When installing your capillary GC column, making accurate connections is essential to ensure trouble free operation. In this technical tip we provide advice on the best way to achieve this.<blockquote>Note: For users with sensitive detectors such as MS and ECD, column conditioning steps should be performed before installing the column to prevent contamination and frequent maintenance of the detector.</blockquote>1) Place the column nut and ferrule past the end of the column and cut a centimeter or two off the end of the column. Be sure that the ferrule is the right size and pointing in the correct direction. Inspect the cut with a magnifier and ensure that the cut is square and smooth. See figure below. Recut if needed.2) Insert the outlet end of the column into the detector exactly the distance prescribed in the instrument manual. Distances will vary between detectors. Tighten the ferrule nut finger-tight then 1/2 turn with a wrench. If the column can still be moved, tighten another 1/4 turn until the column is secure.3) Inspect the column connections for leaks using an electronic leak detector. Leaks at the inlet end may introduce oxygen to the column that will result in increased column bleed and damage to the column phase.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Gas_Chromatography_GC_Column_Installation_Detector_Installation_813b4d81a0.jpg" alt="Phenomenex: Gas Chromatography (GC) Column Installation - Detector Installation: Correctly and Incorrectly Cut GC Capillary." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Gas_Chromatography_GC_Column_Installation_Detector_Installation_813b4d81a0.jpg 245w," sizes="100vw" width="877" height="242">

Gas Chromatography (GC) Column Installation - Detector Installation

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

The effects of gradient on buffer capacity

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?

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 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?

<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?

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

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

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GC Column Selection by Column Dimension

Considerations when working with solid samples and SPE

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