HPLC Hints & Tips Web Page

If you would like to see some of these tips in a tabular format on a single web page, then check out this link:  

   HPLC HINTS & TIPS for Chromatographers:

   http://www.hplctools.com/hplc_hints_tips.htm

   HPLC BLOG link, which is updated more frequently, can be found here: 
  http://hplctips.blogspot.com/


Note: The web page and blog versions present some of the same tips, but some of the tips are only shown on one or the other page so it pays to check them both to stay up-to-date on all of the tips offered.

Common LC/MS ESI Tune Compounds:

ION MODE	COMPOUND NAME	Mw
Positive	Acetaminophen	151
Positive	Caffeine	194
Positive	Diphenylhydramine	255
Positive	Desipramine	266
Positive	Aspartame	294
Positive	Cortisone	360
Positive	Reserpine	608
Negative	Aldicarb	190
Negative	Benzo(a)pyrene	252
Positive	di-Octylphthalate	391

Windows 7 Shortcuts & Tips

Here are some of My Favorite Helpful Shortcuts/Tips For Use With Microsoft Windows 7.0 

  Do you miss the 'Quick Launch Bar' ? The bar is absent in Windows 7, but it can be put back.

1.   Use your mouse to right click on the Windows taskbar and then choose Toolbars; New Toolbar.
2.   The system will ask you for the path of the new folder. Type:   %userprofile%\AppData\Roaming\Microsoft\Internet Explorer\Quick Launch
3.   Select 'folder'. A new link to the quick launch bar will be added to your current task bar. 

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  Are you using a laptop and want to know how the power is being used ? There is a built-in application which will create a file containing a power efficiency report for you.

1. From the command line type ("Folder" is the name you supply where you want the report to go): powercfg -energy -output \Folder\Energy_Report.html

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  Would you like to minimize all of the running application windows in one shot instead of minimizing them one at a time ? Windows 7 has a feature for this called " shake". The window that you are currently using will stay active and the rest will be minimized. Here is how to use it.

1. Click and hold the title bar of the current active window you want to stay on-top. 
2. While holding it with the mouse, shake the item back and forth until all of the other windows are minimized. 
3. Once they are, let go of the mouse button. 
4. Shake the title bar once again to bring them all back.

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.