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10(ish) Ways to Prevent LC-MS Contamination

1) Clean, particle free solvents

Particulates can cause havoc in an LC-MS system by blocking components, while contaminants, such as alkali ions, plasticizers, and surfactants interfere strongly with LC-MS by causing higher background noise and the formation of adduct ions. Non-volatile components will result in a contaminated ion source which needs cleaning more often. 

Therefore, LC-MS grade solvents should always be used for LC-MS applications. LC-MS grade solvents will be pre-filtered by the manufacturer with a 0.2 μm (or smaller) filter.  Further filtration may not be required and could introduce contamination from the filter itself (Figure 1). 

These solvents will also meet the stringent requirements for low levels of impurities (e.g. metal ions).

Figure 1: Effect of rinsing to remove extractables from syringe filters.1

 

2) Use ultra-pure water

Water should never be overlooked as a possible contamination source. The water used in the lab serves a myriad of purposes from washing glassware, to making up standards and blanks, to being a component in the mobile phase. Impurities in water can collect on the column during equilibration with the weak solvent, which could cause damage to the column and affect chromatographic results (Table 1). Therefore, ultra-pure water should be utilized for LC-MS applications.

Ultra-pure water is particle free, chemically clean, and has a resistivity of 18 mΩ. Purification systems for ultra-pure water use reverse osmosis to remove most contaminants, ion exchange to remove ions, carbon filtration for the removal of organics, UV sterilization to kill bacteria, and a pharmaceutical grade 0.2 μm membrane filter to remove particulates.

Contaminants Effects
Organics Noisy or drifting baselines
Ghost peaks
Extensive contamination can result in shifting retention times and distorted peak shapes
Excess background ions in MS
Ions Some ions absorb in the UV range (e.g. nitrites and nitrates)
Metal ions can form adduct peaks in MS detection
Particles Damage HPLC pump and detector
Increase system back pressure
Bacteria Behaves as particulates (increased back pressure)
By-products include organics and ions

Table 1: Contaminants and their effect on HPLC(-MS) systems.

 

3) Prevent microbial growth

Microbial growth can be particularly problematic for UHPLC systems which can be much more sensitive to blockages due to the smaller tubing diameters and column frit porosities. Aqueous mobile phases and water are prone to microbial growth (even over short time periods); this can cause extra peaks in gradient elution and increase background absorbance during isocratic methods. Microbial growth behaves as a particulate and can block filters, frits, and columns, as well as causing check valve malfunctions. All of these problems will result in high pressure which can damage columns and cause system shutdown.

Microbial growth can be prevented by preparing mobile phases fresh each day, filtering, and degassing. To avoid microbial growth when the system is idle, flush all buffer components from the system and column and store both in an appropriate solvent e.g. 60:40 organic:H2O (Figure 2).

Figure 2: HPLC system and column flushing gradient.

 

4) Degas all solvents

Degassed mobile phases will produce steadier baselines and there will be reduced risk of forming bubbles in the system which can adversely affect chromatography (flow rate problems, retention time issues). Even if a system has an inline degasser it is recommended that solvents are degassed prior to use. The optimum method for degassing solvents is vacuum degassing (Figure 3).

Figure 3: Solvent degassing methods.

 

5) Minimize the use of additives

Use the lowest amount of the required additive possible to reduce background noise. Any additives which are being used should be volatile to avoid contamination of the ion source (i.e. use formate or acetate buffers and not phosphate, Table 2).

Some additives cause signal suppression, for example TFA; formic acid can be a good alternative if sensitivity cannot be sacrificed. Furthermore, all additives should be of the highest possible purity, i.e. low concentration of metal ions (Figure 4).

Remember, if a little bit works, a little bit less probably works better - 10 mM or 0.05% v/v is a good place to start.

Figure 4: Acids, bases, and buffers suitable for LC-MS applications.

 
Buffer pKa Buffer Range Formula Buffering
Equilibrium
10 mM Concentration Mobile Phase Preparation* pH Adjustment
(Acid or Base)
Ammonium
acetate pKa1
4.76 3.8-5.8 CH3COONH4 CH3COOH ↔ CH3COO- 0.77 g CH3COOH or NH4OH
Ammonium
acetate pKa2
9.20 8.2-10.2 CH3COONH4 NH4+ ↔ NH3 0.77 g CH3COOH or NH4OH
Ammonium
formate pKa1
3.80 2.8-4.8 NH4COOH HCOOH ↔ HCOO- 0.64 g HCOOH or NH4OH
Ammonium
formate pKa2
9.20 8.2-10.2 NH4COOH NH4+ ↔ NH3 0.64 g HCOOH or NH4OH

Table 2: Properties of acetate and formate buffers for LC-MS.  * Addition of volume or mass per 1 liter.

 

6) Proper solvent storage

Although it is ideal to make up eluents fresh each day to avoid microbial growth, realistically solvents will sometimes be stored. The proper length of time for solvent storage is a much disputed subject in the literature; however, the information below can serve as a good initial guideline. Each method should be monitored and if chromatography starts to deteriorate then solvent storage limits can be reassessed.

Deionized water - The most conservative value is 3 days. With most people replacing after 1 week.

For aqueous/organic solutions (without buffer) - 3 days is a conservative time frame. With one week to one month being the average.

Buffer solutions - 3 days.  Although with UHPLC 1 day has been noted. Also, microbial growth has been shown to alter ion chromatography.

Aqueous solutions < 15% organic - 1 month.

Aqueous solutions > 15% organic - 3 months. Addition of 20 - 30% organic inhibits microbial growth extending shelf life. Evaporation of more volatile solvents over longer periods should also be kept in mind.

For pure organic solvents - these should be stable for extended periods of time and normally the manufacturer will give a use by date (if this is not on the bottle then please contact your supplier for this information). Care should be when using solvents that could produce peroxides (ethers, THF) as these may have shorter shelf lives if exposed to air or other oxidizing components.

For mixtures of organic solvents - The danger here is from selective evaporation of one or other of the solvents which will change the composition of the eluent over time and in turn will affect chromatography. Even if the bottle is well sealed there may be some change in composition and shelf life of 1-3 months is common practice.  Even capped bottles may suffer from some evaporation. 

Most labs will find that they make up eluent frequently as you are limited to the volume that can be stored, and therefore, if you are running your HPLC instrument everyday stocks will be rapidly depleted.

Solvents should be stored in clean glass reservoirs with covers to prevent airborne contamination. Reservoirs should be glass as plastic can promote plasticizer contamination (e.g. phthalates).  Although it should be noted that sodium contamination can originate from glassware.
See this article for more information Controlling Na and K Adducts in LC-MS »

Also remember not to prop up bottles to get last drop out. Apart from the risk of running the pump and column dry, mobile phases evaporate from the surface; therefore the mobile phase at the top of the bottle will have changed composition from the bulk. This portion from the top is exactly what will be running through the column if you use the last drops in the bottle.  Do not top off solvents as the composition may not be correct due to evaporation from the original solvent; instead discard the old solvent, rinse the bottle and filters with new solvent and refill with fresh mobile phase.

 

7) Correct cleaning of laboratory glassware

Soaps and detergents can play mayhem with an LC-MS system, causing ion source contamination and high background noise. Therefore, avoid using soaps and detergents to wash glassware which is to be used for LC-MS. Glassware should not be washed in dishwashers which contain detergent contamination. Glassware should be cleaned by rinsing with organic solvent then water, and then rinsed with the solvent which will be used in the piece of glassware.

For more aggressive cleaning glassware can be sonicated with 10% formic or nitric acid, then water, then methanol or acetonitrile, and finally water. This process can be repeated.

Glassware which contains microbial growth can be treated using an autoclave and all filters and tubing between the mobile phase reservoir and instrument should be replaced.  The instrument itself can be rinsed with acetonitrile or methanol and left to sit overnight.

 

8) Make sure samples are clean

Filter, filter, filter.  Make sure all samples are filtered prior to injection to avoid blockages in tubing and column end frits. Make sure to select the correct filter membrane (i.e. filter material, size, and porosity). The porosity of the syringe filter should be considered in conjunction with the porosity of the column inlet frit and/or packing material diameter so that any particulates which would be large enough to block the column inlet frit, or column itself, are removed prior to reaching the HPLC system. For example if a column is packed with particles which are less than 2 μm a 0.2 μm UHPLC filter should be used.  Syringe filters with a 0.2 or 0.45 μm porosity are suitable for the filtration of samples which will be analyzed using columns packed with particles > 2 μm.

Filtration is important, but only gets rid of particulates. The real bad guys are all the materials that dissolve but are non-volatile. Sample matrix is probably the biggest source of contamination.  For samples which contain high levels of non-volatile matrix components (i.e. salts in biological samples) consider more rigorous sample preparation techniques, such as solid phase extraction - as well as reducing contamination of the LC-MS system removal of these matrix components will reduce background noise and interferences which can improve analytical results.

Any plasticware which is used (e.g. pipette tips, well plates, vials etc.) should be high quality, phthalate free to avoid contamination.

 

9) Use clean fittings and tubing

Any tubing and fittings which come into contact with the sample or mobile phase (e.g. all flow path components - check valves, seals, o-rings, filters etc.) should be as inert as possible, compatible with any components or solvents being used in the mobile phase, and be of high quality. Tubing made of polymers can be made of polymers which may contain plasticizers which may leach into the LC-MS system.

 

10) Wear gloves

There is nothing worse than analyzing a complex mass spectrum and realizing the peaks are keratin (human protein from skin cells, Table 3). MALDI is particularly sensitive to keratin contamination. Or trying to interpret a spectrum which contains a plethora of sodium adducts which can come from handling samples, labware, and instrument parts without wearing gloves.       

897.4140 1179.6010 1365.6399 1838.9149
973.5318 1184.5911 1373.6549 1993.9772
1037.5267 1193.6166 1383.6909 2312.1482
1060.5639 1234.6769 1434.7705 2383.9524
1066.4992 1307.6782 1474.7858 2510.1323
1066.5169 1320.5834 1699.8251 2705.1617
1140.5649 1357.7188 1707.7727 2831.1947
1165.5853 1357.6963 1716.8517 3312.3087

Table 3: Common keratin peaks.2

 
Some Bonus Tips...

11) Look after columns

Many of the points above will help to keep columns contamination free. However, some contamination may be unavoidable due to the type of analytes being analyzed; for example proteins can precipitate and get trapped at the head of the column or organic contaminants which are difficult to remove using sample preparation may also get trapped. Column contamination will reduce column lifetime, can alter chromatographic results, can increase background noise, and cause system pressure issues.

To help remove contaminants run a high organic wash at the end of the analytical method. If columns become badly contaminated follow manufacturer’s cleaning guides to help restore them. If this information is not available then generic cleaning methods can be found at: HPLC Column Cleaning and Regeneration »

Be careful with column chemistries. Some types bleed more or give higher background signals than others (e.g. some polar embedded phases).

 

12) Flow rates and splitting

Contamination can be reduced significantly by using a post-column diversion valve which is automatically set to run to waste at all times except for the retention time period containing the peaks of interest. Early elution (unretained material in the t0 peak) and late eluters at the tail of gradient are the real culprits for clogging sources.

Make sure the temperatures and gas flows in the source are suitable for the mobile phase aqueous content and flowrate. If the temperature is too low, the liquid condenses in the source.

For electrospray, keep the flow low. Use a post column splitter to reduce flow or use a 2.1 mm column with a flow rate of 0.3 mL/min.

 

13) Tune

Optimize/tune MS conditions to get the best sensitivity for the compounds of interest, then use the high sensitivity to reduce the amount of sample injected. It is not just matrix which contaminates - large amounts of actives cause problems too.

 

References

  1. http://www.chromacademy.com/chromatography-Troubleshooting-Filtration.html
  2. http://cgs.hku.hk/portal/files/CGS/Proteomics/MS/ms_signal_exclusion_list.pdf

 

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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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