What type of Water Should I use for HPLC, UHPLC or LC/MS Analysis?

Water is one of the most common solvents used in reversed phase chromatography. HPLC and LC/MS work demands ultra pure quality water be used in all applications which call for it as part of the method. Special types of HPLC analysis, such as amino acid analysis and ion chromatography, demand fresh ultra high quality water be used or artifact peaks may result. Poor quality or low grades of water may lead to "ghost peaks", baseline instability, high background noise or signals, contamination of columns and an inability to obtain reproducible results. Use the freshest and highest purity of water for best results.

A good starting point for describing the type of water suited to liquid chromatography applications is to look at the specification for ASTM Type 1 Reagent grade water. We often exceed this requirement for chromatography applications as several unspecified items such as nitrates and other chemicals present may have a negative effect on our analysis methods.

How does the grade of water affect our chromatography? The grade specified often dictates the amount of organics, bacteria, particulate, residues and overall absorbance the water will have. For example.

(1) Organics: High levels of T.O.C. can accumulate on the particles, inside the pores, or bind to active sites on the support inside the column causing a loss of resolution or sensitivity. *Lower T.O.C. levels are desirable.

(2) Bacteria: Microorganisms can contaminate the buffer solutions used causing ghost peaks, column fouling and the release of additional foreign organic matter into the system. This can result in clogs, ghost peaks, poor reproducibility or loss of resolution and/or sensitivity. *The water should be filtered through a 0.2 micron filter before use. Refrigerate solutions for no more than 3 days to slow growth, then dispose of the solutions.

(3) UV absorbance: High background or interfering ions which absorb can raise the baseline and noise levels seen, decreasing the total dynamic range. *Again, the lowest values, esp. at 200nm, are desirable.

A few of the general requirements for HPLC grade ultrapure Type 1 water can be stated as follows:

   Resistivity :         > 18 MΩ•cm at 25.0 C
   T.O.C. :              < 5 ppb
   UV cutoff :          190nm (as low in absorbance as possible!)
   Filtered :             0.2 micron Filter

*Some suppliers will also specify residue after evaporation (usually < 2 ppm); Trace metal analysis; Optical properties at specified wavelengths and other information. If purchasing by the bottle, request a copy of the lot certification sheet for the water so you can compare the measured values to other products.

Generating your own in-house, reverse osmosis (RO) ultra pure water from potable tap water is one of the best ways to insure you have high quality water for your LC methods. These systems pre-filter the water to remove large particulates then typically use UV lamps and/or multiple resin cartridges to remove the maximum amount of T.O.C.'s from the water plus many trace metals before finally filtering the water through a 0.2 micron membrane as a final polishing step. Various types of systems can be purchased, but for HPLC or LC-MS applications, it is critical that you select a system that provides ultra pure water suitable for your applications. Periodic maintenance of the filter cartridges and monitoring of the main water supply source is critical to their operation (some "tap" water sources may require pre-treatement). *"Water On Demand" systems such as these provide fresh clean water on demand so there is no need to be concerned with storage issues. A number of different vendors offer these lab grade systems for HPLC and LC/MS applications and you can contact them (e.g. Millipore/Sigma Milli-Q® brand) to determine which system will provide you with the volume and quality of water which is appropriate for your application(s).

If you do not have access to an in-house reverse osmosis system, then purchasing HPLC or LC/MS grade water in glass bottles may be another option. A hint, before opening and using them,  clean the outside of bottles of all dust. Date the bottles when you first open them. Bacteria will start to grow once the bottle has been opened. The glass will also slowly leach ions (i.e. Sodium) over time into the water so it is best to use the water quickly.

Never underestimate how the quality of the water you use to perform chromatography can change the results seen in your methods. Water quality is just as critical as any other component in your system so be sure and take the time to monitor it just like you do to any other part of the system.

Hydrophilic Interaction Chromatography (HILIC)

Perhaps you have a polar sample which shows poor or no retention under reverse phase conditions. HILIC may provide you with an alternative method for retention and separation. HILIC is a unique mode of chromatography which uses numerous retention mechanisms. The most important mechanisms involve surface layer liquid-liquid partitioning, adsorption and various types of ionic interactions.

Sometimes referred to as "aqueous normal phase chromatography", this hybrid technique utilizes a stationary phase which is very polar (e.g. silica, amino or a diol column) and a mobile phase which is made up mostly of organic phase with some water added. The retention mechanism is based on the idea that adding a low percentage of polar phase (water in this case) to a polar surface will result in a water layer forming. Typically this hydrophilic layer results when as little as 2 or 3% water is added to the mobile phase. The remainder of the mobile phase is an organic solvent (ACN is the most popular, but many others can be used). The polar charged analyte(s) will partition into and out of this adsorbed water layer (often, a cation exchange process takes place, but their may be a purely electrostatic mechanism going on as well). Unlike conventional reverse-phase chromatography, in HILIC increasing the organic content of the mobile phase increases the retention! Put another way, increasing the water content of the mobile phase and decreasing the organic portion (as in an HILIC gradient method) results in retention and then elution of very polar analytes. 

With the HILIC mode, sample elution (retention) decreases as you increase the polarity of the organic solvent. Based on this information, good HILIC column wash solutions usually use alcohols in place of ACN  (IPA, Ethanol and Methanol; with Methanol being a stronger eluter). For best results, consider incorporating an alcohol wash after each analysis. Allow plenty of time for the column to equilibrate too.

As with other modes of chromatography, the use of additives, buffers and pH can all play a role in retention and separation plus improve reproducibility. When developing methods, be sure and evaluate their role. Because of the low water content of most methods, buffers must be chosen carefully to insure full solubility. Ammonium formate and acetate are popular as are acids such as formic acid. Regarding pH, the low aqueous portion will mean that the actual pH of the final solution will be much closer to neutral.

 

• Caution: Sales and marketing people sometimes stick an HILIC label on an existing silica column to create a new product. No "special" HILIC columns are needed to develop an HILIC method. Since HILIC is a mode of chromatography, not a support type, most any high quality, NP silica column can be used.

 

As some HILIC methods may be hard to reproduce (very sensitive to changes in composition and long equil times) they are best used by more experienced cinematographers, only after conventional methods have been unsuccessful.