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