I am happy to learn that you wish to actually measure the T zero time (by measuring the column void volume) for your HPLC column and method. This is a critical measurement to make before starting any HPLC system method development or analysis. It is needed to determine future K prime values of all samples (aka: Capacity Factor) as well as other key parameters. *Once you have K, then you can input it into the Log P(o/w) formula. Thiourea has a Log P (o/w) of -1.02.

"Dead Time". When using such expressions we need to include a bit more information. "Dead Time" of what ?

• "Column Dead Time" is the time it takes an unretained sample to pass through the HPLC column w/o interacting with the support chemistry. We often refer to this TIME as the Column Dead Time or Column Void Time (also, "T zero time").
• The "System Dead Time" is the time it would take a sample to go through the whole HPLC system and is very different.

Neither of the above TIMES should be confused with similar metrics we use to describe the VOLUMES of these two different measurements. IN fact, the volume measurement is the one we want (Column Volume and System Volume are far more useful #'s). Once we have the volume measurements, then we can use them with the known flow rate (expressed in volume/time) to calculate times, which are more useful when looking at the chromatograms (when flow rate changes, so do the times). "T zero" time is the most common one you will hear being discussed where chromatography is concerned. It is the first thing you check when looking at a chromatogram (*to see if there is retention of the sample(s)).

Now, the data you provided above in your post does not make any sense. A 4.6 mm x 250 mm column packed with traditional porous particles of silica used at a flow rate of 1.00 ml/min should have an approximate column void TIME of ~ 2.9 minutes (Column volume estimated at 2.90 ml and at a flow rate of 1.00 ml/min would result in a T zero time of 2.9 minutes). The "unretained" peak that you observe at 0.538 minutes is not thiourea at all (or any other sample as that would be impossible). You have made an error. An actual measured retention time of 2.508 minutes sounds far more reasonable (as the estimate we calculated of ~ 2.9 minutes, is just that, an estimate).

Does any of this answer your original question(s)?

- - -

• "Determination of HPLC Column Dead Volume / Dead Time (T zero)"; https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
• "K Prime (also known as: Capacity Factor, Ratio or Retention Factor): One of the Single Most Important HPLC Parameters of All"; https://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html

Thanks for the information William Letter.

I was also expecting that T zero will be the retention time of thiourea. But initially i did get confused with RT 0.538 (that might be some impurity). Yesterday, i had calculated the capacity factor of reference standards using 2.508 RT as a T zero, got a good calibration curve and this value is close to calculated value of dead time of thiourea.

"RT 0.538 (that might be some impurity)".

• It can not be an impurity because it showed up before Tzero. It must be noise, artifact, another sample or sourced to contamination in your column or HPLC system.

Thanks for the information William Letter.

Hello @William Letter,

according to the link you shared above to get retention factor, I'm wondering if the estimated column void volume T zero is always equal to the measured one from experiment? or if the estimated T zero obtained using a list of typical HPLC column dimensions and their associated estimated void volumes mentioned in the link can be used as T zero in the formula k1 = T(R) - T(0) / T(0) instead of using measured/calculated T zero).

any suggestion?

"Dushimabarezi": Terminology is critical in science. We estimate when it is useful to have an approximate answer and measure when we need the actual answer.

As stated in the articles, you MUST always MEASURE the column void volume. Once you have an accurate column void volume, you can then calculate T zero (and other values based on T zero). Understanding the concepts behind pore volume allows us to estimate the column void volume. This is useful for initially ESTIMATING the column volume and using that value to estimate T zero.

Will the estimated column void volume (not T zero) equal the measured column void volume? Maybe. As stated in the article, differences in pore volume and support structure/type will effect the estimate. It is usually within +/- 10%, but the purpose of initially estimating it is to help guide us when starting out. We must ALWAYS measure it for every method used (that is the correct and scientific way to do things). We use the measured value for our calculations (K prime etc).