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Lab 6: Capillary Electrophoresis

Electrophoresis is a class of separation techniques in which we separate analytes by their ability to move through a conductive medium—usually an aqueous buffer—in response to an applied electric field. In the absence of other effects, cations migrate toward the electric field’s negatively charged cathode. Cations with larger charge-to-size ratios—which favors ions of larger charge and of smaller size—migrate at a faster rate than larger cations with smaller charges. Anions migrate toward the positively charged anode and neutral species do not experience the electrical field and remain stationary.

There are several forms of electrophoresis. In slab gel electrophoresis the conducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs are formed by pouring the gel between two glass plates separated by spacers. Typical thicknesses are 0.25–1 mm. Gel electrophoresis is an important technique in biochemistry where it is frequently used for separating DNA fragments and proteins. Although it is a powerful tool for the qualitative analysis of complex mixtures, it is less useful for quantitative work.

In capillary electrophoresis, the conducting buffer is retained within a capillary tube whose inner diameter is typically 25–75 μm. Samples are injected into one end of the capillary tube. As the sample migrates through the capillary its components separate and elute from the column at different times.

Introduction

The electrophoretic mobility of an object in an applied electric field is determined by the charge on the molecule via Stokes' Law, the frictional coefficient of the molecule, which depends on size and shape, and the viscosity of the solvent:

\[\mu_{e} = \dfrac{q}{6\pi\eta{r}} \label{6.1}\]

The velocity of the particle in an applied field is \(μ_e \times E\), where \(E\) is the applied field. Slab or gel electrophoresis is commonly used in biochemistry to separate macromolecules, nucleic acids and proteins. Proteins and nucleic acid fragments are separated by differences in mobility through a sieving gel under the force of an applied electric field. Capillary electrophoresis is a technique in which molecules are separated in narrow capillaries under an applied electric field. The electric field rather than gas or solvent flow moves the molecules through the capillary. Molecules in solution will then be separated based on their electrophoretic mobility. Figure 6.1 shows the components of the instrument.

Figure12.59.jpg

Figure 6.1: Schematic diagram of the basic instrumentation for capillary electrophoresis. The sample and the source reservoir are switched when making injections.

There are several different methods used in capillary electrophoresis. All work on the same premise that molecules will travel through the capillary under the influence of the applied electric field.

Capillary Zone Electrophoresis

The simplest CE method is capillary zone electrophoresis (CZE), a method by which molecules, ions, or particles are separated solely by their electrophoretic mobility. The figure below shows the relative velocities of particles with different electrophoretic mobilities. The simplification that holds true for this technique is that the velocity is proportional to the charge to mass ratio. The capillaries are usually made of silica. In uncoated capillaries at pH greater than 3 the SiOH groups are ionized to SiO-. This leads to a phenomenon called electroosmotic flow (EOF).

Capillarywall.gif

Figure 6.2: Ionized Silica Capillary Walls

The negative charge on the capillary wall leads to the formation of a double layer of cations along the wall. The inner layer is tightly bound to the capillary wall and the outer layer is a diffuse layer of cations. A zeta potential forms at the boundary between the inner and outer layers. When an electric field is applied the cations on the diffuse layer move towards the cathode. The cations are more solvated than the anions and pull the bulk solvent towards the cathode. The concentration of positive charges along the capillary wall pulls the bulk solvent towards the cathode (Figure 6.3).

Figure12.56.jpg

Figure 6.2.5: Schematic diagram showing the origin of the double layer within a capillary tube. Although the net charge within the capillary is zero, the distribution of charge is not. The walls of the capillary have an excess of negative charge, which decreases across the fixed layer and the diffuse layer, reaching a value of zero in bulk solution.

 

Figure12.58.jpg

Figure 6.3: Electroosmotic and Electrophoretic flow. Visual explanation for the general elution order in capillary electrophoresis. Each species has the same electroosmotic flow. Cations elute first because they have a positive electrophoretic velocity, νep. Anions elute last because their negative electrophoretic velocity partially offsets the electroosmotic flow velocity. Neutrals elute with a velocity equal to the electroosmotic flow.

The relative mobilities of the particles are the same, but now neutral molecules and negative particles are pulled toward the cathode by the EOF. Neutral molecules will not be separated from one another. But, the negatively charged particles will be separated because the electrophoretic mobility counters the EOF. A solute’s total velocity, \(v_{tot}\), as it moves through the capillary is the sum of its electrophoretic velocity and the electroosmotic flow velocity.

\[ν_{tot} =ν_{ep} + ν_{eof}\]

As shown in Figure 6.2, under normal conditions the following general relationships hold true.

\[(ν_{tot})_{cations} > ν_{eof}\]

\[(ν_{tot})_{neutrals} = ν_{eof}\]

\[(ν_{tot})_{anions} < ν_{eof}\]

Cations elute first in an order corresponding to their electrophoretic mobilities, with small, highly charged cations eluting before larger cations of lower charge. Neutral species elute as a single band with an elution rate equal to the electroosmotic flow velocity. Finally, anions are the last components to elute, with smaller, highly charged anions having the longest elution time.

The EOF is extremely useful for separating molecules with both positive and negative charges. If the EOF is not necessary, or it is desired to completely separate positively and negatively charged particles, the EOF can be abolished by changing the buffer conditions. Using running buffer at very low pH will abolish the EOF. If low pH is a problem for the stability of the samples, the inside of the capillary can be coated with an uncharged layer. Low concentrations of ionic detergent, below the critical micelle concentration will also diminish the EOF. There is no separation of molecules with similar charge to mass ratios. It is frequently desirable to improve or alter the separation. Molecules with similar electrophoretic mobilities can be separated by the addition of carrier compounds to the running buffer.

Micellar Electrokinetic Chromatography

The addition of molecules to the running buffer will separate molecules based on their affinity for those molecules. There are several different ways to do this. The addition of cyclodextrins to the running buffer allows the separation of chiral species.  MEKC, or micellar electrokinetic chromatography, will separate compounds with similar mobilities in CZE experiments by the difference in affinity for detergent micelles that are added to the running buffer. Neutral species will partition between the running buffer and the hydrophobic interior of the micelles. The micelles, which are negatively charged, have a retention time greater than the EOF. Thus as molecules enter the micelles they are slowed down. The stronger an affinity the neutral species has for the micelle, the longer its retention time. The more nonpolar neutral species have the highest affinity for the micelles. Charged particles that have hydrophobic groups will also be retained by interaction with the hydrophobic core of the micelle. Highly positively charged particles will interact with the surface of the micelle and also be retained.

Figure12.64.jpg

Figure 6.4: Micellular Interactions within the Capillary. (a) Structure of sodium dodecylsulfate and its representation, and (b) cross section through a micelle showing its hydrophobic interior and its hydrophilic exterior.

You can see that the separation of the species in the mixture will be changed by addition of detergent to the running buffer. You can tailor your separation to exactly suit your needs by experimenting with different additions to the running buffer.

Procedure

In this experiment you will repeat the analysis you did (or will do) in the HPLC experiment. You will compare two different modes, Capillary Zone Electrophoresis (CZE) and Micellar Electrokinetic Capillary Chromatography (MEKC), to achieve the same separation. Differences in resolution and retention times will be observed and explained. Differences, if any, between the results obtained by HPLC and CE will also be addressed.

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Figure 6.5: Agilent Technologies 7100 Capillary Electrophoresis, UCD Capillary Electrophoresis instrument. Note that multiple vials can be probed sequentially if required.

Use the samples and standards that you prepared for the HPLC experiment. If you haven’t done the HPLC experiment yet, prepare the standards and samples as described in the HPLC experiment. 

Solutions necessary

Write out the recipes for the buffer solutions A and B before coming to lab and have your TA check them before you proceed.  The solutions A and B below have been prepared for you already by the procedure stated below.

  • Solution A: 0.05M borate buffer, pH=9.0. Dissolve boric acid in water; add NaOH until pH = 9.0.
  • Solution B: 0.05M SDS (sodium dodecyl sulfate) in 0.05M borate buffer pH =9.0. Once SDS is added, measure pH to ensure pH = 9.0. Adjust pH with either HCl or NaOH if necessary.

Prepare 100 mL of solution A and then use that to prepare 50 mL of solution B. Use a pH meter identical to that used in Chem 105. The molecular weight of SDS is 288.38 g/mol. The molecular weight of boric acid is 61.83 g/mol.

Filter approximately 10 mLs of each buffer into a clean, labeled vial, just as you did with your samples and standards for the HPLC experiment.  It is imperative that the vials are not filled more than 75% full!  Vial liquid levels at and above the vial shoulder is too much.  Over filling vials can lead to salt build-up and over-pressurization of the capillary.  If arcing or over-pressurization occurs, the capillary is almost certain to be rendered useless.  The proper solvent level is only 1.4 cm, which translates to about a 75% full vial.  As always, ask your TA if you have any questions.

Setting up the instrument

The first experiment you will run is the CZE separation. All the steps are the same for both methods.

The instrument used in this experiment is an Agilent CE.  The software used to control the instrument and collect the data is the Agilent Chemstation, which is also used for the HPLC.

Turn on the instrument, if it is not already on, by pressing the power button at the bottom left of the front face of the CE.  The light in the middle of the button should be green when its on.

Open the Chemstation software if its not already on by double clicking the "Instrument 2 Online" icon with an image of the CE.

You will start in "Instrument View." 

Instrument View shows a diagram of the complete system, with clickable features to make many actions easier.  The menu bar could also be used, but takes more steps per action.

The instrument must first be initialized.  Click the "On" button next to the question mark button on the bottom right of the "CE" window.  Click on the power button icon in the top left corner of the "DAD" (diode array detector) window to "make device ready".. Wait until the system goes to "Ready."  This may take some time if the temperature controlled zones need to stabilize.

When the software goes to "Ready", in the "CE" window, right click "Inlet" and unload by selecting  "Unload Inlet Lifter."  Do the same for the "Outlet." You should then hear the instrument lowering the vials in positions 1 and 2.  Right click the sample wheel and select "Get Vial."  "Vial" should be set to "1" - click "Get." The tray will rotate vial 1 to the front of the instrument.  Open the tray door and remove vials 1 and 2, if they are present.

Fill a new vial with the pH 9 0.05 M borate buffer by filtering it with a syringe filter and label it "Buffer A" with a pen.

Do Not Use Tape to Label Vials!!!!!  It Will Jam the instrument. Use a sharpie pen (ask TA) !!!

Place this vial in position 1.  Place an 50% filled vial in position 2 and label it 'outlet'. Close the tray door. The tray will rotate back to its operating position.

Right click on the regulator pressure icon in the "CE" window and click "Flush" on the menu. Enter a time of "600" seconds and click "OK". The pressure should increase to about 930 mBar and slowly decrease as liquid is pushed from one vial to the other during the flush time.  This will wash out any residues from the previous runs and equilibrate the capillary with the running buffer.

Make sure that the method is "CAFFEINE.M" and that the sequence is "CAFFEINE.S."  The method and sequence names can be found in the drop down boxes in the main menu bar.  Check and make sure the CZE method is using 20 kV by selecting "Method" then "Edit Entire Method". Check with your TA what the run time is for the current capillary.  Press "OK" in the first two windows that appear until you get to the "Setup Method" window.  Select the "CE" tab to check the voltage and run time under "Stop Time." Check that under "Injection," on the right side of the "CE" tab window, there is one row that under "Function" has "Apply Pressure" and under "Parameter" it says "50mbar for 5s (Inlet: Injection Vial Outlet: Oulet Home Vial)."  Then click "OK".   "OK" through or cancel through the remaining windows. 

Make sure you filter your samples into vials and make sure you labeled the vials with a pen.  Right click on the tray icon and select "Get Vial."  Set the "Vial" number to 7, and click "Get."  You can now open the tray door and put all your samples in, starting at position 4 (leave position 3 empty).    Start with the 0.01 g/L standard followed by the rest of the standards in increasing concentration, then the four samples. Make sure you keep track of the order in which the samples are run.  Close the tray door.

On the menu bar, click "Sequence" and select "Sequence Parameters".  Enter an operator name, make sure "Prefix/Counter" is checked, and enter a prefix for your datafile names.  Click "OK".

To run the sequence click the "Sequence" play button above the "DAD" window.

After the sequence has started, watch the "Online Plot."  You should see a peak for your first standard about 2-3 minutes into the run.  The electrical current, which can be monitored in the "CE" window should be 15-16 microamperes.

You can print the the reports after each sample is run.  When the entire sequence has completed, click "Data Analysis" at the bottom left of the window.  Click on "Chem_115" in the data tree and select your sequence.  Select the data you want and print. Repeat for all your other data files.  A prompt to save each trial as a PDF will appear after each trial.  Create a file and save the trials to the file.  Then print the PDFs.

IMPORTANT

Make sure that you get a peak for each standard. You can always abort the run and restart it if there is a problem.

Now do the MEKC run using the SDS-containing buffer, buffer B, as the run buffer. You should flush the capillary with buffer for "300" seconds before the first run.

The parameters are slightly different for the MEKC run. The voltage is 15 kV, rather than 20 kV and run time is longer. Check the exact run time with your TA. Everything else is the same. Select the "CAFFEINE_MEKC.M" method in the menu bar.  Select "CAFFEINE_MEKC.S" sequence in the menu bar.

Make a calibration curve in Chemstation.  Since you have to wait awhile while the data is being acquired it is good to do this during data acquisition.  You can begin to do this even if you have not finished all your runs.  From the "Method and Run Control" view click the "Data Analysis" tab in the bottom left corner.  It will open up another window of "Instrument 2" but it will be "Offline."  Under the "Data Analysis" window on the left there will be a file tree.  Go to the "Chem_115" folder and select your data file.  The sequence runs you have done will appear in the main window.  Double click on your first standard run in the "Sequence" window at the top of the screen.  The line for the first standard run should now appear in bold font. On the menus bar go to "Calibration"  and select "New Calibration Table."  The window "Calibrate: Instrument 2"  will appear.   Select "Automatic Setup" set the "Level" to "1" and put in the concentration of your first run in "Default Amount."  Click "OK."  Then double click on the second run.  Go to "Calibration" on the menu bar and select "Add Level."  Set the "Level" to "2" and enter the concentration in the "Default Amount."  Click "OK."  Repeat for the rest of the standards.  The "Calibration Table" and the "Calibration Curve" windows are at the bottom of the view.

Make sure all reports have been printed.  Using the peak areas of the standards plot a calibration curve in either Chemstation, MatLab or other data analysis software.  From the calibration curve, calculate the concentrations of caffeine in each of the unknowns.

The vials and filter you used should be thrown away.

Report

  1. Include print outs of all your electroferograms for each sample and standard, and the integration reports for each set run. Also print the calibration data.
  2. Explain the difference in retention time for the two different experiments.
  3. What would you expect to happen to the retention time of the caffeine peak if you decreased the run voltage for the first experiment to 10 kV?
  4. Did you get the same answer for the two different CE experiments? Explain.
  5. Did you get the same answer as you did for the HPLC experiment? Is this surprising? If the answers are different, suggest some possible explanations.

References

  1. Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, Fifth Edition; Harcourt Brace: Philadelphia, 1998; 591-621.
  2. Copper, C. L. Capillary Electrophoresis Part I. Theoretical and Experimental Background. J. Chem. Ed. 1998, 75, 343-347. pdf
  3. Copper, C. L.; Whitaker, K. W. Capillary Electrophoresis Part II. Applications. J. Chem Ed. 1998, 75, 347-351. pdf
  4. McDevitt, V. L.; Rodriguez, A.; Williams, K. R. Analysis of Soft Drinks: UV Spectrophotometry, Liquid Chromatography, and Capillary Electrophoresis. J. Chem. Ed. 1998, 75, 625-629. pdf