The overall goal of this method is to improve the efficiency and sensitivity of a separation through the optimization of a curtain flow column. This method can help answer key questions in the field of environmental sciences, forensics, and in areas that require trace sample analysis, where an efficient and highly sensitive separation technique is required. The main advantage of this technique is that it increases the efficiency of a separation and the sensitivity of the analysis compared to conventional methods of HPLC separation.
This method can provide a means to increase the separation power, and thus speed capacity, in an economically and time-efficient way. The curtain flow column is a new concept to practitioners of HPLC, however, its use is not complicated. It simply requires the optimization of appropriate flow ratios.
First, prepare bottles of 100 percent ultra pure water, and 100 methanol for mobile phases A and B of the HPLC instrument. Purge the pumps as per manufacturer requirement. After purging, disconnect the pump line from the injector valve of the auto sampler.
Attach a T piece to the disconnected line. To each port of the T piece, attach tubing of 0.13 millimeter internal diameter and 15 centimeters long. Connect one tube from the T piece to the injector valve of the auto sampler.
Finally, set the pump to a flow rate of one milliliter per minute. Tare two empty collection vessels and label them central and peripheral. For 60 seconds, use the central-labeled vessel to collect the exiting mobile phase from the line coming from the injector.
Re-weigh the central vessel and determine the mass of the collected mobile phase. Repeat this process by collecting the flow through of the peripheral port with the peripheral-labeled vessel. Determine the percentage of flow, or milliliters per minute, from each line using the indicated equations.
The central port should be about 40 percent, and the peripheral port about 60 percent. If the values differ by more than two percent, change the tubing length or diameter to adjust the flow ratio. Then repeat the collection and the calculation steps.
Next, turn the pump flow off and connect the injector line to the central port of the column inlet. Also connect the line from the T piece to the peripheral port of the column inlet. Slowly ramp the flow rate to one milliliter per minute of 100 percent mobile phase B.To finish tuning the system, flow 100 percent methanol at one milliliter per minute for 10 minutes to equilibrate the column.
To begin, use tubing to connect the central outlet port to the UV-Vis detector. Connect the same type and length of tubing to the peripheral outlet port of the CF column. Then, set the pump to a flow rate of one milliliter per minute.
Tare two empty collection vessels and again label one for central collection and one for peripheral. For 60 seconds, use the central-labeled vessel to collect the exiting mobile phase from the UV-Vis detector. Re-weigh the collection vessel to determine the mass of solution eluded.
Repeat this process with the peripheral outlet port and labeled collection vessel. Now determine the percentage flow from each line as performed earlier with the inlet flow. The UV-Vis line should be 21 percent, and the peripheral outlet flow should be 79 percent.
If the flow ratio deviates significantly, adjust the size of the tubing attached to the exit of the UV-Vis detector to alter the central flow percentage. It is important that the flow ratios between the central and peripheral at the inlet and the outlet of the column follow this protocol to ensure optimal performance of the curtain flow column. Active flow technology columns were developed using a specialized frit design in the multiport column end fittings to overcome the column bed heterogeneity and improve separation performance.
A test mixture containing phenetole, butylbenzene, and pentylbenzene was analyzed on conventional 4.6 and 2.1 millimeter internal diameter columns, and a 4.6 millimeter internal diamater curtain flow column. The separation of the mixture illustrates the improved performance in terms of efficiency and sensitivity of a curtain flow column relative to standard columns. The use of a curtain flow column increased the signal for butylbenzene despite the reduced peak volume.
Once mastered, this technique can be set up in less than half an hour if it is performed properly. The curtain flow column setup can be reused over and again, but the flow ratios must be checked routinely. While attempting this procedure, it is important to remember to be as close to prescribed flow ratios as possible.
The curtain flow column has paved the way for the researchers in the field of HPLC to push the limits of the columns and UPLC instruments to obtain faster sample analysis without compromising separation performance. After watching this video, you should understand the importance of curtain flow columns, as they can provide better separation than conventional HPLC columns.
