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

More often than not, GC column problems are traceable to something improperly done during installation. The table below may assist you in identifying and correcting common installation problems highlighted by your Column Performance Test Mixture.&nbsp;<h3>Symptom: Tailing Peaks</h3><h4>Possible Cause &nbsp;&nbsp;/ &nbsp;&nbsp;Suggested Remedy</h4><ul><li>Contaminated or active injector liner or column:&nbsp;<ul><li>Clean or replace injector liner.</li><li>Solvent rinse or replace the column.</li></ul></li><li>Dead volume due to poorly installed liner or column: Reinstall liner and column as necessary.</li><li>Ragged column end: Score the tubing lightly with a sapphire scribe or a ceramic scorer wafer before breaking it. Examine the end (a 20-power magnifier glass is recommended). If the break is not clean and the end square, cut the column again. Point the end down while breaking it, and while installing a nut and ferrule, to prevent fragments from entering the column. Reinstall the column.</li><li>A bad match between the polarities of the stationary phase and the solvent: Change the solvent.</li><li>A cold region in the sample flow path: Remove any cold zones in the flow path. Debris in the liner or column:&nbsp;<ul><li>Clean or replace the liner.</li><li>Cut a centimeter or two off the ends of the column and reinstall.</li></ul></li><li>Injection takes too long: Improve injection technique.</li><li>Split ratio is too low: Increase split ratio.</li><li>Overloading the inlet: Decrease the sample volume.</li><li>Some types of compounds such as alcoholic amines, primary and secondary amines, and carboxylic acids tend to tail:&nbsp;<ul><li>Try a different column.</li><li>Make a derivative of the sample.</li></ul></li></ul>

Peak Shape Problems: Tailing Peaks

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

In this technical tip, we will address a common issue encountered in Gas Chromatography (GC) analysis: Broad solvent peaks/fronts. These distorted solvent’s peak shapes can adversely affect the accuracy and reliability of your results making determination of early eluting compounds very difficult. Let's explore possible causes and suggest remedies:<h3>Symptom: Broad solvent peaks / fronts</h3><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Peak_Shape_Problems_Broad_Solvent_Peaks_Fronts_b4c36131a7.jpg" alt="Phenomenex: Peak Shape Problems: Broad Solvent Peaks/Fronts" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Peak_Shape_Problems_Broad_Solvent_Peaks_Fronts_b4c36131a7.jpg 245w," sizes="100vw" width="627" height="352"><h4>Possible Cause &nbsp;/ &nbsp;Suggested Remedy</h4><ul><li>Bad column installation: Reinstall column.</li><li>Injector leak: Find and fix leak.</li><li>Injection volume too large: Decrease sample size.</li><li>Injection temperature too low: Increase injection temperature so the entire sample is vaporized “instantly”. An injection temperature higher than the temperature limit of the column will not damage the column.</li><li>Split ratio is too low: Increase split ratio.</li><li>Column temperature too low: Increase column temperature. Use a lower boiling solvent.</li><li>Initial column temperature too high for splitless injection: Decrease the initial column temperature. Use a less volatile solvent so the initial column temperature is at least 10°C below the boiling point of the solvent.</li><li>Purge time too long (splitless injection): Use a shorter purge activation time.</li><li>Septum purge plugged or turned off: Remove plug or turn septum purge on.</li></ul>By addressing these potential causes and implementing the suggested remedies, you can easily overcome the issue of broad solvent peaks/fronts, improving the peak shape and the overall accuracy of your GC analysis.

Peak Shape Problems: Broad Solvent Peaks/Fronts

<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

<h4>No Peaks - Some or all peaks are missing from the run</h4>How many times have you found that some or all peaks are suddenly missing from the run?In this month’s GC technical tip, we explore some of the possible causes and solutions to overcome peak disappearance, by evaluating three possible scenarios that should cover all possible real cases.<h3>Symptom: All Peaks Missing</h3><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Peak_Shape_Problems_No_Peaks_b0d5e8c344.jpg" alt="Phenomenex: Peak Shape Problems: No Peaks" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Peak_Shape_Problems_No_Peaks_b0d5e8c344.jpg 245w," sizes="100vw" width="1516" height="943"><h4>Possible Cause &nbsp;/ &nbsp;Suggested Remedy</h4><ul><li>Defective or clogged syringe.: Try a new or proven syringe.</li><li>“Blown” septum or massive leaks at the inlet.: Find and fix leaks.</li><li>Problems with carrier gas flow.: Adjust gas flow.</li><li>Column may be broken or installed in the wrong detector or inlet.: If breakage is close to the beginning or end, cut off the short piece. Breakage in the middle can be repaired with a press-fit connector. For multiple breakages, replace or reinstall the column.<ul><li><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/GC_Column_Instalation_Guide_73f12586a8.pdf">See column installation</a>.</li></ul></li><li>The detector is not functioning or is not connected to the recorder or integrator.: Ensure detector is working properly. (e.g. is the flame in a FID lit?) Check connection to the output device.</li><li>Incorrect injector temperature:<ul><li>Injector too cold: sample is not vaporized.: Cold injector: check injector and oven temperature with an accurate thermometer. If accurate, increase temperature as necessary, but do not exceed the maximum temperature limit of the column. Inject the sample directly onto the column.</li><li>Injector too hot: thermally labile sample is decomposing.: Hot injector: check injector and oven temperature with an accurate thermometer. If accurate, reduce temperature as necessary, ensuring compatibility with sample and column minimum limit.</li></ul></li></ul><h3>Symptom: No Peaks After Solvent Peak</h3><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Peak_Shape_Problems_No_Peaks_2_5f1459f9d3.jpg" alt="Phenomenex: Peak Shape Problems: No Peaks" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Peak_Shape_Problems_No_Peaks_2_5f1459f9d3.jpg 245w," sizes="100vw" width="1481" height="943"><h4>Possible Cause &nbsp;/ &nbsp;Suggested Remedy</h4><ul><li>Sample volume is too high.: Inject less sample or use a higher split ratio.</li><li>For FID detectors, the flame is blown out by the solvent peak.: Check the detector temperature.</li><li>Carrier gas flow is too high.: Reduce the flow rate.</li><li>Incorrect column temperature; column is too hot and sample is eluting in solvent peak.: Check oven temperature with an accurate thermometer. If accurate, reduce temperature as necessary, ensuring compatibility with sample and column minimum limit.</li><li>Column cannot separate components from solvent.: Change solvent or column.</li></ul><h3>Symptom: Some Peaks Missing</h3><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Peak_Shape_Problems_No_Peaks_3_be7fd462a8.jpg" alt="Phenomenex: Peak Shape Problems: No Peaks" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Peak_Shape_Problems_No_Peaks_3_be7fd462a8.jpg 245w," sizes="100vw" width="1453" height="886"><h4>Possible Cause &nbsp;/ &nbsp;Suggested Remedy</h4><ul><li>Activity in the inlet liner or column if the missing peak is an active compound.: Clean or replace the inlet liner. Ensure an inert column is used. If necessary, replace the column. See column installation.</li></ul>When some or all peaks are missing in a GC analysis most causes are related to the detector, which plays a crucial role, but do not dismiss other possible causes, such as a defective syringe, an improper injection or an activated column or liner.See you on the next technical tip, feel free to contact us if you need support in troubleshooting or for your method development.

Peak Shape Problems: No Peaks

Polysaccharide chiral stationary phases are widely used in enantiomeric separations. There are numerous benefits to using a polysaccharide stationary phase which include the wide chiral recognition they offer stemming from the numerous chiral sites found in the sugar molecule used to form the stationary phase. To these sugar molecules polar aromatic groups are bound to enhance the selectivity. This enhancement is which offers greater chiral recognition over chiral stationary phases of a different nature&nbsp;Polysaccharide phases offer good flexibility in terms of the type of mobile phase you can work with and allow the user to use normal phase, polar organic, reversed phase and SFC modes, all with a single chiral column. The stationary phases are strong and robust and offer good loadability as they are silica based.The polysaccharide based chiral selector can come coated or immobilized onto the silica particle and this can affect selectivity, either directly through a change in interaction with the phase or indirectly through the use of different solvents available to you when you use an immobilized phase in place of a coated phase.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_dbbd1cd098.jpg" alt="Phenomenex: Advantages of using immobilized stationary phases in chiral separations" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_dbbd1cd098.jpg 245w," sizes="100vw" width="1709" height="743"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_2_a84f55b812.jpg" alt="Phenomenex: Advantages of using immobilized stationary phases in chiral separations" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_2_a84f55b812.jpg 245w," sizes="100vw" width="1490" height="797">Most polysaccharide-based chiral stationary phases are coated, and this means the stationary phase is not covalently bonded to the underlying silica. A number of chiral selectors in recent years have been immobilized to the silica particle which means they are covalently linked and infer greater stability in a wider range of more aggressive solvents. The ability to use an expanded range of solvents has increased the selectivity options of these chiral selectors and below we give some tips on how to maximise chiral selectivity using immobilised polysaccharide chiral stationary phases.&nbsp;With an immobilised phase you have a wider range of organic solvents at your disposal during method development, this can allow you to resolve notoriously problematic enantiomers by working with stronger organic solvents.&nbsp;Here baseline resolution of Ornidazole is achieved on the immobilised <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2156">Cellulose-5</a> phase by introducing MtBe into the mobile phase and this combination shows greater resolution compared to the coated<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2153"> Cellulose-1 </a>phase.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_3_d35608168c.jpg" alt="Phenomenex: Advantages of using immobilized stationary phases in chiral separations" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_3_d35608168c.jpg 245w," sizes="100vw" width="1321" height="522"><h4>Chiral Separation of Ornidazole on Lux 5 µm i-Celluslose-5</h4><ul><li>Column: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2156">Lux 5µm i-Cellulose-5</a>, LC Column, ea.</li><li>Dimensions: 250 x 4.6 mm ID</li><li>Order No.: 00G-4756-E0</li><li>Elution Type: Isocratic</li><li>Eluent A: 0.1% Ethylamine in MtBE</li><li>Flow Rate: 1 mL/min</li><li>Column Temperature: ambient</li><li>Detection: UV-Vis Abs.-Variable Wave. (UV) @ 254 nm (ambient)</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_4_403e57f26b.jpg" alt="Phenomenex: Advantages of using immobilized stationary phases in chiral separations" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_4_403e57f26b.jpg 245w," sizes="100vw" width="1188" height="581"><h4>Ornidazole on Lux 5 µm Cellulose-1 in NP</h4><ul><li>Column:<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2153"> Lux 5µm Cellulose-1</a>, LC Column, ea.</li><li>Dimensions: 250 x 4.6 mm ID</li><li>Order No.: 00G-4459-E0</li><li>Elution Type: Isocratic</li><li>Eluent A: Hexane / etOH (80:20) DEA (0.1%)</li><li>Flow Rate: 1 mL/min</li><li>Column Temperature: ambient</li><li>Detection: UV-Vis Abs.-Variable Wave. (UV) @ 220 nm (ambient)</li></ul><h3>Injecting using strong organic solvents:</h3>Many applications require the use of a strong organic solvent for injection – with a coated phase these can lead to stationary phase dissolution and loss of selectivity. With an immobilised phase no damage is done and selectivity is maintained throughout the lifetime of the column. Here we see no change in resolution when an immobilised phase is used even with a DMSO injection solvent.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_5_bf95cb7fe4.jpg" alt="Phenomenex: Advantages of using immobilized stationary phases in chiral separations" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Advantages_of_using_immobilized_stationary_phases_in_chiral_separations_5_bf95cb7fe4.jpg 200w," sizes="100vw" width="1009" height="789"><h4>Conditions for all columns</h4><ul><li>Column: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2156">Lux 5µm i-Cellulose-5</a></li><li>Dimensions: 250 x 4.6 mm ID</li><li>Order No.: 00G-4756-E0</li><li>Mobile Phase: Methanol/DEA (100:0.1)</li><li>Flow Rate: 1.5 mL/min</li><li>Detection: UV @ 280 nm</li><li>Temperature: 27 C</li><li>Sample: Laudanosine</li><li>Dilution Solvent: Dimethyl Sulfoxide (DMSO)</li></ul>In summary with polysaccharide chiral stationary phases you have a wide chiral recognition and vast permutations of mobile phase systems and selectivity’s at your disposal. This can help ensure good enantiomeric resolution for almost all chiral molecules when a methodical and thorough screening process is done.

Advantages of using immobilized stationary phases in chiral separations

For most reversed-phase SPE applications using a silica-based SPE media (e.g. those using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1507">Strata™ C18-E</a>), cartridges (or wells in the case of 96-well plates) are initially conditioned using an organic solvent such as methanol, followed by rinsing with water or a buffer. If silica-based SPE media is allowed to dry out for extended periods of time during these conditioning steps, they may exhibit poor retention for target analytes, especially if the target compounds are polar in nature. It is therefore essential that SPE users pay close attention to their methods during this crucial conditioning step in order to obtain accurate and reproducible results.&nbsp;The figure below shows two <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1546">Strata C18-T </a>cartridges (1 gm/6 mL) that were loaded with 4 mL of a blue dye (CuCl2). The cartridge on the left (A) was first conditioned using 12 mL of methanol, then was dried for ten minutes under high vacuum (10” of Hg), followed by 12 mL of water. As you can see from the figure, the blue dye is found uniformly distributed throughout the sorbent bed. In contrast, the cartridge on the right hand side (B) was conditioned with methanol, followed immediately by water without any drying. Under these conditions, the blue dye is visible as a tight, focused band at the head of the sorbent bed. After elution, recovery from cartridge B was far greater than was the recovery from cartridge A (the dried cartridge). This difference in recovery can be attributed primarily to the poor retention of the dye compound due to drying of the sorbent bed.&nbsp;<h3>Tips to Prevent Accidental over-drying to prevent loss of Analyte</h3><ul><li>Don’t allow your bed to dry out at any step (unless it is specifically indicated in the method).</li><li>Use gravity flow as an alternative to vacuum to prevent over-drying.</li><li>Centrifuge at low velocities (e.g. 500 RPM) or use positive pressure.</li><li>Switch to an equivalent polymeric sorbent, such as Strata-X, which do not display shifts in retention and recovery due to periodic drying</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Over_Drying_of_Silica_Based_SPE_Cartridges_Can_Lead_to_Poor_Sample_Recovery_0839e0ef05.jpg" alt="Phenomenex: Over-Drying of Silica-Based SPE Cartridges Can Lead to Poor Sample Recovery: A. Silica C18 sorbent dried after conditioning with methanol; B. Silica C18 sorbent conditioned without drying." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Over_Drying_of_Silica_Based_SPE_Cartridges_Can_Lead_to_Poor_Sample_Recovery_0839e0ef05.jpg 245w," sizes="100vw" width="548" height="312">

Over-Drying of Silica-Based SPE Cartridges Can Lead to Poor Sample Recovery

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