10 Q&As on Preparative HPLC chromatography

Today, Let’s talk about preparative liquid chromatography. Although preparative liquid chromatography is not as widely known as analytical liquid chromatography, this is mainly due to their different uses. However, it is an indispensable part of our research and production work.

Let’s take a look at the ten most common problems with preparative liquid chromatography!

Schematic diagram of preparative liquid chromatography

1. What is Preparative Liquid Chromatography?

Preparative liquid chromatography is a chromatographic technique used for separating and purifying known or unknown substances. It is mainly applied in fields such as chemistry and biology, utilizing the separation capacity of chromatographic columns to remove impurities and obtain high-purity samples. A preparative liquid chromatography system consists of solvent bottles/buckets, high-pressure pumps, sample injection systems, preparative columns, detectors, fraction collectors, and chromatographic data processing systems.

2.What are the Uses of Preparative Liquid Chromatography?

Compared to traditional purification methods such as column chromatography, distillation, and crystallization, preparative liquid chromatography is an effective separation method with simple operation and high sensitivity. Therefore, it is widely used for the extraction and purification of high-value samples, particularly for certain pharmaceutical impurities.

Purification scale	Target mass	Typical applications
Analytical grade micro-purification	µg	Enzyme, peptide, biomolecule, or other compound separation in small-scale pharmaceutical research experiments
Semi-preparative	mg	Enzyme, peptide, biomolecule, or other compound separation in small-scale pharmaceutical research experiments
Preparative	g	Analysis of reference standards, toxicology research, chemical screening, high-purity main component/by-product separation and enrichment
Process	kg	Industrial-scale production of pharmaceuticals and active compounds

Table 1: Chromatography scale, target compound quantity, and related applications

3. What is the difference between preparative liquid chromatography and analytical liquid chromatography?

Preparative liquid chromatography and analytical liquid chromatography are based on the same principles, but their main difference lies in their objectives:

Analytical Liquid Chromatography	Preparative Liquid Chromatography
Qualitative: ☆☆	Purity: ☆☆
Quantitative: ☆	Recovery Rate: ☆☆
Peak Shape: ☆☆	Separation Throughput: ☆
Resolution: ☆☆	Post-processing Convenience: ☆☆
High Sensitivity: ☆☆	Compound Stability: ☆☆
Compliance: ☆☆	Cost: ☆☆

Table 2: Main Differences between Analytical Liquid Chromatography and Preparative Liquid Chromatography

The purpose and function of analytical liquid chromatography are to perform qualitative and quantitative analyses. Therefore, analytical liquid chromatography focuses on separation resolution and sensitivity. It often aims for lower detection limits, with high-sensitivity detectors typically achieving levels in the ng (nanogram) or even pg (picogram) range.

Preparative chromatography refers to the use of chromatographic methods to separate a certain amount of compounds with sufficient purity for subsequent experiments or processing. Preparative liquid chromatography utilizes the separation ability of the chromatographic column to remove impurities and obtain high-purity samples. While balancing separation resolution and sensitivity, it aims for greater preparation efficiency. In summary, our requirements for preparative liquid chromatography are to achieve "more (sample load), fast (preparation speed), good (fraction purity), and economical (solvent use)" experimental results.

Project	Analytical Liquid Chromatography	Preparative Liquid Chromatography
Purpose	Qualitative and quantitative analysis	Separation and purification
Focus	Separation resolution, sensitivity	Preparation efficiency
Function	Analysis	Remove impurities to obtain pure products
Specification	10 mL/min	Semi-preparative: 50 mL/min Preparative: ≥100 mL/min
Configuration	Pump, injector, analytical column, column oven, detector	Pump, injector, preparative column, detector, fraction collector
Subsequent Experiments	N.A.	Further analysis such as NMR Provide samples with sufficient purity for experiments

Table 3: Comparison between Preparative Liquid Chromatography and Analytical Liquid Chromatography

4. What are the principles and experimental process of preparative liquid chromatography?

After sample injection, the mobile phase drives the sample forward. As the components of the sample pass through the stationary phase, they interact with it differently (adsorption, partitioning, exclusion, affinity, desorption). These interactions result in different retention times in the stationary phase, causing the components to elute from the stationary phase at different times, thus achieving separation.

Experimental Process:

（1）Method Reproducibility

Select Equivalent Chromatographic Columns
Confirm Sample
Establish Welch Laboratory Central Control Methods

（2） Method Development

Select Volatile Additives
Sample Stability
Column pH Tolerance Range
Single Injection Run Time

（3） Scale-Up Preparation

Fraction Purity Testing
Single Injection Sample Load
Solution Stability

（4） Post-Processing

Stability

5. What are the key parameters of preparative liquid chromatography experiments?

The three main parameters for preparative chromatography separation are purity, recovery rate, and sample load.

The three parameters form a triangular relationship, and they are optimized based on the separation objectives. Since these parameters are interdependent, it is impossible to simultaneously optimize all three in purification separation. A clever balance is required to achieve the best results.

6. How is preparative liquid chromatography classified?

Based on the separation of samples with different types and purity requirements, preparative liquid chromatography can typically be classified by system pressure into low-pressure, medium-pressure, and high-pressure modes. Generally, low-pressure, flash, and medium-pressure preparative chromatography are collectively referred to as medium-low pressure preparative chromatography.

Low Pressure (<2 MPa): Generally suitable for compounds that are easy to separate, but the long separation time can lead to the decomposition of sensitive compounds.

Flash (<1 MPa): Also known as fast preparative chromatography, Flash is an alternative to low-pressure separation due to its shorter separation time. Although its resolution is not as high as medium pressure, it is often used for simple separation problems.

Medium Pressure (<5 MPa): Medium pressure typically uses a constant flow pump to provide a constant mobile phase flow rate. The packing material particles are smaller, providing higher resolution, and can withstand higher pressure and faster flow rates. The operation is basically the same as low pressure and Flash.

High Pressure (>5 MPa): High pressure is usually used in the final stages of separation preparation for fine purification. It is applied to the separation of difficult samples, with purity that can reach up to 99.9%. It can withstand higher pressures.

7. How should the mobile phase for preparative liquid chromatography be selected?

The mobile phase should ensure good separation and selectivity for the target compound.
Volatile mobile phases simplify post-processing.
The evaporation residue of the mobile phase should be as low as possible to ensure the purity of the separated product.
The viscosity of the mobile phase should be low to ensure low column pressure.
The spectral characteristics of the mobile phase should be compatible with the detector.
The mobile phase should be low cost and chemically inert.

Common Volatile Buffer Salts and Their pH Values
Trifluoroacetic Acid	<2.2
Formic Acid	2.8-4.8
Ethanoic acid	3.8-5.8
Ammonium formate	3.8-5.8
Ammonium acetate	3.8-5.8
Ammonium carbonate	5.5-7.5
Ammonium bicarbonate	8.3-10.3
Ammonia solution	8.2-10.2

In reversed-phase chromatography systems, the order of solvent strength is as follows: water (weakest) < methanol < acetonitrile < ethanol < tetrahydrofuran < isopropanol < dichloromethane (strongest). Except for dichloromethane, which is immiscible with water, the above solvents can be combined with water for reversed-phase HPLC. The most commonly used combinations are acetonitrile/water and methanol/water.

Other choices should consider that there will be extraction steps after chromatography. Avoid using high-boiling solvents, highly toxic solvents, and minimize the use of multi-solvent systems with large differences in density.

8. Common Methods and Issues in Sample Post-Processing

1) Methods of Sample Post-Processing

Direct lyophilization of fractions
Rotary evaporation of fractions
Rotovap followed by lyophilization
Liquid-liquid extraction of fractions followed by rotary evaporation
Solid-phase extraction of fractions followed by rotary evaporation

2) Issues Encountered in Sample Post-Processing

Evaluation of the solution stability of fractions
Evaluation of overall stability of fractions
Removal of inorganic salts from fractions
Light protection for fractions
Conversion of compounds to salts after lyophilization

9. What is concentration or volume overload?

Concentration Overload: This occurs when the sample concentration is too high during a small volume injection (volume not overloaded). The stationary phase near the peak band of the chromatogram becomes saturated, meaning that a component of the sample exceeds the column's capacity, resulting in overload. This typically manifests as peak tailing and reduced column efficiency. In concentration overload, the sample concentration increases while the injection volume remains the same. The capacity factor K′ K'K′ increases, and the peak shape changes from a Gaussian curve to a triangular shape. Concentration overload can only be achieved if the sample has good solubility in the mobile phase.

Diagram of Concentration Overload

Volume Overload: A large injection volume typically leads to broader, flat-topped peaks. Additionally, volume overload can affect the peak retention time, with a greater impact on components with shorter retention times and a smaller effect on peaks eluting later.

Both concentration and volume overloads can reduce the separation efficiency of compounds. Since effective separation requires a certain degree of resolution, optimizing separation efficiency is crucial during method development. Selectivity and the potential for overload are interdependent; improving selectivity will increase the amount of sample that can be separated in a single run.

10. Development and Scale-Up Ratio Calculation for Preparative Liquid Chromatography

In analytical chromatography, the amount of sample added to the chromatographic column is generally at the µg level, or even less, with a compound-to-stationary phase mass ratio of less than 1:100,000. The sample volume is typically much smaller than the column volume (less than 1:100), which allows for sharp and symmetrical peak shapes. In contrast, preparative liquid chromatography requires much higher sample loads.

Purifying large quantities of samples can be achieved by two methods: scaling up the analytical system proportionally or column overload.

Using column overload allows for the separation of milligram-level samples even on analytical columns.

To prepare larger quantities, you can scale up proportionally from this point.

（1）Optimize the separation efficiency of the analytical method

（2） Perform column overload on the analytical column

（3） Scale up to preparative columns

How to calculate scaling up?

When transitioning from a smaller diameter chromatographic column to a larger diameter column, the two parameters that need to be scaled up are flow rate and sample load.

Alright, that's the end of our ten questions and answers for today. Do you have any valuable experiences to share about preparative liquid chromatography?