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Water for HPLC


Impact of Water

HPLC is an established, reliable technique that has become the workhorse of many laboratories. Robust as it may be, HPLC is still subject to problems that could mean waste of time and resources to the researcher. One of the most common problems in HPLC is related to the quality of solvents/mobile phase. Contaminants in the solvent could affect HPLC work in several ways.

Particles may damage pump and injector. They could also plug the column and frits, causing an increase in back pressure. Particulates may also behave as a secondary solid phase which could bind sample constituents.

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)

will also affect chromatographic separations. A modification of the ionic strength of the solution may affect some separations, and if the ionic contaminant is UV-absorbing ions (e.g., nitrates, nitrites), it will come out as peak and make data analysis difficult.

Among the different water contaminants that may affect the quality of water used for sample dilution, standard solutions or mobile phase preparation, organics are probably the most important ones, as the results below demonstrate.

Figure 2 shows chromatograms from an experiment where ultrapure water containing different TOC levels was used as mobile phase A (with acetonitrile as mobile phase B) in a gradient elution on a C18 column without any injection. Notice how increasing levels of TOC results to chromatograms with numerous and high background peaks.1
Figure 2: Chromatograms of ultrapure water containing different TOC levels. Gradient elution on a C18 column, with ultrapure water as mobile phase A and acetonitrile as mobile phase B.

Some chromatographers choose HPLC-grade bottled water when they prepare their aqueous mobile phase. What goes unnoticed though is that most, if not all, of these waters do not give specifications on TOC levels. Off-line TOC measurements of several brands of bottled HPLC-grade water show that TOC levels can be over 700 ppb TOC (Table 1).2 The high levels of TOC adversely affect the chromatograms of the water, as shown in Figure 3. Contaminant peaks are present in most of the HPLC-grade bottled water tested.

Water Source
TOC (ppb)
Bottle Water A 100
Bottle Water B 87.0
Bottle Water C 777
Bottle Water D 16.5
Bottle Water E 32.4
Bottle Water F 25.5
Fresh ultrapure water 7.0

Table 1: TOC levels in bottled HPLC-grade water2
Figure 3. UV (210 nm) chromatograms of different sources of HPLC-grade bottled water and ultrapure water.

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.
[1] S.Mabic, C. Regnault, J. Krol, LCGC North America, (Jan 2005). The misunderstood laboratory solvent: Reagent water for HPLC (LCGC North America, Jan 2005. 
[2] B.M. Stewart and B.L. Williamson, Am Biotechnol, 19 (2001), 16. Evaluation of HPLC reagent water purity via LC-MS and total organic carbon analysis.

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