High Performance Liquid Chromatography

1
High Performance Liquid Chromatography Components
of HPLC instrumentation

• Pumps
• Several types
• Reciprocating
• Syringe
• Constant pressure

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• High Performance Liquid Chromatography
• Pumps
• Reciprocating

• Small volume chamber
• Such pumps can generate very high pressures
  10,000 psi
• The output pressure of a single stage pump
  pulsates as the pump switches from output to
  fill mode
• Dampen pulsations using a diaphragm between the
  pump and the column
• Dampen the pulsations by using three stages
  phased so that two are filling while one is
  pumping
• Use a profiled cam that modulates the compression
  of liquids
• Use a feed back control system to control the cam
  motor and thus the pressure and flow rate of
  liquids

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• High Performance Liquid Chromatography
• HPLC packings and columns
• 1960s - pelicular particles consisting of a
  solid core 40 mm with a porous surface layer
  of 2 mm thickness
• Silica gel
• Alumina
• Polyamide resin
• Today - bonded phase packings are most common
• 3 mm, 5 mm, 10 mm silica particles are available
  having a very narrow distribution in particle
  diameter
• Columns 1 mm to 6 mm ID are available
• There is a trend to smaller diameter columns
• Column efficiency increases so that 1 mm columns
  produce peaks that are 20 x higher than 4.2 mm
  columns
• This improves detection limits
• Column lengths of 25 cm are common, although
  shorter and longer columns are available
• Such columns have 10,000 theoretical plates
• Bonded phases

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• High Performance Liquid Chromatography
• HPLC packings and columns
• R groups
• Polar packings
  non-polar packings

• HPLC detectors supply the output signal related
  to the composition of the solution eluting from
  the column
• Commercially available
• UV-Visible absorption of light Mass
  spectrometry
• Differential refractive index IR
• Fluorescence NMR
• Electrical conductivity Micro heat of
  adsorption
• Volumetric

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• High Performance Liquid Chromatography
• HPLC detectors some designs
• UV-Visible cells
• Fluorescence detectors involve promoting
  electrons in atoms, ions, or molecules to a
  higher energy singlet orbital with a lamp and
  observing the light produced when the electrons
  relax to a lower energy orbital

• Volume 1 - 10 mm
• The use of a UV-Visible detector requires
  analytes with a chromophore that absorbs light

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• High Performance Liquid Chromatography
• HPLC detectors some designs
• Differential refractive index detectors (RI)

• Extremely temperature sensitive
• Detect all components that change the refractive
  index of mobile phase

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• High Performance Liquid Chromatography
• HPLC detectors
• Performance criteria
• Sensitivity response/analyte amount or
  response/analyte concentration
• Different analytes have different sensitivities
• High sensitivity is desirable because small
  differences in analyte amount or concentration
  are easier to detect
• Detection limit minimum amount of analyte that
  can be reliably detected
• Usually defined as the amount or concentration of
  analyte that produces a signal-to-root-mean-squa
  re noise ratio of 31
• Depends on the analyte
• Low detection limits require larger sample size
• The column must be able to accommodate the larger
  samples
• This may mean longer columns are required because
  of the low column efficiencies produced when
  larger samples are used
• Longer columns increase analysis times and
  require higher head pressures
• Should the chromatographic instrument cause zone
  broadening, lower detection limits are required
• Linear range the range of amount or
  concentration of analyte that produces a linear
  calibration plot
• Low end is at the detection limit and high end is
  where the calibration plot becomes non linear
• A large linear range makes quantitative analysis
  easier
• Peak area is used for quantitative analysis to
  avoid the effects of peak broadening

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• High Performance Liquid Chromatography
• HPLC detectors
• Performance criteria
• Reproducibility an absolute requirement for
  quantitative analysis
• The standard deviation should be measured for a
  series of detector responses using identical
  standard samples
• Peak shape varies with type of detector used
• UV-Visible detector faithfully shows the shape of
  zones as they elute if the detector cell has a
  small volume
• All peaks are positive
• RI detector can produce both positive and
  negative peaks which makes computer detection
  and analysis difficult

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• High Performance Liquid Chromatography
• HPLC detectors
• Performance criteria
• Peak spreading in the detector results from
• Mixing of component bands with adjacent mobile
  phase in the detector inlet and flow cell
• Finite response time of the detector which for LC
  is 1 s
• High efficiency HPLC columns can produce peak
  widths of 1 s
• Flow and temperature sensitivity
• RI detectors are very temperature sensitive
• Detectors that have flow sensitivity place
  requirements on the pumping system
• Some detectors translate flow variation
  into a deflection of the baseline pump
  pulsations produce noise in the response curve
  and increase detection limits
• Constant pressure pumps can produce mobile phase
  flow variation due to settling of column
  material or change in mobile phase viscosity

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• High Performance Liquid Chromatography
• HPLC detectors
• Performance criteria for two detectors