Introduction. Developed by: K. Moore, J. Giannini, K. Nordstrom & W. Losert (Univ. of Maryland, College Park) Page 1

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1 TA GUIDE Lab 7: How do charged objects in a fluid interact with each other and respond to external electric fields? Electrophoresis and Charge Screening in Fluids. Introduction In this two-week lab, students explore how microspheres that are suspended in solution move in an electric field. Students discuss that beads charge spontaneously when placed in solution. By varying fluid salinity, the effective charge is changed due to screening of charged objects by the surrounding ions (Debye screening). Using the basic vocabulary of colloidal chemistry, students model the mechanism of electrophoresis, examining how the terminal velocity of the bead's motion in an electric field is related to the effective charge of the bead. The biophysical motivation for this laboratory is the ubiquity of charges in living systems. Large molecules in particular, proteins are often phosphorylated and thus carry net charge. These charges generate electric fields, exerting attractive forces on other molecules, and playing a role in the intricate balance of forces and movements that occur in a living cell! We might ask: how far is the reach of the force field from a charge in a large protein when many ions are present in the surrounding fluid? How fast would a charged protein move in an electric field? These effects are crucial for understanding biological systems at the cellular level. In this two-week lab sequence, students will investigate related questions in a simple model system. They will be investigating charge screening in fluids by employing the technique of electrophoresis. Their investigation will come in two parts: Part 1, an investigation of glass beads in de-ionized water (DI water, pure H 2 O); and Part 2, an investigation of glass beads in two saline solutions of different concentrations. We have been employing solutions of glass beads in water in most of our labs. Students may have noticed that some of the beads are stuck together, forming clumps (also called 'flocs' because their aggregation (coming together) is a form of flocculation), or that the beads sometimes "settle-out" of the fluid (like the sedimentation we observed in the tilted microscope lab last semester). Aggregation (flocculation or coagulation) and sedimentation are behaviors common to colloidal fluids. A colloidal system is one in which one phase of matter is finely dispersed throughout another phase of mater. Our glass beads in water are colloidal fluids because the glass beads (solids) are small particles dispersed throughout the water (fluid). In fact, when the glass beads are submerged in water, they become charged even in DI water! The glass beads, SiO 2, have surface groups of SiOH. When immersed in water, the hydrogen nucleus breaks free (increasing the ph of the fluid due to the roaming H + ) and leaving an SiO - behind. Thus the surface of the glass beads becomes negatively charged each bead carries charge on the order of femto-coulombs (fc). (Lest you think a fc is small, about how many extra electrons are on each bead?) [You might then ask: if all the beads are negatively charged, then why do they stick together? Besides the electric repulsive forces Developed by: K. Moore, J. Giannini, K. Nordstrom & W. Losert (Univ. of Maryland, College Park) Page 1

2 between the beads, there are also attractive van der Waals forces; at close enough distances, the attraction dominates the interaction and the beads will stick together to form a floc.] If a salt (like NaCl) is added to the fluid, it will dissolve and the free cations (or anions, for a positively charged colloid) will group around the negatively charged beads of glass, thus decreasing the 'effective charge' of the unit this is called 'charge screening' or 'Debye screening.' The more ions that are available in the fluid, the greater the charge screening effect will be; thus the concentration of the electrolyte is important. Investigation To investigate this charge screening effect, students will need to determine how charged their glass beads are in DI water (Part 1) and then compare that charge to the 'effective charge' as seen in various concentrations of saline solution (Part 2). They can investigate these charges using the technique of electrophoresis. By applying an electric field (generated by a potential difference between two electrodes) to the fluid (see picture above right), students can cause an electric force on the charged beads that will induce motion through the still fluid. A larger electric field will cause faster motion. Before they begin Part 1, students will need to model the situation: consider what forces act on the bead as it moves through the fluid and determine how changing the potential difference between the electrodes and measuring the resultant speed of the beads will enable them to find the charge on the beads. Students should carefully reflect on what assumptions they are making as they model the situation and think about the implications these assumptions have for the design of their experiment. The experimental set-up available (see picture below right) includes: the microscope (with camera), a power source (the big box on the right with knobs and wires) to create constant voltage of their choosing, two wires with banana plugs on one end and micro-grabbers (for holding electrodes, see larger images in photo above) on the other, short segments of copper wire to use as electrodes, an eight-well chamber slide (fill one of these chambers halfway with solution for each investigation when investigating a different solution, students can simply move to another chamber, or choose to empty the chamber and rinse with distilled water. At the end of the day please have students rinse the chamber with distilled water and leave the chambers upside down on a paper towel to dry), Developed by: K. Moore, J. Giannini, K. Nordstrom & W. Losert (Univ. of Maryland, College Park) Page 2

3 three vials of solution: one of distilled water and two different saline solutions (LOW and HIGH), and other tools (rulers, paper towels, pipettes, etc.). If students have never used a power source before, you should explain basic safety and use instructions. Here are some other details that may help students design their experiment: The maximum voltage the power source can produce is 18 V; students will likely need 4 V or more to see bead motion in DI water. The chambers each hold less than 1 ml of fluid; the vials provided should be sufficient. All solutions contain glass beads that are 5 µm in diameter. The low concentration saline solution is 9 mg/l NaCl; the high concentration saline solution in 90 mg/l NaCl. The viscosities of these solutions are almost identical to that of pure DI water 9.0 * 10-4 Pa-s at 26 C. Students should gather their video data as soon after applying the potential difference to the electrodes as possible (think about why this might be so). Students should be careful to leave the fluid sample on the microscope stage (above the hot bulb) for as short a time as possible before gathering their video (think about why this might be so). Students WILL NOT need to use particle tracking (manual or automated) for these videos; look for the hint at the end of this lab. Though students may not need to track, they will need lots of data encourage them to collect carefully and precisely, collect sufficient sets of data, and consider ways to reduce their uncertainty. What students have been asked to do: Part 1: DI Water Design an experiment to determine the charging of beads in fluid. Consider 'broad stroke' details (e.g., What data are we collecting? How much data is 'enough'?) as well as 'fine stroke' details (e.g., What part of the slide are we viewing? How are we collecting data? How do our assumptions affect our procedure and analysis?). Be sure to consider the qualitative aspects of the motion, as well (e.g., Is this really directed motion? Could it be random motion? Which electrode (+ or -) are the beads moving toward?). Once you have a good experimental design, gather data to determine the charging of our glass beads in DI water. Be sure to note the TIMING used by the video capture program, VirtualDub, for EACH video taken. Part 2: Saline Solutions of Low and High Concentration Now investigate how saline solutions 'screen' the charge on the glass beads. Using your experimental design from Part 1, find the 'effective charge' of our glass beads in a LOW concentration saline solution. Examine the effect of a HIGH concentration saline solution. Before you do either of these, it may help to think about a model for a charged glass bead in an electrolyte solution. *** Be sure that each group member has a copy of all data- and wordprocessing documents created before leaving the lab at the end of week 1. *** Developed by: K. Moore, J. Giannini, K. Nordstrom & W. Losert (Univ. of Maryland, College Park) Page 3

4 ImageJ Analysis Hint: Manual tracking or automated tracking may be 'overkill' for these videos. If you wish, the velocity of a single bead can be determined by measuring the displacement over a corresponding time interval. (What assumptions are implicit in this method? Are they justified assumptions? Under what conditions?) Select a bead and note both the initial frame at which it appears in your video and the final frame before it disappears from your video. In the initial frame, use the 'point tool' (plus shape icon) to click on the location of the bead. Use the scroll bar to advance the video to the final frame; note that the location you clicked on with the 'point tool' remains visible. Using the 'line selection tool' (slanted line icon), draw a line segment from the location left by the 'point tool' to the location of the bead in the final frame. Note the pixel length of this line. (If you let go of the mouse too quickly to note the line's length, you can always choose 'Analyze,' 'Measure' to find the length of the line segment.) Using appropriate pixel to distance conversions and frame to time conversions, you can now state the velocity of this glass bead. For the Lab Report: Beyond the expectations of a normal lab report (journal, procedure, data, analysis, conclusions, etc.), students should be sure to include careful statements of the models they have developed and the assumptions contained within those models. Students should also include qualitative descriptions of observed phenomena to accompany the quantitative analysis of each colloidal fluid. Students should include as much procedural detail, data, and analytical detail as they feel are necessary to prove their points and substantiate their conclusions. Students should also discuss the ways in which the ideas explored here relate to biology and/or chemistry. Introduction to provide to students: Groups of three or four students performing in the Community Lab roles Discuss with students that this lab examines the effect of instantaneous charging of beads in solution and the use of an electric field (potential difference gradient) to determine the charge of the bead (or the effective charge, if in saline solution). Ask the students to consider the following question: Since the beads automatically charge in the fluid (due to changes in the surface chemical composition of the bead), why have we not had to worry about this effect before this lab? (I.e., Why does the charge matter for this lab?) Before students design their experiment, they will need to consider a cluster of questions to model the physical situation. Students should consider how the bead will move when an electric field is applied. Will the electric force cause it to speed up indefinitely? Will it reach a maximum velocity? Have them create an FBD (or a system schema and an FBD) for the bead at terminal velocity. From this pictorial model, have them develop a mathematical model relating electric charge, q, terminal velocity, v, and potential difference, V. Students should consider what other information is needed in their model (e.g., viscosity, bead size, etc). How will they get this information? This mathematical modeling will help them interpret the results from the video analyses. [TA Note: Students should relate E = - V/ x, F E = qe, and F V = -6πµrv; with E the electric field, x the electrode separation, µ the fluid viscosity, and r the bead radius.] Before students collect any videos, they will need to think carefully to design their experiment. They should think about what to measure and why they will measure it. How will these measurements help them determine the bead's charge? What design Developed by: K. Moore, J. Giannini, K. Nordstrom & W. Losert (Univ. of Maryland, College Park) Page 4

5 considerations will help them reduce the experimental uncertainty? [TA Note: One possible experimental design is to vary the voltage difference and find the resultant terminal velocity. The slope of a plot of v vs. V should eventually yield q. This is not the only design that students might consider. Even if you feel the design they have chosen is not ideal (or not how you would do it), so long as the design will work give them the freedom to try it their way and discuss, defend, and critique their choices with their peers.] Remind the students that they need to record the average frames-per-second used by VirtualDub for EACH video. Before depositing a solution from the vial into the chamber slide well, remind students to SHAKE the solutions vial and then draw the sample with a pipette from the MIDDLE of the solution vial. (This will provide the optimum density of the beads in the solution sampled.) Chamber slide wells should be filled 1/3- to 1/2-way with solution. Remind students to turn off the power supply between investigations and to turn off both the power supply and the microscope when they have finished gathering videos. Videos should be taken rapidly once the potential difference is applied (within minutes). Solutions should not be left above the hot microscope bulb for extended periods of time, especially prior to applying the potential difference and gathering the video. Encourage students to split the video harvesting (in ImageJ) and analysis (in Excel) onto multiple computers to complete the analysis. Encourage them to think about HOW they will harvest and analyze the data. They need to make a plan BEFORE they begin the analysis. (E.g., How many beads should they 'track' in each video? (10 at the minimum...) Do they need to use Manual Tracking? If they use the line tool, do the starting (start of line) and ending (end of line) frames matter? How do the pixel distances get converted into physical distances?) Remind students that they should be making a 'back up' copy of their raw Excel/ImageJ data before they begin analyzing the data in Excel that way, if something bad happens to their data they will not have to re-'track' the video. When students leave the lab room today, EACH student in the group should have an electronic copy of ALL of the group's data (via or flash-drive). Briefly review the Skill Goals. Remind students that they are ALL expected to master the skills, so they should take turns and help each other out. Taking notes may not be a bad idea, either. Inform students that a lab report will be due at the end of the lab next week but it is a good idea to write as much as they can of the report this week, so that they don t forget what they did! Summation at the end of the lab period: o Recap the Skill Goals, clarifying any remaining confusion about physics concepts o Discuss the challenges and considerations with the class, if any have not yet been addressed. Ask the students what they found most difficult/challenging about either the qualitative discussion or the initial analysis. o Remind students to save their work (data, Excel files, Word documents, etc.). Developed by: K. Moore, J. Giannini, K. Nordstrom & W. Losert (Univ. of Maryland, College Park) Page 5

6 Physics Skill Goals: Understand uses of electrophoresis; especially with respect to charge, speed, and potential difference. Understand the effect of an electric field (potential difference gradient) on the motion of a charged particle. Understand the relationship between electric fields and potential differences. Understand how Debye screening changes an object's effective charge. Select an appropriate method for determining the uncertainties of the bead velocities. Challenges/Considerations: What is electrophoresis? What are the possible uses of electrophoresis? How are electrophoresis techniques used in chemistry and biology? Does the electric field affect the fluid, the object, or both? How can you tell? To which electrode does the glass bead move? Does this make sense? What will happen to the motion of the bead when the potential difference gradient is increased? What physical quantities must be measured to determine the electric field? How are the vector electric field and the scalar potential difference related? How can the electric field be understood as a gradient? What happens when the free ions in an electrolytic solution encounter a large, charged object? To what extent will these free ions 'screen' or 'shield' the charged object? How large might you expect this effect to be? How will this charge screening affect the motion of the bead? How will the concentration of the saline solution affect the charge screening and the bead's effective charge? Which error analysis method best matches our data analysis decisions? Do we need standard deviations? Do we need error propagation? How does the uncertainty help us? Approximate Timing: Week 1: o Introduction min. o Modeling Situation min. o Designing Experiment min. o Gathering Part 1 Videos min. o Gathering Part 2 Videos min. o Analyzing Videos in ImageJ min. o Start Analyzing Data in Excel... 5 min. Week 2: Introduction min. Finish Analyzing Data in Excel min. Creating Posters/Presentations min. Posters/Presentations min. Class Discussion min. Finalizing Lab Reports min. Developed by: K. Moore, J. Giannini, K. Nordstrom & W. Losert (Univ. of Maryland, College Park) Page 6