Today we will bring “Solutions to solve poor reproducibility between batch to batch”.
How to ensure good reproducibility when a new validated HPLC method is transferred to Q.C. lab?
HPLC column’s reproducibility is very important if a method will be used for a long time. It is necessary to confirm that the column is stable supplied, which means to choose a reliable HPLC column manufacturer and a common stationary phase instead of special or less used stationary phases. Column specifications and batch to batch reproducibility information should be collected from manufacturer as more as possible and judge meticulously.
How to treat reproducibility problem of the column from the same batch?
We should understand that slightly differences exist in retention time, number of plates, column pressure even these columns are produced from the same batch of packing materials.
The changes in retention time is proportional to the packing materials’ density and the column volume. The column volume reproducibility depends on the column ID. After the column ID changes, the retention time changes correspondingly, but the resolution remains unchanged, which indicates that your method reproducibility is good. When we develop a method, elution schedule should be made correctly to avoid the bad reproducibility caused by slightly changes of the column. RSD will reach about 3% in retention time changes caused by column volume changes in most column manufacturer. However, Welch can keep RSD around 1% by hardware control.
The changes in column plate number will also affect the method, especially when it comes to a pair of peaks that can be separated at baseline. In general, the reproducibility of the plate number is about ±10%, and the standard deviation is reduced to 2-3%, but the resolution depends on the square root of the number of plates, so a 2% change in the plate number means that the peak width changes only 1%. You can check the manufacturer’s column plate number control data.
The reproducibility of the column pressure in any HPLC columns produced by most manufacturers is good during the production.
How to treat the reproducibility problem from different batches?
The parameters mentioned above will change in different batches. More importantly, the packing materials of different batches may cause the change of selectivity, lead to the relative retention time changes on the chromatogram, which is not conducive to the determination of the method. This problem can be avoided by collecting information from the manufacturer on the stability of batch reproducibility, generally the physical and chemical parameters used to test the chromatographic performance of packing materials.
In the physical parameters, the most important factor is surface area, as its change will directly leads to the change in retention time. Specific surface area usually changes in the range of ±10% while welch is ±5%.
In the chemical parameters, the change in surface coverage of bonded phase has a greater influence on the retention time. Manufacturers will usually give the number of the carbon loading. There will be a detailed number, usually in the range of ±10% while welch is ±4%.
For testing chromatographic reproducibility, manufacturers are using a mixed solution of uracil, phenol, 4-chloronitrobenzene, and toluene as analyte reagent. The mobile phase should be 75% organic solvent and 25 % water under neutral condition. A better batch to batch reproducibility test is to add an alkali compound (usually amitriptyline) to the test mixture, under optimized condition, it mainly interacts with the free silanol groups on the silica, while the neutral compounds mainly interact with the bonding phases, so the relative retention time between the alkali and neutral compounds is a strict test for batch reproducibility of reverse packing materials. Welch has accumulated this type of batch reproducibility test data and controlled it within a small range.
How to determine whether this column has good batch to batch reproducibility for my experiments?
Manufacturer’s control and requirements for batch reproducibility is just a start of column selection, the final test is the reproducibility test of the project itself. Some manufacturers will provide HPLC column kit in which columns are from different batches for project test, while other manufacturers may provide columns from different batches according to clients’ needs. What we need is the columns with same packing materials from different batches and packed in different time, not the column with same batch packing materials packed in different time. Three different batches of column are suggested to test the method reproducibility. If the test is not influenced by the change of packing materials, then this method reproducibility is ensured in following years.
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