Observance of the fundamentals of chromatography are key to developing high quality HPLC methods. 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"). Sounds rather obvious at first, but you may be surprised to learn that many chromatography 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 been retained and 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. K prime values of greater than 10 are acceptable, but often show minimal improvements to resolution. Try and insure that the earliest eluting peak in your sample has a K Prime of >1.5. Do not develop methods which only result in K Primes of less than 1.5 (an indication of poor quality chromatography).
Note 1: Many regulatory agencies (e.g. 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). 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 System Suitability and fail validation. IOW: 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.
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.
Note 2: 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. Most are RP methods run on popular C18 columns and show the main peak of interest eluting out as a nice sharp peak right at the void volume. These methods often describe the sample analyzed as "100% pure" and are fully validated (because the person doing the work may not have had any HPLC experience or training)! 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.
Note 3: In some cases, when other modes of chromatography are utilized (e.g. ion exchange, size exclusion chromatography (SEC / GPC), K prime is not as relevant. The mode of chromatography can affect the interpretation. For example: This is because size exclusion chromatography relies on the sample's interaction with well defined pores inside the support (inclusion/exclusion) to separate based on molar size. A variety of pore sizes can be used to "filter" the sample. So a large K prime value might be normal for a molecule that is low in molecular weight and spends a lot of time working its way through the column. A high molecular weight sample might just "shoot" through the column due to little or no interaction with the pores. You still need to have retention on the column, but now it is determined by how long it takes the sample to find its way out of the column. SEC columns are bracketed by Pore Size (e.g Mw. Excluding all samples that do not "fit"). With size exclusion columns, determine the Retention and Exclusion times, not the K prime). This article is presenting for the far more common cases where traditional NP or RP modes are used. In these cases, low K prime values indicate no retention took place and the method fails all claims of specificity for the sample (selectivity is absent or poor). HPLC methods with little to no selectivity fail scientifically as no chromatography has taken place.
Is there any reference for k prime acceptance criteria ?
ReplyDeleteDifferent 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.
DeleteSo 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.
GREAT POST :)
ReplyDeleteFor 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.
We are grateful for your many articles and knowledge!!!! Several methods used for purity analysis by our lab (we are Contract laboratory) come from published sources by our customers. Most have been run for many years without any problems. Now, we feel very humble. After reading your articles on column void volume and K prime, we understand that our training in HPLC has been very poor. It turns out that 6 of the regular methods we use from our clients, do not show any chromatography at all. All are reverse phase methods (C8 and 18) and we calculated K primes for the main samples and found all of them are less than 1, and a few are just 0.2. This is understood as no retention on the column and the method developed and used is junk. The scientists who provided these methods work at IVY league university, but their training was extremely poor and perhaps we now know so was ours. Our lab is now going to learn those basic chromatography fundamentals that you write about (and this website is the best place to do so) before we run any more samples. We will also do a better job of reviewing methods submitted to us. We thank you for providing this knowledge to us. Through reading and training, we have the chance to be better.
ReplyDeleteAs they say, better to learn now rather than than later. Knowledge is power. Most training is lifelong and has no end. Stubbornness to change or excuses such as, "we always do it that way" are common reasons of failure. It is wise to always be a bit skeptical of any new information presented (e.g. HPLC methods) before use. Never assume...
DeleteIs a K prime of 19 too large? Is it OK to have peaks with K primes of 20 or 30?
ReplyDeleteThe larger the K prime, the longer the peak retention time (and wider the peak width is). Good chromatography balances a reasonable total analysis time against an acceptable K prime. Optimizing the chromatographic conditions is key to developing a method which has scientifically acceptable K prime values (which demonstrate proper method selectivity has been achieved) and a reasonable run time. A "reasonable" run allows us to complete the analysis quickly, use the least amount of solvent and have the quickest turn-around time to run the next sample. *So yes, you can develop a method which has large K prime values, but the extra peak broadening and drift which are associated with long run times are not desirable (greater error).
Delete