Dichloromethane Safety for Handlers & Environment
Explore key insights about dichloromethane, including its applications, safety measures, and chemical structure.
How Do You Dispose of Acetonitrile
Discover uses, hazards, and safe disposal methods for acetonitrile.
LC-MS Purity and Solvent Contamination Prevention
A significant challenge with maintaining LC-MS performance is preventing contamination, which can easily impact test reliability, cause equipment downtime, and possibly require expensive maintenance to fix. To avoid these adverse impacts, following purity guidelines with your solvents will ultimately minimize unanticipated problems within your LC-MS system that may produce misleading results. By focusing on strategies to prevent contamination, you can optimize your lab's efficiency and data accuracy. Effective solvent management begins with understanding the impurity risks associated with LC-MS systems. Solvent compatibility, purity grade, proper handling, purification techniques, equipment calibration, and regularly maintaining a sterilized lab environment are all necessities to avoid the repercussions of contaminants. Liquid Chromatography-Mass Spectrometry offers some of the most precise analytical tools that modern technology has available today, and with proper knowledge about solvents, your LC-MS analyses will yield more accurate, reliable results. LC-MS Contamination Sources and Impacts Contamination in LC-MS systems often leads to misleading results and reduced instrument efficiency. The ability to maintain accurate and reliable analyses depends on comprehending the technology, chemical interactions, and detection of possible contamination sources ahead of time. Sample integrity remains intact during an LC-MS analysis when researchers create strict environmental controls and follow purity grades. By controlling lab conditions with regular maintenance, airborne contaminants, microbials, and particulate interference can be suppressed for system efficiency and longevity. Sources of Contamination in LC-MS Contamination in LC-MS systems primarily stems from solvents, tubing, and system components. Solvents not specifically graded for LC-MS can introduce high background signals, which interfere with signal interpretation even on the parts-per-billion (ppb) level. Improper handling and transfer of solvents can quickly introduce enough impurities to compromise results, even from airborne particles. For these reasons, filtering solvents can help prevent contaminants from accumulating over time, especially mixing processes with the mobile phase or other additives. Other contamination sources include traces from previous samples, carryover in the system, and degraded components like seals and gaskets. Regular maintenance helps in reducing these risks, such as routine checks for worn parts and flushing a system with solvents that are compatible with the stationary phase and residual analytes. LC-MS grade acetonitrile is a common choice for flushing, given its purity and chemical cleaning properties. Lab Air Quality Control Airborne contaminants, such as particulate matter and volatile organic compounds, are easily overlooked and can cause peak suppression in a final analysis. They affect non-testing tasks, like transferring containers and long-term storage. To minimize airborne particles, consistent air conditions in a lab should be maintained and monitored, including a stable temperature and humidity in an acceptable suitable for your solvents, samples, and other testing equipment. Implement a cleanroom-like environment where air pressure and airflow are regulated with ventilation systems and high-efficiency particulate air (HEPA) filters. These standards will create a controlled setting for testing and prevent sample degradation. Preventing Microbial and Particulate Contamination Microbial growth in a laboratory can severely affect sample integrity and testing performance. Stringent cleaning protocols with disinfectants that are within purity guidelines are a proven way to prevent the accumulation of microbes on work surfaces and equipment. For particulate contamination, maintaining a clutter-free workspace where materials are properly stored can help reduce risks. Consider using cleanroom garments and only in-lab accessories to reduce personal contamination that may enter a lab setting. Regular checks and maintenance of HVAC systems further contribute to maintaining a contaminant-free environment. Solvent Grade and Selection When selecting solvents for LC-MS, it is imperative to opt for a purity with at least an LC-MS grade. These meet stringent purity standards with minimal metal content, organic carbon, and other contaminants that could interfere with results. They also consider factors like conductivity, where lower conductivity indicates fewer ionic impurities. Although some higher grades can be suited for LC-MS, like ACS (American Chemical Society) Grade and Reagent grade, these choices tend to be less economical or practical compared to grades tailored for LC-MS applications. During the purchasing process, make sure to check your seller’s product specifications and manufacturing certifications to guarantee your selection matches your testing grade with reliable standards. Contamination Prevention and Maintenance Acknowledging contamination impacts and sources is the first step toward preventing unexpected hindrances within your LC-MS system. The following tips and techniques can help assure your lab that these conditions are controlled and maintained both during tests and for long-term accommodations. Mixing and Preparing Mobile Phases To prepare your solvents, maintain a strict protocol for using LC-MS grade products that interact with an analysis, including cleaning treatments for glassware, mixtures, and buffers. Mix solvents in a clean environment to avoid introducing impurities. Rinse bottles with only LC-MS grade solvents like isopropyl alcohol before use to remove residual contaminants. Glass bottles are generally used to store solvents, as they minimize interaction with container materials and preserve purity. Amber glass is often used for solvents like dichloromethane and acetonitrile that may create adverse reactions to sunlight during storage. Only use ultra-pure water for aqueous mobile phases. Consider using LC/MS-grade bottled water[1] or LC-MS grade water available with us at Birch Biotech. Always verify that solvents are made under robust quality control processes, and refer to detailed quality documentation provided by the manufacturer for confirmation. Purification and Filtration Methods Effective purification and filtration can address potential contaminants. Filtering solvents through 0.2-micron or smaller filters removes particulate matter, which might otherwise clog instrument components or interfere with analyses. Preventative maintenance includes regular cleaning of solvent lines to counteract issues such as microbial growth. Reverse osmosis is commonly used to produce high-purity water, which helps prevent interference from contaminants. Other purification techniques, like distillation, assure comprehensive results by removing various impurities. When using mobile phases containing additives, a thorough flush with high-purity water or organic solvents that follow standard cleaning guidelines[2] helps guarantee contaminants are minimized. Solvent Transferring and Storage When transferring solvents, always use dedicated equipment like pipettes or dispensers to prevent cross-contamination in a lab controlled environment. Constantly repeat cleaning steps in between their uses. Store solvents in well-sealed containers away from direct sunlight to prevent degradation. Proper storage also involves monitoring conditions like temperature and humidity. Solvent reservoirs should be covered to minimize exposure to the air, which can introduce contaminants. Implement a clear labeling system for mobile phase bottles correct handling and to minimize errors. Cleaning Routines Proper cleaning is a quintessential task for contamination prevention and optimizing LC-MS analysis. To maintain cleanliness, regularly scrub LC-MS vials and rinse all washed glassware thoroughly. Use desalting liquid-liquid extraction methods for efficient sample cleanup. Consider performing a wet prime by flushing your system with water followed by an organic solvent in order to eliminate residual additives. As mentioned other times earlier in this article, only suitable LC-MS grade cleaning supplies should be applied to uphold the purification standards necessary for optimal LC-MS performance. Frequently switch to fresh, clean solvents to minimize any accumulation of contaminants. Instrument Calibration Regular calibration can help guarantee that all LC-MS instruments perform correctly and that detectors accurately analyze mass-to-charge ratios. This will optimize your mass spectrometer, chromatography machinery, ion source, transmission equipment, and other key components of your LC-MS system. Improperly calibrated instruments can lead to inaccurate readings and compromised data quality, so reference your equipment manuals and guidelines for particular action items, techniques, and instructions. Regular Maintenance Regular maintenance not only extends the lifespan of your equipment but also improves the accuracy of your results. Regularly service your vacuum pump, which is typically recommended to change the oil and filters every six months. Consider implementing a shutdown method to maintain your system’s integrity when the equipment is idle. Keep N₂ flowing through your mass spectrometer source to prevent contamination buildup and protect instrumentation. Always use your LC-MS equipment guidelines for recommended maintenance, which can vary based on the products you purchase. Want to learn more about our solvents? Read more of our articles or visit us at Birch Biotech. -- Disclaimer: The content provided on the Birch Biotech blog is for educational and entertainment purposes only. The information offered here is designed to provide helpful insights and advice related to laboratory practices and supplies. Readers are advised to refer to our product-specific quality data sheets and Certificates of Analysis (COAs) available on our website for detailed information on product specifications. It is essential to handle and store all materials according to the safety guidelines and regulatory requirements applicable to your area. While we endeavor to ensure the accuracy and relevance of the information published, it should not be used as a substitute for professional advice or official protocols. We encourage all our readers to consult their institution's guidelines, local regulations, and professional standards before implementing any practices discussed here. Birch Biotech does not accept liability for any actions undertaken based on the information provided in this blog nor for the misuse of our products. Furthermore, Birch Biotech does not guarantee the completeness, reliability, or timeliness of the information contained on this website. This disclaimer is subject to change at any time without notifications. [1] Top 10 things users can do to minimize contamination of LC/MS systems. (n.d.). https://sciex.com/support/knowledge-base-articles/the-top-10-things-users-can-do-to-minimize-contamination_en_us [2] Waters. (2015). Controlling contamination in UltraPerformance LC/MS and HPLC/MS systems. https://www.waters.com/webassets/cms/support/docs/715001307d_cntrl_cntm.pdf
Solvent Selection for Mass Spectrometry: Key Considerations
Selecting a solvent for mass spectrometry impacts the quality of your operation and the choice of components best suited for your testing goals. Aligning solvent properties with these factors will balance your sample and mass spec techniques in a strategic way that will improve the accuracy of your results. Solvent Compatibility with Mass Spec To choose an appropriate solvent for an analysis, certain criteria change depending on your instrumentation and ionization technique. Mass spectrometers revolve around the relationships between a molecular ion source, a mass analyzer, and a detector. Sample molecules are converted into ions and separated with an analyzer based on their mass-to-charge ratio before a detector measures the relative abundance of each ion upon a spectrum. Different MS methods like quadrupole, time-of-flight, and magnetic sector analysis can require different solvents, ionization strategies, and other spectrometry equipment. For these reasons, solvents are chosen based on their compatibility with different ionization techniques. There are several to consider with electron ionization and electrospray ionization known for being versatile choices: Electron ionization (EI) is a common method that involves bombarding a sample with high-energy electrons to ionize and fragment the compounds. Electrospray ionization (ESI) uses a high-voltage electric field to ionize the sample and produce fine droplets that evaporate and release ions. Chemical Ionization (CI) introduces an analyte through a chamber where a reagent gas like methanol facilitate ionization, forming plasma. Field Ionization/Desorption are both techniques with principles centered around the quantum tunneling of of electrons, which generate the cation and generates a high potential gradient. Matrix Assisted Laser Desorption Ionization (MALDI) uses a pulsed laser to desorb molecules and generate singly charged ions and volatilize analytes with extremely high molecular weights. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) uses a plasma torch and electromagnetic induction at high temperatures, which break down analytes into constituent elements. High-purity solvents act as a carrier for mass spectrometry and other tandem techniques, especially liquid chromatography. They dissolve samples into a mobile phase to traverse the stationary phase, mass analyzer, and detector. With ionization at the core concept of mass spectrometry, specific solvent properties become detrimental attributes for compatibility with your mass spectrometer. Solvent Selection Criteria Several different solvent properties play into your selection criteria for your mass spec analysis. Even with advanced technology, selecting the wrong solvent can undermine your analytical results. In order to match their compatibility with proper ionization techniques, consider the key factors below for your selection. Purity Grade The efficiency of your mass spectrometer largely depends on the purity of a solvent. Contaminants often meddle with analyzers and detectors, ultimately creating misleading or compromising results. Given the precision mass spec offers, purity grade can matter even at the parts-per-billion level. Some solvents are manufactured in ISO9001 facilities in order to standardize purity expectations, guarantee quality, and create batch-to-batch consistency. At Birch Biotech, we offer a selection of LC-MS grade solvents best suited for liquid-chromatography mass spectrometry. Polarity and Solubility A solvent's polarity plays a direct role in the ability to dissolve a sample, directly influencing separation efficiency. Matching solvent polarity with the analyte creates the proper solubility needed for high-quality spectra. Different polarity characteristics change how samples may dissolve into a mobile phase or interact with other stationary phase properties. Most solvents are classified into two polarity categories: Protic solvents: methanol and water mixtures, often used due to their ability to donate protons, aiding in positive ionization methods. Aprotic solvents: acetonitrile is popular for its low viscosity and high volatility, making it ideal for nebulization and evaporation in techniques like ESI. The choice depends on whether your target compounds are more soluble in polar or nonpolar environments, which directly affects retention and resolution during analysis. Polar solvents like water can dissolve ionic and polar compounds, enhancing peak resolutions. Conversely, nonpolar solvents like hexane allow for the separation of nonpolar substances. Chemical Compatibility Chemical compatibility assures that a solvent does not react with an analyte or instrumental materials in adverse ways. Several chemicals, such as acetonitrile and dichloromethane, have hazardous properties that may create dangerous reactions if not premeditated. Solvents must be inert to minimize unwanted interactions that could alter the sample or damage equipment, such as clogging or corrosion. Be aware of all solvents' reactivity and interaction with charged analytes, as these can impact ionization efficiency. It is always best to be mindful of any known interactions with your analytes when planning your analyses to guarantee stability and safety. Volatility and Evaporation Rate Volatility affects a solvent’s ability to evaporate, directly impacting its evaporation rate and vapor pressure. A high evaporation rate minimizes residual solvent and impurities reaching the detector, reducing background noise in the measurements. This also enhances sensitivity and accuracy at the detector, making these solvents ideal for certain methods. For efficient LC-MS analysis, volatile solvents like methanol and acetonitrile are commonly preferred due to their quick evaporation rates. When selecting a solvent, consider how its volatility will impact the transition of analytes from a liquid phase to a gas phase effectively. Along with the evaporation rate, boiling point is another indicator of what volatility is best for your analysis. UV Transparency UV transparency impacts how mass spectrometers use solvent-based UV detection methods in conjunction with LC-MS by influencing how ultraviolet light (UV) absorbs, scatters, or passes through certain materials. Assess each solvent’s UV cutoff point and choose one that is transparent across the UV spectrum needed for your analysis. This minimizes interfering absorbance, increasing the accuracy of quantitation in spectroscopic detection. Solvents with higher UV transparency also provide a clearer baseline and improved peak distinction, particularly at low wavelengths. Methanol and acetonitrile have excellent transparency and are often chosen when UV detection is required. Viscosity Viscosity relates to the fluidity of a solvent and the ease of flow through a system, ultimately affecting the flow rate and mixing efficiency. This can be detrimental to other components, especially in chromatography and tests that require passing a mobile phase through a column and stationary phase. Different sample compositions also may require an increase or decrease in viscosity to prepare for spectrometry analysis. pH With ionization being a fundamental function of mass spectrometry, the pH of your solvent is an important consideration. Acidic solutions measure higher concentrations of free hydrogen ions, while basic solutions will have less of their abundance. This directly influences ionization techniques, signal intensity, and solvent compatibility with a particular analyte. Buffers and acidic additives are often used to balance the pH of mixtures and enhance ionization by protonating a sample, thus improving detection in positive mode. Selecting a Solvent for Mass Spec As highlighted above, there are quite a few considerations when it comes to choosing a solvent for mass spectrometry, chromatography, or any analytical test that requires precision and the interaction of many chemical properties. Among these factors, certain solvents have become popular choices due to their ideal nature for these tests based on their particular attributes: Acetonitrile: Highly volatile and compatible with many analytes. Methanol: Offers low viscosity and is versatile. Tetrahydrofuran (THF): Suitable for certain applications, though less common due to chemical instability and compatibility issues. Dichloromethane: Useful in specific scenarios due to its solubility properties, but often limited due to chemical compatibility issues. Water: Can be mixed with other solvents to create optimal conditions for different analytes, including pH and viscosity. Select the correct purity grade to avoid impurities that could introduce contaminants or inaccuracies. Various solvent blends are also available to meet different application needs. They can be purchased pre-made or mixed in a lab. The choice between these blends can influence ionization efficiency, requiring careful optimization and precision. Always consider solvent specifications before purchasing yours, which follow global standards and should be listed on each bottle or in product details. To find the best solvent for your mass spectrometry or chromatography needs, explore our selection at Birch Biotech. Want to learn more about our solvents? Read more of our articles or visit us at Birch Biotech. -- Disclaimer: The content provided on the Birch Biotech blog is for educational and entertainment purposes only. The information offered here is designed to provide helpful insights and advice related to laboratory practices and supplies. Readers are advised to refer to our product-specific quality data sheets and Certificates of Analysis (COAs) available on our website for detailed information on product specifications. It is essential to handle and store all materials according to the safety guidelines and regulatory requirements applicable to your area. While we endeavor to ensure the accuracy and relevance of the information published, it should not be used as a substitute for professional advice or official protocols. We encourage all our readers to consult their institution's guidelines, local regulations, and professional standards before implementing any practices discussed here. Birch Biotech does not accept liability for any actions undertaken based on the information provided in this blog nor for the misuse of our products. Furthermore, Birch Biotech does not guarantee the completeness, reliability, or timeliness of the information contained on this website. This disclaimer is subject to change at any time without notifications.
Mastering Solvent Evaporation in LC-MS: Sample Preparation Techniques and Tips
Learn about solvent evaporation and sample preparation techniques ideal for LC-MS.
Understanding High-Purity Solvent Specs: Essential Insights for Researchers
Discover how high-purity chemical specifications are verified and learn how to use them to guide your research.
Essential Sample Preparation Techniques for LC-MS Analysis
Discover LC-MS sample preparation techniques for reliable, accurate results.
Best Solvents for Peptide Analysis in LC-MS: Preparing Solutions and Overcoming Challenges
Discover how to use solvents for peptide analyses with LC-MS.
What Makes Acetonitrile Ideal for HPLC
Explore the properties, applications, and safe handling of acetonitrile.
How to Create a Balanced Solvent Composition for Effective Solutions
Learn how to create a balanced solvent composition for effective solutions.
How Solvent Attributes Affect Your Analysis: Key Insights Into Their Properties
Discover how solvent attributes impact your analysis and learn key strategies for success.
Ensuring Product Safety with Residual Solvent Testing
Explore best practices needed for testing impurities and residual solvents for various applications.
HPLC Solvent Gradient Optimization: Enhancing Analytical Precision
Learn effective strategies for optimizing HPLC solvent gradients to achieve consistent results in your analyses.
How Solvent Properties Interact: Understanding Solvent-Analyte Dynamics
Explore the interactions between solvents and analytes, understanding how solvent properties influence results.
UV Absorbance for Solvents in Chromatography
Every solvent used for Liquid Chromatography (LC) and Mass Spectrometry (MS) has a UV absorbance to consider. Choosing a low UV absorbance may minimize baseline noise, increase sensitivity, and improve a UV detector’s performance. However, other mechanics may change depending on your solvent’s properties or vice versa. Different wavelengths may be of interest for your testing purposes, while chemical compatibility with your system may be more or less necessary. Using solvents with low particulate contamination can also lead to better UV detection results. Like most HPLC or LC-MS analyses, there are many trade-offs to contemplate, and studying UV absorbance will help sharpen your knowledge of solvents, their interactions, and how to modify them to create better testing in the future. UV Absorbance and Detection Principles In the simplest terms, UV absorbance refers to the capability to reflect or absorb light. Every element or compound produces unique wavelengths that can then be studied. For testing methods like mass spectrometry and UV spectroscopy, measuring light spectrums, intensity, and radiation are some of the fundamental principles for their analysis. Depending on the chemical interactivities between a solvent and a sample along with other system components, UV absorbance becomes a compelling tool to understand what elements and their abundance may be in complex matrices. UV-Vis Spectroscopy and Liquid Chromatography UV spectroscopy is a method that measures the absorbance of UV by a substance, governed by the Beer-Lambert Law. This law describes the linear relationship between absorbance and concentration, using the formula: A = εlc, where ( A ) is absorbance (unitless), ( ε ) is the molar absorptivity with standard units of L/mol-cm, ( l ) is the path length with standard units of cm, and ( c ) is the concentration with standard units of mol/L. For liquid chromatography methods like LC-MS, HPLC, or HPLC-UV, UV detectors measure light absorbance at certain wavelengths, typically between 190 and 400 nm. This range covers both the visible and UV spectrum, allowing for a precise wavelength selection. UV detectors also use principles from the Beer-Lambert Law to quantify certain quantifications of a specific analyte. Role of Solvents in UV Detection Since solvents are typically used to carry analytes through a liquid chromatography system, their UV absorbance properties are just as important to consider. Otherwise, they can meddle with the results you wish to study, for certain solvents that demonstrate strong UV light absorption often disrupt analyte detection processes. Common solvents like methanol and acetonitrile are applied in HPLC due to their predictable cutoff values (methanol: ~205 nm, acetonitrile: ~190 nm) while maintaining compatibility with several stationary phases. However, solvent absorbance close to the detection wavelength can disrupt signals that are being analyzed. Also, contaminants create misleading feedback or unaccounted interference. These reasons further emphasize why high-purity solvents with low UV absorbance are typically the most ideal choices. UV Considerations for Solvents When selecting a solvent, consider how UV absorbance may affect UV detectors, which rely on solvent transparency at specific wavelengths. Additionally, take into account the several properties that may change with UV absorbance, such as polarity and chemical interactivity with the stationary phase, impacting chromatographic separation processes. Solvent UV Cut-Off and Transparency UV cut-off expresses the range within a solvent’s UV absorbance that may impact detectors, while transparency reflects ranges where UV light may pass through. Solvents with a low UV cut-off often increase transparency, which are reasons why they are ideal choices for minimizing light interference. Acetonitrile and methanol represent common solvent choices because they provide high transparency. Their UV cut-off points measure approximately 190 nm for acetonitrile and 205 nm for methanol. Many organic solvents exhibit a wide transparency range suitable for multiple chromatography techniques. However, you should always verify the lot-to-lot variability of solvents, as it can affect UV detection when working near the cut-off wavelength. Solvent Polarity Influences The separation effectiveness in chromatography depends on how solvent polarity influences analyte distribution between the stationary and mobile phases. The polarity of the solvent controls interactions that establish elution strength and selectivity factors column adsorption, which ultimately govern performance. The behavior of polar solvents like methanol and water interact differently compared to non-polar solvents, which impacts their retention and separation capabilities. Normal-phase chromatography often uses non-polar solvents like hexane to create strong interactions between polar analytes and a stationary phase. In reversed-phase chromatography systems, polar solvents aid in retaining non-polar analytes. Adjusting the ratios of solvents in the mobile phase alters its polarity, which impacts both elution strength and selectivity. Accurate retention and resolution, along with reliable analytical results, rely on proper process optimization. Solvatochromic and Stabilization Applying a solvent will directly influence the UV absorbance of analytes. Certain interactivities can create solvatochromic effects, which is a phenomenon known for altering the UV-visible spectrum as certain analytes dissolve into particular solvents. Sometimes this changes their color, but it especially impacts those with conjugated systems or specific functional groups. Additionally, both polar and non-polar choices can create stabilization effects and skew UV wavelengths in different directions. Polar solvents can stabilize excited states in carbonyl-containing compounds, leading to a shift in UV absorbance and longer wavelengths. On the other hand, non-polar solvents can stabilize ground states in aromatic compounds, shifting UV absorbance to shorter wavelengths. Balancing Solvents and UV Performance Balancing UV absorbance factors like UV cutoff and transparency with solvent properties like polarity, viscosity, volatility, and pH can vary from case to case and may require modifications or mixtures for a suitable composition. To match your solvent’s UV range with other samples and testing factors, consider the following for chromatography and spectroscopy techniques: Adjust polarity by creating precise mixtures of different solvents. This can help resolve closely eluting compounds, especially in complex samples. Balance pH with buffer salts to facilitate separation while maintaining a stable pH level for consistent retention times. Control viscosity while dissolving samples and preparing mobile phases. This will ultimately impact your flow rate, column efficiency, and pressure limits. Check for chemical stability between testing materials, including your sample composition, choice of column, and packing material. Stability improves repeatability and reliability from test to test. Avoid adverse reactions from volatile solvents and implement safety protocols for handling hazardous materials. During your selection, consider functional groups in your analyte. For example: in reverse-phase chromatography, small amounts of polar or mixed-polarity solvents like water can be combined with methanol or acetonitrile to modulate the retention and elution of non-polar molecules. The mobile phase composition balances polarity to optimize analyte partitioning between the non-polar stationary phase and the solvent system for efficient separation. This helps prevent issues like peak broadening or tailing when sample mixtures are incompatible with mobile phase solvents, leading to incomplete separation or retention time shifts and affecting results. Analyte-Solvent Compatibility Solvents that do not significantly overlap with an analyte's UV absorption are best for clear signal interpretation, and minimizing particulates and volatile additives can prevent issues with background absorbance and enhance detection sensitivity. Solvents with high UV transparency are ideal, as they allow accurate measurement of analytes with minimal interference. This is also known as molar absorptivity, which relates wavelength to the ability to absorb light. Low molar absorptivity corresponds to high transparency. UV Absorbance with Complex Mixtures For methods like high-performance liquid chromatography (HPLC), analyzing complex mixtures can become complicated and require precise methodologies for analyzing wavelengths. Choosing a specific UV range as a parameter can significantly reduce the potential variables of other compounds. With more knowledge about your analytes, you can focus on even more particular ranges that will produce the absorbance maxima (highest peaks) most relevant to your results. Baseline stability can also present new challenges when assessing complex mixtures, such as pharmaceutical or biological compounds. Methods like gradient elution can adjust separation parameters during a test to compensate for multiple attributes present in a matrix. Employing more advanced equipment and UV detectors with higher sensitivity can also significantly improve sensitivity and produce better peaks and adjust band broadening. Conclusion UV absorbance is one of the fundamental attributes for any analytical method that involves observing a light spectrum, especially for UV spectroscopy, mass spectrometry, and different types of chromatography. Since solvents carry samples through these different tests, its UV cutoff and transparency can either improve your peaks or create misleading results. Moreso, a solvent’s other properties outside UV absorbance may impact several other interactions within your system that should be accounted for or adjusted while testing. There is always more to learn about when it comes to HPLC and LC-MS solvents. To expand your knowledge on other solvent properties and testing techniques, explore our information available at Birch Biotech. Want to learn more about our solvents? Read more of our articles or visit us at Birch Biotech. -- Disclaimer: The content provided on the Birch Biotech blog is for educational and entertainment purposes only. The information offered here is designed to provide helpful insights and advice related to laboratory practices and supplies. Readers are advised to refer to our product-specific quality data sheets and Certificates of Analysis (COAs) available on our website for detailed information on product specifications. It is essential to handle and store all materials according to the safety guidelines and regulatory requirements applicable to your area. While we endeavor to ensure the accuracy and relevance of the information published, it should not be used as a substitute for professional advice or official protocols. We encourage all our readers to consult their institution's guidelines, local regulations, and professional standards before implementing any practices discussed here. Birch Biotech does not accept liability for any actions undertaken based on the information provided in this blog nor for the misuse of our products. Furthermore, Birch Biotech does not guarantee the completeness, reliability, or timeliness of the information contained on this website. This disclaimer is subject to change at any time without notifications. [1] How it works: UV detection for HPLC. (2020, November 13). Chromatography Online. https://www.chromatographyonline.com/view/how-it-works-uv-detection-hplc-0 [2] Taylor, T. (2020, November 12). Important aspects of UV detection for HPLC. Chromatography Online. https://www.chromatographyonline.com/view/important-aspects-uv-detection-hplc [3] SMACgig World. (2023, May 31). Nature of factors impacting UV-VIS spectroscopy. https://www.smacgigworld.com/blog/factors-affecting-uv-vis-spectroscopy.php [4] Wysocki, J., & Dong, M. W. (2022, April 15). Ultraviolet Detectors: Perspectives, principles, and practices. Chromatography Online. https://www.chromatographyonline.com/view/ultraviolet-detectors-perspectives-principles-and-practices
Optimizing Solvent Conditions for HPLC
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. Want to learn more about our solvents? Read more of our articles or visit us at Birch Biotech. Disclaimer: The content provided on the Birch Biotech blog is for educational and entertainment purposes only. The information offered here is designed to provide helpful insights and advice related to laboratory practices and supplies. Readers are advised to refer to our product-specific quality data sheets and Certificates of Analysis (COAs) available on our website for detailed information on product specifications. It is essential to handle and store all materials according to the safety guidelines and regulatory requirements applicable to your area. While we endeavor to ensure the accuracy and relevance of the information published, it should not be used as a substitute for professional advice or official protocols. We encourage all our readers to consult their institution's guidelines, local regulations, and professional standards before implementing any practices discussed here. Birch Biotech does not accept liability for any actions undertaken based on the information provided in this blog nor for the misuse of our products. Furthermore, Birch Biotech does not guarantee the completeness, reliability, or timeliness of the information contained on this website. This disclaimer is subject to change at any time without notifications. [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