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

If you are working with an established method, we would recommend for you to:<ul><li>Check all solutions used for the SPE method to ensure that nothing is expired or has been mislabelled.</li><li>Check the process. Was flow potentially too fast during loading or elution? Could the cartridges have been allowed to dry between equilibration and sample loading?</li></ul>It is generally quickest to simply repeat the extraction, taking care of these points.If, however, you are developing a new method, it might be necessary to “track down” your analyte. The first thing we would recommend is to make up a solution of the analyte without sample matrix (to use as an ideal sample). Then work through the sample method capturing the eluent from each step potentially containing the analyte:<ol><li>Condition the cartridge.</li><li>Equilibrate the cartridge.</li><li>Load the sample and collect the flow-through in a vial.</li><li>Analyze this for analyte content. If analyte is found in this fraction it would suggest:<ul><li>Sample solvent is too strong, or the pH is incorrect.</li><li>Loading flow rate is too high.</li><li>You have breakthrough because either sample load is too high, or sorbent mass is too low.</li></ul></li><li>Wash the cartridge, collecting each wash fraction in a separate vial.</li><li>Again, analyze for analyte content. If analyte is found in any wash fraction, this suggests:<ul><li>Wash solvent is too strong.</li><li>Wash solvent is not at the correct pH.</li></ul></li><li>If the analyte has not been found in the load or wash fractions, then the analyte must be retained on the cartridge.<ul><li>Try using a stronger elution solvent.</li><li>Ensure pH of the elution solvent is correct.</li><li>If the first 2 suggestions don’t help, try using a less retentive sorbent (in reversed phase, try switching a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50&amp;search=Strata">C18 cartridge for a C8</a> if the analytes are very hydrophobic).</li></ul></li></ol>

I am getting low recovery in my SPE method, how do I fix the problem?

It is easy to think that once a sample elutes from Solid Phase Extraction (SPE) it is then ready to be injected onto the analytical column. While the sample is now significantly cleaner, it is not always the case that injecting it directly onto the column will result in perfect chromatography. Solvent mismatch is a known cause of poor peak shape, so care must be given to consider the solvent strength used in the elution step of any SPE being performed on samples.The solvent with which the sample is eluted, the overall concentration of the sample, and the mobile phase conditions of the method should be evaluated when building sample preparation protocols. An injection of 100% organic solvent when your starting Mobile Phase conditions begin with high water (90%), can cause solvent mis-match to occur within the system, as seen in Image 1 below. Ideally, a sample will be in solvent that closely matches the starting mobile phase ratio of the LC method to prevent issues in peak shape from occurring.&nbsp;Remember that not all elution solvent ratios are troublesome to chromatography. A good example of a favourable elution solvent is when <a target="_blank" rel="noopener noreferrer" href="https://app.explore.phenomenex.com/e/er?s=822460770&amp;lid=8512&amp;elqTrackId=AF71A2845D16ED3FEE8AD0E50AD40B88&amp;elq=00000000000000000000000000000000&amp;elqaid=5382&amp;elqat=2&amp;elqak=8AF5AB5B4A0284363E33E235633CBBF8B994CF7C906A90E1BF5C55FDC1EA7D8B80B0">Phree™ Protein Precipitation</a> product is used to prepare samples for analysis in HILIC (hydrophilic interaction liquid chromatography) mode. The Phree protocol calls for samples to be diluted in a 3 : 1 (Acetonitrile : Aqueous) based sample ratio for the protein precipitation step to be successful.<a target="_blank" rel="noopener noreferrer" href="https://app.explore.phenomenex.com/e/er?s=822460770&amp;lid=8513&amp;elqTrackId=58B1E1E14DBED21807DB7913ABA26DA8&amp;elq=00000000000000000000000000000000&amp;elqaid=5382&amp;elqat=2&amp;elqak=8AF503F0D16829D3E24EB118C4BBECBABEFBCF7C906A90E1BF5C55FDC1EA7D8B80B0"> HILIC mode</a> starts at high concentrations of Acetonitrile, but still requires water to be present in the sample for optimal peak shape. The sample eluting from Phree is already in the ideal solvent ratio for HILIC mode and no further processing is needed, but if your analysis is looking to use<a target="_blank" rel="noopener noreferrer" href="https://app.explore.phenomenex.com/e/er?s=822460770&amp;lid=8514&amp;elqTrackId=BC52B3D9F6E8C58C9E6787A59D44EB41&amp;elq=00000000000000000000000000000000&amp;elqaid=5382&amp;elqat=2&amp;elqak=8AF57A92CCD54FE8CC7F340714830182B6BACF7C906A90E1BF5C55FDC1EA7D8B80B0"> Ion Exchange with Rezex™</a>, the presence of Acetonitrile in the sample could cause issues.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_danger_of_mobile_phase_mismatch_2_46cfdb13b7.jpg" alt="Phenomenex: The danger of mobile phase mismatch: Sample in 50% Methanol." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_danger_of_mobile_phase_mismatch_2_46cfdb13b7.jpg 170w," sizes="100vw" width="854" height="787"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_danger_of_mobile_phase_mismatch_3_14e68491b5.jpg" alt="Phenomenex: The danger of mobile phase mismatch: Sample in 100% Methanol." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_danger_of_mobile_phase_mismatch_3_14e68491b5.jpg 166w," sizes="100vw" width="837" height="789">

The danger of mobile phase mismatch

<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

When sensitivity loss occurs, it can significantly impact the accuracy and reliability of analytical results. Several factors can contribute to sensitivity loss, such as column contamination, sample matrix effects, injector and detector issues, or improper sample preparation. This technical note aims to guide analysts in identifying the common causes behind sensitivity loss and implementing effective strategies to address the issue.<h3>Symptom: Decreased Response</h3>Some or all peaks are displaying decreased response.<h4>Possible Cause &nbsp;/ &nbsp;Suggested Remedy</h4><ul><li>Contamination of column and/ or liner can lead to loss of sensitivity for active compounds.:&nbsp;<ul><li>Clean liner.</li><li>Bake out the column. Solvent rinse or replace the column.</li></ul></li><li>Injector leaks reduce the peak height of the most volatile components of a sample.:&nbsp;<ul><li>Find and fix any leaks</li></ul></li><li>If initial column temperature is too high for splitless injection, this prevents refocusing of sample. This affects the more volatile components most.:&nbsp;<ul><li>Lower temperature below the boiling point of the solvent. Decrease the initial column temperature, or use a less volatile solvent</li></ul></li><li>Inlet discrimination: injector temperature is too low. Later eluting and less volatile compounds have low response.:&nbsp;<ul><li>Increase the injection temperature or use on-column injection with direct connect liner</li></ul></li><li>Issues with the sample:&nbsp;<ul><li>Check the sample concentration, any sample preparation procedures, and shelf life. Prepare a fresh sample ensuring the proper concentration</li></ul></li></ul>In conclusion, sensitivity loss in GC is characterized by decreased response in one or more peaks, which can hinder accurate and reliable analysis. It is crucial to maintain a clean column and liner, address any injector leaks, optimize column conditions, and ensure proper sample preparation. Remember to regularly perform maintenance tasks and quality checks to ensure the ongoing performance of the GC system.By implementing these countermeasures, you can overcome sensitivity loss and achieve more accurate and reliable analytical results.

Peak Shape Problems: Sensitivity Loss

Peptides are often considered closer to small molecules when it comes to the development of chromatographic methods for their analysis. However, they bridge the gap between small molecules, (traditional pharmaceuticals) and larger biomolecules (proteins, AAVs, Oligos). When analyzing peptides, we cannot simply consider the properties of their comprising amino acids, as their characteristics depend not only on these building blocks but also the bonding sequence into a peptide chain and also the way in which the peptide chains are coiled or folded in space. For this reason, peptides can behave differently than small molecules when you analyze them through HPLC.Initial method development for peptides should begin with a chemical screening process, and during this, a variety of stationary phases are screened, beginning with a typical alkyl phase. If the profile you achieve does not give the desired selectivity you should move to an alternative phase, ideally with very different properties.The mobile phase gradient is also an important variable that should be considered for peptide analysis. Typically you see much shallower gradients than you would with small molecule work. For gradient optimization in peptide analysis, we would advise starting with a low % B (organic) to focus the peptides onto the front of the column and run a shallow slope up to 50-60% organic in which all peptides should elute (increase of 1% B per minute is typical). ThepH of the sample diluent and mobile phase should also be considered as it affects the physiochemical properties of the peptide, and as a result, their retention time. Finally, the nature of your eluting solvent should be considered, which typically for peptides is acetonitrile.Once you have conducted your initial screen, method optimization can be done by changing other method parameters (Tab. 1). The most dramatic of these in terms of the impact on selectivity is pH (Fig. 1).<h5>Tab. 1 Variable | Solution</h5><ul><li>Flow-rate: Less impactful for peptides than small molecules</li><li>pH: Small change can have large impact</li><li>Gradient Slope: Gradient changes affect selectivity considerably</li><li>Temperature: 40–90ºC</li><li>Buffer Type: Many solutions including TFA, formic acid, HFBA, phosphate buffers</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_A_Systematic_Approach_to_Peptide_Analysis_3_fbd3281d82.jpg" alt="Phenomenex: A Systematic Approach to Peptide Analysis: Figure 1." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_A_Systematic_Approach_to_Peptide_Analysis_3_fbd3281d82.jpg 219w," sizes="100vw" width="784" height="558"><ul><li>Column:&nbsp;<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1512">Kinetex EVO C18 5µm</a></li><li>Dimensions: 250 x 4.6mm</li><li>Order No.: 00D-4790-AN</li><li>Mobile Phase: A: 50mM Ammonium acetate in Water; Acetonitrile (v/v 97:3), pH as indicated&nbsp;</li><li>Mobile Phase: B: 50mM Ammonium acetate in Water: Acetonitrile (v/v 60:40), pH as indicated&nbsp;</li><li>Gradient: 14-34% B, 40 minutes, ramp to 80%</li><li>Flow Rate: 1.5 mL/min</li><li>Temperature:&nbsp;65 ˚C</li><li>Detection:&nbsp;UV-Vis @ 220nm</li><li>Sample:&nbsp;12 AA synthetic peptide with synthetic related impurities</li></ul>Stationary phases play an important part in the development process and it is recommended you explore different column chemistries (Fig. 2).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_A_Systematic_Approach_to_Peptide_Analysis_2_3736b8feab.jpg" alt="Phenomenex: A Systematic Approach to Peptide Analysis: Figure 2." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_A_Systematic_Approach_to_Peptide_Analysis_2_3736b8feab.jpg 245w," sizes="100vw" width="767" height="334"><h4>Conclusion</h4>Developing a systematic approach to method development for the analysis of synthetic peptides will allow you to accurately quantify and purify peptides in a robust and reliable process. Screening column chemistries and pH is of paramount importance and well-buffered and controlled systems are a must. Following a Quality by Design (QbD) process simplifies this strategy and brings together all the important elements during your method development.

A Systematic Approach to HPLC Peptide Analysis

<h3>Negative Peaks - Some or All Peaks Dip Below the Baseline</h3>Negative peaks, where some or all peaks dip below the baseline, can occur in Gas Chromatography (GC) analysis and might impact the accuracy and reliability of your results. In this month's technical tip, we will explore the possible causes and suggest remedies to troubleshoot the presence of negative peaks.<h4>Symptom - Some Peaks Dip Below the Baseline</h4><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Peak_Shape_Problems_1_3aa9313384.jpg" alt="Phenomenex: Peak Shape Problems: Symptom - Some Peaks Dip Below the Baseline" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Peak_Shape_Problems_1_3aa9313384.jpg 245w," sizes="100vw" width="905" height="103"><h5>Possible Cause | Suggested Remedy</h5><ul><li>Detector overload in element specific detectors such as ECD, NPD, FPD, etc. can produce both positive and negative peaks: Have the compounds of interest arrive at the detector at a different time from the solvent or other compounds in high concentration. H₂ produces negative peaks with a TCD and helium carrier gas.</li><li>Dirty ECD detector can give a negative peak after a positive one: Clean or replace the ECD detector.</li><li>Sample contaminants (hydrocarbons or other non-responders) are present when using ECD, PID, or NPD (thermoionic specific) detectors: Improve sample preparation and cleanup methods prior to injection.</li></ul><h4>Symptom - All Peaks Dip Below the Baseline</h4><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Peak_Shape_Problems_2_67ef91aa12.jpg" alt="Phenomenex: Peak Shape Problems: Symptom - All Peaks Dip Below the Baseline" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Peak_Shape_Problems_2_67ef91aa12.jpg 245w," sizes="100vw" width="905" height="103"><h5>Possible Cause | Suggested Remedy</h5><ul><li>Incorrect polarity of the recorder connections results in nearly all peaks being negative: Reverse polarity of recorder connections.</li><li>Recorder-integrator wires reversed: Correct connections.</li><li>Sample injected onto the wrong column for dual-column setups: Reinject the sample onto the correct column.</li></ul><h4>Symptom - Dip after Solvent Peak</h4><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Peak_Shape_Problems_3_374ae71825.jpg" alt="Phenomenex: Peak Shape Problems: Symptom - Dip after Solvent Peak" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Peak_Shape_Problems_3_374ae71825.jpg 245w," sizes="100vw" width="905" height="103"><h5>Possible Cause | Suggested Remedy</h5><ul><li>Detector contamination: Clean or bake out the detector.</li><li>Sample contamination: For PID detectors, check that the sample has not been contaminated with methanol or water. If necessary, prepare a fresh sample.</li><li>Often normal for NPD (thermoionic specific) detectors: No correction necessary.</li></ul>Negative peaks in GC analysis can be addressed by understanding the possible causes and implementing the appropriate remedies. Most of the causes that has been observed are related to the detector that play a crucial role in negative peaks troubleshooting.

Peak Shape Problems in Gas Chromatography

In this technical tip we look at an issue that may occur before you start your gas chromatography run - how to solve offset baseline issues, where sudden unexplained changes in baseline position are seen.<h4>Troubleshooting Baseline Offset in Gas Chromatography</h4>Baseline offset in a chromatogram can result from several factors, including electrical issues, contamination, or improper column installation. The following guide describes common causes and practical steps to resolve the problem.<h5>1. Line Voltage Changes</h5>Fluctuations in the electrical line voltage can cause baseline drift or offset.<ul><li>Remedy: Monitor the line voltage and check for correlation with the observed offset. If the offset coincides with voltage variations, install a voltage regulator to stabilize the power supply.</li></ul><h5>2. Poor Electrical Connections</h5>Loose or corroded electrical connections may introduce noise or baseline instability.<ul><li>Remedy: Inspect all electrical connections in the GC system. Tighten any loose fittings and clean dirty or corroded contacts to ensure consistent conductivity.</li></ul><h5>3. Contaminated Injector</h5>A contaminated or dirty injector can contribute to baseline offset and irregular peaks.<ul><li>Remedy: Perform full injector maintenance — clean the injector body, replace the inlet liner and glass wool, and ensure the system is free of residue or buildup.</li></ul><h5>4. Contaminated Column</h5>Contaminants on the analytical column can cause baseline disturbances.<ul><li>Remedy: Trim approximately 10 cm (4 inches) from the front of the column and bake it out at high temperature according to manufacturer recommendations. If the problem persists, replace the column with a new one.</li></ul><h5>5. Column Inserted Too Far into the Detector</h5>Incorrect column installation can lead to signal instability, especially with FID, NPD, or FPD detectors.<ul><li>Remedy: Reinstall the column according to the instrument manual. Ensure the column tip is inserted exactly the correct distance into the detector to maintain optimal performance.</li></ul><h5>6. Contaminated Detector</h5>Residue or buildup inside the detector can distort the baseline or create false signals.<ul><li>Remedy: Follow the instrument’s detector maintenance procedure to clean or replace contaminated components.</li></ul><h5>7. Gas Generator Issues</h5>Fluctuations in carrier or detector gas pressure may arise from irregular performance of the gas generator.<ul><li>Remedy: Check the supply pressure regulator on the gas generator. For systems with long supply tubing, install a step-down regulator near the GC to stabilize pressure. Adding an additional regulator can also smooth out fluctuations caused by the gas source.</li></ul><h4>Summary</h4>Addressing baseline offset requires a systematic approach — verify electrical stability, cleanliness, and correct installation throughout the GC system. Routine maintenance of injectors, detectors, and gas supplies helps ensure stable baselines and reliable chromatographic performance.<blockquote><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/application::paper.paper/183bdef8-200f-4854-9e39-e61a49cdeded.pdf">Download GC Troubleshooting Guide</a>The complete guide to GC troubleshooting with hints, tips, and tricks for chromatographic remedies.</blockquote>

Phenomenex technical tip for Baseline problems - offset

The clinical utility of oligonucleotides as a means of controlling gene expression makes them of increasing interest as therapeutics. Oligonucleotides have very different chemical properties to typical small molecule drugs, which makes them an interesting target, but also makes their analysis more complicated than many small molecule therapeutics. They are inherently polar compounds and require quite specific conditions to allow the analysts to fully characterize them and their impurities.In this two-part series we will look at the challenges 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: Oligonucleotide" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_oligonucleotide_2_3d8377cbf6.jpg 125w," sizes="100vw" width="505" height="628">Traditional oligonucleotide methods employ the use of an ion-pair agent (typically an alkylamine such as triethylamine) to facilitate their retention on a C18 column. C18 phases are still preferred as they offer the highest level of efficiency when the goal is to resolve closely eluting impurities. In this first part, we will look at the effect of the mobile phase, specifically the alkylamine ion pair selected, on resolution and characterization.In the literature TEA is often listed as a standard alkylamine to be used alongside hexafluoroisopropanol (HFIP) for the analysis of oligonucleotides. However, studies by the Bartlett laboratory would suggest that the different alkylamine types should be evaluated when developing a method for the analysis of oligonucleotides.1Some general guidelines for selecting alkylamine and alkylamine concentration for use in your method are outlined below. Please be mindful these are guidelines to help method development and should form part of good experimental design.Higher concentrations of alkylamine leads to increased retention time and higher ionization efficiency (Fig. 1).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_1_Total_Ion_Chromatogram_cbce86558a.jpg" alt="Phenomenex: Figure 1 - Total Ion Chromatogram" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_1_Total_Ion_Chromatogram_cbce86558a.jpg 220w," sizes="100vw" width="352" height="250"><ul><li>Column: Biozen™ 2.6 µm Oligo</li><li>Dimensions: 100 x 2.1 mm</li><li>Order No.: 00D-4790-AN</li><li>Mobile Phase: A: 12.5 mM HFIP, Hexylamine in Water</li><li>Mobile Phase: B: 12.5 mM HFIP, Hexylamine in MeOH</li><li>Gradient: 25-75% B in 14 minutes</li><li>Flow Rate: 0.3 mL/min</li><li>Temperature: 65 ˚C</li><li>Detection: TOF-MS</li><li>Sample: Nusinersen</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Deconvoluted_Spectra_1e0646f51f.jpg" alt="Phenomenex: Deconvoluted Spectra" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Deconvoluted_Spectra_1e0646f51f.jpg 245w," sizes="100vw" width="755" height="250">The concentration of alkylamine may be a concern, as ion-pair agents can linger around in your LC system and MS interface if you haven’t implemented a proper wash. In such cases, there are other strategies you can evaluate to reduce the amount of alkylamine in the first place. The use of a more hydrophobic alkylamine, such as hexylamine (HA), can allow you to reduce the concentration of ion-pairing agent and run a higher proportion of organic solvent in your gradient (Fig. 2) . Greater retention time, a higher proportion of organic solvent in your mobile phase, and lower concentration of ion-pairing agent will all help to facilitate higher ionization efficiency (Fig. 3).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_2_79c3f75f17.jpg" alt="Phenomenex: Figure 2" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_2_79c3f75f17.jpg 245w," sizes="100vw" width="497" height="178"><ul><li>Column: Biozen 2.6 µm Oligo</li><li>Dimensions: 100 x 2.1 mm</li><li>Mobile Phase:&nbsp;&nbsp;&nbsp;&nbsp;<ul><li>A: 12.5 mM HFIP, HA or TEA (as indicated) in water</li><li>B: 12.5 mM HFIP, HA or TEA (as indicated) in methanol</li></ul></li><li>Gradient: As shown (in % B)</li><li>Flow Rate: 0.3 mL/min</li><li>Temperature: 65 ˚C</li><li>Detection: TUV @ 260 nm</li><li>Sample: BNA</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_3_ce2677219d.jpg" alt="Phenomenex: Figure 3" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_3_ce2677219d.jpg 245w," sizes="100vw" width="1709" height="536"><ul><li>Column: Biozen 2.6 µm Oligo</li><li>Dimensions: 100 x 2.1 mm</li><li>Part No.: 00D-4790-AN</li><li>Mobile Phase A: 12.5 mM HFIP, 1 mM TEA in Water 12.5 mM HFIP, 4 mM HA in Water</li><li>Mobile Phase B: 12.5 mM HFIP, 1 mM TEA in Methanol 12.5 mM HFIP, 4 mM HA in Methanol</li><li>Gradient: 5-75 % B in 14 min</li><li>Flow Rate: 0.3 mL/min</li><li>Temperature: 65 ˚C</li><li>Detection: TOF-MS</li><li>Sample: 2’-MOE Gapmer (200 ng)</li></ul>One final thing you should consider when choosing an ion-pairing agent is solubility. When there is lower solubility of the ion pair in the mobile phase, you can see some enhancement of ion pairing. However, if we take octylamine as a unique additional example, this additive is very much less soluble in highly aqueous mobile phases and requires more of an organic component. The high levels of organic which are required to solubilize this ion-pair agent make it less applicable in all situations, particularly when analyzing shorter oligonucleotides that are not well retained in highly organic mobile phases.In summary, there is no magic bullet when it comes to selecting the best ion-pairing agent for the analysis of oligonucleotides, but following a few simple guidelines can significantly improve the characterization data you are able to obtain from your analysis.<blockquote>Watch our free on-demand webinars<ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3945">Oligonucleotide Analysis in Tissue Sampless</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/2967">Advances in Oligonucleotide Sample Preparation and Analysis</a></li></ul></blockquote>

Method Development Strategies for Oligonucleotides

When it comes to selecting the best GC column for your analysis, understanding the concepts of polarity and selectivity is crucial. While often used interchangeably, they have distinct meanings in the context of gas chromatography. Let's explore how you can make informed decisions for column selection.<h4>Overview</h4>Polarity and selectivity are often regarded as interchangeable, however in practice they are quite different when looking at gas chromatography. Selectivity refers to the difference in retention factors between two peaks whilst polarity is an assigned value specific to a GC column, based on an assignment using solute-solvent properties. With a plethora of GC phases available to an analyst, how can you ensure you are selecting the best stationary phase for your method?<h4>Tip</h4>Resolution between compounds is primarily determined by the selectivity of the stationary phase, and your choice will have the largest effect on this. Column selectivity is unique to column polarity, but it is important to also ensure the polarity of the phase you select is appropriate to the compound types you are analyzing. For example, if you have a selection of mid-polarity compounds generally these will resolve best on a mid-polarity column. What is important when selecting a stationary phase is to consider that two columns with almost equivalent polarity can offer very different selectivities. If we consider the <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products/1576">Zebron ZB-35</a> and <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products/1573">ZB-1701</a>, which have polarity indexes of 18 and 19 respectively, due to their very different stationary phases they work in different ways depending on the class of compound in question.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Zebron_ZB_35_and_ZB_1701_0793537614.jpg" alt="Phenomenex: GC columns Zebron ZB-35 and ZB-1701" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Zebron_ZB_35_and_ZB_1701_0793537614.jpg 245w," sizes="100vw" width="1057" height="238">Using PAHs as an example we can see the different selectivity profiles for these hugely aromatic compounds, with the <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products/1573">ZB-1701</a> and its higher level of aromaticity giving better resolution, specifically of the critical pair of dibenzeno[a,h]acridine and dibenzeno[a,j]acridine (Fig. 1) when run using the same column dimensions and temperature profile.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Zebron_ZB_35_and_ZB_1701_2_eac90b78f7.jpg" alt="Phenomenex: Comparison of GC columns Zebron ZB-35 and ZB-1701" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Zebron_ZB_35_and_ZB_1701_2_eac90b78f7.jpg 245w," sizes="100vw" width="1279" height="727">In GC, 3 types of interactions are important for selectivity:<ol><li>Van der Waals Interactions, which occur between non- polar groups on the phase and non- polar compounds.</li><li>Dipole-dipole interactions, which can assist with separating compounds with similar boiling points where their chemical structures are different.</li><li>Hydrogen bonding, which can be a powerful tool for polar compounds. It can also lead to poor peak shape if not controlled appropriately.</li></ol>When selecting the most appropriate GC column for your application, polarity should be considered first, and using a column which matches your compound polarity is generally advised. Once this is selected, elution order of your compounds is primarily determined by boiling point and in general compounds with lower boiling points will elute earlier than those with higher boiling points. The caveat to this is when the lower boiling compound has more interaction with the stationary phase than the higher boiling compound. For a purely boiling point based separation you would be looking to use a stationary phase such as the <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products/1572">ZB-1 </a>which is 100% dimethylpolysiloxane – where Van De Waals forces form the main interaction between the analytes and the stationary phase.<h4>View On Demand Webinar</h4>🎥 <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/webinars/exploring-fast-gc-solutions-for-priority-pollutants-like-pah-and-pcb-in-food-and-environmental-sampl">Exploring Fast GC Solutions for Priority Pollutants like PAH and PCB in Food and Environmental Samples.</a>

Good Column Selection: Polarity vs Selectivity

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