Impact of Water
Many water contaminants may impact the quality of mass chromatograms and mass spectra. The effect of these contaminants can be traced back to the HPLC part of the LC-MS set-up, while some are more specific for the MS detection.
Particles may damage HPLC pump and injector. They could also plug the column and frits, causing an increase in back pressure.
Organics contamination of the ultrapure water may affect chromatographic separations different ways:
- Organic molecules may accumulate on the surface of the chromatography column beads, slowing down the access of sample and solvent molecules to the binding sites located inside the pores of the beads. This causes mass transfer issues and results in a loss of resolution and ultimately in a shorter column life time.
- Organic molecules in the water used as an eluent may compete with the sample molecules for binding to the active groups on and in the chromatography beads. The consequence is that less sample molecules are bound during the injection and binding phase and therefore less sample molecules are released during the elution process. As a result, the sensitivity of an analytical chromatography method can be reduced by the presence of organics in the water used for chromatographic separations.
- Organics may accumulate at the head of the column and later on elute as contaminant/ghost peak(s).
- If the level of organic contamination is very high, the contaminant(s) may act like a new stationary phase, causing shift in retention time and peak tailing.
- Accumulation of organic material in the column may also lead to back pressure increase.
- For all these reasons, it is critical to monitor accurately the level of organics in water used for HPLC applications and this can conveniently be performed by an on-line TOC monitor.
Colloids may adsorb irreversibly on the stationary phase, thereby losing its original separation efficiency of the column.
Bacteria may plug column and frits and release organic by-products. (See effects of organic contamination above.)
- Ions contamination
Ions contamination will also affect chromatographic separations because modification of the ionic strength of the solution may affect some separations. A significant concern when there is ionic contamination is that analyte molecules could form adducts with metal ions (e.g., Na+, K+) which could data interpretation more challenging.
Among the different water contaminants that may affect the quality of water used form sample dilution, standard solutions or mobile phase preparation, organics are probably the most important ones, as the results below demonstrate.
Off-line TOC measurement of several brands of HPLC-grade water reveal TOC levels as high as >700 ppb (Table 1).1 The same brands of bottled water were used as mobile phase A in a gradient elution on a C18 column (without any injection, mobile phase B was acetonitrile). The high TOC levels, which correspond to high levels of organic contamination, resulted to HPLC mass chromatograms with numerous and high background peaks (Figure 2.)
|Bottled Water A
|Bottled Water B
|Bottled Water C
|Bottled Water D
|Bottled Water E
|Bottled Water F
|Fresh ultrapure water
Table 1: TOC levels in bottled HPLC-grade water1
Figure 2: Electrospray positive mass chromatogram of bottled water from different sources. Gradient elution on a C18 column (no injection) with water as mobile phase A and acetonitrile as mobile phase B.
The ultrapure water referred to in Table 1 and Figure 2 was obtained with a Mill-Q® system equipped with a dual wavelength (254/185 nm) UV lamp that efficiently decreases the organic levels in ultrapure water to 5 ppb. As an illustration of how effectively it works, consider Figure 3 that shows the total ion count (TIC) obtained by mass spectrometry on water purified by a Milli-Q® Gradient with the UV lamp turned on (pink trace) and then off (blue trace). Note the number of peaks present and their relative intensities when the UV lamp is off. Compare this with the chromatogram when the UV lamp is on (pink trace). Peaks have disappeared or reduced significantly in size when the UV lamp is on.
Figure 3. Electrospray positive mass chromatogram of water purified by a Milli-Q® Gradient with UV lamp ON (pink trace) and OFF (blue trace).
Other sources of contamination
- Storage of ultrapure water
Storing ultrapure water will introduce leachables to the water. Plastic containers leach out organics and some ions. Glass containers do not leach out as much organics, but they can introduce ions into the water. Storing ultrapure water would also encourage the proliferation of bacteria.
- Solvent additives
Additives to make buffers, such as salts, acids, and bases, are commonly used. Only the highest grade reagents should be chosen.
 B.M. Stewart and B.L. Williamson, Am Biotechnol, 19 (2001), 16.
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