Handbook of Size Exclusion Chromatography and Related ...

individual dispersion processes inside and outside the column, as shown in
Eq. (21):
s 2 obs ¼s2 col þs2 inj þs2 det þs2 ct
¼s 2 col þ X s 2 ec
(21)
wheres 2 obs istheobservedvarianceoroutputvarianceands2 col isthevariancedue
to column band broadening. The other variances represent the contributions from
injector, capillary tubing, and detector, respectively, and P s2 ec isthe sum of
extracolumn variances. If needed, Eq. (21) can be extended with other variances,
such as those caused by the electronics of the recording system. The validity of
Eq. (21) is limited to random dispersion processes that give rise to aGaussian
distribution.Thisconditionisgenerallyassumedinchromatographicapplications.
The equations describing the individual contributions from extracolumn band
broadening are discussed in detail elsewhere (106–110).
Although ideally the observed variance is equal to the column variance,
mostHPLCsystemsdetractfromthecolumnefficiency.Equation(22)canbeused
to calculate the importance of extracolumn effects:
s 2 obs ¼s2 col þs2 ec
¼s 2 col þu2 s 2 col
(22)
where u 2 isthe fractional increase of the columnvariance caused by extracolumn
effects. A10% loss of column efficiency (or a5% increase in bandwidth) as a
result of extracolumn effects, u 2 ¼0:1, is considered acceptable in practise.
Injection effects as aresultof mass and volumeoverloadingor the injection
technique can detract from column efficiency.As with other extracolumn effects,
injection effects become more critical with smaller bore columns, which require
smaller injectionvolumes and low flow rates; refer to Ref. 109 for adiscussion of
extracolumn effects in microcolumn systems.
Equation (23) relates the maximum injection volume to the column
dimensions,particlesizedp,mobile-phaseporosityeT,u,andreducedplateheight
(110).TheconstantKinjdependsontheinjectiontechnique;K 2 inj ¼12forplugflow
injectionandvariesfrom2to9for mostcommercial injectors (74).Equation(23)
isvalidfor asmallmolecular marker thatelutesinthetotal mobile-phasevolume:
(Vinj) max ¼ 1
4 pKinjeTu(dc) 2 (Lhdp) 1=2
(23)
For areasonably efficient (h ’8) 30 cm 7.5 mm, 10-mm, SEC column,
Eq. (23) predicts amaximum injection volume of 165 mL for Kinj ¼3, u 2 ¼0:1,
andeT ¼0:8.Figure15showsexperimentaldatafortheeffectof injectionvolume
on column efficiency for bovine serum albumin on a 30 cm 7.5 mm, 10-mm,
© 2004 by Marcel Dekker, Inc.