How to Choose the Right Liquid Chromatography Column, SPE, FALSH, or HPLC?

How to understand the chromatographic separation? Put it in short, the more similar the separated substance to the stationary phase, the longer the residence time on it. In this way, different substances in a complex mixed solution can be separated according to time, and the separated substances are sequentially entered into the detector to be detected by the detector, and then the scientific researchers can use these detected signals (chromatographic peaks) to calculate the concentration and content of a substance.

Liquid Chromatography (LC) Column:

  1. Column Type:
    • Choose between normal-phase, reversed-phase, ion exchange, size exclusion, or affinity columns based on the nature of your analytes and separation requirements.
  2. Particle Size:
    • Smaller particle sizes provide higher resolution but may result in increased backpressure. Choose the particle size based on the complexity of the separation and the capabilities of your LC system.
  3. Length and Diameter:
    • Longer columns generally provide better resolution, while the diameter affects the sample capacity. Choose the length and diameter based on the analytical goals and sample volume.

SPE Column:

  1. Sorbent Material:
    • Choose the appropriate sorbent material (e.g., C18, silica, ion exchange) based on the properties of your analytes and the type of interactions required for purification.
  2. Particle Size:
    • Similar to LC, the particle size of the SPE sorbent affects the efficiency of the extraction. Smaller particles offer higher surface area and better extraction, but they may require higher pressure.
  3. Sorbent Bed Mass:
    • Select the sorbent bed mass based on the volume of your sample and the desired level of purification.

Flash Chromatography Column:

  1. Sorbent Type:
    • Similar to SPE, choose the sorbent type based on the desired purification mechanism (normal-phase, reversed-phase, etc.) and the properties of the compounds.
  2. Particle Size:
    • Flash columns typically use larger particle sizes compared to HPLC columns. The choice of particle size depends on the speed of separation required and the pressure limitations of the system.
  3. Column Size:
    • Flash columns are available in various sizes. Choose the column size based on the amount of sample to be purified and the desired resolution.

HPLC Column:

  1. Column Type:
    • Reversed-phase columns are commonly used in HPLC, but there are also other types like normal-phase, ion exchange, and size exclusion. Choose based on the properties of your analytes.
  2. Particle Size and Pore Size:
    • Smaller particle sizes and appropriate pore sizes contribute to higher resolution. Consider the pressure capabilities of your HPLC system when choosing particle size.
  3. Column Length and Diameter:
    • Longer columns provide better resolution, and the diameter affects sample capacity. Choose the length and diameter based on the separation requirements and sample volume.

General Considerations

  1. Sample Characteristics:
    • Consider the nature of your sample, such as its polarity, size, and chemical properties. Different columns and techniques are suited for different types of samples.
  2. Analytical Goals:
    • Clearly define your analytical goals, whether it’s separation, purification, or both. This will guide the choice of the column and technique.
  3. Instrument Compatibility:
    • Ensure that the chosen column is compatible with your chromatography system, including pressure limitations and detection methods.
  4. Budget and Cost:
    • Consider the budget constraints and ongoing costs associated with column replacement or maintenance.
  5. Application Specifics:
    • Different columns are optimized for specific applications (e.g., pharmaceuticals, environmental analysis). Choose a column that aligns with your application needs.

To choose the right liquid chromatography column, firstly we should know the packing matrix and its influencing factors.

Packing matrix

1. Silica gel matrix-pH 2-8 —————— At high or low pH, silica gel will dissolve
2. Al2O3. NH2O-pH1-14 ————- Chemical modification is difficult
3. Polymer matrix-pH1-14 ————- Complex pore structure, uneven pore size results in insufficient column efficiency, organic solvents may cause swelling and damage to the polymer matrix.

Influencing factors

1.Physical factors
Purity of silica gel —Purity of filler silica gel and residual metal ion concentration
Column size — length and inner diameter of packed bed
Particle shape —-spherical or irregular
Particle size — average particle diameter
Surface area—the sum of the outer surface of the particle and the inner pore surface, expressed in m2 / gram
Pore size— The average size of the pores or cavities of the particles, ranging from 80-300 & Aring

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2. Chemical properties
Bonding type — monomer bonding-the bonding phase molecule and the matrix are connected at a single point
Carbon coverage—the amount of bonded phase connected to the matrix material
Capping—After the bonding step, use a short chain to bond the exposed silyl hydroxyl groups and block them.

About the samples and impurities’ type structure, polarity, acidity, molecular weight
Before choosing a column, learn more about your samples and impurities, the type structure, polarity, acidity, molecular weight, etc.
1. The sample is polar and weakly acidic: you can choose C18 for detection under the condition of 100% acidic aqueous solution, that is, select a chromatography column that withstands 100% pure water and retains polar compounds well.
2.Too polar or acidic sample: silica gel column, CN, NH2, HILIC (hydrophilic chromatography) can be adopted. The disadvantage is that the ion-pairing reagent has a long equilibration time, and the pH of the mobile phase is relatively precise. Otherwise, it is difficult to repeat the experiment. In addition, the ion-pairing reagent is difficult to wash off.
3.If the sample is alkaline: choose a high-purity silica gel column (high-purity silica gel lacks metal impurities and the end-capping of the silica gel) or some modified C18 columns (such as polar embedding technology or alkaline deactivation technology), which will reduce the tailing of alkaline compounds. So generally we choose to do under neutral or alkaline conditions, because this can increase the retention of alkaline samples.
4. If the polarity of the basic compound is too strong, or the basicity is too strong: a wide pH C18 column can be selected for high pH detection. The advantage is that the method is simple to develop, and the disadvantage is fewer supplier and high price or choose HILIC column (silica gel column is used under reversed-phase conditions, a classic method to detect alkaline samples).
Selecting strong ion exchange column’s disadvantage is that it cannot be used to analyze other samples, more precise requirements for mobile phase pH, otherwise it is difficult to repeat the experiment. There are also C18 + strong anion pair reagents or strong anion exchange columns.