Proper mobile phase preparation to minimize contaminants

Overview

Mobile phase components (water, solvents and buffers) are not only used for making mobile phase but also for preparation of samples, standards and blanks as well as rinsing of bottles and vials and hence quality of mobile phase components can have a big impact on quality of chromatography. Impurities in any of these cause high level of background signal which leads to noisy baseline and reduction in Limit of quantitation (LoQ) as well as Limit of Detection (LoD)

Solvents:

Most researchers purchase solvents that are certified for use in LC-MS but these certifications vary from vendor to vendor and hence its highly possible that different mass spec certified / grade solvents can show different levels of background signal.

Figure 1 shows TIC (Total Ion current) signal coming from various grades of Acetonitrile. As can be clearly seen, LC-MS certified acetonitrile from two vendors (Vendor A and B) show significantly higher level of background signal compared to even gradient grade (Suitable for HPLC-UV) acetonitrile from MilliporeSigma (LC-MS grade Lichrosolv Acetontrile).

Combined TICs of the blank runs of four different acetonitrile qualities. All solvents were delivered to the MS source via an LC system.

 

Chromatographic conditions
 

System Bruker Esquire 3000+ ion trap MS
Detection Pos. ESI-MS, m/z range 50 – 2000
Flow rate 1.0 mL/min
Mobile phase A: Water (Cat. No. 115333)
B: Various ACN qualities as indicated in the graphics
Gradient 0 min 98% A, 30 min 0% A, 38 min 98% A
Temperature 25°C

Figure 1. Combined TICs of the blank runs of four different acetonitrile qualities. All solvents were delivered to the MS source via an LC system.

Hypergrade and gradient grade solvents minimize contaminant peaks

Figure 1 illustrates the influence of LiChrosolv® acetonitrile quality on the background noise intensity in mass spectra. MilliporeSigma solvents labeled “hypergrade for LC-MS LiChrosolv®” are dedicated for use with MS systems and deliver minimized contaminant peaks, ion suppression, adduct formation and background noise and therefore maximize sensitivity. Gradient grade solvent quality (labeled “gradient grade for liquid chromatography LiChrosolv®”) are suitable for LC-UV gradient runs.

Use ultrapure water (bottled or freshly delivered)

Ultrapure water for LC-MS applications can be either bottled or freshly delivered from a water purification system. The choice is mainly determined by daily consumption. Demineralized tap water is not recommended for use in combination with LC-MS setups because of possible system contamination. The quality of LiChrosolv® bottled water for chromatography and fresh Milli-Q® ultrapure water produced from laboratory water purification systems is consistently high and generally independent of the regularity of use.

As a result, the now-improved water quality is perfectly suitable for the production of mobile phase, buffers, blanks, standards preparation, sample dilution, glassware rinsing or extraction used in these critical applications.

Of course, the prerequisite is careful storage and handling to prevent contamination during drawing. Figure 10 displays total ion currents (TICs) of Milli-Q® water drawn at different points in time: Directly on Monday (after system standby over the weekend), on the same day after discarding several liters prior to ultrapure water collection, and after four days of daily use. Generally, it is recommended to flush the system every morning by drawing and discarding several liters prior to water collection.

TIP

If sharing a Milli-Q® ultrapure water system with molecular biology researchers requiring a BioPak® application-specific polisher at the point of use, we recommend adding a separate point-of-delivery (POD) unit fitted with an LC-Pak® polisher, because water passing through a BioPak® unit may not be suitable for LC-MS applications. The LC-Pak® polisher is optimized for mobile phase, buffers, blanks, standards preparation, sample dilution, glassware rinsing or extraction used in LC-MS.

Exploiting the full potential of Milli-QR systems via proper handling. Top: MS spectra of two samples of water delivered at different points of time; bottom: TICs of the same samples and one additional sample. All analyses were performed via direct injection of the preconcentrated solvents into the MS operated in positive ESI mode.

Figure 2. Exploiting the full potential of Milli-QR systems via proper handling. Top: MS spectra of two samples of water delivered at different points of time; bottom: TICs of the same samples and one additional sample. All analyses were performed via direct injection of the preconcentrated solvents into the MS operated in positive ESI mode.

Solvent storage

Tips for maintaining your solvent purity:

  • Store all eluents (water and organic) in surfacetreated amber glass bottles (original packaging of all MilliporeSigma LC-MS grade solvents) or in borosilicate glass (if solvents have to be decanted).
  • Select a solvent storage system that is appropriate for usage volume and withdrawal frequency (Table 5).
  • Do not use standard glass bottles; silica and alkali dissolve and form adducts [M+X]+ with analytes.
  • Use MilliporeSigma HPLC bottle caps/adapters with tube connections and membrane filter mounted directly on the original brown glass bottle. This protects both solvents and environment.
  • Avoid decanting; it is a possible source of contamination.
  • Avoid improvised repairs for fixing solvent tubing; this may cause leakages and/or release of contaminants to the eluents.
  • Do not use plastic devices (bottles, funnels, etc.) to handle or store solvents, buffers, etc. Solvents extract additives (anti-static agents, stabilizers, plasticizers) from plastic, a source of contaminant ghost peaks and increased background noise.
Solvent storage systems Storage Container Volume
Bottle top adapters for directly connecting solvent bottles to LC system 1 L, 2.5 L, 4 L (for infrequent withdrawal)
Stainless steel barrel with adapter for direct withdrawal 10 L, 30 L (for frequent withdrawal)
Stainless steel barrel directly connected to LC system  
Central storage of stainless steel barrels with adapters to supply solvent to multiple different laboratories  

Table 1. Select solvent storage options based on volume of usage and frequency of withdrawal to minimize contamination.

Using water as a mobile phase?

Keep in mind these additional considerations:

  • Keep 5% organic solvent in your eluent if chromatographic conditions allow. This avoids microbial contamination of bottle, tubing and LC system.
  • Keep 5% of aqueous eluent in the organic mobile phase to avoid buffer precipitation in the system, e.g., in valves, and subsequent tedious cleaning procedures.

Solvent container cleaning: avoid the dishwasher

Dishwashers are standard laboratory equipment, but they are operated using chemicals such as strong bases and surfactants. Strong bases can lead to dissolution of silica and alkali from glassware and cause the formation of adducts [M+X]+ with analytes, while traces of surfactants remain on the glass surface after the cleaning process and decrease MS sensitivity by increasing background noise.

The easiest way to avoid dishwashing is “cleaning” of all equipment via simple evaporation of both solvents and additives. All chemicals dedicated to the application in LC-MS are volatile; therefore, this procedure is straightforward as long as chemicals are highly pure and microbial growth can be eliminated. In case of equipment contamination, flushing with LiChrosolvR or Milli-QR water or organic hypergrade solvents has to be performed to achieve sustainable cleaning.

Buffers and additives

When working with buffer to adjust pH of eluents, keep in mind:

  • Use volatile salts (such as ammonium formate, ammonium acetate, or trimethylamine). Nonvolatile salts (e.g., phosphates, borates, sulfates or citrates) precipitate in and block the MS source, requiring tedious cleaning procedures.
  • Total ionic strength of the eluent should not exceed 20 mM. Adjust buffer concentration in the aqueous solvent accordingly. Buffers for LC-MS should be prepared using the purest salt and acid/base quality available.

Buffer pH is generally adjusted via a titration with the respective acid or base and monitored with a pH electrode. The unavoidable contamination of the buffer solution with alkali ions from the pH electrode can be decreased by using a miniaturized system available from several suppliers. Unlike standard equipment with a diameter of approximately 10 mm, the diameter of miniaturized electrodes is only 3 mm.

Buffers not only adjust the pH and ionize a target molecule [M], they can also form adducts [M+buffer], e.g., with ammonium, alkali, halogens, formate or acetate. This leads to the detection of additional peaks in the MS spectrum. Even a complete suppression of the analyte signal is possible when the vapor pressure of the resulting adduct (mainly alkali) is decreased significantly. As a result of this phenomenon and in order to keep the ESI source clean, volatile buffers are recommended.

TIP

Avoid using TFA. Trifluoroacetic acid (TFA) is widely used as an ion pairing reagent to improve the liquid chromatographic separation of peptides or proteins when using standard UV for detection. However, TFA can cause strong ion suppression in mass spectrometry (mainly in negative ESI mode) and also contaminates the LC-MS system. Formic acid (0.1%) is commonly used instead as a mobile phase modifier that is compatible with LC-MS.

Materials

     

TIP

Avoid equilibrating columns with more than 10 column volumes of mobile phase (or one blank gradient run with subsequent equilibration). Contaminants in solvents and additives can accumulate on a stationary phase. Figure 12 shows this effect for plasticizers dissolved in the eluent on a reversed phase column after equilibration for 0, 15 and 60 minutes. While these compounds would become eluted as very broad peaks under isocratic conditions (and cause an increased background noise), they elute as distinct, intensive peaks under gradient conditions and can interfere with analyte signals. Instead, run samples immediately after two or three blank runs to ensure that the system is stable prior to sample analysis.

 

Chromatographic conditions
 

System Bruker Esquire 6000plus
Detection Pos. ESI-MS, TICs
Flow rate 1.0 mL/min
Mobile phase A: Water (Cat. No. 115333) / acetonitrile (Cat. No. 100029) 95/5 (v/v) +0.1 % formic acid (Cat. No. 100264
B: Acetonitrile +0.1% formic acid
Gradient 0 min 100% A, 3 min to 95% B, 5 min at 95% B, then bacl to 100% A
Temperature 25°C
Sample Plasticizers (*) were added by the immersion of plastic tubing in aqueous solvent B

Figure 3. Accumulation of contaminants on an HPLC column for various periods of time and elution via a gradient profile.