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?

Have you ever considered hidden chemicals in your clothing and home textiles? The latest research of scientists from RECETOX, Masaryk University (Brno, Czech Republic), and the Helmholtz Centre for Environmental Research (Leipzig, Germany) reveals how various fabrics absorb and release harmful aromatic amines (AAs) compounds linked to cancer and other health risks. This research is especially important because by studying the most common fabric types, we can better predict how these chemicals behave in indoor environments.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Recetox_Nova_studie_odhaluje_schopnost_textilii_pohlcovat_a_vypoustet_skodlive_chemikalie_Obrazek_1_Mikroskopicke_snimky_tkanin_A_bavlna_B_polyester_C_vlna_8f78cacccb.jpg" alt="Recetox: New study reveals how textiles trap harmful chemicals: Figure 1. Fabric's microscopic pictures, A – cotton, B – polyester, C – wool" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Recetox_Nova_studie_odhaluje_schopnost_textilii_pohlcovat_a_vypoustet_skodlive_chemikalie_Obrazek_1_Mikroskopicke_snimky_tkanin_A_bavlna_B_polyester_C_vlna_8f78cacccb.jpg 122w," sizes="100vw" width="738" height="944"><h4>What Are Aromatic Amines and Why Should We Care?</h4>Aromatic amines (AAs) are harmful chemicals found in everyday products like hair dyes, rubber materials, and tobacco smoke. “Indoor activities such as protein-enriched foods and smoking release these compounds into the air, directly impacting human health. While regulations restrict some AAs due to their toxicity, others remain widely used . Long-term exposure increases the risk of cancer and other serious health problems,” explains first author of the study Özge Edebali.Surprisingly, textiles can trap AAs from the air and later release them, contributing to indoor pollution. Since we spend most of our time indoors, understanding how different fabrics interact with AAs can help us reduce exposure and improve indoor air quality. Moreover, washing contaminated fabrics can also send these chemicals into wastewater, threatening aquatic ecosystems.“The research tested how different textiles—cotton, wool, and polyester—absorb and release AAs indoors. The test was conducted in a specially designed chamber, where different textiles were exposed to AAs. Afterward, AAs were measured in textiles and in the air as well. The finding is that cotton absorbed the highest levels, making it a significant source of chemical exposure,” says Özge Edebali.These everyday fabrics act as hidden reservoirs, transferring pollutants into indoor air and even our waterways through laundry. Understanding this process is key to reducing exposure and creating safer indoor environments.The project results were created with the financial support of the provider Czech Science Foundation within the project no.GF22-06020K and Deutsche Forschungsgemeinscha (DFG, German Research Foundation) – MU 4728/2-1. The authors thank the RECETOX Research Infrastructure (LM2023069) financed by The Ministry of Education, Youth and Sports for a supportive background. This work was supported by the European Union's Horizon 2020 research and innovation program under grant agreement no. 857560 (CETOCOEN Excellence).<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g">Characterizing the distribution of aromatic amines between polyester, cotton, and wool textiles and air</a></h4>Özge Edebali, Anna Goellner, Marek Stiborek, Zdeněk Šimek, Melis Muz, Branislav Vrana and &nbsp;Lisa MelymukEnviron. Sci.: Processes Impacts, 2025,27, 1054-1062<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1039/D5EM00015G">https://doi.org/10.1039/D5EM00015G</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by-nc/3.0/">CC-BY 3.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.<h4>2 Materials and methods</h4><h5>2.1. Determination of textile–air distribution coefficients&nbsp;</h5>The purpose of the experiment was to determine textile–air distribution coefficients of selected AAs (Table S2<a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g#fn1">†</a>) under laboratory conditions as a ratio of concentration in the textile fabric and concentration in the adjacent air under equilibrium conditions. The experiment was performed by extracting AAs (by trapping on Tenax® TA sorbent) from a defined volume of air that was in contact with a piece of textile containing a range of AAs inside an exposure chamber. Following each textile's exposure to air, concentrations of AAs in the textile sample were determined after solvent extraction of the textile.The experimental apparatus consisted of a textile exposure chamber consisting of a three-litre glass cylinder with a flat bottom and a ground-joint flange lid (<a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g#fig1">Fig. 1</a>). The exposure chamber was designed with a threaded air inlet in the lid, implemented as a steel capillary pierced through a septum, and an air outlet at the bottom. The chamber operated in a flow-through setup, meaning it was not a closed system. We assumed that the dosing rate of the textile (in terms of air volume equivalent per unit of time) was significantly higher than the airflow rate. Consequently, the chamber conditions were maintained close to a textile/air distribution equilibrium. A wire mesh sheet was rolled up inside the chamber, serving as a framework to support a piece of the tested textile. A small fan inside the chamber was used to homogenize the air inside the chamber and minimize the air boundary layer at the surface of the textile. The chamber was kept at a constant temperature of 25 °C through an external thermostatic water circuit.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Environ_Sci_Processes_Impacts_2025_27_1054_1062_Fig_1_final_d2d7ce8fc7.jpg" alt="Environ. Sci. Processes Impacts, 2025,27, 1054-1062: Fig. 1 Scheme of apparatus used for measurement of KTA of AAs; 1- textile pieces before wrapping to the chamber mesh (Fig S1†), 2- glass chamber with a ground joint lid, mesh and airtight seal, 3- steel capillary pierced through a septum for air intake, 4- metal clamp, 5- small fan, 6- two glass tubes filled with Tenax adsorbent, in series, 7- air mass flow meter, 8- low volume air pump." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Environ_Sci_Processes_Impacts_2025_27_1054_1062_Fig_1_final_d2d7ce8fc7.jpg 245w," sizes="100vw" width="1601" height="901">Two glass tubes packed with Tenax® TA (Gerstel, Germany) were connected in series to the chamber outlet to trap AAs from the air extracted from the exposure chamber. Air was drawn from the apparatus through the Tenax tubes using a low-volume air pump connected to a digital air mass flow controller (Omega FMA-A2404, USA) adjustable in the 0–200 standard cubic centimetres per minute (sccm) range.<h5>2.2. Textile and Tenax preparation&nbsp;</h5>Tenax tubes (Tenax® TA thermal desorption tubes, 6 × 60 mm, Gerstel, Germany) were pre-cleaned before each experiment with methyl tert-butylether (MTBE, 12 h, Soxhlet extraction). After solvent cleaning, tubes were placed into a tube conditioner (Gerstel TC2, Gerstel, Germany) and dried for 2 h in a pure nitrogen stream at 300 °C.Three types of textile materials were tested: wool, cotton, and polyester. All textiles were white/undyed materials; detailed information is provided in ESI Fig. S2, Text S1 and Table S1.<a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g#fn1">†</a> The purchased textile fabrics were cut into larger sheets (27 × 32 cm; area of 864 cm2 each), pre-cleaned (cleaned in water in overhead tumbler) and dried (Text S1 and Fig. S3<a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g#fn1">†</a>). Textiles were then dosed with a mixture of 19 primary AA compounds (Table S2<a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g#fn1">†</a>). The dosing procedure in a rotary evaporator was selected after testing different dosing methods to ensure homogeneously dosed textile samples (Text S2 and Fig. S4–S7<a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g#fn1">†</a>). The selected AAs were diluted in acetone by spiking 1 mL of AA standard (for the spiked masses of each analyte see Table S3<a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g#fn1">†</a>) in approximately 200 mL of acetone in a round rotary evaporator flask and mixed by shaking for 5 minutes. Next, the textile sheet was added to the acetone-filled flask. The textile samples were dosed for 6 hours while turning on a rotary evaporator (270 rpm, 25 °C at normal pressure). Subsequently, acetone was evaporated (556 mbar, 40 °C) while continuing the flask rotation, a process taking 1 hour to achieve complete dryness. After dosing, the textile samples were stored in the closed rotary evaporator flask at 4 °C until they were transferred to the exposure chamber.<h5>2.5. Analysis&nbsp;</h5>The chromatographic separation of target compounds in the textile and Tenax extracts was performed using a liquid chromatograph (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=1290+II+LC">Agilent 1290 II LC System</a>, Agilent Technologies, Santa Clara, CA) equipped with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1515">Kinetex F5 Core–Shell HPLC Column</a> and a precolumn (150 × 2.1 mm, 1.7 μm particle size, Phenomenex, Torrance, CA). Extracts and calibration solutions of the standard mixture, prepared in the concentration range of 1.0–250 ng mL−1 in ACN, were diluted, mixed, and injected using a sandwich injection technique. The injection sequence consisted of 3 μL of distilled water, followed by 1 μL of the sample or calibration solution, 0.5 μL of an internal deuterated standard mixture in ACN, and another 3 μL of distilled water. The final composition of the injected sample or calibration solution was 80% water and 20% ACN. Five deuterated AAs, used as internal standards (ISTD) at a concentration of 50 ng mL−1, were selected to span the retention time range of the target amine mixture. The column temperature was set to 35 °C. The mobile phase consisted of water (A) and ACN/methanol (50/50) (B), both containing 0.1% (v/v) formic acid. The gradient program was as follows: 0 min, 10% B; 0–7 min, increased from 10% to 98% B; 7–10 min, held at 98% B; 10–10.3 min, decreased from 98% to 10% B, followed by a re-equilibration at 10% B until 20 min. The flow rate was maintained at 0.3 mL min−1.An Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=6495A+triple+quadrupole">6495A Triple Quadrupole Mass Spectrometer</a> (Agilent Technologies, Santa Clara, CA) with a mass resolution of 0.7 amu (FWHM, autotune) and mass accuracy of 0.1 amu from 5 to 1000 m/z was used to quantify AAs. The ion source parameters were set as follows: gas temperature of 280 °C, gas flow at 15 L min−1, nebulizer pressure at 25 psi, sheath gas temperature at 380 °C, sheath gas flow at 12 L min−1, capillary voltage of 3000 V (positive mode), and nozzle voltage of 300 V. The instrument operated in ESI-positive dynamic multiple reaction monitoring (dMRM) mode. For MS/MS analysis, two transitions were monitored for each analyte. Collision energies for the [M + H]+ precursor ions of both target analytes and ISTDs are provided in Table S4.<a target="_blank" rel="noopener noreferrer" href="https://pubs.rsc.org/en/content/articlelanding/2025/em/d5em00015g#fn1">†</a> Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=MassHunter+Workstation+Software">MassHunter Workstation Software</a>, LC/MS Data Acquisition (version B.08.00) for the 6400 Series Triple Quadrupole, was used for dMRM data acquisition, while AA quantification was performed using Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=MassHunter+Quantitative+Analysis+software">MassHunter Quantitative Analysis software</a>, version B.07.01 for QQQ.

New study reveals how textiles trap harmful chemicals

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

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

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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RECETOX (Research Centre for Toxic Compounds in the Environment) is an independent department of the Faculty of Science of Masaryk University in Brno, which has been established in 1983. RECETOX is focused on the research and education in cross-cutting area of Environment and Health. It addresses interactions among chemicals, environment, and biological systems, including consequences at local, regional, and global levels.

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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

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