Analytical Chemistry Method development
Method on HPLC ( High Performance Liquid Chromatography)
Steps in HPLC Method Development
1]Information on sample,define separation goal
2]Need for special HPLC procedure sample pretreatment etc.?
3]Choose detectors and detector settings
4]Choose LC method; preliminary run; estimate best separation conditions
5]Optimization separation conditions
6]Check for problems or requirement for special procedure
a)Recover purified material b)Quantitative Calibration c)Qualitative method
7}Validate method for release to routine analysis
Column Selection for HPLC Method Development
Reversed-phase and ion-pair method
C18 (Octadecyl orODS)
Rugged, highly retentive, widely available.
C8 (Octyl) Similar to C18 but slightly less retentive.
C3, C4 Less retentive and mostly used for peptide andproteins
C1[trimethylsilyl(TMS)]Least retentive and least stable
Phenyl, phenethyl Moderately retentive and some selectivity differences CN (cyano) Moderately retentive and used for both reversed and normal phases
NH2 Amino Weak retention, used for carbohydrates and lessstable
Polystyrene Stable with 1<pH<13 mobile phase and better peak shape and longer column life for some
separations.
Normal-phase method
CN Rugged and fairly polar general utility
OH More polar than CN
NH2 Highly polar and less stable.
Silica Very rugged and cheap, less convenient to operate , used in prep LC
Mobile phase:
Mobile phases in reversephase chromatography contains mixture of aqueous phase and organic phase, buffer is not required for neutral samples. Whenever acidic or basic samples are separated, it is strongly advisable to control mobile-phase pH by adding a buffer. It is strongly recommended that the pH of the buffer be adjusted before adding organic. In selecting buffer several considerations should be kept in mind. Buffer capacity is determined by pH, buffer pKa, and buffer concentration. As for the case of sample compound, buffer ionization occurs over a range in pH given by pKa + 1.5 and buffer can be effective only in this pH range. Buffers selected for a particular separation should 16 be used to control pH over a range pKa +1 and concentration of 10 to 50 mM is usually adequate for reversed phase separation. A mobile phase with marginal buffer capacity will give less reproducible separation for compounds that are partially ionized at the pH of the mobile phase. In this case, retention may change from run to run, and distorted peaks may result.
Method on HPLC ( High Performance Liquid Chromatography)
Steps in HPLC Method Development
1]Information on sample,define separation goal
2]Need for special HPLC procedure sample pretreatment etc.?
3]Choose detectors and detector settings
4]Choose LC method; preliminary run; estimate best separation conditions
5]Optimization separation conditions
6]Check for problems or requirement for special procedure
a)Recover purified material b)Quantitative Calibration c)Qualitative method
7}Validate method for release to routine analysis
Column Selection for HPLC Method Development
Reversed-phase and ion-pair method
C18 (Octadecyl orODS)
Rugged, highly retentive, widely available.
C8 (Octyl) Similar to C18 but slightly less retentive.
C3, C4 Less retentive and mostly used for peptide andproteins
C1[trimethylsilyl(TMS)]Least retentive and least stable
Phenyl, phenethyl Moderately retentive and some selectivity differences CN (cyano) Moderately retentive and used for both reversed and normal phases
NH2 Amino Weak retention, used for carbohydrates and lessstable
Polystyrene Stable with 1<pH<13 mobile phase and better peak shape and longer column life for some
separations.
Normal-phase method
CN Rugged and fairly polar general utility
OH More polar than CN
NH2 Highly polar and less stable.
Silica Very rugged and cheap, less convenient to operate , used in prep LC
Mobile phase:
Mobile phases in reversephase chromatography contains mixture of aqueous phase and organic phase, buffer is not required for neutral samples. Whenever acidic or basic samples are separated, it is strongly advisable to control mobile-phase pH by adding a buffer. It is strongly recommended that the pH of the buffer be adjusted before adding organic. In selecting buffer several considerations should be kept in mind. Buffer capacity is determined by pH, buffer pKa, and buffer concentration. As for the case of sample compound, buffer ionization occurs over a range in pH given by pKa + 1.5 and buffer can be effective only in this pH range. Buffers selected for a particular separation should 16 be used to control pH over a range pKa +1 and concentration of 10 to 50 mM is usually adequate for reversed phase separation. A mobile phase with marginal buffer capacity will give less reproducible separation for compounds that are partially ionized at the pH of the mobile phase. In this case, retention may change from run to run, and distorted peaks may result.
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