Beckman Coulter JCF-Z Instructions For Use Manual

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Page 1

Instructions For Use

Beckman Coulter, Inc. 250 S. Kraemer Blvd. Brea, CA 92821 U.S.A.

JCF-Z

Zonal and Continuous Flow Rotor

PN JCFZ-IM-12AB June 2019

Page 2

JCF-Z

Beckman Coulter Eurocenter S.A. 22, rue Juste-Olivier Case Postale 1044 CH - 1260 Nyon 1, Switzerland Tel: +41 (0) 22 365 36 11

Zonal and Continuous Flow Rotor

PN JCFZ-IM-12AB (June 2019)

Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.

All other trademarks, service marks, products, or services are trademarks or registered trademarks of their respective holders.

If you have any questions, contact our Customer Support Center.

• Worldwide, find us via our website at

www.beckman.com/support/technical.

• In the USA and Canada, call us at 1-800-369-0333.

• Outside of the USA and Canada, contact your local

Beckman Coulter Representative.

Find us on the World Wide Web at: www.beckman.com

May be covered by one or more pat. - see www.beckman.com/patents

Glossary of Symbols is available at beckman.com/ techdocs (PN C24689).

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Revision History

This document applies to the latest software listed and higher versions. When a subsequent software version changes the information in this document, a new issue will be released.

Initial Issue, 02/08

Issue AA, 03/14

Updates were made to the document format. Changes were made to Specifications, Installing the Zonal Interlock Accessory, Run Procedure For Pelleting Sample (Continuous Flow Cores), Standard Zonal Core, and Zonal Bracket Kit Contents.

Issue AB, 06/19

Changes were made to CHAPTER 3, J2 and J6 Series Centrifuges, CHAPTER 6, Supply List.

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Revision History

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Safety Notice

Read all product manuals and consult with Beckman Coulter-trained personnel before attempting to operate instrument. Do not attempt to perform any procedure before carefully reading all instructions. Always follow product labeling and manufacturer’s recommendations. If in doubt as to how to proceed in any situation, contact your Beckman Coulter Representative.

Alerts for Danger, Warning, Caution, Important, and Note

WARNING

WARNING indicates a potentially hazardous situation which, if not avoided, could result in death or serious injury. May be used to indicate the possibility of erroneous data that could result in an incorrect diagnosis.

CAUTION

CAUTION indicates a potentially hazardous situation, which, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. May be used to indicate the possibility of erroneous data that could result in an incorrect diagnosis.

IMPORTANT

Following the advice in the Important adds benefit to the performance of a piece of equipment or to a process.

NOTE NOTE is used to call attention to notable information that should be followed during installation, use,

or servicing of this equipment.

This safety notice summarizes information basic to the safe operation of the rotor described in this manual. The international symbol displayed above is a reminder to the user that all safety instructions should be read and understood before installation, operation, maintenance, or repair of this equipment is attempted. When you see the symbol on other pages of this manual, pay special attention to the specific safety information presented. Observance of safety precautions will also help to avoid actions that could damage or adversely affect the performance of the rotor. This rotor was developed, manufactured, and tested for safety and reliability as part of a Beckman Coulter centrifuge/rotor system. Its safety or reliability cannot be assured if used in a centrifuge not of Beckman Coulter’s manufacture or in a Beckman Coulter centrifuge that has been modified without Beckman Coulter’s approval.

IMPORTANT is used for comments that add value to the step or procedure being performed.

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Safety Notice

Alerts for Danger, Warning, Caution, Important, and Note

Handle body fluids with care because they can transmit disease. No known test offers complete assurance that such fluids are free of micro-organisms. Some of the most virulent —Hepatitis (B and C)) viruses, HIV (I–V, atypical mycobacteria, and certain systemic fungi—further emphasize the need for aerosol protection. Handle other infectious samples according to good laboratory procedures and methods to prevent spread of disease. Because spills may generate aerosols, observe proper safety precautions for aerosol containment. Do not run toxic, pathogenic, or radioactive materials in a rotor without taking appropriate safety precautions. Biosafe containment should be used when Risk Group II materials (as identified in the World Health Organization Laboratory Biosafety Manual) are handled; materials of a higher group require more than one level of protection.

The rotor and accessories are not designed for use with materials capable of developing flammable or explosive vapors. Such materials (such as chloroform or ethyl alcohol) should not be centrifuged in or handled near the centrifuge.

Always connect a pressure gauge between the pump and the rotor. If pressure exceeds 25 psi during acceleration or rises gradually during centrifugation, there is an air block in the system.

Always turn the pump off before clamping a line.

Always keep two lines to the rotor unclamped when the pump is on.

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Safety Notice

Alerts for Danger, Warning, Caution, Important, and Note

WARNING

The processing of large volumes of pathogenic materials in the JCF-Z rotors can produce extremely potent concentrations. Agents that are relatively harmless in their naturally occurring dilute state may become infectious when concentrated. Biological hazards must be carefully assessed and precautions taken. Consideration must be given to the air flow in the room; consult your laboratory safety officer.

WARNING

Protect your hands when handling bowl and lid threads, which become very sharp with prolonged use. Do not touch the rotor while the centrifuge is operating.

WARNING

Never attempt to block the open end of any pressurized tubing or coupling with the figures or hand, as injection of liquid under the skin could occur.

CAUTION

The rotor lid is designed to have liquid support. Never run an empty or partially empty rotor to full speed. Do not run the rotor above 10 000 rpm unless it is completely full of liquid.

CAUTION

The seal assembly (used with the standard zonal and all continuous flow cores) requires liquid lubrication for cooling. Do not stop liquid flow into the rotor for longer than 2 minutes during centrifugation. Do not run the seal assembly above 3000 rpm without fluid.

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Safety Notice

Alerts for Danger, Warning, Caution, Important, and Note

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Revision History,iii

Safety Notice,v

Alerts for Danger, Warning, Caution, Important, and Note,v

CHAPTER 1: The Process of Continuous Flow and Zonal Centrifugation,1-1

Continuous Flow Centrifugation,1-1

ZONAL CENTRIFUGATION,1-3

CHAPTER 2: Description,2-1

Rotor Components,2-1

Specifications,2-6

CHAPTER 3: Assembly and Installation,3-1

Installing the Zonal Bracket (Avanti J Series Centrifuges Only),3-1

Installing the Zonal Interlock Accessory,3-1

Tools and Supplies,3-1

Tools Provided,3-1 Supplies Required But Not Provided,3-2

Assembling the Rotor,3-3

Step 1,3-3

Step 1a,3-3 Step 1b,3-4 Step 1c,3-4

Step 1d,3-5 Step 2,3-6 Step 3,3-7 Step 4,3-8 Step 5,3-9 Step 6,3-10

Step 6a,3-10

Step 6b,3-12 Step 7,3-13

Standard zonal and continuous flow rotors.,3-13

Standard Seal Housing,3-13

High Flow Seal Housing,3-14 Step 8,3-14

Standard zonal and continuous flow rotors,3-14

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Contents

Step 9,3-15

Step 9a,3-15 Step 9b,3-16

Step 10,3-17 Step 11,3-18 Step 12,3-18 Step 13,3-19

CHAPTER 4: Preparing for Operation,4-1

Determining the Rotor Speed,4-1

Determining The Flow Rate: Large And Standard Pellet Cores,4-2

Setting The Temperature,4-5

Avanti Series Centrifuges,4-5 Microprocessor-controlled J2 And J6 Series Centrifuges (Models J2-

MI, J6-MI, J2-21M, J2-MC, J6-MC, and J2-21 M/E),4-5

Analog J2 And J6 Series Centrifuges (Models J2-HS, J2-21, J2-HC, J6-

HC, and J-6B),4-6

Determining The Liquid Pressure,4-6

Determining Appropriate Gradients,4-6

CHAPTER 5: Operation,5-1

Operating Precautions,5-1

Continuous Flow Operation,5-2

Pelleting Technique,5-2 Run Procedure For Pelleting Sample (Continuous Flow Cores),5-3

Purging Air From The System (Continuous Flow Rotor Only),5-5

Density Gradient Operation (Continuous Flow Cores),5-6

Gradient Or Cushion Separation Techniques,5-6

Forming an Air Block,5-7 Run Procedure for Separating in a Gradient or Cushion

(Continuous Flow Cores),5-9

Zonal Operating Techniques And Run Procedures,5-9

Standard Zonal Core,5-9 Reorienting Gradient (Reograd) Zonal Core,5-13

Ending The Run,5-14

Continuous Flow Core Containing Pellet,5-14 Continuous Flow Core Containing Gradient or Cushion,5-18 Standard Zonal Core,5-19 Reograd Zonal Core,5-20 Flushing The Tubing Lines,5-21

CHAPTER 6: Care and Maintenance,6-1

Maintenance,6-1

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Cleaning,6-1

Decontamination,6-2

Sterilization And Disinfection,6-3

Inspecting The Bearings,6-3

Smoothing (Lapping) The Rotating Seal,6-4

Returning a Rotor,6-4

Supply List,6-5

CHAPTER 7: Troubleshooting,7-1

Leakage,7-1

Rotating Seal/Stationary Seal Interface,7-1 Scavenger Line,7-1 Other Areas,7-1

Mixing of Inlet and Outlet Liquid,7-1

Contents

Vibration,7-2

APPENDIX A: Assembling the Rotating Seal,A-1

Assembling the Rotating Seal,A-1

APPENDIX B: Using the JCF-Z Continuous Flow/Zonal

Rotor Bracket (Avanti J Series Centrifuges Only),B-1

Description,B-1

Zonal Bracket Kit Contents,B-2

Assembly Instructions,B-3

Step 1. Install the Rotor,B-3 Step 2. Attach the Zonal Bracket to the Centrifuge Chamber

Wall,B-3 Step 3. Insert the Tubing Through the Door Gasket,B-3

Size 16 Tubing,B-3

Sizes 14 and 15 Tubing,B-4 Step 4. Attach the Tubing to the Zonal Bracket,B-5 Step 5. Attach Tubing to the JCF-Z Seal Assembly Housing,B-6 Step 6. Attach a Scavenger Line (Optional),B-7

JCF-Z Rotor Warranty And Seal Assembly Warranty

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Illustrations

1.1 Cross Section of a Continuous Flow Rotor,1-2

1.2 Cross Section of Zonal Rotor with Standard Zonal Core,1-3

2.1 Rotor Components.,2-2

2.2 Seal and Transfer Assemblies,2-3

2.3 Identifying O-Rings. O-rings requiring occasional replacement are shown, actual size.,2-5

3.1 Tools Provided in Tool Kit 335143.,3-2

4.1 Speed and Flow Rate Nomogram, Standard Pellet Core.,4-4

4.2 Radius Versus Volume for Standard Pellet Core, Standard Zonal Core, and Reograd Core.,4-7

5.1 Pelleting Technique, Continuous Flow Cores,5-2

5.2 Rotor Installation,5-3

5.3 Line Connections, Continuous Flow Cores. Using this tubing arrangement for basic pelleting against the rotor wall.,5-4

5.4 Gradient or Cushion Separation Technique, Continuous Flow Cores. Technique B, described in Table 5.1, is shown.,5-8

5.5 Sucrose Gradient after One Hour, Monitored by Refractometer. Diffusion between the discontinuous layers of the step gradient results in a near-linear gradient.,5-9

5.6 Line Connections, Standard Zonal Core,5-10

5.7 Operating Procedure, Standard Zonal Core,5-11

5.8 Removing and Reinserting Fittings and Liquid Lines,5-12

5.9 Line Connections, Reograd Zonal Core,5-13

5.10 Operating Procedure, Reograd Zonal Core,5-15

5.11 Lowering Rotor onto Plug. Rotor contents will spill out as soon as the core is removed if the plug and plug O-ring are not in place.,5-16

5.12 Disassembling the Rotor. Disassemble as shown to remove sample (arrows show direction of tool use). See CHAPTER 6, Care and Maintenance for instructions on inspecting, cleaning, and replacing the bearings.,5-17

5.13 Removing Small Pellet. Lift out canoe-shaped pellet container and scrape out pellet.,5-18

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Illustrations

A.1 Transfer Tube Assembly Components,A-1 A.2 O-ring (870656) and Silicone Vacuum Grease (335148),A-2 A.3 Fully Assembled Transfer Tube Assembly,A-3 A.4 Transfer Tube Assembly and Rotating Seal

(Ready for Assembly into Rotor),A-3 A.5 Transfer Tube Beveled End,A-4 A.6 Correct Position of the Rotating Seal,A-4 A.7 Incorrect Position of the Rotating Seal (Wrong Spring

Tension),A-5 A.8 Bearing Housing Assembled,A-5 A.9 Stationary Seal Housing Mated to Bearing Housing

(Seal/Bearing Housing),A-6 A.10 Pushing Down on Seal/Bearing Assembly to Tension Spring,A-7 A.11 Finishing Rotor Assembly,A-7 B.1 Avanti J Series Centrifuge Chamber with JCF-Z Rotor and Zonal

Bracket Installed,B-1 B.2 Attaching the Bracket to the Chamber Wall,B-3 B.3 Inserting Size 16 Tubing through the Gasket,B-4 B.4 Attaching Tubing to the Adapter,B-5 B.5 Inserting the Standard-Flow Feed Fitting into the Tubing,B-5 B.6 Attaching the Tubing Clamps to the Bracket,B-6 B.7 Inserting Tubing and Feed Fittings into the JCF-Z Seal

Assembly,B-6 B.8 Attaching a Scavenger Line,B-8

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Tables

1.1 JCF-Z Continuous Flow Applications,1-2

1.2 JCF-Z Zonal Applications,1-4

2.1 Specifications,2-6

2.2 Different Core Specifications,2-7

4.1 J2 and J6 Series Centrifuge Temperature Settings. All settings are approximate.,4-6

5.1 Two Techniques for Forming an Air Block During Density Gradient Operation,5-7

6.1 Supply List,6-5

B.1 Basic Kit 363843 or 366431,B-2 B.2 Kit 363844 Contents (for Size 14 Tubing),B-2 B.3 Kit 363845 Contents (for Size 15 Tubing),B-2

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The Process of Continuous Flow and Zonal

The JCF-Z rotor, which can be used in Beckman Coulter J2, J6, and Avanti J series centrifuges, has interchangeable cores that enable it to perform both continuous flow and zonal centrifugation. This section describes these processes and their applications.

Continuous Flow Centrifugation

In continuous flow centrifugation, solutions are pumped through the rotor continuously while it is spinning in the centrifuge (Figure 1.1). Particles sediment out of the flowing stream, which then emerges from the rotor as a particle-depleted effluent. This process continues until the particlecontaining capacity of the rotor is reached, or until the starting material (sample) is completely processed. Thus the amount of sample that can be processed in one run is determined by the sample’s volume and concentration of particles.

CHAPTER 1

Centrifugation

Continuous flow processing in the JCF-Z rotor allows efficient processing (pelleting, purification, or concentration) of large sample volumes containing particles with 600 S or higher sedimentation

coefficients large subcellular particles such as membranes, and mitochondria. It is also frequently used to clear virus-containing culture media of cellular debris before final isolation of the virus.

(Table 1.1). Continuous flow processing is suitable for sedimenting bacteria, yeast,

PN JCFZ-IM-12AB

* The sedimentation coefficient is usually expressed in Svedberg units (S), where 1 S = 1 ´ 10-13 seconds.

1-1

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The Process of Continuous Flow and Zonal Centrifugation

Continuous Flow Centrifugation

Figure 1.1 Cross Section of a Continuous Flow Rotor

1. Edgeline Outlet

2. To Center Inlet

3. Rotating Seal Assembly

4. Lid

5. Milled Slot

7. Bowl Wall

8. Core Taper Volume

9. Core

10. Channel Leading to Central Passage

11. Bowl

6. Upper Radial Channel

Particles may be concentrated in one of three ways: by pelleting onto the wall of the rotor bowl, by sedimenting onto a cushion of dense liquid such as concentrated sucrose, or by banding in a gradient. Use of a cushion gradient is appropriate if your solution contains biologically active particles that could be damaged by pelleting against the rotor wall. Banding in a gradient is appropriate when it is necessary to separate a certain particle from contaminants of different sedimentation velocities in the solution.

Table 1.1 JCF-Z Continuous Flow Applications

Core Type Typical Sample Type

Large pellet

large viruses (s 600 S),

pellet, concentrate 400 to 800 mitochondria, tissue homogenates, bacteria

Standard pellet large viruses,

mitochondria,

pellet, purify, or

concentrate tissue homogenates, bacteria

Small pellet mitochondria, bacteria, algae,

pellet 50 to 200 clay

a. Pellet refers to sedimenting particles onto the rotor wall. Concentrate refers to sedimenting particles onto a cushion of dense

solution (e.g., sucrose). Purify refers to sedimenting particles through a step gradient.

Result

Recommended Pellet

Size Range (mL)

200 to 400

1-2

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The Process of Continuous Flow and Zonal Centrifugation

ZONAL CENTRIFUGATION

The amount of pellet anticipated or the necessity to sediment onto a cushion or into a step gradient determines which of the three available cores is appropriate for your application.

• The

standard core rotor configuration has a total capacity of 660 mL. Of this volume, about 400

mL can be occupie d by pellet, step gradient, or cushion. This co re is suitable if no more than 400 mL of pellet is anticipated.

large pellet core rotor configuration has a total capacity of 1250 mL, of which

approximately 800 mL can be occupied by pellet. This core is well suited for centrifuging fluids with high solid-to-liquid ratios, when a large pellet is expected. Note that when processing a solution of high solid-to-liquid ratio, the core fills with particles more quickly and must be unloaded more often than during processing of a less concentrated solution.

small pellet core rotor configuration has a total capacity of 240 mL, of which about 200 mL

can be occupied by pellet. It is designed for processing large amounts of material with a low solid-to-liquid ratio. In order to minimize resuspension of sedimented materials and to facilitate sample recovery, this core has six individual cavities, each holding removable containers in which the pellets are collected.

ZONAL CENTRIFUGATION

In density gradient separation, sample capacity is limited by the area in the sample cavity over which the sample spreads. The only practical way to significantly increase the sample capacity is to increase the area of the sample cavity. When density gradient separation is performed in a zonal rotor, gradient and sample are loaded directly into the rotor cavity; no tubes or bottles are used (Figure 1.2). Cylindrical or bowl-shaped zonal rotors have a capacity 50 to 100 times that of the typical swinging bucket rotor; thus zonal centrifugation enables large quantities of sample to be processed in much less time than in other types of rotors.

In the JCF-Z rotor, the zonal technique is useful in separating large particles, such as mammalian viruses and subcellular organelles, from plant or animal tissue homogenates (see Table 1.2).

Figure 1.2 Cross Section of Zonal Rotor with Standard Zonal Core

1. Rotating Seal Assembly

2. Lid

3. Core

4. Bowl

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1-3

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The Process of Continuous Flow and Zonal Centrifugation

ZONAL CENTRIFUGATION

Two zonal cores are available with the JCF-Z: standard and reorienting gradient (also known as reograd). These cores are divided into sector-shaped compartments by vanes attached to the core; the rotor is enclosed by a threaded lid. A rotating seal assembly allows fluid to be pumped in and out of the standard zonal core cavity (for loading and unloading only) while the rotor is spinning.

Table 1.2 JCF-Z Zonal Applications

Core Type Typical Sample Type

Standard zonal tissue homogenates, small or

large viruses, chloroplasts, bacteria, algae, whole cells

Reorienting gradient (reograd) zonal

a. Density gradient may refer either to sedimentation velocity (rate zonal) centrifugation or to equilibrium sedimentation

(isopycnic banding) centrifugation. Concentrate refers to sedimenting particles onto a cushion of dense solution (e.g, sucrose).

• The

standard zonal core has a capacity of 1900 mL. When this core is used, the rotor is loaded

tissue homogenates, small or large viruses, chloroplasts, bacteria, algae, whole cells, and especially fragile material

density gradient

concentrate

density gradient

concentrate

Result

Recommended Sample

Volume (mL)

59 to 500 (1400 to 1850 mL is gradient)

1650 (250 mL is cushion)

50 to 500 (1250 to 1700 mL is gradient)

1500 (250 mL is cushion)

and unloaded at a speed of 2000 rpm. Gradient solution is pumped in at the periphery of the rotor; the sample and overlay solutions are pumped in through the center. After centrifugation, the rotor contents are unloaded by introducing heavy displacement fluid at the periphery and collecting fractions at the center.

• The

reograd zonal core has a capacity of 1750 mL. When this core is used, the rotor is loaded and

unloaded at rest. To prevent mixing of the rotor contents, slow acceleration to and deceleration from 1000 rpm is recommended. This core is recommended for use with long-stranded, fragile materials that might be damaged by passing through a rotating seal assembly.

1-4

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This section describes the JCF-Z rotor components and lists rotor specifications.

Rotor Components

The basic components of the JCF-Z rotor are the rotor bowl and lid, the rotor core, the transfer tube, and the rotating seal assemblies. The cores are interchangeable. Continuous flow cores are available in the following sizes: standard pellet, large pellet, and small pellet. Two types of zonal cores are available: standard and reorienting gradient (also known as reograd). Each core, except the continuous flow small pellet, has four baffles dividing the rotor interior into four quadrants (the small pellet core has six cavities). Baffles in continuous flow cores are removable; in zonal cores they are not. All of the cores have holes drilled through them to enable fluid to flow into or out of the rotor. The transfer tube separates the sample and effluent channels of fluid flow and transfers these liquids into and out of the rotor.

CHAPTER 2

Description

The rotor bowl and lid are made of titanium; the rotor cores are made of Noryl plastic. (Components and materials used in the rotor assemblies are detailed in Figure 2.1 and Figure 2.2) Tygon tubing feed lines attach to the rotor for fluid introduction and removal. A drive pin in each core seats it into the centrifuge drive hub to prevent core slippage during acceleration. Depending on which core is used, the fully loaded JCF-Z rotor weighs 12 to 14 kg (26 to 31 lb). The seal assembly (except bearings and rotating seal) is warranted for 1 year; other rotor components are warranted for 7 years. See the Warranty for details.

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2-1

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2-2

Figure 2.1 Rotor Components.

Description

Rotor Components

PN JCFZ-IM-12AB

The seal and transfer assemblies are described in Figure 2.2. Letters indicate O-rings made of Buna N or of Viton. Buna N: (a) 815473, (d) 870315, (e) 870688, (f) 854519 or 870528 (Viton), and (g) 812715. Viton: (b) 870526 x 4 and (c) 817033. See also Figure 2.3.

1. Seal Assembly (Standard 335142; high flow

335134)

2. Transfer Assembly (part of 350585)

3. Lid (336282)

4. Small Pellet Core (350601)

5. Standard Core (335130)

6. Large Pellet Core (350641)

7. Standard Zonal Core

(335141)

8. Reorienting Gradient Core (350585)

9. (354006)

10. (354005)

11. (354005)

12. Continuous Flow

Rotors

13. Zonal Rotors

Page 21

Figure 2.2 Seal and Transfer Assemblies

Description

Rotor Components

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2-3

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Description

Rotor Components

1. Standard Flow (SF) Seal Housing

2. Feed Fitting (Standard Flow) (335146)

3. Feed Fitting O-ring (824464)

4. Manifold

5. Fitting Retaining Clamp (344108)

6. Manifold Screw (870690)

7. Washer (853118)

8. Standard Flow Housing (336080)

9. Scavenger Drain Tube (336286)

10.High Flow (HF) Seal Housing Assembly

(335133)

11.Manifold O-ring (870655)

12.Manifold O-ring (868638)

13.Stationary Seal (335145)

14.Rotating Seal (335144)

15.Rotating Seal O-ring (824412)

16.Rotating Seal O-ring

(870656; in bottom of seal)

17.Sample Transfer Tube

19. Spring (870654))

20.Retaining Ring (870689)

21. Flat Washer (853391)

22.Retaining Seal Mount (336284)

23.Fitting Retaining Clamp (344108)

24.Retaining Ring (870689)

25.Feed Fitting O-ring (824464)

26.Feed Fitting (Std. Flow)(335146)

27.Housing

28.Transfer Tube Assy O-ring (870656)

29.Retaining Ring (870689)

30.Transfer Assembly (reograd only)

31. Seal Assembly (all other cores)

32.Bearing Housing (336079)

33.Flat Washer (878279, nylon)

34.Bearings screw (804063)

35.Spring Washer (870658)

36.Bearing (366190)

37.Bearing Spacer (336285)

(335120 [SF] or 335132 [HF]; and 306653)

18.Transfer Tube Assy O-ring (870656)

a. Standard-Flow Manifold consists of part numbers 870690, 853118, 336080, 870655, 868638, and 335145. High-Flow

Manifold is part of High-Flow Seal Housing Assembly, which consists of part numbers 870690, 853118, 338672, 2 x 335136, and 336286.

2-4

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Figure 2.3 Identifying O-Rings. O-rings requiring occasional replacement are shown, actual size.

Description

Rotor Components

1. Edge of Rotor Bowl (seals lid) (870688)

2. Bottom of Rotor Bowl (854519 or 870528 [Viton])

3. Zonal Core (870315)

4. Continuous Flow Core (817033)

5. Rotor Plug (812715)

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6. Rotating Seal (824412)

7. Rotor Lid Stem (815473)

8. Transfer Tube Assembly and Bottom of Rotating Seal (870656)

9. Large Pellet Core Vane (870526)

10. Seal Housing Fitting (824464)

2-5

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Description

maximum speed

1.45g mL⁄

maximum density at rotor wall

--------------------------------------------- -------------------------- ----- 20 000 rpm×=

Specifications

Only values with tolerances or limits are guaranteed data. Values without tolerances are informative data, without guarantee.

Table 2.1 Specifications

Maximum rated speed

Minimum speed

NOTE Necessary for adequate vertical positioning of the rotor contents,

except when the reograd core is used. The reograd core is loaded and unloaded while the rotor is at rest and is accelerated and decelerated slowly to prevent mixing of the contents.

Materials

rotor bowl and lid

rotor cores

Maximum permissible average density of rotor contents at maximum speed

NOTE When liquids of higher average density than this are centrifuged, the

maximum rotor speed must be reduced. Calculate the speed reduction as follows:

Speed reductions may also be required when sedimenting unusually heavy pellets, such as liquids containing metal or clay particles.

Maximum continuous flow rates:

with standard-flow (SF) seal assembly

with high-flow (HF) seal assembly

Allowable pH range for liquids

20 000 rpm

2 000 rpm

titanium

Noryl plastic

1.45 g/mL

45 liters/hour

100 liters/hour

pH 4 to 10

Acceleration/deceleration time

Maximum gauge pressure tolerated by the rotating seal

Vacuum trap for scavenger line

J2 and J6 series centrifuges

Avanti J series centrifuges

Rotor code (for microprocessor-controlled J2 and J6 series centrifuges) 20.9

a. Maximum pressure limit may be reached before maximum continuous flow rates are achieved at higher rotor speeds.

2-6

6 to 7 minutes each

30 psig (210 kPa)

5 psia (35 kPa)

3 psia (20 kPa)

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Table 2.2 Different Core Specifications

RCF

rω

---------=

RCF 1.12r

RPM 1000

-------------





Description

Specifications

Rotor

Type

Cont.

Flow

Core Type

large

pellet

standard

pellet

small

min

(mm)

top: 56

bottom: 51

top: 76

bottom: 71

max

(mm)

Max RCF

(x g)*

at r

max

39 900

36 300

Rotor

Capacity

(mL)

1250

660

240

Pellet

Capacity

(mL)

800

400†

204

k Factors

(outermost r

at 20 000 rpm)

293

100

281

min

pellet

Zonal

Standard

Zonal

reograd

top: 20

bottom: 29

39 900

39 000

1900

1750

filled rotor

—

710

779

1/2-filled

rotor‡

260

277

* Relative Centrifugal Field (RCF) is the ratio of the centrifugal acceleration at a specified radius and speed (rω 2) to the standard acceleration of gravity (g) according to the following formula:

where r is the radius in millimeters, ω is the angular velocity in radians per second (2 π RPM /60), and g is the standard acceleration of gravity (9807 mm/s

). After substitution:

†If you are using gradients rather than pelleting, about 500 mL of the capacity will be gradient.

‡If you are only partly filling the rotor with gradient, be sure to fill the balance of the rotor with plain buffer. Do not run an empty or partially empty rotor at full speed.

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Page 26

Description

Specifications

2-8

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Page 27

Assembly and Installation

This section describes JCF-Z components and accessories for both continuous flow and zonal operation, and the appropriate assembly and installation procedures for each.

Installing the Zonal Bracket (Avanti J Series Centrifuges Only)

When the JCF-Z rotor is used in any of the Avanti J series centrifuges, a zonal bracket must be installed. See APPENDIX B, Using the JCF-Z Continuous Flow/Zonal Rotor Bracket (Avanti J Series

Centrifuges Only) for complete information.

Installing the Zonal Interlock Accessory

CHAPTER 3

Before using the standard zonal or reograd core in a J2-HS, J2-21, J6-HC, or J-6B centrifuge, you must install a zonal key safety interlock (343348). Instructions for installation are shipped with the interlock.

Tools and Supplies

Tools Provided

A set of rotor assembly tools ships with the rotor (tool kit 335143). The components of this kit are shown in Figure 3.1 and their use is described in this section. Other supplies provided by Beckman Coulter are listed in CHAPTER 6, Care and Maintenance.

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Assembly and Installation

Tools and Supplies

Figure 3.1 Tools Provided in Tool Kit 335143.

1. Rotor vise (335127)

2. Plug (335149) and O-ring (812715)

3. Large spanner wrench (870667)

NOTE See Section 3 for complete instructions on when and how to use these tools.

Supplies Required But Not Provided

• Tubing connectors for 3/16-in. tubing

• Clamps or hemostats

• Auxiliary timer

• vacuum trap for scavenger line, 5 psia (35kPa)

• Pressure gauge

• Pump that meets the following requirements: (a) has a continuously adjustable flow rate from

10 mL to 1,000 mL/min for #15 tubing or 17 mL to 1,700 mL/min for #24 tubing; (b) has a flow rate controllable to within ±1.5%; (c) has a flow rate independent of back pressure up to 40 psi; and (d) has pulsation less than 5% of flow rate.

We recommend the Cole-Parmer Masterflex L/S pump head model EW-07518-12 (accepts L/S 15 and L/S 24 tubing), 10 ft (3 m) of Tygon LFL L/S 24 tubing part number EW-06440-24 (flow range of 28 to 1700 mL/min), and 10 to 600 rpm standard digital drive model EW-07523-40 (115 VAC) or EW-07523-47 (230 VAC).

4. Small spanner wrench (870666)

5. 3/16-in. T-bar hex wrench (001311)

6. Etched glass plate (870671)

3-2

Pump and pump head model numbers can change, so contact Cole-Parmer directly to receive current information before ordering. See the Cole Parmer website at www.coleparmer.com for additional information.

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Assembling the Rotor

Step 1

Assembly is much the same for continuous flow and zonal rotor configurations; assembly steps for both configurations are covered in steps 1 through 13 below. Some steps are for particular cores only and are so identified. Before beginning assembly, make sure that

• all parts are clean and dry, and

• the rotor vise is bolted to a sturdy table.

Step 1a

Large and Standard pellet— Put the rotor in the centrifuge. Install the four baffles in the core,

flat end up. and install the core O-ring (817033; see Figure 2.1 and Figure 2.3).

Large pellet only: lubricate and install the four O-rings (870526; see Figure 2.3). Lubricate

Assembly and Installation

Assembling the Rotor

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Assembly and Installation

Assembling the Rotor

Step 1b

Small pellet only— Install and align the canoe-shaped pellet containers.

NOTE

During the run, the small pellet containers may rotate slightly. This rotation can often be prevented by applying a small patch or two of silicone vacuum grease (335148) to the outside of the container before installing it in the core. Rotation may cause slightly less pellet to be retained, but any difference in pellet size from container to container is not sufficient to cause rotor imbalance.

Step 1c

Standard Zonal only— Lubricate and install the core O-ring (870315, shown in Figure 2.1).

3-4

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Assembly and Installation

Assembling the Rotor

Step 1d

Reorienting Gradient only— Fit the core and base together, aligning the pin in the core with the hole

in the base.

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Assembly and Installation

Assembling the Rotor

Step 2

Coat both of the bowl O-rings with silicone vacuum grease and install them in the rotor as shown: one in the bottom of the rotor and one around the upper edge. Set the rotor into the rotor vise (which has been bolted to a sturdy surface). Apply Spinkote lubricant to the metal threads around the outside of the bowl.

1. O-ring (870688)

2. O-ring (854519)

3. Vise

3-6

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Step 3

Assembly and Installation

Assembling the Rotor

Install the core in the rotor bowl. Note that the “up” side of each core has a small hole in the center. The “down” side has a large hole in the center with a metal drive pin in or across it.

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Assembly and Installation

Assembling the Rotor

Step 4

Screw the lid onto the rotor and tighten it with the large spanner wrench. Align index marks on the lid and rotor (the lid may be tightened past the bowl mark).

1. Large Spanner Wrench (870667)

2. Index Marks

3-8

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Step 5

Assembly and Installation

Assembling the Rotor

Coat the O-rings on the transfer tube or transfer tube assembly (reograd core) with silicone vacuum grease. Check the Tygon tubing on the transfer tube to be sure it is clean. Replace this tubing as necessary.

1. Transfer Tube Assembly

2. O-ring

3. Transfer Tube

4. Tygon Tubing (306653)

5. O-ring (870656)

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Assembly and Installation

Assembling the Rotor

Step 6

Step 6a

1. Spanner Wrench (870666)

2. Seal Mount (336284)

3. Bearing Assembly

4. O-ring (815473)

5. Bearing Housing (336079)

6. Nylon Washer (878279)

Standard zonal and continuous flow rotors. Assemble and align the bearing assembly as follows:

Unscrew the bearing housing with the T-bar hex wrench.

Loosen and remove the seal mount so that you can remove the bearings.

Inspect the bearings and replace them, if necessary (see CHAPTER 6, Inspecting The Bearings).

Place the two bearings, the spacer, and the spring washer on the rotor in the order shown in the illustration below. Install the bearing housing.

7. Bearing Screw (804063)

8. 3/16-in. T-bar Hex Wrench (001311)

9. Bearing (366190)

10. Spring Washer (870658)

11. Spacer (336285)

12. Bearing (366190)

3-10

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Assembly and Installation

Assembling the Rotor

Screw the seal mount into place counterclockwise (it has a left-hand thread) and tighten it with a spanner wrench (870666).

Tighten the bearing screw using T-bar hex wrench (001311).

CAUTION

To avoid damaging the bearings, never press on the bearing assembly with your hand when tightening the bearing screw.

Loosen the seal mount slightly, spin the assembly several times, and then retighten the seal mount firmly.

Loosen the bearing screw with the hex wrench and spin the assembly around again several times. Retighten. Do not press down on the housing while tightening it.

NOTE

Proper alignment of the bearing assembly is essential for good rotor performance.

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Assembly and Installation

Assembling the Rotor

Step 6b

Reograd zonal rotor

Coat the rotor lid stem O-ring with silicone vacuum grease and slip it onto the rotor lid shaft. Apply Spinkote lubricant to the threads of the shaft. Insert the transfer tube into the shaft and turn it counterclockwise onto the lid until it is firmly seated. Go to Step 9b.

1. O-ring (815473)

3-12

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Step 7

Assembly and Installation

Assembling the Rotor

Standard zonal and continuous flow rotors.

Before rotor assembly can be completed, the proper seal housing assembly should be selected and prepared. Your choice depends on the required flow rate: maximum rate for the standard assembly is 45 liters/hour, and for the high flow, 100 liters/hour. Except for the fitting retaining clamp, it is a permanent assembly and does not ordinarily require disassembly. (If it is damaged, replace with new parts. See Figure 2.2 standpipe. To effectively overcome the chamber vacuum, connect the standpipe to a 5-psia (35-kPa) vacuum trap (with J2 and J6 series centrifuges) or to a 3 psia (20-kPa) vacuum trap (with Avanti J series centrifuges). Lubricate the threads on the seal housing base with Spinkote.

footnote for part numbers.) Use a screwdriver to remove or install the

Standard Seal Housing

Standard Zonal

Rotate retainer to remove fittings before centrifugation or to attach after. Standpipe must be removed.

1. Fitting Retaining Clamp

2. Feed Fitting

3. Feed Fitting

Continuous Flow

Standpipe must be hooked up to a vacuum to keep bearings dry.

1. Standpipe

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Page 40

Assembly and Installation

Assembling the Rotor

High Flow Seal Housing

Continuous Flow

Standpipe must be hooked up to a vacuum to keep bearings dry.

1. Standpipe

Step 8

Standard zonal and continuous flow rotors

Check the rotating seal to be sure it is not damaged; the sealing surface should be smooth and shiny. Lubricate O-ring (824412) with silicone vacuum grease and put it around the rotating seal. Lubricate the two stationary seal O-rings (870656 and 868638) with silicone vacuum grease and place them in the inside grooves of the upper seal housing. The upper seal housing containing the stationary seal is then screwed to the left (counterclockwise) onto the lower bearing assembly.

1. Undamaged

2. O-ring (824412)

3-14

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Step 9

Assembly and Installation

Assembling the Rotor

Step 9a

Standard zonal and continuous flow rotors

If the seal assembly is already assembled and aligned, assemble the rotor as shown here. Be sure that all O-rings, as well as the threads and center shaft of the lid, are lightly coated with silicone vacuum grease. Use the small spanner wrench to tighten the seal mount counterclockwise onto the rotor.

NOTE

Proper assembly of the rotating seal is critical to successful rotor operation. See APPENDIX A,

Assembling the Rotating Seal for detailed instructions.

PN JCFZ-IM-12AB

1. Rotating Seal

and O-rings

2. Small Spanner Wrench

(870666)

3. Lid and Bowl

4. Seal Housing

(from Step 7)

5. Transfer Tube and O-ring

6. Spring

7. Metal Washer

8. Seal Mount

9. Bearing Assembly (from Step 6a)

10. O-ring

11. Vise

3-15

Page 42

Assembly and Installation

Assembling the Rotor

Step 9b

Reograd zonal rotor

Install the assembled rotor on the centrifuge drive hub.

CAUTION

The centrifuge drive hub can be bent if the rotor is dropped onto it or forced sideways. Install the rotor by centering it over the hub and carefully lowering it straight down.

3-16

1. Zonal Seal Cap (part of assembly 350585)

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Step 10

Assembly and Installation

Assembling the Rotor

Load the rotor with gradient and sample according to the appropriate procedure in CHAPTER 5,

Operation. Then unscrew the transfer tube assembly from the rotor and replace it with the zonal

seal cap.

1. Drive Hub

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Assembly and Installation

Assembling the Rotor

Step 11

Avanti J Series Centrifuges

Refer to APPENDIX B, Using the JCF-Z Continuous Flow/Zonal Rotor Bracket (Avanti J Series Centrifuges

Only) for complete information.

J2 and J6 Series Centrifuges

Remove the access panels on the sides of the centrifuge with a Phillips screwdriver. Remove the rubber stoppers from two or three portholes, as necessary.

Step 12

3-18

1. Rubber Stoppers

Avanti J Series

A zonal bracket is needed to support the tubing lines in Avanti J series centrifuges. Refer to

APPENDIX B, Using the JCF-Z Continuous Flow/Zonal Rotor Bracket (Avanti J Series Centrifuges Only) for

complete installation instructions.

J2 and J6 Series Centrifuges

Thread 1/8-in. inside-diameter Tygon tubing lines (144664) to the rotor through the ports as shown (J2-21 porthole configuration shown). Tubing lines should be long enough to reach all necessary pumps and facilities, but no longer than necessary.

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Assembly and Installation

Assembling the Rotor

Use the #1, one-hole rubber stoppers provided to hold the tubing in the ports as shown. Connectors for 3/16-in. tubing (for a tight fit) are needed on the lines as shown.

Step 13

1. This port not needed for JCF-Z use.

2. Tubing Connector

Assemble and install the pressure gauge between the pump and rotor.

3. Scavenger line (for continuous flow rotors only)

4. Vacuum (psia)

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Page 46

Assembly and Installation

Assembling the Rotor

3-20

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Page 47

Set system variables according to the parameters presented in this section.

RPM 20 000

1.45 g/mL

density of contents

------------------------------------------ ----=

Determining the Rotor Speed

The maximum allowable run speed for the JCF-Z rotor is 20 000 rpm, as long as the mean liquid density in t he rotor is 1 .45 g/mL or l ess. If the m ean solution density in the rotor is greater than 1.45 g/mL, the maximum allowable rotor speed must be reduced according to equation (1):

Equation (1)

Whether particles of a certain size (and all larger particles) are retained in the rotor or not depends on a combination of flow rate and rotor speed. The speed determined with equation (1) is the maximum speed at which the rotor may be run, and should not be exceeded. In addition, the rotor should not be used at speeds slower than 2000 rpm (the minimum speed required for adequate vertical positioning of the rotor contents).

CHAPTER 4

Preparing for Operation

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4-1

Page 48

Preparing for Operation

F πshω

max

–

1n r

maxri

⁄()

------------------------------







rtrb–

1n r

trb

⁄()

------------------------=

Determining The Flow Rate: Large And Standard Pellet Cores

The equations below may be used to determine approximate flow rate F or rotor speed in RPM for specific samples. Figure 4.1 has been generated from equation (3a) for the standard pellet core.

Equation (2)

whereπ = 3.412 h (height) = 8.2 cm ω = 0.10472 x RPM

= 8.9 cm

max

For the standard pellet core r

= 7.6 cm, and rb = 7.1 cm, reducing ri to 7.35 cm and Equation (2) to

Equation (3a):

Equation (3a)

F = 2.23 x 10

-10

srRPM

For the large pellet core, rt = 5.6 cm and rb = 5.1 cm. Thus ri becomes 5.35 cm and Equation (2) reduces to Equation (3b):

Equation (3b)

F = 1.69 x 10

-10

srRPM

F is in milliliters per minute; sr, in Svedberg units, is the sedimentation coefficient adjusted for run conditions. Equation (3a) is illustrated in Figure 4.1for ready calculation of flow rate in the standard

pellet core.

NOTE

The above equations assume that the density and viscosity of the liquid in which the particles are suspended are similar to water at 20°C. If this is not so, an adjusted sedimentation coefficient (sr) should be calculated.

4-2

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Preparing for Operation

D2ρpρr–()

18η

------------------------------=

srs

20 w,

ρpρr–

pρ20 w,

–

--------------------------





20 w,

--------------





Determining The Flow Rate: Large And Standard Pellet Cores

Before you use the nomogram or equation (3), however, sr must be calculated from the known sedimentation coefficient or the known diameter of the particle of interest. If the diameter (D) is

known, use equation (4):

Equation (4)

whereρp = density of the particles in g/mL ρ

= density of the liquid containing your particles in g/mL

= viscosity of the liquid in mPa•s or in cp

If the sedimentation coefficient (for example, s

) of your particle is known, it may be used —once

20,w

adjusted to the conditions of your run—to calculate sr.

Equation (5)

Equation (5) is used to adapt any known sedimentation coefficient to the conditions of your centrifuge run. Substitute the known values for the “20,w” variables in the equation.

Once s

is known, it may be used in Figure 4.1 to determine flow rate.

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Preparing for Operation

Determining The Flow Rate: Large And Standard Pellet Cores

Figure 4.1 Speed and Flow Rate Nomogram, Standard Pellet Core.

4-4

Connect two known scale values with a straight-edge. Read the desired information where it intersects with the third scale. Values vary with viscosity and density: use equation (4) or (5) to determine sr before using this figure to estimate flow rate with the standard core.

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Setting The Temperature

To ensure that the rotor reaches the required temperature during the run, follow the appropriate instructions below for the model of J centrifuge being used.

Precooling the rotor is recommended whenever temperature control of the sample is critical. Place the rotor, with the core installed, in the centrifuge and run it at 3000 rpm for 30 minutes. For J2 and J6 series centrifuges, use the temperature and compensation settings shown in Table 4.1. At the end of this time, remove the rotor, complete the assembly procedures, and return it to the centrifuge.

Gradient material should also be precooled if the sample is temperature sensitive.

Temperature control will also depend on ambient conditions when the JCF-Z is run above 18 000 rpm. The lowest temperatures which the rotor can be controlled are about 5°C at 19 000 rpm and 15°C at 20 000 rpm.

Avanti Series Centrifuges

Preparing for Operation

Setting The Temperature

Enter the run temperature according to the instructions in your centrifuge instruction manual. No additional input is required.

Microprocessor-controlled J2 And J6 Series Centrifuges (Models J2-MI, J6-MI, J2-21M, J2-MC, J6-MC, and J2-21 M/E)

Enter rotor code 20.9, then follow the steps below.

Press the sample temperature.

Find the compensation value in Table 4.1 that corresponds with the set temperature and run speed.

NOTE

Press flashes.

TEMP

key on the centrifuge control panel and then use the keypad to enter the

(

)

The temperature compensation settings in Table 4.1 are based on zonal operation. For continuous-flow operation at low flow rates, add one to two units to these figures to compensate for heat generated in the bearings.

COMP ADJ

(

. The word COMP flashes below the TEMPERATURE display and the display

)

PN JCFZ-IM-12AB

Use the keypad to enter the required compensation value. Press the sign; pressing it again will remove the minus sign.

Check the temperature display. (If the entry is incorrect, press

key to enter a minus

(±)

and reenter the digits.)

(CE)

4-5

Page 52

Preparing for Operation

Determining The Liquid Pressure

When the entry is correct, press

NOTE

To clear a COMP ADJ entry, press (COMP ADJ), (0), and (ENTER/RECALL).

Table 4.1 J2 and J6 Series Centrifuge Temperature Settings. All settings are approximate.

ENTER/RECALL

(

)

Speed

(rpm)

20 000

18 000

15 000

10 000

–20° –10° 2° 5° 10° 20° 40°

Required Rotor Temperature (°C)

–max

–10

–6

–max

–9

–6

–max

–10

–8

–5

–max

–9

–7

–4

–10

–8

–6

–3

Analog J2 And J6 Series Centrifuges (Models J2-HS, J2-21, J2-HC, J6-HC, and J-6B)

Enter the required run temperature and the appropriate temperature compensation units (see

Table 4.1) on the centrifuge control panel as follows.

Turn the SET knob to the required sample temperature.

Find the compensation value that corresponds to the required temperature and run speed. Set the COMP dial to that setting. (Interpolate if intermediate values are required.)

Determining The Liquid Pressure

A line that delivers liquid to the rotor should be set up with a pressure gauge between the pump and the centrifuge. At flow rates up to 250 mL/min, pressure may be as high as 10 psi. Above 500 mL/min, 15 to 20 psi is allowable. Unless your liquid is quite viscous, higher pressures probably indicate that air is trapped in the passages of the rotor. Trapped air can block liquid flow and cause tubing to separate from connectors when the pump is operating. The flow rate should not decrease with increased gauge pressure. (See CHAPTER 5, Purging Air From The System (Continuous

Flow Rotor Only) for instructions on removing an air block.)

Determining Appropriate Gradients

Figure 4.2 provides an aid to banding sample in a gradient using the standard pellet core or either

zonal core. According to the figure, the radius at which a zone bands can be determined by

4-6

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Preparing for Operation

Determining Appropriate Gradients

measuring the liquid volume it takes to displace the sample out of the rotor. The illustration relates each recovered fraction to its radial position in the rotor. For example, with the standard zonal core, if the desired band begins to emerge when 1000 mL of heavy displacement liquid has been pumped into the rotor, then the inner edge of the band is at a radius of 67.5 mm. The centrifugal force (RCF) at this point can be calculated by inserting the run speed and the radius (mm) into the

equation RCF = 1.12r(RPM/1000)

Figure 4.2 Radius Versus Volume for Standard Pellet Core, Standard Zonal Core, and Reograd Core.

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Page 54

Preparing for Operation

Determining Appropriate Gradients

4-8

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Page 55

This section describes operating procedures for the rotor and each of its cores. It contains general information as well as items specific to either continuous flow or zonal operation. Some steps pertain only to a particular core or cores and are so noted; follow only those steps appropriate to the core you are using. If a problem arises during operation, see CHAPTER 7, Troubleshooting. Use this manual in combination with the appropriate centrifuge instruction manual for complete operating information.

Operating Precautions

WARNING

CHAPTER 5

Operation

WARNING

Protect your hands when handling bowl and lid threads, which become very sharp with prolonged use. Do not touch the rotor while the centrifuge is operating.

WARNING

Never attempt to block the open end of any pressurized tubing or coupling with the figures or hand, as injection of liquid under the skin could occur.

CAUTION

The rotor lid is designed to have liquid support. Never run an empty or partially empty rotor to full speed. Do not run the rotor above 10 000 rpm unless it is completely full of liquid.

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Page 56

Operation

Continuous Flow Operation

CAUTION

While the rotor is moving, do not remove any portion of the seal assembly except the fitting retaining clamp and the two push-pull feed fittings.

NOTE

The rotor will automatically stop at the end of a run if the centrifuge timer is set to anything but HOLD. Some JCF-Z uses require unloading while the rotor is spinning. To prevent inadvertent rotor shutdown, always set the centrifuge to HOLD and time centrifugation with an auxiliary timer.

Continuous Flow Operation

Pelleting Technique

Although the JCF-Z can also perform separations in a gradient or cushion, the rotor is most commonly used for pelleting. Below is a description of pelleting technique which can be used for each of the three continuous flow cores. This technique is illustrated in Figure 5.1.

Figure 5.1 Pelleting Technique, Continuous Flow Cores

5-2

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Run Procedure For Pelleting Sample (Continuous Flow Cores)

Calibrate the pump flow rate in the range to be used for separation.

CAUTION

If you use your pump for more than one application, always disconnect and rinse the line in the pump and check that you have set the correct flow rate before installing and using the pump.

Operation

Continuous Flow Operation

NOTE

Assemble the rotor according to CHAPTER 3, Assembly and Installation. Then refrigerate or warm the rotor as appropriate prior to use.

Prepare buffer and sample, refrigerating or warming as appropriate.

When automatically calibrating pumps with digital controllers, flow must not be interrupted for

more than 15 seconds if the rotor is running above 2,000 RPM.

CAUTION

Do not allow rotor contents to freeze, or threads between bowl and lid may be damaged.

Install the rotor in the centrifuge: set it straight down on the drive hub so that the rotor base is flush with the lip of the drive spindle (Figure 5.2). Hook up lines according to Figure 5.3 or

Figure 5.6, and label the lines with masking tape: lower or edge fitting, upper or center fitting,

and standpipe.

Figure 5.2 Rotor Installation

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1. Drive spindle lip must be

even with bottom of rotor

5-3

Page 58

Operation

Continuous Flow Operation

Figure 5.3 Line Connections, Continuous Flow Cores. Using this tubing

arrangement for basic pelleting against the rotor wall.

• To fill rotor, clamp C

• To load sample, clamp B

1. Lower Fitting

2. Upper Fitting

3. Buffer

4. Sample

5. Supernatant

6. Standard and Large Pellet Cores

Set all centrifuge controls, including setting the timer on HOLD* and the speed to 1000 rpm. If you are using a J2 or J6 microprocessor-controlled centrifuge, enter rotor code

20.9. See

CHAPTER 4, Preparing for Operation for establishing run parameters. (See your centrifuge

instruction manual for complete centrifuge operating instructions.)

Use the pump to pull sample up through the valve that splits the sample inlet line and the buffer

NOTE

inlet line. Sample should be drawn through the valve so that when flow is switched from buffer to sample, air in the valve is not pumped into the rotor.

Begin to pump buffer into the rotor, at the flow rate to be used during the separation, as you accelerate to 1000 rpm.

When you reach 1000 rpm, if air bubbles are no longer coming out of the rotor, reverse the direction of the flow (most pumps have a flow direction switch).

When bubbles no longer exit the rotor, accelerate the rotor to 5000 rpm.

Switch the flow direction back to the forward direction.

5-4

There may be a slight amount of back pressure (approximately 5 psi) if flow rates are high. If

NOTE

the pressure is greater than 7 psi, repeat steps 7 through 9.

* Use an auxiliary timer to time centrifugation runs.

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Operation

Purging Air From The System (Continuous Flow Rotor Only)

If gauge pressure exceeds 25 psi (175 kPa) during acceleration or rises gradually during centrifugation, you have at least a partial air block. To remove the block:

a. Start decelerating the rotor to 2000 rpm. b. Change the exit line to collect effluent, because at lower rpm sample particles will no

longer be retained.

c. Save the effluent to reintroduce after the air block has been cleared. d. If lowering the speed does not clear the air block, reverse the flow direction as described in

Purging Air From The System (Continuous Flow Rotor Only), below.

You may want to switch flow from sample to buffer during the air cleaning operation. Before changing the flow direction, make sure that the inlet tubing is submerged in a reservoir of liquid (sample or buffer).

When the rotor reaches run speed, switch to sample flow.

See Ending The Run, later in this section.

Purging Air From The System (Continuous Flow Rotor Only)

Air in the system can cause a rise in system pressure which may eventually interfere with fluid flow. Air can enter the system accidentally through an inlet tubing line, or can accumulate in the rotor as a result of outgassing of the sample under centrifugal force.

At the beginning of the run, when the rotor is loaded and ready to be accelerated to operating speed, follow the steps below to verify that there is no air in the tubing lines:

Accelerate the rotor to 1000 rpm.

Pump buffer into the center line at 50 mL/min and let the buffer circulate through the system; then reverse the pump direction

opposite direction.

Switch the pump back to its original direction and let the buffer recirculate through the system.

Accelerate the rotor to 5000 rpm and repeat steps 2 and 3.

and let the buffer circulate back through the system in the

PN JCFZ-IM-12AB

* See the instructions accompanying the pump.

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Page 60

Operation

Density Gradient Operation (Continuous Flow Cores)

Be sure that a pressure gauge is connected between the pump and the rotor to monitor system pressure while the centrifuge and rotor are operating. If gauge pressure exceeds 25 psi during acceleration or rises gradually during centrifugation, the system has an air block which is affecting fluid flow.

To remove the block:

Turn valve to switch reservoirs from sample to buffer.

Start decelerating the rotor to 2000 rpm. (Continue with steps 3 through 5 as the rotor is decelerating.)

Collect the outflow in a separate receptacle so that it can be reprocessed after the air block is removed.

Pump buffer into the center line at 50 mL/min and let the buffer circulate through the system; then reverse the pump direction and let the buffer circulate through the system in the opposite

direction.

Switch the pump back to its original direction, and let the buffer recirculate through the system.

Begin accelerating back up to run speed.

To prevent air from entering the inlet lines:

•

Do weight the end of the tubing that sits in the reservoir so that the end doesn’t come out

accidentally.

•

Don’t let a buffer or sample reservoir run dry.

•

Don’t lift the inlet tubing line out of the buffer/sample reservoir.

Density Gradient Operation (Continuous Flow Cores)

Gradient Or Cushion Separation Techniques

The JCF-Z can separate particles in a gradient or cushion. These techniques are described in

Table 5.1 and technique B is illustrated in Figure 5.4.

5-6

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Operation

Density Gradient Operation (Continuous Flow Cores)

Forming an Air Block

During unloading, the rotor contents are displaced toward the center by a very dense liquid pumped to the rotor wall. An air block is needed to cause the displacing solution to enter at the periphery instead of at the top of the gradient. This bubble is extremely difficult to dislodge and thus forces the displacing liquid to enter the passages across the top of the core to the periphery of the rotor.

Before forming the air block, begin decelerating the rotor to 2000 rpm. Then perform the following steps:

Cut a piece of 1/8-in. inside-diameter tubing approximately 40 inches (1 meter) long. This

tubing must be dry on the inside

disconnect the line from the core edge, at the rotor and at the T-connector. Replace with the dry tubing.

Pump dense solution at 50 mL/min. to form the air block. Watch the gauge as you pump. If it registers 15 to 17 psi, the air block is successful. Continue pumping to displace the rotor

contents (the pressure will drop slightly to about 12 to 15 psi). If an air block did not form, stop the pump and repeat step 1 with dry tubing.

. When the rotor reaches 2000 rpm, clamp the tubing and

Continue pumping dense liquid until the rotor contents have been displaced.

Table 5.1 Two Techniques for Forming an Air Block During Density Gradient Operation

Step 1 Step 2 Step 3 Step 4

Technique A Buffer in, verify no air in

system, then cushion in

Technique B Buffer in, then gradient

in, light end first

In Figure 5.4, the arrow from the left indicates that solution enters the rotor from the left; the arrow from the right indicates liquid entry from the right. Technique B is illustrated.

sample in air block (8 mL) dense solution

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Operation

Density Gradient Operation (Continuous Flow Cores)

Figure 5.4 Gradient or Cushion Separation Technique, Continuous Flow Cores.

Technique B, described in Table 5.1, is shown.

5-8

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Operation

Zonal Operating Techniques And Run Procedures

Run Procedure for Separating in a Gradient or Cushion (Continuous Flow Cores)

Follow steps 1 through 11 in Run Procedure For Pelleting Sample (Continuous Flow Cores), earlier in this section, noting the following changes in steps 3 and 6 that pertain to separation in a gradient or cushion:

STEP 3: Be sure to cool or warm the gradient or cushion as appropriate.

STEP 6: Begin to pump buffer into the rotor, at the flow rate to be used during the separation, as the

rotor accelerates to 1000 rpm. If you are using a gradient or cushion, switch from buffer to the gradient or cushion and pump at 30 mL/min. (A typical step gradient is shown in Figure 5.5.)

Figure 5.5 Sucrose Gradient after One Hour, Monitored by Refractometer. Diffusion between the discontinuous layers of the step gradient results in a near-linear gradient.

Zonal Operating Techniques And Run Procedures

Zonal operation of the JCF-Z is described below and illustrated in Figures Figure 5.7 and Figure 5.8.

WARNING

Practice the procedure shown in Figure Figure 5.8 before using this core.

Standard Zonal Core

Calibrate the pump flow rate in the range to be used for operation.

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Operation

Zonal Operating Techniques And Run Procedures

Assemble the rotor according to CHAPTER 3, Assembly and Installation. Then refrigerate or warm the rotor and solutions as appropriate prior to use.

Install the rotor in the centrifuge. Set it straight down on the drive hub (see Figure 5.2) so that the rotor base is flush with the lip of the drive spindle. Hook up lines according to Figure 5.6,

and label the lines with masking tape: upper or center line, and lower or edge line.

Figure 5.6 Line Connections, Standard Zonal Core

Set the centrifuge to ZONAL mode using either the centrifuge keyswitch or touchscreen interface (see your centrifuge manual for details). On analog J2 and J6 models (J2-HS, J2-21, J6HC, and J-6B), the keyswitch is located on the zonal key safety interlock accessory.

Earlier J-21 centrifuges have no key lock; the door may be opened when the rotor is turning by removing a stopper from one of the chamber portholes, which releases chamber vacuum. A stopper must also be removed to open J2 series centrifuge doors.

Set all centrifuge controls, including setting the timer on HOLD,† the speed to 2000 rpm, and the temperature to 30°C. If you are using a J2 or J6 microprocessor-controlled centrifuge, enter

rotor code

Pump in gradient at 30 to 50 mL/min (see Figure 5.7). Then pump in the sample solution at 15

20.9.

to 25 mL/min. About 50 mL of an overlay solution (buffer) may be layered over the sample if desired, to move the sample away from the center of rotation. Pump the overlay in at 15 to 25

mL/min.

* If you have not already installed a zonal key safety interlock, contact Beckman Coulter to order one.

Publication J-TB-025 accompanying the interlock provides complete installation instructions.

† Use an auxiliary timer to time centrifugation runs.

‡ See Griffith, O. M. and Wright, H. Resolution of components from rat liver homogenate in reorienting density gradients.

Anal. Biochem, Vol. 47, No. 2, 575-583 (June 1972) for a description of a discontinuous gradient. (Reprints available from Spinco Business Center of Beckman Coulter.

‡

5-10

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Figure 5.7 Operating Procedure, Standard Zonal Core

Operation

Zonal Operating Techniques And Run Procedures

PN JCFZ-IM-12AB

Stop the pump and clamp the lines.

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Operation

Zonal Operating Techniques And Run Procedures

Rotate the fitting retaining clamp as described in Figure 5.8 and remove the fittings and liquid lines from the chamber. Reinstall stoppers in all portholes. Reset the centrifuge controls as

necessary.

Figure 5.8 Removing and Reinserting Fittings and Liquid Lines

5-12

1. Fitting Retaining Clamp (344108)

Before Accelerating to Run Speed:

Rotate the fitting retaining clamp counterclockwise to unlock the fittings and slip them out. Lift off the retaining clamp.

After Decelerating from Run Speed:

After the seal assembly comes to a stop, slide the retaining clamp down over the seal housing and reinstall the fittings. Turn the retaining clamp clockwise to lock it in place.

Close the centrifuge door.

Accelerate the rotor to speed and set the auxiliary timer.

See Ending The Run, later in this section.

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Reorienting Gradient (Reograd) Zonal Core

Calibrate the pump flow rate in the range of 15 to 50 mL/min.

Assemble the rotor according to CHAPTER 3, Assembly and Installation. Then refrigerate or warm the rotor and solutions as appropriate prior to use.

The rotor may be filled either in the centrifuge or in a cold room. Hookup the lines as shown in

Figure 5.9. Pump in gradient first (Figure 5.10) at 30 to 50 mL/min. Then pump in sample at 15

to 25 mL/min.

Figure 5.9 Line Connections, Reograd Zonal Core

Operation

Zonal Operating Techniques And Run Procedures

PN JCFZ-IM-12AB

Remove the lines and transfer tube and cap the rotor with the zonal seal cap. Install the rotor in the centrifuge: set it straight down on the drive hub so that the rotor base is flush with the

lip of the drive spindle (see Figure 5.2). Then close the centrifuge door.

Set all centrifuge controls as required. If you are using a J2 or J6 microprocessor-controlled centrifuge, enter rotor code

Accelerate the rotor to speed using the slowest acceleration rate possible for the centrifuge.

See Ending The Run, below.

* See Griffith and Wright, op. cit.

20.9.

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Page 68

Operation

Ending The Run

Continuous Flow Core Containing Pellet

Push the

Turn the pump flow rate down.

CAUTION

Do not turn off the pump completely until the rotor has stopped. Without some flow through the spinning rotor, the bearing housing may overheat and shorten the life of the bearings. Overheating the bearing housing may also dislocate the feed fitting, resulting in significant leakage.

STOP

button on the centrifuge control panel.

(

)

5-14

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Figure 5.10 Operating Procedure, Reograd Zonal Core

Operation

Ending The Run

PN JCFZ-IM-12AB

When the rotor has stopped, turn off the pump. Flush the tubing lines (see Flushing The Tubing

Lines, later in this section).

Clamp the lines. Remove the rotor from the centrifuge and set it in the vise.

Remove the rotating seal assembly.

Loosen the rotor lid one half turn, using the large spanner wrench (870667). It may be necessary to tap the wrench handle with a hammer to loosen the lid. Remove the rotor lid.

5-15

Page 70

Operation

Ending The Run

Figure 5.11 Lowering Rotor onto Plug. Rotor contents will spill out

Take the rotor off the vise.

Put the plug on the vise. (If the plug is not in the vise, the rotor contents will leak as soon as you take the core out.)

Lower the rotor onto the plug (see Figure 5.11). The plug prevents leakage from the rotor bowl, and raises the core for easier removal.

as soon as the core is removed if the plug and plug O-ring are not in place.

5-16

1. Plug (335149)

2. Plug O-ring (812705)

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Operation

Ending The Run

Siphon out the liquid. Take the core out of the rotor. Scrape the pellet from the core and bowl (small pellet, see Figure 5.13). Don’t use metal tools to lift the pellet container or to scrape out

the pellet, as they can scratch the rotor.

Figure 5.12 Disassembling the Rotor. Disassemble as shown to remove sample

(arrows show direction of tool use). See CHAPTER 6, Care and Maintenance for instructions on inspecting, cleaning, and replacing the bearings.

PN JCFZ-IM-12AB

1. Small Spanner Wrench

2. Large Spanner Wrench

3. Plug/O-ring

4. Vise

5-17

Page 72

Operation

Ending The Run

Figure 5.13 Removing Small Pellet. Lift out canoe-shaped pellet container and

scrape out pellet.

1. Toothpick

Finish disassembling the rotor (see Figure 5.12).

See CHAPTER 6, Care and Maintenance for cleaning instructions.

Continuous Flow Core Containing Gradient or Cushion

When the auxiliary timer indicates that the run is over, turn the pump off and clamp the sample inlet line.

Begin decelerating the rotor to 2000 rpm. Switch the pump to a reservoir of dense displacement solution; purge the pump and fill lines with displacement solution. Stop the pump again and

install air-block tubing as described in Forming an Air Block, earlier in this section.

5-18

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Operation

Ending The Run

While the rotor is spinning at 2000 rpm, pump dense displacement solution at 30 mL/min into the rotor. This first forces the air from the dry tubing into the rotor, creating an air block as

shown in Figure 5.1. The dense solution then forces the rotor contents out. Collect fractions, manually or with a fraction collector, as liquid exits the rotor.

When sample has been collected, turn off the pump. Flush the tubing lines (see Flushing The

Tubing Lines, later in this section).

Clamp and disconnect the lines, and stop the rotor. Remove the rotor from the chamber and set it on the plug in the rotor vise (Figure 5.11)

Disassemble the rotor (Figure 5.12) and see CHAPTER 6, Care and Maintenance for cleaning instructions.

Standard Zonal Core

Reattach feed fittings and tubing lines. Attach one line to a pump and reservoir of heavy displacement liquid, and pump the line and fitting full of liquid. Place collection vessels at the

end of the other line.

At the end of the run, decelerate the rotor to 2000 rpm.

Remove stoppers from chamber portholes to release the chamber vacuum.

Open the chamber door (the centrifuge must be in ZONAL mode).

Install inlet and outlet tubing lines (step 1 above) and fittings by passing them through the portholes into the chamber. Use holed stoppers to hold them just within reach of the seal

assembly.

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Operation

Ending The Run

After the seal housing comes to a stop, install and rotate the plastic fitting retainer so that fittings and liquid lines can be reattached.

CAUTION

Make sure there is no air in the displacement liquid line.

Attach the liquid-filled line and fitting into the lower fitting hole and the collection line into the upper fitting hole. Rotate the plastic retainer on the lower fitting hole back so that the fittings

are locked in place. Let go of the seal housing.

Unclamp the pump line and begin pumping displacement liquid at 30 mL/min. Collect sample as it exits.

When all fractions have been collected, turn off the pump. Flush the tubing lines (see Flushing

The Tubing Lines, later in this section).

Clamp the lines and stop the rotor. Detach tubing lines and remove the rotor from the chamber, setting it onto the plug in the rotor vise (Figure 5.11).

Disassemble the rotor (Figure 5.12) and see CHAPTER 6, Care and Maintenance for cleaning instructions.

Reograd Zonal Core

Decelerate the rotor (it should take approximately 5 minutes to accelerate to and decelerate from 1000 rpm) and then push the centrifuge

When the rotor has stopped, remove the zonal seal cap and reinstall the transfer tube assembly. Then attach tubing and pump to the vertical fitting and collect rotor contents through that

fitting.

STOP

(

button.

)

5-20

Flush the tubing lines (see Flushing The Tubing Lines, below).

Unscrew the transfer tube assembly and complete disassembly as appropriate (see Figure 5.12). See CHAPTER 6, Care and Maintenance for cleaning instructions.

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Page 75

Flushing The Tubing Lines

After every run, flush the tygon tubing lines according to the following procedure. The rotor can then be disassembled and the components can be cleaned and sterilized according to the procedures in CHAPTER 6, Care and Maintenance. (After a continuous flow run in which pellet is collected, flush the tubing before clamping the lines. After a continuous flow run using gradient or cushion, or after a zonal run [standard or reograd core], flush the tubing after all fractions have been collected.)

Set the pump at approximately 30 mL/min. Pump at least 200 mL of Solution 555, diluted approximately 1 to 10 with water, through the system.

To remove the detergent, pump at least one liter of sterile distilled water through the rotor. Stop the pump.

Remove the rotor from the centrifuge. Remove the lid and core (see Figure 5.13) and rinse all parts thoroughly with distilled water to remove any remaining detergent residue. See

CHAPTER 6, Care and Maintenance for detailed cleaning instructions.

Operation

Ending The Run

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Operation

Ending The Run

5-22

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Page 77

This section describes procedures for handling, inspecting, cleaning, and sterilizing the rotor. It also tells how to return a rotor to the factory for repair, if necessary.

Maintenance

NOTE Do not use sharp tools on the rotor that could cause scratches in the rotor surface. Corrosion begins

in scratches and may open fissures in the rotor with continued use.

CHAPTER 6

Care and Maintenance

WARNING

Protect your hands when handling the bowl and lid threads, which become very sharp with prolonged use. Do not touch the rotor while the centrifuge is operating.

Cleaning

• Periodically (at least monthly) inspect the rotor for rough spots or pitting, white powder

deposits (frequently aluminum oxide), or heavy discoloration. If any of these signs are evident, do not run the rotor. Contact your Beckman Coulter representative for information about the Field Rotor Inspection Program and the rotor repair center.

• Regularly lubricate the metal threads in the rotor with a thin, even coat of Spinkote lubricant (306812). Failure to keep these threads lubricated can result in damaged threads.

• Regularly apply silicone vacuum grease (335148) to the O-rings. Replace O-rings about twice a year or whenever worn or damaged.

• Routinely apply Spinkote lubricant (306812) to the centrifuge drive spindle hub to prevent the rotor from sticking (not required on Avanti J series centrifuges).

Refer to Chemical Resistances (publication IN-175) for chemical compatibilities of rotor and accessory materials. Your Beckman Coulter representative provides contact with the Field Rotor Inspection Program and the rotor repair center.

Wash the rotor body, cores, and other rotor components immediately if salts or other corrosive materials are used or if spillage has occurred. Do not allow corrosive materials to dry on the rotor.

Under normal use, wash the rotor frequently (at least weekly) to prevent buildup of residues.

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6-1

Page 78

Care and Maintenance

Decontamination

With the plug in place (Figure 5.11) to prevent the rotor from leaking when the core is removed,

disassemble the rotor according to Figure 5.12.

NOTE

After a pelleting run, remove the liquid and scrape out the pellet. See Figure 5.13 for instructions on removing the pellet from the small pellet core. After gradient separation, the

liquid contents may be siphoned out.

Clean the emptied rotor with Solution 555 (diluted 10 to 1 with water). Inject the solution into all liquid passages of the seal assembly and the core. Air-dry components thoroughly before

reassembling.

After each run, dismantle the rotating seal assembly, thoroughly rinse the components with distilled water, and air-dry.

The bearing screw must be removed before the seal housing can be removed. Remove O-rings

by slipping a narrow spatula along their edges. Reograd core: unscrew the transfer tube assembly.

Clean metal threads every 6 months, or as necessary. Use a brush and concentrated Solution 555. Rinse and dry thoroughly, then lubricate lightly but evenly with Spinkote to coat all threads.

Periodically remove the O-rings and wipe clean as necessary, then reapply a light film of silicone vacuum grease. Clean the O-ring grooves with a cotton-tipped swab.

Decontamination

If the rotor (and/or accessories) becomes contaminated with radioactive material, decontaminate it using a solution that will not damage the anodized surfaces. Beckman Coulter has tested a number of solutions and found two that do not harm anodized aluminum: RadCon Surface Spray or IsoClean

Solution (for soaking),

IsoClean can cause fading of colored anodized surfaces. use it only when necessary and remove it promptly from surfaces.

While Beckman Coulter has tested these methods and found that they do not damage components, no guarantee of decontamination is expressed or implied. Consult your laboratory safety officer regarding the proper decontamination methods to use.

and Radiacwash.†

6-2

* In U.S.A., contact Nuclear Associates (New York); in Eastern Europe and Commonwealth States, contact Victoreen

GmbH (Munich); in South Pacific, contact Gammasonics Pty, Ltd. (Australia); in Japan, contact Toyo Medic Co. Ltd. (Tokyo).

† In U.S.A., contact Biodex Medical Systems (Shirley, NY); internationally, contact the U.S. office to find the dealer nearest

you.

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Page 79

Sterilization And Disinfection

WARNING

Flammability Hazard. Do not use Ethanol near operating centrifuges.

The rotor— except for the baffles on the standard and large pellet cores—can be autoclaved at 121°C for up to 1 hour. However, autoclaving the Noryl plastic cores is not recommended. We recommend cold sterilization methods only, such as Ethanol (70%).

the cores and of the bearings

To autoclave, empty all liquid from the rotor. Set the rotor and lid (separately) upside down in the autoclave. Run a dry cycle at the end of the autoclave cycle.

While Beckman Coulter has tested these methods and found that they do not damage components, no guarantee of sterility or disinfection is expressed or implied. When sterilization or disinfection is a concern, consult your laboratory safety officer regarding proper methods to use.

Care and Maintenance

Sterilization And Disinfection

Repeated autoclaving will shorten the life of

Inspecting The Bearings

Before each use, inspect the bearings by disassembling the bearing assembly using the 3/16-in. T-bar hex wrench (001311). Hold each bearing between your thumb and forefinger and spin it. If a bearing is noisy or spins roughly, it should be replaced. To reassemble the bearings, follow the alignment procedure in CHAPTER 3, Step 6a.

Some JCF-Z rotors with new or older bearings may be misidentified in Avanti J series centrifuges. If the rotor is misidentified, stop the run and follow one the steps below. (JCF-Z rotors manufactured after March, 1997 have factory-installed magnets that prevent misidentification.)

On the first few uses of a new JCF-Z rotor, or in an older JCF-Z rotor with newly replaced bearings,

•

run the rotor from 0 to 5000 rpm and back to 0 rpm three times. This procedure will distribute lubricant around the bearings in the rotating seal assembly, reducing drag.

•

In older JCF-Z rotors with bearings that have not been replaced recently, follow the same

procedure as for a new JCF-Z rotor (perform three runs from 0 to 5000 rpm and back to 0 rpm) to ensure that the bearings are properly lubricated. If the rotor is misidentified after the third run, when the bearings are lubricated, this indicates that the bearings are worn and need to be replaced. Replace the bearings following the instructions CHAPTER 3, Step 6a.

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Page 80

Care and Maintenance

Smoothing (Lapping) The Rotating Seal

With normal use, the rotating seal (335144) will give several thousand hours of service. However, the seal is made of carbon graphite and can be nicked, scratched, or unevenly worn in the presence of abrasive samples; such damage may eventually allow leakage between the inlet and the outlet lines. To repair damage, rub the damaged surface in a figure-eight motion against the etched glass plate (870671) provided with the rotor. Rotate the seal as you press it lightly. The raised rings (lands) on the seal mu st be at lea st 1.5-mm ( 1/16-in.) high for s urfaces to seal prope rly. If the l ands are wo rn beyond this, replace the seal. Properly lapped lands should reflect light smoothly and evenly. Note that the glass plate will eventually wear out and should be replaced.

Returning a Rotor

Before returning a rotor or accessory for any reason, prior permission (a Returned Goods Authorization form) must be obtained from Beckman Coulter, Inc. The RGA form may be obtained from your local Beckman Coulter sales office. It should contain the following information:

• rotor serial number,

• history of use (approximate frequency of use),

• reason for the return,

• original purchase order number, billing number, and shipping number, if possible,

• name and phone number of the person to be notified upon receipt of the rotor or accessory at

the factory, and

• name and phone number of the person to be notified about repair costs, etc.

To protect our personnel, it is the customer’s responsibility to ensure that the parts are free from pathogens and/or radioactivity. Sterilization and decontamination must be done before returning the parts. Smaller items (such as tubes, bottles, etc.) should be enclosed in a sealed plastic bag.

All parts must be accompanied by a note, plainly visible on the outside of the box or bag, stating that they are safe to handle and that they are not contaminated with pathogens or radioactivity.

this notification will result in return or disposal of the items without review of the reported problem.

Use the address label printed on the RGA form when mailing the rotor and/or accessories.

Customers located outside the United States should contact their local Beckman Coulter office.

Failure to attach

6-4

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Supply List

Many of the parts listed are pictured in Figure 2.1, Figure 2.2, and Figure 2.3. Refer to these Figures for easy identification. Refer to APPENDIX B, Using the JCF-Z Continuous Flow/Zonal Rotor Bracket

(Avanti J Series Centrifuges Only) for parts required for the zonal bracket.

Table 6.1 Supply List

Bearing housing (anodized aluminum) 336079

Bearing (pressed steel) 355161

Bearing (stainless steel) 366190

Bearing screw, hex head (stainless steel) 804063

Bearing spacer (stainless steel) 336285

Core Kit, continuous flow, standard pellet 335130

Core Kit, continuous flow, large pellet 350641

Core Kit, continuous flow, small pellet 350601

Core, reorienting gradient, and sample transfer assembly 350585

Care and Maintenance

Supply List

Core Kit, standard zonal 335141

Feed fitting, high flow (stainless steel)

Feed fitting, standard flow (stainless steel) 335146

Fitting retaining clamp (Noryl) 334108

Housing, high flow stationary seal (stainless steel) 338672

Housing, standard flow stationary seal (stainless steel) 336080

Key interlock, zonal 343348

Mount, rotating seal (stainless steel) 336284

O-ring (Viton), continuous flow lid 870526

O-ring (Viton), continuous flow lid 817033

O-ring, rotating seal, standard flow manifold 868638

O-ring, rotating seal 824412

O-ring, rotor bowl bottom (minimum order 6) 854519

O-ring, rotor bowl edge 870668

O-ring, rotor lid stem (minimum order 6) 815473

O-ring, rotor plug (minimum order 12) 812715

O-ring, seal housing feed fitting 824464

O-ring, standard flow manifold (minimum order 6) 870655

335136

PN JCFZ-IM-12AB

O-ring (Viton), transfer tube assembly and rotating seal 870656

O-ring, zonal lid 870315

Retaining ring (stainless steel) 870689

Rotating seal (carbon-graphite) 335144

6-5

Page 82

Care and Maintenance

Supply List

Table 6.1 Supply List

Sample transfer tube, standard flow seal assembly

335120

(stainless steel)

Sample transfer tube, high flow seal assembly (stainless steel) 335132

Scavenger drain tube (stainless steel) 336286

Screw, manifold (stainless steel) 870690

Seal assembly, high flow 335133

Seal assembly kit, high flow (for continuous-flow operation) 335134

Seal assembly kit, standard flow (for zonal and continuous-flow operation) 335142

Silicone vacuum grease, 1 oz 335148

Solution 555 rotor cleaning concentrate (1 qt) 339555

Spinkote lubricant (2 oz) 306812

Spring (stainless steel) 870654

Stationary seal (aluminum) 335145

Stationary seal housing, high flow, with brazed fitting 393290

Stoppers for tubing lines (#1, one-hole) 336403

Tygon tubing 1/8-in. inside diameter (1/4-in. outside diameter) 144664

Tygon tubing for transfer tube 306653

Washer, flat (nylon) 878279

Washer, high-flow seal 853118

Washer, flat (stainless steel) 853391

Washer, spring (spring steel) 870658

Took Kit

335143

Includes

Rotor vise Plug Plug O-ring Spanner wrench, large Spanner wrench, small Hex wrench, 3/16-in. T-bar Glass plate

a. Use loctite grade “T” primer and #35 retaining compound to bond fitting to seal housing.

335127 335149 812715 870667 870666 001311 870671

6-6

PN JCFZ-IM-12AB

Page 83

This section lists possible malfunctions, together with probable causes and corrective actions required. If a problem persists, contact Beckman Coulter (1-800-742-2345 in the United States; customers outside the U.S. should contact their local Beckman Coulter office).

Leakage

Rotating Seal/Stationary Seal Interface

Leakage from the rotating seal/stationary seal interface can be caused by a damaged rotating seal or by insufficient spring pressure on the rotating seal. Inspect the rotating seal for signs of wear or damage and replace if necessary. See APPENDIX A, Assembling the Rotating Seal for detailed instructions on assembling the rotating seal.

CHAPTER 7

Troubleshooting

Scavenger Line

Excessive leakage from the scavenger line can result from either excessive back pressure in the system or a damaged rotating seal. Check the gauge to see if there is excessive system pressure (about 30 psi); if so, follow the instructions in CHAPTER 5, Operation to purge air from the system. Inspect the rotating seal for damage and replace, if necessary.

Other Areas

Leakage from under the rotor lid indicates that the lid is not fastened down properly. Check O-rings and resecure the lid to the rotor body, making sure that the lid has been tightened to or past the index marks (see CHAPTER 3, Step 4).

Leakage from the rotor bottom indicates that the bottom O-ring (854519) is missing. Reinstall the O-ring, making sure that it is lightly coated with silicone vacuum grease.

Mixing of Inlet and Outlet Liquid

Such mixing is due to cross-leakage over the face of the rotating seal. Remove the rotating seal and inspect all of its surfaces. See CHAPTER 6, Care and Maintenance for instructions on smoothing the seal.

PN JCFZ-IM-12AB

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Page 84

Troubleshooting

Vibration

Rotor vibration can be caused by defective or misaligned bearings (see CHAPTER 3, Step 6a for bearing alignment instructions) or by an improperly assembled rotor. Review assembly steps 1 through 13 in CHAPTER 3, Assembling the Rotor, if necessary.

7-2

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Page 85

Assembling the Rotating Seal

Use the following assembly procedures when assembling the rotating seal. They incorporate instructions for proper tensioning of the rotating seal and ensure proper operation of the seal at high speeds and high operating pressures. Following these procedures will avoid rotating seal leakage problems, which are the main cause of reduced operational efficiency and performance of this rotor. At properly tensioned spring pressures, the rotating seal is capable of up to two liters per minute of sample throughput in the JCF-Z.

Figure A.1 illustrates the components of the transfer tube assembly: the transfer tube (335120,

standard flow; 335132, high flow), the tygon tubing (306653), the spring (870654), the spring washer (853391), and the O-ring (870656).

Figure A.1 Transfer Tube Assembly Components

APPENDIX A

Assembling the Rotating Seal

PN JCFZ-IM-12AB

1. Tygon Tubing (306653

2. Transfer Tube

Standard Flow (335120) High Flow (335132)

Before each use, lubricate the O-ring sparingly with silicone vacuum grease (Figure A.2).

3. O-ring (870656)

4. Spring Washer (853391)

5. Spring (870654)

A-1

Page 86

Assembling the Rotating Seal

Figure A.2 O-ring (870656) and Silicone Vacuum Grease (335148)

A-2

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Assembling the Rotating Seal

After lubricating the O-ring, assemble the transfer tube assembly for use in the rotor as shown in Figure A.3.

Figure A.3 Fully Assembled Transfer Tube Assembly

The transfer tube and rotating seal are shown in Figure A.4. Place the transfer tube in the rotor stem, inserting the spring end in the down position.

Figure A.4 Transfer Tube Assembly and Rotating Seal

(Ready for Assembly into Rotor)

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Assembling the Rotating Seal

The beveled end of the transfer tube should protrude out of the seal housing as shown in

Figure A.5.

Figure A.5 Transfer Tube Beveled End

Place the rotating seal on the transfer tube assembly as shown in Figure A.6. Push down on the rotating seal until the transfer tube spring bottoms out and then release pressure on the

rotating seal. It should immediately return to its original position as shown in Figure A.6.

Figure A.6 Correct Position of the Rotating Seal

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Assembling the Rotating Seal

If the rotating seal remains in the seal housing (Figure A.7), it is not properly tensioned. (This condition has been virtually eliminated by the new design transfer tube assembly which has a

pre-tensioned spring.) Repeat steps 1 through 6 to ensure that the seal is pushed out of the seal housing when spring tension is released (see Figure A.6).

Figure A.7 Incorrect Position of the Rotating Seal (Wrong Spring Tension)

When proper spring tension is achieved, assemble the bearing housing as shown in Figure A.8.

Figure A.8 Bearing Housing Assembled

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Assembling the Rotating Seal

Assemble the stationary seal housing together with the bearing housing and check to see that the entire seal/bearing housing assembly sits on the rotating seal and rides up on the bearings.

This happens because the transfer tube spring supports the weight of the seal/bearing housing when it is properly tensioned (Figure A.9).

Figure A.9 Stationary Seal Housing Mated to Bearing Housing (Seal/Bearing Housing)

1. Note height

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Assembling the Rotating Seal

Push down on the seal/bearing assembly until the transfer tube spring bottoms out (Figure A.10).

Figure A.10 Pushing Down on Seal/Bearing Assembly to Tension Spring

While continuing to bear down on the seal/bearing housing, tighten the seal/bearing assembly using the T-handle wrench supplied with the rotor (001311). Properly tightened, the seal/

bearing assembly will remain in the bottomed-out position, keeping tension on the transfer tube spring (Figure A.11).

Figure A.11 Finishing Rotor Assembly

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Assembling the Rotating Seal

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Description

The zonal bracket is designed for use in Avanti J series centrifuges when the JCF-Z Continuous Flow/ Zonal Rotor is used. The bracket provides support for the inlet and outlet tubing lines, keeping them stable and out of the way of the spinning rotor during high-speed operation. The complete installation is shown in Figure B.1.

Figure B.1 Avanti J Series Centrifuge Chamber with JCF-Z Rotor and Zonal Bracket Installed

APPENDIX B

Using the JCF-Z Continuous Flow/Zonal

Rotor Bracket (Avanti J Series Centrifuges

Only)

PN JCFZ-IM-12AB

1. Avanti J Centrifuge Chamber

2. Zonal Bracket

3. JCF-Z Rotor

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Using the JCF-Z Continuous Flow/Zonal Rotor Bracket (Avanti J Series Centrifuges Only)

Description

Zonal Bracket Kit Contents

Two zonal bracket kits are available for use with standard Cole-Parmer size 16 tubing (6.4-mm, 1/4-in. O.D.). Kit (363843) is used with Avanti JXN-30, J-30I, and J-25 series centrifuges; kit (366431) is used with Avanti JXN-26, J-26S XP, J-26 XP, and J-20 series centrifuges.

Table B.1 Basic Kit 363843 or 366431

Item Quantity Part Numbers

Zonal Bracket 1 363818

(Avanti JXN-30, J-30I,

and J-25 series)

366430

(Avanti JXN-26, J-26S XP,

J-26 XP, and J-20 series)

Thumbscrew, stainless steel, M4 (thread size) x 19 mm

Thumbscrew, stainless steel, M4 (thread size) x 12 mm

Cable Clamp, nylon,

6.4-mm (1/4-in.) I.D.

2 893412

2 893411

10 000499

Customers using Cole-Parmer size 14 (4.8-mm, 3/16-in. O.D.) or size 15 (9.5-mm, 3/8-in. O.D.) tubing must use one of the other two kits described below in addition to the basic kit 363843 or 366431.

Table B.2 Kit 363844 Contents (for Size 14 Tubing)

Item Quantity Part Numbers

Tubing Adapter, stainless steel, for size 14 (4.8-mm, 3/16-in. O.D.) tubing

Cable Clamp, nylon,

4.8-mm, (3/16-in.) I.D.

2 363830

10 003343

B-2

Table B.3 Kit 363845 Contents (for Size 15 Tubing)

Item Quantity Part Number

Tubing Adapter, stainless steel, for size 15 (9.5-mm, 3/8-in. O.D.) tubing

Cable Clamp, nylon,

9.5-mm (3/8-in.) I.D.

2 363831

10 00596

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Using the JCF-Z Continuous Flow/Zonal Rotor Bracket (Avanti J Series Centrifuges Only)

Assembly Instructions

Step 1. Install the Rotor

Install the assembled JCF-Z rotor into the Avanti J chamber. See CHAPTER 3, Assembly and

Installation, for instructions.

Step 2. Attach the Zonal Bracket to the Centrifuge Chamber Wall

Insert the two 19-mm thumbscrews (the longer ones) through the holes in the bracket into the rivnuts in the chamber wall (Figure B.2). Finger-tighten until secure.

Figure B.2 Attaching the Bracket to the Chamber Wall

Assembly Instructions

Step 3. Insert the Tubing Through the Door Gasket

Size 16 Tubing

You will need two pieces of tubing, one for the inlet line and one for the outlet line. Size 16 tubing can be inserted directly through the tubing holes in the left side of the door gasket, resulting in a snug fit.

Remove the plugs from the two center tubing holes in the door gasket.

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Using the JCF-Z Continuous Flow/Zonal Rotor Bracket (Avanti J Series Centrifuges Only)

Assembly Instructions

To make it easier to insert the tubing, first carefully cut the end at an angle with scissors or a sharp blade. Insert a piece of tubing through one of the tubing holes, from

centrifuge (Figure B.3). After the tubing is inserted, recut the tubing straight across. Pull about eleven inches of tubing into the centrifuge chamber. Leave enough tubing outside

the centrifuge to reach your sample and buffer containers.

Figure B.3 Inserting Size 16 Tubing through the Gasket

1. Door Gasket

outside to inside the

Attach the other tubing end to a vacuum trap outside the centrifuge.

Sizes 14 and 15 Tubing

Sizes 14 and 15 tubing require the use of tubing adapters, which fit snugly inside the tubing holes. The adapter ends are sized to fit the smaller- and larger-diameter tubing.

Remove the plugs from the two center tubing holes in the door gasket.

Insert an adapter through one of the holes until it is centered, with equal lengths of adapter on either side of the tubing holes (Figure B.4). Repeat this step with the second adapter in the other

hole.

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Using the JCF-Z Continuous Flow/Zonal Rotor Bracket (Avanti J Series Centrifuges Only)

Figure B.4 Attaching Tubing to the Adapter

1. Door Gasket

Assembly Instructions

Step 4. Attach the Tubing to the Zonal Bracket

Sizes 16 and 14 tubing: insert a standard-flow feed fitting into each tubing end, as shown in

Figure B.5. (For the larger size 15 tubing, which is used with the JCF-Z high-flow seal assembly,

feed fittings are not used; see Figure B.7.)

Figure B.5 Inserting the Standard-Flow Feed Fitting into the Tubing

1. Standard Flow Feed Fitting

2. Tubing Clamp

PN JCFZ-IM-12AB

Place one tubing clamp over each piece of tubing, about one inch behind the end of the fitting (Figure B.5). The raised numbering on the clamp, indicating the O.D. of tubing used with the

clamp (in inches), should face up.

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Using the JCF-Z Continuous Flow/Zonal Rotor Bracket (Avanti J Series Centrifuges Only)

Assembly Instructions

Place one of the tubing clamps, with tubing pointing inward, over one of the holes in the bracket (Figure B.6). Insert one of the shorter (12-mm) thumbscrews through the hole and turn

slightly to fasten. Do not tighten. Repeat with the other piece of clamped tubing. Leave both thumbscrews slightly fastened. You will tighten them securely in step 5, number 2.

Figure B.6 Attaching the Tubing Clamps to the Bracket

Step 5. Attach Tubing to the JCF-Z Seal Assembly Housing

Sizes 16 and 14 tubing: press one standard-flow feed fitting into each of the holes in the side of

the seal housing.

Size 15 tubing: press one tubing end over each fitting. Size 15 tubing is used with the JCF-Z high-

flow seal assembly, in which the fittings are welded to the housing (Figure B.7).

Figure B.7 Inserting Tubing and Feed Fittings into the JCF-Z Seal Assembly

1. Standard Flow Assembly

2. High Flow Assembly

3. Seal Housing

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Make sure that the two sections of tubing from the gasket to the rotor are of even length, so that the rotor is not being pulled to either side. Then finger-tighten the thumbscrews (shown

in Figure B.6) down securely.

You are now ready to complete setting up the JCF-Z flow system as described in this manual. To attach a scavenger line (optional), follow the instructions in step 6 below.

Step 6. Attach a Scavenger Line (Optional)

Make sure that the seal assembly is oriented with the scavenger port facing the centrifuge gasket portholes (see Figure B.8).

Remove the plug from the gasket hole nearest the front of the centrifuge.

Assembly Instructions

Route a piece of tubing through the hole, as shown in Figure B.8, from outside to inside the centrifuge. This tubing must be long enough to reach from the scavenger port to a

20-kPa (3-psi) vacuum trap outside the centrifuge. Carefully cut the tubing at an angle to make insertion easier, and then cut it straight across after insertion.

CAUTION

Do not route the scavenger line through the hole with the slit in the rubber (nearest the rear of the centrifuge), or the door seal will be compromised, affecting the vacuum level inside the chamber.

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Assembly Instructions

Figure B.8 Attaching a Scavenger Line

1. Scavenger Line

2. Scavenger Port

Attach the tubing to the scavenger port. Gently pull the tubing back through the porthole until there is no excess tubing inside the centrifuge.

Attach the other tubing end to a vacuum trap outside the centrifuge.

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Beckman Coulter JCF-Z Instructions For Use Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Revision History

Safety Notice

Alerts for Danger, Warning, Caution, Important, and Note

Contents

Illustrations

Tables

Continuous Flow Centrifugation

ZONAL CENTRIFUGATION

Rotor Components

Specifications

Installing the Zonal Bracket (Avanti J Series Centrifuges Only)

Installing the Zonal Interlock Accessory

Tools and Supplies

Tools Provided

Supplies Required But Not Provided

Assembling the Rotor

Step 1

Step 1a

Step 1b

Step 1c

Step 1d

Step 2

Step 3

Step 4

Step 5

Step 6

Step 6a

Step 6b

Step 7

Standard zonal and continuous flow rotors.

Standard Seal Housing

High Flow Seal Housing

Step 8

Standard zonal and continuous flow rotors

Step 9

Step 9a

Step 9b

Step 10

Step 11

Step 12

Step 13

Determining the Rotor Speed

Determining The Flow Rate: Large And Standard Pellet Cores

Setting The Temperature

Avanti Series Centrifuges

Microprocessor-controlled J2 And J6 Series Centrifuges (Models J2-MI, J6-MI, J2-21M, J2-MC, J6-MC, and J2-21 M/E)

Determining The Liquid Pressure

Analog J2 And J6 Series Centrifuges (Models J2-HS, J2-21, J2-HC, J6-HC, and J-6B)

Determining Appropriate Gradients

Operating Precautions

Continuous Flow Operation

Pelleting Technique

Run Procedure For Pelleting Sample (Continuous Flow Cores)

Purging Air From The System (Continuous Flow Rotor Only)

Density Gradient Operation (Continuous Flow Cores)

Gradient Or Cushion Separation Techniques

Forming an Air Block

Zonal Operating Techniques And Run Procedures

Run Procedure for Separating in a Gradient or Cushion (Continuous Flow Cores)

Standard Zonal Core

Reorienting Gradient (Reograd) Zonal Core

Ending The Run

Continuous Flow Core Containing Pellet

Continuous Flow Core Containing Gradient or Cushion

Standard Zonal Core

Reograd Zonal Core

Flushing The Tubing Lines

Maintenance

Cleaning

Decontamination

Sterilization And Disinfection

Inspecting The Bearings

Smoothing (Lapping) The Rotating Seal

Returning a Rotor

Supply List

Leakage