Pickering Laboratories Pinnacle PCX User manual

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APPROVED
OperatOr Manual
Post-Column
Derivatization
Instrument
1280 Space Park Way
Mountain View
CA 94043
USA
800-654-3330 • 650-694-6700
650-968-0749 fax
www.pickeringlabs.com
[email protected]
http://pickeringlabs.blogspot.com
Cat. No. 0101-0008
Version 10, April 2016
Copyright © 2005 by Pickering Laboratories, Inc. All rights reserved. No Portion of this manual or
any artwork contained herein may be reproduced in any shape or form without the
express written consent of Pickering Laboratories, Inc.

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Pinnacle Operators Manual
Pickering Laboratories Inc.
table Of COntents
1Getting Started
How to Use this Manual 1
Read this First! 2
Symbols and Warnings 2
Specifications 2
Site Requirements 4
1.1 Section 1 Introduction
What is Post-column Derivatization? 1.1
Requirements for a Successful Post-column Method 1.1
Design of a HPLC system 1.2
Designing a Post-column System 1.4
Design of the Pinnacle PCX 1.6
2.1 Section 2 Overview
Introduction 2.2
Check Valve 2.3
Column Connections and Column Oven 2.3
Reagent Pump 2.4
Reagent Valves 2.5
Reservoir Tray / Bottles 2.5
Fluidics Panel 2.7
Quick Change Reactor 2.8
Detector Connections 2.8
Pump Compartment 2.9
Gas Manifold 2.9
Display Module 2.10
Electronics Compartment 2.11
Back Panel 2.11
Safety Features in the Pinnacle PCX 2.13
Standard Configurations of Pinnacle PCX 2.14

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3.1 Section 3 Installation
Site Requirements 3.2
Instrument Unpacking and Preparation 3.4
Unpacking 3.4
Gas Connections to Back Panel of Pinnacle PCX 3.5
Computer Connections 3.6
Software Installation 3.6
Pump and Autosampler Connections 3.8
Eluant Priming 3.10
Column and Guard Installation 3.10
Detector Connections 3.11
Reservoir Connections 3.12
Reagent Pump Preparation 3.13
Running a Chromatogram 3.14
Shutdown 3.15
4.1 Section 4 Pinnacle PCX Operation
Software Overview 4.1
Status Window 4.2
Menu Options/Functions 4.4
File
Control
Method
Sequence
Instrument
Configuring your Pinnacle PCX 4.6
Log Files 4.7
Creating and Editing Methods 4.7
Creating and Editing Sequences 4.10
Tutorials 4.11
Running post-column analysis using Pinnacle PCX 4.14
STOP/RESET Pinnacle PCX 4.17
Pinnacle Shutdown 4.17

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5.1 Section 5 Maintenance
Maintenance Suggestions 5.1
Operating Suggestions 5.2
Proper Shutdown Procedures 5.3
Basic Maintenance Procedures 5.6
Reagent Filter(s) Replacement 5.7
Ambient Reactor Replacement 5.8
Replacement of Heated Reactor Cartridge 5.8
Valve Maintenance 5.9
Pump Seal Replacement 5.12
Fuse Replacement 5.16
Tubing Kit Guides 5.17
6.1 Section 6 Applications
Amino Acids 6.2
Introduction 6.2
Background 6.2-1
Basic Sample Preparation 6.2-2
Reagent Preparation 6.2-3
Post-Column Conditions 6.2-4
Procedure 6.2-5
Sample Chromatograms and Gradient Programs 6.2-6
Precautions 6.2-18
Carbamates 6.3
Introduction 6.3
Background 6.3
Basic Sample Preparation 6.3-2
Reagent Preparation 6.3-3
Post-Column Conditions 6.3-5
Analytical Procedure 6.3-5
Sample Chromatograms and Gradient Programs 6.3-5
Precautions 6.3-12

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Glyphosate 6.4
Introduction 6.4
Background 6.4-1
Basic Sample Preparation 6.4-1
Reagent Preparation 6.4-2
Analytical and Post-Column Conditions 6.4-4
Analytical Procedure 6.4-4
Sample Chromatograms 6.4-5
Precautions 6.4-6
7.1 Section 7 Troubleshooting
Contact Pickering Laboratories For Support 7.1
Instrument Parameter Log 7.2
General Troubleshooting Advice 7.2
Common System Problems 7.3
Common Chromatography Problems 7.4
Common Column Problems 7.6
Application Specific Troubleshooting 7.7
• Amino Acids
• Carbamates
• Glyphosate
Amino Acid 7.7
Software Troubleshooting 7.8
Procedures 7.11
• To Remove Silica Deposits From Reactor
• To Remove Mineral Deposits In The Reactor From Hard Water
• To Remove Grease Deposits
• If Reagent Backflows Onto Column
• If TRIONE Backflows Onto Column
• If NaOH Is On Column
• To Remove Iron Contamination From Column
• To Pump RESTORE Through The Glyphosate Column

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8.1 Appendices
Installation/Operational Qualitication of the Pinnacle PCX 8.2
Installation/Operational Qualitication of the Pinnacle PCX Data Sheet 8.7
Performance Qualifications of the Pinnacle PCX Data Sheet 8.8
Sample Instrument Parameter Log 8.15
Flow Diagram – Simplex 8.16
Flow Diagram – Duplex 8.17
Parts List for Pinnacle PCX 8.18
Recommended Consumables and Spare Parts 8.21
Limited Warranty 8.24
References 8.25
9.1 Index

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Pickering Laboratories Inc.
GettinG started
How to Use this Manual
The Pinnacle PCX manual is designed to contain all of the information necessary for the installation, operation,
maintenance and troubleshooting of the Pinnacle PCX. It is designed to contain any and all information that you
may require during the lifetime of your Pinnacle PCX.
In addition to the general operation information, this manual also contains sections devoted to a particular
application. Since Pickering Laboratories provides the complete solution, we included the chromatograms,
operating conditions, and some troubleshooting information for our most commonly supported methods.
As well as instructional information, this manual also contains a section for recording service information such
as serial numbers, installation dates, service dates etc.
Each section of the manual is divided by a tab with the title of that section. For easy reference, simply select
the tab that you require based on the information you are looking for. For example, if you are running Amino
Acids, and have a question about the gradient program, simply go to the section titled “Amino Acids”.
1How to Use this Manual
2Read this First!
2Symbols and Warnings
2Specifications
4Site Requirements

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Read this First!
Before attempting to install the Pinnacle PCX post-column derivatization instrument, it is vitally important that
you read this manual first, and attend to site, HPLC, and accessories requirements:
HPLC – Page 5
Gas Supply Requirements – Page 5
Reagent Reservoir bottles – Page 5
Computer – Page 5
Symbols and Warnings
Caution – this symbol indicates that caution must be used when dealing with this part.
Hot – this symbol is located on the Heated reactor, which can reach scalding temperatures.
Specifications
INSTRUMENT
Dimentions
21.50 H x 10.63 W x 18.25 D inches (54.0 x 26.7 x 46.4 cm), instrument only, doors closed
Weight
77lbs for Dual-pump systems
67lbs for Single-pump systems
Reagent Pumps
Max operating pressure 500 psi (35bar)
Flow rate range 50μl –1500μl/minute
Refill cycle 60 seconds
Heated Reactor
5°C above ambient to 130°C
Thermal Safety switch limits temperature to 150°C
Stability +/- 0.5°C
Accuracy +/- 1°C
!
!

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Electrical
720 W
120 VAC +/- 10%, 240 VAC +/- 10%
5 A maximum at 108 VAC
47 – 63 Hz
Installation over voltage category: Pinnacle PCX complies with Class B Emission Test Specifications.
Fuses
2 ea, 5mm x 20 mm, 6 A, time lag
LCD
Backlit, positive mode, high contrast, viewing area – 125mm (L) x 75mm (W)
COMMUNICATION
USB
Requires a USB port.
Relay
Any machine that drives this relay input shall provide a relay contact pair that is electrically isolated from all
other electrical devices. The relay contacts must be capable of switching 1mA at 24 +/- 2 Vdc.
Ethernet (optional)Connector: RJ45
Connection Speed: 10/100-BASE-T
Ethernet Protocol: TCP/IP
IP Address: User set or DHCP (DHCP requires the Network Administrator to “Reserve” an IP address with the
DHCP server)
Note: Recommend connection to a Network Switch or Router, or direct connection to a dedicated Network
card in the PC.
ENVIRONMENTAL
Indoor use only
Altitude up to 6500 ft (1981 m)
Ambient Temperature – 40°C
Relative Humidity up to 80% at 31°C

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Radio Frequency Pinnacle PCX complies with IEC 61000-4-3
This device complies with Part 15 CHECK Class B of the FCC Rules. Operation is subject to the following
two conditions: (1) This device may not cause harmful interference, and (2) this device must accept any
interference received, including interference that may cause undesired operation.
WETTED MATERIALS
• PEEK, PTFE, Air Barrier Tubing
• 99.9% Ceramic
• Perlast®
• Borosilicate glass
• EPR
SOFTWARE
PCX Control Software runs on Microsoft Windows XP, Vista, or Windows 7.
10Mb hard disk space
PCX History Log file: Maximum file Size on the computer: 1.2 Mb
Site Requirements
INSTRUMENT
Bench Space
33 H x 17 W x 21 D inches (84 x 56 x 59 cm), both doors fully opened, with bottles and electrical connections
in place.
Minimum 3 inches clearance at back of instrument for venting.
Note: Space quoted above is for Pinnacle PCX.
The total space requirement depends on the brand and model of HPLC.
Electrical Outlet
One grounded outlet must be provided for the Pinnacle PCX.

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Computer
IBM-compatible
PCX Control Software runs on Microsoft Windows XP, Vista, or Windows 7.
Ethernet or USB port
Available Memory: Minimum 10Mb
Ethernet (optional)Connector: RJ45
Connection Speed: 10/100-BASE-T
Ethernet Protocol: TCP/IP
IP Address: User set or DHCP (DHCP requires the Network Administrator to “Reserve” an IP address with the
DHCP server)
Note: Recommend connection to a Network Switch or Router, or direct connection to a dedicated Network
card in the PC.
Relay
For synchronization, the HPLC system must be capable of sending a relay signal to an external instrument.
No relay connection is needed for Agilent 1100 or Agilent 1200. Pinnacle PCX software directly synchronizes
with Chemstation version 9.0 or higher.
Gas Supply
High purity Nitrogen, 45-75 psi (min - max)
Outlet of regulator must connect to 1/8” OD tubing
Reagent Reservoir Bottles
The Pinnacle PCX includes one pressurized reagent reservoir for the one reagent system and two for the two
reagent system.
Note: For your safety, the bottles are coated with a tough plastic film and rated to a maximum of 15 psig
(1 bar). Do not use uncoated bottles.
HPLC Pump
Binary gradient for glyphosate, carbamate applications.
Quaternary for all others.

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Autosampler
Minimum injection volume 10μl, preferably by full-loop injection
For drinking water, minimum injection volume 200μl
Tefzel rotor seal required for all applications using eluants with pH>10
PEEK needle seat required for all applications using eluants with pH>10
Detector
Pressure rating of flow cell must be > 110 psi
Inlet capillary must be > 0.17 mm ID
MISCELLANEOUS SUPPLIES
For Amino Acid analysis, a Dead-Head kit is required. This can be purchased from Pickering Laboratories.
Chemistry
The user must check the chemistry requirements for the specific application.
For Carbamate Analysis
HPLC Grade Methanol
HPLC Grade Water
Materials for calibration standards
Carbamate hydrolysis reagent (Cat. No. CB910)
Carbamate OPA diluent (Cat. No. CB130 or CB130.2)
o-phthaladehyde (Cat. No. O120)
Thiofluor™(Cat. No. 3700-2000)
For Glyphosate Analysis
5% Sodium hypochlorite solution
Materials for calibration standards
Methanol for OPA reagent preparation
Glyphosate Eluant, pH2.0 (Cat. No. K200)
Glyphosate Regenerate (Cat. No. RG019)
Glyphosate Hypochlorite diluent (Cat. No. GA116)
Glyphosate OPA diluent (Cat. No. GA104)
o-phthaladehyde (Cat. No. 0120)
Thiofluor™ (Cat. No. 3700-2000)

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For Amino Acid Analysis
A: Fluorescence detection
• Methanol for OPA reagent preparation
• 5% Sodium hypochlorite if using the 2-reagent method
• Brij 35 solution for OPA reagent preparation
• DI Water
• Pickering sodium or lithium elution buffers
(see application section for cat. nos.)
B: UV – Visible detection
• DI Water
• TRIONE®Ninhydrin reagent (cat. no. T1OOC or T200)
• Pickering sodium or lithium elution buffers
(see application section for cat. nos.)

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Notes

1.1
Section 1
IntroductIon
Pinnacle Operators Manual
Pickering Laboratories Inc.
Section 1
intrOduCtiOn
1.1
1.1
1.2
1.4
1.6
What is Post-column derivatization?
Requirements for a Successful Post-column Method
Design of a HPLC system
Designing a Post-column system
Design of the Pinnacle PCX
What is Post-column Derivatization?
This is a method which renders visible certain compounds that are normally invisible. Since this reaction
occurs after, or post-separation, it is referred to as post-column deriviatization. The analytes of interest are
separated on the column first, and then reacted with a chemical that will render them detectable at a desirable
wavelength, voltage, or any number of various means of detection.
Post column derivatization enhances the sensitivity of HPLC by several means:
1) Most reagents are selective for a particular class of substances, so analytes of that class are more easily seen
against a complex background.
2) Since the separation is performed first, the matrix of the sample is either washed off of the column before
the analytes, or is retained by the column. This leaves a very pure sample of analyte to react. This eliminates
the need for extensive sample clean-up, and provides a very reproducible reaction because there are no matrix
interferences.
The Pinnacle PCX post-column derivatization instrument automatically mixes the stream of effluent flowing
from the HPLC column with a stream of reagent solution. The mixture flows through a reactor to allow enough
time for the chemical reactions to complete. In many cases, the reaction is very slow at room temperature. For
this reason, the reactor can be heated. There are some methods that require two or more reagents added in
sequence. This is done by the addition of a second reagent pump. In many cases, the second reaction occurs
at a much faster rate, and can be efficiently accomplished at room temperature. After the reaction is complete,
the derivatives flow into the detector, where the absorbance or the fluorescence (usually) is measured by the
HPLC system. These two means of detection are the most common, but they are certainly not the only means of
detection.
Requirements for a Successful Post-column Method
There are many things to take into consideration when developing a method and instrument for post-
column derivatization. For example, many pumps have a periodic motion when drawing and dispensing that

1.2
Section 1
IntroductIon
Pinnacle Operators Manual
Pickering Laboratories Inc.
will manifest itself in the baseline of a chromatogram unless it is properly dampened. Below are the basic
requirements for a successful automated post-column method:
1) Reagent Stability. 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.
2) Reaction Speed. The analytical separation is complete when the reagent is mixed with the column effluent.
Therefore it is important that the analyte react as quickly as possible. The longer the reaction time, the larger
the reactor volume required. With larger volumes, the peak shape will become distorted. To minimize band
spreading, it is important to keep the overall time (and therefore volume) as low as possible between the
column and detector. If the reaction is slow (in excess of one minute), an elevated temperature can be used to
decrease the reaction time.
3) 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 flow rate 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. Therefore, it is important that the pumps maintain a constant flow rate, and that the reactor maintain a
constant temperature. It is also very important that the column be maintained at constant temperature to ensure
that the analytes are properly separated and identified.
4) 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.
5) Solubility. All species must remain in solution, including the combined components of the eluants and the
reagent(s), as well as the newly formed derivative(s). Precipitates can block capillary tubes, burst reactors, and
foul detector flow cells.
6) Uniformity of flow. The baseline noise is a function of the flow-noise in the eluant and reagent pumps.
Non-uniform flow causes non-uniform mixing leading to modulation of the background signal which appears
as noise. Refractive index noise can be even more objectionable than absorbance noise. Common techniques
for evening the flow of the pumps is the addition of a pulse dampener, or the use of a syringe pump.
Design of a HPLC system
This next section is a simplified view of a HPLC, followed by the ideas behind a post-column system. This
section is intended to help novice HPLC operators.

1.3
Section 1
IntroductIon
Pinnacle Operators Manual
Pickering Laboratories Inc.
In order to understand post-column HPLC, we need to understand the design of an HPLC. If we connect an
HPLC pump directly to a detector (with nothing in between), the baseline from the detector shows a periodic
noise (Figure 1-1); the time period is equivalent to the pump stroke.
Now add a commercial pulse dampener. The baseline is still not smooth; the periodic noise is still there
although less pronounced (Figure 1- 2). The pulse dampener absorbs most of the pulses from the pump, but
the flow requires more stabilization.
A restriction inline will cause the flow at the outlet of the restriction to be constant. In an HPLC system, this is
accomplished with the analytical column. Actually, the column does more than separation; it creates a back-
pressure. It is the combination of the pulse dampener and the column that creates a smooth baseline.
(Figure 1-3)
Eluants
LC Pump
Injector
Detector
Waste
Baseline
Eluants
LC Pump
Injector
Detector
Waste
Baseline
Commmercial
Pulse
Dampener
(capacitor)
FIGURE 1-1
FIGURE 1-2

1.4
Section 1
IntroductIon
Pinnacle Operators Manual
Pickering Laboratories Inc.
An analogy will help us understand the concept. Let us use a river as an example. If it rains; the river swells.
If it stops raining; the level goes down. As the level fluctuates, it is equivalent to a periodic noise. To obtain a
constant flow, we need to add a reservoir (pulse dampener) and a dam (column). The flow downstream from
the dam is constant (smooth baseline).
Designing a Post-column System
The same principals that are used in HPLC can be applied to the post-column system. What happens if we
simply add a post-column pump, a mixing tee, and a reactor? The periodic noise returns to the baseline
(created by the post-column pump; Figure 1-4).
Pulse
Damper
Eluants
LC Pump
Injector Pre-column
Check Valve
Thermostatted
Column & Guard
Detector
Waste
Baseline
FIGURE 1-3