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.

Techniques To Enhance Negative Mode ESI in LC/MS

Many sample types require MS analysis using negative ion electrospray mass spectrometry (ES MS) mode. Sensitivity can be less in this mode, compared to positive mode, as they employ mostly non-polar solvents which do not promote ion formation. In the past we discussed how adduct formation can be employed to enhance ion formation. There are other techniques which can be used as well. Here are a few tips which can be used to improve the quality of the signal obtained under negative mode ES MS conditions.

(1) Negative ion formation and signal response can be improved by choosing the right solution chemistry. One method to improve solvent desolvation and reduce the electrical discharge (noise) is to add isopropyl alcohol (2-Propanol) to the mobile phase. Reported in the literature nearly twenty years ago, as little as a 10% solution has been found to increase the signal level and decrease the noise level under many conditions. The addition of methanol to the mobile phase also can improve the signal, though usually not to the same degree as IPA.


(2) Another technique used to improve ionization involves the pH of the solution or mobile phase used. Higher pH values are often better. Post column addition of a basic solution which adds proton acceptors to the mix, such as ammonium hydroxide (20 or 30 mM), can also improve signal response and stability.


(3) APCI mode: If running nonpolar to semi-polar small molecular weight compounds, especially those which are volatile, this is still the source of choice. *Conventional ESI mode is best for polar to neutral molecules in small to large Mw ranges.

Popular Microsoft Windows® Short Cuts:

Here is a short list of some of the most popular Windows short cuts. Hopefully, you will spot a few that you do not know about and can use.

• Windows Keyboard Logo Symbol + L: Lock the computer (without using CTRL+ALT+DELETE).

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A Full List of Microsoft Short Cut Commands Can Be Found In This Key Directory (Listed by Application): http://support.microsoft.com/search/default.aspx?query=Microsoft%20shortcut%20commands&catalog=LCID%3D1033

Adduct formation in LC-MS Analysis (esp. ESI)

Almost everything you analyze by Electrospray ionization mass spectrometry will create an adduct with something in the system. Normally, hydrogen is the most common adduct formed (M+1), but other chemicals, often in trace amounts may form adducts with your sample too. Sometimes we can take advantage of this fact and introduce our own adduct into the system (post column) to increase signal sensitivity or help us isolate one signal from another (the addition of an adduct can sometimes increase the signal seen for one species, but not the other). 

One of my favorite elements to form an adduct with is sodium (Na+). Two common forms are; sodium citrate and sodium acetate. Both have PKA’s between 3 and 6 so a variety of buffered solutions can be prepared for use. However, it is very important that we keep the concentration of sodium as low as possible so as to not clog the mass detector or suppress ionization completely (and see nothing BUT Sodium for weeks …). My suggestion is to initially prepare the buffers such that the solution is less than or equal to 3 mM in concentration. The lowest concentration should be used that yields reproducible results. Ranges from 0.1 mM to 5 mM are common. Only use the highest purity, volatile buffers (some manufacturer’s use names such as “ultra” to describe them) when preparing these ‘doping’ solutions for post-column addition and be sure and filter them through a 0.2 micron filter before use. A syringe pump can be used to deliver the solution during the run. A low flow rate should be used to infuse the adduct solution into the main inlet of the detector. Make sure you have a simple way of controlling the pump through the system (e.g. ‘On’ / ‘Off’, contact closure) so the flow can be turned off when you are not acquiring data. Be sure to not only monitor the baseline, but also measure true peak S/N values of a standard when evaluated the results (decreasing baseline noise may also mean the signal is decreasing too).

Ammonium (NH4) is another popular adduct to add to the system, often in the form of ammonium acetate. It reduces the chances of adding more sodium ions to the system (from glassware). Whichever adduct you use in your system, always start off testing as low a concentration as possible. Monitor the baseline carefully for noise and also to see if the addition of the compound is suppressing or enhancing the signal generated for your compound. Careful use of adducts in your system can provide you with another means to selectively enhance the signal of some compounds without changing the original chromatography method.

I must again emphasize to use the lowest concentration of doping agent. Proper pH control and mode choice are also very important. Use of a syringe pump for infusion, post column can help you to quickly optimize the fragmentor settings in real time.

• For additional information you may want to take a look at this useful Adduct Calculator webpage: "Mass Spectrometry Adduct Calculator(ESI)".