summary

The preparation process is widely used in many fields, such as selectively screening candidate compounds or lead compounds from newly synthesized compounds, or for structural analysis of impurities in drugs and components with specific functions in natural substances. Preparative supercritical fluid chromatography (abbreviated as preparative supercritical fluid chromatography)SFC has the advantages of short analysis time and simple post-processing, and has been widely used in the pharmaceutical industry and many other fields. For analysis with limited number of chromatographic peaks, such as the separation of chiral isomers, "superposition injection" can improve the efficiency of preparation and purification. This report describes an example of using the "superposition injection" function of a Nexera UC prepared supercritical fluid chromatograph to improve the efficiency of preparation operations.

keywordsPreparation type SFC, superimposed injection

1useSFC to shorten analysis time

Due to the low viscosity and high diffusion coefficient of supercritical carbon dioxide, even at high flow rates,The chromatographic column pressure of SFC is also very low. This means that analysis speed can be improved without sacrificing chromatographic column efficiency. Therefore, its analysis time is much shorter than that of high-performance liquid chromatography.

Taking the chiral separation of * as an example, prepare usingThe comparison between LC and the time required for preparing SFC is shown in Figure 1. The time required for preparing SFC is only 1/4 of the time required for preparing LC, which improves the analysis efficiency.

imageComparison of HPLC and SFC for * Chiral Resolution (Preparation Type)

table1. Analysis conditions

2Superimposed injection

'Superposition injection' is a standard configuration forThe continuous injection technology of SIL-40 automatic sampler and FRS-40 fraction collector utilizes the time interval of chromatographic peak retention for continuous injection, thereby saving analysis time and improving separation efficiency. Its working principle is shown in Figure 2. When setting up the "superposition injection", special attention should be paid to the following points:

• Only applicable to isometric analysis

• The chromatographic peaks will not overlap with each other

imageWorking principle of "superposition injection"

3Method for setting up "superimposed injection"

The 'superimposed injection' function can be used inEasy setup in LabSolutions workstation software. By setting parameters such as "injection interval", "injection frequency", and "waiting time for the next preprocessing" (Figure 3), and simulating the results of a given injection interval using a single run result (chromatogram) (Figure 4), it is easy to confirm whether there is chromatographic peak overlap. If continuous injection is required, an appropriate waiting time must be set so that after the sample flows out of the sample loop (Figure 5), the sample loop can switch back to the LOAD state (on the right side of Figure 5).

image. 3 Set the "superimposed injection" parameter (for Figure 4)

image4. Simulation results of "superposition injection" (LabSolutions)

imageSample valve action (FRS-40)

For data collection time, input a value greater than the duration of a single analysis plus the product of the sample interval and the number of injections. For example, toIf the injection interval is 0.8 minutes and the sample is injected continuously 9 times, the input value should be at least the duration of a single analysis plus 7.2 minutes (Figure 6).

imageData collection time (SPD-M40)

After each injection cycle, return the fractionation valve to its initial position to collect the same peak in the same bottle. Therefore,The "superposition injection" method can collect all peaks of the same compound in the same collection bottle. The collection of fractions will be carried out according to a time program. Only the time program for a single analysis needs to be entered, and then the workstation can automatically calculate the fraction collection time based on the injection interval (Figure)7 and 8)

imageReset time for "superposition injection"

imageThe relationship between reset time and preparation interval

4Superposition injection "is used for chiral sample separation

Here is an introduction to a practical useA case study of separating chiral drugs using "additive injection", with the sample being10mg/mL * solution (methanol). The analysis conditions are shown in Table 2, and the obtained chromatogram is shown in Figure 9. The results showed that within 10.5 minutes of analysis time, the number of injections could be increased from 3 for conventional injections to 9 for "superimposed injections", improving the efficiency of the preparation operation.

table2 Analysis conditions

image9 * Separate instances