Bioequivalence and Bioavailability Forum

Dear Vishal!

❝ In column specification we have a calcuation for capacity facotor K'

❝ (formula is k'=tr-to/to) and as a 'to', first retained peak of the

❝ combination is consider.

I think that you missed one pair of perentheses; the formula should read:
k'= (t_R - t₀)/t₀

❝ kindly give me the actul procedure to calculate the dead volume

❝ and also discribe how it is effect my chromatogram if the

❝ value of k' is >10, < 1 and >30 ?

One should distinguish between theoretical dead volume (the interstitial volume between the particles of the package material + the porous volume of the packing material) and the experimental dead volume, which includes the volume of the entire LC system (injector, mixing chamber, fittings, connecting capillaries, etc).
For a glossary of terms see this article from LCGC Europe (Feb 2002).
The one compound we used in getting an experimental value for t₀ in RP LC is thiourea - SC(NH₂)₂; others are uracil and acetone.
You may calculate the dead volume (V_M) from the geometry of your column (length, diameter), the flow rate and the retention time.
V_M = t₀ × v, where v=velocity of the mobile phase.

An approximate formula uses the column's inner diameter d [cm] and column length L [cm], as well as the flow rate F [ml/min]:
V_M = 0.5 × d² × L, and t_M = V_M / F

Example:
d=0.46cm, L=15cm, F=1.5mL/min
V_M=1.6mL, t_M=1.1min

You will hardly find a compound with a t_R < t₀ (besides dissolved oxygen...).
In practical LC applications I would not go with a method with a k' of less than 3-5. The higher the k' the better the separation, but you are loosing sensitivity (peaks are getting broader). Therefore k' >10-15 is also not a good idea.