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

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

The development of hybrid materials has greatly extended the high pH stability of HPLC sorbents. This new development allowed users to use high pH to deprotonate basic compounds, increasing their hydrophobicity (and hence retention) while also removing the possibility of ion exchange.This approach is particularly useful when considering polar basic compounds as the combination of good peak shape with enhanced retention is useful, especially in LC-MS/MS where elution under higher organic mobile phase conditions has been shown to be beneficial for peak response. Columns such as <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/gemini-hplc-column">Gemini™</a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=48&amp;manufacturerItems=50&amp;search=evo">Kinetex™ EVO</a> can be used in the pH range 1-12.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_lidocaine_8aa6d60638.jpg" alt="Phenomenex: Lidocaine (peak 3) run at pH 12 on a Twin technology, high pH stable material." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_lidocaine_8aa6d60638.jpg 245w," sizes="100vw" width="677" height="280"><h4>Read the related technical notes:</h4><h5>Basic Compounds by LC/MS using a High pH Mobile Phase</h5>We investigate the effectiveness of using high pH mobile phases for the quantitation of basic compounds in ESI+ LC/MS/MS by comparing limits of detection (LOD), limits of quantitation (LOQ), linearity ranges, precision, and accuracy observed in high and low pH mobile phases.<ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/5847_l_c21fdc6602.pdf">Download HERE</a></li></ul><h5>Using pH-LC to Control Selectivity of Acidic and Basic Compounds by HPLC</h5>We investigate the effectiveness of using pH-LC techniques in the separation of a mixture of acidic (naproxen), basic (amitriptyline), and neutral (toluene) compounds using a Gemini 5 um NX-C18 column<ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/4241_l_db8b374e06.pdf">Download HERE</a></li></ul>

Basic Analytes and the Use of High pH Stable Stationary Phases

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

Basic Analytes and the Use of High pH Stable Stationary Phases

A Systematic Approach to HPLC Peptide Analysis

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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