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

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