Advantages of Mixed Mode HPLC for Polar Compounds

It was a relief when I used the brand new Cogent UDA™ columns for analysis of closely related polar compounds. I used them in the Aqueous Normal Phase (ANP) retention mode with additional selectivity of the Ion exchange chromatography capacity.
                With the Cogent UDA 2.o™ column, you can have the best of both worlds by using each retention mode together to obtain excellent separations. In the latest study from our laboratories, we demonstrated the ion exchange characteristics of the Cogent UDA 2.o™ column by separations of three test solutes. With a carboxylic acid group at one of the bonded ligand, ion exchange propertied can be either activated or inactivated by selection of the mobile phase pH.  Under acidic conditions, the carboxylic acid is protonated and neutral and no ion exchange occurs. At mild pH, the group becomes de-protonated and anionic. When the group was ionized, increased retention and separation of the three basic test analytes were observed.

                You can read about the full investigation here.

Separation of the three analytes under ion exchange conditions

An Interesting Compound found in Green Tea

Green tea is more than just a tasty beverage. It also has a number of compounds with potential health benefits. For example, catechins found in tea are known antioxidants. Another such interesting compound is L-theanine. It is a psychoactive compound and is believed to exhibit various beneficial effects on the brain. These may include increased alertness, decreased anxiety, and general sense of well-being.

                It is a polar compound though and may be difficult to analyze by reversed phase chromatography. With the Cogent Diamond Hydride 2.o™ column, it can be well-retained using an Aqueous Normal Phase approach. There are not many other polar compounds in green tea (catechins are fairly hydrophobic), so you can obtain a nice L-theanine peak which is well-separated from the others.

                We estimated L-theanine content in a typical home-brewed green tea sample using this column and found concentrations consistent with those reported in the literature (around 10 mg/L). See the full study for more information!

 

How much caffeine is in your coffee?

The amount of caffeine in various beverages can vary widely. Drip-brewed regular coffee has about 555–845 mg/L caffeine content but a shot of espresso can have about 1,691–2,254 mg/L. What some people may not know is that decaffeinated coffee has caffeine as well. The caffeine is chemically removed by a series of extraction procedures (solvents used can include benzene, supercritical CO₂, or others). However, even after 8–12 extractions there is still some caffeine remaining. Therefore, decaffeinated coffee has about 24–72 mg/L caffeine.
                This can be studied with HPLC methodology. Here a Cogent Bidentate C18 2.o™ column is used to separate caffeine from other matrix components in three types of common coffee products. The caffeine peak is the most prominent of all the peaks in the espresso sample but is only a minor peak with the decaf sample. Using a caffeine reference standard, a calibration curve could be made which allows for an estimation of the caffeine content in the samples. The calculated values were: espresso: 964 mg/L, regular: 539 mg/L, and decaf: 85 mg/L. These values were mostly close to the literature ranges for each coffee beverage, although the espresso sample had somewhat lower caffeine content than expected.
                Click the link for more info!

How to Obtain a Flat Baseline in Gradient Mode

You developed a nice gradient method for your pharmaceutical impurities analysis. The peaks are sharp and symmetrical. The separation is good. The only problem is the baseline, which slopes and makes accurate quantitation of your peaks difficult. There are a couple of ways you can avoid this problem and you might find one easier than the other depending on your situation.

                 The first way is to run a blank. Typically this will be your sample diluent with nothing else in it. You only need to do this once for the method and then you can use it for each sample. Most modern chromatography software has a feature called “blank subtraction.” Here, you would load your sample chromatogram with the sloping baseline and then apply the blank subtraction operation. Every chromatographic feature in the blank will be subtracted from your sample data, resulting in a completely flat baseline. Some analysts may find this cumbersome though because it requires you to run an additional sample. Still, many protocols may require the analyst to run a blank anyway to demonstrate that they don’t have any “ghost peaks” in their sample chromatograms.         

                The second method doesn’t require an additional run but it can be more tricky to initially develop. This technique is called “absorbance matching.” The reason the gradient slope occurs is because of a difference in UV absorbance between the A and B solvents. You don’t notice it in isocratic methods because the solvent composition never changes. Generally, the B solvent will absorb more because of the organic solvent (e.g. acetonitrile, methanol, etc.). In this approach, the goal is to achieve identical absorbance between the two solvents. The two don’t have to have the same absorbance across the whole UV spectrum, just the wavelength you are operating at. To achieve this, add a UV-absorbing species to the A solvent until you don’t see any change in the baseline. Once you know the correct amount, the process should be easy to write into your SOP. Generally though, determining the proper amount is accomplished by trial and error. The additive should be unretained and should not interact with or affect the sample. Examples include nitrate, nitrite, azide compounds, etc.

An example of blank subtraction

AQ™ Brand Polypropylene Vial Study (LC-MS) compared to market leading vial

Many scientists use a very common 300µL plastic (polypropylene) vial for chromatography (HPLC) when they have compounds that stick to glass. With the advancement of LCMS, HILIC and ANP chromatography these vials are not useable because they produce spurious peaks. The peaks are due to common chemicals used to make these vials at a low cost. The peaks shown below are from injection mold release agents, clarifiers and antioxidants are added to manufacturing process.

The MicroSolv Advanced Quality™ (AQ) LCMS compatible vials, both screw top and snap top, are made from a proprietary blend of virgin polypropylene that cannot be made with the same speed as our process does not include mold release agents, clarifiers or antioxidants. The higher cost of production is due to slow speed of production but the resulting product is much cleaner than others and can be used confidently with LCMS and HPLC solvents.