Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

The overall goal of this method is to improve the efficiency and sensitivity of high performance liquid chromatography post column derivatization, or PCD method, through the use of a reaction flow column. This method can help answer key questions in fields such as pharmaceutical, biomedical, and environmental sciences, where compounds that have low response to HPLC detectors are analyzed. The main advantage of this technique is that no reaction coils are needed.

As mixing of the column effluent and derivatizing reagent occurs more efficiently than conventional methods. This method has been used to provide insight into antioxidants, amino acids, and phenols. It can also be applied to other class compounds, such as thiols, metals, antibiotics, and toxins.

Although the entire sample is not derivatized, due to lower band broadening, the concentration of analyte within the effluent flow stream is higher than in conventional post column derivatization analysis. To begin this procedure, prepare the HPLC instrument with 100%water on Line A and 100%methanol on Line B as the mobile phase, purging the pumps as per the manufacturer’s requirements. Set up the HPLC instrumental components and the additional derivatization pump.

Following this, set the UV/VIS detector to analyze at a wavelength of 520 nanometers. Connect the inlet of the reaction flow, or RF, column to the HPLC instrument. Connect an outlet peripheral port to the UV/VIS detector using a 15 centimeter length of 0.13 millimeter inner diameter tubing.

Next, connect the DPPH pump line to a peripheral port on the outlet of the RF column. Block the unused peripheral port on the outlet of the RF column using a column stopper. Connect a 15 centimeter length of 0.13 millimeter inner diameter tubing to the outlet central port of the RF column.

Bring the flow rate of the HPLC pump to one millimeter per minute at 100%methanol. Then equilibrate the column with 100%methanol for 10 minutes. At this point, prepare a 0.1 milligram per milliliter solution of DPPH and methanol.

Sonicate the flask containing the DPPH reagent for 10 minutes. Purge the DPPH pump with the prepared DPPH reagent as per the manufacturer’s requirements. Following this, take two dry and clean vessels and label one as central”and the other as peripheral”Accurately weigh the two vessels.

Collect the effluent exiting the central port into the vessel labeled central”for one minute. After reweighing the central port vessel, calculate the weight of the flow from the central port. Repeat the previous steps for the effluent exiting the UV/VIS that is attached to the peripheral port of the RF column.

Calculate the weight for the peripheral port vessel. Next, calculate the percentage of the flow coming from the central and peripheral ports. Repeat the previous steps until the flow ratio is correct, then set the flow rate of the DPPH pump to 0.5 milliliters per minute.

Once the run has finished, stop the derivatization reagent pump flow. Remove the DPPH reagent pump line from the peripheral port and stopper the port. Equilibrate the column with the mobile phase in which it is to be stored, allowing the mobile phase to pass through the column at one millimeter per minute for 10 minutes.

Then, stop the flow of the bubble phase pump on the HPLC instrument. Finally, replace the DPPH reagent with methanol and purge the additional pump. Two chromatograms of a ristretto coffee sample, derivatized with a DPPH radical using both conventional PCD and RF-PCD instrumentation, are shown here.

The calculated limits of quantitation and detection for each of the amino acids analyzed in both RF-PCD and conventional PCD modes are listed here. A chromatogram of the four amino acids analyzed using the conventional PCD method, the RF-PCD method, and the un-derivatized stream from the RF-PCD method is shown here. A comparison of the signals obtained for the peaks due to glycine and leucine using both the conventional PCD and RF-PCD methods is displayed here.

A comparison of the peak width of the tryptophan peak when analyzing using the conventional PCD method, the RF-PCD method, and the un-derivatized stream from the RF-PCD method is shown here. A 21-component test sample that contained some components that show a response to the derivatization scheme and some that do not were separated, derivatized, and detected. The same mixture was also separated and detected un-derivatized for comparison.

A comparison of the peak shape of para-Cresol, both derivatized using the RF-PCD column and un-derivatized, is shown here. Once mastered, this technique can be set up in the same time as conventional post column derivatization analysis. When performing this procedure, it is important to remember to be as close to the prescribed flow ratios as possible.

Following this procedure, other post column derivatization reagents, such as OPA, ninhydrin, or halogens can be used in order to analyze other compounds. After watching this video, you should have a good idea of how to set up and tune a reaction flow column.