With regard,
i want to know that what is the basic behind the capacity factor. what is the role of capacity factor in analysis? as we can say migration rate of analyte on column about this, but when we compromise a limit about this than what is the goal to set a capacity factor parameter in our analysis?
Thank you
Hi Dilip,
Capacity factor = (Tr - To) / To
Here, Tr = Retention time of your desired analyte
To = First baseline disturbance
So, if Tr = To, then capacity factor will be zero (0), indicates co-elution (no interaction with the stationary phase). This will surely generate erroneous result.
But if capacity factor is more than 1, it indicates a certain level of interaction. To analyze an ingredient, we want this type of interaction. High capacity factors (greater than 20) mean that elution takes a very long time. Ideally, the capacity factor for an analyte is between 1 to 5.
Hi Dilip,
Capacity factor = (Tr - To) / To
Here, Tr = Retention time of your desired analyte
To = First baseline disturbance
So, if Tr = To, then capacity factor will be zero (0), indicates co-elution (no interaction with the stationary phase). This will surely generate erroneous result.
But if capacity factor is more than 1, it indicates a certain level of interaction. To analyze an ingredient, we want this type of interaction. High capacity factors (greater than 20) mean that elution takes a very long time. Ideally, the capacity factor for an analyte is between 1 to 5.
Capacity factor is an indication of how long a compound can be retained by the stationary phase. It is calculated as, k = (Tr - To)/To, where Tr is the retention time of the target and To is the unretained peak time. If Tr = To, then the sample is not retained by the stationary phase. Higher the value for k, longer the retention.
Dilip: The role of Capacity Factor ( K prime ) is to provide a calculation or formula which defines how much interaction the sample (peak) has had with the chromatography support material. 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.
No such thing as an ideal K prime, but a K prime of between 2.0 and 10.0 would be a reasonable range for a guideline.
K primes that exceed a value of 20 show no real benefit in resolution, just peak widening and longer delays.
K prime values below 1.5 may demonstrate that the method used is not selective for the sample under analysis (IOW: invalid method used or fails most HPLC validation requirement per regulatory authorities).
Please refer to these free HPLC training articles for more information:
- "K Prime (also known as: Capacity Factor or Retention Factor): One of the Single Most Important HPLC Parameters of All"; http://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html
- "Determination of HPLC Column Dead Volume / Dead Time (T zero)"; http://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
The guideline that we teach in our HPLC classes (and which are also used by most regulatory authorities such as the FDA in the USA) is for most NP/RP methods to have a minimum K prime value of 2.0 for each sample. Some consider a value of 1.5 as a minimum, but 2.0 is better. Remember, a value of "1" is too small.
If your peak is coming out at or near the column void volume, then you have no chromatography and an invalid HPLC method (Method fails all validation). *There are thousands of published HPLC methods in the journals with samples eluting at or near the void volume! The authors could have simply placed their samples in a spectrophotometer and gotten the same result as no chromatography was used.
The whole point of this technique (chromatography) is to develop a method which retains the sample on the column (it must interact with the support) for a long enough time to be resolved apart from any potential impurities or other compounds (to show that the method used is 'selective' for the sample). Failure to do so means you did not use the column in your "chromatography" method. *Calculating Column Void Volume allows you to determine K prime for your peak. **Note: K prime is less relevant in some modes of chromatography such as Size Exclusion (SEC/GPC) which rely on pore inclusion/exclusion mechanisms of the sample.
to find capacity factor for a analyte. inject mixture of unretained and retained compounds.
note down the retention times T1 and T2(Retained).
Capacity factor=(T2-T1)/T1.
To know the void volume, it is useful.
Regards,
Bujji Reddy. Kanchi.
Mahmood. How much interaction? Enough to obtain a minimum K prime value of 2.0.
Shubhendu: "Limit"? Not sure what you mean, but probably none. However, the goal is to complete the analysis, so we try to develop methods that we can complete in a reasonable amount of time.
The most important parameter for all cinematographers. Nicely defined by researchers before me.
In other words it says whether something binds to the column or not. For low k primes, the binding is none or to weak to call it "binding".
Subrata Bhadra, I would not say: co-elution, but rather, flow through elution. Also, I would not say that low k' will lead to generation of erroneous result. Result is a result, the question whether we like it or not is another thing.
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