A Case of Changing Solution pH. Formic Acid Stability in Solution (Methanol)

Real life examples help to better illustrate problems that I am called in to troubleshoot for clients. As a professional scientific consultant, many of my clients have spent months (sometimes years) trying to solve an analytical problem on their own before I am brought in to make the diagnosis and propose a solution. Many years of working in a wide range of scientific fields allows me to identify problems quickly and efficiently saving clients the most money and allowing them to resume work on their projects.

This was the case during a recent consult for a major cannabis testing laboratory. They were having a great deal of difficulty obtaining reproducible results for their analytical testing screens (14 compounds in their analysis with a need for repeatable and accurate results). Variations from 25% to 50% were observed run-to-run over the course of seven days. They assured me they were doing everything in the same way. To begin the troubleshooting process, we started by looking at the actual data gathered and the actual method(s) used to acquire the data. These were evaluated to see if they followed good practices and techniques, also to make sure they had SOP's in place which were clear. Good SOP's must include enough detail to allow anyone reviewing them to prepare samples, standards and/or solutions in the exact same way. Additionally, the HPLC instrumentation was checked and tested to verify it was performing as designed.

After reviewing their training and methodologies on-site, a number of areas of concern were quickly identified. One of the most likely reasons for the variation in values over time was found to be caused by a common mistake in the preparation of mobile phase solutions for the HPLC system. To save time, the client's scientists prepared all organic solvent solutions in advance (~ one month or more), then filtered and stored them at room temperature. For example, their solutions of 0.1% formic acid in HPLC grade Methanol were pre-mixed and stored in glass one liter bottles. These bottles were then put aside, for an average of one month before use. This finding proved key as someone with proper HPLC training would be aware of a well known problem when formic acid is left in pure organic solvent, especially methanol, over time (less so with ACN). Briefly, the formic acid content degrades quickly over time and is often found to be only half of what it was initially after just three or four days (If you have not done so already, this is a simple and useful experiment to run in your lab, monitoring the acid level by titration, not with a pH meter, over time at room temperature in methanol)! This degradation continues over time reducing the amount of acid in solution. If the acid is added to the solution to enhance ionization (i.e. LC-MS; LC-MS/MS) or provide acidification to maintain the sample in a fully ionized form, then as the level of acidification decreases, so does the solution's ability to maintain it. In other words, your HPLC method may change over time (resulting in an in-valid method).

•  I have always promoted the importance of making and using freshly prepared mobile phase solutions (daily), especially where any aqueous solutions are used (to prevent degradation of additives and/or bacterial or fungi growth). However, this precaution does not normally apply to many pure organic solvents, but there are a few very important exceptions to this, formic acid and methanol in this example. 

Changes were made to their SOP's to insure that future solutions of formic acid in methanol were not prepared in advance, but instead, fresh on the day needed only. This coupled with a few basic improvements to their column washing, equilibration and overall training resulted in %RSD of only 0.3% for future analysis runs.

 
As a side note, I have been asked why solutions of formic acid in methanol are sold commercially for HPLC use? I have no answer to this, but respectfully remind everyone that just because something is offered for sale, does not mean it should be purchased. Ask yourself if the item is appropriate for your application? It may not be suitable for your use or application. 

BTW: Please be sure to flush your HPLC system of all organic acids (e.g. acetic, formic) after use and do not leave them in the HPLC system overnight. Even 1% levels of organic acids may be corrosive to stainless steel. 

Preparation of Phosphate Buffered Saline (PBS)

PBS

While not commonly used in liquid chromatography, PBS solution is commonly used in preparing samples. Because chemical forms and grades vary, there are several recipes on the web. By popular request, I am provided a common laboratory recipe for a 1X PBS solution here.

To make Phosphate Buffered Saline (PBS), 1x pH 7.4 solution:

Method:

1. To a 1-liter Beaker, add the following four anhydrous salts:

a. 200 mg KCI

b. 8,000 mg NaCI

c. 240 mg KH₂PO₄

d. 1,400 mg Na₂HPO₄

2. Add about 850 ml of deionized water and stir to dissolve the salts. Adjust the pH to 7.4 with 10% phosphoric acid (phosphate solutions should be adjusted with phosphoric acid only). Transfer to a 1 L volumetric flask (class-A). Fill to the “line” with more deionized water. Cap and mix a final time to insure a uniformly mixed solution.

3. Filter the final solution through a suitable 0.22 micron filter before use.

Note: pH increases with decreasing temperature.

HPLC Solvents, Acetonitrile and Methanol, Key Differences and Properties

Widely used in RP HPLC method development, Acetonitrile (ACN) and Methanol (MeOH) are the two most common solvents you will use with water or aqueous buffers to develop methods. So, besides the fact that Acetonitrile is well know to have a higher elution strength / capacity than Methanol [*but NOT at high organic concentrations (e.g. 95% Methanol vs 95% ACN) where Methanol has a higher elution strength than Acetonitrile does], what other properties should chromatographer's be aware of? Let's discuss a few that all chromatographers should know.

PREPARATIONS of MIXTURES (A/B):
First, a few comments about the preparation of mobile phase solutions. 

• Only the purely aqueous portion can be correctly adjusted for pH. Do not try and measure or adjust the pH of the organic or organic solution mixture. 

     There are two common methods of preparing binary mixtures (V/V) of mobile phase solutions.

• Method #1 is to fill a volumetric flask with a specific volume of the "A" solution, then fill the flask up to the line with the "B" solution.

• Method #2 is to fill a graduated cylinder (or volumetric flask) with a specified amount of "A" solution; fill a second graduated cylinder (or volumetric flask) with a specified amount of the "B" solution and then mix the contents of both together.

Whichever method you use, please fully document it in your HPLC method so anyone reading it will be able to accurately reproduce it. The two methods described above are both correct in design, but will result in solutions with different properties.

ABSORBANCE of UV LIGHT:
For HPLC grade solvent (*we should always use HPLC or LC-MS grade solutions in HPLC analysis), ACN has the lowest absorbance (~ 190 nm) of the two making it well suited for low UV applications. HPLC grade MeOH has a slightly higher UV cut-off, around 205-210 nm, limiting its use in the very low UV ranges. *Methods which require low UV wavelengths (<230 nm) should not use Methanol as the primary solvent.

SOLVENT SOLUBILITY:
There is a significant difference between ACN and MeOH in their ability to dissolve many types of buffer salts AND samples. These differences may be critical during method development as higher salt concentrations could lead to plugs, clogs or precipation. 

Solubility of the Mobile Phase:

• A common reason for gradient runs to show poor reproducibility or to fail may be associated with running high concentrations of buffer combined with high concentrations of organic solvent. Most aqueous / organic solutions containing salt solutions of less than 10 mM concentration are not likely to precipitate under most gradient conditions (running to a max of 95% organic, not 100%). If high percentages of organic solvent are mixed with more concentrated buffer solutions, then the higher salt concentrations may precipitate out of solution during the analysis (resulting in clogs, leaks, plugs and/or inaccurate results). Be cautious when mixing organic solvents and buffers together for gradient analysis. Make sure the solutions used will stay in solution and be stable at all concentrations used. Also verify that the buffering capacity is still present when high organic concentrations are used (as your buffer will be diluted). *Not sure if the salt will stay in solution? Just mix up a sample at the same concentration for a test. Look at it. Is there any turbidity or particulate visible? You should have your answer.

• Methanol's overall better solubility characteristics (better than ACN) mean that it often does a better job of dissolving most salts (esp NH4, K and Na) at higher concentrations resulting in better performance and less precipitation.

Solubility of the Samples (changes to Peak Shape, Selectivity & Retention):

• A fundamental requirement of liquid chromatography is that the sample fully dissolves in the mobile phase (initial mobile phase). Dissolve the sample in the mobile phase or in a slightly weaker strength solution (not a stronger solution) before analysis. This insures it will be loaded onto the head of the column as a concentrated slug improving peak shape and RSD. If the sample does not fully dissolve in the mobile phase then you are not in fact analyzing the whole sample. Another area where Methanol may be superior to ACN can be found in its ability to fully dissolve more types of samples. This improved solubility may result in better overall peak shape. Methanol also has different selectivity, often better than ACN (not just the elution strength) which may result in peaks eluting at different retention times than expecting. This is another reason why we always try different mobile phase mixtures containing either ACN or MeOH when developing RP methods. Please never assume that one solvent will be better than the other. Too many novice chromatographer's use only ACN as their main organic solvent for method development. Please don't make their mistake as such a strategy indicates a lack of practical experience and knowledge. You must first try them both separately (ACN & MeOH) to evaluate the results with your own sample (best to start with comprehensive gradients at different pH values, as applicable). You will be rewarded for putting in the initial time to test both types of solutions as no simulator has yet been developed which can predict a truly accurate result with your own sample(s). You may be surprised to learn how many samples show better peak shape and performance using MeOH solutions. If no improvement is seen, document it and move forward with more confidence.

BACKPRESSURE & OUTGASSING:

• ACN is less viscous than MeOH ( 0.34 vs. 0.54 viscosity, respectively) and if used alone will result in lower column and system back-pressures overall. Less gas will dissolve into ACN vs MeOH. Mixtures of ACN and Water will also exhibit an endothermic reaction (cooling the solution) which can trap gas inside the solution. If you pre-mix your mobile phase, let it rest for several minutes after preparation.Mixtures of ACN and Water will show a pressure max around 70% ACN (*This is an unusual characteristic well worth learning).
  

• MeOH is more viscous than ACN alone. It also has an unusual property where a 50/50 mixture of MeOH and Water will result in a much higher system and column back pressure than either MeOH or Water alone will (*ACN has a similar property, but the peak pressure occurs between 60-70%). The effect with methanol is very Gaussian with a peak pressure observed with a 50/50 mixture. An exothermic reaction results from an initial mixture of the two solutions (MeOH and Water) releasing some gas. When preparing solutions it is best to allow the solution to rest for a few minutes to out-gas before topping off or using in the HPLC system.

I hope that this short discussion about some of the differences between these two popular HPLC solvents will aid you in developing better quality HPLC and LC-MS methods.

Reference: Table of HPLC Solvent Properties

pH Measurement of HPLC Mobile Phase Solutions and Buffers

Several times each month I am asked how to "correctly check and adjust the pH of an HPLC buffer solution which has an organic solvent component"? Well, the answer is to always check and adjust the pH of the purely aqueous solution first. Only pure aqueous solutions can be correctly adjusted for pH in the laboratory. Do not mix any organic solvent into the water based solution until after you have correctly adjusted the pH. The addition of an organic solution will throw off the final reading. Once the aqueous portion of your solution has been correctly adjusted to the desired pH value, then you can mix the solutions (or run an organic solvent gradient against the aqueous portion) as needed.

*This procedure also serves to make sure that all solutions used in chromatography are prepared in the same manner. It is true that the pH of the final mobile phase mixture (aqueous and organic mixture) may not be the same anymore, but the prepared stock solutions from which they were made will be the same each time, insuring reproducible results. Developing and describing chromatography methods and procedures which are highly reproducible equates to good scientific technique.

Adduct formation in LC-MS Analysis (esp. ESI)

Almost everything you analyze by Electrospray ionization mass spectrometry will create an adduct with something in the system. Normally, hydrogen is the most common adduct formed (M+1), but other chemicals, often in trace amounts may form adducts with your sample too. Sometimes we can take advantage of this fact and introduce our own adduct into the system (post column) to increase signal sensitivity or help us isolate one signal from another (the addition of an adduct can sometimes increase the signal seen for one species, but not the other). 

One of my favorite elements to form an adduct with is sodium (Na+). Two common forms are; sodium citrate and sodium acetate. Both have PKA’s between 3 and 6 so a variety of buffered solutions can be prepared for use. However, it is very important that we keep the concentration of sodium as low as possible so as to not clog the mass detector or suppress ionization completely (and see nothing BUT Sodium for weeks …). My suggestion is to initially prepare the buffers such that the solution is less than or equal to 3 mM in concentration. The lowest concentration should be used that yields reproducible results. Ranges from 0.1 mM to 5 mM are common. Only use the highest purity, volatile buffers (some manufacturer’s use names such as “ultra” to describe them) when preparing these ‘doping’ solutions for post-column addition and be sure and filter them through a 0.2 micron filter before use. A syringe pump can be used to deliver the solution during the run. A low flow rate should be used to infuse the adduct solution into the main inlet of the detector. Make sure you have a simple way of controlling the pump through the system (e.g. ‘On’ / ‘Off’, contact closure) so the flow can be turned off when you are not acquiring data. Be sure to not only monitor the baseline, but also measure true peak S/N values of a standard when evaluated the results (decreasing baseline noise may also mean the signal is decreasing too).

Ammonium (NH4) is another popular adduct to add to the system, often in the form of ammonium acetate. It reduces the chances of adding more sodium ions to the system (from glassware). Whichever adduct you use in your system, always start off testing as low a concentration as possible. Monitor the baseline carefully for noise and also to see if the addition of the compound is suppressing or enhancing the signal generated for your compound. Careful use of adducts in your system can provide you with another means to selectively enhance the signal of some compounds without changing the original chromatography method.

I must again emphasize to use the lowest concentration of doping agent. Proper pH control and mode choice are also very important. Use of a syringe pump for infusion, post column can help you to quickly optimize the fragmentor settings in real time.

• For additional information you may want to take a look at this useful Adduct Calculator webpage: "Mass Spectrometry Adduct Calculator(ESI)".

HPLC Solvent Properties Table (e.g. Polarity, Density, Boiling Point...)

Here is a link to a table listing common chromatography solvents and their physical properties (e.g. viscosity, polarity, UV absorption...). 

http://www.hplctools.com/lcsolvent.htm