Typical Commercial Strengths of Common Acids and Bases Used in HPLC

CHEMICAL NAME	MOLECULAR WEIGHT	MOLES / LITER	GRAMS / LITER	PERCENT by WEIGHT	SPECIFIC GRAVITY
Acetic Acid	60.05	6.27	376	36	1.045
Acetic Acid, Glacial	60.05	17.4	1045	99.5	1.05
Formic Acid	46.02	23.4	1080	90	1.21
Hydrochloric Acid	36.5	11.6	424	36	1.18
Nitric Acid	63.02	15.99	1008	71	1.42
Perchloric Acid	100.5	11.65	1172	70	1.67
Phosphoric Acid	98	14.7	1445	85	1.70
Sulfuric Acid	98.1	18.0	1766	96	1.84
Ammonia (in H20)	17.0	14.8	252	28	0.898
Potassium Hydroxide	56.1	13.5	757	50	1.52
Sodium Hydroxide	40.0	19.1	763	50	1.53

Data obtained from The Merck Index, 11^th edition (1989).

Popular LC/MS and HPLC Volatile Mobile Phase Modifiers

For applications which utilize an Evaporative Light Scattering Detector (E.L.S.D.), Charged Aerosol Detector (CAD) and/or Mass Spectrometer Detector with Electrospray Ionization source (e.g. LC/MS, MSD or LC/MS/MS), a fully volatile buffering system is usually required. Many of the common HPLC buffers such as sodium or potassium phosphate are not compatible.Use the smallest amount of buffer which provides buffering under the analysis conditions (e.g. 10mM). *Select a buffering agent (or modifier) which are within 2 pH units (+/- 1) of the sample's pKa and 2 pH units away from any acid's pKa. 

• For LC/MS applications: Positive ion mode favors acidic mobile phases and Negative ion mode favors basic mobile phases. However, feel free to experiment using both ionization modes and don't forget about using adducts (e.g. ammonium and sodium) with all types of samples to improve signal response. *Maintain these buffers at or below 10 mM. Adjust the pH of the mobile phase to be 1 to 2 units away from your sample's pKa.

Table 1:  Popular examples of useful volatile mobile phase buffers, modifiers and/or additives.

BUFFERING/MODIFIER AGENT                                   USEFUL pH RANGE

• Ammonium formate                                 2.8 - 4.8; 8.2. - 10.2
• Formic Acid                                            3.3 - 4.3
• Pyridine/Formic Acid                               3.3. 4.3, 4.8 - 5.8
• Trimethylamine/Formic Acid                     3.3 - 4.3, 9.3 - 10.3
• Ammonium Acetate                                  3.8 - 5.8; 8.2 - 10.2
• Acetic Acid                                              4.3 - 5.3
• Trimethylamine/Acetic Acid                      4.3 -5.3, 9.3 - 10.3
• Ammonia/Formic Acid                              3.3 - 4.3, 8.8 - 9.8
• Ammonia/Acetic Acid                               4.3 - 5.3, 8.8 - 9.8
• Ammonium Bicarbonate                           5.9 - 6.9,  8.8 - 9.8
• Ammonium Carbonate                              5.9 - 6.9, 8.8 - 9.8  
• Carbonic Acid                                            6 - 8 (pKa 6.37/pKb 7.63)
• 1-Methylpiperidene                                   10.0 - 12.0  

• Trifluoroacetic Acid (TFA)                        pKa = 0.3 (WARNING when used with MS

                                                                                     systems!).  See notes #2 and #4 below.           

*Notes: (1) Formic acid (3.75) is slightly stronger and more volatile than Acetic acid (4.75). Formic acid is often available in higher purity grades and absorbs less in the UV region making it a better choice for most chromatography applications. It works well in positive mode LC/MS analysis, esp at 0.1%. (2) Trifluoroacetic acid (TFA, pKa = 0.3) is very strong and volatile, but we do not recommend its use in LC/MS applications as it can increase the background signal levels (esp. in Negative Mode) LC/MS (m/z 113), be very hard to remove from the source and result in long term instrument contamination. Difluoroacetic acid (DFA) and ammonium formate are other alternatives as they offer good ion pairing capacity with less ion suppression problems. (3) Triethylamine (TEA, pKa 11) is volatile, strong and very stable, but causes similar contamination problems resulting in high background signals when used in Positive Mode LC/MS (m/z 102). (4) Many ion-pairing reagents suppress ionization, bind to the plastics and metals used and contaminate the flow path. If you must use them, please do so using the lowest possible concentrations levels and thoroughly decontaminate the entire flow path of the system after use (or dedicate the MS system to use with them only). Minimize further contamination by labeling and using a dedicated column for the application (Do not use that same column exposed to ion pairing compounds for any other methods or applications). (5) Acids and bases alone provide little "buffering" so should be used with a secondary buffering species to resist change in pH.

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)".