By M Paciolla on Wednesday, January 17, 2001 - 08:10 am:
Can anyone suggest a standard protocol for equilibrating a new colum? Assume the packing conditions are ACN/H2O (75:25) and the mobile phase is KH2PO4/MeOH (70:30) containing 10mM ion-pairing reagent. The column is either a C8 or C18.
By Anonymous on Wednesday, January 17, 2001 - 09:49 am:
Traditionally 10-15 column volumes is the rule of thumb. However, modern RP columns on highly pure silica with very high ligand-density or endcapping can be re-equilibrated with much lower volumes. Ion-pair reagents are another matter. Equilibration generally requires much longer. I've known it to take all day to initially equilibrate a column for one ion-pairing method. For that reason, it is suggested to put the same concentration of ion-pairing agent in all your mobile phase components and to dedicate your column to the analysis.
By bill tindall on Thursday, January 18, 2001 - 03:14 pm:
Your specific question has an easy answer. Fire up your autosampler and shoot standards, under the conditions you will run samples, until the retention time for components of interest becomes as consistent as you need it to be. There isn't a theoretical answer to this question that can be applied to most situations because everyone has a different tolerance for retention wanderings.
Also test to be sure you need ion pair reagent. Many old fashion separations can be done on modern columns with out ion pairing. As an example, it is possible to get adequate retention of sulfonated isophthalic acid on some reversed phase columns without it.
The suggestion to dedicate the column to ion pairing is sage advice.
By Anonymous on Friday, June 4, 2004 - 06:58 am:
Can anyone explain why methylene selectivity for columns is a function of surface coverage and not surface area please
By Anonymous on Saturday, June 5, 2004 - 09:50 am:
One word: "silanols".
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