HPLC gradients are a fantastic means of analyzing a sample for which the analytes exhibit a wide range of retentive strengths along the stationary-phase of your preferred column. Gradients are not always accommodating towards buffers when attempting to maintain a specific pH. If the buffer is limited to your aqueous mobile-phase while the organic mobile-phase remains strictly an organic solvent, then the gradient is prone to solubility limitations and inconsistent buffering strength as the ratio of organic solvent increases during the gradient. This may be overcome by applying an even distribution of the buffer towards both the “weak” and “strong” mobile-phases of your method, for which we will review some approaches and examples.How about spiking both the “weak” and “strong” mobile-phases with an acidic or basic additive, such as the formic acid (HCO2H) at 0.1% (v/v)?The spike in both mobile-phases will ideally maintain a consistent level of the additive in the overall mobile-phase as the ratio of “weak” and “strong” mobile-phases alters during the gradient. The shortcomings of such a spike are that the additive may be prone to volatilization from the mobile-phase mixture, while also inducing inconsistent strengths of protonation or deprotonation towards analytes. Perhaps some of us can admit to having been less than precise in our preparation of (v/v) spikes at one time or another. Alternatively, a proper buffer with the corresponding salt for a specific additive will precisely maintain the consistent pH at which it is prepared without the risk of the additive volatilizing.Ammonium formate (NH4HCO2) and ammonium acetate (NH4CH3CO2) buffers are the most amenable towards gradients, as these salts are soluble in methanol, while the corresponding aqueous buffers may be mixed with high ratios of acetonitrile. The necessary buffering strength typically ranges only 5 – 10 mM in each mobile-phase, but compare this with an isocratic method of 60:40 “20 mM buffer”/acetonitrile, for which the buffer concentration relative to the overall mobile-phase is only 12 mM. Figure 1 features an analysis of Mycotoxins on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1514">Kinetex™ Biphenyl</a> with 5 mM ammonium acetate buffers comprising both the aqueous and methanol mobile-phases, and for which the ammonium acetate was dissolved directly in the methanol.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_effects_of_gradient_on_buffer_capacity_3_788ba526cf.jpg" alt="Phenomenex: The effects of gradient on buffer capacity: Figure 1 - Mycotoxin Analysis on Kinetex Biphenyl (App ID 21951)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_effects_of_gradient_on_buffer_capacity_3_788ba526cf.jpg 245w," sizes="100vw" width="904" height="475">Acetonitrile will need to be mixed with an aqueous buffer of common buffering reagents to maintain the solubility of the buffering salt. Figure 2 features the HILIC-mode analysis of underivatized amino acids on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1542">Biozen™ Glycan</a>, for which a stock buffer of 100 mM ammonium formate buffer (pH 3.1) was spiked into either water or acetonitrile in a 9:1 ratio of solvent/buffer. The resulting mobile phases each featured overall buffer concentrations of 10 mM ammonium formate, allowing the gradient to maintain a consistent overall buffering strength.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_effects_of_gradient_on_buffer_capacity_2_bb25c9ae6a.jpg" alt="Phenomenex: The effects of gradient on buffer capacity: Figure 2 - Underivatized Amino Acids on bioZen Glycan (Tech Note 1264)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_effects_of_gradient_on_buffer_capacity_2_bb25c9ae6a.jpg 245w," sizes="100vw" width="987" height="486">Ammonium fluoride (NH4F) is often included in small amounts (0.1 – 0.2 mM) in the overall mobile-phase to generate highly sensitive fluoride adducts [M+F]- during ESI-negative mode LC/MS, or to encourage the generation of either [M+H]+ or [M+NH4]+ ions in ESI-positive mode. Figure 3 demonstrates the use of a third solvent line by which a 1 mM NH4F buffer is constantly delivered at a ratio of 10% of the overall mobile-phase, corresponding to 0.1 mM in the overall mobile-phase throughout the gradient. Ternary and quaternary pumps facilitate this method, but an initial isocratic hold is recommended at the beginning of the method to accommodate the transferability of the method among instruments. Ternary and quaternary gradients are subject to dwell volumes that are specific to each instrument, and can be a concern even when one of the solvent lines is maintained at a constant percentage of the overall gradient.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_effects_of_gradient_on_buffer_capacity_dd2c7475e7.jpg" alt="Phenomenex: The effects of gradient on buffer capacity: Figure 3 - Microcystins on Luna Omega Polar C18 (App ID 23568)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_effects_of_gradient_on_buffer_capacity_dd2c7475e7.jpg 245w," sizes="100vw" width="1071" height="550">Be mindful of solubility considerations when attempting to incorporate either ammonium bicarbonate buffers or phosphate buffers into a gradient, particularly when working with phosphate buffers. These buffers run the risk of precipitation when blended with high percentages of organic solvents, and a solubility study is recommended. Lastly, be sure to include your intended mobile-phase buffer as a portion of your sample diluent to align the pH of your mobile phase, which will benefit the peak shape and reproducibility. Using a gradient where the mobile phase has a consistent buffering strength and pH value will ultimately improve the reproducibility of the LC method.

The effects of gradient on buffer capacity

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

The scientific team of Dr. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/articles?search=miloslav+polasek">Miloslav Polášek</a> at IOCB Prague has developed a new method of separating the rare earth elements, or lanthanides, which are widely used in the electronic, medical, automotive, and defense industries. The unique method allows metals such as neodymium or dysprosium to be purified from used neodymium magnets. The environmentally friendly process precipitates the rare earths from water without organic solvents or toxic substances. The results were published in the Journal of the American Chemical Society (JACS) at the end of June.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/IOCB_Prague_Tomas_Bellon_Dr_Miloslav_Polasek_head_of_the_Coordination_Chemistry_research_group_at_IOCB_Prague_and_Kelsea_Grace_Jones_Ph_D_student_in_Dr_Miloslav_Polasek_s_group_c94ff60915.jpg" alt="IOCB Prague / Tomáš Belloň: Dr. Miloslav Polášek, head of the Coordination Chemistry research group at IOCB Prague, and Kelsea Grace Jones, PhD student in Dr. Miloslav Polášek’s group. " srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_IOCB_Prague_Tomas_Bellon_Dr_Miloslav_Polasek_head_of_the_Coordination_Chemistry_research_group_at_IOCB_Prague_and_Kelsea_Grace_Jones_Ph_D_student_in_Dr_Miloslav_Polasek_s_group_c94ff60915.jpg 234w," sizes="100vw" width="1348" height="900">Global demand for rare earths is driven primarily by their use in extremely strong neodymium magnets, which enable efficient conversion of motion into electrical energy and vice versa. They are essential to manufacturers of electric cars, wind power plants, mobile phones, computers, and data centers. As these industries develop, demand for rare earths will continue to grow. However, the process of mining and purifying these elements is highly energy intensive and produces large amounts of toxic and radioactive waste.The rare-earth market is dominated by China, giving it leverage over Europe and North America. It is therefore strategically advantageous to focus on so-called urban mining, i.e. the recycling, renewal, and reuse of materials from discarded equipment, such as electric vehicles, as a significant domestic source of rare earths.“In the future, we won’t be able to cover the growing consumption of rare earths with primary mining. We know that within ten years at the latest, it will be necessary to manage these materials more carefully. In order to achieve this, the development of new technologies must start now,” explains Miloslav Polášek, head of the Coordination Chemistry group. “Our method solves the fundamental problems of recycling neodymium magnets. We can separate the right elements so that new magnets can be produced. Our process is environmentally friendly, and we believe that it will work on an industrial scale. Fortunately, unlike plastics, chemical elements don’t lose their properties through repeated processing, so their recycling is sustainable and can compensate for traditional mining.”<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/DSC_2799_2_d804034e7a.jpg" alt="IOCB Prague / Tomáš Belloň: A product of separation carried out using a method developed in Dr. Miloslav Polášek’s lab. He and his team processed a magnet from an electric car and obtained 99.7% pure neodymium." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_DSC_2799_2_d804034e7a.jpg 234w," sizes="100vw" width="1348" height="900">The topic, which Polášek’s group has been working on for a long time, is part of Kelsea G. Jones’s doctoral thesis. “We’ve developed a new type of chelator, which is a molecule that binds metal ions. This chelator specifically precipitates neodymium from dissolved magnets, while dysprosium remains in solution, and the elements are easily separated from each other. The method is also adaptable for the other rare earths found in neodymium magnets,” says Jones. “The separation is done in water and generates no hazardous waste. We achieve the same or better results than current industrial methods that rely on organic solvents and toxic reagents.”The new technology is patented and responds to a fundamental global problem at the right time. “We’re impatiently awaiting the results of a feasibility study, which will help us direct this research from the laboratory into practice. I believe that in cooperation with the investors and business partners we’re approaching, this new technology from IOCB Prague has the potential to influence a wide range of industrial sectors,” says Milan Prášil, director of the transfer company <a target="_blank" rel="noopener noreferrer" href="https://www.uochb.cz/en/iocb-tech">IOCB Tech</a>.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/DSC_0502_2_449fa66802.jpg" alt="IOCB Prague / Tomáš Belloň: Milan Prášil, director of the transfer company IOCB Tech." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_DSC_0502_2_449fa66802.jpg 234w," sizes="100vw" width="1348" height="900">This research has also yielded another important finding: namely, that the element holmium is used in neodymium magnets of newer electric cars. Scientists from Polášek's team discovered this by analyzing samples from the electric motors of European and Chinese cars. However, professional publications have not yet mentioned this fact, and most recycling projects do not take it into account when processing waste from electric cars. These findings will undoubtedly influence other development and recycling projects, even beyond the automotive industry.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/DSC_2816_2_80e809d242.jpg" alt="IOCB Prague / Tomáš Belloň: Kelsea Grace Jones, PhD student in Dr. Miloslav Polášek’s research group" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_DSC_2816_2_80e809d242.jpg 234w," sizes="100vw" width="1348" height="900"><blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/jacs.5c04150">Macrocyclic Chelators for Aqueous Lanthanide Separations via Precipitation: Toward Sustainable Recycling of Rare-Earths from NdFeB Magnets</a></h4>Kelsea G. Jones, Tomáš David, Martin Loula, Stanislava Matějková, Jan Blahut, Anatolij Filimoněnko, Miroslava Litecká, Jan Rohlíček, Jiří Böserle, Miloslav Polasek*J. Am. Chem. Soc. 2025<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/jacs.5c04150">https://doi.org/10.1021/jacs.5c04150</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.<h4>Abstract</h4>Rare-earth elements (REEs) are critical materials in modern industry, but their production has a significant environmental footprint. Environmentally friendly separation methods would enable efficient, sustainable recycling of REEs. This work introduces a class of cyclen-based macrocyclic chelators that induce significant differences in solubility for REE chelates, enabling their selective precipitation from pH-neutral aqueous solution. The process was refined using simple coordinating additives (e.g., acetate) to form ternary coordination compounds to fine-tune these chelate solubilities. Conditions were optimized for the REEs found in NdFeB magnets, allowing separations of even adjacent lanthanides by repeated precipitations. Separation factors comparable to those of industrial solvent extraction methods were achieved without organic solvents. Analysis of NdFeB magnets from current electric car motors revealed an unexpected presence of holmium as a supplement and/or replacement for terbium and dysprosium, suggesting shifting industrial trends with implications for future recycling efforts. In a case study, one such automotive magnet was processed to obtain a 99.7% pure neodymium product. Scalable, tunable, and entirely aqueous, this approach advances the sustainable use of REEs toward a circular economy.<h4>Materials and Methods</h4>Liquid chromatography: Analytical HPLC experiments were performed on <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=1260+Infinity+II">1260 Infinity II </a>with UV (DAD, part number G7115A, 190 – 400 nm) and MS (single quadrupole, G6125B) detectors from Agilent (referred to as LC-MS or HPLC) equipped with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1529">Luna Omega Polar C18 column</a> (5 µm, 100 Å, 150 × 4.6 mm) using H2O–MeCN gradients (1 mL min−1 flow rate) either with additives or without additives (specified where relevant). The UV-absorption profiles given for the characterization of the chelators and/or chelates were obtained from these chromatograms. Preparative HPLC experiments were performed on 1260 Infinity II (Agilent) equipped with YMC-Actus Triart C18 column (5 µm, 100 Å, 250 × 20.0 mm) using H2O–MeCN gradients (20 mL min−1 flow rate) with either TFA (0.1%) or no additional additive.High-resolution mass spectra: HRMS (with ESI ionization) were recorded on an Agilent 5975C MSD Quadrupole, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?instrumentationItems=29-32&amp;manufacturerItems=5&amp;search=Q-Tof">Q-Tof micro</a> from Waters or <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=2&amp;search=LTQ+Orbitrap+XL">LTQ Orbitrap XL</a> from Thermo Fisher Scientific.Elemental analysis: CHN elemental analysis was performed on PE 2400 Series II CHN Analyzer from Perkin Elmer. Fluorine elemental analysis was performed by combustion of the sample in quartz vessel, followed by adsorption of HF in H2O and determining its concentration by potentiometry using F- -selective electrode. Lanthanide content was determined by ICP-OES (SPECTRO Arcos Multiview from SPECTRO Analytical Instruments). All EA data are presented as: calcd. (found).NMR spectroscopy: 1H and 13C NMR spectra were recorded on a Bruker Avance III™ HD 400 MHz spectrometer (401.0 MHz for 1H, 100.6 MHz for 13C) equipped with a broad-band Prodigy cryo-probe with ATM module (5 mm CPBBO BB-1 H/19F/D Z-GRD) or on an Avance II™ 500 MHz (Bruker, 499.9 MHz for 1H, 125.7 MHz for 13C) spectrometer equipped with a 5 mm TBO probehead. The measurement temperature is indicated for the respective chelator. Chemical shifts are in ppm and coupling constants in Hz. Spectra were referenced using the residual DMSO-d6 solvent signal (2.50 ppm in 1H; 39.52 ppm in 13C), or to the signal of t-BuOH external standard (1.25 ppm in 1H; 32.43 ppm in 13C) for spectra measured in D2O. Integrals in showcased NMR spectra were rounded to integers for clarity; obscured signals that cannot be exactly integrated due to overlap with peak from solvent are coloured grey. Signals of the cyclen macrocycle are abbreviated mc.X-ray powder diffraction: Data were acquired using the Debye-Scherrer transmission configuration on the powder diffractometer Smartlab (Rigaku) equipped with a Cu X-ray source (Cu/Kα radiation; λ = 1.5418 Å), focusing mirror (CBO-E), capillary holder and D/tex ultra 250 detector. The sample was ground, placed in a 0.5 mm borosilicate-glass capillary, and measured at 298±5 K over 18 h from 3° to 65° 2θ with 0.01° step size and with variable counting time.

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One of the most common causes of short column lifetime is column contamination, often resulting in high back pressure and a so called “plugged” inlet frit.This technical tip aims to explain what is happening, and also how column contamination can be avoided.&nbsp;In the manufacture of particulate based HPLC and UHPLC columns, frits are used to prevent extraneous material entering the column, and to prevent the HPLC packing material from leaving the column. These frits are discs of porous metal, generally 0.5, 1.0 or 2.0 μm in porosity. If samples contain undissolved material, it will be filtered out of the mobile phase solvent stream, sitting on the outside of the inlet frit. As this process continues, the amount of the frit available for mobile phase to pass through becomes smaller, restricting the flow of mobile phase which in turn increases the back pressure. Eventually it becomes impossible to push solvent through the frit and the HPLC pump shuts down.This process can be averted by the filtration of samples prior to injection onto the HPLC system. It is generally advised to use 0.45 μm porosity syringe filters for samples that will be run on 5 μm columns, 0.2 μm filters for 3 μm columns and smaller. Care should be taken as ideally the sample diluent should match the mobile phase. If it does not, you may find that some sample components are soluble in the sample diluent allowing them to pass through the syringe filter. When they are injected into the mobile phase they may precipitate causing frit blockage. This can be avoided by either dissolving the sample in mobile phase, or alternatively installing an inlet frit, or a guard cartridge as a sacrificial device to prevent the inlet of the column becoming fouled.Alternatively, we have this <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/tools/security-guard-selection#bybrand">web tool</a> which will guide you through guard cartridge selection.&nbsp;

Column contamination – what is happening, and how it can be avoided

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The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences (IOCB Prague) is a leading scientific institution in the Czech Republic, recognized internationally. Its primary mission is basic research in the fields of chemical biology and medicinal chemistry, organic and material oriented chemistry, chemistry of natural compounds, biochemistry and molecular biology, physical chemistry, theoretical chemistry, and analytical chemistry. 

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