High-Performance Liquid Chromatography (HPLC) systems are known for their versatility in separating complex mixtures, but their ability to tailor an analysis around a sample’s unique attributes comes with several testing variables to consider. Different interactions between the chromatography column, stationary phase, mobile phase, and chemical compounds of your analyte create more scenarios than can be accounted for, especially when you are searching for one precise result. In order to guide your HPLC tests to your goal, understanding the many components and their variables is a necessary step toward chromatographic performance. Among these factors, one of the most important is optimizing solvent conditions.
HPLC Components
The process of optimizing solvent conditions requires a strategic approach. Understanding the nature of your sample will require choosing an appropriate chromatographic method. Although HPLC focuses on a high-pressure system for particularly precise analysis, there are types of techniques to consider, such as normal phase, reversed phase, ion exchange, and size exclusion. These systems typically consist of a solvent reservoir where the mobile phase is stored, a pump to move the phase through the system, an injector for introducing samples, a stationary phase that separates samples, and a detector to identify and quantify separated compounds.
These factors influence the size and dimensions of an HPLC column, which may be packed with different materials for the stationary phase, such as functionalized silica, polymers, ceramics like zirconia, or a combination of these options. These parameters will significantly affect the solvent selection for a mobile phase to carry a sample through an analysis.
Solvent Properties in HPLC
Solvent properties like polarity, pH, and ionic strength are some of the most pivotal factors that coincide with your other HPLC components. Common choices include acetonitrile, water, and methanol, although less popular options like dichloromethane and isopropyl alcohol serve specific functions for unique tests.
Ionic Strength, Polarity, and Selectivity
Solvent strength refers to the ability of the mobile phase to elute analytes from the stationary phase, which directly impacts retention times and the selectivity of compounds. Solvent strength depends on the relative polarity between the mobile phase and the stationary phase. Weaker solvents generally increase retention times, while stronger solvents can reduce them, impacting the desired retention factor and separation efficiency.
Increasing solvent strength by adding more polar solvents like alcohols or acetonitrile reduces retention by disrupting interactions between polar analytes with the stationary phase. Normal-phase chromatography involves the use of organic solvents like hexane or toluene with polar stationary phases, which retain polar compounds. In contrast with reversed-phase chromatography, polar solvents like water are paired with non-polar stationary phases in order to keep non-polar analytes attached. Through precise solvent composition modifications, solvent strength can be a tool to fine-tune your retention factor for the most effective separation process.
Mobile Phase pH
Mobile phase pH directly affects the ionization state of analytes and influences the stability of the stationary phase, especially with pH-sensitive columns like silica. Proper pH adjustments help create a balance between analyte-stationary phase interactions, particularly for ionizable compounds.
To achieve stable pH levels during separation, additives like buffers are used to control separation conditions. They either pair a weak acid with a conjugate base or a weak base with a conjugate acid to resist pH changes. Buffers like formic acid/ammonium formate remain a preferred choice for HPLC applications due to their volatility and pH range compatible with most UV and MS detectors.
Solvent Purity
No matter your solvent selection, always consider a purity grade appropriate for your LC system. Otherwise, even small traces of contaminants will have detrimental consequences for your analysis. HPLC-grade solvents provide a purity level needed for HPLC systems, which we specialize in manufacturing at Birch Biotech.
Preparing HPLC for Solvent Conditions
HPLC method development involves selecting appropriate stationary and mobile phases based on sample chemistry and desired analyte separation. This includes selecting solvent compositions, strength, gradients, pH, and buffer concentration. Adjusting these parameters can drastically improve analyte separation and peak shape. Fine-tuning conditions such as flow rate and temperature ensure optimal analysis times and minimal solvent consumption. Efficient method development results in improved resolution and analyte detection tailored to sample complexity and desired outcomes.
Sample Preparation
As one of the most preliminary considerations in your HPLC testing process, sample preparation conditions help clean samples, improve analyte recovery, and reduce adverse inference before combining with a solvent into a mobile phase. Techniques like filtration, centrifugation, solid-phase extraction, liquid-phase extraction, and protein precipitation are commonly used to stabilize and concentrate sample components before an analysis. They can also minimize matrix effects and enhance the efficiency and reliability of subsequent HPLC procedures.
Stationary Phase Properties
The way the chemical properties of a sample may determine the selection of your column can also impact your stationary phase’s particle and pore sizes. Identifying an appropriate balance between particle size, pore size, and back pressure.
Pore size determines which analytes can be separated and their interactions with the stationary phase. Smaller pores provide higher surface area for interactions, making them ideal for small molecules, while larger pores are necessary for retaining larger analytes like proteins. In size-exclusion chromatography, pore size directly controls separation based on molecular size.
Particle size increases the interaction time between analytes and the stationary phase, influencing column efficiency and direct factors like flow rate and pressure. Smaller particles generally provide higher separation efficiency due to their increased surface area and reduced interparticle diffusion distances. However, this can also lead to increased back pressure, which is an adverse result of pressure build-up from a mobile phase that can lead to possible column damage and compromised results.
Column Selection
Sample properties like molecular size, weight, and polarity may change what column selection is best for your analysis goals. Various column materials are available depending on your choice of chromatography technique, such as silica functionalized with C18, phenyl/cyano groups, polymers, or ceramics like zirconia. Column selection typically impacts several compatibility factors for the sample, solvent, and stationary phase.
Optimizing HPLC Performance
After determining the best solvent properties and HPLC components in preparation for your test, monitoring the following factors will help optimize performance during your analysis for clear, reliable results.
Temperature Control
As a solvent carries a sample through a column, temperature can become detrimental for accurate results. It reduces the viscosity of the mobile phase, increases the flow rate, and reduces back pressure. Additionally, temperature influences analyte-stationary phase kinetics and thermodynamics, impacting retention times, selectivity, and resolution. Precise temperature control enhances reproducibility by minimizing variability in retention and peak shape. Adjusting the temperature can also optimize the retention factor and improve resolution. A thermostatted column oven is a common way labs maintain a consistent temperature.
Flow Rate and Pressure
By adjusting the flow rate, the interaction time between analytes and a stationary phase can be controlled. This represents the speed and volume of the mobile phase as it passes through a chromatography column. A higher flow rate may lead to shorter retention times but can also reduce resolution and increase pressure drop. Flow rate can help determine factors about your column size while also becoming a calibration method when already in place. This is often illustrated by the Van Deemter Equation, which calculates the relationship between flow rate and separation efficiency.
Pressure affects column performance and influences the flow rate and solvent properties in an HPLC system. It is important to note how it affects equipment longevity, as operating at high pressures can reduce the lifespan of columns, packed beds, and stationary phases. It also stresses system components, increasing the risk of failure. Like flow rate, pressure can be used as a variable to sharpen your HPLC conditions during your analysis.
Gradient Elution
Gradient elution in HPLC involves changing the solvent composition during analysis. By varying the gradient slope, analysts can make adjustments to varying polarities in real time for a better separation of complex mixtures.
The choice of gradient steepness can affect resolution and run time. A steeper gradient can shorten analysis time, but may compromise the resolution between peaks. An ideal gradient slope selection improves HPLC efficiency by increasing both separation speed and selectivity.
Enhancing Detector Sensitivity
Detector calibration improves chromatographic signals, retention times, and other beneficial conditions specific to the solvent and sample during analysis. Precise adjustments with your flow rate, pressure, temperature, and gradient significantly boost detector sensitivity, band broadening, peak resolution, precise metrics, and clean testing results.
Troubleshooting Common Issues
Recognizing and addressing common issues in HPLC can help prevent improper solvent preparation, which often lead to inadequate separations, peak tailing, or misleading results. Another frequent issue to avoid is column degradation, which can affect the performance and longevity of liquid chromatographic systems. Regular maintenance, including cleaning and replacing columns, helps reduce or avoid system-related issues, analytical inaccuracy, and the use of broken equipment. For troubleshooting, evaluate the conditions and optimization steps mentioned above, starting with solvent pH, proper mixing, and mobile phase composition.
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[1] Crawford Scientific. (n.d.). The theory of HPLC chromatographic Parameters. https://www.inacom.nl/wp-content/uploads/2020/04/theory_of_hplc_chromatographic_parameters__1_.pdf
[2] Dolan, J. W. (2020, November 12). The perfect method, v: changing column selectivity. Chromatography Online. https://www.chromatographyonline.com/view/perfect-method-v-changing-column-selectivity
[3] Today, C. (n.d.). What is the Van Deemter Equation? Chromatography Today. https://www.chromatographytoday.com/news/gc-mdgc/32/breaking-news/what-is-the-van-deemter-equation/31273