Using Freshly Delivered Ultrapure Water With Low TOC Improves Chromatographic Performance

Wednesday, January 27, 2016

Analytical instruments have become increasingly sensitive, requiring a higher level of reagent and solvent quality, including water. In HPLC, the importance of water quality becomes more obvious when running gradient elution using reversed-phase columns, since it requires equilibration with several column volumes of the aqueous solvent. The organic contaminants in the aqueous solvent tend to adsorb at the head of the column and elute as peaks in subsequent gradient runs.

According to McMaster,1 most of the difficulties encountered in HPLC are due to column contamination problems, with contaminated water often being the cause. As a result, spurious peaks, humps and dips in the baseline can be observed.2–5 Experienced chromatographers typically use HPLC-grade solvents that are filtered and degassed prior to use. However, HPLC-grade bottled water can contain up to several hundred μg/L (parts per billion [ppb]) of total oxidizable carbon (TOC). TOC is a measure of the total organic species present in water.6

Two sets of experiments were performed to study the impact of organic contamination of water on HPLC analyses. The first involved preconcentrating the aqueous solvent (water) on a column, followed by gradient elution onto an analytical column. In the second set of experiments, gradient elution was used to separate the components of a drug mixture. The mixture was injected 1310 times to examine the long-term effects of using either HPLC-grade bottled water or freshly delivered ultrapure water as the aqueous solvent.

Materials and methods

For baseline comparisons, two sources of water were compared: freshly purified ultrapure water from a Milli-Q system (MilliporeSigma, Billerica, Mass.) and HPLC-grade bottled water. In the second experiment, a mixture containing 0.200 μg/mL of chromatographic drug standard was prepared. Water and acetonitrile were used as mobile phases and prepared fresh every day. Two sources of water were used: HPLC-grade water from a prominent vendor and freshly delivered ultrapure water from a Milli-Q Advantage A10 system.

An Alliance 2695 HPLC system (Waters Corp., Milford, Mass.) was used for the baseline comparisons. Water was first preconcentrated on a Waters C18 XTerra column (4.6 × 30 mm, 3 μm) for 60 min at 1 mL/min with the flow directed to waste. Next, a Waters C18 Atlantis column (2.1 × 150 mm, 3 μm) was connected to the XTerra column and the following gradient elution profile was used to elute any organic molecules that were adsorbed: 100% to 0% water for 30 min; hold at 0% water for 10 min. The flow rate was 0.25 mL/min.

For the long-term study, Millex-LCR (MilliporeSigma) filter units (0.45 μm, 13 mm) were used to filter the drug mixture prior to 25-μL injections. A Waters 510 HPLC pump with Symmetry Shield RP18 columns (4.6 × 150 mm, 3.5 μm) and a photodiode array (PDA) detector were used.

The following gradient profile was employed: 80% to 30% water for 15 min, 30% to 80% water for 1 min, and hold at 80% water for 4 min. Flow rate was 1 mL/min. Separate yet identical columns were utilized for the two different types of water used as mobile phase.

Results and discussion

Baseline comparison of HPLC-grade water and low-TOC ultrapure water

 Figure 1 – Chromatograms at (a) 254 nm and (b) 214 nm of preconcentrated HPLC-grade bottled water and freshly produced ultrapure water. The latter gives much cleaner baselines. Note the difference in scale between (a) and (b).

In the first set of experiments, during the 1-hr preconcentration step, it was anticipated that any organic contaminant present in the water would adsorb at the head of the column. In the elution step, these organic species would elute off the column and come out as peaks in the chromatogram. Figure 1 illustrates the level of organic contamination in HPLC-grade bottled water. Figure 1a (at 254 nm) and Figure 1b (at 214 nm) show that more peaks were present when HPLC-grade bottled water was used, with peaks as much as 15 times larger than in preconcentrated freshly delivered ultrapure water.

Long-term effects of using HPLC-grade bottled water and low-TOC ultrapure water in the mobile phase

For the second set of experiments, a significant difference in baseline drifts was seen. Although baseline drifts are expected in gradient elution— especially at low wavelengths—the large variable drifts observed when using HPLC-grade bottled water (Figure 2a) suggest a buildup of contaminants in the column over time. On the other hand, when fresh ultrapure water was used (Figure 2b), no such variable shift in baseline was observed. Another difference between the two sources of water is the appearance of extraneous peaks when using HPLC-grade bottled water, an additional indication of organic contamination in the aqueous mobile phase. The appearance of extraneous peak(s) in a chromatogram could be mistaken as a sample component, or may coelute with an analyte, which could pose a problem in quantification. When using HPLC-grade water, extraneous peaks appear in the chromatograms at 254 nm (at ~5 min, injection 770), as demonstrated in Figure 2a, and 214 nm (at ~15 min, injection 530) (data not shown). The area of the extraneous peak appearing at ~5 min in Figure 2a is more than three times larger than the phenobarbital peak (peak 3) and more than two times larger than phenytoin (peak 5).

 Figure 2 – Chromatograms (254 nm) of a drug mixture when the aqueous solvent was a) HPLC-grade bottled water and b) freshly produced ultrapure water. HPLC-grade acetonitrile was used as the organic solvent. Peaks: 1) acetaminophen, 2) acetazolamide, 3) phenobarbital, 4) carbamazepine, 5) phenytoin, 6) secobarbital, 7) nabumetone.

The results of both experiments suggest that freshly delivered ultrapure water is an excellent source for HPLC mobile phase preparation. In this water, the level of organic contamination is kept to a minimum (≤5 ppb) and monitored using an on-line TOC monitor. In contrast, organic levels in bottled water can vary significantly from one manufacturer to another, with some bottles tested containing levels as high as 777 ppb of TOC.7,8

A typical reversed-phase HPLC gradient elution requires equilibration of the column to the initial conditions with several column volumes of the weak (aqueous) solvent. Organic contaminants in the aqueous solvent adsorb at the head of the column and cause interferences, such as baseline shifts and extraneous peaks, in the succeeding chromatograms.

Unwanted background peaks due to mobile phase contamination can reduce the accuracy, precision and/or sensitivity of the method, compromising peak identification or quantification.

Obtaining ultrapure water with low TOC

The combination of technologies in water purification systems ensures the delivery of water with consistently low levels of organics. Reverse osmosis eliminates 80–90% of the incoming organic load. Electrodeionization efficiently removes charged species (inorganic and organic ions) and complements reverse osmosis for organic removal by eliminating small organic acids and amines that can pass through the pores of the membrane. Adsorption of organics onto activated carbon is a third way to decrease the organic concentration in water. Another means to reduce the organic concentration in water is UV photooxidation with a dualwavelength (185/254 nm) UV lamp.9

Finally, for successful HPLC experiments, other sources of contamination must be avoided. Trace contaminants in solvents (acetonitrile, methanol) and modifiers (trifluoroacetic acid) have been shown to contribute to background peaks.4 Careless preparation of the mobile phase solution (use of plastic containers or stoppers, paraffin film, gloves) can introduce phthalates and other UV-absorbing species.

Conclusion

Freshly produced ultrapure water with consistently low TOC levels can be used reliably to prepare HPLC mobile phases. When HPLC-grade bottled water was preconcentrated on a C18 column with subsequent gradient elution, several large peaks appeared. These peaks were not present when freshly produced ultrapure water was used, confirming that the former source of water contains far higher levels of organic contaminants than the latter. When HPLC-grade bottled water was used as the aqueous solvent to separate a drug mixture, large shifts in baselines occurred and extraneous peaks appeared over time. These manifestations may be attributed to the presence of organic contaminants in the water; they were not observed with fresh ultrapure water, resulting in more consistent baselines and minimized risk of extraneous peaks.

References

  1. McMaster, M.C. HPLC and column maintenance. LC/MS: A Practical User’s Guide: John Wiley & Sons: Hoboken, N.J., 2005.
  2. Nelson, M.D. and Dolan, J.W. LC troubleshooting: gradient background peaks—a case study. LC·GC 1998, 16(11), 992–6.
  3. Oehme, M.; Berger, U. et al. Trace analysis by HPLC-MS: contamination programs and systematic errors. Trends Anal. Chem. 2002, 21(5), 322–31; doi:10.1016/S0165-9936(02)00503-4.
  4. Ringo, M.C.; Allen, J.R. et al. Mobile-phase cleanup using solidphase extraction disks. LC·GC North America 2003, 21(2),168– 78; http://images.alfresco.advanstar.com/alfresco_images/pharma/2014/08/22/585b2ca8-fad1-46bf-8f60-5c454ea00f56/article-47802.pdf.
  5. Mihailova, A.; Lundanes, E. et al. Determination and removal of impurities in 2-D LC-MS of peptides. J. Sep. Sci. 2006, 29(4), 576–81; doi: 10.1002/jssc.200500496.
  6. Stewart, B.M. and Williamson, B.L. Evaluation of HPLC reagent water purity via LC-MS and total organic carbon analysis. Am. Biotechnol. Lab. 2001, 19(13), 16–25.
  7. Majors, R.E. Column watch: current trends in HPLC column usage. LC·GC Europe 2012, 25(1), 31; http://www.chromatographyonline.com/current-trends-hplc-column-usage-0
  8. Snyder, L.R.; Kirkland, J.J. et al. Practical HPLC Method Development, 2nd ed.; John Wiley & Sons: Hoboken, N.J., 2012.
  9. Regnault, C.; Kano, I. et al. Ultrapure water for liquid chromatographymass spectrometry studies. J. Chromatogr. A. 2004, 1030(1–2), 289–95.

Cecilia Devaux is head of laboratory and analytical group manager, R&D; Dr. Estelle Riche is senior scientist, Application Group; and Dr. Stephane Mabic is worldwide application and training manager. All three work for the Lab Water business field of Millipore SAS, 1 Rue Jacques Monod, 78280 Guyancourt, France; tel.: +33 (0) 825 045 645; e-mail: [email protected]; www.emdmillipore.com. The authors are grateful to Maricar Tarun Dube and Coralie Monferran for their technical support and contribution to this article.

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