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Transcript
Sample Exam Questions
1. _____ Which type of liquid chromatography separation depends on
interactions between solute, mobile phase, and immobilized liquid
stationary phase?
A. Adsorption
B. Partition
C. Ion-exchange
D. Size exclusion
2. _____ This functional group is regularly employed in the stationary
phase of cation-exchange chromatography.
A. Trimethylammonium, -N(CH3)2+
B. Peroxide, -O2C. Sulfonic acid, -SO3D. Magnesium, -Mg+
Liquid Chromatography
1.
What is it? Separation of liquid-phase molecules
2.
Physical principle? Partition of analyte between liquid
mobile phase and immobilized liquid stationary phase;
partition, adsorption, ion-exchange, or size exclusion
3.
How is the measurement made? Sample introduced as
small liquid aliquot into column, elution measured at end
of column
4.
What data is obtained? Peak areas proportional to
concentration and characteristic retention times
5.
What are the applications? Any mixture of liquids or
liquid-soluble components
6.
What innovations are possible? Development of general
use detectors, decreasing dimensions (small samples)
1
HPLC Instrument Diagram
He
sparge gas
Solvent
reservoirs
Backpressure
regulator
Pulse
damper
Filter
Pressure
transducer
Injector
valve
Pump
Solvent
mixing valve
Column
Outlet
check
valve
Inlet
check
valve
Detector
Figure 28-4
Instrument
28C
Mobile Phases / Solvent Systems
Each reservoir contains 200 to 1000 mL solvent (mobile phase)
Solvents normally degassed (air bubbles removed) – vacuum pumping
system, vacuum filtration, distillation, heating, stirring, sonication, or
sparging (gases swept away by inert, low solubility gas – He); prevents
band spreading, detector interference
Isocratic elution – uses single solvent as mobile phase
Gradient elution – mixture of 2 or more solvents that varies during
separation, normally by using mixing chamber; could use multiple pumps
and mix at high pressures; shorter elution times w/o loss of resolution (Fig.
28-5)
Instrument
28C
2
Pumping Systems
Requirements:
1) Generate pressures up to 6000 psi (400 bar)
2) Pulse-free output
3) Flow rates from 10 mL/min. to less than 1 µL/min.
4) Flow control / reproducibility of 0.5% relative or better
5) Corrosion-resistant components
Three main types:
Reciprocating Pumps – most common (90%); piston & check valves
Displacement / Syringe Pumps – screw-driven plunger; viscosity & back
pressure-free flow rates; pulse-free; limited solvent capacity (~250 mL)
Pneumatic Pumps – driven by compressed gas; flow rate depends on
viscosity & back pressure; inexpensive; pulse-free; limited capacity
Instrument
28C
Reciprocating Pumps
Reciprocating Pumps – small internal volume (35-400 µL); high output
pressures (up to 10,000 psi); accommodate gradient elution; constant
flow rates; pulsed output
To column
seal
outlet
Ball check valves
Reciprocating piston
inlet
Solvent from reservoir
found in ~90% of commercially available systems
Instrument
28C
3
Syringe Pump – Gilson 402
Assures accuracy in sample transfer,
dilution, reagent addition, mixing and
more. Offers speed and reliability for
repetitive liquid handling tasks.
• Delivers volumes from 1.0 µL to 25
mL with excellent precision
• Adjustable flow rates to compensate
for different sample viscosities
• Single-and dual-syringe with tee
junction and dual-syringe with valve
modules available
• Dual-syringe models provide extreme
flexibility for quick and easy access to
syringes, allowing you to create the
configuration that will precisely fit your
application
http://www.gilson.com/Products/product.asp?pID=17
Microfluidic (Pneumatic) Flow Control
Rather than utilizing positive
displacement pistons or syringes
to regulate flow, Eksigent HPLC
systems rely on Microfluidic Flow
Control (MFC) technology. This
entirely new pumping principle
uses continuous feedback to an
electronically controlled pressure
source to maintain precise
nanoscale flow rates as low as
20 nL/min without flow splitting.
flow meter
column
electronically controlled
pressure source
control
processor
By combining true microfluidic pump control with direct feedback of flow rate, the system
eliminates the flow inaccuracies and plumbing problems caused by splitter-based systems.
•Ability to pump against substantial back pressures (to 10,000 psi or more)
•Active feedback for identification and prediction of leaks or blockages
•Virtually instantaneous response to step changes in flow rate setpoint
http://www.eksigent.com/innovation/technology/index.asp#MFC
4
Sample Injection Systems
“Load”
“Inject”
Loop
Loop
From pump
To column
From pump
To column
To waste
To waste
Allow reproducible injection volumes from 5 to 500 µL;
0.5 to 5 µL for micro injection valves
6 and 10-port valves commonly used
Instrument
28C
Chromatographic Columns
Stainless steel or glass tubing – various sizes / packings; ~$200-$500 each
Length (~10-30 cm), inner diameter (~4-10 mm), and particle size (3, 5, or
10 µm) all vary – most common is 25 cm long, 4.6 mm id, 5 µm particles
40,000-60,000 theoretical plates/meter
New high-performance columns – smaller (3-7.5 cm x 1-4.6 mm id, 3 or 5
µm particles) – faster, 100,000 plates/meter (Fig. 28-8)
Guard columns – small disposable columns w/ same packing as analytical
column, larger pores – protect analytical column from clogging,
contamination
Thermostats – ambient to ~150 °C; regulates temp to +/- 0.2 °C for better
separations
Packings – pellicular (30-40 µm nonporous glass or polymer) for guard
columns; porous (3 to 10 µm silica) w/ stationary phase attached
Instrument
28C
5
Detection Systems – Ideal
1. Adequate Sensitivity (varies from 10-8 to 10-15 g solute/s)
2. Good stability & reproducibility
3. Linear response over several orders of magnitude
4. Short response time – independent of flow rate
5. Reliable / Easy to use – foolproof for inexperienced users
6. Same response to all solutes / predictable selective response
7. Nondestructive to sample
8. Minimal internal volume – prevent peak broadening
• No universally applicable detector for LC, such as FID & TCD for GC
• Two types: bulk property (refractive index, dielectric constant, density)
and solute property (UV absorbance, fluorescence, diffusion current)
Instrumentation
27B
Detector Systems
Absorbance detectors – use Z-shaped flow cell (Fig. 28-9)
UV absorbance detectors – w/ filters or monochromators to select λ or
diode-array to collect spectra (~70%)
FTIR absorbance – NaCl or CaF2 windows, limited by solvent absorption
Fluorescence – Hg or Xe excitation sources; solute must fluoresce - dansyl
chloride deriv. of primary & secondary amines, amino acids, phenols (15%)
Refractive Index – response to wide range of solutes; temperature sensitive
(mK); poor sensitivity (5%)
Evaporative Light Scattering (ELSD) – effluent nebulized to fine mist,
mobile phase evaporated, passed through laser beam to measure
scattered radiation; broadly applicable; better sensitivity than RI
Electrochemical – amperometry, polarography, coulometry, conductometry;
broad applicability; good sensitivity (4%)
Mass spectrometry – atmospheric pressure chemical ionization,
electrospray ionization; solute identification
Instrument
28C
6