Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, June 27, 2015

K Prime (also known as: Capacity Factor or Retention Factor): One of the Single Most Important HPLC Parameters of All

The role of Capacity Factor (K prime) in chromatography is to provide a calculation or formula which defines how much interaction the solute (sample peak) has had with the stationary phase material (the relative time interacting with support vs. the mobile phase). 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. 

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.
Example #1: 
 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.

Example #2:
 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.

Example #3:
 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.


  1. Is there any reference for k prime acceptance criteria ?

    1. Different regulator authorities establish different criteria. Yes. One example of an authority which does establish an acceptance value is the US Food and Drug Administration (FDA). *K prime greater than or equal to 2.0.

      So many methods that I am asked to review have K primes of less than 1.0 ! This means that many types of potential impurities can easily co-elute or be hidden under the main peak since very little effort has been made to separate it.

  2. GREAT POST :)
    For several years, our lab has used a published method which we use for the determination of the purity of a clients sample. We have always wondered about the quality of the method used, but no one here is really an expert at HPLC so it is used and accepted because it is "published". After reading your post and learning more about lc, we now have evidience that the peak comes out at the VOID vol. As I understand it, that mean no separation at all. the purity values obtain are always 100% and now we know why. You are correct, just because it is published in a respected journal means ZERO. no one checks and the data and purity are worthless. Not sure what we can do, but we will not run that sample using that bad method anymore. We will find a new method.