FDA) require that K prime values for HPLC separations be equal to or greater than 2.0 to meet Specificity acceptance criteria (System Suitability/Method Validation). Do not develop methods which only result in K Primes of less than 1.5 (an indication of poor quality chromatography). Try and insure that the earliest eluting peak in your sample has a K Prime of >1.5. K prime values of greater than 10 are acceptable, but often show minimal improvements to resolution. Your final method should baseline separate all compounds apart and, if properly developed, each sample p eak will often have K Prime values between 2.0 and 10.0. Once you have this T(0) value, you can then determine the retention factor (the "K Prime") of your actual sample(s) using the simple formula below. If your HPLC method does not retain the sample on the column long enough past this time, then you are not allowing any chromatography to occur. The time it takes an unretained compound to elute off the column is critical to know. This establishes what we often refer to as the 'T' zero time, or T(0). Must calculate the column void volume AND inject a sample which will not be retained by the column toĭetermine what time an unretained sample will be eluted off the column. Knowing the column void volume allows you to determine the retention time of an unretained sample and the resulting retention factor (K prime) of each sample eluted after it. Calculation and/or measurement of the Column Void Volume should be one of the very first chromatography method development tasks you learn to perform. Knowing a sample's retention or capacity factor allows us to be confident that it has been retained and eluted past this critical point, but to calculate it we first need to know the column's void volume. Sounds rather obvious at first, but you may be surprised to learn that many chromatography methods fail this test of retention and are invalid. For most modes of HPLC separation, highest on this list of fundamentals is that the sample(s) be retained on the HPLC column used and not eluted out at or near the column void volume ( we often refer to this time in minutes as, " T-zero"). Observance of the fundamentals of chromatography are key to developing high quality HPLC methods. It must be long enough to demonstrate that the method developed is specific to the sample and shows good selectivity for the sample analyzed. If this interaction is too short, then no chromatography has taken place and you have just developed a "flow-injection" method (no column used) instead of a chromatography method. The role of Capacity Factor / Ratio ( K prime) in chromatography is to provide a calculation or formula which defines how much interaction the solute (sample peak) has with the stationary phase material (the relat ive time interacting with the support vs.
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