Q: What is the gradient retention volume of liquid phase system? What impact does it have on analysis?
Answer: the gradient retention volume is the volume from the gradient mixing point to the column inlet. This volume leads to a certain delay in the gradient, so it is also called gradient delay volume.
At present, many liquid phase systems are single pump low-pressure gradient systems, where, the gradient is mixed upstream of the pump. The first part of the gradient delay volume is the volume of the gradient mixer. In this way, you have to add the connection volume between the gradient mixer and the pump head. The second is the volume of the pump head, and then the connecting pipeline of the injector. Usually, this volume is increased a little to fully mix the mobile phase and smooth the gradient a little. The volume from the injector to the injector and the column head is another part. Look, in the usual low-pressure gradient system, there are quite a lot of places where the gradient stays
The gradient can also be generated at the high pressure side. This requires a minimum of 2 pumps to minimize the gradient delay volume. However, the high-pressure gradient system is not set up like this. Usually, the connection position is before the injector. The advantage is that the sample can enter the column regardless of the flow rate of which pump. It is better to use the initial mobile phase to dissolve the sample, so that the gradient delay volume can be completely forgotten. A small disadvantage of this technique is that the sample will be diluted a little by the second component of the mobile phase, but if the initial mobile phase is 90% mobile phase a, the problem is very small, only 10% diluted. Another problem is that most of the summit is usually gathered at the stigma, so that only a small part of the peak is eluted in the initial isocratic elution. At this time, we should ensure that the volume between the injector and the column head is small enough. High pressure gradient system has no such problem.
Now let’s discuss how the gradient delay volume affects the analysis! Since it takes time for the gradient to reach the column, the gradient is delayed at the beginning, and the starting time is equal degree running. The first effect is that the retention time of the previous peak may be different after changing the system. If the gradient delay time is obvious, the peak in the early stage of the chromatogram will be significantly different, while the late stage of the gradient is not very related to the delay volume. However, the elution time will still change with the gradient delay time. These situations are very troublesome. This is why gradients are avoided in QC laboratories. After all, the sample is determined by retention time. If the retention time is different after changing the instrument, it is difficult to judge. Of course, it can also be judged by the retention time of the standard, but it is not complex
Q: We agree. It’s a little complicated, but it’s not Insurmountable. Is there anything else to consider?
Answer: if the mixing volume is large in a low-pressure system, the gradient map you observe will not be very sharp. This usually does not affect the standard linear-gradient, but if it is a stepped gradient, it will affect the elution. Gradient systems with small delay volumes are usually used. Now many HPLC systems have very small gradient delay. For very fast gradients with small cycle time, even a small delay volume is not enough. At this time, delayed injection technology can be used. Theoretically, the gradient still operates as usual, but the sample is not injected until the gradient reaches the injector. This is like the delay volume without gradient. Of course, the volume of the injection ring and the pipeline from the injector to the column head still has a certain delay, but the small ones can be ignored.
When the gradient runs, the column will start balancing again, which is also delayed. So we don’t have to wait until the column is rebalanced before starting a new gradient. We just need to know the design procedure and the operation of the pump is different from that of the column head. The gradient operation and column rebalancing are offset by the emptying gradient delay volume. This solution allows us to implement a fast gradient without delay in a single pump system. In addition, if the same gradient is run on different instruments, the effect of delay volume will be very small. So we can repeat the gradient on different instruments. Of course, the above assumes that the gradient generator works stably, repeatable and can work as required.
Q: How to measure the gradient delay volume?
How to determine that the gradient of the system is really what you want?
Answer: A standard test is very useful. Refer to the gradient verification in JJG 705 2014 verification regulation of liquid chromatograph. Without connecting the column, add a small amount of UV absorber 0.1% acetone to mobile phase B, and then run the gradient. This allows you to observe the real gradient. The gradient delay volume of the system can be detected from the difference between the program and the practice. If you use delayed injection, you must understand this. The more you know about the system, the less time you waste in gradient analysis.
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