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LCHPLC - ANSHigh Performance Liquid Chromatography
-components of a liquid sample are separated as they partition between a MP and SP contained
in a column
Typical HPLC Modes Used - ANS-reversed phase
-normal phase
- ion-exchange
-size exclusion
-bioaffinity
Differentiate between Normal and Reversed Phase HPLC - ANSRate of elution is dependent on
the polarity of the analyte
Normal: more polar analyte elutes slower
- polar stationary phase
- nonpolar mobile phase
Reverse: more non-polar analyte elutes slower
- nonpolar stationary phase
- polar mobile phase
Polar Index (PI) - ANSmeasure of the ability of the solvent to interact with various test solutes
- increases with solvent polarity
ex. hexane is non-polar, has ~0 PI, and therefore will not readily interact with a polar MP
Equation:
P' = ΦaPa + ΦbPb
Methods of care for MP - ANS- use sterile solvent bottles
- Filter solvents through filters (< 0.45 μm).
- Replace the solvents every two days (do not re-filter).
- Avoid exposure to direct sunlight
Differentiate between Isocratic and Gradient Elution - ANS- Isocratic: elution with single
solvent/mixture, where solvent composition remains constant
- Gradient: Solvent composition changed continuously or in steps
Components of HPLC - ANSa) solvent reservoirs
b) detector (DAD in lab)
c) column thermostat
d) autosampler
e) degasser and quat-pump
, Solvent Reservoirs - ANS- made of materials that will not contaminate MP: Teflon, glass, or
stainless steel.
- Solvent typically contained within glass jar
- Solvent is delivered from the reservoir to the pump by means of Teflon tubing -- called the
"inlet line" to the pump.
- cap to prevent particulate matter from contaminating MP
- closing cap too tightly prevents MP from flowing the pump, creating "vapor lock" within pump.
Autosampler - ANS- sample is introduced by an automated syringe drawing samples from vials
in a motorized tray
- introduce [inject] sample into the continuously flowing MP stream that carries the sample into
the HPLC column
Degasser (3) - ANSremove excess dissolved gas from MP solvents
-Degas solvents prior to adding modifiers
1) Sonication: MP reservoir placed in ultrasonic bath, sound waves promote the coalescence of
small bubbles which can escape more easily by allowing the excess gas to escape before it
enters the pump
2) Vacuum filtration: of the MP to remove both dissolved gas and particulate contamination
3) Helium sparge: stream of helium bubbles sweep dissolved air out of liquids (helium is virtually
insoluble in most HPLC solvents)
Characteristics of an ideal HPLC solvent - ANSa) high purity
b) boiling point higher than operating temperature
c) relatively low viscosity
d) chemically inert
e) non-toxic
f) miscible with water (except for normal phase)
Common mobile phase components (2) - ANS- organic modifiers
- pH buffers
Column (Reversed Phase) - ANS- mode dependent
- Nonpolar
- C8, C18
- Increase polarity with various groups - phenyl, CN
- pH range 2 - 12
- prefer end-capped columns to avoid problems due to free - -OH groups which can interact with
analytes and affect peak shape
Chiral chromatography - ANS- enables separation of racemic mixture into its enantiomeric parts.
- Chiral resolving agent: MP additive/SP preferentially complexes one of the enantiomers to
diastereomers
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