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.

Method Development Hint: Use your HPLC Diode Array Detector (DAD or PDA) as a Spectrophotometer

One of the many useful features of a UV/VIS scanning diode array detector is that it can be employed in flow injection mode to scan a sample and provide you with some useful data about the absorbance characteristics of the sample (which probably contains a mixture of components). Unlike a spectrophotometer, you only need about 1 ul of sample instead of a 1ml cuvette and only 15-20 seconds of time to gather the data.

Why do this? I use this feature often when I receive a new and unfamiliar sample for method development. I set up the detector to scan and store all wavelengths, in steps of 2nm, from 210nm to 450nm and inject the sample in flow injection mode (that means no-column is present and I easily do this using the By-Pass position on my column selector). In a very short amount of time I can view the resulting spectra of the sample which aids me in selecting the initial discreet wavelengths to monitor. For example: If I notice that the sample shows some absorbance at 410nm using the flow injection run, then notice while developing the analysis method that none of the peaks seen show absorbance near 410nm, then I can assume that I may still have some components retained on the column.

Setup Hints:
(1) For this to work well, you should have a high performance, low volume switching valve or automated column selection system (e.g. The LC Spiderling Column Selection System) installed so you can easily by-pass your column (otherwise, remove your column and place a high pressure, low volume union in its place).
(2) Set the diode array detector to a high sampling rate because the sample is going to fly through the flow cell quickly. Use a sampling rate that is faster than you would use if a column was there to disperse the sample and slow down the peaks.
(3) Choose a wide range of wavelengths to scan and store. If the sample appears colorless to the eye in solution and I am running in a UV transparent solvent such as acetonitrile, then I often use a range of 210 to 450nm.

Proper Wavelength Selection for HPLC Method Development (or Purity Determination)

Selecting the best HPLC wavelength(s) to monitor during an analysis method for use in quantitation and/or purity determination requires both knowledge and careful attention. Here is the basic procedure to use:

Step 1. Create the Method. To determine which UV/VIS detector wavelength(s) should be chosen for the analysis of your sample, you will first need to create a general HPLC Method which retains and resolves the compound(s) of interest on the column (goal is a K prime of >2.0, less than 10.0). Be sure and utilize a scanning diode array detector in full scan mode (often referred to as a photo-diode array detector, PDA or DAD) to scan all relevant wavelengths of your samples (e.g. 210 to 450nm). Note: Your choice of mobile phase and detector settings will effect the S/N values.

Step 2. Determine the lambda max of the sample's spectra using the Data analysis software. Once you have completed the analysis, review the spectral data to determine which prominent peak wavelengths have the maximum signal to noise (S/N) ratio. These “peaks” can be used as the individual wavelengths for integration and purity determination. By sure and double check that any detector options which use a “reference wavelength" are turned ‘OFF’ when running these methods (more info on “reference wavelengths” can be found on this blog in another post). With the wavelength selected, chose an appropriate bandwidth for use (narrow).

Step 3. Edit the HPLC method to use the discreet wavelengths found in step 2. Whenever you run a real sample, continue to use the full scanning mode of the detector so you will know about any other components which absorb at wavelengths far away from and/or near the peak wavelengths. These compounds can add or subtract signal from the main peak making it appear to be more or less concentrated (or more or less pure) than it actually is. If you only monitored the sample with a single wavelength detector, then you would miss this vital information and make errors in your purity or concentration determinations.

Conclusion. (1) Using a multi-wavelength, scanning HPLC detector such as a DAD is one of the most important tools you can use to create accurate and reliable chromatography methods. Always use a scanning DAD for method development to prevent errors. (2) Learning how to correctly use and set up the detector's settings, parameters, special features and options may prevent false or misleading results. Only after you have developed a reliable and repeatable method with good sample retention and peak shape can you begin to report accurate integration and concentration values (and/or make UV/VIS "purity" determinations).

Troubleshooting HPLC Injectors (Manual and Automated)

Sample injectors are a critical component of a chromatography system. Understanding how they operate as well as the proper techniques to use and maintain them are fundamental skills needed to operated an HPLC system. Lets briefly discuss some of these fundamentals as applied to a standard manually operated HPLC injection valve and also in an automated mode as found in an autosampler. *Note: You should always refer to the specific manufacturer's product specifications, operation, servicing documentation or support personnel before servicing any injector.

MANUAL INJECTION VALVE Notes:

These valves allow you to use a high precision syringe to manually fill a fitted "loop" with a sample and then, by turning a valve handle, introduce the sample to the high pressure flow stream directed toward the column inlet. Sample loops are available in a wide range of volumes and take just minutes to install. The injector valve has very tiny openings inside which are moved between two different positions (LOAD and INJECT). The LOAD positions allow the valve to seal off the internal high pressure flow from the loop to allow it to be safely filled with sample at atmospheric pressure. The INJECT position introduces the liquid contained inside the loop to the main flow path (under high pressure). The parts must be clean and seal well to insure proper function. Leaks from all areas (except the vent) are not acceptable and indicate a problem. Here are a few tips regarding the use of manual injectors in HPLC.

• Use the correct type of sample syringe. Usually these are high precision glass syringes with Teflon plungers. The needle tip style is the most critical item! Most injectors are designed to only work with a needle which has a squared off tip (NOT a point as is commonly used in GC!). The most common gauge used is #22. Always check with the valve manufacturer to determine the correct style and gauge of needle before use.
• Leave the valve in the INJECT position during the entire run to flush it clean of sample and stay equilibrated with your method. Switch it back to LOAD only when you are ready to load a new sample.
• For high reproducibility and accuracy within one HPLC system, fill the loop with at least three times the volume of the loop with sample to insure that the entire path is full of sample. This is known as the complete or over-filled loop method. *Choose your loop volume size with this in mind. Loading the same volume as the loop will often result in poor accuracy.
• Loops often do not contain the exact volume stated on them. They can be off by ~25% so consider this when injecting partial volumes and not using the standard over-filled loop method.
• Types of common leaks: (1) Leaks at the needle port (needle seal worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

AUTOMATED INJECTION VALVE (auto-injectors) Notes:

These valves use a high precision syringe or high pressure pump to fill a fitted "loop" with a sample and introduce the sample to the high pressure flow stream, all automatically. Most function exactly the same as described above, though some are based on true high performance liquid chromatography pumps so have no "syringe" at all (e.g. Agilent 1100/1200 designs) Here are a few tips regarding the use of automated injectors in HPLC.

• Regular maintenance is even more critical with auto-injectors since you often can not see what is going on during the injection cycle. Leaks, if present can be harder to find so make it a habit to visually check all of the areas around the injector regularly.
• Needle and Needle Seats are normal wear items on these instruments. As such, they require routine checking for leaks or damage and replacement when worn.
• Vial Caps: If you make multiple injections from one vial (or large volume injections) with a tightly sealed vial cap, a vacuum can form inside the vial causing volumetric errors to occur in your samples (resulting in you injecting less sample each time). Leave the caps slightly loose to avoid this problem. Multiple injections into the same vial can also cause the needle hole to become enlarge over time allowing the sample or solvent to evaporate over time, changing the concentration of the sample (more concentrated). Replace the cap and seal with a new one if used multiple times. Always leave the cap slightly loose.
• Loop volume: Autoinjectors often incorporate one large loop to handle a wide range of sample volumes. This is a trade off of accuracy for convienence. Accuracy is often poorest at the very low end of the range and best near the middle to high end. Always verify the reproducibility of the injector to inject a specific volume through statistical analysis of repetitive injections.
• Types of common leaks: (1) Leaks at the needle seat (needle seat worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

These are just a few tips related to HPLC injectors. Please consult the service documentation for your specific instrument to better understand how the system works and what areas you should be monitoring. Understanding HOW these systems operate (and can fail) is one of the most important skills you can learn as a chromatographer. Take the time to understand the complete flow path of your system before using it.