Prepared for Chemistry and Biochemistry Fall 2003 Kevin Olsen

So What’s the Big Deal with Those Little Tubes?Capillary Electrophoresis Exercises for MSU Students Prepared for Chemistry and Biochemistry Fall 2003 Kevin Olsen

Program • General principles • Safety • Obtaining good results • Example application, CE in drug discovery • Running your samples on the Beckman Coulter model P/ACE

System Schematic Applied Electric Field. 1000 Volts / Centimeter Anode + Cathode - Detector (UV, PDA or LIF) Velocity = (Field Strength) (Electrophoretic Mobility)

Beckman P/ACE System

Electrophoretic Mobility Z Uep = --------------- 6 n r Where: Z = Net charge on the analyte n = Viscosity of the medium r = Stoke’s Radius

Variables Governing Electrophoretic Mobility • Charge • Radius* • Mobility *The Stoke’s radius is related to molecular mass. Heavier molecules will have a greater radius.

Electroosmotic Flow(EOF) This flow is a phenomena resulting when a solution is contained in a capillary with fixed charges along its wall. This is also known as the Electroendosmotic Flow.

Where does the Electroosmotic flow come from? Anode + Cathode - Detector

Where does the Electroosmotic flow come from? Applied Electric Field. 1000 Volts / Centimeter Anode + Cathode - Detector

Where does the Electroosmotic flow come from? The interior wall of the capillary contains charged sites that are created by the ionization of Silanol groups on the fused silica. Anode + Cathode - Detector

Where does the Electroosmotic flow come from?

This is where the Electroosmotic flow comes from. What happens to the + cations when we turn on the power?

pH, Silanol Population, and the rate of EOF flow. • At very low pH, not many silanols are ionized and the EOF is slow. • As pH increases the number of ionized sites also increases. The EOF speed rises steadily. • At very high pH values, a maximum number of ionized sites is reached. The EOF speed also reaches a maximum.

The apparent velocity of any analyte (u) will be a combination of its electrophoretic velocity and its movement in response to the EOF. u = (Uep + Ueo) E

How does apparent velocity help us? EOF Analytes with a net positive charge will move faster than EOF EOF Analytes with no net charge will move at the same speed as the EOF. (This is a useful tool that helps us to measure the EOF.) EOF Analytes with a net negative charge will move slower than EOF

Separation Efficiency (apparent mobility) (Voltage) N = ---------------------------------------- 2 (diffusion coefficient)

Separation Efficiency (Y) and Diffusion Coefficient (X) • Note the very dramatic drop in separation efficiency with increasing diffusion coefficient. • This means that in some cases, there is no real advantage over conventional HPLC for smaller molecules.

Injections There are two principle methods: • Pressure differential works by applying a pressure across the capillary while it it is dipping into the sample solution. • Electrokinetic injection works by applying a voltage and allowing ions to migrate into the capillary because of their charge. Injection volumes are typically very small: • Typically if injection volumes exceed 1% of the column volume, separation efficiency severely suffers. • Sample volume can be increased by focusing the ions inside the capillary. This technique uses a combination of additives to the medium and selectively applied charges.

Preconcentration to Increase Sensitivity • Attached to front of column • Contains a selective binding agent • Allows several capillary volumes to pass • Analytes of interest are then eluted

Pressure and Electrokinetic Injections + + + + + + + + - - • One additional advantage of electrokinetic injections is that if appropriate conditions are set, extended injection times allow analytes to be concentrated without overloading the column.

Setting up the Capillary Column • Cut the ends cleanly. • Load capillary into the cartridge • Place the clear portion in the detector window.

The Capillary Column’s Cartridge • Allows the column to be moved from vial to vial. • Contains a cooling medium. • Contains gas and vacuum connections. • Holds electrodes that place a charge on the sample vials.

The Advantages of CE are: • The number of theoretical plates is typically in the hundreds of thousands. • There is no mass transfer between mobile and stationary phases as with HPLC and GC, therefore the analytes remain in a “plug” instead of spreading as a result of laminar flow. (Peaks can still broaden however.) • Altering column conditions allows focusing or concentration of samples.

SAFETY • Chemical and Biological • Remember that solvents will be flowing under high pressure inside an electrically powered device. • Aerosols may be generated, work in appropriate enclosure. • Take all normal safety precautions when working with toxic, pathogenic, or radioactive materials. • Electrical • Never remove covers and expose the electronics. • Under certain conditions the chemist may have to be grounded for protection against static electricity. • Mechanical • The CE unit features a robotic autosampler with many moving parts and a sharp needle. Keep hands out of the sample compartment while the unit is running.

Obtaining Reproducible (Good) Results • Column condition. • Composition and pH of the medium. • Viscosity of the medium. • Operating temperature. • Adequate sample volume. • Use of internal standards.

The pH must be tightly controlled to obtain reproducible EOF flow. • Remember that the percentage of silanols that are ionized is dependent on the pH.

Column Condition • As time goes on, certain molecules will block or otherwise neutralize the ionized silanol sites. This will change the EOF and alter retention times. • It is also very important to condition the column properly before use. Follow the directions in the published method.

Internal Standards • The main advantage of an internal standard is that it is subject to the same conditions as the analyte.

Courtesy of Cetek Corporation

20,000 Compounds Tested per Day 6,000,000 Tested since 1998 Courtesy of Cetek Corporation

Other Applications • Analysis of molecules that are not suited to HPLC. • Chiral separations of enantiomers. • Determination of drug molecules in biological fluids. • Separating bacteria. • Expect the unexpected.

Setting Up Methods • Use File|Method|New from the menu bar to create your method. • When finished use File|Method|Save as from the menu bar.

Tray Layout B6 C6 E6 F6 A6 D6 B5 C5 E5 F5 A5 D5 B4 C4 E4 F4 A4 D4 B3 C3 E3 F3 A3 D3 B2 C2 E2 F2 A2 D2 B1 C1 E1 F1 A1 D1

Manipulating Tray Layouts B1 C1 E1 F1 B1 C1 E1 F1 A1 D1 A1 D1 B1 C1 E1 F1 B1 C1 E1 F1 A1 D1 A1 D1 B1 C1 E1 F1 B1 C1 E1 F1 A1 D1 A1 D1 B1 C1 E1 F1 B1 C1 E1 F1 A1 D1 A1 D1 B1 C1 E1 F1 B1 C1 E1 F1 A1 D1 A1 D1 B1 C1 E1 F1 B1 C1 E1 F1 A1 D1 A1 D1

Manipulating Tray Layouts Blue = Rinse Green = Multiple use Orange = Fraction collection Dark purple = Sample Light purple = Other injection Red = Selected vial Yellow = Separation B1 C1 E1 F1 A1 D1 B1 C1 E1 F1 A1 D1 B1 C1 E1 F1 A1 D1 B1 C1 E1 F1 A1 D1 B1 C1 E1 F1 A1 D1 B1 C1 E1 F1 A1 D1

Manipulating Tray Layouts • Vials positions can be designated under “instrument set-up”. Afterwards, you will be prompted to save the changes to your method. • Vial positions can also be designated while setting up the “Timed events” table in your method. • In either case, the software commands and procedures are the same.

P/ACE System Control • Select your method from the drop-dow method under FILE • You may then change the tray layouts but all changes become part of the method. • In this class, we will have two control options: 1. Direct Control 2. Single Run

THE EXERCISES • Each team will create one method that will rinse and condition the column, then inject a sample. • Each team’s method parameters will be slightly different. • Your instructor will string the methods together for an overnight run. • When the class meets again, the results will be compared. Each student will submit a report comparing and contrasting the results.

Generic Test Solution Method • Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi. Destination = waste vial. • Rinse: 1.5 minutes, Run buffer A, 20 psi. Destination = waste vial. • Inject: 10 Seconds, Test mix, 0.5 psi. Destination = Run buffer A on outlet tray. • Separate: 7 minutes, Run buffer A, 25 kV. Ramp time 0.2 minutes. Destination = Run buffer A on outlet tray.

Team 1, Variation • Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi. Destination = waste vial. • Rinse: 1.5 minutes, Run buffer A, 20 psi. Destination = waste vial. • Inject: 10 Seconds, Test mix, 0.5 psi. Destination = Run buffer A on outlet tray. • Separate: 7 minutes, Run buffer A, 25 kV. Ramp time 0.2 minutes. Destination = Run buffer A on outlet tray.

Team 2 Variation • Rinse: 0.5 minutes, Regenerator sol’n A, 20 psi. Destination = waste vial. • Rinse: 1.5 minutes, Run buffer A, 20 psi. Destination = waste vial. • Inject: 10 Seconds, Test mix, 0.5 psi. Destination = Run buffer A on outlet tray. • Separate: 7 minutes, Run buffer A, 25 kV. Ramp time 0.2 minutes. Destination = Run buffer A on outlet tray.

Prepared for Chemistry and Biochemistry Fall 2003 Kevin Olsen