Dear All,
How to practically find Unretained Component and the Unretained Time?
Shall we inject a sample which will not be retained by the column to determine what time an unretained sample will be eluted off the column? If the answer is yes for this point, how can we select such a compound? Are there general common compounds to do this job?
OR
Shall we add a compounds to our mixture which we absolutely know that it does not retain in our mobile phase system, then find out its peaks, which is supposed to be the first one, then take the time of this peak as an Unretained time (t0)?
OR
just pick up the first peak (which is usually appears within the front of the mobile phase) and take it as an Unretained time (t0)? In the appended picture, can we consider the first peak (labeled with red arrow) as the Unretained Time (Void volume)?
OR
Finally, just pick up the t0 values that provided in the literature (depending on the column's dimensions)?
Please, I need a very clear example which explains this fundamental to me.
Best regards
Saddam
1. Yes.
Obviously it is column and mobile phase dependent. One common way is to make an injection of a solvent that is transparent to the detector and check to see where you get an initial dip in the signal (for example, water if your mobile phase is water/methanol or water/acetonitrile).
I imagine that is what you are seeing in the chromatogram, but in the absence of crucial information that you have omitted I cannot be certain.
My question is why do you care? 'Real' chromatography occurs when the k or capacity factor is between 2-10. For a 5 cm column operating at 1 mL/minute this is 5-10 minutes or a 25 cm column flowing at 0.5 mL/minute 10-15 minutes.
The answer to your question is one of the most fundamental aspects of chromatography. Learning what the Tzero time (column dead volume) of the method is AND how to properly measure and estimate it is one of the most important basic skills you must learn before you start to use or develop HPLC methods. You will need to know this value to calculate Resolution, K prime, Separation and other critical values. *Always calculate AND measure it before starting.
Please refer to this short article to find out HOW to Estimate AND how to measure it (with examples of compounds you can use). "Determination of HPLC Column Dead Volume / Dead Time (T zero)"
http://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
Dear Mark,
Thank you so much for your generous answer.
Dear Bill,
You are my favorite scholar in HPLC, always your answer are highly qualified and they do help me.
All the best for all of you.
Saddam
Injection of an unretained peaki is best.....for reverse phase columns I used to inject potassium nitrate....nitric ion has a strong UV at around 210 and as long as you are not using ion pair reagents it will be pretty well unretained.
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