Bio-Rad Rotofor and Mini Rotofor Cells User Manual

Rotofor®System

Instruction Manual

For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

Table of Contents

Page

Section 1 General Information ......................................................................1

1.1 Introduction................................................................................................1

1.2 Specifications ............................................................................................2

1.3 Isoelectric Focusing ...................................................................................3

1.4 Safety ........................................................................................................4

Section 2 Description of Major Components ...............................................5

Section 3 Setting Up For A Run ....................................................................6

3.1 Equilibration of the Ion Exchange Membranes ...........................................6

3.2 Assemble the Electrodes ...........................................................................7

3.3 Assemble the Focusing Chamber ..............................................................9

3.4 Prepare the Focusing Chamber ...............................................................10

3.5 Load the Sample......................................................................................10

3.6 Seal the Loading Ports.............................................................................10

3.7 Remove Air Bubbles ................................................................................11

Section 4 Running Conditions ....................................................................11

4.1 Starting the Fractionation .........................................................................11

4.2 Power Supply ..........................................................................................12

4.3 Fraction Collection ...................................................................................13

4.4 Refractionation.........................................................................................13

4.5 Final Purification ......................................................................................14

Section 5 Disassembly and Cleaning.........................................................14

Section 6 Sample Preparation.....................................................................15

6.1 Salt Concentration ...................................................................................15

6.2 Clarification..............................................................................................15

6.3 Solubility ..................................................................................................15

Section 7 Optimizing Fractionation ............................................................16

7.1 Ampholyte Choice....................................................................................16

7.2 Sample Capacity......................................................................................17

7.3 Power Conditions.....................................................................................17

7.4 Cooling ....................................................................................................17

7.5 Electrolytes ..............................................................................................18

7.6 Pre-running the Cell .................................................................................18

7.7 Prefocusing..............................................................................................18

7.8 Refractionation.........................................................................................19

Section 8 Analysis of Results .....................................................................19

8.1 Fraction Analysis .....................................................................................19

8.2 Separation of Ampholytes From Proteins.................................................19

Section 9 Troubleshooting Guide...............................................................20

9.1 Solubility and Precipitation of Proteins .....................................................20

9.2 Factors Affecting the pH Gradient ............................................................21

9.3 Recovery of Biological Activity .................................................................22

9.4 Maximizing Resolution .............................................................................23

9.5 Power Related Conditions........................................................................24

9.6 Uneven Harvesting ..................................................................................25

9.7 Mechanical Problems...............................................................................25

Section 10 Maintenance Guide .....................................................................26

10.1 Vent Buttons ............................................................................................26

10.2 O-rings.....................................................................................................26

10.3 Cooling Finger O-rings.............................................................................26

10.4 Membrane Core.......................................................................................26

Section 11 Rotofor References.....................................................................27

Section 12 Rotofor Application Notes ..........................................................38

Section 13 Application for Preparative Two Dimensional

Electrophoresis System .............................................................39

13.1 Introduction..............................................................................................40

13.2 Methods...................................................................................................41

13.3 Results ....................................................................................................44

Section 14 Product Information ....................................................................45

Note

To insure best performance from the Rotofor cell, become fully acquainted with these operating instructions before using the cell to separate samples. Bio-Rad recommends that you first read these instructions carefully. Then assemble and disassemble the cell completely.

Bio-Rad also recommends that all Rotofor cell components and accessories be cleaned with a suitable laboratory cleaner (such as Bio-Rad Cleaning Concentrate, catalog number 161-0722) and rinsed thoroughly with distilled water before use.

Warranty

Bio-Rad Laboratories warrants the Rotofor cell against defects in materials and workmanship for 1 year. If any defects occur in the instrument during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts free. The following defects, however, are specifically excluded:

1. Defects caused by improper operation.

2. Repair or modification done by anyone other than Bio-Rad Laboratories or an

authorized agent.

3. Use of fittings or other parts supplied by anyone other than Bio-Rad

Laboratories.

4. Damage caused by accident or misuse.

5. Damage caused by disaster.

6. Corrosion due to use of improper solvent or sample.

For any inquiry or request for repair service, contact Bio-Rad Laboratories. Be prepared to provide the model and serial number of your instrument.

Model

Catalog No.

Date of Delivery

Warranty Period

Serial No.

Invoice No.

Purchase Order No.

Section 1 General Information

1.1 Introduction

Bio-Rad’s unique Rotofor System fractionates complex protein samples in free solution using preparative isoelectric focusing. The Rotofor system is designed for the initial clean up of crude samples and for use in purification schemes for the elimination of specific contaminants from proteins of interest that might be difficult to remove by other means.

The Rotofor cell provides up to 500-fold purification for a particular molecule in less than 4 hours. Because electro-focusing is carried out in free solution, fractions from an initial run can be easily collected, pooled and refractionated, resulting in up to 1000-fold enrichment for a particular molecule. Purification using isoelectric focusing is especially advantageous when protein activity needs to be maintained. Bioactivity is maintained because the proteins remain in solution in their native conformation.

The Rotofor cell incorporates a cylindrical focusing chamber with an internal ceramic cooling finger. Rotation at 1 rpm around the focusing axis stabilizes against convective and gravitational disturbances. Nineteen parallel, monofilament polyester screens divide the focusing chamber into 20 compartments, each holding one fraction. After focusing, the solution in each compartment is rapidly collected without mixing using the harvesting apparatus supplied with the unit.

The Rotofor system is designed to accommodate a range of sample volumes using interchangeable focusing chambers. The Mini Rotofor chamber is used for sample volumes of 18 milliliters containing micrograms to milligrams of total protein. The large Rotofor chamber is used for samples of 35 to 60 milliliters containing milligrams to grams of total protein.

The Rotofor cell is used to purify a wide range of proteins. These include monoclonal antibodies, cell surface receptor proteins, integral membrane proteins, cytosolic and secreted enzymes, chemotactic factors, and recombinant proteins. It has been used to separate isoenzymes, lipoproteins, and apolipoproteins.

Should a final purification step be required, we recommend the Model 491 Prep Cell. The Prep Cell is a continuous elution gel electrophoresis device that uses SDS-PAGE or Native-PAGE to completely purify individual proteins of interest. For examples of published Rotofor cell applications, please refer to the Rotofor Technical Folder (request Bulletin 1555A).

*Patent No. 4,588,492

1.2 Specifications

Construction

Focusing chambers Acrylic Vent buttons Porous polytetrafluoroethylene (PTFE)

membrane in molded plastic

Gaskets Silicone rubber O-Rings Fluorocarbon elastomer Cooling finger Ceramic Housing Polycarbonate and acrylic Harvest box and lid Polycarbonate and acrylic Tubing Polyvinyl Needle array Stainless steel and acrylic Electrodes Platinum, 0.010 inch diameter Membrane Core Molded polyethylene with polyester mem-

branes

Chemical The Rotofor cell components are not com- compatibility patible with chlorinated hydrocarbons

(

e.g.

chloroform), aromatic hydrocarbons

(

e.g.

toluene, benzene), or acetone.

Use of organic solvents voids all warranties.

Shipping weight 9 kg Overall size 45.7 cm (L) x 16.5 cm (W) x 22.8 cm (H) Cell voltage limit 3000 VDC Cell power limit 15 W Cooling The Rotofor cell must be run with cooling or

excessive heating may occur, damaging the unit. A refrigerated circulating water bath is recommended to keep the coolant temperature at 4 °C.

Maximum coolant 12 L/minute flow rate Minimum coolant 50 ml/minute flow rate Sample volume 18–58 ml Electrical 3 wire cord connection Input power 120 V Model: 100-120 VAC, 50/60 Hz, 12W Requirements 240 V Model: 220-240 VAC, 50/60 Hz, 12W Fuses 250 mA Type T (1 required, 1 spare)

1.3 Isoelectric Focusing

Isoelectric focusing (IEF) is a gentle, non-denaturing technique; antibodies, antigens, and enzymes usually retain their biological activities. IEF is also a high resolution technique capable of resolving proteins that differ in pI by fractions of a pH unit. IEF in the Rotofor has the added advantage that the proteins can be easily recovered once they are focused.

Separation of proteins by isoelectric focusing is based on the fact that all proteins have a pH-dependent net charge. The net charge is determined both by the amino acid sequence of the protein and the pH of the environment. When a protein is electrophoresed through an established pH gradient, it will migrate until it reaches the pH where the net charge on the protein is zero; at that point it will stop migrating and is said to be focused at its isoelectric point or pI.

Ampholytes which are small, charged buffer molecules are used to establish the pH gradients increasing in pH from anode to cathode. When voltage is applied to a system of ampholytes and proteins, all the components migrate to their respective pIs. Ampholytes rapidly establish the pH gradient and maintain it for long periods allowing the slower moving proteins to focus.

A protein with a net positive charge, for example, in a particular region of the pH gradient will tend to migrate toward the cathode while concurrently giving up protons. At some point, the net charge on the molecule will be zero and the protein will cease to migrate. If the protein diffuses into a region of net charge, the resultant electrical force on it will drive it back to its pI, so that the molecule becomes focused at that point.

Fig. 1.1. Acidic Protein “Focusing” in a pH gradient.

ode

(+)

(

)

(+2)

(0)

(-2)

COOH

COO

4 5 6 7 8 9 1

net char

athod

1.4 Safety

This instrument is intended for laboratory use only.

This product conforms to the “Class A” standard for electromagnetic emissions intended for laboratory equipment applications. It is possible that emissions from this product may interfere with some sensitive appliances when placed nearby or in the same circuit as those applicances. The user should be aware of this potential and take appropriate measures to avoid interference.

Power to the Rotofor preparative IEF cell is to be supplied by an external DC voltage power supply. This power supply must employ a safety isolation transformer to isolate the DC voltage output with respect to ground. All of Bio-Rad’s power supplies meet this important safety requirement. Regardless of which power supply is used, the maximum specified operating parameters for the cell are:

3000 VDC maximum voltage limit 15 Watts maximum power limit 50 °C maximum ambient temperature limit

Current to the cell, provided by the external power supply, enters the unit through the lid assembly, providing a safety interlock. Current to the cell is broken when the lid is removed. Do not attempt to circumvent this safety interlock, and always turn the power supply off before removing the lid, or when working with the cell in any way.

Important: This Bio-Rad instrument is designed and certified to meet IEC1010-1* safety standards. Certified products are safe to use when operated in accordance with the instruction manual. This instrument should not be modified or altered in any way. Alteration of this instrument will:

• Void the manufacturer’s warranty

• Void the IEC1010-1 safety certification

• Create a potential safety hazard

Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not performed by Bio-Rad or any authorized agent.

*IEC1010-1 is an internationally accepted electrical safety standard for laboratory instruments.

Section 2 Description of Major Components

Fig. 2.1. Rotofor components. Harvesting apparatus (1), safety cover (2), housing (3), cooling finger (4), electrode assemblies (5), O-rings (6), ion exchange membranes (7), vent buttons (8), sealing tape (9), membrane core (10), focusing chamber (11), cell covers (12), test tube rack (13).

Focusing chambers - Two focusing chambers are available with the Rotofor

cell. The Mini focusing chamber holds 18 ml of sample and should be used for fractionating micrograms to milligrams of total protein. The Mini chamber is also ideal for refractionation. The standard chamber holds from 35 to 60 ml of sample and is used to fractionate milligrams to 3 grams of total protein. The focusing chambers are machined acrylic cylinders 120 mm long. Twenty evenly-spaced ports are bored in opposite sides for sample filling and collection.

Membrane core - The membrane core divides the focusing chamber into 20

compartments. The core assembly is a stack of 19 membrane units made from monofilament polyester screens of 10 µm nominal pore size. This assembly is inserted in the focusing chamber to stabilize the zones of focused proteins.

Electrode assemblies - There are two electrode assemblies. The assemblies

hold the cathode and anode electrolyte solutions and provide electrical contact between the focusing chamber and the power supply. They are not interchangeable; alignment pins prevent improper assembly. Ion exchange membranes, inserted in the assemblies, isolate the electrolytes from the sample in the focusing chamber while allowing establishment of an electrical field across the chamber. A plastic gear mounted on the cathode assembly engages the drive motor to rotate the focusing chamber.

Ion exchange membranes - Ion exchange membranes are used in the electrode

assemblies to separate the electrolytes from the sample while allowing current

flow. The anion-exchange membrane is notched to fit only the cathode assembly (black button) and the cation exchange membrane will fit only the anode assembly (red button).

Before the initial use, the membranes must be equilibrated overnight in the appropriate electrolyte. Once wetted they cannot be allowed to dry. If they dry out, membranes should be discarded. Membranes generally last 4–5 runs.

Anion Exchange Membranes are equilibrated in 0.1 M NaOH.

Cation Exchange Membranes are equilibrated in 0.1 M H3PO4.

Gaskets - Four grey colored silicone rubber gaskets are provided to seal the

ion exchange membranes within the electrode assemblies. These will fit either electrode assembly.

Vent buttons - Both electrode assemblies have filling ports. Vent caps containing integral, gas-permeable, PTFE membranes provide pressure relief form the gases which build up in the electrolyte chambers during the run. The vent buttons will fit either electrode assembly.

Housing - The stand supports the assembled focusing chamber during the run and houses the rotation motor. Focusing power is transmitted to the focusing chamber through brass contacts that are spring-loaded to maintain constant electrical contact between the focusing chamber and the housing. The assembled focusing chamber fits on the stand, with the anode (red) compartment to the left. If assembled correctly, the cathode electrode assembly will engage with the gear on the housing. If any connections are loose, the unit will not fit. Electrical contact to the case is through jacks on the safety cover. The safety cover must be in place for safe operation of the Rotofor cell.

Harvesting apparatus - A test tube rack which holds 20 test tubes (12 x 75 mm culture tubes) is enclosed in the harvesting box. This box has a fitting for connection to a vacuum source. House vacuum is usually sufficient for harvesting. Stainless steel tubes on the lid of the box are connected to an array of needles by flexible tubing. Individual fractions are collected through the tubing into the test tubes.

Cooling finger - The ceramic cooling finger extends through the focusing chamber and the electrode assemblies. The cooling finger is in contact with the sample and provides efficient heat dissipation up to 20 W.

Section 3 Setting Up For A Run

Assemble the anode and cathode electrolyte chambers first. Alignment pins prevent misassembly of the two electrodes. The anion-exchange membrane is notched to fit only the cathode compartment (black button) and the cation exchange membrane will fit only the anode assembly (red button). The four silicone rubber gaskets can be used in either electrode assembly. The procedure is identical for assembly of both the mini focusing chamber and standard focusing chamber.

3.1 Equilibration of the Ion Exchange Membranes

Ion exchange membranes are used in the Rotofor cell to separate the sample from the electrolyte while allowing current flow. The ion exchange membranes used in the Rotofor cell are of two types: cation exchanger and anion exchanger. The cation exchanger is negatively charged and repels negatively charged ions, preventing them from contaminating the anolyte. The anion exchanger works in the opposite way; it is positively charged and repels positive ions.

Using the ion exchange membranes gives a concentration gradient of the corresponding ions at the respective ends of the sample chamber. The highest concentration of negative ions will be next to the cation exchanger and the highest concentration of positive ions will be next to the anion exchanger.

Prior to assembly, the ion exchange membranes must be equilibrated overnight in the appropriate electrolyte solution. Ion exchange membranes are used for 4–5 runs prior to replacement.

Anion Exchange Membranes: These membranes are lighter in color than the cation exchange membranes when dry. The color of the two membranes is similar when wet. These membranes are equilibrated in 0.1 M NaOH. They are stored in distilled water or electrolyte between runs.

Cation Exchange Membranes: These membranes are darker colored than the anion exchange membranes when dry. These membranes are equilibrated in

0.1 M H3PO4. They are stored in distilled water or electrolyte between runs.

Note: The membranes can be stored indefinitely when dry. After rehydration, they must be kept moist. If the membranes dry out, they should be discarded.

3.2 Assemble the Electrodes

1. Examine the inner portion of an electrode assembly. For the Standard Rotofor

there should be a small O-ring in the central hole on the flat side, and a large

O-ring seated in the large groove around the central shaft on the other side. For

the mini chamber, the outer portion contains only one large O-ring. Place a gasket

over the alignment pins and seat it on the flat surface of the inner assembly. The

three oblong holes in the ion-exchange gaskets should align with the six holes

of the electrolyte chamber. When properly aligned, the gasket should not

obstruct the six holes in any way.

Fig. 3.1. Outer and inner portions of the electrode assemblies. Arrows indicate O-rings. Electrolyte buffer should just cover the central shaft when completely assembled. For the mini focusing chambers, the six holes in the inner portion of each electrode assembly are much smaller in diameter than six holes in the inner portion of the electrode assemblies used with the larger focusing chamber. In addition the six holes for the mini chamber are drilled at a distinct angle to the central axis of the assembly. These parts are not interchangeable!

Central shaft

Large o-ring

Inner portion

Outer portion

2. Place the proper ion exchange membrane on the gasket by aligning the notches

in the membrane around the pins, and complete a “sandwich” with a second gasket

on top of the membrane. The cathode holds one anion exchange membrane and

the anode holds one cation exchange membrane.

Fig. 3.2. Ion exchange membrane and gasket sandwich on inner portion of electrode assembly.

3. Make sure that there is a small O-ring inset in the central shaft of the large,

outer portion of the electrode assembly and fasten the halves together with the

captive, threaded sleeve.

4. Repeat the assembly process for the second electrode.

5. Fill the electrode chambers with electrolytes immediately after assembly to prevent

the membranes from drying. Filling is most easily accomplished with the assembled

focusing chamber mounted on its stand. The anode (+) electrode assembly (red

button), containing the cation exchange membrane, is filled with acidic electrolyte,

usually 0.1 M H3PO4. The cathode (–) electrode assembly (black button), containing

the anion exchange membrane, is filled with basic electrolyte, usually 0.1 M

NaOH. To fill the compartments, remove the vent buttons, add 25–30 ml of the

appropriate electrolyte to each chamber, so that the chambers are about 65% full,

and replace the buttons. The electrolyte should just barely cover the central shaft

of the chamber. Excessive electrolyte does not provide sufficient air space to allow

gases to escape. Pressure may build up inside the electrode assembly and cause

leaking from the vent buttons or ion exchange membranes.

The vent buttons are interchangeable and can be used with either electrode

assembly. The life of these buttons is usually 4–5 runs. After 4–5 runs, electrolyte

may begin to leak from the vent buttons during the run. If a vent button is

inadvertently perforated or, if during focusing an inordinate amount of electrolyte

leaks from the filling port, stop the run and replace the vent cap.

When the cell is used for the first time, the electrode assemblies will contain fresh

electrolyte. If the cell has been run previously, the distilled water or electrolyte

solutions must be left in the electrode assemblies between runs to maintain

hydration of the ion exchange membranes. Use fresh electrolytes for each run. If

the membranes are allowed to dry, they must be replaced. Empty the electrode

assemblies and fill with fresh electrolyte solution before each focusing run.

3.3 Assemble the Focusing Chamber

1. Slide the assembled anode electrode assembly over the ceramic cooling finger

so that the two protruding screw heads fit into the holes in the black plastic

base of the cooling finger support assembly.

Fig. 3.3. Anode electrode assembled on the cooling finger.

2. Slide the membrane core onto the ceramic cooling finger, making sure the core

abuts the acrylic ridge on the anode chamber.

3. Slide the focusing chamber over the membrane core, inserting the metal pin

into the small hole in the anode chamber. Position the focusing chamber so

that each membrane screen lies between two adjacent ports. These ports must

not be blocked by the membrane screens at either side, load or harvest. If the

ports are blocked, remove the focusing chamber, and slide it once more over

the membrane core. Tighten the black, nylon retaining screws. Check again to

make sure the membrane screens do not block the ports of the chamber.

Fig. 3.4. Slide the focusing chamber over the membrane core.

4. Slide the assembled cathode compartment over the cooling finger, aligning the

metal pin and hole in the cathode chamber, and tighten the nylon retaining

screws.

Fig. 3.5. Assembled focusing chamber.

5. Mount the assembled focusing chamber in the stand. The gear on the cathode

electrode assembly should be fully engaged with the gear on the stand. If the

focusing chamber does not slide in easily, remove it to check that all parts are

properly assembled.

6. Attach the power cord to the back of the unit and connect it to an electrical outlet.

3.4 Prepare the Focusing Chamber

With the cell mounted on the stand, rotate the focusing chamber so the 20 collection ports, identified by the two metal alignment pins, are facing up. Cover the ports with a piece of the sealing tape provided with the cell. Reinforce the taped ports with one of the two acrylic cell-cover blocks, and finger tighten the screws. We recommend pre-running the cell with pure water for the first use or after cleaning the components in the focusing chamber with NaOH. Pre-running the cell with water for 5 minutes at 5 watts constant power will remove residual ionic contaminants from the membrane core and ion exchange membranes before addition of the sample.

3.5 Load the Sample

Rotate the cell so the filling ports face up. This is easily accomplished by flipping the toggle switches to ON and HARVEST. In the harvest mode the focusing chamber will automatically stop with the filling ports facing up and the collection ports facing down. Fill the cell with sample through the ports using a 50 ml syringe with a 1-1/2 inch 19-gauge needle. Typically, every other port is filled, and the sample spreads into the adjoining compartments. For the large focusing chamber, the minimum sample volume must be sufficient to cover the cooling finger. For the mini focusing chamber load the maximum sample volume of 18 ml.

3.6 Seal the Loading Ports

A. Mini Rotofor chamber: Place the grey rectangular, silicone gasket in the slot

containing the loading ports then place the second cell cover block over the

gasket (tape is unnecessary), and the Rotofor cell is ready for operation.

B. Standard Rotofor chamber: Seal the filling ports with only the second cell

cover block (tape is unnecessary), and the Rotofor cell is ready for operation.

3.7 Remove Air Bubbles

During filling, air bubbles can become trapped in the 6 ports of the electrolyte chamber. This is especially true with the mini focusing chamber. If the bubbles are not removed, they will produce occasional fluctuations in the voltage and currents due to the discontinuity they create in the electrical field. Some power supplies, such as the Bio-Rad Power Pac 3000, have safety sensors that may trip and shut off the voltage in response to the resistance change that occurs when a bubble rotates into the electrical circuit. Thus, bubbles must be eliminated prior to commencing electrophoresis. Remove the assembled, loaded cell from the stand, turn it vertically and tap the electrode chamber to dislodge the bubbles. Then turn the cell 180° and tap the other chamber. If any air bubbles remain in the 6 ports between the sample and the ion exchange membranes, repeat this process. When all the bubbles are eliminated from the electrode ports, return the cell to the stand and start the fractionation.

Fig. 3.6. Loading the sample.

Section 4 Running Conditions

4.1 Starting the Fractionation

Excessive heating may denature proteins and damage the Rotofor cell. Connect the ports of the cooling finger to a source of recirculating coolant and begin coolant flow. The ports are interchangeable, so either one may be connected to the coolant inlet. It is usually sufficient to set the chiller at 4°C. For more critical temperature control, the chiller can be adjusted accordingly. At 12 W constant power (normal operating mode) the coolant temperature should be set at 10°C less than the temperature desired for the sample. In other words, if the coolant is -6°C than the sample temperature will be maintained at about 4°C. Attach the cover of the unit, mating its jacks to the plugs on the base. Allow the system to come to thermal equilibrium at the cooling temperature before beginning the run, approximately 10–15 minutes.

4.2 Power Supply

1. Never operate the Rotofor cell with the cover removed. When focusing power

is applied to the jacks without the cover in place, several high voltage elements

become exposed. To avoid personal injury due to accidental contact with these

elements, always operate the cell with the cover in place.

2. Attach the high voltage leads to the power supply, and the Rotofor cell is ready

for use. To begin rotation, flip the toggle switches to ON and RUN.

3. Power supply:

Standard Rotofor chamber - Set the supply to 15 W constant power and begin

the run.

Mini Rotofor chamber- Set the supply to 12 W constant power and begin the run.

The starting voltage and current will vary depending on the salt concentration of the

sample. For example, if the salt concentration of the sample is 10 mM, the starting

voltage will be 300–500 V, and the current will be 24–40 mA. The maximum power

that can be dissipated is about 15 W for an initial fractionation when the Rotofor cell

is operated at 4°C. If more than 15 W is applied to the cell, overheating can damage

the cell. The applied power is too high if the current increases or remains constant,

rather than decreases, during a run. If a constant power supply is not available,

check the graph in Figure 4.1 to determine the optimum starting voltage and

increase the voltage manually in increments over time. The voltage should be

increased as the run progresses to keep the power at a constant 12 W.

4. A typical run is completed in 3–5 hours. To monitor the progress of a run under

conditions of constant power, observe the voltage increase over time. The run

is complete when the voltage stabilizes. At that point, allow the run to continue

for 30 minutes before harvesting. The total run length should not exceed

6 hours. Longer run times do not tighten the focusing and may begin to break

down the gradient.

Fig. 4.1. The maximum power that should be applied to the Rotofor cell is 12-15 W. The graph shows the voltage and current readouts for setting a constant voltage power supply. If the current reading is too high at the set voltage (in the danger zone), reduce the voltage until a safe power level is obtained. Watts = voltage x current.

m Amps

12 W

2000

1500

1000

Volts

500

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