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

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

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

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

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How can capacity factors drive method robustness?

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