HPLC K Prime. Also known as: Retention Factor, Capacity Factor): One of the Single Most Important HPLC Parameters of All

The role of Capacity Factor / Ratio (K prime) in liquid chromatography is to provide a calculation or ratio which defines how much interaction the solute (sample peak) has with the stationary phase material relative to the mobile phase (IOW: the relative time the sample spends interacting with the support vs. the mobile phase). If this interaction is too short (i.e. K prime less than 1.0), then no chromatography has taken place and you have just developed a "flow-injection" method (the same as if no column was used) instead of a chromatography method. The method fails all validation and is NOT fit for purpose. Sample Retention must be long enough to demonstrate that the method developed is specific to the sample and shows good selectivity (retention) for the sample analyzed. This is true for most, but not all modes of liquid chromatography (3). 

• New and ever 'experienced' users lacking training in HPLC often make this error, developing methods where the sample has little to no retention on the column. We routinely review methods where the actual K prime of the key sample is measured to be at or near 0, failing to show any chromatography took place in the method (*The Journals are filled with thousands of examples of invalid HPLC methods of analysis). This mistake is made because the author(s) have no formal training in liquid chromatography and do not understand the basics of the technique. 
• Note: Slowing down the flow rate to "show" a later elution time DOES NOT increase K prime (a very common novice mistake) !!! 
  

Observance of the fundamentals of chromatography are key to developing high quality HPLC methods. For most modes of HPLC, highest on this list of basic fundamentals is that the sample(s) be retained on the HPLC column used and not eluted out at or near the column's void volume (we often refer to this time in minutes as, "T-zero" or "t0"). Many chromatography methods fail this simple test of retention and are invalid as written. Knowing what a compound's retention or capacity factor is allows us to be confident that it has been retained and eluted past this critical point, but to first calculate K prime, we first need to know the HPLC 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 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 ratio 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. The USA FDA) requires 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, K1 (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.


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 specific to thr more common cases where traditional HPLC 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.

HPLC Peak Tailing - Some of the Most Common Reasons For it

Three easy ways to minimize chromatography peak tailing:

(1) Tailing often results from using “Type – A” HPLC silica. Type-A silica often contains more acidic silanol groups and metal impurities than Type-B. To improve peak shape, use modern “Type – B” silicas which are of higher overall purity, have less metal contamination and feature minimal silanol ionization under higher pH conditions.

(2) Minimize ionic interactions and utilize a buffer or ion pairing agent (e.g. TFA 0.02%) in your mobile phase. Select a buffer that is at least 2.0 pH units away from your sample's pKa and use the smallest concentration or amount that gets the job done. For LC/MS or MS/MS applications, remember to only use volatile buffers and avoid the use of ion pairing agents unless absolutely necessary (and if used, use at the lowest possible concentration to avoid source contamination).

(3) Always use a freshly washed and equilibrated column. Is the column fouled or the inlet frit dirty? If the head of the column is fouled from sample overloading or from a failure to wash off strongly retained compounds from many runs (much more common problem), then your peak shape and reproducibility will suffer. Incorporate a washing step in between your analysis methods which utilizes a solvent which is stronger (in concentration) than your mobile phase to wash off any strongly retained material after each run. For example, if you normally end a method with an 80% concentration of ACN, utilize a separate wash method which has 95% ACN in it. Allow enough wash time for this work.

Standard Addition HPLC Calibration Method (Spiking)

The standard additions calibration method is used to determine the concentration of an analyte which is in a sample matrix such as is commonly found when working with clinical, biological or food samples. To rule out interference from other components in the sample matrix, the sample is “spiked” and the detector response is monitored for any change which is not due to the change in sample concentration. The fortified standards created by spiking the original samples is useful when no version of the sample matrix is available without the analyte for use as a true blank. 

Common “Spiking” Standard Addition Procedure Example:

Divide up your sample evenly into FIVE volumetric flasks, representing five different final concentrations of spiked samples. Into four of the flasks add the same volume of increasing linear levels of the analyte to create four different calibration levels (e.g.  spiked amounts such as 25, 50, 75 and 100 units). Into the fifth flask add the same volume of diluent, without any spiked component, to serve as the ‘0’ or blank equivalent std. Next, extract all samples, inject and analyze the data per the usual method. A calibration curve of this new data set should show a linear relationship. If so, and your method is validated, you may be able to use just one spiked sample along with your regular samples to show that the reported concentration of your sample is not effected by the matrix and save yourself a lot of work (because you would need to prepare these standards and run a calibration curve each time).

Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems

"Carry-over" is a term used to describe a type of sample contamination which causes sample peaks to re-appear in later runs which do not actually contain the sample (e.g. blank runs). The contamination can last for several sequential runs, often decreasing in amount after each injection (which is a key observation when troubleshooting). When proper instrument training has been provided, modern HPLC system designs make carryover extremely rare, but when it does appear, the contamination can be due to: (1) A lack of HPLC maintenance; (2) Overloading samples which foul the column; (3) Poor Wash Vial Usage and/or Sample Vial Selection; (4) Inadequate operator training in how to set-up and use the chromatography system. *Note: Proper operator training greatly reduces the chances of contamination and is the most overlooked reason for the problem.

The Types of HPLC Carry-Over Contamination; Why They Occur and How To Reduce Them:

(1) A Lack of HPLC Maintenance: Most auto-injector valves rely on a rotary seal to move the sample from the needle loop to the flow path of the system. The components within these valves wear out and should be inspected at least every 6 months and replaced when needed. Also, always check the needle seat and needle for signs of wear or leaking. Note: Look for signs of leaks by the injector. Leaks always indicate a problem and should be fixed immediately. Don't run samples when you have leaks. Your method and data obtained will be invalid. Any worn parts should be replaced and the system performance tested. One of the most common causes of carry-over is due to a worn sample injector valve rotary seal. A worn seal can allow sample to be retained in the worn grooves, in-between injections, resulting in sample peaks appearing in subsequent runs. Additionally, buffer salts can lodge between the seals causing leaks or carryover. Routine HPLC service and, if applicable, flushing of all buffers/salts every day can eliminate these issues.

(2) Column Fouling / Overloading of Sample: If you inject too high a concentration of sample and overload your column with material, then it is possible that your column will continue to bleed sample long after the analysis is over. This also happens when the sample has a high affinity for the support you have chosen too. Failure to regularly flush and clean your HPLC column on a regular basis can also result in a similar problem as retained material is released from the column over time. Avoid this problem by performing a loading study to determine how much material can be effectively loaded on to the column. Next, create a wash method which utilizes a stronger solvent than your method (often utilizing a gradient) which will wash away any strongly retained material in between runs. This is critical if you are running an isocratic method as material will be retained on the column and must be washed off at frequent intervals using a stronger wash solution. *When using only isocratic methods, people often do not initially observe carry-over problems (because the sample just sticks to the column and accumulates over time). When the solvent strength is changed or the method is revised to a gradient, then the problems start... Avoid the problem by selecting the right column (which retains, then elutes ALL of the sample), not overloading the column (do a loading study) and washing the column down with a stronger solution that fully dissolves (not precipitates out) any remaining material off the column after each run.

(3) Wash Vial Usage and/or Sample Vial Selection: If you are using a modern high-pressure, "Flow-Through" design autoinjector (e.g. Agilent 1100, 1200, 1260, 1290), then carryover is rarely an issue as these modern injectors use a high pressure pump to aspirate and inject the samples directly into the flow path, reducing the need for any wash stage. The entire HPLC's injection flow path is continuously washed with mobile phase during the analysis run. This dramatically reduces the chances of any sample re-appearing in later runs. The need for a separate wash vial is nearly eliminated in this way as the needle, needle seat, loop, injector pump and valve are all flushed clean during each method. Many older auto-injector designs utilize either a low pressure injector (glass syringe) or injector pump which is not part of the main flow path. These injectors benefit from a separate wash vial as they are not continuously cleaned. Effective cleaning requires that a wash vial be employed in these cases. It should be filled with mobile phase or a solution which will dissolve any remaining material which might still be in the system.

When sticky sample solutions are used, separate Wash Vials can be used to reduce contamination with either older or newer injector designs . Sometimes these sticky samples can adhere to the outside of the needle while it is being withdrawn from a vial which has a septa which has been punctured many times. High puncture rates tend to open up the hole resulting in a lack of "wiping' of the needle surface upon withdrawal. *For vials that are punctured many times, it is critical to replace the septa OR use septa materials which seal for a long enough time frame to reduce this effect. Septa needle wiping eliminates some of this contamination. Two types of contamination can occur from this problem. (a) When the needle is dipped into a vial (same or different one) which also has a large septa opening, it can carry some of the sample with it and deposit it into the new vial (or onto the septa of the vial). (b) The contamination can also run down the needle itself and drip onto the needle seat at the time of injection resulting in contamination of the seat or sample.

One of the easiest solutions to reduce external needle contamination involves incorporating a wash vial which contains a solution which is optimized to quickly dissolve the sample into solution. This sounds simple, but many chromatographer's choose wash solutions which do not enhance the cleaning aspect of the needle at all. For example: Mobile phase, which is normally ideal, but does not work in some cases. Samples such as peptides, proteins, fats, oils and/or lipids can be troublesome as their solubility can be at odds with the mobile phase chosen. For the wash vial to be effective, it must quickly dissolve the material. The needle can be first "dunked" (dipped) into one vial containing the solution and withdrawn, followed by an aspiration and wash in a second wash vial. If needed, you take this cleaning one step further and use additional aspiration steps to serially dilute any remaining material. These wash vials must be changed frequently (easily done by having several wash vial positions programmed in the system). Additionally, the caps should be left OFF the wash vials to reduce pickup contamination each time they are used (this step is critical).

Lastly, if you are analyzing sticky materials which are known to interact with metals found in chromatography systems, consider using a system which incorporates bio-compatible materials such as titanium, tantalum and/or polymers such as PEAK. You can also utilize plastic sample vials (e.g. PP) or plastic vial inserts too.

(4) Inadequate Operator Training: Good chromatography requires a complete understanding of the hardware used and the fundamentals of HPLC. You must be able to troubleshoot the complete flow path of the system and understand the concepts of chromatography as used in method development. This is not a technique best learned by trial and error, but rather through mentoring using logical steps. Depending on your skill set, troubleshooting a "carry-over" problem in an HPLC system can take minutes to months to diagnose and solve. We learn these skills through hands-on experience and training. Reading many of the better books and articles on the subject matter helps too. Get as much practical hands-on training as you can. Ask your supervisor or manager(s) to invest in you by purchasing professional training for you in this field so you can learn on your own systems. You will learn far faster this way and spend less time troubleshooting problems and more time running samples, accurately in less overall time. Training also costs just a fraction of what the instrumentation and your salary are. If you have acquired the fundamental skills, a skilled teacher can impart about one years worth of practical knowledge to you in as little as one week of training.

Summary: The two most common reasons for sample carry-over contamination in an HPLC or LC/MS system are due to: lack of operator training and/or lack of system maintenance (most commonly manifested as a worn injector rotor seal).

 Note: This article specifically addresses carry-over contamination as it relates to the most commonly used HPLC, UHPLC and LC-MS autoinjectors (or autosampler modules).

You may wish to read a related article on "Troubleshooting HPLC Injectors (Manual and Automated)" found at this link: http://hplctips.blogspot.com/2013/06/troubleshooting-hplc-injectors-manual.html