Pickering Laboratories PCX 5200 User manual

Operation Manual
PCX 5200
Post-column
Derivatization Instrument
Pickering Laboratories, Inc.
1280 Space Park Way
Mountain View, CA 94043
(800) 54-3330
International: ( 50) 94- 700
Fax: ( 50) 9 8-0749
Website: http://www.pickeringlabs.com
E-mail: [email protected]
Cat. No. 0101-0001
Version , January 2002
© Pickering Laboratories, Inc.
PI
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PCX 5200 User’s Manual Contents

PCX 5200 User’s Manual Contents
Contents
Please Read ..................................................................................................BYS-1
Symbols.........................................................................................................BYS-1
Chapter 1 • Introduction
Why Post-coIumn Derivatization? ...................................................................... 1-1
How Does It Work?............................................................................................. 1-1
What Are the Requirements? ............................................................................. 1-1
Pickering PCX5200 ............................................................................................ 1-2
Reagent Delivery......................................................................................................................1-2
Column Protection....................................................................................................................1-2
Supplement—The Elements of an HPLC with Post-column System.................. 1-4
Design of an HPLC ..................................................................................................................1-4
A Simpler View of an HPLC .....................................................................................................1-5
Designing a Post-column System ............................................................................................1-5
Chapter 2 • PCX5200 System Components
PCX5200 Post-column Instrument ..................................................................... 2-1
Getting to Know Your PCX5200 ......................................................................... 2-1
Front Panels .............................................................................................................................2-1
Internal .....................................................................................................................................2-3
Rear Panel ...............................................................................................................................2-4
Column Oven ...........................................................................................................................2-6
Reagent Reservoirs .................................................................................................................2-8
External Reagent Pressurization System ................................................................................2-9
Keypad and Liquid Crystal Display User Interface ................................................................2-10
Keypad Examples............................................................................................. 2-13
Loading a Preset ....................................................................................................................2-13
Saving a Preset......................................................................................................................2-15
Flow Diagrams.................................................................................................. 2-18
PRESET Key..........................................................................................................................2-18
PROG Key..............................................................................................................................2-18
Specifications ................................................................................................... 2-20
Wetted Materials, PCX5200...................................................................................................2-20
PCX5200 PEEK .....................................................................................................................2-20
Ratings ...................................................................................................................................2-20
RS232 Serial Port .................................................................................................... ....2-21
Downloading firmware............................................................................................................2-21
Radio frequency .....................................................................................................................2-21

PCX 5200 User’s Manual Contents
Chapter 3 • Installation
Unpacking........................................................................................................... 3-1
Site Requirements .............................................................................................. 3-1
HPLC System Requirements ...................................................................................................3-1
Space Requirements................................................................................................................3-2
Electrical Requirement .............................................................................................................3-2
Inert Gas...................................................................................................................................3-2
Miscellaneous Supplies............................................................................................................3-2
Installation of the PCX5200 ................................................................................ 3-2
Layout of the HPLC and PCX5200 ..........................................................................................3-2
Note About Fittings...................................................................................................................3-3
Inert Gas...................................................................................................................................3-3
Reagent Reservoir Connections ..............................................................................................3-3
Pressure-interlock Connections ...............................................................................................3-4
Injector Connections ................................................................................................................3-6
Detector Connections...............................................................................................................3-6
Guard & Analytical Column Installation....................................................................................3-7
Reagent Pump .........................................................................................................................3-8
Piston-wash System.................................................................................................................3-8
Priming the Reagent Pump......................................................................................................3-9
System Testing & Verification...................................................................................................3-9
Shutdown.......................................................................................................... 3-10
Short-term ..............................................................................................................................3-10
Medium-term .......................................................................................................................... 3-11
Long-term ...............................................................................................................................3-12
Chapter 4 • Routine Maintenance & Troubleshooting
Initial System Testing.......................................................................................... 4-1
Test Chromatogram..................................................................................................................4-1
Paramater Log .........................................................................................................................4-1
Precautions & Problem Prevention..................................................................... 4-2
Mobile Phase ...........................................................................................................................4-2
Back-flow Prevention ...............................................................................................................4-2
Reactor Precautions.................................................................................................................4-2
Electrical Precaution ................................................................................................................4-2
Routine Maintenance.......................................................................................... 4-3
Reagent Pump .........................................................................................................................4-3
Pump Seal Replacement .........................................................................................................4-3
Check-valves............................................................................................................................4-6
Cleaning Check-valves ............................................................................................................4-6
Removing Check-valves ..........................................................................................................4-7
Installing New Check-valves ....................................................................................................4-7
Rebuilding Check-valves .........................................................................................................4-8
Pre-column Filter......................................................................................................................4-9
Reagent Filter.........................................................................................................................4-10
Ambient Reactor (not applicable to single reagent PCX5200) .............................................. 4-11

PCX 5200 User’s Manual Contents
Heated Reactor ...................................................................................................................... 4-11
Fuse .......................................................................................................................................4-12
Troubleshooting Guide ..................................................................................... 4-12
Advice.....................................................................................................................................4-12
When Priming the Reagent Pump is Difficult.........................................................................4-13
Interpretation of Pressures ............................................................................... 4-14
Two-reagent PCX5200...........................................................................................................4-14
One-reagent PCX5200...........................................................................................................4-14
Cleaning the Post-column System ................................................................... 4-15
Fittings....................................................................................................................................4-15
External ..................................................................................................................................4-15
Internal ...................................................................................................................................4-15
Internal Spill ...........................................................................................................................4-15
Appendix A
PCX5200 Parameter Log ...................................................................................A-1
Analytical Conditions ..........................................................................................A-2
Post-column Band-spreading Test......................................................................A-3
Appendix B
Spare Parts.........................................................................................................B-1
References
Limited Warranty
Index

Before You Start
Please Read
BYS-1
• Page 2-8 for the safety requirement of coated-bottles; they must not be substituted!
• Page 3-1 for the HPLC System Requirements, especially for glyphosate or amino
acid analysis! For most ion-exchange applications, the HPLC components must be
compatible with high pH regenerant.
• Pages 2-3 and 3-8 about the piston-wash system of the post-column pump
A note supplies supplementary information which may be helpful or necessary for
better understanding of the material.
The caution calls attention to an operating procedure, practice, or the like, which if not
correctly done or adhered to, could result in loss of information, or damage to, or
destruction of part or all of the equipment. Do not proceed beyond a caution sign until
the indicated conditions are fully understood and met.
Symbols
Please Read

PCX 5200 User’s Manual Before You Start
BYS-2
The following symbols appear on the PCX5200 or its accessories.
This warning sign denotes a hazard. It calls attention to a procedure, practice, or the like,
which if not correctly done or adhered to, could result in injury or loss of life. Do not
proceed beyond a warning sign until the indicated conditions are fully understood and met.
Ce symbole est un signal de danger. Il indique qu’une manipulation, si elle n’est pas
respectée ou effectuée correctement, risque d’entrainer des blessures, voir la mort.
Dieses Warnsymbol kennzeichnet eine Gefahr. Es macht aufmerksam auf einen Vorgang,
eine Handhabung oder ein Vorhaben, die bei unkorrektem Befolgen der Vorschriften zu
einer Verletzung oder einer lebensgefährlichen Situation führen können.
El signo de atención indica un riesgo. Requiere atención sobre un procedimiento, práctica,
o similar, que, si no se ejecuta correctamente o se sigue minuciosamente, prodía producir
heridas o muerte. No continúe a partir de un signo de atención hasta que no se hayan
entendido y alcanzado completamente las condiciones indicadas.
Questo avvettimento informa del pericolo. Molto attenzione riguardante il moto di usare
questa macchina émolto inportante altrimenti risulterádanni e anche morte. Non
continuare di piu affinchéle condizioni e instruzioni sono completamente chiare.
!

PCX 5200 User’s Manual Before You Start
BYS-3
This warning sign denotes a hot surface, a high temperature hazard. It calls attention to a
column heating block hotter than 70°C. For your safety, wear insulating gloves when the
column oven is warm.
Ce symbole indique une surface brûlante. Il signifie que la résistance chauffante de la
colonne a atteint une température supérieure à70°C. Pour votre sécurité, prière de porter
des gants isolants.
Dieses Warnsymbol kennzeichnet eine heiße Oberfläche oder eine Gefahr durch hohe
Temperaturen. Es macht aufmerksam auf den Heizblock des Saulenofens, der heißer als
70°C sein kann. Zu Ihrer Sicherheit sollten Sie isolierende Handschuhe tragen, wenn der
Saulenofen warm ist.
Este signo de atención indica una superficie caliente, un riesgo de alta temperatura. Pide
atención sobre un bloque calefactor de columnas por encima de 70°C. Para su seguridad
use guantes aislantes cuando el horno de columnas estécaliente.
Questo avvettimento informa della temperatura molto alta che possibilmente potrebbe
bruciare. Molto attenzione énecesaria specialmente al blocco caldo della colonna che é
superiore ai 70°C. Per essere protetti énecesario usare guanti insulanti per questa
applicazione.

PCX 5200 User’s Manual Before You Start
BYS-4
Power On
En marche
An
Escendido
Acceso
Power Off
Éteint
Aus
Apagado
Spento
Fuse Specification
Spécification du fusible
Spezifikation der Sicherung
Fusible
Valvole Specificazioni
Protective Ground
Prise de terre
Erdung
Masa de protección
Protezioni a terra

1-1
Chapter 1
Introduction
Post-column derivatization, also known as post-column reaction, renders visible certain
compounds that are normally invisible. This trick is accomplished after the separation
by performing a chemical reaction on the substances that gives them an easily
detectable physical property. Typically you use a reaction that produces a strong color or
makes a fluorescent product. You can increase the sensitivity of detection by several
orders of magnitude in favorable cases. Most reagents are selective for a particular class
of substances, so analytes of that class are more easily seen against a complex
background. So, post-column derivatization is used to increase sensitivity and selectivity
in HPLC analysis.
The post-column reaction system mixes the stream of effluent flowing from the HPLC
column with a stream of reagent solution. The mixture usually flows through a reactor
to allow enough time for the chemical reactions to complete. If the reaction is slow, the
reactor may be heated to speed things up. Some reactions need two or more reagents
added in sequence. Finally, the mixed streams pass into the detector, typically UV/VIS
absorbance or fluorescence. Of course, a practical system requires metering pumps,
pulse damper, thermostats, and safety systems to give reliable results.
• Stability of Reagent. The minimum reagent stability sufficient for routine work is one
day. This means that the yield and signal-to-noise ratio for a given sample must
remain constant for at least 8 hours.
• Speed of Reaction. The analytical separation is complete when the reagent is mixed
with the column effluent. Therefore, in order to minimize band spreading, it is
important to keep the time (therefore volume) small between the mixing tee and the
detector. If the reaction is slow (in excess of one minute), an elevated temperature can
be used to decrease the reaction time.
• Reproducibility. Because the reaction is occurring “on the fly,” as the combined
column and reagent stream flows toward the detector, the reproducibility is linked to
the flowrate precision of the pumps and to the temperature. Accordingly, even an
incomplete reaction will be as repeatable as the retention time for any given species.
The completeness of the reaction, then, is not strictly necessary for reproducibility,
but it is important for maximum sensitivity.
• Minimal Detector Response of Reagents. The color or background fluorescence of the
reagent (or its by-products) represents a continuous noise source. Because the reagent
is present in excess relative to the analyte, the analyte’s signal could be obliterated by
the reagent’s strong background signal. The baseline noise is proportional to the
background signal.
Why Post-coIumn
Derivatization?
How Does
It Work?
What Are the
Requirements?