1. High Performance Liquid
Chromatography (HPLC)
S What is HPLC?
S Types of Separations
S Columns and Stationary Phases
S Mobile Phases and Their Role in Separations
S Injection in HPLC
S Detection in HPLC
S Variations on Traditional HPLC
S Ion Chromatography
S Size Exclusion Chromatography
2. What is HPLC?
HPLC is really the automation of traditional
liquid chromatography under conditions which
provide for enhanced separations during
shorter periods of time!
Probably the most widely practiced form of
quantitative, analytical chromatography
practiced today due to the wide range
of molecule types and sizes which can
be separated using HPLC or
variants of HPLC!!
6. Partitioning
S Separation is based on the analyte’s
relative solubility between two liquid
phases
Mobile Phase Stationary Phase
Solvent Bonded Phase
11. What does the analyst do?
S Select the correct type of separation for the
analyte(s) of interest, based on the sample type
(among other factors).
S Select an appropriate column (stationary phase)
and mobile phase
S Select an appropriate detector based on whether
universal or compound-specific detection is
required or available
S Optimize the separation using standard
mixtures
S Analyze the standards and sample
12. What does the analyst do?
S Select the correct type of separation
for the analyte(s) of interest, based on
the sample type (among other
factors).
16. HPLC - Modes
S Normal Phase.
- Polar stationary phase and
non-polar solvent.
• Reverse Phase.
- Non-polar stationary phase and
a polar solvent.
17. Common Reverse Phase Solvents
Methanol CH3OH
Acetonitrile CH3CN
Tetrahydrofuran
Water H2O
18. What does the analyst do?
Select an appropriate column
(stationary phase) and mobile phase
19. Columns and Stationary Phases
HPLC is largely the domain of packed columns:
S some research into microbore/capillary
columns is going on.
S Molecules move too slowly to be able to
reach and therefore “spend time in” the
stationary phase of an open tubular column
in HPLC.
SIn solution, not the gas phase
SLarger molecules in HPLC vs. GC
(generally)
20. Columns and Stationary Phases
Stationary phases are particles which are usually
about 1 to 20 µm in average diameter (often
irregularly shaped):
S In Adsorption chromatography, there is no
additional phase on the stationary phase
particles (silica, alumina, Fluorosil).
S In Partition chromatography, the stationary
phase is coated on to (often bonded)
a solid support (silica, alumina,
divinylbenzene resin)
21. Columns
S Solid Support - Backbone for bonded phases.
S Usually 10µ, 5µ or 3µ silica or polymeric
particles.
S Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
S Extremely stable
S Reproducible
S Guard - Protects the analytical column:
S Particles
S Interferences
S Prolongs the life of the analytical column
25. Stationary Phases
S Polar (“Normal” Phase):
S Silica, alumina
S Cyano, amino or diol terminations on the bonded phase
S Non-Polar (“Reversed Phase”)
S C18 to about C8 terminations on the bonded phase
S Phenyl and cyano terminations on the bonded phase
S Mixtures of functional groups can be used!!
S Packed particles in a column require:
S Frits at the ends of the column to keep the particles in
S Filtering of samples to prevent clogging with debris
S High pressure pumps and check-valves
S Often a “Guard Column” to protect the analytical column
26.
27. The Mobile Phase in HPLC...
S Must do the following:
S solvate the analyte molecules and the solvent they are in
S be suitable for the analyte to transfer “back and forth”
between during the separation process
S Must be:
S compatible with the instrument (pumps, seals, fittings,
detector, etc)
S compatible with the stationary phase
S readily available (often use liters/day)
S of adequate purity
S spectroscopic and trace-composition usually!
S Not too compressible (causes pump/flow problems)
S Free of gases (which cause compressability problems)
30. Polarity Index for Mobile Phases…..
S The polarity index is a measure of the relative polarity
of a solvent. It is used for identifying suitable mobile
phase solvents.
S The more polar your solvent is, the higher the index.
S You want to try to choose a polarity index for your solvent (or
solvent mixture) that optimizes the separation of analytes
S usually the index is a starting point
S the polarity of any mixture of solvents to make a mobile phase
can be modeled to give a theoretical chromatogram
S Usually, optimization of solvent composition is experimental
S A similar number is the Eluent Strength (Eo]
S Increasing eluent strength or polarity index values
mean increasing solvent polarity.
S Remember, the analyte(s) and samples must be
mobile phase and stationary phase compatible!
31.
32.
33. Optimization of Mobile Phase Polarity…
Changing the mobile phase composition alters the
separation.
34. Isocratic versus Gradient Elution
S Isocratic elution has a constant mobile phase composition
S Can often use one pump!
S Mix solvents together ahead of time!
S Simpler, no mixing chamber required
S Limited flexibility, not used much in research
S mostly process chemistry or routine analysis.
S Gradient elution has a varying mobile phase composition
S Uses multiple pumps whose output is mixed together
S often 2-4 pumps (binary to quarternary systems)
S Changing mobile phase components changes the
polarity index
S can be used to subsequently elute compounds that
were previously (intentionally) “stuck” on the
column
S Some additional wear on the stationary phase
S Column has to re-equiluibrate to original conditions
after each run (takes additional time).
35.
36.
37.
38. Injection in HPLC
S Usually 5 to 1000 µL volumes, all directly onto the
column
S not much worry about capacity since the columns have a
large volume (packed).
S Injector is the last component before the column(s)
S A source of poor precision in HPLC
S errors of 2-3 %RSD are due just to injection
S other errors are added to this
S due to capillary action and the small dimensions/cavities
inside the injector
S 6-PORT Rotary Valve is the standard manual injector
S Automatic injectors are available
S Two positions, load and inject in the typical injector
S Injection loop internal volume determines injection
volume.
39. LOAD (the sample loop)
Inject (move the sample
loop into the mobile
phase flow)
40.
41. What does the analyst do?
S Select an appropriate detector based
on whether universal or compound-
specific detection is required or
available
42. Detectors
S UV
SSingle wavelength (filter) [610, 8330]
S Variable wavelength (monochromator)
[8316, 8325]
SMultiple wavelengths (PDA) [555]
S Fluorescence [610]
S Electrochemical [605]
S Mass Spectrometric [8325]
47. Detection in HPLC
S Numerous Types (some obscure)
S Original HPLC Detectors were common laboratory
instruments such as spectrophotometers, etc.
S you can even use a SPEC 20!
S Usually a narrow linear range (1E3, usually)
S Must be solvent -compatible, stable, etc.
S Universal
S respond to all analytes
S Analyte Specific
S respond to specific properties of analytes
S Non-destructive
S most
S Destructive
S ELSD, MS and a few others.
48.
49. Standard Absorbance Detector….
S Single Beam UV-VIS instrument with a flow-through cell
(cuvette)
S Can use any UV-VIS with a special flow cell
S Extra connections lead to band-broadening if UV-VIS is far
from HPLC column exit.
S Usually utilize typical UV-VIS lamps and 254 nm default
wavelenth
S Can be set to other wavelengths (most)
S Simple filter detectors no longer widely used
S adjustable wavelength units are cost-effective
S Non-destructive, not-universal
S not all compounds absorb light
S can pass sample through several cells at several different
wavelenghts
S Usually zeroed at the start of each run using an electronic
software command. You can have real-time zeroing with a
reference cell.
50.
51. Diode Array Detector (DAD)
S The more common tool for research-grade
HPLC instruments
S quite versatile...
S Advances in computer technology since
~1985 or so have lead to the development
of Diode Array instruments
S Non-destructive, non-universal
S DAD scans a range of wavelengths every
second or few seconds. At each point in
the chromatogram one gets a complete
UV-VIS spectrum!
S Huge volumes of data
S Detailed spectra for each peak and each
region of each peak
52.
53.
54. Refractive Index Detector
S One of a very few Universal HPLC detectors.
Non-destructive
S Responds to analytes changing the RI of the
mobile phase
S requires a separate reference flow of mobile phase
S Extremely temperature sensitive, usually
heated
S sensitive to temp changes of +/- 0.001 °C
S No longer really widely used
S Absorbance detectors are relatively cheap.
S Useful for process work, on-line monitoring,
etc.
55.
56.
57. ELSD (Evaporative Light Scattering
Detector)
S Universal, destructive
S Useful for very large molecules, and a wide linear
range
S Analytes are de-solvated in the detector
S Molecules pass through what is essentially a large
cuvette for a UV-VIS instrument
S The reduction in light intensity detected (due to
scattering by the analytes) is measured
S The larger and more concentrated a particular
molecule is, the greater the scattering.
58.
59. What does the analyst do?
S Optimize the separation using
standard mixtures and then for
samples.
60. Optimization of Separations in HPLC
S Correct choice of column so the above
equilibrium has some meaningful (non-infinity,
non-zero) equilibrium constants.
S Correct choice of mobile phase
S Decision on the type of mobile phase
composition
S constant composition = isocratic
S varying composition = gradient elution
S Determination if flow rate should be constant
S usually it is
S Decision on heating the column
S heating HPLC columns can influence the
above equilibrium….
61. Types of HPLC Separations (partial list)
S Normal Phase: Separation of polar analytes by
partitioning onto a polar, bonded stationary phase.
S Reversed Phase: Separation of non-polar analytes by
partitioning onto a non-polar, bonded stationary
phase.
S Adsorption: In Between Normal and Reversed.
Separation of moderately polar analytes using
adsorption onto a pure stationary phase
(e.g. alumina or silica)
62. Types of HPLC Separations (partial list)
S Ion Chromatography: Separation of organic and
inorganic ions by their partitioning onto ionic
stationary phases bonded to a solid support.
S Size Exclusion Chromatography: Separation of large
molecules based in the paths they take through a
“maze” of tunnels in the stationary phase.
63.
64.
65. Advantages of LC compared to GC:
1.) LC can be applied to the separation of any compound that is
soluble in a liquid phase.
‚oLC more useful in the separation of biological compounds,
synthetic or natural polymers, and inorganic compounds
2.) Liquid mobile phase allows LC to be used
at lower temperatures than required by GC
‚ LC better suited than GC for separating
compounds that may be thermally labile
66. 3.) Retention of solutes in LC depend on their interaction with
both the mobile phase and stationary phase.
‚ GC retention based on volatility and interaction with
stationary phase
‚ LC is more flexible in optimizing separations change
either stationary or mobile phase
4.) Most LC detectors are non-destructive
‚ most GC detectors are destructive
‚ LC is better suited for preparative or process-scale
separations
Disadvantage of LC compared to GC:
1.) LC is subject to greater peak or
band-broadening.
‚ much larger diffusion coefficients of solutes
in gases vs. liquids
Editor's Notes
Mobile Phases - Component solvents/mobile phases to make up gradient Gradient Controller - Sets up gradient - linearity, steps, ramps, number of solvents/mobile phases (binary, ternary, quaternary). Pump - Dual piston, Pulse free, Able to deliver 4000PSI, Precision flow rates of 0.001mL/min, Flow range 0.001-10.001 mL/min. Injector - How do you inject a sample in to a flowing sream at 4000PSI? If you tried you’d have the syringe plunger go thru a wall! The sample injector utilizes a set of valves in which a sample loop switchs in-and-out the flowing stream. You introduce the sample by injecting it into the sample loop which has a fixed volume. The fixed volume injected replaces the contents of the loop so therefore for manual injection you should have enough injected to completely replace the previous loop contents. The sample is automatically introduced into the flowing stream by valve switching.
555- hydrolysis prior to LC; conc col in-line (C-18) (~SPE); gradient 25mM H 3 PO 4 :MeCN 90:10 > 10:90, PDA uses 2ºλ confirmed by response ratio. Typical cmpd -Cl 5 phenol has BP=310ºC -- not volatile. 605- Electrochemical - 4,4’-Diaminobiphenyl > Diphenoquinone. V=+0.8v 0.1M pH 4.7 OAc : MeCN. BP= 402ºC -- not volatile. Also true electrochemical VA determination. 610 - LC separates all 16. UV for Naphthalene, Acenaphthene, Acenaphthalene and Fluorene, rest Fluorescence; gradient MeCN:H 2 O . MeCN. Also GC/FID (4 sets of unresolved pairs) method. FL then UV, λ=254 8316 - Direct H 2 O inj. MP= H 2 O, UV=195nm. GC can run direct H 2 O inj but not recommended. GC run about 20 min. HPLC run about 12 min. 8330 - >4PPM Direct H 2 O inj. C-18 confirm with CN; MeOH: H 2 O. λ=254. TNT BP= 240ºC (explodes)! 8331 - Tetrazene- MP with ion-pairing (MeOH: H 2 O (C 10 -SO 3 H)(HOAc)), λ=280. 8332 - Nitroglycerine - MP= MeOH: H 2 O(3:2)-CN col. Confirm C-18 (1:1), λ=254. 8325 - LC/PB/MS - Benzidines and N-containing pests/herbs (Carbaryl, Siduron (Tupersan)). Also UV λ=230.
TNT - show numbering system Acrylonitrile = vinyl cyanide Naphthyl Methyl Carbamate
- “Like dissolves like”. In Normal Phase the analyte will be partitioned preferentially in the mobile phase and provide little interaction with the stationary phase. This is not desirable since selective retention on the column will be very hard to control. It can be controlled by modifying the stationary phase, a very time consuming and expensive proposition even if feasible. In Reverse Phase the opposite is true. The analyte will be partitioned preferentially in the stationary phase ( “Like dissolves like” ) . and by simply modifying the mobile phase, by adjusting the polarity, ionic strength or pH, selectivity can be virtually fully controlled.
It is common practice to make up these organic solvents as mixtures with water or, in a lot of cases, have each pure solvent mixed under instrument control and changed at a certain rate with time (gradient). Gradients can be simple or complex. Simple - as a linear gradient (ramp); Complex as steps (start and hold) and ramps together. You can have a solvent or several solvents being controlled at the same time with a changing modifier such as a pH buffer. Methanol - Most common solvent. Close to water in structure. Miscible in all proportions with H 2 O so that for less polar organics you can have the power of 90% Methanol with 10% H 2 O. Acetonitrile - Highly polar, very low UV absorbance. Also completely miscible with H 2 O but lacking in hydrogen bonding capability thus affording a different partitioning effect. Tetrahydrofuran - Molecule has high dipole moment. More soluble with non-polar compounds. Water - Also a very common solvent. Used to make up solvent modifiers to adjust pH (buffers) as well as ion-pairing reagents. Emphasize degassing (“bends”- bubble in detector) and for particle-free (dust could be up to 10X size of particles of solid support). 555- 25mM H 3 PO 4 :MeCN(90:10)->10:90 605 - 0.1M pH4.7 OAc: MeCN (1:1) isocratic. 610 - MeCN:H 2 O -> MeCN 8316 - 100% H 2 O 8330 - MeOH:H 2 O isocratic 8331 - MeOH:H 2 O(HOAc-C 10 SO 3 H) 8332- MeOH:H 2 O (3:2-CN), (1:1-C-18) 8325-A=MeCN:0.01M OAc (75:25), B= MeCN -> 60%MeCN total. λ= 190 for MeCN, λ= 205 for MeOH, λ= 190 for H 2 O, λ~ 290 for THF, λ~ 255 for 1% HOAc and λ~ 260 NH 4 OAc (1M)
Here is a perfect example of why we should have a confirmatory column. Look at peaks 8, 9,10 and 11 on the C-18 chromatogram and notice the poor resolution of the group primarily due to peak 11: 2,6-dinitrotoluene. Now notice the CN chromatogram. See how 2,6-Dinitrotoluene is separated from the two amino-dinitrotoluene compounds and the 2,4-dinitrotoluene allowing baseline resolution for all four. Also note peak 14, HMX. It takes almost 50 min to get out while it was the first (< 2.5 min) to come out on C-18.
Here is a perfect example of why we should have a confirmatory column. Look at peaks 8, 9,10 and 11 on the C-18 chromatogram and notice the poor resolution of the group primarily due to peak 11: 2,6-dinitrotoluene. Now notice the CN chromatogram. See how 2,6-Dinitrotoluene is separated from the two amino-dinitrotoluene compounds and the 2,4-dinitrotoluene allowing baseline resolution for all four. Also note peak 14, HMX. It takes almost 50 min to get out while it was the first (< 2.5 min) to come out on C-18.
Here are two chromatograms which very dramatically show the effect of column particle size and flow rate. Note that resolution has, for the most part, (peaks 3 and 4) NOT deteriorated. You would think that the pressure needed to run chromatogram B would be extrememly high. However, the pressure needed to run chromatogram B, due to new porous particles making up the solid support, was only 2000 PSI (about 50% higher than for A).
Here are two chromatograms which very dramatically show the effect of column particle size and flow rate. Note that resolution has, for the most part, (peaks 3 and 4) NOT deteriorated. You would think that the pressure needed to run chromatogram B would be extrememly high. However, the pressure needed to run chromatogram B, due to new porous particles making up the solid support, was only 2000 PSI (about 50% higher than for A).
