When looking at data generated from HPLC analysis, it is natural to focus on retention time, peak asymmetry and efficiency. These parameters will allow the user to identify and quantify analytes: the more precise this information is the greater the confidence in peak identity and analyte concentration.Capacity factor is often an overlooked parameter of separations, but when utilized correctly can guide the user to produce more robust methodology. The capacity factor serves to normalize the retention time of an analyte to the dimensions of the analytical column, and is defined as:<h5 style="text-align:center;">K’ = tr - t0/t0</h5>Where tr is the retention time of the analyte, and t0 the elution time of an unretained compound.The importance of capacity factor can best be illustrated by an example: &nbsp;<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_How_can_capacity_factors_drive_method_robustness_2_f6553ef593.jpg" alt="Phenomenex: How can capacity factors drive method robustness? 1. Oxalic acid, 2. Tartaric acid, 3. Glycolic acid, 4. Formic acid, 5. Pyruvic acid, 6. Malonic acid, 7. Acetic acid, 8. Maleic acid, 9. Citric acid." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_How_can_capacity_factors_drive_method_robustness_2_f6553ef593.jpg 245w," sizes="100vw" width="547" height="345">Here we have a separation of organic acids, run on a C18 column which is stable to 100% aqueous conditions. The mobile phase is 20 mM potassium phosphate buffer at pH 2.9, with a flow rate of 0.7 ml/min. At first glance oxalic acid is retained at almost 4 minutes, but upon looking at this a little more closely the t0 for this column is approximately 3.7 minutes. When this is considered it is clear that oxalic acid, a very polar diacid, is barely retained by the column at all.<h5 style="text-align:center;">K’ = 3.9 - 3.7/3.7 = 0.054</h5>This indicates that oxalic acid spends almost no time associating with the stationary phase during its transit through the column. In such cases analyte retention will be greatly influenced by small, unintentional changes in parameters including:<ul><li>Mobile phase composition</li><li>Mobile phase pH</li><li>Sample diluent strength</li><li>Temperature</li></ul>Such a method is therefore unlikely to be robust, with oxalic acid retention varying from run to run, column to column and from one batch of mobile phase to another.This leads to the recommendation, whenever methods are being developed, that capacity factors should lie in the range of 2 - 10. We have seen the potential for problems with capacity factors of less than 2, but why stop at a capacity factor of 10?During an isocratic run, peak width increases with increasing retention time due to diffusion. Whilst the use of smaller particle columns and optimal flow rates will help to reduce this diffusion, it cannot be eliminated. Additionally, high capacity factors ultimately result in long run times, which are generally undesirable in a productive laboratory environment.&nbsp;<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_How_can_capacity_factors_drive_method_robustness_22b7c55c44.jpg" alt="Phenomenex: How can capacity factors drive method robustness? 1. Hydrocortisone, 2. Corticosterone, 3. 11-a-Hydroxyprogesterone, 4. Cortisone acetate, 5. 11-Ketoprogesterone." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_How_can_capacity_factors_drive_method_robustness_22b7c55c44.jpg 245w," sizes="100vw" width="685" height="339">Here we have an example of steroids run on a 150 x 4.6 mm column at 1 ml/min. 11-etoprogesterone elutes at around 17.5 minutes, with a capacity factor of approximately 10.6. Peak broadening is evident in this isocratic run, indicating that the latest eluting peak is being retained on column for an excessive period of time.Understanding the guideline (capacity factors of between 2 and 10) is helpful, but leaves us with the question, how can they be adjusted?The capacity factor is inversely related to the elutropic strength of the mobile-phase. A higher elutropic strength will lower the capacity factor, and vice versa. When low capacity factors cannot be increased by reducing the elutropic strength of the mobile phase then it may be necessary to switch to a stationary phase that can offer greater retention of the analytes, for example by using a more polar stationary phase for polar analytes.

How can capacity factors drive method robustness?

Silica used to pack HPLC columns is a polymer of silicon and oxygen with Si-OH groups (silanols) at the surface. To these silanols, functional groups are bonded using a silane reagent to functionalize the base media.When HPLC columns are functionalized, it is impossible to ensure 100% phase coverage and react all available silanol groups (free hydroxyls on the surface of the silica) with the bonded phase. Sterically, as you begin to bond a phase you increase the hindrance to bonding making subsequent bonding stages more difficult than the previous ones.Without end-capping, a column would be left with significant numbers of accessible, highly active residual silanols which have the potential to alter the selectivity of the stationary phase. These residual groups have the potential to form strong ionic and polar interactions (often called “secondary interactions”) with polar and basic groups on the compounds introduced to them. So, reducing the number of free silanols after bonding the stationary phase through end-capping is imperative.Historically, end-capping was done with a small hydrophobic group, similar in nature to a trimethyl silane. In recent years several manufacturers have also introduced other groups to add a <a target="_blank" rel="noopener noreferrer" href="https://app.explore.phenomenex.com/e/er?s=822460770&amp;lid=6998&amp;elqTrackId=D5A4A87A868E75FA6693FC5A1E9B3C89&amp;elq=00000000000000000000000000000000&amp;elqaid=5674&amp;elqat=2&amp;elqak=8AF5A07C3CDAC28222BF387672C6F595E7A5CF7C906A90E1BF5C55FDC1EA7D8B80B0&amp;_gl=1*edplpz*_ga*MTM5ODI2NzM3NS4xNzQyMjc3MDM0*_ga_790WGKEF4G*czE3NDk5ODYwMzUkbzI0JGcxJHQxNzQ5OTg3NjQwJGo2MCRsMCRoMA..">different selectivity element</a> to the column.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_Role_of_End_Capping_in_RP_a458911946.jpg" alt="Phenomenex: The Role of End-Capping in RP." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_Role_of_End_Capping_in_RP_a458911946.jpg 245w," sizes="100vw" width="767" height="214">If the reagent used for end-capping contains charged functionality, for example, this will impart ion exchange functionality to the surface and give the column ion exchange properties together with the <a target="_blank" rel="noopener noreferrer" href="https://app.explore.phenomenex.com/e/er?s=822460770&amp;lid=8525&amp;elqTrackId=63166B02CED6893A95911CAE6AA3AE95&amp;elq=00000000000000000000000000000000&amp;elqaid=5674&amp;elqat=2&amp;elqak=8AF5B477F9C56C31BC7CAC9FBFB946361A9ECF7C906A90E1BF5C55FDC1EA7D8B80B0&amp;_gl=1*edplpz*_ga*MTM5ODI2NzM3NS4xNzQyMjc3MDM0*_ga_790WGKEF4G*czE3NDk5ODYwMzUkbzI0JGcxJHQxNzQ5OTg3NjQwJGo2MCRsMCRoMA..">reversed phase</a> interactions from the C18 ligands. Likewise, a polar moiety will give polar characteristics to the surface.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_Role_of_End_Capping_in_RP_4_e1b1855040.jpg" alt="Phenomenex: The Role of End-Capping in RP." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_Role_of_End_Capping_in_RP_4_e1b1855040.jpg 245w," sizes="100vw" width="786" height="246">Now that we have understood the role of end-capping, let’s review the main benefits of silica end-capping depending on the chemical groups used.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_Role_of_End_Capping_in_RP_3_be82757ab7.jpg" alt="Phenomenex: The Role of End-Capping in RP." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_Role_of_End_Capping_in_RP_3_be82757ab7.jpg 137w," sizes="100vw" width="640" height="728">In summary, new end-capping technologies can significantly improve the analysis of compounds that were once considered challenging. For example, nowadays, great peak shape and shorter run times can be achieved for the analysis of basic drugs using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=341&amp;manufacturerItems=50&amp;search=ps">PS-C18 columns</a>. The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=341&amp;manufacturerItems=50&amp;search=polar">Polar C18</a> chemistry has been proven to best retain compounds such as metabolites, and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=341&amp;manufacturerItems=50&amp;search=XB">XB-C18</a> chemistry has shown excellent chromatography for peptide analysis, with enhanced retention of small polar peptides.

The Role of End-Capping in RP

One of the most common causes of short column lifetime is column contamination, often resulting in high back pressure and a so called “plugged” inlet frit.This technical tip aims to explain what is happening, and also how column contamination can be avoided.&nbsp;In the manufacture of particulate based HPLC and UHPLC columns, frits are used to prevent extraneous material entering the column, and to prevent the HPLC packing material from leaving the column. These frits are discs of porous metal, generally 0.5, 1.0 or 2.0 μm in porosity. If samples contain undissolved material, it will be filtered out of the mobile phase solvent stream, sitting on the outside of the inlet frit. As this process continues, the amount of the frit available for mobile phase to pass through becomes smaller, restricting the flow of mobile phase which in turn increases the back pressure. Eventually it becomes impossible to push solvent through the frit and the HPLC pump shuts down.This process can be averted by the filtration of samples prior to injection onto the HPLC system. It is generally advised to use 0.45 μm porosity syringe filters for samples that will be run on 5 μm columns, 0.2 μm filters for 3 μm columns and smaller. Care should be taken as ideally the sample diluent should match the mobile phase. If it does not, you may find that some sample components are soluble in the sample diluent allowing them to pass through the syringe filter. When they are injected into the mobile phase they may precipitate causing frit blockage. This can be avoided by either dissolving the sample in mobile phase, or alternatively installing an inlet frit, or a guard cartridge as a sacrificial device to prevent the inlet of the column becoming fouled.Alternatively, we have this <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/tools/security-guard-selection#bybrand">web tool</a> which will guide you through guard cartridge selection.&nbsp;

Column contamination – what is happening, and how it can be avoided

<h3>The Importance of the HPLC Method</h3>Enhancing performance for all column separations can be achieved through the following method-related optimization tips. Specifically, these suggestions can significantly boost your method's performance when using core-shell columns.When optimizing HPLC methods, our main efforts should focus on ensuring the minimization of sample dispersion in order to mitigate peak broadening and improve the sensitivity and resolution of your peaks. This entails properly concentrating the sample as a tight band at the head of the column when injecting and then working to minimize sample dispersion through the column.Adjusting injection volumes and selecting a proper sample diluent can greatly help reducing injection-related sample dispersion. Additionally, integrating a simple injector programming into the method can reduce dispersion due to undesired extra-column volume. These suggestions will greatly improve peak shape and efficiency, especially for rapid core-shell LC separations.<h3>Problem: All Peaks are Broad</h3><h4>1. Check Sample Volume</h4>Errors in sample loading may negate the performance gain the core-shell particle can achieve. If you inject the sample in the same organic composition as the mobile phase under isocratic conditions, the initial bandwidth on column will be directly proportional to the injection volume. As such, a large volume sample is injected as a wide band on the column and gives low peak efficiency.Solution: To reduce the efficiency loss of large volume injections, it is possible to pre-concentrate the sample on the head of the column by using a weaker diluent than the mobile phase. You can also reduce your sample injection volume. When high efficiency columns are used often, lower sample amounts are needed to get good signal response.<h4>2. Check Sample Diluent</h4>If the organic strength of the diluent is greater than the HPLC mobile phase, the sample will load as a diffuse band at the head of the HPLC column. This may occur in either isocratic or gradient methods and can negate much of the performance advantages that the column delivers.<ul><li>Gradient methods where the sample is injected in a stronger injection solvent than the mobile phase, the compound exhibits surfing effects which will cause peaks to broaden and split.</li><li>Isocratic methods where the diluent is stronger than the mobile phase will result in excessive sample dispersion.</li></ul>Solution: Inject a weak solvent. It is best to use a diluent either equal to or weaker than the organic strength of the mobile phase. This focuses the sample on the head of the column, resulting in sharper peaks (i.e., higher efficiency).<h3>Goal: Further shorten the runtime and peak capacities in gradient methods</h3><h4>Check Injector Program</h4>Extra-column volume before the column will cause dispersion of the sample on the way to the column; the injector loop is a major source of extra-column volume.Solution: It is possible to bypass the loop with an injector program during the analysis method (after the sample has left the injector loop). Table 1 and Figure 1 show an example where the injection loop is bypassed after injection to reduce system dwell volume.<h4>Steps | Commands | Comments</h4>1 | DRAW | Draw volume of sample (injection volume) from vial. 2 | INJECT | Introduce sample into flow path. 3 | WAIT | Flush sample loop after injection (wait time = 6× (injection volume + 5 µL) / flow rate). 4 | VALVE bypass | Direct flow from pump to column, bypassing injection valve to exclude delay volume ~200 – 500 µL from auto-injector path 5 | WAIT 1.5 min | The period of VALVE bypass time (Wait time = Run time – 1 min). 6 | VALVE reinject | Switch valve from bypass to injection position.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_LC_System_Optimisation_for_UHPLC_Performance_on_any_HPLC_0bd7e07b05.jpg" alt="Phenomenex: LC System Optimisation for UHPLC Performance on any HPLC" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_LC_System_Optimisation_for_UHPLC_Performance_on_any_HPLC_0bd7e07b05.jpg 245w," sizes="100vw" width="464" height="251">* Mainpass peak is an injector artifact due to switching the injector valve back to include the injector loop in the flow (to flush injector in preparation for the next injection).

LC System Optimisation for UHPLC Performance on any HPLC

The clinical utility of oligonucleotides through controlling gene expression makes them of increasing interest as therapeutics. Oligonucleotides have very different chemical properties when compared to typical small molecule drugs, making them an interesting target, while also complicating their analysis when compared to many small molecule therapeutics. Oligonucleotides are inherently polar compounds, and require specific conditions to allow analysts to fully characterize them and their impurities. In this two-part series we will look at the challenges analytical scientists face in the analysis of oligonucleotides and some of the method development levers they could pull when working on their analysis.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/oligonucleotide_2_3d8377cbf6.jpg" alt="Phenomenex: Oligonucleotides" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_oligonucleotide_2_3d8377cbf6.jpg 125w," sizes="100vw" width="505" height="628">In <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6254">Part 1</a> we began by looking at some method development strategies for the analysis of oligonucleotides. In this second part we will explore the effect HPLC column hardware has on the analysis, specifically looking at resolution, sensitivity and adduct formation.<h4>Improving Resolution</h4>It is well documented that large biomolecules such as proteins may adhere to stainless steel, such that there is a requirement for column priming when stainless steel hardwere is used when separating proteins. To reduce the degree of column priming needed, non-adsorbing, inert column hardware, such as the BioTi™ seen in our <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=48&amp;manufacturerItems=50&amp;search=biozen">Biozen™ line</a>, can be used. When analyzing oligonucleotides, a similar effect can be seen due to their phosphorylated backbones, which makes them susceptible to adsorption onto stainless steel hardware. When BioTi hardware is used, significant improvements in chromatography are seen for oligonucleotide analysis.In Figure 1 we compared the results for the analysis of a double stranded siRNA using two different columns that have been packed with media from the same batch of core-shell, organosilica (hybrid) packing material,. One was packed into stainless steel hardware and the other into BioTi. The first clear observation is poor peak shape, including a later eluting shoulder for the anti-sense strand. There is also a clear loss of resolution between the two strands to stainless steel hardware.&nbsp;<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_1_3524e03533.jpg" alt="Phenomenex: Figure 1" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_1_3524e03533.jpg 245w," sizes="100vw" width="1422" height="600">This is further supported by the spectra seen in Figure 2 which shows the bimodality of the antisense strand. Conversely, with BioTi hardware we are able to achieve good resolution and sharp peaks. The spectra shown in Figure 3 further demonstrates that BioTi hardware provided a clear separation between the sense and antisense strands.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_2_bd0aa7e66d.jpg" alt="Phenomenex: Figure 2" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_2_bd0aa7e66d.jpg 245w," sizes="100vw" width="1424" height="450"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_3_bdc9ba4043.jpg" alt="Phenomenex: Figure 3" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_3_bdc9ba4043.jpg 245w," sizes="100vw" width="1419" height="437"><h4>Increasing Sensitivity</h4>When considering hardware, sample losses due to adsorption can hinder analytical sensitivity. This is particularly important if you are working with low levels of samples, such as for a biodistribution study. In Figure 4 we compare the differences in response level (signal) between the two columns. In this case, new columns were loaded with 12.5 mg of two different oligonucleotides, BNA and 5amC12, and run under the same analytical conditions. The BioTi column generated a formidable signal for both oligonucleotide types. However, with stainless steel hardware, we observe complete adsorption.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_4_9a31caf063.jpg" alt="Phenomenex: Figure 4" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_4_9a31caf063.jpg 245w," sizes="100vw" width="1419" height="495"><h4>Reducing Adduct Formation</h4>Another known problem in LC-MS analysis of oligonucleotides is adduct formation. It is generally recommended to carry out system passivation by flushing the system and column with 0.1% formic acid. This is due to the fact that analytical LC-MS approaches for oligonucleotides use mobile phases containing additives that generate basic conditions, and residual metals from the mobile-phases, stationary phase and hardware can accumulate over time. The accumulation of metals can reduce sensitivity, and ultimately the quality of the spectral data. In Figure 5 we compare the spectra of nusinersen, analyzed on both a column featuring stainless steel hardware and a separate column featuring BioTi hardware. When we look at the deconvoluted spectra, we see a 62 daltons adduct that is only observed in stainless steel.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_5_def677c204.jpg" alt="Phenomenex: Figure 5" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_5_def677c204.jpg 245w," sizes="100vw" width="1424" height="726">In summary, when analyzing oligonucleotides, bio-inert hardware such as BioTi can significantly improve separation, sensitivity, and spectral data quality.

The Use of Bio-Inert Hardware in the Analysis of Oligonucleotides

Join host David Oliva from Organomation for an in-depth conversation with Sean Orlowicz, Principal Marketing Development Manager at Phenomenex, about the analytical challenges and innovative solutions for GLP-1 peptide therapeutics like semaglutide and tirzepatide.In this episode, presented in collaboration with Separation Science—a premier learning platform for analytical scientists—Sean shares over 22 years of chromatography expertise covering critical topics including:<h5>Key Discussion Topics:</h5><ul><li>Unique analytical challenges in separating GLP-1 analogues and related impurities</li><li>Column selectivity and efficiency optimization for complex peptide separations</li><li>The role of Aeris Peptide XB-C18 and core-shell particle technology</li><li>Method robustness, transferability, and regulatory considerations for sterile injectables</li><li>Size exclusion chromatography for aggregate analysis</li><li>High-resolution mass spectrometry applications in peptide analysis</li><li>Preparative chromatography for API purification at commercial scale</li><li>Common pitfalls and troubleshooting tips for peptide method development</li><li>The genericization of GLP-1 therapeutics and global market trends</li></ul><h5>About the Guest:</h5>Sean Orlowicz brings extensive experience from both laboratory and global pharmaceutical market perspectives at Phenomenex, specializing in peptide analysis workflows from API manufacturing to analytical testing.This episode is brought to you in collaboration with Separation Science, the leading online educational platform providing chromatographers with expert tutorials, webinars, application notes, and learning modules across LC, GC, MS, and separation technologies.Special thanks to Phenomenex for their continued innovation in developing chromatography solutions that advance pharmaceutical analysis, including specialized columns and methods for GLP-1 peptide therapeutics.Resources Mentioned:<ul><li>Phenomenex GLP-1 Applications Notebook</li><li>Biozen dSEC-1 SEC columns for aggregate analysis</li><li>Technical notes on semaglutide and tirzepatide analysis</li><li>Visit www.phenomenex.com for application support</li><li>Explore www.sepscience.com for chromatography education and training</li></ul><h3>Video Transcription</h3>Sean Oritz (Phenomenex) has worked in chromatography for 22+ years and is now Principal Marketing Development Manager for the pharmaceutical sector, helping shape global strategy for pharma customers—currently with a major focus on GLP-1 therapeutics.He explains that GLP-1 analytical methods are challenging because impurities and degradants are extremely similar to the main peptide (single amino-acid deletions, oxidations, isomers), so achieving reliable separation and quantitation is difficult. For reverse-phase peptide workflows, he highlights two key drivers of performance: selectivity (secondary interaction mechanisms beyond pure hydrophobicity) and efficiency (highly efficient particles improve resolution and integration of low-level impurities). He notes the Aeris Peptide XB-C8 is widely used because it combines peptide-friendly selectivity with high efficiency (core-shell platform).Regulatory scrutiny is high (sterile injectables, global distribution), so robustness and method transferability matter: methods must work across instruments and labs worldwide, and long shallow gradients can amplify system differences (dwell/gradient delay volumes, hardware configurations). He recommends investing effort upfront (temperature control, understanding instrument effects, testing multiple column batches, strong vendor support) and being patient—methods are often long (45–100 min).He also points out the growing role of mass spectrometry (including triple quad and HRMS) for specificity—sometimes compensating for chromatographic limitations—and the increasing need for SEC in GLP-1 drug products to monitor aggregation (monomer/dimer/trimer). Finally, he emphasizes not “copy-pasting” generic conditions (e.g., TFA/standard columns) for peptides, and he notes Phenomenex is supporting the field through education, real-sample application work, and new product development (including SEC columns tailored to GLP-1s).<blockquote>This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.</blockquote><blockquote><h4>Concentrating on Chromatography Podcast</h4>Dive into the frontiers of chromatography, mass spectrometry, and sample preparation with host David Oliva. Each episode features candid conversations with leading researchers, industry innovators, and passionate scientists who are shaping the future of analytical chemistry. From decoding PFAS detection challenges to exploring the latest in AI-assisted liquid chromatography, this show uncovers practical workflows, sustainability breakthroughs, and the real-world impact of separation science. Whether you’re a chromatographer, lab professional, or researcher you'll discover inspiring content!You can find Concentrating on Chromatography Podcast in podcast apps:<ul><li><a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL">Spotify</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podcasts.apple.com/tz/podcast/concentrating-on-chromatography/id1788429185">Apple podcast</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podscan.fm/podcasts/concentrating-on-chromatography">podscan</a></li></ul>and on <a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@ChromatographyTalk/videos">YouTube channel</a></blockquote>

GLP-1 Peptide Analysis: Chromatography Challenges & Solutions with Phenomenex's Sean Orlowicz

Solvent switching is the process of changing the solvent contained within a liquid chromatography (LC) column. This procedure must be performed carefully to avoid irreversible damage to the column.The topic originates from a common customer question:<blockquote>“My column arrived in hexane, but I need to use it with water and acetonitrile. How do I switch the solvents in the column?”</blockquote>This article explains the solvent switching process, how to determine the solvent currently inside a column, how to select an appropriate transition solvent, and why proper solvent switching is essential.<h2>Step 1: Determine the Shipping Solvent</h2>Before changing solvents, it is important to know which solvent or mobile phase is already present in the column.This information can typically be found in:<ul><li>The column care guide</li><li>The Certificate of Quality Assurance (CQA)</li></ul><h4>Using the Column Care Guide</h4>The column care guide is usually available on the manufacturer's website under the specific column brand.<ul><li>In the Shipping Solvent section, users can identify the solvent in which each phase is supplied.</li><li>The Mobile Phase Compatibility section can be used to determine whether the phase has any mobile-phase limitations beyond pH restrictions.</li></ul><h4>Using the Certificate of Quality Assurance (CQA)</h4>The CQA also provides useful information.Under the Test Conditions section, the mobile phases used to test the column are listed.<ul><li>In the example shown in the video, a Luna column was tested using:<ul><li>75% acetonitrile</li><li>25% water</li></ul></li></ul>The testing conditions are often the same as the shipping solvent conditions, although this is not always the case.If uncertainty remains, the recommendation is to contact the column manufacturer directly.<h3>Step 2: Check Solvent Miscibility</h3>Once the shipping solvent is known, the next step is to verify whether it is miscible with the mobile phases used in the analytical method.A solvent miscibility chart can be used for this purpose.<h4>Water and Immiscible Solvents</h4>The video highlights that water is immiscible with several commonly used LC solvents, including:<ul><li>Hexane</li><li>Dichloromethane (DCM)</li><li>Heptane</li></ul>These solvents are commonly used under normal-phase conditions.<h4>Isopropanol as a Transition Solvent</h4>The video identifies isopropanol (IPA) as a solvent that is miscible with all LC solvents shown in the chart.Because of this property, IPA is commonly used as an intermediate or transition solvent when switching between:<ul><li>Water and hexane</li><li>Water and heptane</li></ul><h3>Step 3: Perform the Solvent Switching Procedure</h3>After selecting an appropriate transition solvent, the solvent exchange can begin.The procedure described in the video consists of the following steps:<h4>1. Flush with the Transition Solvent</h4>Flush the column with the selected transition solvent for:<ul><li>30–60 column volumes</li></ul><h4>2. Flush with the Pure Starting Solvent</h4>After the transition solvent, flush the column with:<ul><li>10 column volumes of the pure starting solvent</li></ul><h4>3. Equilibrate with the Method Mobile Phase</h4>Finally, equilibrate the column using the mobile phases required for the analytical method.<h3>Example Solvent Switching Protocol</h3>The video provides the following example.<h5>Initial Column Solvent</h5><ul><li>The column arrives packed in:<ul><li>99% hexane</li><li>1% acetonitrile</li></ul></li></ul><h5>Transition Solvent</h5><ul><li>Use:<ul><li>Isopropanol (IPA)</li></ul></li><li>Flush the column with IPA for:<ul><li>30–60 column volumes</li></ul></li></ul><h5>Intermediate Step</h5><ul><li>Flush with:<ul><li>10 column volumes of acetonitrile</li></ul></li></ul><h5>Final Equilibration</h5><ul><li>Equilibrate with one of the following:<ul><li>50% acetonitrile / 50% water</li><li>50% acetonitrile / 50% buffered water</li></ul></li></ul><h3>Why Proper Solvent Switching Is Important</h3>The video emphasizes that improper solvent switching can cause irreversible column damage.Potential issues include:<h4>Formation of Voids and Pockets</h4>Immiscible solvent transitions can create:<ul><li>Pockets within the packed bed</li><li>Voids at the column inlet</li></ul>These defects can permanently damage column performance.<h4>Salt Precipitation</h4>If buffered mobile phases are involved and pure solvents are not used appropriately during the transition, salts may precipitate from solution.<h4>Damage to Pressure-Sensitive Polymer Media</h4>Pressure-sensitive polymer-based media may burst during improper solvent transitions.<h4>Stripping of Coated Phases</h4>Columns containing coated stationary phases or coated ligands may experience removal of these coatings during unsuitable solvent changes.<h3>Irreversibility of Column Damage</h3>According to the video, once damage caused by immiscible solvent exposure occurs, it is extremely difficult—if not impossible—to reverse.For this reason, solvent changes should always be performed through an appropriate intermediate solvent whenever required.<h3>Conclusion</h3>Successful solvent switching begins with identifying the solvent in which the column was shipped. The miscibility between the shipping solvent and the intended mobile phases must then be evaluated. When immiscible solvents are involved, an appropriate transition solvent such as isopropanol should be used.Following a controlled sequence of flushing, intermediate solvent exchange, and equilibration helps prevent permanent column damage and ensures safe operation of the chromatographic column.

Solvent Switching in Liquid Chromatography Columns: How to Change Column Solvents Safely

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