Observance of the fundamentals of chromatography are key to developing high quality HPLC methods. 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. Sounds rather obvious at first, but you may be surprised to learn that many methods fail this test of retention and are invalid. Knowing a sample's retention or capacity factor allows us to be confident that it has eluted past this critical point, but to calculate it we first need to know the column's void volume. 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 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. To do this, you must calculate the column void volume AND inject a sample which will not be retained by the column to determine what time an unretained sample will be eluted off the column. This establishes what we often refer to as the 'T' zero time, or T(0). The time it takes an unretained compound to elute off the column is critical to know. 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. 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. Your final method should baseline separate all compounds apart and, if properly developed, each sample peak will often have K Prime values between 2.0 and 10.0. Try and insure that the earliest eluting peak has a K Prime of >1.5 and do not develop methods which only result in K Primes of less than 1.5 (poor quality chromatography).
Note: Many regulatory agencies (e.g. FDA) require that K prime values be equal or greater than 2.0 to meet Specificity acceptance criteria. After all, if it elutes at or near the void volume, then your method is not specific for anything. Besides being unscientific in design, your method will fail validation if it does not meet this basic requirement.
- K Prime (Capacity Factor or Retention Factor) Formula:
- k1 = T(R) - T(0) / T(0)
(where T(R) equals the retention time of the peak in minutes and T(0) is
the retention time of an unretained peak).
- *The 'K Prime' of your sample must be > 1.00. A value greater than 1.5 should be your goal.
T(0) found to be 2.90 minutes and the sample elutes at 5.80 minutes. k1 = 5.80 - 2.90 / 2.90. k1 = 1.00.
T(0) found to be 2.90 minutes and the sample elutes at 9.10 minutes. k1 = 9.10 - 2.90 / 2.90. k1 = 2.13.
T(0) found to be 1.75 minutes and the sample elutes at 1.74 minutes. k1 = 0. No retention and no chromatography have taken place at all. The method is invalid.
*I see and read published HPLC methods (including "Validated Methods" !) every week which ignore this fundamental requirement and present data showing little to no retention of the primary sample on the column. These methods often describe the sample analyzed as "100% pure" and fully validated! A mixture will always look like a single peak by HPLC when no 'chromatography' is employed to separate out all of the possible components. The sample must be retained on the column for a period of time before we can conclude anything about its purity by the method employed.