A buffer is an AQUEOUS solution containing a weak acid and its conjugate base (or a weak base and its conjugate acid). The pH of a buffer solution changes very little when a small amount of strong acid or base is added to it and can help to negate condition changes in an analytical method when small environmental factors vary. Buffer solutions are used to maintain the pH at a nearly constant value. They are resistant to pH change due to the presence of an equilibrium between this weak acid (HA) and its conjugate base (B).How a buffer worksWhen some strong acid (more H+) is added to an equilibrium mixture of the weak acid and its conjugate base, the equilibrium is shifted to the left, in accordance with Le Chatelier's principle. Similarly, if a strong base is added to the mixture, the hydrogen ion concentration decreases by less than the amount expected for the quantity of base added.When preparing a buffer there are a few general guidelines you should adhere to:<ul><li>Calibrate pH meter; ideally at pH values above and below target for your buffer preparation. There are calibrated buffer solutions sold for this purpose</li><li>Weigh out buffer salt to give desired buffer concentration</li><li>Add to volumetric flask containing HPLC grade water and mix thoroughly to dissolve</li><li>Using a pH probe and pH meter, measure the pH of the solution</li><li>Adjust the pH to the desired value by additions of small volumes of strong acid and/or strong base</li><li>Once the pH is correct, dilute the solution to the final desired volume and mix well</li></ul>In HPLC, buffers are typically used to control secondary interactions which are generally regarded as ‘unwanted’ in the chromatographic world. They are also used to control the ionization state of the analyte to ensure it is not present in more than one ionized state. The pKa of a compound presents the pH at which it is 50% ionized and 50% neutral. If you work at this pH you will see split peaks and variable retention times. All methods should strive to work at least two pH units away from the pKa of their target analyte.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Vyznam_spravne_pripravene_mobilni_faze_822a0786d0.jpg" alt="Phenomenex: Figure 1" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Vyznam_spravne_pripravene_mobilni_faze_822a0786d0.jpg 245w," sizes="100vw" width="960" height="521"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Vyznam_spravne_pripravene_mobilni_faze_2_e5b5a8b4c3.jpg" alt="Phenomenex: Figure 2" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Vyznam_spravne_pripravene_mobilni_faze_2_e5b5a8b4c3.jpg 245w," sizes="100vw" width="960" height="320">When preparing isocratic mobile phases there are a few further considerations to ensure good method robustness<ul><li>Pre-Mix your mobile phase where possible, this will eliminate error from the pump</li><li>Always use the same method for preparation; for example, if you add the water first, you should always add it first. If you measure the individual components and then mix, you should always do it in this way. Do not change this process as the results can vary significantly in terms of analyte retention time.</li></ul>Below is an experiment which shows this variability when the mobile phases were mixed in the following ways<ol><li>Water first: 400 mL water in graduated cylinder, add to 1-Liter volumetric flask, dilute to mark with MeOH</li><li>MeOH first: 600 mL MeOH in graduated cylinder, add to 1-Liter volumetric flask, dilute to mark with Water</li><li>MeOH and Water measured individually: 600 mL MeOH in graduated cylinder, transfer to mobile phase reservoir; 400 mL Water in same graduated cylinder and transferred to mobile phase reservoir</li><li>Allow pump to mix A = Water with B = MeOH</li></ol><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_Vyznam_spravne_pripravene_mobilni_faze_3_51ea3293e9.jpg" alt="Phenomenex: Figure 3 - Kinetex™ 5µm XB-C18 150 x 4.6mm; 60:40 MeOH: H20 Flow rate 1.25mL/min Inj Vol 2µL" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_Vyznam_spravne_pripravene_mobilni_faze_3_51ea3293e9.jpg 213w," sizes="100vw" width="960" height="703">Buffer concentrations are also important, it is generally regarded that concentrations below 10mM do not offer the buffering capacity required to control silanol activity, or ionization state of a compound and 10mM would be considered the minimum effective concentration for reversed phase and HILIC. For other techniques such as size exclusion, significantly higher concentrations are required and it is not uncommon to see concentrations is excess of 100mM for these methods. Buffer concentration is something which must be carefully balanced to ensure solubility in all conditions can be achieved. When working under reversed phase conditions, you may need to limit the concentration of the buffer you are working with if the method contains high levels of organic (for example across a gradient) to ensure no precipitation of the buffer occurs.The mobile phase contributes as much to selectivity as your stationary phase and care with selection and preparation is as important as batch testing stationary phases when developing robust HPLC methods.

The importance of a well-prepared mobile phase

Each SPE mechanism/phase has a general set of solvent conditions under which SPE may be performed. Use the solvents/pH conditions listed below, volumes as determined in <a target="_blank" rel="noopener noreferrer" href="https://discover.phenomenex.com/0623-spe-technical-tip-part2-en?elqTrackId=E9D53E5725E641C0655E0409B9A065B7&amp;elq=00000000000000000000000000000000&amp;elqaid=4878&amp;elqat=2&amp;elqCampaignId=&amp;elqak=8AF59AC35AC6CFF8178E68A8DF72C3B8D482CF7C906A90E1BF5C55FDC1EA7D8B80B0">Method and Sorbent Volume Selection</a>.<h4>Reversed Phase SPE Method</h4><h5>Sorbent - SDB-L, C18, C8, PH, CN, X, XL</h5><ul><li>Analyte Properties: Low to moderate polarity (or nonpolar) Hydrophobic Neutralized/uncharged – Pharmaceuticals, Pesticides, Herbicides</li><li>Sample/Matrix: Aqueous, diluted with buffer – Biological fluids, Water</li><li>Conditioning Step<ul><li>Solvation – polar organic solvents – Methanol</li><li>Equilibration – aqueous, buffers – Water or buffer</li></ul></li><li>Wash Step: Aqueous buffers with 5 to 50 % polar organic solvent – Methanol:Water (5:95)</li><li>Elution Step: Polar or non-polar organic solvent(s) with or without water, buffer and/or strong acid or base – Methanol:Acetonitrile (50:50)</li></ul><h4>Normal Phase SPE Method</h4><h5>Sorbent - Silica, Florisil®, NH2, CN</h5><ul><li>Analyte Properties: Moderate to high polarity compounds (neutralized/uncharged) – Pesticides</li><li>Sample/Matrix: Non-polar organic solvents or moderately polar organic solvents – Hexane, chloroform, petroleum ether, toluene or methylene chloride</li><li>Conditioning Step<ul><li>Solvation – polar organic solvents (optional) – Methanol (optional)</li><li>Equilibration – sample/matrix solvent – Hexane or chloroform</li></ul></li><li>Wash Step: Non-polar organic solvents with a low concentration (1 to 5 %) of moderate to low polarity organic solvents – Hexane with 1 % THF, ethyl acetate, acetone, acetonitrile or IPA</li><li>Elution Step: Non-polar organic solvents containing higher concentrations (5 to 50 %) of moderate to high polarity organic solvents – Hexane with 10 % THF, ethyl acetate, acetone, acetonitrile or IPA</li></ul><h4>Strong Ion-Exchange SPE Method</h4><h5>Sorbet:&nbsp;Anion exchange: Screen-A, SAX, X-A, X-AW, XL-A, XL-AW Cation exchange: Screen-C, SCX, X-C, X-CW, XL-C, XL-CW</h5><ul><li>Analyte Properties: Ionized/charged compounds – Anion exchange: Acidic analytes Cation exchange: Basic drugs</li><li>Sample/Matrix: Aqueous; Low ionic strength buffers (&gt;30 mM), pH adjusted – Biological fluids plus buffer</li><li>Conditioning Step<ul><li>Conditioning – polar organic solvents – Methanol</li><li>Equilibration – low ionic strength buffers, pH adjusted – 25 mM Tris-OAc, pH 7</li></ul></li><li>Wash Step: Aqueous buffers of low salt concentrations with or without organic solvent<ul><li>Anion exchange: Buffer pH 7: Methanol (50:50)</li><li>Cation exchange:<ul><li>Buffer pH 6</li><li>1 M acetic acid</li><li>3. Methanol</li></ul></li></ul></li><li>Elution Step:<ul><li>Neutralize the charge on the weak anion or cation</li><li>Increase the ionic strength and counter ion concentration</li><li>Add a strong counter ion displacer<ul><li>Anion exchange: Hexane:ethyl acetate (75:25) + 1 % glacial acetic acid</li><li>Cation exchange: Methanol + 5 % NH₃</li></ul></li></ul></li></ul><blockquote>NOTE: These are general starting conditions. Please <a target="_blank" rel="noopener noreferrer" href="https://discover.phenomenex.com/customersupportEU?elqTrackId=9FD41A83311DD308567C001F2CC9485A&amp;elq=00000000000000000000000000000000&amp;elqaid=4878&amp;elqat=2&amp;elqCampaignId=&amp;elqak=8AF530A7A08EE95226A9F3C49828A74B76F7CF7C906A90E1BF5C55FDC1EA7D8B80B0">contact </a>your Phenomenex technical consultant for more assistance.</blockquote>

Generic SPE Method by Retention Mechanism

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

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