A 1276, 3346 (2013). 18, 19 x 100 mm, 5 m Part Number: the Waters Focused Gradient 186005421 Ammonium acetate, 10 M. BCIP, 5% (w/v) DEPC (diethylpyrocarbonate)-treated solutions. However, one could argue that the eluent produced in Case B is consistent with the description in the sense that after combining the aqueous buffer with the acetonitrile there will be 10 millimoles of ammoniaammonium present in 1 L of eluent (that is, the ammoniaammonium has been diluted by a factor of 10 by addition of the acetonitrile). The most common way to do this is to weigh out the ammonium acetate as described above (0.308 g), then use its liquid acid or base to adjust the pH as needed. The changes in pKa with the addition of different levels of acetonitrile are shown in Table I for some common buffering agents and simple analytes. Tindall, LCGC North Am. 36(1), 1824 (2018). Scenario 2: mobile-phase A: 5:95 (v/v) 100 mM ammonium acetate, wwpH 6-acetonitrile; mobile-phase B: 20:80 (v/v) 25 mM ammonium acetate wwpH 6-acetonitrile; mobile-phase gradient: 0-100% B in 2.5 min; flow rate: 1.2 mL/min; oven temperature: 35 C. In this second installment on the topic of eluent preparation for HILIC separations, we discuss some of the other practical factors to consider concerning buffers used in the aqueous component of the eluent. The effect of mobile phase buffer pH on the acetonitrile/100 mM ammonium formate. Temperature. A 1037, 283298 (2004). The ammonia solution had a concentration of 0.16 molar, and when we mix 100 milliliters of the ammonia solution with 100 milliliters of the solution of ammonium chloride, the total volume would double. Prepare 800 mL of distilled water in a suitable container. With this result in hand, we most commonly do our method development work using the scheme described in Scenario 1. a* Buffer Range Formula Buffering Equilibrium 10 mM Concentration Mobile-Phase Preparation** pH Adjustment (Acid or Base) Ammonium Acetate pK a 1 4.76 3.8-5.8 CH 3COONH 4 CH 3COOH CH 3COO-0.77 g CH 3COOH or NH 4OH . Case B: In this case we could prepare 1 L of buffer by adding 100 millimoles of ammonium acetate to 900 mL of water, adjusting the w w pH to 6.0 by addition of acetic acid, and then add water to bring the buffer to a final volume of 1 L. Then, we would combine 100 mL of this buffer with 900 mL of acetonitrile to produce the 90:10 organic . @!EW~f[T|rlcThIw{[#1n][xi]*\P^ $uwq,NJ.l)@56S&0)Tui;POjq"?~_Ma+D5mj(>oa`FBTD{>stream This buffer calculator provides an easy-to-use tool to calculate buffer molarity and prepare buffer solutions using the formula weight of the reagent and your desired volume (L, mL, or L) and concentration (M, mM, or nM). The fundamental aspects underlying answers to this question have been discussed at length over the years in LCGC (35) and elsewhere (6). (2) J.W. dNTPs: dATP, dTTP, dCTP, and dGTP . Here too, different methods of preparing buffers can have a significant impact on retention, selectivity, and reproducibility of HILIC separations. McCalley, J. Chromatogr. Recipes for cell culture media and reagents are located elsewhere in the manual. It is likely that this type of comparison carried out on different high performance liquid chromatography (HPLC) systems with different pump and eluent mixer designs will yield different retention patterns and different degrees of variation between the two scenarios. Stability: 6 months; Inspect daily for contamination. It is common to see a description of an eluent in the experimental section of a paper describing a HILIC separation that goes something like this: Description: 90:10 acetonitrilebuffer, with 10 mM ammonium acetate at pH 6. Dilute to mark with DI water. On the other hand, with Scenario 2 the ammonium acetate concentration delivered to the column stays constant at 5 millimoles per liter of eluent over a gradient that runs from 100% B to 100% A. Visually it is obvious that while the two scenarios produce different retention patterns, the repeatability of each separation is really very good. (7) A. Kumar, J.C. Heaton, and D.V. This appendix describes the preparation of buffers and reagents used in the manipulation of nucleic acids. However, ammonium acetate is a dual component buffer system, which buffers around pH 4.75 1 (acetate) and pH 9.25 1 (ammonium), but it has no significant buffer capacity at pH 7, contrary to common misconception. Clearly, these effects are important to HILIC separations particularly because much higher levels of acetonitrile are used in the eluent compared to what is normally used in reversed-phase LC. Citric Acid - Sodium Citrate Buffer Preparation, pH 3.0-6.2. (1) C.A. Add 25.703 g of Sodium Citrate dihydrate to the solution. Tindall, LCGC North Am. . 100 mM Acetate Buffer (pH 4.5): Dissolve 2.93 g sodium acetate trihydrate in 400 mL D.I. We see that whereas there is very little impact of the buffer concentration on the neutral compounds inosine and guanosine, the effect on the retention of amitriptyline is most pronounced with the bare silica column. RECIPES: Acids, concentrated stock solutions; Ammonium acetate, 10 Mobile Phase Buffers in LC: Effect of Buffer Preparation Method on Retention Repeatability, LCGC North America Volume 37, Issue 7, July 2019 . . The mobile phase buffer pH was measured before the addition of acetonitrile The mobile phase buffer pH can also affect the stationary phase charge state; this, for example is the case for silica phases, where the degree of silanol ionisation is dependent on the pH. This makes a 100 mM solution. Add 2.421 g of Citric Acid to the solution. Ammonium acetate "solution" should be used instead. 2. The way I prepared my 0.1M acetate. It will be mildly basic. Centrifuge each sample and measure activity and UV absorbance at 280 nm in the supernatant. Note that the two conditions were run at different times and the differences in peak height are simply a result of different preparations of the sample mixture. Let's say that according to the pKa of your analytes, you need to prepare a solution with a pH of 4.0. She developed a study focused on column re-equilibration in HILIC under Professor Dwight R. Stoll's supervision while she was a visiting postdoctoral fellow at Gustavus Adolphus College, in St. Peter, Minnesota. Mobile phase B: 50/50 ACN/water with 10 mM ammonium . Stoll, LCGC North Am. This appendix describes the preparation of buffers and reagents used in the manipulation of nucleic acids. pH Ranges of Selected Biological Buffers Chart (25 C, 0.1 M) Tris or Trizma Buffer Preparation - pH vs. Ammonium acetate, 10 M. BCIP, 5% (w/v) DEPC (diethylpyrocarbonate)-treated solutions. Adjust pH to 4.5 0.1 with 100 mM sodium acetate or 100 mM acetic acid. "WzV1& m@w`/`e#(ty[je *] oMg}n`FwNn%yb6Ebzqt^8Xn'jGN We close by reiterating the suggestion of Tindall that no matter what method of buffer preparation is used for your HILIC separation, be sure to describe your work using a complete, unambiguous description of how exactly the buffer was prepared so that others can reproduce the results of your work (5). How do I prepare a 0.1M acetate buffer? 'np8.XWqo'NhANywl]AGSaj,#WN *-:9$U4 Dolan, LCGC North Am. In many cases there is no single "right" answer, but we think data of the type shown here can inform the adoption of a set of best practices that will make method development for HILIC separations more robust, simple, and repeatable. The final solution pH was 9.0. This differential effect on pKa that depends on the chemistry of the compound can have a significant effect on the behavior of a separation. Figure 1: Retention time under gradient elution conditions for 5-methylcytidine and nortriptyline on two different HILIC columns: (a) bare silica (Ascentis Express HILIC); and (b) OH5 (Ascentis Express OH5). He is also a member of LCGC's editorial advisory board. Required components Prepare 800 mL of distilled water in a suitable container. To calculate the amount of buffer needed, please select a buffer from the Selection menu. Other. The 200 mM ammonium formate buffer was prepared by weighing out approximately 120 12.6 g of ammonium formate into a 1 L volumetric flask, adding 900 mL of water and 25 mL of 121 formic acid, and filling to the mark with water to a total volume of 1 L . As described by John Dolan in an article on buffers recently, systematic variation of the eluent pH should certainly be a part of method robustness testing to make sure that the method does not use a pH where analyte retention is sensitive to small changes in pH (2). 21(1), 2832 (2003). This appendix describes the preparation of selected bacterial media and of buffers and reagents used in the manipulation of nucleic acids and proteins. The dissociation constants of acids (that is, pKas) in water are influenced by the presence of organic solvents like acetonitrile and methanol. or of weak alkalis and their salts. 20(11), 10281032 (2002). Figure 1 shows the impact of pH on the retention of two compounds on two different stationary phases under HILIC conditions. . Lucy, C.B. Figure 2: Retention time under gradient elution conditions for different probe compounds on (a) bare silica (Ascentis Express HILIC); and (b) OH5 (Ascentis Express OH5) HILIC columns with either 10 or 100 mM ammonium acetate, wwpH 6 buffer used in the preparation of an acetonitrile-buffer eluent. Column: 100 mm 2.1 mm, 2.7-m Supelco Ascentis Express HILIC. . The salt concentration of ammonium acetate buffer is typically 10-20mM but can be varied up to 100mM. dNTPs: dATP, dTTP, dCTP, and dGTP . 331 0 obj <>stream Understanding these differences will be especially important in situations where a method will be transferred to a laboratory running different instrumentation. Here, we briefly review the main points, because they remain very important to the general topic of using buffers in HILIC separations. And, the consequences of the difference can be significant in the case of HILIC separations. We see that 5-methylcytidine, which is not ionized in water at pH values below about 12, is similarly retained on the two columns, and the retention is nominally unaffected by pH. (e.g., from water oxidation ) will lower the pH of a 10 mM ammonium acetate solution from pH 7 to 5.8. Nortriptyline, on the other hand, is much more retained on the bare silica column, and the dependence of retention on mobile-phase pH is much stronger than it is on the OH5 column. Dwight Stoll is the editor of "LC Troubleshooting." Mobile-phase A: 100 mM or 10 mM ammonium acetate, adjusted to wwpH 6.0 with acetic acid; mobile-phase B: acetonitrile; mobile-phase gradient: 95-80% B in 2.5 min for the bare silica column, and 3.27 min for the OH5 column; flow rates: 1.2 mL/min (bare silica) and 0.9 mL/min (OH5); column temperature: 35 C. Test the stability and occurrence of proteolytic activity by leaving an aliquot of the sample at room temperature overnight. What is Chemical Composition Distribution in Polyolefins, and Why Should It Be Part of Your HT GPC Analysis? (5) G.W. In reversed-phase liquid chromatography (LC), the pH of the eluent can significantly influence analyte retention, especially if the analyte contains one or more ionizable functional groups (2). This result can be rationalized by understanding that nortriptyline is present primarily in the protonated and positively charged form in water at pH values below about 9, and that deprotonated and negatively charged silanol groups on the exposed silica surface can interact strongly with the positively charged analyte through ionion interactions. Clearly, there is a difference in the properties of the eluents produced in these two cases. The most commonly used salting out agents in HIC are ammonium sulfate, sodium sulfate, and ammonium acetate. doi:10.1016/j.chroma.2012.12.037. H 2O; add 1.62 mL glacial acetic acid. Now, let's come back to the question we started with: should the pH be measured in the aqueous component of the eluent, or in the aqueousorganic mixture? Required components Prepare 800 mL of distilled water in a suitable container. Our view is that measuring or even using swpH calibrants in aqueousorganic mixtures to obtain the sspH is definitely useful for mechanistic studies of HILIC retention. All rights reserved. We discussed how different methods of preparing eluents composed of acetonitrile and aqueous components can lead to very different retention times and selectivities, and that we can help our colleagues by providing more detail when documenting eluent preparation procedures, whether for standard operating procedures (SOPs) or journal articles. We see that for some compounds the pKa values increase upon addition of acetonitrile, meaning the compounds become less acidic, whereas for others the pKa values decrease. Store the solution in tightly sealed bottles at 4C or at room temperature. Craven, C. Seidl, and D.R. 2 M ammonium sulfate, 50-100 mM sodium . Phosphate Buffer Preparation - 0.2 M solution. When possible, one should anticipate these effects during method development and decisions involving eluent pH. Add 7.721 g of Sodium Acetate to the solution. Claudia Seidlis a postdoctoral fellow in the Chemistry Department at the University of So Paulo, Brazil. Rinse vial with another 10 mL of 100 mM ammonium acetate pH 7 and transfer to the 150-mL flask/beaker. For an ammonium acetate buffer in water, the eluent pH used for separation should be 3.8 to 5.8 when using formic acid as the pH modifier and 8.5 to 10.5 when ammonia is used to adjust the eluent pH. Calibrating the pH electrode using aqueous calibrants and then measuring the pH of the aqueous buffer alone to obtain wwpH provides a useful reference point for the pH of the eluent. endstream endobj 332 0 obj <>stream Hello, I am in the process of making mobile phases for my HPLC research and I had one question about buffer. For example, to prepare a 100 m M ammonium acetate solution at p H 9, you'd first prepare a 100 m M ammonium acetate, and then add ammonium hydroxide dropwise until the desired p H was achieved (a titration, using a pH meter to determine endpoint). Certain details related to mobile-phase preparation that can be assumed in reversed-phase LC separations can be much more consequential in HILIC. We note here that these retention times were obtained under gradient elution conditions, and so the near doubling of retention time for amitriptyline is even more striking. 2023 MJH Life Sciences and Chromatography Online. Sample clarification In the January installment of LC Troubleshooting (1), we addressed several practical issues related to the preparation of eluents for hydrophilic interaction chromatography (HILIC) separations. Citric Acid - Na 2 HPO 4 Buffer Preparation, pH 2.6-7.6. New Liquid Chromatography Columns and Accessories: What to Know for 2023, New Sample Prep Products and Accessories for 2023. For example, 1 L of ammonium acetate (buffer 20, mM pH 4.0) will be prepared the following way: 100 mmol of ammonium acetate salt is dissolved in a little less than 1000 mL of water, the acidity of the solution is adjusted to pH=4.0 by (adding) acetic acid and total volume is adjusted to 1000 mL with water. We also discussed the effects of different buffer cations (for example, Na+ and K+) on retention and selectivity in HILIC separations. Potassium acetate buffer, 0.1 M. Potassium phosphate buffer, 0.1 M . Test the temperature stability in +10 C steps from +4 to +40 C. Potassium acetate buffer, 0.1 M. Potassium phosphate buffer, 0.1 M . The columns used to obtain the data shown here were provided by Dave Bell at MilliporeSigma. Ammonium acetate, also known as spirit of Mindererus in aqueous solution, . Mobile-phase A: 100 mM ammonium acetate adjusted to wwpH 6.0 with acetic acid, 100 mM ammonium formate adjusted to wwpH 3.0 with formic acid, or 100 mM ammonium bicarbonate adjusted to wwpH 9.0 with ammonium hydroxide; mobile-phase B: acetonitrile; mobile-phase gradient: 95-80% B in 5 min with a hold for 1 min at 80% B; flow rate: 0.6 mL/min; column temperature: 35 C. However, if we add acetonitrile to the eluent such that only 20% of the eluent is acetate buffer, the swpH (this nomenclature means that the pH is being measured in a solution containing organic solvent, while the pH standards are still in aqueous solution) will increase to about 8, whereas the pKa of the pyridinium ion will decrease to about 4. Stoll is an associate professor and co-chair of chemistry at Gustavus Adolphus College in St. Peter, Minnesota. Table 1. It is hoped that the habit of referring to pH 7 solutions as ammonium acetate "buffer" will disappear from the literature. (phenylmethanesulfonyl fluoride), 10 mM. In Scenario 1, the number of moles of ammoniumammonia per liter of eluent delivered to the column will actually increase from 5 to 20 millimoles per liter over the course of a solvent gradient program that runs from 95% to 80% B solvent. Scenario 1: mobile-phase A: 100 mM ammonium acetate at pH 6 adjusted to wwpH 6.0 with acetic acid; mobile-phase B: acetonitrile; mobile-phase gradient: 95-80% B in 2.5 min; flow rate: 1.2 mL/min; column temperature: 35 C. Therefore, since we're doubling the volume, we're halving the concentration of ammonia. So we went from 100 milliliters to 200 milliliters. His primary research focus is on the development of 2D-LC for both targeted and untargeted analyses. %sUTa/3? $LiI-^Mw1Ta]94k]zRWCMi^a n Popular answers (1) Adam B Shapiro Innoviva Specialty Therapeutics Prepare a solution of sodium acetate that is a little more concentrated than desired. Case B: In this case we could prepare 1 L of buffer by adding 100 millimoles of ammonium acetate to 900 mL of water, adjusting the wwpH to 6.0 by addition of acetic acid, and then add water to bring the buffer to a final volume of 1 L. Then, we would combine 100 mL of this buffer with 900 mL of acetonitrile to produce the 90:10 organicaqueous eluent-the same as in Case A. A\f9*Htal. Our point here is not that the method of eluent preparation described in Case A or B is better per se, but that the method used can have a significant impact on retention, and that descriptions of experimental conditions used for these separations should be written explicitly to avoid ambiguity. However, these steps are not necessarily required for practical work. Case A: We could prepare 1 L of buffer by adding 10 millimoles of ammonium acetate to 900 mL of water, adjusting the wwpH to 6.0 by addition of acetic acid, and then add water to bring the buffer to a final volume of 1 L. Then, we would combine 100 mL of this buffer with 900 mL of acetonitrile to produce the 90:10 organicaqueous eluent. We typically recommend about 1.5-2 M ammonium sulfate as a starting point. This post will focus on preparing high salt mobile phases and buffer conditions needed for HIC separations. Figure 2 shows the retention times obtained for neutral, acidic, and basic probe compounds on two different HILIC columns using either 10 or 100 mM ammonium acetate, wwpH 6.0 in the preparation of a 95:5 organicaqueous eluent. Suppose the eluent delivered to our HILIC column is nominally 90:10 acetonitrilebuffer-where the buffer alone is 100 mM in ammonium acetate-but that we would like to do gradients from more to less acetonitrile to accommodate analytes in our sample with a range of hydrophilicity. Set the. A buffer solution is prepared as a combination of weak acids and their salts (sodium salts, etc.) On the other hand, in Scenario 2 there are fewer mobile phase variables changing inside the column during the gradient, but making adjustments to the A and B solvents during method development is very tedious. Acetic Acid CH 3COOH 0.1% 1.0 mL 1 -- Yes Ammonium Hydroxide NH4OH 0.1% 1.0 mL 1 -- Yes Ammonium Hydroxide NH 4OH 0.2% 2.0 mL 1 -- Yes Ammonium Hydroxide NH 2 mM Ammonium Acetate buffer, pH-adjusted to 4.0. In our own work, we have compared the repeatability of HILIC separations carried out under the two scenarios described here. Adjust solution to final desired pH using HCl or NaOH Buffer Stock Solution (100 mM Ammonium acetate aqueous solution): Weigh 3.854 g ammonium acetate and quantitatively transfer to a 500 mL volumetric flask. 35(1), 2228 (2017). As we stated at the beginning of this series of column installments, we have been motivated to discuss the details of eluent preparation for HILIC separations in part because of the ambiguity we find in many descriptions of eluent conditions in the chromatographic literature. To prepare L of Acetate Buffer (pH 3.6 to 5.6): Change the value in the textbox above to scale the recipe volume Table 1. 118 formate (pH~ 3), 200 mM ammonium acetate (pH~9), and 100 mM ammonium acetate 119 (pH~6.4). (6) M. Ross, J. Chromatogr. (4) G.W. Some details related to mobile phase preparation that can be assumed or taken for granted in reversed-phase LC separations with little consequence can be much more consequential in HILIC separations. High-Throughput Analysis of Volatile Impurities in Sustainable Packaging using SIFT-MS, Automated Sample Prep in Regulated and Nonregulated Labs Just Got Easier. (phenylmethanesulfonyl fluoride), 10 mM. Dilute to 500 mL with D.I. %PDF-1.6 % However, given that the change in pKa because of the organic solvent is unknown, or at least not available in the literature, for many compounds of interest, simple scouting experiments are the most reliable way to understand the impact of eluent pH on retention of an ionizable compound under HILIC conditions. The question here is, should we prepare A and B solvents such that the ammonium acetate concentration of the eluent delivered to the column is constant over the duration of the gradient, or can we just have the ammonium acetate in the A solvent, which will result in not only an acetonitrile gradient but also an ammonium acetate gradient? If using a Binary Solvent Manager system, prepare the high pH A and B mobile phases as follows: Mobile phase A: 90/10 ACN/water with 10 mM ammonium acetate and 0.04% ammonium hydroxide: Add 50 mL of 200 mM stock buffer and 50 mL of MilliQ water to 900 mL of ACN. Mobile Phase Preparation Guide 132 Mobile Phase Formula Concentration Volume or Mass Preparation pH Adjustment MS Chemical Name (per 1 L) Procedure Number* Acid/Base Compatible? For a 100 mM solution the pH will only drop to 6.5 under . Now, the eluent produced in Case A is consistent with the description in the sense that the ammoniaammonium concentration in the buffer before adding it to the acetonitrile would be 10 mM. H 2O. Tindall, LCGC North Am. Ronald J. Midura, . Clearly the chemical dynamics inside the column will be much more complex in Scenario 1, but the upside of this approach is that the A and B solvents can be used very flexibly during method development. Given that the pKa of the pyridinium ion in water is about 5.2, roughly 80% of the pyridine injected into a column containing only the acetate buffer will be present in the column as pyridine and 20% will be present as the protonated pyridinium ion. Adjust solution to final desired pH using HCl or NaOH Add distilled water until the volume is 1 L. C. Seidl acknowledges support from the So Paulo Research Foundation - FAPESP - Process number 2016/02941-5 for her contributions to this article. The same is true for HILIC separations, and the effects can be even more pronounced than those in reversed-phase LC if a significant component of the retention of an analyte is dependent on electrostatic (that is, ionion) interactions. The other kinds of buffers, such as sodium or potassium phosphate buffers, sodium acetate buffers, formate or citrate buffers, can also be used. The last topic we'd like to touch on here concerns gradient elution for HILIC separations, particularly the composition of the two solvents used in the gradient elution program. Storage: 25 C in glass or plastic. Under these conditions, virtually all of the pyridine injected into the mobile phase will be present as the deprotonated pyridine free base, which in turn will significantly affect the way the analyte interacts with the stationary and mobile phases, especially relative to other analytes. doi:10.1016/j.chroma.2003.12.063. . Peaks: 1 = acenaphtalene, 2 = pyridine, 3 = deoxyuridine, 4 = benzoic acid, 5 = inosine, 6 = amitriptyline, 7 = methylguanosine. Sodium Acetate - Acetic Acid Buffer Preparation . When swpH can be measured reproducibly it is helpful for estimating the ionization state of the analyte in the eluent. The problem with descriptions like these is that they are ambiguous. Table II shows two possible scenarios that reflect this difference in operation. Suppose we use an acetate buffer with a measured wwpH of 6.0 (the wwpH nomenclature means that the pH is being measured in a completely aqueous solution, and that the pH electrode has been calibrated using pH standards that are aqueous solutions), and pyridine is one of our analytes.

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