YMC Pilot PLUS User Manual

Original-

Manual

Low pressure chromatography column

YMC Pilot

PLUS

In order to avoid personal injuries and damage to equipment, please read this manual carefully and

act on the recommendations given. Keep this manual accessible for the user at all times.

Version 1.3

26.03.2018

YMC Europe GmbH

Schöttmannshof 19
46539 Dinslaken, Germany

[phone]

[e-mail]

www.ymc.de

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YMC Europe GmbH

Contents

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Contents

1 Information on manual ........................................................................................... 5

1.1 General ................................................................................................................ 5

1.2 Explanation of warnings ....................................................................................... 5

1.3 Structure of instructions ....................................................................................... 6

1.4 Guidelines ............................................................................................................ 6

1.5 Figures ................................................................................................................. 6

2 Safety ....................................................................................................................... 7

2.1 Intended use ........................................................................................................ 7

2.2 General safety instructions .................................................................................. 7

2.3 Personnel requirements ....................................................................................... 7

2.4 Personnel protective equipment .......................................................................... 8

3 Specifications and overview .................................................................................. 9

3.1 Fields of application ............................................................................................. 9

3.2 Specifications ....................................................................................................... 9

3.3 Chemical Resistance ......................................................................................... 10

3.4 Overview ............................................................................................................ 11

3.4.1 Adjustable end piece .................................................................................... 12

3.4.2 Column body................................................................................................. 13

3.4.3 Column stand................................................................................................ 14

4 Delivery .................................................................................................................. 15

4.1 Delivery contents ............................................................................................... 15

4.2 Delivery inspection ............................................................................................. 15

5 Requirements for Operation ................................................................................ 16

5.1 Pilot column set up ............................................................................................ 16

5.2 Pressure test ...................................................................................................... 16

6 Operation ............................................................................................................... 18

6.1 Removal of adjustable end piece ...................................................................... 18

6.2 Pilot column packing .......................................................................................... 19

6.2.1 Slurry preparation ......................................................................................... 19

6.2.2 Slurry column packing under flow................................................................. 20

6.2.3 Slurry column packing with sedimentation ................................................... 24

6.3 Column test and release criteria ........................................................................ 27

6.4 Pilot column unpacking ...................................................................................... 28

7 Cleaning and maintenance .................................................................................. 30

7.1 Notes for Cleaning-In-Place (CIP) ..................................................................... 31

7.2 Cleaning and maintenance plan ........................................................................ 32

7.3 Cleaning ............................................................................................................. 33

7.4 Replacement of spare parts............................................................................... 36

7.4.1 Frit replacement in the adjustable end piece ................................................ 36

7.4.2 Replacing the glass body ............................................................................. 37

7.4.3 Frit replacement column body ...................................................................... 40

8 Troubleshooting .................................................................................................... 42

9 Storage, packaging and transport ...................................................................... 44

9.1 Storage .............................................................................................................. 44

9.2 Transport............................................................................................................ 44

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10 Disposal ................................................................................................................. 45

11 Appendix ................................................................................................................ 46

11.1 Spare parts column stand .................................................................................. 46

11.2 Spare parts adjustable end piece ...................................................................... 46

11.3 Spare parts column body 70 – 100 mm ID ........................................................ 47

11.4 Spare parts column body 140 – 200 mm ID ...................................................... 48

12 Index ....................................................................................................................... 49

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Information on manual

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DANGER

WARNING

CAREFUL

ATTENTION

1 Information on manual

1.1 General

This manual includes important massages and information for the intended use of this
pilot column. The manual must be kept accessible for the user at all times.

In order to avoid injuries and damages, read this manual carefully prior to use and act
on the recommendations given.

1.2 Explanation of warnings

Warnings are highlighted by coloured signal word fields. Always read the full text of
the warning in order to effectively protect yourself from damage!

The following signal word fields marked by different colours and signal words distinct
security levels:

Disregarding this warning will result in death or serious injuries.

Disregarding this warning may result in death or serious injuries.

Disregarding this warning may result in minor or moderate injuries.

Disregarding this warning can result in damage to surrounding property.

Warnings are always in the same structure. They include signal word, type and source
of danger, consequences of neglect and security measures / avoidance.

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Example for additional information

1.3 Structure of instructions

The instructions relate directly to an activity. They are structured in a task oriented
manner. Always perform the individual steps in the order in which they are given.

Instructions are structured as follows and are marked by the corresponding levels:

 Aim of the instruction

1. Action step
 Impact of the action step to check whether the step has been performed

correctly.

2. Another action step

Figures within instructions:
A figure is only related to the action step which is listed directly below the figure.

1.4 Guidelines

The following guidelines are available in this manual:

• Contents at the beginning

• Index in the end

• Name of the main Chapter in the header

• Links to other Chapters, for example “Information in this manual“ ( Chapter 1, S.

5)

• Additional information and hints indicated with an i-symbol

1.5 Figures

Figures can differ in the graphical presentation from the product.

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Safety

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2 Safety

This section gives an overview of all the important safety aspects for the protection of
personnel and for the safe and trouble-free handling of the YMC Pilot
Other task-specific safety requirements are contained in the sections relating to the
respective operation. In addition, the national and local legal and other applicable
safety regulations must be observed.

2.1 Intended use

The pilot columns are only to be used for the separation of substances via
chromatographic techniques.

The intended use of this product must be performed with regards to the specifications

Chapter 3.2, S. 9. The product is only for use with liquids that have a pressure <0.5
bar above the atmospheric pressure at the intended maximum temperature (40°C).
Every use which is outside these specifications is defined as not intended use. The
intended use includes the compliance with the advice and terms in the manual.

PLUS

column.

2.2 General safety instructions

In order to avoid accidents and injuries,

• operate the pilot column only within its intended use.

• follow all general and special safety instructions as well as all warnings in this

manual.

• follow danger signs and sign giving instructions.

• follow the rules and regulations for prevention of accidents at the site of operation.

• comply with all inspection and maintenance intervals.

2.3 Personnel requirements

The various tasks described in this manual make different demands on the
qualifications and training of the persons carrying them out.

The user must:

• have competent and proficient training.

• be trained in dealing with pressure devices.

• read and fully understand this manual before using this YMC Pilot

PLUS

column.

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Symbol

PPE

Phase of life

Action

Protective gloves

Operation

• Column packing

• Column unpacking

Initiation

• Cleaning

Safety goggles

Operation

• All actions

Initiation

• Cleaning

• Pressure testing

Dust mask

Operation

• Slurry preparation

Protective clothing

Operation

• All actions

Initiation

• Cleaning

• Pressure testing

2.4 Personnel protective equipment

In order to reduce the risks during working on the column, always use the
recommended personnel protective equipment.

The following protective equipment is required:

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PED* fluid group:

Group 1 referred to EU Guideline 2014/68/EU

PED* safety requirement:

Article 4.3 of EU Guideline 2014/68/EU

Column body:

Borosilicate glass 3.3
Calibrated precision glass (Duran KPG®)

Frits:

PE

Seals:

75 Fluoroprene XP41 blue

Connection:

FEP tubing ¼" OD

Wetted parts:

Borosilicate glass 3.3, FEP, PE, PTFE, PEEK, 75
Fluoroprene XP41 blue

Approved temperature range

4 – 40°C

Length Glass Body
[mm]

500

850

Inner Diameter
[mm]

70

100

140

200

70

100

140

200

Bed Length
[mm]

100 - 4501

250 - 8001

Volume [mL]

0.4
–

1.7

0.8
–

3.5

1.5
–

6.9

3.1
–

14.1

1.0
–

3.0

2.0
–

6.2

3.8
–

12.3

7.8
–

25.1

Pressure Limit [bar]

16

10 7 5

16

10 7 5

Total Weight Empty
Column [kg]

19

20

37

38

23

24

45

47

Footprint [mm]

620

750

620

750

Maximum Height [mm]

1,700

1,750

2,250

2,300

3 Specifications and overview

3.1 Fields of application

The YMC Pilot
is compatible with common BioLC separation modes (IEX, SEC, Affinity, IMAC, HIC,
MMC) and aqueous conditions. Due to the metal-free and the minimal dead volumes,
the column is perfectly suitable for the separation of biomolecules (e.g. proteins,
oligonucleotides or nucleic acids).

3.2 Specifications

PLUS

pilot column is especially designed for pilot scale use. The column

*Pressure Equipment Directive

1

Depending on packing material and packing method

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If the desired solvent is not listed, YMC will be pleased to advise you.

Material

Borosilicate

glass 3.3

FEP

75

Fluoro-

pren

XP41

PE

PTFE

PEEK

Lösungsmittel

Acetone

Good

Good

Unstable

Good

Good

Good

Acetonitrile

Good

Good

Limited

Good

Good

Good

Dihydrogen phosphate

Good

Good

Good

Good

Good

Good

Cyclohexane

Good

Good

Good

Limited

Good

Good

Dichloromethan

Good

Good

Limited

Unstable

Good

Unstable

0.1M EDTA (3%)

Good

Good

Good

Good

Good

Good

1M Acetic acid (6%)

Good

Good

Good

Good

Good

Good

Ethanol

Good

Good

Good

Good

Good

Good

Ethyl acetate

Good

Good

Unstable

Good

Good

Good

n-hexane

Good

Good

Good

Limited

Good

Good

Isopropanol

Good

Good

Good

Good

Good

Good

Methanol

Good

Good

Good

Good

Good

Good

2M Sodium hydroxide

(8%)

Good

Good

Good

Good

Good

Good

1M Hydrochloric acid

(4%)

Good

Good

Good

Good

Good

Good

8M Urea (36%)

Good

Good

Good

Good

Good

Good

1M NaCl

Good

Good

Good

Good

Good

Good

0,5M Na2SO4

Good

Good

Good

Good

Good

Good

3.3 Chemical Resistance

The chemical resistance depends on the wetted materials used Chapter 3.2, S. 9.

YMC recommends avoiding the following chemicals:

• Strong oxidizing agents

• Halogenated compounds

• Chlorinated solvents

• Aromatic compounds (e.g. toluene)

• High concentrations of strong acids

• THF

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A parts list with all wetted parts and non-wetted parts is in the appendix.

Adjustable end piece

Column body

Column stand

3.4 Overview

The YMC Pilot

PLUS

column consists of the adjustable end piece, the column body and
the column stand. These parts can be assembled whilst operating the column without
any tools.

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1

Tubing

4

Star screw

2

Spindle

5

Closing disc (AEP)

3

Rotary Handle

6

Connecting flange

1

Spindle

4

Frit

2

Connecting flange

5

Profile gasket

3

Plunger (AEP)

3.4.1 Adjustable end piece

The adjustable end piece consists of a closing disc and the piston. The piston consists
of the spindle, the connecting flange, the plunger, the frit and the profile gasket. A
tubing goes through the spindle to the connecting flange. With the rotary handles in
the closing disc, the piston can be adjusted up and down. The closing disc is attached
with star screws to the column body.

Piston front

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1

Cylinder screw

6

Connector

2

Column disc

7

Glass body

3

Connector drainage tubing

8

Rod 4 Drainage tubing

9

Holding bar

5

Closing disc (CB)

3.4.2 Column body

The glass body is fixed between the column disc and the closing disc by the rods. The
glass body has a visible scale. Excessive solvent is removed during piston insertion
via the connector drainage tubing. The fixed end piece is assembled with the closing
disc (CB). The fixed end piece consists of the connector, the sealing washer, the
plunger (CB), the ring gasket B, the frit (CB) and the ring gasket A. The column body
can be assembled in the stand in two different positions.

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1

Connector without cap nut

4

Frit (CB)

2

Plunger (CB)

5

Ring gasket A

3

Ring gasket B

1

Levelling feet

3

Frame disc

2

Main pillar

4

Locking bolts

Frit column body

3.4.3 Column stand

With the locking bolts in the frame disc the column body is attached to the column
stand. With the adjustable levelling feet, the pilot column can be aligned vertically.
Alternatively, the column stand is available with casters.

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YMC recommends retaining and storing the original packaging and the
foam cushion so that the column can always be shipped safely.

4 Delivery

4.1 Delivery contents

• Pilot column

• Documentation folder:
– Brochure YMC Glass Columns
– Manual
– Overview drawings
– YMC Quality Control / Leakage Tests
– Certificate of conformity FDA/BSE/TSE/USP Class VI

4.2 Delivery inspection

Delivery inspection

 Delivery inspection is performed as follows:

1. Check the delivery for visible transport damage.

2. Check the delivery for the completeness of parts.

3. Compare the column description on the glass body with the data of the delivery
note.

Transport damages

 Procedure, if you observe any visible transport damage:

1. Refuse or reserve acceptance of the delivery.

2. Record the transport damage on the delivery note of the transporter or in the
transport documents.

3. Inform YMC Europe GmbH and/or the responsible supplying agent.

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WARNING

WARNING

5 Requirements for Operation

5.1 Pilot column set up

Required devices:

• Drainage tubing

• Collecting vessel

 The pilot column is set up as follows:

1. Make sure that the pilot column is standing on fixed ground.

2. Make sure that the pilot column is standing vertical in the final operating place.

3. Connect the drainage tubing to the drainage tubing connector.

4. Put the drainage tubing in the collecting vessel.

5.2 Pressure test

Operation of pressure tests

Eye or skin injuries possible due to solvent leakage or incorrectly sealed connectors.

► Perform the pressure test behind a safety screen.
► During work on the pilot column, wear the prescribed personnel protective

equipment.

Pilot column under high pressure

Eye or skin injuries possible due to remaining pressure in the tubing or in the pilot
column.

► Stop the pump during work on the pilot column.
► During work on the pilot column, wear the prescribed personnel protective

equipment.

A pressure test is used to check the tightness of both the seals in the pilot column and
also the tubing connectors. Before using the column for chromatographic applications,
it is recommended that a pressure test is performed to verify the correct assembly.
Prior to delivery of the column, the column has been tested according to the standard
procedure listed below. The relevant certificate is located in the documentation folder.

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1

Pilot column

4

Pressure gauge

2

Back pressure regulator

5

Gate valve

3

Gate valve

6

Pump

If a back pressure regulator is used, the maximum pressure is reached
easier.

Leakages at the piston of the adjustable end piece are often related to
impurities trapped in the seal.

Standard procedure pressure test

 The pressure test should be performed as follows:

1. Assemble the system according to the schematic overview of a pressure test.

2. Turn the piston of the adjustable end piece in the DOWN direction as far as it
will go.

3. Fill the pilot column with isopropanol.

4. Increase the testing pressure in the pilot column by slow and stepwise
adjustment of the flow rate at the pump.

 The testing pressure is the pressure limit of the column plus 10% (70

mm ID = 17.6 bar | 100 mm ID = 11 bar | 140 mm ID = 7.7 bar | 200
mm ID = 5.5 bar)

5. Keep the testing pressure constant for 10 minutes.

6. If leakages occur, check the connections. If required, replace tubing and/or
connectors.

 If the testing pressure was constant for 10 minutes, the column is ready for use.

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WARNING

6 Operation

Risk of explosion

Severe injuries possible due to glass splinters.

► Never put the glass column under gas pressure!

In order to guarantee safe operation, the pilot column must be used for the intended
use only. ( Chapter 2.1, S. 7).

6.1 Removal of adjustable end piece

Required devices:

• Clean and soft surface

 The adjustable end piece is removed as follows:

1. Turn the piston of the adjustable end piece in the UP direction as far as it will
go.

2. Remove the star screws from the closing disc (AEP).

3. Remove the adjustable end piece from the glass body and put it on a clean
and soft surface.

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CAREFUL

CAREFUL

The use of a more dilute liquid slurry simplifies the column packing. The
total volume of the slurry should not exceed the maximum volume of the
column.

The removal of fines decreases the column back pressure and so
improves the performance of the packed column.

6.2 Pilot column packing

Packing material

Eye and skin injuries possible through contact with packing material.

► Wear the prescribed personnel protective equipment.

Two different procedures are common for slurry packing the column:

• under flow,

• with sedimentation.

6.2.1 Slurry preparation

Formation of dust with dry packing material

Lung irritation possible if packing material is inhaled.

► Read the material safety data sheet from the packing material manufacturer.
► Wear a dusk mask.

 The slurry is prepared as follows:

1. Mix the solvent or buffer with the appropriate amount of packing material in a
suitable container (see manufacturer’s recommendations).

 The total volume of the slurry must be less than the maximum column volume.
 The amount of packing material determines the final bed length.

2. Mix the slurry with a stirring rod until the slurry is homogenous and avoid
introduction of air bubbles.

3. Check if the material is suitable for ultrasonication (see manufacturer’s
recommendations).

 If the material is suitable for ultrasonication, degas the slurry in an ultrasonic

bath.

 If the material is not suitable for ultrasonication, let the material settle.

4. Let the degassed slurry settle.

5. If fines are visible on the surface of the slurry, remove them.

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6.2.2 Slurry column packing under flow

 The column is packed with the slurry method under flow as follows:

1. Remove the adjustable end piece Chapter 6.1, S. 18.

2. Check the drainage tubing for correct connection.

3. Check the drainage tubing outlet for correct position in the collecting vessel.

4. Connect the pump with the tubing at the column body.

5. Start the pump.

6. Fill the glass body with solvent or buffer until 2-3 cm above the frit in the
column body (bottom-up).

 This ensures that no air is trapped under or is inside the frit.

7. Close the valve in the tubing on the column body.

8. Connect the pump with the adjustable end piece.

9. Flush the piston of the adjustable end piece with slurry solvent until the frit of
the adjustable end piece is wetted completely.

10. Disconnect the pump from the adjustable end piece.

11. Pour the homogenous and/or degassed slurry into the glass body.

12. Rinse the inner wall of the column body with slurry solvent for removal of
particles.

 By removing particles from the inner wall of the column body, a correct seal

with the profile gasket is guaranteed.

13. Fill the pilot column up to the edge of the drain function with slurry solvent.

14. Turn the closing disc of the adjustable end piece in the UP direction as far as it
will go.

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15. Turn the closing disc of the adjustable end piece half turn in the DOWN
direction.

 The adjustable end piece is ready for insertion into the column body.

16. Put the piston of the adjustable end piece in a smooth angle opposite the drain
function on the column body.

17. Bring the piston of the adjustable end piece slowly to the vertical position.

 Excessive slurry solvent will flow into a reservoir through the drainage tubing.

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18. Align the marks on the closing disc (AEP) and column disc.

19. Screw the 4 star screws continuously in a diagonal pattern to each other.

20. Open the valve in the tubing on the adjustable end piece.

21. Turn the piston of the adjustable end piece in the DOWN direction until the
tubing is completely filled with liquid.

 The tubing is now free of air.

22. Connect the pump with the tubing of the adjustable end piece.

23. Open the valve in the tubing on the column body.

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24. Start the flow (top down).

25. Increase the flow rate stepwise until the desired pressure / flow rate is
reached. Wait after every step until the pressure is constant. Afterwards, the
flow rate can be further increased.

 Consider the manufacturer’s recommendations for the packing material.
 The flow rate should be considerably (> 20%) higher than the operating flow

rate.

 The column bed is visually compressed.

26. Check and mark the bed length frequently.

27. If the final bed length is reached, stop the pump.

28. Turn the piston of the adjustable end piece in the DOWN direction until the
column bed is reached.

29. Start the pump.

30. Check, if the column bed is further compressed. A dead volume would occur.

31. If a dead volume occurs, stop the pump and repeat step 28 to 30.

32. If no dead volume occurs, the pilot column is released Chapter 6.3, S. 27.

33. Remove liquid above the connecting flange through the two holes in the
adjustable end piece.

 YMC recommends flushing this area if high salt concentrations are used.

34. Equilibrate the pilot column with the desired eluent and test the column
performance.

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6.2.3 Slurry column packing with sedimentation

 The column is packed via the sedimentation slurry method as follows:

1. Remove the adjustable end piece Chapter 6.1, S. 18.

2. Check the drainage tubing for correct connection.

3. Check the drainage tubing outlet for correct position in the collecting vessel.

4. Connect the pump to the tubing of the column body.

5. Start the pump.

6. Fill the glass body with solvent or buffer until 2-3 cm above the frit in the
column body (bottom-up).

 This ensures that no air is trapped under or is inside the frit.

7. Close the valve in the tubing of the column body.

8. Disconnect the pump from the column body.

9. Connect the pump to the adjustable end piece.

10. Flush the piston of the adjustable end piece with slurry solvent until the frit in
the adjustable end piece is wetted completely.

11. Disconnect the pump from the adjustable end piece.

12. Pour the homogenous and/or degassed slurry into the glass body.

13. Rinse the inner wall of the column body with slurry solvent in order to remove
particles.

 By removing particles from the inner wall of the glass body, a correct seal with

the profile gasket is guaranteed.

14. Do not move the pilot column anymore.

 YMC recommends covering the open column body during sedimentation with

some plastic film.

15. Wait until the packing material has settled (sedimentation).

 Depending on the packing material, this can take some hours.

16. Fill the pilot column up to the edge of the drain function with slurry solvent.

17. Turn the closing disc of the adjustable end piece in the UP direction as far as it
will go.

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18. Turn the closing disc of the adjustable end piece half turn in the DOWN
direction.

 The adjustable end piece is ready for insertion into the column body.

19. Put the piston of the adjustable end piece in a smooth angle opposite the drain
function on the column body.

20. Bring the piston of the adjustable end piece slowly to the vertical position.

 Excessive slurry solvent will flow into a reservoir through the drainage tubing.

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21. Align the marks on the closing disc (AEP) and column disc.

22. Screw the 4 star screws continuously in a diagonal pattern to each other.

23. Open the valve in the tubing on the adjustable end piece.

24. Turn the piston of the adjustable end piece in the DOWN direction until the
column bed is reached.

25. Connect the pump to the tubing of the adjustable end piece.

26. Open the valve in the tubing at the column body.

27. Start the flow (top-down).

28. Increase the flow rate stepwise until the desired pressure / flow rate is
reached. Wait after every step until the pressure is constant. Afterwards, the
flow rate can be further increased.

 Consider the manufacturer’s recommendations for the packing material.
 The flow rate should be considerably (> 20%) higher than the operating flow

rate.

 The column bed is visually compressed.

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29. Check and mark the column bed length frequently.

30. If the final bed length is reached, stop the pump.

31. Turn the piston of the adjustable end piece in the DOWN direction until the
column bed is reached.

32. Start the pump.

33. Check if the column bed is further compressed. A dead volume would occur.

34. If a dead volume occurs, stop the pump and repeat step 31 to 33.

35. If no dead volume occurs, the pilot column is released. Chapter 6.3, S. 27.

36. Remove liquid above the connecting flange through the two holes in the
adjustable end piece.

 YMC recommends flushing this area if high salt concentrations are used.

37. Equilibrate the pilot column with the desired eluent and test the column
performance.

6.3 Column test and release criteria

The column test and the release criteria are selected with regards to the later
application field. The preparation of the required specifications for the final usage of a
method or for the purification of a target molecule is determined individually. YMC will
gladly support you.

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WARNING

CAREFUL

6.4 Pilot column unpacking

Pilot column under high pressure

Eye or skin injuries possible due to remaining pressure in the tubing or in the pilot
column.

► Stop the pump during working on the pilot column.
► While working on the pilot column, wear the prescribed personnel protective

equipment.

Packing material

Eye and skin injuries possible through contact with packing material.

► Wear the prescribed personnel protective equipment.

 The column is unpacked under flow as follows:

1. Close the valve in the tubing of the column body.

2. Disconnect the pump from the column body.

3. Ensure that the valve at the adjustable end piece is open.

4. Disconnect the pump from the adjustable end piece.

5. Turn the piston of the adjustable end piece in the UP direction until the profile
gasket does not touch the glass body anymore.

 The loosening of the profile gasket from the glass body is noticeable.

6. Remove the adjustable end piece Chapter 6.1, S. 18.

7. Put a collecting vessel below the column stand.

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8. Loosen the locking bolts and lock them by turning slightly.

9. Lift the column body vertical from the column stand using two holding bars
which are vertically above each other.

10. Pour any unpacked material and/or solvent into a collecting vessel.

11. If no material now remains in the column body, clean the column body
Chapter 7.3, S. 33.

12. If material remains in the column body, put the column body into the stand with
the open end facing down.

13. Turn the column body slowly in the frame disc until the locking bolts lock.

 You will hear a noticeable “click” when the locking bolts are locked.

14. Connect the pump with the tubing at the column body.

15. Open the valve in the tubing of the column body.

16. Start the pump.

 Because of the flow, the packing material is pressed out of the column body

into the collecting vessel.

17. Clean the pilot column Chapter 7.3, S. 33.

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WARNING

WARNING

WARNING

Consumable parts lists are included in the appendix.

7 Cleaning and maintenance

Incorrect maintenance

Severe injuries when used after incorrect maintenance is possible, if the column or
column parts do not withstand the operating pressure.

► Only authorised and qualified personnel should carry out the maintenance.

Incorrect replacement of spare parts

Severe injuries due to breakage possible if unauthorised spare parts are used.

► Use only the manufacturer authorised spare parts.

Faulty maintenance of the pilot column

Severe injuries due to deterioration possible.

► Meet maintenance intervals.
► Perform the maintenance as described.

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CAREFUL

ATTENTION

7.1 Notes for Cleaning-In-Place (CIP)

Packing material

Eye and skin injuries possible through contact with packing material.

► Wear the prescribed personnel protective equipment.

Cleaning with inappropriate solvents

Material damage possible due to corrosion, leaching or deformation.

► Check the chemical resistance of the wetted parts for the cleaning method before

the CIP procedure.

With the cleaning-in-place procedure, a packed pilot glass column can be cleaned
without unpacking or dismantling. Depending on the contamination, three different
procedures can be used:

1. Regeneration,

2. Sterilisation,

3. Depyrogenation.

Regeneration

Regeneration removes inorganic and organic contamination, which binds to the
chromatography matrix and substantially affects the capacity and resolution of the
column. Commonly these are: lipids, pyrogens, protein aggregates, pigments,
polyphenols or metal complexes.

Sterilisation

Sterilisation is the removal and/or destruction of microorganisms and spores that will
contaminate the purified product. The most common method is the treatment with
sodium hydroxide, acetic acid or ethanol solution.

Depyrogenation

Depyrogenation includes the removal of endotoxins, which can be bound on the
chromatographic material or the column hardware (frits, tubing, etc.) and which can
contaminate the purified product.

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Treatment

Type of

contamination

Sterilisation

Depyrogenation

Pepsin, pH 1.5 - 2

Proteolysis of adsorbed
proteins

No

No

Non-ionic detergents (Triton X­100; Tween 80)

Removal of hydrophobic
proteins and lipids

No

No

Cationic detergents, pH 9 - 11

Removal of hydrophobic
proteins and lipids

No

Partly

Urea, 6 - 8 M

Removal of protein
aggregates

No

Unknown

1 - 100 mM EDTA, pH
depending on the stationary
phase

Removal of metal
complexes

No

No

0.1 - 1 M NaOH

Removal of bounded
hydrophobic proteins,
lipopolysaccharides

Good

Good

0.5 - 1 M acetic acid in 60%
ethanol

Removal of lipids,
pigments,
lipopolysaccharides, and
other lipophilic
substances

Very good

Good

Interval

Maintenance

After every operation

• Clean the pilot column.

Daily

• Check the column bed, adjust the piston if necessary.

• Check all tubing and connections regarding leakage Chapter 5.2,

S. 16.

• Fix loosened fixing screws again or replace tubing and connectors.

Every 3 months

• Check the profile gasket in the adjustable end piece.
If damaged, replace it.

• Check the ring gasket of the column body.
If damaged, replace it.

Every 6 months

• Check the frit in the adjustable end piece.
If damaged, replace it Chapter 7.4.1, S. 36.

• Check the frit in the column body.
If damaged, replace it Chapter 7.4.3, S. 40.

Cleaning methods for CIP

7.2 Cleaning and maintenance plan

The specified maintenance plan here is to be regarded as a general recommendation.
Depending on the frequency of use and specific application, the appropriate
maintenance plan and the intervals must be defined locally.

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WARNING

CAREFUL

7.3 Cleaning

Pilot column under high pressure

Eye or skin injuries possible due to remaining pressure in the tubing or in the pilot
column.

► Stop the pump while working on the pilot column.
► When working on the pilot column, wear the prescribed personnel protective

equipment.

Packing material

Eye and skin injuries possible through contact with packing material.

► Wear the prescribed personnel protective equipment.

Required devices:

• Wetted and lint-free rags

• Collecting vessel

• Solvent for column body and adjustable end piece cleaning Chapter 3.3, S. 9

• Cleaning agent for cleaning the outer wall of the column body

Authorised cleaning agents for cleaning the outer wall of the
column body:

• Commercially available dishwashing cleaners

• Aliphatic hydrocarbons

• Aliphatic alcohols

• Aromatic hydrocarbons

• Aldehydes

• Diluted and weak acids

• Bases

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 The pilot column is cleaned as follows:

1. Remove the adjustable end piece Chapter 6.1, S. 18.

2. Clean the outer wall of the column body.

3. Clean the column body with an authorised cleaning agent and a wetted, lint­free rag.

4. Put a collecting vessel below the column stand.

5. Loosen the locking bolts and lock them by turning slightly.

6. Lift the column body vertical from the column stand using two holding bars
which are vertically above each other.

7. Put the column body with the open end facing downwards in the column stand.

8. Turn the column body slowly in the frame disc until the locking bolts lock.

 You will hear a noticeable “click” when the locking bolts are locked.

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For a rigorous cleaning, dismantle the pilot column.

9. Connect the pump to the tubing in the column body.

10. Start the pump.

11. Flush the column body with an appropriate solvent and/or 1 M sodium
hydroxide.

12. Stop the pump.

13. Disconnect the pump from the tubing in the column body.

14. Loosen the locking bolts and lock them by turning slightly.

15. Lift the column body vertical from the column stand using two holding bars
which are vertically above each other.

16. Insert the column body in the starting position again.

17. Turn the column body slowly in the frame disc until the locking bolts lock.

 You will hear a noticeable “click” when the locking bolts are locked.

18. Clean the adjustable end piece with an appropriate solvent and/or 1 M sodium
hydroxide.

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ATTENTION

7.4 Replacement of spare parts

Faulty assembly

Damage to the glass body possible due to faulty assembly.

► Take care during the assembly of the glass body that the maximum torque of 5

Nm for columns with 70 and 100 mm ID respectively 10 Nm for column with 140
mm and 200 mm ID are not exceeded.

► Assemble the cylinder screws in a diagonal pattern and always tighten on the

opposite sites.

► Take care during the assembly of the glass body that all parts are correctly

aligned.

7.4.1 Frit replacement in the adjustable end piece

 The frit in the adjustable end piece is replaced as follows.

1. Remove the adjustable end piece Chapter 6.1, S. 18.

2. Remove the profile gasket.

3. Remove the frit.

4. Insert the new frit. The distribution profile faces the plunger.

5. Reattach the profile gasket and press firmly in position.

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ATTENTION

7.4.2 Replacing the glass body

Faulty assembly

Damage to the glass body possible due to faulty assembly.

► Take care during assembly of the glass body that the maximum torque of 5 Nm

for columns with 70 and 100 mm ID respectively 10 Nm for column with 140 mm
and 200 mm ID are not exceeded.

► Assemble the cylinder screws in a diagonal pattern and always tighten on the

opposite sites.

► Take care during assembly of the glass body that affected parts are correctly

aligned

Required devices:

• Cylinder screw key / torque wrench

 The glass body is dismantled as follows:

1. Remove the adjustable end piece Chapter 6.1, S. 18.

2. Loosen the cylinder screws stepwise half turn. Do it diagonally: Loosen
cylinder screws always on opposite sites.

3. Remove cylinder screws.

4. Remove column disc from the column body.

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5. Remove flat gasket.

6. Remove glass body carefully.

7. Position the new glass body on the ring gasket A.

8. Check that the glass body is concentric with the gaskets.

9. Put the flat gasket corresponding to the structure on the top edge of the glass
body.

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10. Adjust the column disc over the rods and lower it onto the glass body.

11. Check that the flat gasket is in the groove in the column disc.

12. Check that the glass body is concentric with the column disc.

13. ATTENTION Damage to the glass body is possible due to faulty

assembly. Note the maximum torque during the assembly of the glass
body.

Insert the cylinder screws in a diagonal pattern and tighten them by gradually
increasing the torque to screws on the opposite sides. Adjust to a maximum

torque of 5 Nm for columns with 70 and 100 mm ID respectively 10 Nm for
columns with 140 and 200 mm ID.

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ATTENTION

7.4.3 Frit replacement column body

Faulty assembly

Damage to the glass body is possible due to faulty assembly.

► Take care during assembly of the glass body that the maximum torque of 3 Nm is

not exceeded.

► Assemble the cylinder screws in a diagonal pattern and always tighten on the

opposite sites.

► Take care during assembly of the glass body that affected parts are correctly

aligned.

Required devices:

• Cylinder key/torque wrench

 The frit in the column body is replaced as follows:

1. Remove the adjustable end piece Chapter 6.1, S. 18.

2. Dismantle the glass body Chapter 7.4.2, S. 37.

3. Remove the union nut of the connector.

4. Press the fixed end piece with the connector out of the closing disc (CB).

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5. Remove the ring gasket A.

6. Remove the frit.

7. Insert the frit. The distribution profile faces the plunger.

8. Reattach the ring gasket A and press firmly in position.

9. Replace the fixed end piece into the closing disc (CB).

10. Fix the union nut at the connector.

11. Assemble the glass body Chapter 7.4.2, S. 37.

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WARNING

WARNING

Fault description

Possible cause

Solution

Dead volume occurred

Exceeding the maximum
allowable pressure of the
column packing

► If dead volume occurs, proceed as
follows:

1. Stop the pump.

3. Readjust the piston of the

adjustable end piece.

Stressed column packing

Peak shape of the eluted
substances deteriorates

Dead volume at the column
inlet

See fault description “Dead volume
occurred”

Partially clogged inlet frit

Frit replacement Chapter 7.4.1, S. 36

Partially clogged outlet frit

Frit replacement Chapter 7.4.3, S. 40

Separation performance of
the stationary phase altered
by impurities

Wash the stationary phase. Contact
also the manufacturer of the packing
material Chapter 7.1, S. 31

Stationary phase
mechanically destroyed

Replace packing material

Air in the column

Degassing or evaporation of
solvent during storage

New column conditioning. Possibly
new packing material is necessary.

Exceptional pressure rise
during operation

Valve circuit incorrect

Check valve positions

Clogged frit

Frit replacement Chapter 7.4, S. 36

Over-tightened fixing
screws

Replace fixing screws and clamping
tubing, cut the end of the tubing

Pressure drop during
operation

Leakage of tubing or fitting
between pump and column

Check tubing and connections
Depleted solvent reservoir

Refill solvents

8 Troubleshooting

Incorrect maintenance

Severe injuries during operation after incorrect maintenance possible if the column or
column parts do not withstand the operating pressure.

► Only authorised and qualified personnel should carry out the maintenance.

Incorrect replacement of spare parts

Severe injuries due to breakage possible, if not authorised spare parts are used.

► Use only the manufacturer authorised spare parts.

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Fault description

Possible cause

Solution

Solvent drips from the
column

Column body leaks

► If the column body leaks, proceed
as follows:

1. Visual check, if solvent leaks
between glass body and flat
gasket.

2. Check the contact surface of the
glass body and the sealing
surface for impurities.

3. Check the torque and the
concentric arrangement of the
parts.

Tubing connection leaks

► If the tubing connection leaks,
proceed as follows:

1. Tighten the fixing screw.

2. Check tightness of tubing
connection.

3. New column conditioning.

If faults occur which are not listed here, please contact YMC for support.

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WARNING

For storage of packed columns, the chemical resistance of the wetted
parts and of the packed stationary phase (please contact the
manufacturer) should be considered.

9 Storage, packaging and transport

Solvents in the pilot column

Dangers possible due to liquids leaking from the column during transportation.

► Unpack and clean the column before transport.

9.1 Storage

The pilot column should be stored as follows:

• unpacked

• cleaned

• dry

• empty

9.2 Transport

We recommend the use the original wooden box for transportation. If you are no
longer in possession of the box, please pack the column in a similar container.

 The column is packaged as follows:

1. Unpack the pilot column Chapter 6.4, S. 28.

2. Clean the pilot column Chapter 7.3, S. 33.

3. Insert the adjustable end piece.

4. Wrap the glass body in a bubble wrap.

5. If existing, fix the wheels to the base of the wooden box.

6. Make sure that there is a minimum distance of 100 mm from the outer wall.

7. Fix the pilot column in the wooden box with bubble wrap.

8. Attach appropriate warning signs to the box (e.g. “Caution breakable glass”
and “Top”).

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10 Disposal

For the disposal of the pilot column it has to be disassembled and disposed according
to the national and other applicable environmental regulations.

YMC Europe GmbH

Appendix

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Pos.

ID [mm]

Part-No.

Description

Material

1

70 - 100

00128

Sliding disc

Teflon (PTFE)

2

140 - 200

000203

Sliding disc

Teflon (PTFE)

Pos.

ID [mm]

Part-No.

Description

Material

1

70

000014

Profile gasket

75 Fluoroprene

XP41

2

70

000022

Frit (AEP)

Poroplast UHMW-

PE

3

100

000114

Profile gasket

75 Fluoroprene

XP41

4

100

000122

Frit (AEP)

Poroplast UHMW-

PE

5

140

000146

Profile gasket

75 Fluoroprene

XP41

6

140

000142

Frit (AEP)

Poroplast UHMW-

PE

7

200

000204

Profile gasket

75 Fluoroprene

XP41

8

200

000208

Frit (AEP)

Poroplast UHMW-

PE

11 Appendix

11.1 Spare parts column stand

11.2 Spare parts adjustable end piece

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Appendix

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Pos.

ID [mm]

Length glass

body [mm]

Part-No.

Description

Material

1

70 000006

Flat gasket

EPDM white

(0082E)

2

70

500

000007

Glass body

Borosilicate

glass 3.3

3

70 SI15,0LM21,0

Drainage tubing

Silicone

4

70 000024

Ring gasket A

75 Fluoroprene

XP41

5

70 000023

Frit (CB)

Poroplast

UHMW-PE

6

70 000015

Ring gasket B

PTFE (USP)

7

70

850

000057

Glass body

Borosilicate

glass 3.3

8

100 000106

Flat gasket

EPDM white

(0082E)

9

100

500

000107

Glass body

Borosilicate

glass 3.3

10

100 SI15,0LM21,0

Drainage tubing

Silicone

11

100 000130

Ring gasket A

75 Fluoroprene

XP41

12

100 000123

Frit (CB)

Poroplast

UHMW-PE

13

100 000115

Ring gasket B

PTFE (USP)

14

100

850

000157

Glass body

Borosilicate

glass 3.3

11.3 Spare parts column body 70 – 100 mm ID

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Appendix

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Pos.

ID [mm]

Length glass

body [mm]

Part-No.

Description

Material

1

140 000141

Flat gasket

EPDM white

(0082E)

2

140

500

000147

Glass body

Borosilicate

glass 3.3

3

140 000140

Ring gasket A

75 Fluoroprene

XP41

4

140 000143

Frit (CB)

Poroplast

UHMW-PE

5

140 000162

Ring gasket B

PTFE (USP)

6

140 SI20,0LM24,0

Drainage tubing

Silicone

7

140

850

000148

Glass body

Borosilicate

glass 3.3

8

200 000211

Flat gasket

EPDM white

(0082E)

9

200

500

000200

Glass body

Borosilicate

glass 3.3

10

200 000216

Ring gasket A

75 Fluoroprene

XP41

11

200 000213

Frit (CB)

Poroplast

UHMW-PE

12

200 000201

Ring gasket B

PTFE (USP)

13

200 SI20,0LM24,0

Drainage tubing

Silicone

14

200

850

000250

Glass body

Borosilicate

glass 3.3

11.4 Spare parts column body 140 – 200 mm ID

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Index

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12 Index

C

CIP 31
Cleaning 33
Cleaning-In-Place 31

D

Delivery contents 15
Disposal 46

F

Frit replacement

adjustable end piece 36
column body 40

G

Glass body replacement 37

P

Packaging 45
Packing 19
Presonnel protective equipment 8
Pressure test 16
Protective equipment 8

R

Resistance, chemical 10

S

Signal word 5
Signal word field 5
Slurry

Slurry preparation 19

Slurry method sedimentation 24
Slurry method under flow 20
Storage 45

T

Transportation 45

U

Unpacking 28

W

Warnings 5
Warning messages 6