Normal phase SPE uses polar interactions as retention mechanism. These interactions include hydrogen bonding, dipole-dipole, induced dipole, π-π, etc. The interactions are between polar functional groups of analytes and polar sorbent chemistries.Reversed phase uses nonpolar, hydrophobic interactions as retention mechanism. These interactions include Van-der-Waals, London dispersion, π-π, etc. These interactions are between carbon rich, hydrophobic parts of both analytes and sorbent.When dealing with sample matrices that are only soluble in organic solvents, one would use a normal phase method. Examples of such matrices are oil, grease, or butter.Ideally, the sample is diluted with a nonpolar solvent (i.e. hexane) prior to sample loading. The sample can then be washed with hexane or 5% methylene chloride in hexane (analogous to washing with 5% MeOH in water in RP SPE methods to remove matrix interferences). Elution will be done with a polar solvent that can interrupt polar interactions between analytes and sorbent.Fractionation can be done by applying solvents in the order of increasing polarity or mixtures of solvents with different polarities.Below is a quick reference of solvents in ascending polarity, for Normal Phase SPE methods.<h3>Solvents in Polarity Order</h3><h5>From low to high&nbsp;</h5><ul><li>Hexane</li><li>Isooctane</li><li>Carbon tetrachloride</li><li>Toluene</li><li>Benzene</li><li>t-butyl methyl ether</li><li>Chloroform</li><li>Dichloromethane</li><li>Diethyl ether</li><li>Ethyl acetate</li><li>Tetrahydrofuran (THF)</li><li>Acetone</li><li>Acetonitrile</li><li>40% MeOH in acetonitrile</li><li>20% MeOH in diethyl ether</li><li>20% MeOH in dichloromethane</li><li>Isopropanol</li><li>Methanol</li><li>Water</li><li>Acetic acid</li></ul>

How is normal phase (NP) SPE different from reversed phase (RP) SPE? How do I decide which one to use?

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?

When considering the mass of sorbent required to perform a solid phase extraction it is critical to consider the sample matrix (serum, plasma, urine, saliva, food, river water, soil etc). This information will allow you, with the use of a suitable look up table to determine the mass of SPE sorbent to use. The tables are produced to reflect the relative levels of interfering endogenous material present in the samples.&nbsp;The SPE device must have enough capacity to retain all analytes of interest together with any endogenous materials capable of retaining on the sorbent with the same type of interactions as the analyte. If there is insufficient sorbent, it is possible some analyte may pass unretained through the SPE device during sample loading, leading to lower recoveries and a loss of consistency of recovery between samples.<h3>Sample Volume</h3><h4>Determines Bed Mass/ Solvent Volumes</h4><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_How_much_sample_can_I_load_onto_my_SPE_device_Is_it_linked_to_the_concentration_of_my_analyte_of_interest_12a9ab171c.jpg" alt="Phenomenex: How much sample can I load onto my SPE device? Is it linked to the concentration of my analyte of interest?" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_How_much_sample_can_I_load_onto_my_SPE_device_Is_it_linked_to_the_concentration_of_my_analyte_of_interest_12a9ab171c.jpg 154w," sizes="100vw" width="983" height="992"><h3>Generic Loading Capacities:</h3><h4>Polymeric: 10 - 15% of Total Bed Mass | Silica: 5% of Total Bed Mass</h4>If we compare plasma and urine, we can load 500 µl of plasma onto a 60 mg polymeric sorbent such as <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?manufacturerItems=50&amp;search=Strata-X">Strata™-X</a>. Using the same 60 mg SPE tube 2 mL of urine could be loaded, indicating that urine contains significantly less endogenous material than plasma.For sample types not included in the table, the use of generic loading capacity, 5% of sorbent mass for silica materials, or 10-15% for polymeric materials are advised. The difference between silica and polymeric sorbent loading capacity is a function of available surface area, which is higher for polymeric materials and also ligand density – the entire surface of a polymeric material is essentially stationary phase, while on a silica material only the phase bonded to the silica takes part in the extraction mechanism for reversed phase or ion exchange extractions.

How much sample can I load onto my SPE device? Is it linked to the concentration of my analyte of interest?

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

<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50&amp;page=2">Solid phase extraction</a> is routinely selected as a sample preparation technique prior to HPLC and GC analysis. It provides:<ul><li>Clean-up;</li><li>Analyte pre-concentration;</li><li>Solvent switching.</li></ul>This works well for any homogeneous liquid samples, but clearly a solid sample cannot be applied directly to an SPE device. Here we will consider how best to approach the handling of solid samples prior to SPE.The first stage of sample pre-treatment involves homogenising the solid sample with a suitable solvent. The solvent selected for this purpose will:<ul><li>Be effective at solubilising the analyte.</li><li>Be effective at solubilising the sample matrix, although this is not necessary providing the sample is blended well with the solvent.</li><li>Ideally it will be a weak solvent when it comes to the SPE stage. This may not be possible, and it may be necessary to dry down and reconstitute the sample prior to loading on to the SPE cartridge.</li></ul>Having formed a suspension of the sample in a suitable solvent the sample should be vortexed to promote thorough mixing, and then centrifuged to pellet the insoluble components of the matrix. The solvent should then be removed for further treatment.At this point the SPE mechanism needs to be selected:&nbsp;<h3>Reversed Phase SPE Method</h3>Mechanism / Weak Solvent / Strong Solvent<ul><li>Reversed phase – Water/buffer – Acetonitrile/Methanol (hexane strongest)</li><li>Normal phase – Hexane/Heptane – Mid polar organic such as DCM, MTBE, IPA (strongest is water)</li><li>Ion exchange – Aqueous buffer at a pH which promotes ion exchange interactions between analyte and SPE sorbent – Organic solvent, typically methanol with pH adjustment to disrupt the ion exchange interactions.</li></ul>Analyte properties and sample diluent both need to be considered at this point. Reversed phase extractions are effective for moderately polar to moderately hydrophobic analytes, but only if they are dissolved in a weak solvent such as water. If the sample is dissolved in a mid-polar solvent the sample will either need to be diluted, or alternatively dried down and reconstituted. The same is true for normal phase, where samples need to be dissolved in hexane/heptane for analytes to be successfully retained during sample loading onto the SPE device.These examples will hopefully illustrate this concept.<h4>The extraction of PCBs from soil.</h4>PCBs are hydrophobic and will dissolve readily in a non-polar solvent. Soil can be dried, weighed and then extracted using hexane (or heptane). Once thoroughly mixed and centrifuged the resulting solution can be applied directed to a conditioned silica SPE tube allowing for a normal phase extraction to be conducted.<h4>Tylosin from meat.</h4>Tylosin is an antibiotic, with a log P of 1.0. Extracting from meat with a mixture of acetonitrile and water (80:20) will break any hydrophobic interactions releasing the tylosin. After centrifugation the resulting liquid sample will require further dilution with water to reduce the loading solvent strength prior to a reversed phase extraction.<h4>Enilconazole from oranges.</h4>Enilconazole is a fungicide widely used in agriculture and is often used with citrus fruits. It can readily be extracted from oranges using acetonitrile. As enilconazole is basic, the addition of HCl to the acetonitrile extract will promote protonation of the analyte, allowing for it to be directly retained on a strong cation exchange SPE material. This greatly simplifies the extraction process as no dilution of the sample is required (in contrast to reversed phase, or normal phase extraction where 100% acetonitrile would cause the analyte to be unretained during loading).

Considerations when working with solid samples and SPE

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

Due to their nature, bioanalytical samples often require a pretreatment step prior to further cleanup by solid phase extraction (SPE). Each sample matrix poses its own unique challenges such as the removal of proteins from plasma and serum, the disruption of red blood cells in whole blood, hydrolysis of glucuronidated analytes in urine, and homogenization of tissue samples. This technical note outlines common sample pre-treatment procedures for bioanalytical samples.<h4>Plasma/Serum</h4>Plasma and serum pre-treatments are analyte dependent. If the analyte of interest is an acid, 2 % phosphoric acid can be used (20 µL 85 % H3PO4 to 1 mL of plasma or serum) to disrupt the drug-protein interaction. If the analyte of interest is basic, 0.1 M sodium hydroxide can be used to disrupt the drug-protein interaction. After addition of acid or base, the sample should be vortexed for 20-30 seconds followed by centrifugation. The supernatant is now ready for further analysis.<h4>Whole Blood</h4>There are several pre-treatment strategies that can be followed for whole blood. If the target analyte is present in red blood cells, a hemolysis step is necessary<ul><li>Hemolysis: To 0.2 mL whole blood (spiked with analytes and internal standard) in a 1.2 mL centrifuge tube, add 400 µL of 2 % zinc sulfate/80 % methanol. Vortex for 10-20 seconds followed by centrifugation at 14,000 rpm for 10 minutes. Collect the supernatant for further analysis. Preparation of zinc sulfate/methanol: Into a 100 mL volumetric flask add 20 mL water and 3.6 g ZnSO4, 7H2O. After the solution is clear and the salt crystals have dissolved, add 100 % methanol. Refrigerate the solution at 2-8 °C for 7 days.</li><li>Osmotic breakdown: To 1 mL of whole blood add internal standard and 4 mL of distilled water. Mix/vortex and let stand for 5 minutes. Centrifuge at 670 g for 10 minutes and discard the pellet. Adjust the pH of the supernatant accordingly with the addition of a buffer solution.</li><li>Sonication: Sonicate 1 mL whole blood for 15 minutes at room temperature. Add 3-6 mL of an appropriate pH buffer (such as potassium phosphate buffer). Mix/vortex. Let stand for 5 minutes. Centrifuge at 670 g for 15 minutes. Analyze supernatant.</li></ul>Note: A comparison of the above pre-treatment techniques for whole blood was performed for acidic, basic, and neutral drugs. Recoveries were generally the highest when the whole blood sample was diluted with buffer and subjected to physical denaturing (sonication) rather than chemical means. In fact, the sonication process disrupts the cell membranes to the extent that no clogging was observed when the procedure listed above was followed.1<h4>Saliva</h4>No hydrolysis is required for oral fluids and the generic protocol used for plasma/serum pre-treatment may be followed.<h4>Urine</h4>Enzymatic hydrolysis is necessary in case of conjugated forms (sulfated or glucuronide form) of the analyte present. Enzymatic hydrolysis requires specific pH (pH 4-5) and temperature ranges. An acid or base hydrolysis can be performed as well, depending on the stability of the compound.<ol><li>Enzymatic hydrolysis: To 500 µL sample (spiked with analyte and internal standard) add 100 µL acidic buffer (see below) and 20 µL beta-glucuronidase. Vortex for 5-6 seconds. Incubate in a water bath at 63 °C for 30 minutes. Transfer sample to a 96-well collection plate or autosampler vial. Seal and centrifuge for 10 minutes at 2,000 rpm. Preparation of acidic buffer (1.0 M acetate buffer, pH 4.0): Dissolve 3.0 g of glacial acetic acid and 4.1g of sodium acetate in a 1 L volumetric flask.</li><li>Base hydrolysis: To 1 mL urine (spiked with analyte and internal standard) add 100 µL 10 N KOH. Mix, vortex, and hydrolyze for 20 minutes at 60 ˚C. Cool and adjust pH to 3.5- 4.0 (by adding 200 µL glacial acetic acid).</li><li>Acid hydrolysis: To 1 mL urine add 0.25 mL HCl in a screw capped test tube. Screw the tube top on loosely and heat in a boiling water bath for 60 minutes. Adjust to pH 7 (or as needed) with 1.0 N NaOH.</li></ol><h4>Tissue</h4>Homogenize with organic or aqueous solvent depending upon analyte solubility. Settle, decant, centrifuge or filter supernatant. Perform direct Matrix Solid Phase Dispersion (MSPD) extraction on tissue.

Sample Pre-treatment Procedures for Bioanalytical Samples

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

In the world of biotherapeutics and biosimilars, regulatory agencies have characterization requirements that are primarily focused around the physicochemical properties of the therapeutics that affect efficacy, safety, and immunogenicity. Chromatography plays an important role in three specific areas of the physicochemical property analysis, being higher order structure, primary structure, and post translational modifications. One key technique to determine primary structure is peptide mapping, which typically involves a serine protease digestion of a purified or isolated protein followed by analysis using HPLC. The most common protease for these types of digestions is trypsin, however even within the digestion step there are many important practices we can apply to improve the result, and these will be discussed in the tech tip below.Most protein digests are done using trypsin due to its well defined specificity. Trypsin will only hydrolyze peptide bonds in which the carbonyl group is followed by either an arginine (Arg) or lysine (Lys) residue, with the following notable exceptions: when Lys and Arg are N-linked to negatively charged amino acids, or if proline (Pro) is positioned on the C terminal side Lys or Arg.Trypsin is also favored as the resulting peptides have basic residues at the C-terminus, making them easier to ionize by mass spec, which is typically the preferred method of detection for these applications. One final benefit of using trypsin is the mass range of the resulting peptides, which is typically between 600 and 4000 Da which is ideal for subsequent analysis when using either LC/UV LC MS/MS.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Data_Apex_Peptide_Mapping_Sample_Preparation_Good_Practices_for_Tryptic_Digests_3_87830e6064.jpg" alt="Phenomenex: Peptide Mapping Sample Preparation - Good Practices for Tryptic Digests" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Data_Apex_Peptide_Mapping_Sample_Preparation_Good_Practices_for_Tryptic_Digests_3_87830e6064.jpg 245w," sizes="100vw" width="1375" height="595"><h4>A basic tryptic digest comprises of a number of steps, for which a few key points of the process are summarized below:</h4><h5>Denaturation and reduction</h5>A chaotrope is necessary to completely unfold the protein such that it accessible for trypsin, for which urea or guanidine are commonly used. Urea is known to reduce sequence coverage as it is can break down and subsequently carbamylate both lysines and cysteines, and so will be avoided in some laboratories. The easiest reagent to use for denaturing is guanidine hydrochloride (GnHCl), however it must be removed with buffer exchange prior to digestion, as it inhibits trypsin.<h5>Alkylation</h5>There are two common alkylating agents, Iodoacetamide (IAM) and Iodoacetic acid (IAA). IAM is the most common as it reacts quickly, however IAA is also used, as it shows less non-specific alkylation. With both reagents the addition of dithiothreitol (DTT) will also help prevent further non-specific alkylation.<h5>Buffer exchange</h5>Once alkylation has taken place, it is critical to then remove residual alkylating reagents as well as any denaturing components prior to the digest. There are three main approaches to this type of sample clean up: dialysis, molecular weight cutoff spin filters, and desalting columns. Dialysis leads to long clean up times, but there is very little loss of sample. Spin filters and desalting columns are susceptible to loss, but do offer a more rapid method for sample clean up over dialysis.<h5>Digestion</h5>Here we are looking at the considerations for digestion using trypsin. Tryptic peptides can be relatively short, and there is also the possibility missed cleavages will occur. Porcine trypsin is most commonly used for digestion, and this enzyme has a preference for Arg, with a lower efficiency for Lys. Other missed cleavages include Asp or Glu next to Lys or Arg on the C terminus, as well as Proline (Pro) on the N terminus. Trypsin alone can only cover a restricted portion of the proteome, and parallel proteases also need to be used, including AspN or GluC in many cases for complete sequence coverage.When sequence coverage is critical orthogonal proteases can be used, and are summarized below.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Data_Apex_Peptide_Mapping_Sample_Preparation_Good_Practices_for_Tryptic_Digests_b0e11e0ee0.jpg" alt="Phenomenex: Peptide Mapping Sample Preparation - Good Practices for Tryptic Digests" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Data_Apex_Peptide_Mapping_Sample_Preparation_Good_Practices_for_Tryptic_Digests_b0e11e0ee0.jpg 245w," sizes="100vw" width="1232" height="529">Below is a summary of the type of parameter restrictions you should look to follow when digesting proteins with trypsin.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Data_Apex_Peptide_Mapping_Sample_Preparation_Good_Practices_for_Tryptic_Digests_2_f12992abfa.jpg" alt="Phenomenex: Peptide Mapping Sample Preparation - Good Practices for Tryptic Digests" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Data_Apex_Peptide_Mapping_Sample_Preparation_Good_Practices_for_Tryptic_Digests_2_f12992abfa.jpg 245w," sizes="100vw" width="1233" height="393">In conclusion, it is not always the analytical method or HPLC analysis which fully dictates sequence coverage when looking at primary structure of a protein. The steps and precautions taken during sample preparation can also play a vital role, and some of these are summarized above. Where appropriate steps are taken in the digestion of the protein, the subsequent HPLC or LCMS analysis becomes a much easier process to ensure adequate coverage is obtained.

Peptide Mapping Sample Preparation - Good Practices for Tryptic Digests

<ul style="list-style-type:disc;"><li>How do I select the correct chiral stationary phase for my analyte?</li><li>When looking at analyte structure, how do I determine which column to use?</li><li>I need a reversed phase chiral column to separate X?</li></ul>Chromatographic separations occur when a combination of stationary phase and mobile phase provide an environment where selectivity between analytes with different chemical properties can be expressed. In reversed phase chromatography for example analytes are retained hydrophobically by the stationary phase, and then eluted in order of increasing hydrophobicity. When looking at enantiomers the chemical and physical properties are identical and therefore some form of chiral recognition is required in order to achieve a separation.The mechanism for enacting chiral separations involves the formation of a diastereomeric complex, either between a mobile phase additive and the analyte of interest, or between the stationary phase and the analyte. Once formed it is possible to separate the enantiomers, either because the diastereomeric complex has different properties, or due to the stability of the complex formed, with one enantiomer being preferentially retained over the other. The use of chiral modifiers in the mobile phase, whilst possible is generally not adopted in HPLC (although is used in capillary electrophoresis) as the cost of such additives renders this option economically inviable in most cases. The types of chiral stationary phase utilized over the years include:<ul><li>Pirkle phases – Fully synthetic phases bases on functionalized amino acids, they provide chiral recognition by offering 3 points of interaction. Materials are generally bonded, so robustness is good. These types of phases generally exhibits, low hit rates but offer good selectivity when they work.</li><li>Cyclodextrin based phases – Fully synthetic bonded phases working on an exclusion principle with one enantiomer able to fit more precisely within the “chiral bucket” structure than the other.</li><li>Protein based phases – Proteins such as BSA are coated onto silica, offering a broader range of chiral interactions, resolution perhaps not as good as for Pirkle or cyclodextrin phases but these columns offer a broader range of selectivity. Care needs to be taken with protein bases phases not to poison the phase, or to wash it off the silica support during use.</li><li>Ligand exchange – The mechanism of ligand exchange uses penicillamine in combination with a mobile phase which contains copper II ions in a fairly specific way. The ions form complexes with amino acids allowing for them to be separated.</li><li>Polysaccharide based phases – These are based on the naturally occurring chiral polymers cellulose or amylose, the polymers are derivatised to provide additional interactions. Depending on the solvents used the polymers swell to different degrees which creates “chiral pockets” within the structure allowing analytes partition in and out of effecting a separation. Recent developments to these have included the immobilisation of such phases allowing for a broader range of solvents to be used than could be previously. Solvents such as DMSO and DMF can be utilized&nbsp;which can be helpful when considering preparative separations where analyte solubility may be challenging.</li></ul>Given the flexibility that polysaccharide phases offer by allowing normal phase, reversed phase, polar organic and SFC separations to be conducted with high efficiency, these phases have become the “go to” column choice for many users conducting chiral separations. The phases offer high hit rates which in turn increases the probability of finding a set of conditions what will provide an effective separation. Due to the complex nature of interactions between enantiomers and a chiral stationary phase it is not possible to accurately predict which stationary phase will provide a good separation. It is necessary therefore to screen different stationary phase and mobile phase compositions in order to test out the best combination for use in a separation. When screening our range of polysaccharide phases, Lux™, we screen all 3 modes normal phase, reversed phase and polar organic mode in tandem. Lux columns can also be successfully used under SFC conditions if users have access to this technology. We screen the following phases:<ul><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/lux-chiral-lc-column/lux-cellulose-1">Lux Cellulose 1</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/lux-chiral-lc-column/lux-cellulose-2">Lux Cellulose 2</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/lux-chiral-lc-column/lux-cellulose-3">Lux Cellulose 3</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/lux-chiral-lc-column/lux-i-cellulose-5">Lux i-Cellulose 5</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/lux-chiral-lc-column/lux-amylose-1">Lux Amylose 1</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/lux-chiral-lc-column/lux-i-amylose-1">Lux i-Amylose 1</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/lux-chiral-lc-column/lux-i-amylose-3">Lux i-Amylose 3</a></li></ul>Screening can also be conducted in LC-MS friendly conditions only if required. Precise details of screening can be found by viewing the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/Lux_Chiral_Screening_Poster_b5f9a0e291.pdf">chiral screening poster</a>. Once screening has been completed it is then necessary to review the results obtained, taking the most promising one or two forwards for optimization if required. This has been found to be the most effective way to develop chiral separations with good resolution and robustness.

A Systematic Approach to Chiral Screening and Method Development

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