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

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

When 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?

<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

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

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

<h3>A Deep Dive into Waters Oasis, Agilent Bond Elut, and Phenomenex Strata Workflows</h3><a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/what-is-solid-phase-extraction-spe">Solid phase extraction (SPE)</a> has become a widely used sample preparation technique across pharmaceutical, environmental, and food safety laboratories worldwide. SPE is used to separate and isolate analytes of interest from a matrix, utilizing the analytes' differential affinities between a solid stationary phase and a liquid mobile phase. The process involves four key steps: conditioning the solid phase; loading the sample onto the solid phase; washing the sample to remove interfering compounds; and eluting the analytes of interest. Following this, the now purified and concentrated sample will usually undergo further analysis such as GC-MS, LC-MS, or HPLC [1]. An often overlooked element within this method of sample preparation is the <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/article/5392">nitrogen blowdown evaporation</a> that bridges SPE extraction and instrumental analysis. This discussion explores the critical role of nitrogen blowdown in enhancing the performance of three widely used SPE platforms: Waters Oasis, Agilent Bond Elut, and Phenomenex Strata.<h3>Why Nitrogen Blowdown Matters in SPE Workflows</h3>In the final step of SPE, elution, an organic solvent like methanol, acetonitrile, or acetone is used to remove the analytes of interest from the solid phase. This solvent must then be removed before <a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/what-is-reconstitution">reconstitution</a> and analysis [1]. Nitrogen blowdown provides controlled, gentle evaporation that concentrates analytes while preventing thermal degradation and sample loss that can occur with other drying methods.<h3><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/215">Waters Oasis SPE</a>: Precision Evaporation for Enhanced Sensitivity</h3><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/215">Waters Oasis SPE cartridges</a> and plates are renowned for their broad analyte retention capabilities, in particular for polar compounds. Their SPE products consistently deliver consistent and pure quality for sample analysis [2]. Many of their protocols consistently emphasize the importance of proper solvent evaporation following elution [3].<h4>Critical Nitrogen Blowdown Steps in Oasis Protocols:</h4>In Waters' documented procedures for pesticide analysis, the protocol states: "Blow down the acetone extract with nitrogen to ca. 0.2 ml then make to 2 ml with pure water" [4]. This concentration step is essential for achieving the required detection limits of pesticide compounds like diuron and its metabolites. When using the Oasis HLB cartridges to process larger sample volumes (2 L versus 200 mL), the method requires: "Blow down the acetone extract with nitrogen to ca. 0.8 ml then make to 2 ml with pure water" [4]. This increased degree of sample pre-concentration dramatically enhances the analytical sensitivity and is particularly important when analyzing trace-level environmental contaminants. The Waters protocols emphasize that this evaporation step must be performed under controlled conditions to aid in the prevention of analyte loss, making nitrogen blowdown the preferred method over thermal evaporation techniques.<h3><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=bond+elut">Agilent Bond Elut</a>: Streamlined Workflows with Controlled Evaporation</h3>With over 40 different sorbent capabilities, Agilent's Bond Elut product line offers versatile solutions for a wide range of sample types and applications [5]. The company's method development guides consistently reference evaporation steps as critical for optimal results. Whether processing <a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/environmental-sample-preparation">environmental samples</a>, pharmaceutical matrices, or targeting polar and nonpolar analytes, the evaporation step remains a consistently critical factor in ensuring a method's success [5].<h4>Nitrogen Evaporation in Bond Elut Protocol:</h4>For Bond Elut PPL applications targeting polar compounds found within drinking water, Agilent's protocol specifies: "Dry the eluent under in 35 °C water bath, then reconstitute with 2 mL 10:90 MeOH/H2O" [6]. This controlled evaporation at moderate temperature ensures complete solvent removal while maintaining analyte integrity. The Bond Elut methodology emphasizes that the nitrogen drying step is particularly important when transitioning from organic elution solvents to aqueous-based mobile phases for <a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/preparing-samples-for-lc-ms/ms-analysis">LC/MS/MS analysis</a> [6]. This solvent exchange is impossible without complete evaporation of the initial organic eluent.<h3><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50">Phenomenex Strata</a>: Advanced Polymeric Performance Enhanced by Proper Evaporation</h3>Phenomenex Strata products, particularly their <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50&amp;search=strata+x">Strata-X</a> polymeric phases, have revolutionized SPE through water-wettable sorbents designed to work effectively with aqueous-based solutions with a simplified protocol [7]. However, even these advanced materials require proper post-extraction handling to achieve optimal results.<h4>Strata-X Protocol Requirements:</h4>Phenomenex documentation for their Strata-X PRO plates specifically instructs: "Dry: <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/article/5411">Evaporate the extract to dryness</a> under a gentle stream of nitrogen at room temperature" [8]. This room-temperature evaporation is particularly important for thermally labile compounds that might degrade under increased temperatures.<h4>Specialized Applications:</h4>For Strata Activated Carbon applications targeting nitrosamines and other polar analytes, the protocol requires: "Evaporate solvent under Nitrogen to required volume and reconstitute with Methylene Chloride" [9]. This controlled evaporation is essential for achieving the trace detection limits required for these potential carcinogens.The matrix removal technology within the Strata product line makes the nitrogen blowdown step even more critical. This step ensures complete removal of interfering matrix components from the eluent that could compromise downstream analysis [10].<h3>Technical Considerations for Optimal Results</h3><ul><li>Temperature Control: All three manufacturers emphasize controlled temperature during nitrogen evaporation. Waters and Agilent protocols frequently specify 35°C water baths, while Phenomenex often recommends room temperature evaporation [4, 6, 8].</li><li>Flow Rate Management: Gentle nitrogen flow is recommended across many manufacturers to reduce and prevent analyte loss through splashing or mechanical carryover. Excessive flow rates can lead to incomplete evaporation or sample loss.</li><li>Timing Optimization: Complete solvent removal is essential, but over-drying can lead to analyte degradation or potentially irreversible adsorption to container walls. Protocols typically specify evaporation "to dryness" or to specific volumes [4, 8].</li></ul><h3> A Nitrogen Blowdown Solution for the Leading SPE Platforms</h3>The importance of nitrogen blowdown in an SPE workflow has been well established. To support this critical step, Organomation offers the following solutions:<h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?manufacturerItems=540&amp;search=n-evap">N-EVAP Nitrogen Evaporator</a></h4><ul><li>Analyte Preservation: The gentle nitrogen blowdown helps preserve heat-sensitive analytes that may degrade when exposed to high temperatures.</li><li>Solvent Removal Flexibility: This system is compatible with a range of tube sizes, enabling researchers to process varying sample volumes tailored to specific laboratory needs.</li><li>Controlled Evaporation: Individual gas flow control at each sample position ensures precise and consistent sample preparation, which is essential for obtaining reliable data.</li><li>Temperature Control: Water bath models are available with a wide temperature range enabling optimization for use of different solvents needed for varying SPE sorbents.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Organomation_45_Position_N_EVAP_Nitrogen_Evaporator_5_20078e3969.jpg" alt="Organomation: 45 Position N-EVAP Nitrogen Evaporator" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Organomation_45_Position_N_EVAP_Nitrogen_Evaporator_5_20078e3969.jpg 156w," sizes="100vw" width="1400" height="1400"><h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?manufacturerItems=540&amp;search=multivap">MULTIVAP Batch Evaporator</a></h4><ul><li>Batch Capability: Some systems can process up to 100 samples simultaneously, making them essential for large-scale laboratory operations.</li><li>Sample Standardization: Provides consistent evaporation conditions across samples, reducing sample variability which can skew interpretation.</li><li>Analyte Integrity: Gentle nitrogen dry down protects delicate analytes from degradation.</li><li>Efficiency: This system significantly reduces sample preparation time, allowing for faster results and turnaround time in clinical settings.</li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Organomation_48_Position_MULTIVAP_Nitrogen_Evaporator_2_b46408a225.jpg" alt="Organomation: 48 Position MULTIVAP Nitrogen Evaporator" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Organomation_48_Position_MULTIVAP_Nitrogen_Evaporator_2_b46408a225.jpg 156w," sizes="100vw" width="1400" height="1400"><h4>Integration with Modern Laboratory Workflows</h4>Modern laboratories increasingly rely on automated nitrogen evaporation systems that can process multiple samples simultaneously while also maintaining precise control over temperature, flow rate, and timing. Additionally, these systems integrate seamlessly with the 96-well plate formats offered by all three manufacturers, enabling high-throughput sample preparation. The combination of advanced SPE materials from Waters, Agilent, and Phenomenex with controlled nitrogen blowdown creates a powerful sample preparation platform that delivers the sensitivity, reproducibility, and throughput required for today's analytical challenges.<h4>Conclusion</h4>Nitrogen blowdown is not merely an optional step in SPE workflows, it is a critical component that enables the full potential of Waters Oasis, Agilent Bond Elut, and Phenomenex Strata products. Nitrogen blowdown ensures optimal analyte concentration, solvent exchange, and method sensitivity through gentle evaporation. Its versatility makes it applicable to diverse fields, including environmental monitoring and pharmaceutical analysis.Laboratories investing in high-quality SPE products should equally prioritize incorporating proper nitrogen evaporation equipment and protocols to maximize their analytical capabilities and ensure consistent, reliable results. Organomation is a trusted provider of precision nitrogen evaporators, offering reliable solutions tailored to laboratory needs.

The Critical Role of Nitrogen Blowdown in SPE Sample Preparation

The combined effect on signal response of all components of the sample, other than the analyte itself, is called the matrix effect. Sample matrix components that co-elute with the analytes of interest could cause the analytes signals to be enhanced or suppressed, compared with those measured with a standard solution of analytes (so, in the absence of matrix). This discrepancy could bring significant issues in accuracy, when not assessed and/or adjusted for.To determine matrix effect, one can do a simple experiment by comparing the extracted analyte response to the non-extracted analyte response.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Matrix_effect_formula_3b77ff748f.jpg" alt="Phenomenex: Matrix effect formula" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Matrix_effect_formula_3b77ff748f.jpg 245w," sizes="100vw" width="1175" height="134">The most frequent approach to omit undesirable matrix effects is removing the matrix through sample preparation techniques (see Figure 1). These approaches can be as simple as sample filtration, protein precipitation and liquid-liquid extraction, or more sophisticated such as solid phase extraction and immunoaffinity capture. Matrix effects can in some cases be reduced or eliminated through LC method development, by using gradients or alternative column selectivities. However, sample preparation is often preferred because it helps to extend the LC column's lifetime.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_1_Common_sample_preparation_approaches_listed_in_order_of_lower_to_higher_selectivity_61e1f39c43.jpg" alt="Phenomenex: Figure 1 Common sample preparation approaches listed in order of lower to higher selectivity." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_1_Common_sample_preparation_approaches_listed_in_order_of_lower_to_higher_selectivity_61e1f39c43.jpg 220w," sizes="100vw" width="428" height="304">A common example of the matrix effect is the signal suppression observed when analyzing clinical samples, such as plasma or serum, with mass spectrometry detectors. In general, these samples undergo protein precipitation as the simplest clean up approach prior to analysis, which helps reduce possible protein build up on the LC column but does not remove other contaminants. Matrix components, especially phospholipids, may have an overall negative impact on LC analyses, and could even degrade system components over time.In Figure 2: we compare the interfering signal coming from phospholipids in: (A) a serum sample that has undergone protein precipitation only and (B) a sample that has been further cleaned up using solid phase extraction with Strata™-X PRO.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Figure_2_Phospholipid_traces_obtained_from_human_serum_samples_after_protein_precipitation_7e049e93c4.jpg" alt="Phenomenex: Figure 2 Phospholipid traces obtained from human serum samples after protein precipitation." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Figure_2_Phospholipid_traces_obtained_from_human_serum_samples_after_protein_precipitation_7e049e93c4.jpg 245w," sizes="100vw" width="677" height="386">After Strata-X PRO clean up, we observed a ten-fold reduction of the interfering signal from phospholipids. This polymeric sorbent was specifically designed for enhanced matrix removal, and as demonstrated above, can remove phospholipids and harmful particulates from the sample, improving analyte recoveries as well as reproducibility.We hope this tip was useful! Check out our tool and products highlighted below.<ul><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/products/strata-x-pro-solid-phase-extraction-products">Strata-X PRO SPE Products</a></li></ul><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Strata_X_PRO_SPE_Products_7d659c74c2.jpg" alt="Phenomenex: Strata-X PRO SPE" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Strata_X_PRO_SPE_Products_7d659c74c2.jpg 239w," sizes="100vw" width="387" height="253"><ul><li><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/tools/spe-method-development">SPE Method Development Tool</a></li></ul>

Sample Preparation Tip - What is the Matrix Effect?

This technical tip explores some issues encountered with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50">Solid Phase Extraction</a> and in particular to Analyte Recovery. Recovery relates to the percentage of analyte captured and retrieved through SPE.&nbsp; A couple of other commonly used terms relating to recovery, which may be of interest are:Relative Recovery: Indirect determination of analyte capture and retrieval during the extraction process based on the recovery of an internal standard (which is a compound with a structure similar to the target analyte) that is added to the crude sample/matrix prior to the actual extraction procedure.Absolute Recovery: Quantitative method for the determination of the actual amount of analyte captured and retrieved during the extraction process. Analyte recovery is based on an external standard that is added to the eluant at the end of the extraction procedure (rather than being based on an internal standard that is co-extracted with the analyte).<h5>Symptom</h5><ul><li>Analyte recovery is less than 100%.</li></ul><h5>Clue</h5><ul><li>Analysis of the unretained fraction (void volume) indicates that the analyte is present in the “flow through”.</li></ul><h5>Problem</h5><ul><li>Analyte binding is not quantitative during the sample loading step.</li></ul><h5>Cause /&nbsp;Solutions / Suggestions</h5>A. Column conditioning is improper, incorrect, or not optimized.:<ol><li>Condition column with methanol or isopropanol.</li><li>Use sufficient methanol or isopropanol to wet the entire sorbent bed; allow more than two column volumes to percolate slowly under low vacuum.</li><li>Follow the methanol or isopropanol directly with one column volume of a solution with a composition similar to the actual sample (pH adjusted) but without the analyte(s).</li><li>Do not use too much of this second “conditioning solvent” in (3) above, or allow it to remain in the column for too long.</li><li>Do not over-dry the column during or after conditioning (use low vacuum for ~1 minute).</li></ol>B. Sample/matrix is in (or contains) a solvent which is “too strong”.:<ol><li>Dilute the sample in a “weaker” solvent/solution to promote binding (less polar for Normal Phase, more polar for Reversed Phase, less salt, buffered and/or pH adjusted for Ion-Exchange.</li><li>Increase the sample dilution with a “weak” solvent.</li><li>Load less sample.</li><li>Increase the sorbent mass.</li><li>Decrease the flow rate during sample loading.</li><li>Use a “stronger” sorbent with a higher affinity for the analyte.</li><li>Add an organic modifier (or adjust the pH) to enhance binding affinity for the analyte(s).</li><li>Adjust the sample pH so that the analyte is neutral for Reversed Phase or charged for Ion-Exchange.</li><li>Add salt (5 to 10% NaCl) to increase the solvent polarity and to enhance the retention of highly polar analytes in Reversed Phase.</li><li>Add an ion-pair reagent to enhance binding of charged analytes in Reversed Phase.</li></ol>C. Column “mass overload” (column is too small and/or the total mass of bound solutes/components is too large).:<ol><li>Decrease the volume of sample loaded.</li><li>Increase the sorbent mass.</li><li>Use a sorbent with higher surface area.</li><li>Use a “stronger” sorbent.</li><li>Decrease the flow rate during loading (to enhance “diffusion”).</li><li>Decrease the column inner diameter but use the same sorbent mass (to increase the pressure drop, reduce flow, and enhance “diffusion”).</li><li>Dilute the sample in a “weaker” solvent to improve capacity.</li></ol>D. Flow rate is too high during the sample loading step.:<ol><li>Decrease the flow rate during loading.</li><li>Increase the sorbent mass.</li><li>Decrease the column inner diameter to reduce flow.</li><li>Use a sorbent with a higher surface area.</li></ol>E. Sorbent is “too weak” (has poor or low affinity for the analyte relative to the sample/matrix and/or the dilution solvent).:<ol><li>Switch to a “stronger” sorbent, to one with a higher ligand density, from a non-endcapped to an endcapped sorbent (or vice versa).</li><li>See “Solutions/Suggestions” for Cause C (“Column mass overload..”), above.</li><li>See Solutions/Suggestions for Cause B (“Sample/matrix is in (or contains) a solvent which is “too strong””), above.</li></ol>F. Conditioning solvent is “washed away” with large sample volumes.:<ol><li>Add 2% methanol or isopropanol to the sample to prevent alkyl chain collapse during the sample loading step.</li></ol>

Troubleshooting SPE

When performing analyte extraction with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50">SPE</a> we expect 100% recovery. If recovery is lower, this would suggest there is an issue with retrieval of the analyte from the sorbent. But what are some of the reasons you may see a recovery greater than what you were expecting (or over 100%)?<h3>Common problems</h3><ul><li>Co-eluting interferences</li><li>Contaminants from the solvent or sorbents selected</li><li>Internal standard is compromised making your reference point inaccurate</li><li>Recovery calculations are inaccurate</li></ul>Let's examine each of these common problems.<h4>Co-eluting interferences</h4>These are typically present in the matrix and the goal of the SPE step is to remove them. If you are experiencing recoveries greater than 100%, the first step would be to check the eluent using an orthogonal analytical technique to identify that the increased recovery is a result of a co-elution.&nbsp;Where a co-elution is identified, there are a few possible solutions.<ol><li>Modify the wash steps of the SPE method to selectively remove the impurity during washing</li><li>Select an alternative SPE sorbent which will better remove these impurities. This is easier if the analyte / impurity can be selectively ionized, as an ion-exchange sorbent will very often afford cleaner extracts than either reversed or normal phase solutions</li><li>Adjust your analytical method to resolve the 2 peaks analytically, negating the necessity to remove this impurity during the SPE step.</li><li>Run a blank plasma sample; this will allow you to ascertain the impurities present and use this blank in the calculations to subtract from your recovery</li></ol><h4>Contaminants from the solvent or sorbent</h4>These will typically be impurities present in the starting materials which are similar in nature to your compound of interest, and as a result co-elute with it during SPE and subsequent analytical analysis. You can follow the steps above to remove co-eluting impurities, or alternatively as this is not an impurity present in your matrix, you can identify the level present in the sorbent using blank runs and subtract this from your main peak during the calculation. Prior to addition of your sample if you equilibrate your sorbent with the elution solvent this should flush out potential contaminants from the sorbent. For impurities introduced from the solvent, typically switching the solvent should resolve this.<h4>Internal standard is compromised</h4>If your internal standard is not showing 100% recovery, your subsequent calculations will be inaccurate. It is always good practice to use an internal standard similar in nature to your compound of interest as this will give the more consistent reference point for your method.&nbsp;If there is concern your internal standard is not showing good recovery from the SPE material using your method, you can use an external standard which is added to the elution solvent to calculate absolute recovery.<h4>Recovery calculations are inaccurate</h4>It is important to follow this step wise to ascertain where the errors are appearing.<ol><li>Calculation Errors - Check all calculations and account for all dilution factors</li><li>Integration Errors - Reintegrate all peaks and use manual calculations to confirm</li><li>Analytical method errors – Ensure the analytical method used allows correct integration and calculation. Ensure no strong solvents are used for injection and run a calibration across a number of dilutions to ensure linearity. Troubleshoot your analytical method to determine any factors which may lead to poor reproducibility</li><li>Ensure detections selected are appropriate and working correctly.</li></ol>We hope this tip was useful! Check out our resources on <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/techniques/solid-phase-extraction">solid phase extraction</a>. See you next month for another tip!

Why are my recoveries greater than 100%?

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