Q1: I want to start developing a new HPLC method. After validation, the method will be transferred to QC laboratory and will be used for many years. I am worried about the long-term repeatability of this method, especially the long-term repeatability of columns. How can we ensure good repeatability?
Answer: First of all, We would like to commend you for taking this aspect into account when starting this method. If you can predict that this method will be used for several years, you need to do some homework in selecting columns. If you want to use the column within the foreseeable application period of this method, you need to choose some large manufacturers. In addition, choose a standard surface chemical (such as C18 or C8) instead of those novel or rarely used.
The next criterion is the repeatability of the column. If you or your colleagues have used a column with good repeatability before, this is a good starting point. At the same time, we should also consider the information provided by the manufacturer. Recently, some manufacturers began to print their instructions and the results of repeated experiments in different batches. You can get this information from the manufacturer and compare it carefully.
Q2: What should I look for from this information?
Answer: Let’s first explain the different aspects of repeatability between different columns and batches. Now let’s talk about inverse fillers, because they are more common.
If you use different columns with the same batch of packing, you will get different number of trays (peak width), different column pressure and different retention time. The retention time varies in proportion to the packing density and column volume, which depends on the inner diameter of the column. This change should have no effect on your method, because the retention time of all peaks changes proportionally, and the resolution remains unchanged. The RSD of retention time change caused by this reason is usually about 3%, but if the manufacturer has good control over the hardware of the column, the RSD can also be as low as 1%. Changes in the number of column trays will affect the method, especially when doing bimodal, they rarely achieve baseline separation. Although the standard deviation of 2-3% of the number of trays can be achieved, it is usually within ± 10%. Remember that the number of trays is a square number, and the resolution only depends on the root of the number of trays. Therefore, the deviation of 2% of the number of trays is equivalent to the deviation of 1% of the peak width. You should confirm whether the column manufacturer has the upper and lower limits of the number of trays or only one lower limit. Both the upper and lower limits are quite satisfactory.
The repeatability of column pressure of columns with the same filling procedure is quite good, and the change of the same batch shall not exceed ± 5%.
Q3: What about the repeatability between batches?
Answer: If you pay attention to the chromatographic parameters between batches, you will find that the same parameters will be different, and more importantly, the selectivity of separation between batches will also change. That is, the relative retention of peaks will change, such as their relative position in the chromatogram, which is unfavorable to the certainty of your method.
You should try to avoid this change, so you should get some information from the manufacturer about how they ensure the repeatability between batches, including some physical, chemical and chromatographic tests. In the physical test, the most important item is the filling surface area, and the change of this parameter will directly lead to the change of retention time. So you need to know the range of surface area change between batches approved by the manufacturer. Generally, the amplitude is ± 10%, but ± 5% can also be achieved. The most important chemical parameter is the surface coverage of the bonded phase. How much do you usually use μ Mol / m2. Many manufacturers did not directly give this parameter, but only explained the percentage content of C. We hope to see this alternative description and a compact range, usually ± 10%, which can also be achieved if it is lower than ± 4%.
Finally, you should know the chromatographic repeatability test of the manufacturer. Usually, the chromatographic column will be attached with a chromatogram to detect the mixture of some simple neutral compounds, such as toluene, naphthalene and anthracene. But this is not a good inter batch repeatability test. Because the relative retention repeatability of these neutral hydrophobic compounds is quite good, they are quite insensitive to the change of surface coverage during loading. A good inter batch repeatability test should also add strong alkali compounds to the mixture. In carefully designed tests, selected basic compounds mainly interact with residual silicon hydroxyl groups, while neutral compounds mainly interact with bonding phases. Only repeatability tests containing two compounds are convincing. If a manufacturer tests like this and has good repeatability, his column will be more reassuring.
Q4: I don’t know the relationship between the manufacturer’s test and my compound test. How can I ensure a good batch repeatability in my test?
Answer: You’re right. The above information can only give you a greater chance to ensure repeatability. But in the end, you must use your own test to do the repeatability test. Some manufacturers provide a complete set of equipment, including columns of different filling batches to do this test, while others provide columns of different batches as required. You need to know what you get. The chromatographic column of the same filling batch is useless. What you need is the column filled with different bonding reactions. If the same batch is filled in different days, and then the column number is different, you’d better ask twice to ensure that the manufacturer gives you what you want.
You need three different batches of chromatographic columns to test the repeatability of your method. If the change between batches does not affect your test, your method should have a good repeatability in the next time.
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