In this technical tip we are pleased to bring you some valuable guidelines for Gas Chromatography (GC) related to installation, from column conditioning to installation testing and through leak checks. Ensuring proper column installation and performance is crucial for accurate and reliable analysis. Let's dive into some important good practices:<h4>Column Conditioning</h4><ul><li>Allow sufficient time for the carrier gas to flow through the column to purge any oxygen that may be in the system.</li><li>Raise the temperature of the column to the maximum isothermal operating temperature that is listed on your individual GC Column Test Report. Maintain this temperature until a constant baseline is achieved. Conditioning times will depend on the phase identity and thickness, with thicker films taking longer to stabilize. In order to minimize the downtime of the instrument, columns can be conditioned overnight at the maximum isothermal temperature.</li></ul><h4>Installation Testing</h4><ul><li>Inject a detectable unretained sample, such as methane for an FID, to determine dead volume time and linear gas velocity at the desired column temperature. Adjust gas pressure for optimal flow depending on carrier gas selection.</li><li>The non-retained peak must have ideal peak shape or installation is faulty and needs to be redone.</li></ul><h4>Checking for Leaks</h4><ul><li>Use a thermocouple-thermometer to check for leaks. It is highly sensitive to H2, He, and N2 and will not contaminate the instrument or column. Liquid leak indicators are not recommended for capillary columns. There is the risk of drawing the liquid into the column or fittings and contaminating the system.</li><li>Any carrier gas that is leaking from the column will alter the conductivity across the thermocouple. As such, just watch for fluctuations within the temperature reading, which would indicate a possible leak near the location of the thermocouple. This prevents any contact and possible contamination of the column.</li></ul>Note: If Vespel® ferrules are being used, leakage can occur after the initial heating phase due to ferrule deformation. Be sure that the fitting is re-tightened after this initial heating phase, then carefully check all corrections for leaks.Remember, following these practices will contribute to the optimal performance and longevity of your GC columns, reducing downtime due to system maintenance.

Gas Chromatography (GC) Column Conditioning, Testing and Checks

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?

<h4>1) Polarity&nbsp;</h4>Some indication of polarity will usually be provided by the GC column manufacturer, the value provided is normally determined using Kovats retention time indices together with McReynold’s numbers. The general advice is that stationary phase polarity should closely mirror the polarity of the analytes of interest, so a <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products?category=355&amp;manufacturerItems=50">non-polar phase</a> such as a ZB-1 (rated as 5 on the polarity scale) would be a good choice for hydrocarbon analysis whilst a <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products?category=7&amp;manufacturerItems=50&amp;search=WAX">ZB-Wax</a> (57 on the polarity scale) is often chosen for alcohols.<h4>2) Selectivity&nbsp;</h4>This is controlled by the different functional groups present within the polymeric stationary phase, the base of which is either polysiloxane or polyethylene glycol. Polysiloxane phases will typically have methyl groups, phenyl groups or cyanopropyl groups attached to the polysiloxane chains which allow for a variety of different interactions to occur:<ul><li>Methyl groups: van der waals or London forces,</li><li>Phenyl groups: π-π interactions,</li><li>Cyano groups: π-π and dipole-dipole interactions.</li></ul>Increasing the percentage of phenyl or cyanopropyl substituents in the stationary phase increases polarity. Polyethylene glycol (PEG)phases offer sites for dipole-dipole and hydrogen bonding interactions to take place, PEG phases are typically some of the most polar phases available.<h4>3) Length</h4>Increasing the length of the column increases the number of theoretical plates, which aids resolution, however increasing column length also increases the run time of any analysis conducted. A 30 m column is generally a good starting point for general method development whereas 60 m columns are typically used for the fingerprint analysis of complex mixtures. 15 m columns are generally utilised when the rapid separation of less complex mixtures is required.<h4>4) ID</h4>The choice of column ID impacts both column capacity (how much analyte you can inject) and efficiency. The narrower the column ID the higher the efficiency, but the lower it’s capacity. A good place to start with method development is a 0.25 mm ID column, which show a good compromise between efficiency and capacity. If you are converting a method from an old-style packed GC column to capillary GC columns then 0.53 mm ID columns are often chosen due to their higher analyte capacity.<h4>5) Film thickness</h4>Thin films provide the highest efficiency but have lower analyte retention. Thicker films have a higher capacity but lead to more bleed at high temperatures. If choosing to use a thin film (0.1 - 0.25 µm) with active analytes it is preferential to select a column which is highly deactivated to prevent peak tailing. Such columns generally provide lower bleed too.

What parameters should I consider when selecting a GC column for my application?

<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

This month’s technical tip focuses on a basic and crucial topic: How to choose the correct GC column dimensions? When starting a new project or when improving an existing method, it’s important to evaluate the contribution that each column dimension (Length, Internal Diameter (ID) and film thickness) brings to the method resolution, sensitivity, and robustness.Another factor to keep in mind, when investing in GC columns, is to choose a reliable source. Manufacturers perform multiple tests on their columns before they find their way to customers. These tests include resolution of critical pairs, column bleed test, retention factor of probe analytes, efficiency of the column and so on. Make sure to select a manufacturer that provides columns that are individually QC tested.To maximize efficiency in GC, column length, internal diameter (ID) and film thickness, each has a significant effect. Knowing the effect of column dimensions on a GC separation will help not only to select the right column dimension, but also to pick an alternate dimension to achieve efficient chromatographic separation, as well as possibly reducing the analysis time.Let’s see how these parameters influence separation in detail.<h4>Column Length</h4>The length of your column affects the rate of resolution improvement. As the length of the column increases, so does the resolution improvement. However, it will also increase run times. Under isothermal conditions, doubling column length only increases resolution by 41 %, but it doubles the run time! Choose a column length that balances efficiency with acceptable run times.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_GC_Column_Selection_by_Column_Dimension_1_8c584730ec.jpg" alt="Phenomenex: GC Column Selection by Column Dimension: Column Lenght." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_GC_Column_Selection_by_Column_Dimension_1_8c584730ec.jpg 245w," sizes="100vw" width="1702" height="598"><h4>Internal Diameter</h4>Column internal diameter (ID) has a major impact on both resolution and sample capacity. Unlike column length, using smaller ID columns can lead to faster run times, because the column length required with a small ID is often shorter due to increased efficiency.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_GC_Column_Selection_by_Column_Dimension_2_c95f77d2fa.jpg" alt="Phenomenex: GC Column Selection by Column Dimension: Internal Diameter." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_GC_Column_Selection_by_Column_Dimension_2_c95f77d2fa.jpg 245w," sizes="100vw" width="1740" height="583"><h4>Film Thickness</h4>Film thickness determines solute retention and plays an important role in column sample capacity. Thin film columns are faster and provide higher resolution, but lower sample capacity. In most instances, choose the thinnest film possible that still provides adequate retention. When working with active samples, using a slightly thicker film can significantly improve peak shape.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_GC_Column_Selection_by_Column_Dimension_3_8fb68c44cb.jpg" alt="Phenomenex: GC Column Selection by Column Dimension: Film Thickness." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_GC_Column_Selection_by_Column_Dimension_3_8fb68c44cb.jpg 245w," sizes="100vw" width="1740" height="583">

GC Column Selection by Column Dimension

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

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 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

<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

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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