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

In this technical tip, we will address the common issue of peak tailing encountered when working with basic analytes and “type A” silicas and offer two strategies to overcome it.<h4>Introduction:</h4>Early reversed phase columns were based on “type A” silicas, these materials had substantial metal contamination, causing the silanol groups on the surface of the silica to have greater and more variable activity. Manufacturers endcapped the materials as far as possible after bonding with C18 reagents, in an attempt to reduce the number of sites where ion exchange could take place. However basic compounds would generally tail badly. Let’s explore how we can overcome this limitation.<h4>Strategies for peak shape improvement:</h4><ul><li>Working at low pH can help neutralise the silanol groups on the silica surface, preventing ion exchange from taking place. A 10-20 mM phosphate buffer, pH 2.5 is ideal for this purpose, although care must be taken if using acetonitrile as the organic solvent, to avoid precipitating the phosphate buffer when running a gradient.</li><li>Adding a competing base (or silanol suppressor) to the mobile phase. Mobile phase additives like TEA (triethylamine) at 5 mM concentration would typically be used. The idea here is that the competing base associates with the anionic silanol groups, reducing their availability to interact with analytes. This approach, whilst successful, generally leads to short column lifetime as silanol suppressors also tend to cause more rapid column stationary phase and endcapping hydrolysis, which in turn exposes more silica allowing for more tailing to occur.</li></ul>These strategies will help diminish the tailing effect but may not completely remove it as demonstrated in the below example chromatograms (Figure A and B).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Overcoming_Peak_Tailing_of_Basic_Analytes_in_Silica_Type_A_Stationary_Phases_in_RP_4c1effe7d3.jpg" alt="Phenomenex: Overcoming Peak Tailing of Basic Analytes in Silica Type A Stationary Phases in RP: A) Mobile Phase: 20 mM potassium phosphate buffer pH= 2.5 : Methanol (70:30) Flow rate: 1 mL/min Detection: UV-Vis @254 nm; B) Mobile Phase: 20 mM potassium phosphate buffer pH= 3 with 400uL/L Triethylamine : Acetonitrile (63:37) Flow rate: 1 mL/min Detection: UV-Vis @230 nm." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Overcoming_Peak_Tailing_of_Basic_Analytes_in_Silica_Type_A_Stationary_Phases_in_RP_4c1effe7d3.jpg 245w," sizes="100vw" width="939" height="332">Figure A shows the moderate effect of using an acidic pH to mitigate tailing for benzylamine, while Figure B shows the resulting chromatography after adding triethylamine as a modifier.<blockquote>We recommend the use of type B (ultra-pure) silica in the development of new separations. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=341&amp;manufacturerItems=50&amp;search=omega">Luna™ Omega</a> fully porous and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=339&amp;manufacturerItems=50&amp;search=Kinetex">Kinetex™ Core-Shell</a> columns provide excellent peak shape for challenging compounds.</blockquote>

Overcoming Peak Tailing of Basic Analytes in Silica Type A Stationary Phases in RP

Silica-based stationary phases are currently the most frequently used phases in liquid chromatography. Traditional C18 columns often suffered from poor peak shape and tailing of basic analytes at low pH and limited stability due to gradual silane hydrolysis at high pH. Through cross-linking and end-capping technologies, silica‘s undesired secondary interactions and limited stability have been significantly improved.The most recent development introduced for analysis of basic analytes at low pH levels is the production of C18 columns with a residual positive charge on the silica support. This combination allows for 100% aqueous conditions to be used, enhancing retention of basic compounds. It also minimizes (ionic) secondary interactions between positively charged bases and silanol groups due to charge repulsion at the silica surface. The result is that basic compounds can be retained and eluted with good peak shape under acidic conditions, which are generally favored. Figure 1 shows an example of enhanced peak shape of chlorpheniramine, a basic antihistamine, using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1513">Kinetex™ PS C18 column</a> with this technology.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Basic_analytes_and_the_benefits_of_modified_silica_supports_in_reversed_phase_liquid_chromatography_56f739e383.jpg" alt="Phenomenex: Basic analytes and the benefits of modified silica supports in reversed-phase liquid chromatography: Figure 1. Improved reversed-phase retention and peak shape of chlorpheniramine with the Kinetex PS C18." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Basic_analytes_and_the_benefits_of_modified_silica_supports_in_reversed_phase_liquid_chromatography_56f739e383.jpg 245w," sizes="100vw" width="1090" height="356"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Basic_analytes_and_the_benefits_of_modified_silica_supports_in_reversed_phase_liquid_chromatography_2_74f367d25c.jpg" alt="Phenomenex: Basic analytes and the benefits of modified silica supports in reversed-phase liquid chromatography: Figure 2. Analysis of nicotine and other metabolites with the Kinetex EVO C18 column." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Basic_analytes_and_the_benefits_of_modified_silica_supports_in_reversed_phase_liquid_chromatography_2_74f367d25c.jpg 245w," sizes="100vw" width="1207" height="387">

Basic analytes and the benefits of modified silica supports in reversed-phase liquid chromatography

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

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://www.phenomenex.com/documents/2020/09/24/13/48/hplcuhplc-selectivity-poster">different selectivity element to the column</a>.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_tms_endcapping_b72bb2084c.jpg" alt="Phenomenex: TMS endcapping" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_tms_endcapping_b72bb2084c.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 reversed phase 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_c18_polar_endcapping_b5f9adde86.jpg" alt="Phenomenex: C18 polar endcapping" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_c18_polar_endcapping_b5f9adde86.jpg 199w," sizes="100vw" width="314" 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.<h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1511">C18</a> – Standard Trimethylsilyl End-Capping</h5><ul><li>End-capping type: Trimethylsilyl group</li><li>Key benefits:<ul><li>Minimizes the presence of active silanol groups.</li><li>Provides stable, reproducible retention for a wide range of neutral and nonpolar compounds.</li></ul></li></ul><h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1513">PS-C18</a> – Positively Charged and Trimethylsilyl Groups</h5><ul><li>End-capping type: Positively charged group with trimethylsilyl group</li><li>Key benefits:<ul><li>Combines ion-exchange secondary interactions with hydrophobic interactions.</li><li>Delivers excellent peak shapes for basic analytes.</li><li>Reduces the need for complex mobile phases or ion-pairing agents.</li><li>Stable under 100% aqueous conditions.</li></ul></li></ul><h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1509">Polar C18</a> – Polar Group and Trimethylsilyl Group</h5><ul><li>End-capping type: Polar group with trimethylsilyl group</li><li>Key benefits:<ul><li>Provides both polar and hydrophobic selectivity.</li><li>Enhances retention and resolution of polar analytes.</li><li>Reduces reliance on complex mobile phase compositions.</li><li>100% aqueous phase stability for extended method flexibility.</li></ul></li></ul><h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1510">XB-C18</a> – Sterically Protected Phase</h5><ul><li>End-capping type: Di-isobutyl side chains with trimethylsilyl group</li><li>Key benefits:<ul><li>Bulky side chains sterically shield residual silanols, minimizing secondary interactions.</li><li>Produces improved peak shapes for basic compounds.</li><li>Offers increased retention for acidic analytes.</li></ul></li></ul>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 PS-C18 columns. The Polar C18 chemistry has been proven to best retain compounds such as metabolites, and XB-C18 chemistry has shown excellent chromatography for peptide analysis, with enhanced retention of small polar peptides.<h4>Troubleshooting LC</h4><ul><li>Problems with your chromatogram and Injector</li><li>Key problem areas and preventative maintenance</li><li>Leaks and Abnormal Pressure</li></ul><blockquote><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/gu61160424_w_d5803022e3.pdf">Download HPLC Troubleshooting Guide</a></blockquote>

The Role of End-Capping in Reversed-Phase

In preparative chromatography there are three goals. How you balance these will dictate how you approach your scale up from analytical to preparative scale for large scale HPLC purifications.<h4>Part 1: Optimizing Separation</h4>In preparative HPLC there is a balance between purity, yield, and throughput, and often you must sacrifice one to achieve the other two. The three diagrams below show different outcomes depending on the purification objectives:<ul><li>Left: you could have a balanced triangle, where all three components are achieved, albeit none of these reach their maximum,</li><li>Middle: you could prioritize purity and yield at the cost of longer sample processing times, or</li><li>Right: you could prioritize sample processing speed and purity, sacrificing purification yield.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Part_1_Optimizing_Separation_4767cb5f67.jpg" alt="Phenomenex: Part 1 - Optimizing Separation" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Part_1_Optimizing_Separation_4767cb5f67.jpg 245w," sizes="100vw" width="1425" height="487"><h4>Before you begin you would want to consider:</h4><ul><li>What is the required final purity?</li><li>Is the desired final product a specific salt form, a solid or a solution?</li><li>How can you balance processing time and yield?</li></ul>Once your objectives are outlined, a suitable analytical method must be established to evaluate purification conditions. Often a screening method is carried out to quickly evaluate large numbers of samples with key results confirmed by the full analytical method. During analytical screening multiple sets of conditions are typically tested to determine the most effective for preparative purification. Below we can see the effect of different solvent systems on the separation of two peaks required for preparative purification.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Part_1_Optimizing_Separation_peptides_68cd160f93.jpg" alt="Phenomenex: The effect of different solvent systems on the separation of two peaks required for preparative purification." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Part_1_Optimizing_Separation_peptides_68cd160f93.jpg 159w," sizes="100vw" width="853" height="838">Typically, with crude samples the impurities can lie close to the peak of interest. Although the desired resolution may be achieved analytically, it is important to ascertain, during the development process, how much of your sample you can load before resolution is lost. Shown below is an example of column screening for liraglutide where four stationary phases were screened under various conditions to ascertain optimal separation of impurities. Once you complete your screening you are able to select the most appropriate separation for scale up (covered in Part 2 - our next week's technical tip).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Kinetex_C8_ae2ccb9d05.jpg" alt="Phenomenex: Kinetex C8 - comparison of different solvents" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Kinetex_C8_ae2ccb9d05.jpg 245w," sizes="100vw" width="1030" height="593"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Kinetex_C18_e8610074c5.jpg" alt="Phenomenex: Kinetex C18 - comparison of different solvents" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Kinetex_C18_e8610074c5.jpg 245w," sizes="100vw" width="1076" height="318"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Kinetex_phenyl_hexyl_eceef0e8d3.jpg" alt="Phenomenex: Kinetex phenyl-hexyl - comparison of different solvents" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Kinetex_phenyl_hexyl_eceef0e8d3.jpg 245w," sizes="100vw" width="1064" height="339"><h5>Preparative Chromatography meets UHPLC: Axia™ Packed Kinetex Columns</h5>The power of Kinetex core-shell technology with the performance of piston packed prep columns.<ul><li>Download brochure <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/br11940313_w_a1926e1f93.pdf">HERE</a></li></ul><h5>An Overview of Preparative Chromatography</h5><ul><li>Watch webinar<a target="_blank" rel="noopener noreferrer" href="https://discover.phenomenex.com/recorded-webinars-en"> HERE</a></li></ul>

What is the most appropriate way to approach scaling up your analytical method when transferring it to preparative chromatography scale? Part 1

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What is the most appropriate way to approach scaling up your analytical method when transferring it to preparative chromatography scale? Part 1

The Role of End-Capping in Reversed-Phase

LC System Optimisation for UHPLC Performance on any HPLC

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

Basic analytes and the benefits of modified silica supports in reversed-phase liquid chromatography

Overcoming Peak Tailing of Basic Analytes in Silica Type A Stationary Phases in RP

The Role of End-Capping in RP

How can capacity factors drive method robustness?

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Understanding quantification algorithms

36th MassSpec Forum Vienna 2026

Analytical Strategies for Peptide & Protein Therapeutics

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High-throughput stability screening strategies for antibody formulation

Big Scale, Big Wins: Prep Chromatography Explained

PFAS in Food & Food Contact Material