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?

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

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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