By Celia on Thursday, January 30, 2003 - 08:59 am:
Hello friends,
once more iīm here asking to you all my doubts..
This time iīm looking for any mathematical expression that could express the influence of sample mass in assimetry and efficiency in HPLC..
Thanks
Celia.
By Anonymous on Thursday, January 30, 2003 - 10:00 am:
Dear Celia,
With increasing sample mass a column can overload, i.e. peaks broaden (i.e. efficiency decreases) and peak assymetry increases.
The 'column saturation capacity (Ws)' is a measure for sample mass overload of a column. You will see that the equation for 'Ws' includes the efficiency parameter.
Find the equation for 'Ws' in:
Snyder, L.R.; Kirkland, J.J.; Glajch, J.L. Practical HPLC Method Development, Wiley, New York, 1997.
or in e.g.:
McCalley D.V., J.Chrom A 793 (1998) 31-46. (page 38).
Note: For heavily overloaded peaks (right angled-triangle shape) the efficiency should be measured
according to:
Snyder, L.R.; Cox, G.B.; Antle, P.E. Chromatographia 1987, 24, 82-96.
Regards,
Stephan
By Uwe Neue on Thursday, January 30, 2003 - 03:41 pm:
My book on "HPLC Columns" has a set of quantitative equations that describe the effect of volume overload, mass overload and column performance. Pages 276 ff "Preparative Chromatography.
This is not meant to be an advertisement....
By B.Buglio on Sunday, February 2, 2003 - 08:27 am:
Celia
In "Troubleshooting LC Systems" Dolan & Snyder
define Ws as "contribution to bands-spreading from
volume of injection" (couldnt find Ws in
"Practical HPLC Method Development"). Mass
overload can sometimes be treated in a manner
similar to volume overload however it really is
more complex. An equation dealing with mass
overload has to deal with nonlinear isotherms and
changing polarity of the mobile phase. Guiochon
took on the challenge in the late 80s. For
references (in addition to those from above) see:
Chromatographia, 24(1987) 165
A.C. 60(19
By B.Buglio on Sunday, February 2, 2003 - 08:30 am:
last reference didnt post.Its A.C.60(1988) 1856
By Anonymous on Monday, February 3, 2003 - 03:55 am:
B.Buglio, Celia,
Please find the equation of consideration in Pract. HPLC Method Development, page 629 under '13.4.1 General Relationships'
Regards,
Stephan
By B.Buglio on Monday, February 3, 2003 - 11:52 am:
Stephan,
Thanks for reference details. I must have a much
older edition - it only has 260 pages. Time to get
to a bookstore.
By Anonymous on Monday, February 3, 2003 - 03:43 pm:
No guys - Snyder's equation on 629 has the plate count in it BEFORE mass overload... If I understand Celia right she wants plate count after mass overload.
By Anonymous on Tuesday, February 4, 2003 - 02:55 am:
Anon,
Yes, however, equ(629) contains N for an overloaded peak, too; this in form of peak width of an overloaded peak and k (for small sample). Similarly we find this in N-equations.
This becomes more obvious looking at the two separate equations e.g. given in the paper I mentioned above (McCalley). One of these contains N for an overloaded signal. (Together they give equ(629)).
If Celia wants N-overload only: Snyder, L.R.; Cox, G.B.; Antle, P.E. Chromatographia 1987, 24, 82-96.
If I remember right, you use baseline method to calculate N-overload. Take the retention time obtained from a small sample mass peak. The width ranges from the peak start of the overloaded peak (which almost equals the retention time for a right-angled triangle peak shape) to the retention time of the small sample mass peak. But rather look it up Celia.
Best wishes,
Stephan
By Celia on Monday, February 17, 2003 - 11:49 am:
Hello guys, (sorry , perhaps itīs too coloquial...), What i really wanted was any formula to calculate the maximun mass possible to inject in a chromatographic column.Iīm working in high speed HPLC, and i couldīnt find any mathematical explanation to avoid overloading...
Thank you very much to all of you..
I canīt go to a ciber everyday, so iīm not very fast answering
By Uwe Neue on Monday, February 17, 2003 - 07:50 pm:
Here is a good rule of thumb for standard porous packings: inject less than 1 mg of analyte per g stationary phase. If you want to be really careful, less than 0.1 mg...
The loadability of compounds depends on a range of things, mostly importantly their ionization. The loadability of ionic compounds can be up to a factor of 100 lower than the loadability of nonionic compounds.
Are you running gradients for your high-speed HPLC? The loadability under gradient conditions is about a factor of 10 higher than in isocratic HPLC.
As you see, there are many factors. Maybe 0.1 mg per gram of stationary phase is a good guess to start...