Water for UHPLC

Impact of Water

• 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. 

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 like trifluoracetic acid (TFA) are often added to the solvent to enhance ionization in LC-MS. Salts to make buffers are also commonly used. Only the highest quality reagents should be chosen. 

More Information

• Chromatography Online: Fast and Ultrafast HPLC on sub-2 μm Porous Particles — Where Do We Go From Here? http://chromatographyonline.findpharma.com/lcgc/article/articleDetail.jsp?id=333246&pageID=1&sk=&date=
• Other articles, user guides, and links for UHPLC: http://www.forumsci.co.il/HPLC/lcms_page.html#UPLC
• Investigating organic contamination in high purity water using UHPLC with ultrasensitive diode array detection and LC/MS, Agilent Technologies: http://www.chem.agilent.com/en-US/Search/Library/_layouts/Agilent/PublicationSummary.aspx?whid=71884&liid=5921 

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