents like acetonitrile or methanol are added, heat reactions during their mixing can cause significant baseline fluctuations. Using a certain proportion of organic solvent as mobile phase B effectively reduces the impact of mobile phase shock.

Principle 2: Gradual Addition of Organic Phase in the Gradient In the exploration of gradient methods, it is advisable to introduce the organic phase gradually. The following is a case study from the author’s own experiment during the exploration of gradient methods, let’s go straight to the figure.

Figure 4-1 Gradient 25 spectrum

Gradient Method 25 Elution Procedure

Figure 4-2 Gradient 26 spectrum

Gradient Method 26 Elution Procedure

Gradient method 27 elution method
Figure 4-3 Gradient 27 spectrum

Gradient method 27 elution method

The comparison of the above chromatograms and gradient elution programs reveals that a slow addition of the organic phase can make the gradient peaks smoother, improving the separation of various impurity peaks.

Regarding the occurrence of gradient ghost peaks, when the pure buffer salt phase is mixed with the organic solvent phase, a significant mutual solubility shock effect occurs. Even if commonly used ghost peak trapping columns are installed, the baseline may still not be very smooth. Therefore, I recommend using a solution of a certain ratio of the organic phase and the salt phase as the mixed salt phase. Also, the mixture of the organic phase with a certain proportion of water can be used as the organic phase for gradient elution. The use of a ghost peak trapping column in combination can make the baseline smoother.

  • Selection of Buffer Salts

Firstly, it is important to understand why buffer salts are added. One of the purposes of buffer salts is to enhance the buffering capacity of the mobile phase. Commonly used inorganic buffer salts such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, ammonium acetate, etc., are employed to adjust the buffering capacity of the mobile phase. What is buffering capacity? Buffering capacity refers to the degree of pH change in the mobile phase when other solutions or substances are added. The smaller the pH change in the mobile phase, the stronger its buffering capacity, and vice versa.

In some standards, you may see two types of buffer salts used in the preparation of the same mobile phase. This is because using a combination of salts as the mobile phase results in stronger buffering capacity compared to using a single salt. Some analytes are prone to hydrolysis in neutral or alkaline environments, such as functional groups containing -OH. To prevent hydrolysis of this type, adding phosphate salts to adjust the pH to an acidic environment can suppress hydrolysis, thus preventing the appearance of double-headed peaks in the chromatogram.
The addition of buffer salts serves another purpose, which is to increase the retention time of the analyte. Typically, for substances with amino groups or amino-containing groups such as -NH₂, -NHR, etc., to enhance the retention of these substances, ion-pair reagents containing -SO₃H groups, like sodium pentanesulfonate or sodium octanesulfonate, can be added to increase retention.

In general, buffer salts do not cause peaks in chromatograms. I once used water with a pH of 3.0 as the mobile phase and a solution of potassium dihydrogen phosphate with a pH of 3.0 as the buffer salt solution. The chromatograms obtained using these two conditions showed excellent agreement in gradient peaks. Therefore, colleagues need not worry about buffer salts causing gradient peaks. The direct cause of gradient peaks is the impact of mixed mobile phases.

  • Selection of Detection Wavelength

Regarding the selection of the detection wavelength, it is necessary to comprehensively consider the UV absorption spectra of multiple impurities. It is preferable to choose the wavelength at the peak position of the UV absorption characteristic spectrum as the detection wavelength. However, it is also essential to consider whether other impurities have UV absorption at this wavelength.

Figure 6-1 UV absorption characteristic spectrum of impurities

With the three impurities in the chromatogram as an example, their maximum absorption peaks are observed at 203.3, 204.5, and 204.5 nm, respectively. Additionally, the range of 205-230 nm can be considered as an alternative wavelength. Therefore, considering these three impurities, the tentative detection wavelength can be set at 205 nm.

Points to consider in wavelength selection:

  • Exercise caution in selecting wavelengths within the range of 190-210 nm. This wavelength range belongs to the lower spectrum and is prone to more interference, especially influenced by the purity of reagents in the mobile phase. In cases of impure reagents or poor water quality, a “peak” similar to a gradient peak may appear. Such peaks are often smoother and not as sharp as true impurity peaks. They persistently appear at a specific position regardless of adjustments to the gradient elution program. If this “peak” happens to affect the impurity peak, elimination can be challenging.

The author has encountered a project entangled with such peaks for a considerable time. Eventually, the issue was resolved by replacing with higher-grade reagents, using purer water, multiple instrument washes, and column changes. In experiments using a diode array detector (DAD), wavelengths of 265 nm, 225 nm, and 205 nm were tested. The undesired “peak” did not appear at 265 nm and 225 nm, emphasizing the cautious selection of 205 nm as the detection wavelength.

Solvent Selection

For isocratic elution, it is preferable to use the mobile phase as the solvent. Generally, using the mobile phase directly as a diluent is sufficient to meet daily analysis requirements. However, for gradient elution, it is best to choose a solvent solution with a concentration similar to the initial gradient concentration. Regardless of whether it is isocratic or gradient elution, several considerations need attention.

Firstly, the solvent’s ability to dissolve the sample is crucial. If the selected solvent has insufficient solvating power for the sample, it may result in either weak or absent peaks or, in severe cases, instrument and column blockage.

Secondly, it is essential to consider whether the solvent peak will impact the separation of target peaks. If the solvent peak interferes with the appearance of the target peaks, the chosen solvent is likely unsuitable.

When selecting a solvent for gradient elution, the proportion of organic phase to salt phase in the solvent can be adjusted step by step. The chromatogram can be observed to choose a suitable solvent. Additionally, attention should be paid to the solvent’s pH and buffering capacity to make it as similar as possible to the mobile phase.

The term “solvent peak” has been a mystery for many researchers who have conducted experiments for several years without fully understanding it. The solvent peak arises due to the difference in the organic phase between the solvent and the initial mobile phase after injection. The dissimilarity in polarity results in different absorbances at the same wavelength, causing the appearance of a “peak.” Generally, changing the detection wavelength can mask the occurrence of the solvent peak.

In practical method development, some individuals may underestimate the importance of solvent selection. The following is an unfortunate incident from a project I worked on due to improper solvent selection.

Figure 7-1 System suitability map

When peak 4 elutes, there is often a solvent peak, causing significant interference when determining quantitative limits and detection limits. Below are chromatograms of blank solvent solutions with different ratios of mobile phase A and mobile phase B after injection.

Figure 7-2 Solvent spectrum of mobile phase A:B=100:0
Figure 7-3 Solvent spectrum of mobile phase A:B=95:5
Figure 7-4 Solvent spectrum of mobile phase A:B=90:10
Figure 7-5 Solvent spectrum of mobile phase A:B=85:15
Figure 7-6 Solvent spectrum of mobile phase A:B=80:20

From the above chromatograms, it can be observed that the solvent gradient peaks mixed with different ratios of mobile phase A and B are different. In preliminary experiments, it is advisable to try various solvents with different concentrations or types, considering comprehensively which solvent should be chosen as the final one.

The road ahead is long, and the journey is challenging! I wish all colleagues the words they most want to hear: smooth experiments!

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