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7. HPLC. Adv. Chrom. Revision. 1. The P in HPLC is often used as an abbreviation for pressure. Explain why . pump uses high pressure to form liquid through column 2. Draw a block diagram of the components of a typical HPLC. 3. How does separation by HPLC differ from that by GLC?

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7 hplc


Adv. Chrom


1. The P in HPLC is often used as an abbreviation for pressure. Explain why.

  • pump uses high pressure to form liquid through column

2. Draw a block diagram of the components of a typical HPLC.

3. How does separation by HPLC differ from that by GLC?

  • temperature not involved
  • polarity attraction to both phases
  • liquid mobile phase

4. What general rules apply to the selection of the two phases in HPLC?

  • SP similar to analytes
  • MP different but not too different
7 1 injection systems













7.1 Injection systems
  • operate at pump pressures of 2 MPa and higher (10 x atmospheric pressure)
  • how to inject the sample against such a pressure
  • how to maintain constant pressure without surges and drop-offs
  • need pressure isolation: rheodyne



7 2 pumps
7.2 Pumps
  • desirable properties
    • stable pulse-free flow
      • avoids baseline fluctuations
    • reproducible flow rates
      • consistency between runs
    • a range of flow rates from 0.5-5 mL/min
      • flexibility in separations
    • against very high pressures (up to 100 atm)
      • needed to get liquid through column in reasonable time
    • without being affected by different solvents (aqueous and non-aqueous)
      • allows range of mobile phases to be used
reciprocating piston pump
Reciprocating piston pump

(a) Do you think this pump by itself would create pulse-free flow?

  • no

(b) What could be done to fix the problem?

  • two pumps
  • working 180o out of phase
mobile phase
Mobile phase
  • must be filtered and degassed to remove:
    • fine particles which would block the check valves in the pump or the column
    • dissolved gases, which may produce bubbles within the column or detector
  • reservoirs should be covered to avoid dust fallout
  • should be HPLC grade
    • guaranteed not to contain stabilisers added to other “pure” solvents
    • may not be as pure as AR grade, e.g. HPLC grade hexane may contain some heptane
  • made from stainless steel to withstand the internal pressure
  • 15-30 cm in length
  • not coiled because there is no need to save space
    • pressure problems created by coils
  • internal diameter of the column is typically 4-5 mm
  • smaller bore packed columns and capillary columns are now available for HPLC
  • packing has decreased in size to < 10 um
  • stationary phase chemically bonded to Si-OH on packing surface (or capillary walls)
stationary phases
Stationary phases
  • guard column - a short version (1-2 cm) of the main column between the injection system and the main column
  • intended to be “disposable” – catches any fine particles resulting from precipitation between sample and mobile phase
  • most HPLC detectors based on familiar lab instruments

Ex. 7.3 - Why?

  • because HPLC uses solutions
  • universal response (or selective in a predictable way)
  • high sensitivity
  • low noise
  • wide linear range
  • response independent of instrumental variations
  • small internal volume #
  • flow-through design #
  • non-destructive
  • stable response
  • relatively inexpensive
  • UV/VIS
  • refractive index
  • conductivity
  • mass spectrometry
  • evaporative light scattering (ELS)
  • fluorescence
  • infrared
  • flame emission
  • potentiometric
  • polarographic


  • some adjustment that can be made to pick out specified analytes and ignore others
  • some UV-VIS detectors are fixed wavelength (universal), others allow choice of wavelengths
flow through design

from column

to waste

UV beam


quartz windows

Flow through design
  • the flow from the column is continuous
  • the “sample” cell in which the detector measurement is made, must be flow-through
    • fresh eluant comes in, pushing out that which is already measured
  • the simplest cell has two openings, one for the inlet, and one for the outlet
  • where cell pathlength is important for sensitivity (eg UV/VIS, fluorescence, conductivity)
    • inlet and outlet must as far apart as possible
    • without increasing the cell volume
internal volume
Internal volume
  • a dead volume where no separation occurs
  • impacts on the ability to the ability to resolve close-together peaks


  • flow rate of 1 mL/minute
  • a detector with an internal volume of 1 mL
  • will take 1 minute to fill or to change over the contents
  • any reading from the detector is an average of the eluant from the last minute
  • a changeover time of 1 second
  • maximum flow rate of 3 mL/minute
  • volume required will be no more than 50 µL
  • detector cell volumes of less than 10 L are not uncommon
solvent response correction









Solvent response correction

Which do our instruments use?

  • mobile phase response at the detector must be zeroed
    • like a reference beam/cell in spectroscopy
  • solvent response will mask small variations due to the low levels of analyte eluting from the column

Type 1 – split before column

Adv– copes with changes in solvent response during gradient elution

Disadv– two detectors may respond differently, half the flow rate lost

Type 2 – diversion after column

Adv– no dead time or flow rate problems

Disadv– two detectors respond differently, can’t cope with changes in response by detector during gradient elution

Type 3 – no diversion

Adv– no detector variation, dead time or flow rate problems

Disadv– can only be zeroed initially, can’t cope with changes in response by detector during gradient elution

ion chromatography
Ion chromatography
  • used to separate mixtures of ions
    • either anions or cations, but not both at the same time
  • mobile phase is aqueous with ionic or polar organic compounds dissolved (modifiers)
    • these provide competition for the SP sites so that the analyte ions don’t stick to the SP
    • higher modifier conc = lower RT
  • cation columns have negatively charged sites, vice versa for anion columns
  • anion chromatography is very important because of the difficulty in analysing anion mixtures at < 100 mg/L
  • cation chromatography less important because of all the various ways of analysing metals
retention factors
Retention factors
  • size - the smaller the ion, the less the attraction
    • retention time for I- > Br- > Cl- > F-
  • charge - the smaller the charge, the less the attraction
    • retention time for Al3+ > Ca2+ > Na+
  • most commonly used detector is conductivity
  • presence of modifiers in MP causes a background conductance
    • makes it difficult to detect small increases due to the analytes (loss of sensitivity)
  • unsuppressed – low modifier concentrations => long RTs
  • suppressed – treatment of eluent between column & detector to remove background conductivity
  • mobile phase is a sodium carbonate/hydrogen carbonate solution
  • suppressor column is a cation exchange resin, loaded with hydrogen ions on the exchange sites
  • Supp-SO3H + Na+ (aq) Supp-SO3- Na+ + H+ (aq)
  • H+ + HCO3-H2CO3
size exclusion
Size exclusion
  • separates molecules by their molecular size
    • by ability (or lack of it) to pass into the porous structure of the stationary phase
  • unusual in that there is no polarity attraction to the stationary phase
  • the mobile phase simply transports the mixture
  • only works with large and very large molecules (FW > 2000)
  • typical analytes are carbohydrates, proteins, plastics
  • relationship between size/mass and retention volume (measured instead of time) can be determined

Which comes out first?

  • Larger molecules
choice of phases



Reverse phase



Normal phase





Ion exchange

Size exclusion


Choice of phases

Exercise 7.7

  • sugars in grape juice
  • normal
  • fluoride in toothpaste
  • anion
  • FW of plastics
  • size exclusion
  • steroids in blood
  • reverse
  • mobile must have some attraction to analytes
  • likely to be medium polarity
elution modes in hplc
Elution modes in HPLC
  • isocratic – same mobile phase throughout
  • gradient elution – gradual change in MP (equivalent of temp programming in GC)
  • 2nd MP means 2nd pump

Class Exercise 7.8

  • column: reverse phase
  • MP: ethanol
  • two well-resolved peaks at 2 & 3 minutes & a broad peak at 10 minutes
  • How could this be improved, using gradient elution?
  • the 10 min peak must be strongly attracted to the SP => it is non-polar
  • must make the MP less polar
  • begin introducing less polar solvent after 1st peak emerges, i.e. 2.5 mins