Hplc column classification

1978 USP XIX Fourth supplement : L1 designation for C18 column
1980 USP XX : 7 columns were classified
-Classified HPLC column to 56 descriptions
-More than 220 columns currently available in the worldwide market can
be classified as L1
-How to select column for a particular application ?
USP Approach
-Use NIST Standard Reference Material (SRM) 870 to evaluate C18 column
-SRM 870 : the mixture of 5 organic compounds in methanol ( uracil, toluene,
ethylbenzene, quinizarin and amitriptyline) -The components in SRM 870 are provide a board characterization of column performance in a single, simple test Parameters used in the characterization of the columns
1. Hydrophobicity (capacity factor of ethylbenzene)2. Chelation (tailing factor of quinizarin)3. Activity toward bases (silanol activity, capacity factor factor Chromatographic condition
Mobile phase : 80% methanol and 20% phosphate buffer pH 7.0Column temperature : 23°C ± 1°C Chromatographic properties
- Peak width (efficiency: theoretical plates)- Peak symmetry (As)- Absolute retention (k´)- Selectivity factor (relative retention (k´1/ k´2) Uracil
-Indicator of the void volume (unretained volume)
-To calculate the retention factor
-The selective factor αE/T= k´ethylbenzene/ k´toluene : Characterize differences
among C8 and C18 column- Marker for calculation of column efficiency (theoretical plates, N) Quinizarin
- Metal chelating agent
- Indicative of the present or absence of metals in HPLC system
- Low activity toward chelating reagent
- High activity toward chelating reagent - Embedded polar functional groups column, quinizarin elute last with good Amitriptyline
-Basic cpd. (pKa 9.4)
-High silanol activity elution of organic bases with severe
Development of SRM870 with 41 commercial C18 columns
- No two columns exhibit identical retention behavior, similarities do
- k´ of ethylbenzene : 0.2-2.8- αE/T : 1.26-1.45 - Retention of quinizarin, k´ :1- 23.6 (peak asymmetry : 1.1-5.7)- Retention of amitriptyline, k´ :1.4-72.9 (peak asymmetry : 1.0-11) The Snyder/Dolan column test -A series of standard mixtures mixture 1 : thiourea, amitriptyline, 4-butylbenzoic acidmixture 1a : N,N-diethylacetamide, 5-phenyl-1-pentanol, mixture 2 : N,N-dimethylacetamide,5,5-diphenylhydantoin, mixture 2a : nortriptyline, acetophenone, mefenamic acidmixture 3 : p-nitrophenol, anisole, 4-hexylanilinemixture 3a : cis/trans chalcone, benzonitrilemixture 4 : berberine Chromatographic condition
Mobile phase : 50%acetonitrile/buffer: pH 2.8 and 7.0
Column temperature : 35°C
- hydrophobicity (H)- steric interaction (S*)- hydrogen-bond acidity (A)- hydrogen-bond basicity (B)- relative silanol ionization or caion-exchange capacity (C) Column hydrophobicity (H)
- Compression of the ends of the alkyl chains in small-pore packing Column steric interaction (S*)
-Crowded bonded phase S* increase
-Increased chain length / concentration of bonded phase
- Compression of the ends of the alkyl chains in small-pore packing -Significant effect on column selectivity, especially for molecules Column hydrogen-bond acidity (A)
-non-ionized silanols increases with column acidity
-More acidic column, greater values of A
-For end-capped column, the number of accessible and unreacted
silanols decrease
-non-ionized basic molecules (amines, amides, aliphatic derivatives) Column hydrogen-bond basicity (B)
-Various function gr. Within the bonded phase B increase
- Columns with larger values of B preferentially retain acidic cpd.
Silanol ionization (C)
-Results in a negative charge on column, attract ionized bases and repel
ionized acids
-pH oh mobile phase increase C increase
-end-capping, decrease access to ionized silanols
Equivalent columns
= similar values of the six column selectivity parameters Column comparison function, Fs
Fs = {[12.5(H2-H1)]2 + [100(S*2-S*1)]2 + [30(A2-A1)]2 +
[143(B2-B1)]2 +[83(C2-C1)]2}1/2 Fs < 3 = equivalent selectivity and band spacingFs > 3 = equivalent separation may still be achieved but less certain - No acids or bases in sample , ignore term C2-C1 -No carboxylic acid in sample, ignore term B2-B1 Conclusion
USP approach
-Provides column performance characterization (theoretical plate
count, good peak symmetry, etc.)
-produces five data points to describe the column
PQRI approach
-provides selectivity characterization (relative retention times)
-produces a list of suitable columns ordered by the Fs factor

Source: http://www.hplc.cz/Tip/HPLC%20Column%20Classification.pdf

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