Date: Tue, 7 Jan 1997 13:47:39 -0500
Message-Id: <v01540b00aef7fde54d57@[10.0.2.15]>
From: nestgrp@world.std.com (Amos Heckendorf)
Subject: Re: Resolution in Chromatography
To: Recipients of ABRF List <abrf@aecom.yu.edu>
>During the preparation of a lecture on chromatography I have found two
>different expressions for the calculation of the resolution:
>
>1. R = 0.25 (alpha-1) (square root of N) (k/k+1)
>
>2. R = 0.25 (alpha-1/alpha) (square root of N) (k/k+1)
>
>In both cases the definition of alpha is the same (k2/k1). Can anybody
>explain me if and how these two equations are correlated to each other?
>Which is the "right" one?
Peter:
I think there is a typographical error to equation #1. Equation #2 is the
correct one.
As selectivity increases, the peaks move further apart from coincidence.
Thus (k2/k1-1)/k2/k1 moves from no resolution (1-1/1)=0, to increasing
positive numbers.
Note also that the resolution equation encourages the use of selectivity to
promote resolution more than it does retention time (capacity). As capacity
factors (K') approaches 10 the rate of increase decreases along the way,
while the opposite is true for selectivity (just a little dig at those
small pore affectionados who think the way to getting better separation is
easier going to higher plate count (smaller particle) or smaller pore
columns (higher capacity factors), rather than changing the pH, the buffer
additive, or the column chemistry.).
For peptides, if you increase the pH from 2.0 to 4.4 you are between the
pKa's of glutamic and aspartic acid, which can promote different
selectivity. Then if you go to pH 6.0 you are above those pKa's and this
gives a potentially different picture. Use a 10 mM phosphate buffer
(triethyl amine or simply sodium) made from a 100 mM stock of pH 4.4
diluted and adjusted with either phosphoric acid or sodium hydroxide.
Best of luck teaching those chromatographers. They are a difficult lot!
Regards,
Amos
Amos Heckendorf (nestgrp@world.std.com) 800-347-6378 ; 508-481-6223
The Nest Group , Value Added Resellers
HPLC Operating Instructions and Molbiol. Protocols on WWW URL=
HTTP://world.std.com/~nestgrp