Pickering Laboratories Pinnacle PCX Operator's 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
[e-mail]
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.

table Of COntents

1 Getting 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

I

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

Pinnacle Operators Manual

Pickering Laboratories Inc.

II

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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Pickering Laboratories Inc.

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

III

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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Pickering Laboratories Inc.

IV

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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Pickering Laboratories Inc.

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

V

9.1 Index

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VI

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GettinG started

How to Use this Manual

1
2 Read this First!
2 Symbols and Warnings
2 Specifications
4 Site Requirements

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.

1

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”.

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2

!

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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)

3

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)

5

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

For Glyphosate Analysis

(Cat. No. 3700-2000)

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)

Pinnacle Operators Manual

Pickering Laboratories Inc.

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

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 1

intrOduCtiOn

1.1

Section 1

In tr od uc tI on

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 post­column derivatization. For example, many pumps have a periodic motion when drawing and dispensing that

Pinnacle Operators Manual

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1.2

Section 1

In tr od uc tI on

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.

Reproducibility. Because the reaction is occurring “on the fly,” as the combined column and reagent

3)
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.

Minimal Detector Response of Reagents. The color or background fluorescence of the reagent (or

4)
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.

Solubility. All species must remain in solution, including the combined components of the eluants and the

5)
reagent(s), as well as the newly formed derivative(s). Precipitates can block capillary tubes, burst reactors, and
foul detector flow cells.

Uniformity of flow. The baseline noise is a function of the flow-noise in the eluant and reagent pumps.

6)
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.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 1

In tr od uc tI on

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.

Injector

Detector

1.3

LC Pump

Eluants

Baseline

Waste

FIGURE 1-1

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.

Injector

Commmercial
Pulse
Dampener
(capacitor)

LC Pump

Baseline

Eluants

Detector

Waste

FIGURE 1-2

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 back­pressure. It is the combination of the pulse dampener and the column that creates a smooth baseline.
(Figure 1-3)

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Pickering Laboratories Inc.

1.4

Eluants

Section 1

In tr od uc tI on

Injector

Pulse
Damper

LC Pump

Pre-column
Check Valve

Detector

Thermostatted
Column & Guard

Baseline

W aste

FIGURE 1-3

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).

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Section 1

In tr od uc tI on

1.5

Pulse
Damper

LC Pump

Eluants

Injector

Filter

Baseline

Thermostatted
Column & Guard

Heated
Reactor

Post-column

1

Pump

Reagent

Bottle

Detector

Back-pressure
Regulator

Waste

FIGURE 1-4

However we do not need to invent anything new; we just need a pulse dampener and a column. In the
generation of Post-column systems prior to the Pinnacle PCX, the restriction performed by the column was
achieved using a Restrictor, which is packed with very inert material. With this “flow conditioner” in place, the
baseline is now acceptable.

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1.6

Section 1

In tr od uc tI on

Design of the Pinnacle PCX

The Pinnacle PCX has taken the post-column system to a new level. The introduction of a syringe pump has
eliminated the need for a pulse-dampener and restrictor by:

• The Pickering syringe pumps complete a filling cycle prior to the injection of a run, and deliver reagent
during the run at a constant rate.

• There is a valve between the pump and reactor which also helps to regulate the reagent flow, by opening key
ports at the appropriate time.

• There is also a pressure transducer which we have added in line before the valve to determine if there are
any blockages in the reactors.

Now you are ready for Section 2.

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Section 2

Overview

2.2 Introduction

2.3 Check Valve

2.3 Column Connections and Column Oven

2.4 Reagent Pump

2.5 Reagent Valves

2.5 Reservoir Tray / Bottles

2.7 Fluidics Panel

2.8 Quick Change Reactor

2.8 Detector Connections

2.9 Pump Compartment

2.9 Gas Manifold

2.10 Display Module

2.11 Electronics Compartment

2.11 Back Panel

2.13 Safety Features in the Pinnacle PCX

2.14 Standard Configurations of Pinnacle PCX

2.1

Section 2

Ov er vi ew

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2.2

Section 2

Ov er vi ew

Introduction

This chapter is designed to familiarize you with the
components, layout, and function of the Pinnacle PCX.
Here you will find descriptions of each key component
of the instrument and what it does.
At the most basic level, the Pinnacle PCX performs
three main tasks:
1 .Regulates the temperature of the analytical column

2. Delivers the reagent

3. Heats the reaction

In addition to accomplishing the above three tasks, the
Pinnacle PCX has various features to make the analysis
more reliable, convenient, and simple. It also contains
features to protect the instrument from accidental
damage.

R1

FLUSH

FROM

HPLC

COLUMN

FILTER

FILTER

WASTE

PUMP 2

HEATED

REACTOR

PUMP 1

TRANSDUCER 2

2

3

TRANSDUCER 1

2

3

1

VALVE 2

4

AMBIENT
REACTOR

1

VALVE 1

4

R2

6

5

6

5

TO DETECTOR

FLUSH

R1

Flow Path – Dual Pump Figure 2-2

2

3

FILTER

VALVE 1

1

6

4

FROM

HPLC

COLUMN

HEATED

REACTOR

TRANSDUCER

PUMP

Flow Path – Single Pump Figure 2-1

Pinnacle Operators Manual

Pickering Laboratories Inc.

The flow path of the Pinnacle PCX is extremely inert,
rendering it very versatile. The same instrument can be
used for many different applications, and will tolerate a
high percentage of reagents.

5

WASTE

TO DETECTOR

Section 2

Ov er vi ew

2.3

Check Valve

The column check valve is located just outside of the
column oven (figure 2-3). The check valve is designed
to protect the column against backflow of the reagents in
case of unexpected HPLC shutdown.

Figure 2-3

Column outlet tubing

Column oven door

Rubber foam seal

Column Connections and Column Oven

The column oven contains the analytical column and
guard column (figure 2-3). The oven is a convection air
oven. The column heater utilizes re-circulating air flow
technology to provide quick, uniform column heating.
Fast column cooling is assisted by the introduction of
fresh air flow into the chamber by means of two fans
that are controlled by the Pinnacle PCX control software.
The temperature range holds within ± 1˚C resolution
from 5˚C above ambient to 75˚C. A temperature gradient
can be performed on the column to effect an increase
in the speed of the separation. The temperatures can
be programmed for a gradient with as many steps as
required for fine-tuning an analysis.

The heater block is designed to receive the analytical
column (5, 10, 15, and 25 cm long) and the guard
column when they are connected via a column easy­connect fitting (included with the instrument, and sold
separately by Pickering, Cat. #3102-3064).
The outlet end of the column hangs in the slot at the top
of the column oven. The inlet to the guard column is at
the bottom of the column oven door.
The column oven door contains a pocket to fit around
the analytical column and guard. There is a groove in
the door that is lined with Santoprene foam rubber cord

to Guard

Column

Blue Peek Tubing

PEEK Nut

(3101-0011)

Check Valve

(3106-1007)

that seals the door to the chassis and provides a stable
insulated heating environment.
The last part of the lead-in capillary is located inside the column oven to preheat the eluant for a more uniform
temperature within the column.

Analytical column

Inlet end

Coupler/column connector

Guard column

Check valve

(1452-0226)

from HPLC

PEEK Nut

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Pickering Laboratories Inc.

2.4

Section 2

Ov er vi ew

Make sure that the column door is securely closed before starting operation.

Warning. The column heating block may become hotter than 75°C. For your safety, wear insulating gloves
when the column oven is warm.
Attention. La résistance chauffante de la colonne peut dépasser une température de 75°C. Pour votre sécurité,
prière de porter des gants isolants lorsque le four de la colonne est chaud.
Warnung! Der Heizblock des Säulenofens könnte heißer als 75°C werden. Für Ihre Sicherheit sollten Sie
isolierende Handschuhe tragen, wenn der Säulenofen warm ist.
Atención. El bloque calefactor de columnas puede estar por encima de 75°C. Para su seguridad use guantes
aislantes cuando el horno de columnas esté caliente.
Avvertimento. Il blocco della colonna potrá diventare molto caldo e superare ai 75°C. Per la sua protezione
usa guanti con insulazione per questa applicazione.

Reagent Pump

The reagent pump is a syringe pump. Refer to figure 2-4 and 2-8 (items 4 and 10). The syringe has a volume
of 70 ml. The flow rate can be programmed from 0.05 ml/min to 1.5 ml/min. Once programmed, the syringe
pump delivers at a constant speed. Since there is no need for constant refilling and dispensing (as with a
reciprocating pump), there is a very high flow precision that does not require external pulse dampening
features.

The syringe pump cylinder and head is made from a single piece of 99.9% Alumina for ruggedness and non­reactivity. The piston surface is made from PEEK with an inert o-ring seal.

The pump contains a piston wash for extended seal life. The piston wash is automatically performed with the
movement of the piston.

For purposes of fast refill and flushing, the pump
can refill or dispense (to waste) at 55 ml/min.

Syringe pump
cylider

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Figure 2-4

Reagent Valves

Reagent Valves are placed immediately after
the pump. The valves have 5 possible operating
positions:

REAGENT: From Reagent Bottle to Pump (3-4)

1)

REACTOR: From Pump to Reactor (1-2)

2)

WASTE: From Pump to Waste (1-6)

3)

FLUSH: From Flush Bottle to Pump (5-4)

4)

BLOCKED: The valve ports are blocked.

5)

The valves are connected to a Flush Bottle which
can be used to flush the reactor in the event of a
shutdown or to aid in flushing the system. This will
reduce the likelihood that the reactor becomes
blocked. The valves are also connected to the
reagent bottles and are used for filling the syringe pump.

Pump

2

3

Section 2

Ov er vi ew

1

6

5

4

Figure 2-5

2.5

Valve

Reservoir Tray / Bottles

This is the tray located on top of the instrument. It holds the
reagent bottles, wash bottle and flush bottle. It is removable
for cleaning. It will hold 1L of liquid and as such is considered
secondary containment. It does not seal any openings at the top
of the instrument in case of heavy spillage.

There are 4 bottles located here:

1. Reagent Reservoir 1

2. Reagent Reservoir 2

3. Flush Bottle – contains water or 80-20 water/alcohol (IPA or
Methanol) for flushing the instrument.

4. Piston Wash bottle – contains 90-10 water/alcohol (IPA or
Methanol). This bottle is connected via 1/4 inch OD flexible
tubing to the piston wash of each pump in the instrument.
Change Wash and Flush solutions at least once a week to prevent
contamination.

The pressurized reagent reservoir serves two purposes:
It protects air-sensitive reagents from oxidation.
It helps the syringe pump fill consistently and quickly by
providing a source of pressure.

Flush

Reagent

Pinnacle Operators Manual

Pickering Laboratories Inc.

Piston
Wash

Figure 2-6

2.6

Section 2

Ov er vi ew

Reagent tubing is pre-connected to the pump at the factory. All
Reagents lines are Air Barrier tubing. Reagent 1 is labeled R1,
and Reagent 2 is labeled R2 throughout the instrument. Nuts
and ferrules (1/4-28 ) are provided for connecting the tubing
to the Reservoir caps.

The reservoir cap (Figure 2-7A) has two connecting ports
on the top – one for connecting reagent line and one for
connecting the gas line. The port for the gas line has a one-way
valve to prevent backflow of the reagent into the gas manifold in
case of a drop in gas pressure. Use nuts and ferrules (1/4-28)
to make the connections. There are two stopcocks, one is for
the vent port and the other is for the reagent line. Keep reagent line stopcock open for operation. Open the vent
stopcock to sparge the reagent and close it to pressurize the reservoir. This is the normal operating position.

When changing reagent, first turn off the gas using the toggle valve on the fluidics panel. Then vent the reagent
bottle by opening vent stopcock. Now you can safely remove the cap. It is convenient to have extra bottles
so that you can simply transfer the cap without setting it down and risking
contamination.

Figure 2-7A

If you have a reservoir cap shown on figure 2-7B, the large white knob is the
valve; pull it up for CLOSED, and push it down for OPEN. If the gas is turned on,
opening the vent valve will sparge the reagent. Closing the valve will pressurize the
reservoir; this is the normal operating position. On the side of the cap, away from
the on-off valve, there is a 1/4-28 fitting; you may optionally connect a tube here
to carry vapors to an exhaust vent.

Warning. For your safety, the bottles are coated with a tough plastic film and are
rated to a maximum of 15 psig (1 bar). Do not use uncoated bottles.
Attention. Pour votre sécurité, les bouteilles sont recouvertes d’un film de plastique dur, et sont calibrées à un
maximum de 10 psig (0.7 bar). Ne pas utiliser les bouteilles non recouvertes.
Warnung! Für Ihre Sicherheit wurden die Reagenzienflaschen mit einem festen Schutzüberzug aus Kunststoff
versehen. Die Flaschen sind bis max. 0.7 bar (10 psig) zugelassen Flaschen mit beschädigtem Schutzüberzug
dürfen nicht mehr benutzt werden Verwenden Sie keine Flaschen ohne Schutzüberzug!
Atención. Para su seguridad, las botellas están recubiertas con una resistente película plástica, y están
constrastadas a 10 psig (0.7 bar). No utilice botellas sin recubrimiento.
Avvertimento. Per la sua protezione, le bottiglie sono construite forti con un percentuale du plastica, e sono
usabili per un massimo di 0.7 Bar (10 psi). Non usare bottiglie normali.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Valve

Figure 2-7B

Section 2

Ov er vi ew

There are three types of Reservoir/Cap assemblies used on the Pinnacle PCX:

1) Reagent Reservoir is 1L, 45-430, safety-coated. These are shipped with blue caps with white valve assembly.
These can be pressurized.

2) The wash Reservoir is 1L, 38-430, clear glass. These are shipped with caps with 1ea 1/4” opening and 2ea
1/8” openings. These bottles cannot be pressurized, and do not contain threads for tubing connections.

3) The flush Reservoir is 1L, 38-430, clear glass. These are also shipped with caps, but with 3 each 1/8”
openings. These bottles cannot be pressurized, and do not contain threads for tubing connections.

Note: The two types of caps cannot be interchanged because the necks of the bottles are different sizes.

Fluidics Panel

The fluidics panel is the busiest area of the
instrument. Everything that you will need is
located on the front of the panel. The door can
be removed if desired by the user for easy­access. Refer to figure 2-8 for parts identification
throughout the next section.

5

3

2.7

Starting at the valves, the parts of the Fluidics
Panel are numbered counter-clockwise.

1. Valve 2 (on dual systems only)

2. Pressure Transducer 2 (on dual systems only)

3. Gas is controlled by the toggle valve. Lever ON

pressurizes the manifold.

4. Pump 2 (on dual systems only)

5. Column Outlet

6. Mixing Manifold 2 (with integrated reagent

filter) (on dual systems only)

7. Heated Reactor

8. Mixing Manifold 1 (with integrated reagent

filter and over-pressure relief valve)

9. Over pressure Relief valve

10. Pump 1

11. Pressure Transducer 1

12. Valve 1

13. Ambient reactor (on dual systems only)

14. Reactor outlet and union to connect to

detector

2

4

1

6

7

8

9

10

13

11

12

14

Figure 2-8

Pinnacle Operators Manual

Pickering Laboratories Inc.

2.8

Section 2

Ov er vi ew

Quick Change Reactor

The standard reactor is a PTFE capillary
tube 0.011” I.D. wrapped on a heated
mandrel. The narrow diameter reduces
band-spreading, and the PTFE is corrosion
resistant. The reactor can be easily switched
if it becomes blocked or if a different
volume is required. The electronics of the
reactor are contained within the Pinnacle PCX, making
replacement affordable and easy.

Figure 2-9

Volumes ranging from 0.15 ml to 3 ml are available. The most commonly used volumes are available off the
shelf, but if you require a volume that is not in our price list, we can make it for you.

There is a 500 psi relief valve in case there is a blockage in the reactor or detector (Item 9 on Fluids Panel).

Detector Connections

from Detector Exit

There is a 100 psi (5 bar) back-pressure regulator
(cat. # 3102-9025) on the exit line from the detector; it
suppresses boiling inside the hot reactor and prevents
bubbles from forming in the detector flow cell. This in turn
provides for a smooth baseline and therefore high-sensitivity.

Flow direction

to Waste

Pinnacle Operators Manual

Pickering Laboratories Inc.

Figure 2-10

Section 2

Ov er vi ew

2.9

Pump Compartment

This compartment is located on the right
side of the Pinnacle PCX. It contains the
reagent pump(s), gas manifold, and
electronics for the heated reactor and
valves(figure 2-11).

The only time you will need to access
this panel is for pump piston seal
replacement. All other times, qualified
personnel should make any repairs.

The Pump, Valves, and Heated Reactor
are described in other sections.

Here you will see the Piston Wash feature
for the reagent pumps. The Piston wash
is a 1/4” OD tubing connected to the
back chamber of the syringe.

Valve
Motor

Heated
Reactor

Pump 2

Pump 1

Gas Manifold

Piston
Wash

Figure 2-11

Gas Manifold

The “Gas Inlet” fitting is a 1/4-28 fitting
located on the back panel of the Pinnacle PCX
(reference fig 2-15). The inert gas source is
connected via a 1/8” OD Air Barrier tubing,
and the pressure is regulated to 5psi before
pressurizing the reagent reservoirs.

The gas regulator requires an input pressure
of 45–75 psi (3–5 bar) to function properly.
The manifold has a safety relief valve that opens
at about 10 psi to prevent dangerous over­pressurizing of the reagent reservoirs.

Figure 2-12

Pinnacle Operators Manual

Pickering Laboratories Inc.

2.10

Section 2

Ov er vi ew

Display Module

The display module (figure 2-13) is the
information panel of the Pinnacle PCX.
It displays:
the flow rate of each pump, the set
point and current temperatures of
the column and reactor the Pump
pressure(s), which are also indicators
of the pressure inside the post-column
system and status information.

Figure 2-13

In addition to the electronic information on
the LCD, the display module contains 3 buttons and one LED.
STOP - this button is used for an instant stoppage of the instrument in the case of an emergency. If this button is
pushed, the instrument will cease all activity, regardless of what is happening.

Stop LED

Stop

Mode

Select

The STOP LED will illuminate when the Pinnacle PCX has been stopped manually by the user. When STOP is
pressed, the LED will glow red, indicating that there has been an emergency manual stoppage. To reset the
instrument after emergency stop, turn instrument OFF and then turn it back ON.

MODE - Used during initial installation to determine the IP address. This function is for instruments that are
connected to a company network.
SELECT - Used during initial installation to determine the IP address. This function is for instruments that are
connected to a company network.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Electronics Compartment

This compartment contains the central nervous system
of the Pinnacle PCX – the main PC board. From here, all
communication is coordinated.

This compartment is separate from the pump compartment for
two reasons:

1) To segregate the liquid end form the electronic end to avoid
any damage to the PC board by reagents and liquids

2) To keep the board cool from the warm action chamber of the
pumps

2.11

Section 2

Ov er vi ew

This compartment also contains:
Power supply
Cooling fans
Column oven cooling mechanism

Figure 2-14

Since this compartment is very sensitive to liquids, and shocks, it is strongly recommended that no repairs are
attempted by the user.

If there is a need for the user to open this panel,
please do so only under the recommendation/
instruction of qualified Pickering support personnel.

Back Panel

This panel contains:
Communication Ports
Gas Inlet

Gas Inlet

USB Port

Ethernet Port

Relay Input

Power
Fuses
Vents and other openings

COMMUNICATION PORTS

USB Port

Figure 2-15

Power Switch
and Main Fuse

The USB port on the Pinnacle PCX connects directly to one of the USB ports on the computer.

Pinnacle Operators Manual

Pickering Laboratories Inc.

2.12

Section 2

Ov er vi ew

Relay Ports

The relays will be explained in the operations chapter under Site Requirements.

Ethernet Port

This port is used for connecting the Pinnacle PCX to a network.

POWER SWITCH AND MAIN FUSE

The power connector is a standard IEC 320 type connector. Use the appropriate power cord for your local wall
outlet and electrical code.

The 120V version comes with a standard North American cord set.

The 240V version comes with a cord set used in much of continental Europe.

Your local reseller may have provided the correct local cord set. If your local power outlets are different, you
will need to obtain the appropriate grounded cord set.
The main power switch is located in the power connector assembly.

The fuse holder is located in the power connector assembly. To change the fuse, first remove the power cord
from the connector. Carefully pry out the fuse clip with a small screwdriver. Replace with the specified-type
fuse: 2 ea, 5mm x 20 mm, 6 A, time lag.

Warning. Ensure that the power cord is disconnected before replacing a fuse. Use only the specified-type fuse.
Attention. Assurez vous que le cable secteur n’est pas connecté avant de changer un fusible.
Warnung. Sicherungen dürfen nur bei nicht angeschlossenem Netzkabel ersetzt oder gewechselt werden.
Cuidado. Asegúrese que el cable de red está desconectado antes de instalar o cambiar un fusible.
Attenzione. Assicuratevi che il cavo di alimentazione sia scollegato prima di installare o sostituire un fusible.
Waarschuwing. Zorg dat de voedingskabel losgekoppeld is, voordat een zekering wordt geplaatst of vervangen.
Avvertimento. Fare atenzione che la corda del voltaggio sia staccata prima di cambiare valvole. Usa solo

valvole di capacitá precisata dalla fattoria.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Safety Features in the Pinnacle PCX

The Pinnacle PCX system has features
designed into the instrument and operation
that will prevent reagent back-flow onto the
column and bursting of reactor tubing due to
a blockage.

Post-column reagent can immediately damage
the analytical column if the reagent flow is
diverted in the wrong direction by a lack of
HPLC flow.
This is prevented by:

Inline
Check valve

2.13

Section 2

Ov er vi ew

Over-pressure
relief valve

1) In-line Check Valve

Figure 2-16

A one-way check valve placed
before the column prevents the
flow of liquid in the direction of
the column.

2) Over-pressure Relief Valve
In the rare event there is a blockage in the post-column system, an integrated 500 psi over-pressure relief valve
will open and divert the liquid into the drip tray. This will prevent a bursting of tubing or fittings. By relieving
the pressure, this will give you the opportunity to correct the blockage rather than having to replace the heated
reactor.

Pinnacle Operators Manual

Pickering Laboratories Inc.

2.14

Section 2

Ov er vi ew

Standard Configurations of Pinnacle PCX

The Pinnacle PCX is available in standard configurations for one-reagent or two-reagents, standard or micro
volume, 120V or 240V operation, and is shipped completely assembled, calibrated, and tested. Custom
instruments are also available; contact Pickering Laboratories for a quote.

LCD

Valve

Ambient
Reactor

Transducer

Check
Valve

Dual Reagent System Figure 2-17

Pump

Pump

Single Reagent System Figure 2-18

LCD

Transducer

Valve

The one-reagent instrument consists of a single reagent pump, valve, heated reactor, column heater, backflow
and over-pressure safety devices, filters, reagent reservoir, gas manifold, Air Barrier gas tubing, and other
accessories.

The two-reagent Pinnacle PCX contains two reagent pumps, two valves, heated and ambient reactors, column
heater, backflow and over-pressure safety devices, filters , reagent reservoirs, gas manifold, Air Barrier gas
tubing, and other accessories.

The Pinnacle PCX includes one pressurized reagent reservoir for the one-reagent system and two for the two­reagent system.

The instrument also includes a 1L wash bottle and a 1L flush bottle for the piston wash and flush feature.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 3

installatiOn

3.2 Site Requirements

3.4 Instrument Unpacking and Preparation

3.5 Gas Connections to Back Panel of Pinnacle PCX

3.6 Computer Connections

3.6 Software Installation

3.8 Pump and Autosampler Connections

3.10 Eluant Priming

3.10 Column and Guard Installation

3.11 Detector Connections

3.12 Reservoir Connections

3.13 Reagent Pump Preparation

3.14 Running a Chromatogram

3.15 Shutdown

3.1

Section 3

In st al la tI on

Installation Checklists (see Appendix)

The Pinnacle PCX instrument is shipped in one carton. Application Kits may be shipped in one or more cartons
each. Report any carton damage to the carrier. Unpack all cartons and review the contents using the Packing
List to ensure that your order is complete. If any items are missing, immediately contact Pickering Laboratories
at [phone] or by fax at [phone].

Store any standards in the freezer or refrigerate immediately upon arrival. Pickering Amino Acid, Carbamate,
and Glyphosate columns are shipped with test mixtures. Remove the vials from the box and freeze upon arrival.

While installing the Pinnacle PCX complete the Installation Checklist located in the Appendix. When the
installation is completed, fax or mail the checklist back to Pickering Laboratories and place in the front of the
Operation Manual under the tab labeled Installation. If the customer requires it, complete the IQ/OQ procedure
(also located in Appendix) and add that to the tab labeled Installation.

Read all installation instructions and material safety data sheets (MSDS’s) before operating your post-column
derivatization instrument and HPLC system.

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Pickering Laboratories Inc.

3.2

Section 3

In st al la tI on

Note: Before the Pinnacle PCX can be installed and qualified properly, the HPLC must be completely installed
and in good working order (including pump, injector, detector and data collection system). The user prior
to Pinnacle PCX installation must remove all organic compounds that are immiscible with the Pickering
Laboratories’ eluants as well as any hazardous chemicals.

The Pinnacle PCX uses three main styles of fittings.

1. Lite-touch fittings
These are 10-32 nut with 2-part ferrules for 1/16" OD tubing. These fittings are found at the column
connections, and at the inlet to the reagent filters. (nut: 1452-0118, ferrule: 1452-0117)

2. Long PEEK fittings
These are 1/4-28 nuts with 2-part ferrules for 1/8" OD tubing. These fittings are found at the inlet and outlet of
the reagent pumps. Low-pressure gas and reagent fittings are 1/4-28 x 1/8 inch size. (nut: 1452-0116, ferrule:
1452-0115)

3. Short PEEK fittings
These are 1/4-28 nuts with 2-part ferrules for 1/16" OD tubing or 1/8" OD tubing. These fittings are found at
the connections to the electronic valves and transducers. (nut: 1452-0113, ferrule: 1452-0115)

Pickering Laboratories supplies all the matching nuts and ferrules needed for normal installation.

Site Requirements

PINNACLE PCX SITE REQUIREMENTS

The minimum bench top space required for the Pinnacle PCX system is approximately 32H x 16W x 20D inches
(81 x 41 x 51 cm), both doors fully opened, with bottles and electrical connections in place. The Pinnacle
PCX weighs approximately 67 lbs (30kg) for simplex systems, and approximately 77 lbs (35kg) for duplex
systems. The minimum bench space does not include the HPLC system. The total space requirement depends
on the brand and model of HPLC.

For most cases, it is best to place the LC pump and injector system on the left side of the Pinnacle PCX, and the
detector on the right.

In addition to the power outlets required for the HPLC system, one grounded outlet will be needed.

Nitrogen is required to pressurize the reagent reservoir(s). The Pinnacle PCX requires gas pressure of
45-75 psi (3-5 bar) at the gas inlet. An adaptor from the gas regulator to 1/8 inch OD tubing is required.

®

To minimize oxidation of the TRIONE
gas supply line (Air Barrier or metal).

ninhydrin or OPA reagent, use oxygen-impermeable tubing for the entire

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Pickering Laboratories Inc.

Section 3

In st al la tI on

Note: If TRIONE® is to be used for Reagent 1, Nitrogen must be used to prevent out-gassing.

A waste container should be provided for the waste lines from the Pinnacle PCX and the HPLC detector.

HPLC SYSTEM REQUIREMENTS

Since every HPLC is different, the following procedure has been generalized. Before attempting to connect any
tubing, examine the HPLC setup, and determine the best possible means of making the connections. Small ID
tubing (0.011") should be used wherever the sample is in the flow path.

Important! If the system will be used for amino acids, glufosinate, glyphosate, polyamines, or diquat &
paraquat analysis, be aware that the column regenerant is strongly alkaline. Any polymers or other materials in
the HPLC pump, injector, needle seat, and detector must be compatible. For example, the standard rotor seal in
Rheodyne injector valves is Vespel® polyimide, which is not recommended at pH >9; a Tefzel® or PEEK rotor
seal must be installed.

3.3

For all applications, the pressure rating of the detector flow cell must be > 110 psi (7.5 bar)

FOR AMINO ACID ANALYSIS

Pump

Minimum ternary gradient elution
Piston wash capability is preferable

Injector

Tefzel or PEEK rotor seal for injector valve
Tefzel or PEEK needle seat if it is an autosampler

FOR GLYPHOSATE ANALYSIS

Pump

Minimum binary gradient elution
Piston wash capability is preferable

Injector

Tefzel or PEEK rotor seal for injector valve
Tefzel or PEEK needle seat if it is an autosampler
For water samples, at least 200 μl injection

Pinnacle Operators Manual

Pickering Laboratories Inc.

3.4

Section 3

In st al la tI on

FOR CARBAMATE ANALYSIS

Pump

Minimum binary gradient elution

Injector

For water samples, at least 200μl injection

For all other applications, review the method notes for chemistry requirements.

HPLC RELAY REQUIREMENTS

For HPLC systems other than Agilent 1100, or 1200, the Software and system must be capable of sending a relay
signal to an external piece of equipment to achieve synchronization.

Chemstation version 9.0 or higher is needed for Agilent 1100 or Agilent 1200. Pinnacle PCX software will
communicate with Chemstation directly – no relay connection is needed.

Any machine that drives this relay input shall provide a relay contact pair that is electrically isolated from all
other electrical devices. The relay signal must have:

Relay detection voltage 24 +/- 2 V
Relay detection current Approximately 1 mA

COMPUTER REQUIREMENTS

For the Installation, we strongly recommend installing the USB cable.
Use of the network connection is optional at the user’s discretion.
The computer must have:
Microsoft Windows XP, Vista, and Windows 7.
Minimum of one extra USB port for installation
Available Memory: Minimum 10Mb

Instrument Unpacking and Preparation

UNPACKING

Unpack all cartons and review the contents using the Packing List to ensure that all of the items are present. If
any items are missing, immediately contact Pickering Laboratories, Inc.

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Pickering Laboratories Inc.

Section 3

In st al la tI on

Toll Free: [phone]
International: [phone]
Email: [e-mail]
Internet: www.pickeringlabs.com

Unpack the instrument and place it on the bench. Place it so there is enough clearance between the Pinnacle
PCX and the HPLC, detector, and the edge of the bench.

Ensure that there is enough room to open the column oven door and the fluidics door.

Ensure that there is at least 3 inches clearance between the outlet vent at the back of the Pinnacle PCX and any
walls or other instruments. This is very important for proper cooling of the column oven.

3.5

PREPARATION AND INSPECTION

Place the Pinnacle PCX on the bench.
Examine the external chassis for damage.
Open the column oven door. Check that it is straight. Check that the latch is tight and undamaged.
Open the Fluidics chamber door. Check that it is straight.
Examine the connections on the face of the fluidics panel. Check that there are no broken, bent, or loose
tubings.
Remove the right side panel. Check that the pump/s is/are firmly attached to the chassis. Uncoil the 1/4" C-flex
tubing. This tube is for the piston wash. Place the tubing on top of the instrument in the Reservoir tray.

Check that the piston wash tubing is firmly connected to the Y fitting, see figure 2-11.

Replace the right side panel.

Connect the power cord to the outlet in the back of the Pinnacle PCX, just above the power switch.

Gas Connections to Back Panel of Pinnacle PCX

Connect the Inlet Gas line to the back of the Pinnacle PCX.
[Set the regulator on the inert gas supply to between 45-75 psi] Using either 1/8" OD Air Barrier tubing, or
1/8" copper tubing, connect the gas supply to the Gas Inlet fitting on the back panel of the Pinnacle PCX with
the 1/4-28-1/8" nuts and ferrules (PN 1452-0181 and 1452-0180).

Pinnacle Operators Manual

Pickering Laboratories Inc.

3.6

Section 3

In st al la tI on

Do not connect the 1/8" Air Barrier tubings to the bottles yet. Lay them carefully in the Reservoir tray. The gas
connections will be completed later in the installation.

Carefully turn on the main gas supply. Switch the toggle valve to the ON position to start gas flow. Let the gas
system purge for about one minute. Check for leaks at the Gas Inlet connection, and check that there is gas
flowing out of the 1/8" tubes.
Switch the toggle valve OFF.

Slide the Pinnacle PCX into place on the bench. Allow a minimum of 3 inches of space for venting at the back of
the instrument.

Computer Connections

RELAY CONNECTIONS

Relay Input Connector can be used to trigger Pinnacle PCX operations.

Connect the relay cable to the Relay Input Connector on the back of the Pinnacle PCX. The opposite end of the
cable will be connected to the HPLC system’s location for relay output. Refer to the HPLC operation manual for
further information.

POWER-UP

Turn on the power switch, but do not operate the buttons.

Upon power-up, the valves will find their home positions. Ensure that this happens.
Check that the LCD displays the status of the instrument.

Software Installation

Insert the CD into the computer and follow the on-screen instructions. The Installation wizard will guide you
through the installation and configuration process.

MULTI INSTRUMENT SUPPORT

Select the number of Pinnacle PCXs that will be connected to the same PC. For each Pinnacle an icon will be
created on the desktop. Configure each instrument separately.

COMMUNICATION METHOD

Select Network or USB cable. These settings can be modified later in the Configuration window of the Pinnacle
PCX software.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 3

In st al la tI on

SECURITY LEVEL

Select who can access the Pinnacle PCX program. “Everyone” will allow access to many users while “Just me”
will only one user.

REGISTER

Complete the registration form and e-mail directly to support @pickeringlabs.com or print and fax to
(650) 968 – 0749.

OPTIONAL

Connecting the Pinnacle PCX to the network

It is at the user’s discretion if they would like their Pinnacle PCX placed on their company network. This will
involve working closely with the Company’s IT personnel, to determine proper address and their network
security procedures.

3.7

A network cable is provided with the Pinnacle PCX

Connector RJ45
Connection Speed 10/100-Base-T
Ethernet protocol TCP/IP
IP address User set or DHCP

DHCP IP address is assigned by a DHCP server. The network administrator must configure the DHCP server so
that the same address is reserved for and always given to Pinnacle PCX.
User set or Static IP address is set in the Pinnacle PCX. The network administrator must provide an address
specifically for Pinnacle PCX.

Note: We recommend connection to a Network Switch or Router, or direct connection to a dedicated Network
card in the PC.

Setting up or Changing User set (Static) IP address Follow the chart below:

Note: Use the MODE button to go through options and the SELECT button to select the option. Messages appear
at the bottom of the main LCD screen.

Turn Pinnacle PCX ON. While instrument is initializing press the STOP button and hold till the message reads:
BOOT: 1-04 CPLD: 2.5
DHCP IP: 192.168.111.111

Pinnacle Operators Manual

Pickering Laboratories Inc.

3.8

Section 3

In st al la tI on

Press MODE button. The message will read:
RUN MAIN F/W
DHCP IP: 192.168.111.111

Follow the chart below to make changes in IP address.

Main LCD Display

DHCP IP: 152.168.111.111

Select

RUN MAIN F/W

DHCP IP: 192.168.111.111

Mode

NETWORK

IP FROM DHCP

Mode

DHCP IP: 192.168.111.111

MAC: 00:50:C2:44:30:45

Mode

PRESSURE UNITS

PSI

Restart the instrument after completing the steps above.

Pump and Autosampler Connections

Note: Do not fit the analytical column and guard yet.

Mode

Select

Select

NETWORK

STATIC IP

Mode

STATIC IP:192.168.111.111

MAC:00:50:C2:44:30:45

Select

EDIT IP: 192.168.111.111
Select changes number

Mode moves next to position

PRESSURE UNITS

BAR

Replace any mobile phase that is more than 2 weeks old, especially DI water. Rinse the reservoir bottles
thoroughly with soap and water. Wipe down the dip tubes on the caps with methanol and a clean, lint-free
cellulose tissue. Avoid touching the tubing or the interior of the reservoir with your skin and do not leave caps
and lines dangling without a reservoir because this can cause contamination.

Fill reservoirs with 80/20 Water/Methanol

Open the prime-purge valve on the HPLC. Purge each line in the system with a water/methanol mixture to flush
the system. Set the flow rate to the maximum and purge at least 25 ml through each line.

Pinnacle Operators Manual

Pickering Laboratories Inc.

outlet check valve

from solvent
switching valve

purge valve

3.9

Section 3

In st al la tI on

pulse dampener

Te e

Plug

inlet check valve

To autosampler

Figure 3-1

Drop the flow rate to 1 ml/min and close the prime-purge valve to flush the lines to the injector.

If this is an amino acid system, it is highly recommended that you dead-head the pulse dampener of the pump.
This will provide reliable gradient formation, and will prevent corrosion of the low-grade steel used in most
pulse dampeners.

Use one of the two methods below for making the connections. Amino Acid systems must be connected
according to method 2.

1.Connect the outlet of the injector to the inlet of the pre-column check valve assembly.
OR

2.Connect a tee to the inlet of the pulse dampener. Connect a line from the outlet of the pulse dampener to a tee
and place a high-pressure plug in the outlet. Connect the inlet and outlet of the pump through the tee (figure
3-1).

Connect the outlet of the injector to the Check Valve assembly. Use 0.011" ID tubing and a PEEK nut and ferrule.
(PNs 1452-0118, 1452-0117, 2104-0210, 1452-0226)

Place the open end of the check valve tubing into a beaker.

Flush line from injector for 5 minutes at 1 ml/min with 80/20 Water/Methanol.

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3.10

Section 3

In st al la tI on

Eluant Priming

Before proceeding, check for and repair any leaks between the pump and the pre-column check valve. Once
you are certain there are no leaks, do not open the connections between the pump and injector.

IMPORTANT! If any application other than Carbamates is to be used, remove water/methanol and replace
with water. Flush at least 10 ml through lines and flush
injector line. This is to prevent any precipitation issues
between Methanol and the buffers and to prevent any
organic solvents from entering the column. Pickering
Laboratories cation exchange columns for Amino Acids
and Glyphosate analysis will be damaged by organic
solvents.

Figure 3-2

Column outlet tubing

Column oven door

Rubber foam seal

Fill the reservoirs with the appropriate eluants/mobile
phases and again flush at least 25 ml OF EACH ELUANT
with the purge valve in the open position.

Close the purge valve, and pump each eluant at 2 ml/
min for 5 minutes.

If buffers will be used as the eluant, use pH paper to
measure the pH of the solution coming through the
tubing.

Column and Guard Installation

Set the HPLC to 100% of the storage solution in the
column.
Install the pre-column check valve and female nut
inline before the column over door. Reference the
Flow Diagram in section 8.5 for complete parts list of
the pre-column check valve.
Start the flow rate to 0.2 ml/min.
Connect the line from the outlet of the pre-column
check valve to the inlet of the guard column.
When liquid exits the guard column, connect the
“column connector/coupler” provided in the packing
kit. When liquid exits the connector, connect the

to Guard

Column

Blue Peek Tubing

PEEK Nut

(3101-0011)

Check Valve

(3106-1007)

Analytical column

Inlet end

Coupler/column connector

Guard column

Check valve

from HPLC

PEEK Nut

(1452-0226)

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Pickering Laboratories Inc.

Section 3

In st al la tI on

analytical column. Connect the Column Outlet tubing (pre-formed and cut to the right length at Pickering).
Hang the column in the column oven, use care to ensure the tubing passes through the guide hooks at the top of
the column oven. The guard column should be at the bottom, and the exit of the column at the top.

NOTE: If the application is one that uses sodium or lithium buffers, use deionized water to clean any stainless
steel fittings that have come into contact with buffer to prevent corrosion.

Monitor the pressures and stop the HPLC pump when the pressure stabilizes.

Detector Connections

Connect a 1/16 inch x 0.020" ID tubing from the outlet of the Detector
to the external 100psi back-pressure regulator (PN 3102-9025) using a
1/4-28 nut with a 1/16 inch reversed-ferrule. There is an arrow on the back
pressure regulator indicating direction of flow. Insure that the arrow is
pointing away from the detector and toward the waste line.

3.11

from Detector Exit

Connect the 0.020" ID PTFE tubing provided in the packing kit (PN 2101-

0225) to the outlet of the external 100 psi back-pressure regulator. Place
the other end in an appropriately labeled waste container.

Connect the PEEK union to the outlet of the Ambient reactor (or Heated
reactor if it is a single-pump system) using a Lite-touch Nut and Ferrule.

Connect a 0.011" ID tubing from the outlet of the union at the exit of the

Figure 3-3

fluidics panel on the right-hand side of the instrument to the inlet of the
detector flow cell.

Set the time constant on the detector to 2–4 seconds.

The pressure rating of the detector flowcell must be >110 psi (7.5 bar). If your detector flowcell is rated lower,
consult Pickering Laboratories.

Special Note to Hewlett-Packard 1046A end-users: Replace the 0.12 mm ID inlet tubing (red) and heat­exchanger from the left side of the detector to the flowcell (behind the front panel of the detector) with a 0.25
mm ID (blue) tubing (HP Cat. No. 79881-67302) to reduce the back-pressure.

Caution! The 100psi back-pressure regulator is directional. Do not reverse flow!

Flow direction

to Waste

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3.12

Section 3

In st al la tI on

Reservoir Connections

Wash all of the Pickering Pinnacle PCX reservoirs with laboratory detergent and hot water.

Rinse with methanol then with deionized water.

Wipe down the dip tubes on the caps with methanol and a clean, lint-free cellulose tissue. Avoid touching
the tubing or the interior of the reservoir with your skin and do not leave caps and lines dangling without a
reservoir because this can cause contamination.

There are three types of Reservoir/Cap assemblies used on the Pinnacle PCX:
Reagent Reservoirs are 1L, 45-430, safety-coated. These are shipped with blue caps with white valve assembly.
These can be pressurized.
Wash Reservoir are 1L, 38-430, clear glass. These are shipped with blue caps with 1ea 1/4" opening and 2ea
1/8" openings. These bottles cannot be pressurized, and do not contain threads for tubing connections.
Flush Reservoir, is 1L, 38-430, clear glass, from Wheaton. These are also shipped with caps, but with 3ea 1/8"
openings. These bottles cannot be pressurized, and do not contain threads for tubing connections.

The two types of caps cannot be interchanged because the necks of the bottles are different sizes.

REAGENT RESERVOIR CONNECTIONS

Connect 1/8" Air Barrier lines from “Gas Supply OUT” port on the back of the Pinnacle PCX to the CHECK
VALVE on the top of the reagent cap. Use a 1/4-28 Nut and ferrule (PN 3101-0005 and 3101-0006). Don’t over
tighten!

Connect 1/8" Air Barrier lines from the Reagent ports of the valves (No 3) ports at the front of the Pinnacle
PCX to the empty port on the top of the reagent cap. Use a 1/4-28 Nut and ferrule (PN 3101-0005 and
3101-0006). Don’t over tighten!

Put the cap on the bottle.

Toggle the Gas switch to ON and check that gas is flowing through the reagent cap. Close the stopcock under
the vent hole to pressurize the bottle. Open the stopcock under the reagent line before refilling the pump.

Caution! The reagent bottles are specially coated with a protective polymer to ensure operator safety if the
reservoirs should become over-pressurized. Non-coated bottles must not be substituted in the Pinnacle PCX
system. Replacement 1 L, 2 L, or 5 L reagent bottles may be ordered directly from Pickering Laboratories.

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Section 3

In st al la tI on

Reservoirs should be labeled with an appropriate label using the GLP of the laboratory.
Fill the Flush reservoir with water or 80-20 water/alcohol (IPA or Methanol)
Fill the Wash reservoir with 90-10 water/alcohol (IPA or Methanol).
Change Wash and Flush solutions at least once a week to prevent contamination.

Rinse the Reagent reservoir(s) with a small amount of reagent or diluent, and then if necessary prepare the
post-column reagents as described in the appropriate Application section of this manual.

FLUSH RESERVOIR CONNECTIONS

Take the 1/8" FEP tubes leading from the Flush (No. 5) position on the valves and slide the tubing through the
1/8" openings on the proper cap. This will be a snug fit to prevent slipping.

3.13

(PISTON) WASH RESERVOIR CONNECTIONS

Take the 1/4" C-flex tubing leading from the piston wash of the reagent pump(s) and slide it through the 1/4"
opening in the proper cap. This will be a snug fit to prevent slipping.

Reagent Pump Preparation

All of the reservoirs should now be filled with the appropriate solutions.
Ensure that the Flush and Piston Wash lines are immersed in liquid.

The Pinnacle PCX has a maximum of two waste lines that must be fed into a waste container. They can either be
fed into a separate container, or into the waste stream of the HPLC. These are the 1/8" OD clear FEP tubes that
are connected to the Waste port (No. 6) on the valves.

Dual pump systems will have two lines, and single pump systems will have one.

In the Pinnacle PCX software, select Empty pump(s) under the Control menu. Select Both pumps for
2-pump system. The valve(s) will move to waste (1-6) position and pump(s) will dispense all the liquid.

In the Pinnacle PCX software, select Flush pump(s) under the Control menu. Select Both pumps for
2-pump system. The system will flush pumps using Flush solution. The pump(s) will be empty at the end of
Flush cycle.

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3.14

Section 3

In st al la tI on

Running a Chromatogram

Look in the column box for the gradient program and conditions.

Set the HPLC pump to run the starting eluant conditions, and while the system is equilibrating, set up the
gradient method in the HPLC software. We recommend that equilibration is set up at the end of each run (as a
post-run, if possible). Equilibration time should be at least 5 min long to give Pinnacle PCX enough time to refill
between the runs. If using relays to synchronize with Pinnacle PCX set up relay signal at time 0.0 in the HPLC
method. Contact your HPLC support representative if you have questions about setting up relay signal.

Create a sequence for the HPLC system.

Set up method and sequence for Pinnacle PCX (refer to section 4 OPERATION of this manual). Load the
sequence. Make sure correct sequence name and method name are displayed in the status bar at the bottom of
the Pinnacle software (If sequence and method fail to load: close Pinnacle PCX software, reopen Pinnacle PCX
software, reload sequence and method).

In the Pinnacle PCX software, select Enable in the Control menu. Column and reactor heater will be turned
ON. Wait for temperatures to reach the set point.

Select Refill pump(s) in the Control menu. Select Both pumps for 2-pump system. The valve(s) will move
to reagent (3-4) position and the pump(s) will refill enough reagent for one run.

NOTE: Pinnacle software will calculate needed reagent volume based on your run time and reagent flow rate.
Some extra volume will be added to that to ensure pumps will not run out of reagent during operation.

Select Pump(s) ON in the Control menu. Select Both pump(s) for 2-pump system. The valve(s) will move
to reactor (1-2) position and pump(s) will start dispensing reagent at set flow rate. Wait until the pressures are
stable.
Select Sequence – Start Sequence. Run time (0.0) will be displayed in the Status Bar at the bottom of the
Pinnacle PCX software. The reagent pump(s) will start dispensing reagent. Pinnacle now waits for injection
signal from HPLC in order to start running the method.

®

For TRIONE

Amino Acid Analysis:

Set the detector wavelength(s) to 570 and 440nm (if applicable).

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Section 3

In st al la tI on

Do four runs of the Amino Acid Standard that was provided with the chemical kit. Inject 10μl.
Discard the first injection.
Compare the chromatogram with that of the QC test of the system and column.
Verify that the system is functioning by using the IQ/OQ documents as reference (See Operation Manual,
Appendix)

For Carbamate and Glyphosate Analyses:
Set the detector excitation wavelength to 330 nm and the emission wavelength to 465 nm
Do at least four runs of the appropriate test mixture. Inject 10ul.
Discard the results of the first injection.
Compare the chromatogram with that of the QC test of the system and column.
Verify that the system is functioning by using the IQ/OQ documents as reference (See Operation Manual,
Appendix)

3.15

Shutdown

Upon completion of the analyses, use one of the following two procedures to shut down the Pinnacle PCX
system properly. These procedures can prevent potential column damage, reaction coil blockage, high
background fluorescence, reagent precipitation, or other problems.

The Pinnacle PCX must be flushed out at the end of a series of injections, and the reactors must be cooled
down. If the instrument is simply stopped, with reagent inside the reactor in a hot state, with no movement, then
the heated reactor will become blocked. It is very important for a long useful life that the reactors be flushed
out until the temperature is cool.

MANUAL SHUTDOWN

You may shutdown the Pinnacle PCX manually by pressing/selecting the Disable function in the Control menu
of the PCX control software.
If you choose this function, allow the HPLC to pump for at least 30 minutes to allow for the reactor to cool and
to flush reagent from the reactor.

OR
AUTOMATIC SHUTDOWN

Create a Shutdown Method for Pinnacle PCX according to section 4 of this manual. Set it up as the last
method in the Pinnacle PCX sequence. Make sure pump flow rate is set to 0 mL/min and reactor temperature
is set close to room temperature. You can leave the column at the operating temperature or set it at room
temperature.

Create corresponding slowdown Method for HPLC. Set it up as the last method in the HPLC sequence. Make
sure HPLC flushes column and reactor with column storage eluant for at least 30 min.

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3.16

Section 3

In st al la tI on

RECOMMENDED ACCOMPANYING HPLC SLOWDOWN METHOD

Set the HPLC to 100% Storage Eluant (see application section for proper eluant for your column), and set the
HPLC pump at the normal analytical flow rate. Choose an eluant that elutes contaminants from the column; for
example, methanol for a reversed-phase column and regenerant for an ion-exchange column.

Time (min) % Storage Eluant Flow (ml/min)

0 100 Analytical flow rate*

30 100 Analytical flow rate*

30.1 100 0.0 or 0.02mL/min

*Follow instructions that come with the column.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 4

pinnaCle pCx OperatiOn

4.1

Section 4

Pi nn ac le PcX OPerati On

4.1

4.2

4.4

4.6

4.7

4.7

4.10

4.11

4.14

4.17

4.17

Software Overview
Status Window
Menu Options/Functions
Configuring your Pinnacle PCX
Log Files
Creating and Editing Methods
Creating and Editing Sequences
Tutorials
Running Post-column Analysis Using Pinnacle PCX
Stop/Reset Instrument
Pinnacle Shutdown

Software Overview

Pinnacle PCX can be controlled only with PC using Pinnacle PCX software. The system can be connected to PC
through USB or Ethernet cable.

Pinnacle PCX software controls column and reactor heaters, electronic valves and pumps. It allows to create,
store and run post-column methods and sequences. It monitors reactor and column temperatures, pump
pressures and flow rates. It keeps logs that records system parameters and HPLC flags.

COMPUTER REQUIREMENTS

Microsoft Windows XP, Vista, or Windows 7 operating environment
Minimum one free USB port or network connection
Available memory: 10 Mb

HPLC SYNCHRONIZATION

Pinnacle PCX needs to receive injection signal from HPLC to synchronize operation.

For HPLC systems other then Agilent 1100 and Agilent 1200 the HPLC software and system must be capable of
sending a relay signal to an external piece of equipment to achieve synchronization.

Agilent Chemstation version 9.0 or higher is needed for Agilent 1100 or Agilent 1200. Pinnacle PCX software will
read injection signal from Chemstation directly – no relay connection is needed. If any other software program
is used to control Agilent 1100 or Agilent 1200, relays board should be installed on the Agilent pump.

Pinnacle Operators Manual

Pickering Laboratories Inc.

4.2

Section 4

Pi nn ac le PcX OPerati On

Any machine that drives the relay output shall provide a relay contact pair that is electrically isolated from all
other electrical devices. The relay signal must have:

Relay detection voltage 24 ±2V
Relay detection current Approximately 1 mA

Status Window

The Status window is the main screen of the Pinnacle PCX software (Figure 4-1). The Status window can be
moved, or minimized into an icon at the bottom of the PC screen. It has a menu bar on the top, status bar at the
bottom and icons for the Pinnacle PCX components.

ICONS

Pump 1:

Displays the actual volume in ml
of liquid in the syringe. The status bars will
increase/decrease with volume. The actual
volume is displayed at the bottom of the icon.

Pump 2: Displays the actual volume in ml

of liquid in the syringe. The status bars will
increase/decrease with volume. The actual
volume is displayed at the bottom of the icon.

Column oven temperature: Displays the

Set and actual temperature in ºC. The actual

Figure 4.1

temperature is displayed at the bottom of the icon. The status bars on the column oven icon will fill in and
increase as the column heats. The reverse is true when the column cools. The icon shows the temperature of
the column oven to the nearest degree. The recommended maximum temperature for the column heater is
75°C. A thermal safety switch limits the heater at ca. 80°C.

Reactor temperature: Displays the Set and actual temperature in ºC. The actual temperature is displayed

at the bottom of the icon. The reactor temperature is measured to the nearest degree. The status bars on the
reactor oven icon will fill in and increase as the reactor heats. The reverse is true when the reactor cools. The
recommended maximum temperature for the heated reactor is 130°C. Above this temperature the reaction coil
begins to lose strength. A thermal safety switch limits the heater at ca. 150°C.

Pressure: Displays the actual pressure in bar or psi. Pump 1 pressure corresponds to the actual pressure on

Pump 1, and through post-column system beginning at the first mixing tee. Pump 2 pressure corresponds to
the actual pressure on Pump 2, and through the post-column system beginning at the second mixing tee.

Flow: Displays the actual flow rate in ml/min of the reagent pumps. When the pump is dispensing at the

analytical flow rate, the black square in the icon will move slowly in a clockwise direction. When the pump is
refilling, the icon changes to an alternating segments that rotate in a counter-clockwise direction.

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Section 4

Pi nn ac le PcX OPerati On

MESSAGE AREA

The message area gives details about the status of the instrument. This area describes s, and actions. Some
sample messages include:
Enabled Heaters are On
Instrument Stopped
Sequence Stopped
Finishing Current Run
Sequence Done
No Relay Signal

STATUS BAR

The status bar at the bottom of the screen displays the running status (elapsed run time, refilling, ready),
loaded method, loaded sequence. The status bar gives very basic information for a quick glance for method
loaded and run time.
If in ‘Run’ mode:
Run Status, i.e. 3 of 7
Elapsed run time in minutes

4.3

BUTTONS

The buttons located on the right side of the Main Window emulate the buttons on the right of the LCD on the
Pinnacle PCX instrument.

Stop: This will stop the instrument. It does not matter what task the instrument is performing when this is

pressed. The Pinnacle PCX will stop whatever it is doing and will not resume until the user has Reset the system.
The button activates a red LED when the instrument is stopped. This is an emergency function designed to
allow the user to fix any catastrophic problems before they do damage or make a mess.

Mode and Select buttons are used during installation and function only on the instrument.

Pinnacle Operators Manual

Pickering Laboratories Inc.

4.4

Section 4

Pi nn ac le PcX OPerati On

Menu Options/Functions

FILE

Import/Export

another instance of the Pinnacle PCX software.

Exit closes Pinnacle PCX software.

CONTROL

Enable

selecting Enable will cause the reactor and column heaters to begin heating. This will not
start the pumps. The Enable button changes to Disable when pressed. Press Disable for the
instrument to stop heating. Instrument must be enabled before turning pump(s) ON or
starting the sequence.

Refill pump(s) this option will refill pumps to the volume calculated for the currently loaded method. If no

method is loaded the full syringe will be refilled. For two-pump instrument there is a choice
of refilling pump1, pump2 or both pumps. The pump(s) must be refilled before the
sequence is started. The pump(s) will refill automatically between the runs.

Flush pump(s) this option will flush the pumps using solution from the flush bottle. For two-pump

instrument there is a choice of flushing pump1, pump2 or both pumps.

Empty pump(s) this option will empty pump(s). For two-pump instrument there is a choice of empting

pump1, pump2 or both pumps.

Pump1 ON turns on Pump1. This option changes to Pump1 OFF when selected.
Pump2 ON turns on Pump2. This option changes to Pump2 OFF when selected.
Both pumps ON turns on both pumps. After this option is selected, Both Pumps OFF option becomes

available.

exports existing methods and sequences into a file. The file then can be imported to

CONTROL menu choices are not available when sequence is running

METHOD

Edit/Delete

this option opens Method editing screen where Methods can be created, edited, saved or

deleted.

Print this will print Method information to default printer.

SEQUENCE

Edit/Delete

this option opens Sequence editing screen where sequences can be created, edited, saved,

loaded or deleted.

Print this will print Sequence information to default printer.
Load this loads the selected sequence.

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Pickering Laboratories Inc.

Section 4

Pi nn ac le PcX OPerati On

Start sequence starts the loaded sequence. Instrument must be enabled and pump(s) must be refilled

before starting the sequence. After Start Sequence is pressed the following choices become
available:

Stop sequence after this button is pressed the Pinnacle PCX finishes current run then stops the pump(s)

and turns the heaters OFF.

Pause sequence this option allows the Pinnacle PCX to finish current run and then stops the pump(s). It

leaves the heaters ON. The sequence will continue after Resume Sequence is selected.

INSTRUMENT

Configuration
Maintenance gives the selection of the following operations:

Flush Instrument flushes the pumps and the instrument using solution from the flush
bottle. Create and load a method for Pinnacle PCX with suitable reagent flow rates, reactor
and column temperature before executing this command. Make sure HPLC is running to
avoid backflow into the column.
Change seals prepares the pump(s) for seal change. Follow directions on the screen to
complete this procedure
Prepare for storage/shipping flushes the pumps and the instrument in preparation for
long term storage or shipping. Follow directions on the screen to complete this procedure.
Pump and valve seal pressure test & system pressure test. Builds up pressure to test
the integrity of the seals after a PM.

opens the configuration screen.

4.5

INSTRUMENT menu choices are not available when sequence is running.

STOP/RESET This option performs the same action as Stop button on the front of the instrument. This

option is used for an instant stoppage of the instrument in the case of an emergency. The
instrument will cease all the activities, regardless of what is happening. The pump(s) and
heaters will be turned OFF and all the loaded methods and sequences will be cleared. After

STOP is selected the option will change to RESET. Select RESET function when you are

ready to start running the instrument again.

HELP

Index this will open searchable Help features for the Pinnacle PCX software.

Send Log to
Support

saves logs to file that can be e-mailed to [e-mail] . Logs carry
information about instrument status from the last 3 days and are necessary to troubleshoot
software and instrument problems.

About gives software version number.

Pinnacle Operators Manual

Pickering Laboratories Inc.

4.6

Section 4

Pi nn ac le PcX OPerati On

Configuring your Pinnacle PCX

Select Instrument then Configuration to open configuration window (figure 4-2)
The tabs open corresponding windows that allow you to configure the Pinnacle PCX.

Instrument Tab:

Network:

PCX will be connected to the computer
through a company’s network. Enter
the network IP address in the spaces
provided.

USB Port: Select this option if the

Pinnacle PCX will be connected directly to
the computer via a USB cable.

Column Tab:

Temperature Range:

displays the current minimum and maximum column temperature range for the Pinnacle PCX.

Reactor Tab:

Volume, ml:

Pinnacle PCX. The temperature range depends on the volume of the reactor. Enter the volume of the reactor
that you are using.

Pump Tab:

Max Volume:
Flow Rate: 0.05 – 1.5 ml/min: This is the flow rate range for the installed Reagent pump(s).
Pressure in: Bar or psi: Select bar if you would like to see the pressure displayed in bars, or psi if you

would like to see the pressure displayed in pounds per square inch.

More Tab:

HPLC type: Select the brand of HPLC that you will be using with Pinnacle PCX. This will determine the type
of synchronization between Pinnacle PCX and HPLC system.

Select this option if the Pinnacle

This window

This window displays the current minimum and maximum reactor temperature range for the

70 ml: This is the volume of the syringe installed on the Reagent pumps(s).

Figure 4.2

For HPLC systems other then Agilent 1100 and Agilent 1200 the HPLC software and system must be capable
of sending a relay signal to an external piece of equipment to achieve synchronization. Agilent Chemstation
version 9.0 or higher is needed for Agilent 1100 or Agilent 1200. Pinnacle PCX software will read injection
signal from Chemstation directly – no relay connection is needed. If any other software program is used to
control Agilent 1100 or Agilent 1200, relays board should be installed on the Agilent pump.

If your HPLC does not have Relay capabilities, select “No connection to HPLC”.

If you need to connect two Pinnacle PCX to the same PC you need to select multiple instruments during the
software installation. For each instrument a separate icon will created on the desktop that will open a main
Pinnacle software window. Configure each instrument before starting operation.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 4

Pi nn ac le PcX OPerati On

Log Files

Pinnacle PCX records detailed information about the instrument parameters in the Log files. Pump(s) flow
rates and pressures, reagent volume, column and reactor temperature, s and flags from HPLC are all stored
for 3 days before being overwritten. Log files are necessary to troubleshoot Pinnacle PCX system and software
and should always be collected after a problem is detected. To collect Log files go to Help, select Send Log to
Support and select file from the day the problem happened. Save the file with the date stamp and e-mail it to
[e-mail]. If you are not sure about the time collect all 3 log files.

Creating and Editing Methods

To run post-column analysis using Pinnacle PCX a post-column method must be created and executed in the
Pinnacle PCX software. To create or edit a post-column method select Method then Edit/Delete. This will open
Methods editing screen (figure 4-3). To execute methods (even as a single run) they should be part of the
sequence.

4.7

Methods can be edited when they are currently running. Once edited the method should be saved and the
sequence reloaded. New methods and methods not currently running can be added/edited/deleted at any time.

Status Bar This area is located at the top of the

Method Edit screen. It displays messages regarding
the saving, loading and deleting of methods. For
example, when a method called Test 1 is saved, this
area will display Method “Test 1” saved.

Method Box This area displays all of the methods

currently in memory of the Pinnacle PCX software.
Here you can Edit an existing method, Delete a
method, or Add a new method. To perform an
action on an existing method, click on the method
name.

Add Click here to add a new method. The status

bar will display Method “name” added.

Delete To delete a method, click once on the

method name, then click Delete. The Status bar will display Method “name” deleted.

PUMPS TAB Timetable for both pumps (one table for both). Pump 2 flow rate column is grayed out for

1-pump systems. If timetables are empty the initial conditions are the conditions for the whole run.

Time, min: Enter the time in minutes to the nearest tenth, but not greater than the Runtime. (For example,

0.5 minutes)

P1, ml/min Enter the desired flow rate for Pump1, between 0.05 and 1.5 ml/min. (For example, 1.3 ml/min.

This means that Pump 1 will begin pumping 1.3 ml/min at 0.5 minutes)

Figure 4.3

Pinnacle Operators Manual

Pickering Laboratories Inc.

4.8

Section 4

Pi nn ac le PcX OPerati On

P2, ml/min Enter the desired flow rate for Pump2, between 0.05 and 1.5 ml/min. (For example, 0.08 ml/min.

This means that Pump 2 will begin pumping 0.08 ml/min at 0.5 minutes). Enter 0.0 ml/min if you want to stop
pumping reagent at a set time during the run.

Add This will add a new line below the currently selected line in the table.
Insert This will insert a new line above the currently selected line in the table.
Delete This will delete the currently selected line in the table.

COLUMN TAB Timetable for the column oven. If timetables are empty the initial conditions are the conditions

for the whole run.

Time, min Enter the time in minutes to the nearest tenth, but not greater than the Runtime. (For example,

15.5 minutes)

Temp, °C Enter the desired temperature, in whole numbers, in ºC. (For example, 53ºC. This means that the

column will reach 53ºC at 15.5 minutes)

Add This will add a new line below the currently selected line in the table.
Insert This will insert a new line above the currently selected line in the table.
Delete This will delete the currently selected line in the table.

INITIAL SETTINGS In this section, set up the initial conditions for the Pinnacle PCX. If no pump timetable or

column timetable is required, Pinnacle will use this information for analysis. The items marked with * indicate
required parameters.

*Run Time, min In this window, enter the total run time for the Pinnacle PCX method. This must be a whole

number, greater than 1

*Equilibration Time, min Enter the time between runs. The Pinnacle PCX will use this information to

determine when to begin pumping again prior to the next injection. Minimum time is 5 minutes.

*Column t, °C Enter the initial column temperature in ºC. Enter a whole number between 30ºC and 75ºC.
Column Type Enter the column type that is used for this application. Any characters are acceptable. There is

no limit on number of characters, but 20 are visible in the window at a time.

*Reactor t, °C Enter the reactor temperature for the analysis. Enter a whole number between 30ºC-130ºC for

reactor volumes ≤2.0ml, or between 30ºC-80ºC for reactor volumes >2.0 ml. (The reactor volume is set in
Configuration).

Reactor Volume, ml Enter the volume of the reactor used in this application.
*Pump 1 Flow rate, ml/min Enter the initial flow rate for Pump 1. Enter a number between 0.05-1.50 ml/

min, or 0

Reagent 1 Enter the name or description of Reagent 1. Any characters are acceptable. There is no limit on

number of characters, but 20 are visible in the window at a time.

*Pump 2 Flow rate, ml/min Enter the initial flow rate for Pump 2. Enter a number between 0.05-1.50 ml/

min, or 0. Put 0.0 for a single-pump system.

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Reagent 2 Enter the name or description of Reagent 2. Any characters are acceptable. There is no limit on

number of characters, but 20 are visible in the window at a time.

ADVANCED SETTINGS

This tab allows you to designate currently open method as a Flush Method (figure 4-4). This method can be
set as a last method in the Pinnacle PCX sequence in order to flush the reagent pumps and the Pinnacle PCX
reagent lines and reactor. Pinnacle PCX will use
solution in the Flush bottle to do this. Always
set Flush Method as the last method in the
sequence and make sure corresponding HPLC
method is created and set as the last method in
the HPLC sequence. Flushing is not necessary
after every sequence and only recommended
before changing applications and long time
storage or in the case of extremely aggressive
reagents.

4.9

ACTION TABS

Save

This will save the current method

Save As This will save the current method

Figure 4.4

information using a different name.

Cancel This will close the window and cancel any changes. No changes will be made to the method.
Print This will print the method to the default printer. The information printed will be: print date, operator,

method name, save date, initial conditions, pump time table, column time table.

Help This will open the searchable Help features for the Pinnacle PCX software.
Close This will close the window.

IMPORTANT PRACTICAL CONSIDERATIONS

In order to start executing the method Pinnacle PCX needs to receive injection signal from HPLC. To avoid
missing the signal it is very important to match Pinnacle PCX method run time and equilibration time to that of
your HPLC method.

HPLC pump uses equilibration to return to original conditions before the next analysis. HPLC software
programs have different ways of setting up equilibration. Most often used are the following:

- as a PostRun;

- at the end of the pump gradient table;

- as additional time before the injection (set as a PreRun or as negative time in the pump gradient table).

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Even if isocratic method is used and no equilibration time is required for HPLC pump Pinnacle PCX still needs
at least 5 min of Equilibration time to refill reagent pumps and stabilize the baseline signal before the next
injection.

If equilibration for HPLC pump is set up as PostRun or PreRun match Equilibration time in the Pinnacle PCX
method to respectively PostRun or PreRun time of your HPLC method. The run time of your HPLC method
(the actual time of the analysis) should be the same as Pinnacle PCX Run time. Pinnacle PCX delivers
reagents only during the Run time set up in the Pinnacle PCX method.

If returning to initial conditions is set as part of the HPLC pump gradient table (at the end or as a negative time
step in the beginning) consider how long the actual analysis and equilibration steps are. Match analysis time to
Run time of the Pinnacle PCX and match time of the equilibration step to Equilibration time of the Pinnacle PCX.

Creating and Editing Sequences

To create or edit a post-column sequence select
Sequence then Edit/Delete. This will open Sequence
editing screen (figure 4-5).

Status Bar This area is located at the top of the

Sequence Edit screen. It displays messages regarding the
saving, creative and deleting of sequences. For example,
when a sequence called Test 1 is saved, this area will
display Sequence “Test 1” saved.

Select/Edit Sequence Box This area displays all of

the sequences currently in memory of the Pinnacle PCX
software. Here you can Edit an existing sequence, Delete
a sequence, or Add a new sequence. To perform an
action on an existing sequence, click on the sequence
name.

Create Click here to add a new sequence
Delete To delete a sequence, click once on the sequence name, then click Delete.

SEQUENCE TABLE WINDOW

Method name Number of runs

Figure 4.5

Select the method name from
the drop down menu

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Add This will add a new line below the currently selected line in the sequence.

Insert This will insert a new line above the currently selected line in the sequence.
Delete This will delete the currently selected line in the sequence.

The end of the sequence should be with a shutdown method or a flush method.

ACTION TABS

Save

This will save the current sequence.

Save As This will save the current sequence information using a different name.
Load This will load the current sequence into the Pinnacle PCX software.
Cancel This will close the window and cancel any changes.
Print This will print the sequence to the default printer. The information printed will be: print date, operator,

sequence name, sequence table (with method names), save date.

Help This will open the searchable Help features for the Pinnacle PCX software.
Close This will close the editing window.

4.11

EDITING THE RUNNING SEQUENCE:

- select the Sequence name from the Sequence Box.

- change number of runs of the currently running Method or add a new line to the Sequence anywhere below
the Method in progress.

- select Save and then Load from the Actions tabs.

- check number of runs on the bottom of the main software window to confirm that changes has taken affect.

Tutorials

CREATE A METHOD

The following is a tutorial on how to create a method and start a run on the Pinnacle PCX. It is not intended to
be used for a real run because the flow rates and temperatures are not representative. If you like, you may use
this tutorial as a guide to create your own methods.

This example is a fictitious method and is not intended to analyze samples.

Double-click on the Pinnacle PCX icon on your desktop. This will open the main Status Window of the Pinnacle
PCX software.
Go to Method, then select Edit/Delete. This will open the Method Edit Screen. Here you will set the initial
conditions, flow rates, temperatures for your method.

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Click Add Method.

Type a Name for your method. For this example, type “Tutorial”, and click OK.

The Pinnacle PCX software has now added the method “Tutorial” to the list of available methods. Highlight this
method if it is not already done so.

Under Initial Settings Tab, type:
Run Time, min: 60
Equilibration Time, min: 5
Column t, °C: 38
Column Type: 1154150 Na cation exchange
Reactor t, °C: 36
Reactor Volume, ml: 0.5
Pump 1 Flow rate, ml/min: 0.3
Reagent 1: Hypochlorite
Pump 2 Flow rate, ml/min: 0.3
Reagent 2: OPA

Click on the Pumps Tab, then click once in the box under Time. The cursor should now be flashing here. Type in
your desired Times and flow rates below. Use the Add button to add lines to the table. Use the data from the table
below as a guide. Time is the elapsed time in minutes. P1 and P2 are the flow rates of each pump in ml/min.

Time, min P1, ml/min P2, ml/min

1.0 0.3 0.5

1.6 0.8 0.0

3.8 1.0 0.05

12.5 0.0 0.05

30 0.1 0.0

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Next, click on the Column Tab, then click once in the box under Time. The cursor should now be flashing
here. Type in your desired Times and temperatures below. Use the Add button to add lines to the table. Use
the data from the table below as a guide. Time is the elapsed time in minutes. Temperature is in °C and is the
desired temperature of the column at the time specified.

Time, min Temp, °C

5.0 45

9.5 48

25 56

50 75

Now Click Save. This has saved the conditions you have created in the method “Tutorial”.

4.13

Click Close to close the Edit Method Window.

Next, we will go through the Create Sequence Tutorial.

CREATE A SEQUENCE

As with the above method, this example sequence is not designed to be used for analysis. It will guide you
through the steps and you may use your own method for the Sequence Table.

From the main Status Window, go to Sequence, then select Edit/Delete.

This will open the Sequence Edit Screen. Here you will set the method names, and the number of injections for
each method.

Click Create Sequence.

Type a Name for your Sequence. For this example, type “Tutorial 1” and click OK.

The Pinnacle PCX software has now added the Sequence “Tutorial 1” to the list of available sequences. Highlight
this sequence if it is not already done so.

Under Method Name, there is a drop down menu. Choose the Method “Tutorial”.

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Next, under the Runs, type in the number of injections you plan to make using this method. In this example, it
will be 3.

Your table should now look like:

Method Name Runs

Tutorial 3

The number of Runs should match the number of runs you plan to make on the HPLC using that method. In this
example, your system will perform 3 injections using the Pinnacle PCX method “Tutorial”. The sequence table
on your HPLC system should also have 3 injections programmed.

Now Click Save. This has saved the conditions you have created in the sequence “Tutorial 1”.

Click Close to close the Edit Sequence Window.

Running post-column analysis using Pinnacle PCX

To start running post-column analysis using Pinnacle PCX follow the steps below:

Create analytical method(s) for your HPLC system using gradient conditions and detector settings from
Pickering Laboratories application notes or other sources. For any other HPLC software except Agilent
Chemstation, set the relay start signal close to time 0. Depending on the software the relay stop signal
can be either a few minutes after injection or at the end of the run. Contact your HPLC support representative if
you have any questions about setting up relay signal in the HPLC method.

Create post-column method(s) for your
application using post-column conditions from
Pickering Laboratories application notes or
other sources.

Set up HPLC sequence. Contact your HPLC
support representative if you have any questions
about creating and running HPLC method or
sequence.

Start HPLC pump. Increase the flow rate
gradually to avoid overpressuring the analytical column.

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Figure 4.6

Section 4

Pi nn ac le PcX OPerati On

Create sequence for Pinnacle PCX using your post-column method(s). Set up the same number of runs as in the
HPLC sequence.

Select Save to save Pinnacle PCX sequence.

Select Load to load Pinnacle PCX sequence. Sequence name and the first method name will appear in the status
bar at the bottom of the Pinnacle PCX software.

Select Control – Enable to turn column and reactor heaters ON. Set and actual temperatures will be
displayed on the heaters icons. Wait until the actual temperature reaches the set point.

Select Control – Flush both pumps if this is the first run after the installation or if the instrument was not in
use for a long time.

4.15

Select Control – Refill Pump(s) to fill pumps with reagent. Empty pumps first if there is any old reagent left
or if instrument was turned OFF earlier. The pump icon should show partially filled syringe and actual volume
of reagent inside (Figure 4-6).

NOTE: If the instrument was turned OFF with partially filled pumps the icons will show alternated black and
white segments and no volume data will be displayed. This means that the instrument does not know the
position of the piston inside the syringe. Select Control – Empty Pump(s). This will allow Pinnacle PCX to
find empty position.

Once reactor and column temperatures are stable and analytical column is equilibrated select Control – Both
pumps ON (or Pump ON for single-pump instrument) to start pumping reagents. Wait until the pressures are
stable.

NOTE: Pinnacle PCX software will calculate needed reagent volume based on your run time and reagent flow
rate. Some extra volume will be added to that to ensure pumps will not run out of reagent during operation. Do
not run pumps for more then 5 min before starting the sequence to avoid running out during the analysis.

Select Sequence – Start Sequence. Run time (0.0) will be displayed in the Status Bar at the bottom of the
Pinnacle PCX software. Pinnacle now waits for injection signal from HPLC in order to start running the method.

Start HPLC sequence. Once an injection is made Pinnacle PCX software will receive an injection signal via relays
or directly from Agilent Chemstation. After that Pinnacle will start the post-column method. Check the timer at
the bottom of the Pinnacle PCX software to make sure it matches the elapsed time of the HPLC run.

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NOTE: after starting a new sequence always make sure that first injection signal was received normally by
checking that Pinnacle PCX timer matches elapsed time of the HPLC run. Pinnacle PCX will stop the sequence if
no injection signal is received from HPLC in 15 min from selecting Start Sequence.

After the post-column method Run time is done Pinnacle PCX turns the pump(s) OFF , checks temperature and
flow rate settings for the next run and automatically refills the reagent. Pinnacle PCX will turn the pump(s) ON
5 min before the end of the Equilibration time.

Once next injection signal is received Pinnacle PCX starts the second method in the sequence. The number
of the current run and total number of runs in the Sequence are shown on the bottom of the Pinnacle PCX
software.

After Sequence is complete Pinnacle PCX turns OFF the pump(s). Message “Sequence done. (Runs n of N)” is
displayed in the software window.

Important Practical Considerations

Always match number of runs in HPLC and Pinnacle PCX sequences. If more samples are added to HPLC
sequence or samples are removed edit Pinnacle PCX sequence accordingly. Total number of samples displayed
by HPLC software often does not include calibrators and controls. Make sure you count total number of runs in
HPLC sequence not just number of samples before creating Pinnacle PCX Sequence.

To edit the running Sequence:

- select Sequence – Edit/Delete

- select the Sequence name from the Sequence Box.

- change number of runs of the currently running Method or add a new line to the Sequence anywhere below
the Method in progress.

- select Save and then Load from the Actions tabs.

- check number of runs on the bottom of the main software window to confirm that changes has taken affect.

We recommend restarting Pinnacle PCX software after Sequence is completed.

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STOP/RESET Pinnacle PCX

In a case of an emergency there are two ways of stopping Pinnacle PCX:

On the front of the instrument press STOP button. To reset turn the instrument OFF and ON again.
In the Pinnacle PCX software select STOP. Once selected this option changes to RESET. Select RESET once the
emergency is cleared.

These option are used for an instant complete stoppage of the instrument. Pinnacle PCX will cease all the
activities, regardless of what is happening. The pump(s) and heaters will be turned OFF and all the loaded
methods and sequences will be cleared. When ready (after the problem is fixed and you have rinsed the
analytical column) load a sequence again and follow steps to start post-column analysis.

4.17

Pinnacle Shutdown

Pinnacle can be shut down at the sequence by either using a Shutdown method or a Flush method. The latter is
recommended if the instrument is be stored for a long time or if reagents will be changed.

Upon completion of the analyses, use one of the following two procedures to shut down the Pinnacle PCX
system properly. These procedures can prevent potential column damage, reaction coil blockage, high
background fluorescence, reagent precipitation, or other problems. The Pinnacle PCX must be flushed out at
the end of a series of injections, and the reactors must be cooled down. If the instrument is simply stopped,
with reagent inside the reactor in a hot state, with no movement, then the heated reactor will become blocked.
It is very important for a long useful life that the reactors be flushed out until the temperature is cool.

MANUAL SHUTDOWN

You may shutdown the Pinnacle PCX manually by pressing/selecting the Disable function in the Control menu
of the PCX control software.
If you choose this function, allow the HPLC to pump for at least 30 minutes to allow for the reactor to cool and
to flush reagent from the reactor.

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AUTOMATIC SHUTDOWN

Create a Shutdown Method for Pinnacle PCX using the following settings as an example:

Run time: 30 min
Equilibration time: 5 min
Reactor temperature: 35 C
Column temperature: 35 C
Pump(s) flow rate: 0 mL/min

Set it up as the last method in the Pinnacle PCX sequence. Make
sure pump flow rate is set to 0 mL/min and reactor temperature
is set close to room temperature. You can leave your column at
your operating temperature or set it at room temperature.

Create corresponding slowdown Method for HPLC. Set it up as

Figure 4.7

the last method in the HPLC sequence. Make sure HPLC flushes
column and reactor with column storage eluant for at least 30 min.

RECOMMENDED ACCOMPANYING HPLC SLOWDOWN METHOD

Set the HPLC to 100% Storage Eluant (see application section for proper eluant for your column), and set the
HPLC pump at the normal analytical flow rate. Choose an eluant that elutes contaminants from the column; for
example, methanol for a reversed-phase column and regenerant for an ion-exchange column.

Time (min) % Storage Eluant Flow (mL/min)

0 100 Analytical flow rate*

30 100 Analytical flow rate*

30.1 100 0.0

*Follow instructions that come with the column.

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Section 5

MaintenanCe

5.1

Section 5

Ma in te na nc e

5.1

5.2

5.3

5.6

5.7

5.8

5.8

5.9

5.12

5.16

5.17

Your Pickering Pinnacle PCX will require some routine maintenance to stay in top condition. Ordinarily, little
maintenance is needed beyond good operating procedures. In this section you will find some suggestions for
maintaining your Pinnacle PCX, as well as Proper Shutdown procedures for typical, Long-term, and Storage of
the Pinnacle PCX.

Maintenance Suggestions
Operating Suggestions
Proper Shutdown Procedures
Basic Maintenance Procedures
Reagent Filter Replacement
Ambient Reactor Replacement
Replacement of Heated Reactor Cartridge
Valve Maintenance
Pump Seal Replacement
Fuse Replacement
Tubing Kit Guides

Maintenance Suggestions

Make copies of the blank forms in the Appendix and complete the parameter log on the photocopy.

Record the pressures for the system equilibrated under initial conditions. Keep a daily log of Column, Pump
1 and Pump 2 (if applicable) pressures for diagnostic use. Include all the settings for the pump, injector,
detector, and integrator.

Record any repairs, or problems in the Instrument Log.

Keep copies of the QC chromatograms and logs, and of the initial chromatograms generated at installation.

Create a maintenance schedule for your laboratory to ensure that the instrument is kept in good working order.

Always use PEEK nuts and ferrules in the Pinnacle PCX fluidics lines. Use of stainless steel nuts and
ferrules in the PEEK ports will void the warranty. Stainless steel nuts and ferrules will cut the threads at
the bottom of the ports, causing them to leak. If this happens, the entire item may need replacing.

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Ma in te na nc e

Operating Suggestions

Below are several helpful suggestions and bits of information which will help keep your Pinnacle PCX, columns,
and reagents in top condition. The following information is broken down into sections for convenience.

There are two items that should be part of any good laboratory practices, regardless of what equipment is being
used:

1) Always wear safety glasses or goggles, laboratory coat, gloves, and other appropriate safety-clothing.

2) Read and understand the instructions in the MSDS’s shipped with the chemicals. If the MSDS’s are missing,
please contact Pickering Laboratories and we can fax you a copy, or you can download them from our website
at www.pickeringlabs.com.

PINNACLE PCX

Check for leaks daily at the column fittings; the eluants can be corrosive.
Frequently observe and record the pressures and check for leaks. You may find a problem before it becomes serious.
Always use the correct fuse. Use of the incorrect fuse can seriously damage the PC board and even the valves,
reactor and pump. Use of an incorrect fuse will void the warranty.
Disconnect the power cord before removing the case of the Pinnacle PCX.
Always follow the proper shutdown procedures. See Below.
Use the proper start-up and shutdown procedures consistently (see Section 4).
Periodically Flush the instrument to ensure long life.
Do not operate the heated reactor above the boiling point of the eluant unless the back-pressure regulator is
connected to the waste line of the detector. Boiling inside the reactor can cause precipitates to block the reactor.

Operating above the boiling point without a back pressure regulator will void your warranty.

Thoroughly clean any leaks from fittings with water and dry with paper towels, especially if the solution is a
buffer or hydroxide. Standing salt and hydroxide solution are corrosive.

Soak up spills with rags, paper towels, or sponge. Clean spill-area with a wet towel and thoroughly dry. Do not
spray water directly into the instrument.

Rinse out the drip tray periodically.

MAINTENANCE SCHEDULE

Check for leaks around all fittings and record column and reagent pressures daily.
Change the reagent filter every 3 months.
Change valve and pump seals every 12 months.
Change the heated reactor and all tubing every two years.

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PICKERING COLUMNS

Do not operate with a column pressure above 2800 psi (193 bars) for an extended period of time. Isolate the
source of the high pressure and replace those items.
Never disconnect any fittings between the pre-column check valve and the column until the post-column system
has been shut down and depressurized (loosen the detector inlet fitting first). If the column pressure drops
below the post-column pressure, it is possible for reagent to back-flow onto the column.

When removing the column, remove the outlet fitting first.

Never pump organic solvent through an ion-exchange column. This will swell the resin and over-pressure the column.
Never pump high pH buffers or hydroxide through a silica column. This can dissolve the silica and cause
extensive damage to the post-column system by permanently blocking tubing.
When switching a system between ion-exchange and reversed-phase applications, be sure to flush the HPLC and
injector with water before connecting the column. Eluants for one analysis may damage the column for the other.

5.3

ELUANTS AND REAGENTS

Thiofluor is extremely hygroscopic. Always keep it in a tightly closed container.
Sodium borate (any grades) contains excessive amounts of heavy metal contaminants and insoluble matter.
These impurities will eventually precipitate in the reactor and flowcell. The one year warranty does not

cover damage caused by these contaminants.

If you must prepare your own borate buffer for the OPA reagent, do not use sodium tetraborate as
suggested by the EPA methods. Instead, use molar equivalents of boric acid and sodium hydroxide, because
they are available in higher purity (ACS-grade or better) and have very little insoluble matter.
Use Pickering Laboratories reagents and eluants. The quality of the chemicals is guaranteed and the cost is low
relative to the worth of your analytical results. The one year warranty does not cover damage caused by

poor-quality reagents and eluants not purchased from Pickering Laboratories.

Do not touch the interior of the mobile phase reservoirs and the dip tubes with your fingers. Amino acids in
fingerprints will cause contamination. Gloves are suggested.
Do not leave caps and lines dangling without a reservoir. To fill reservoir, transfer caps and lines into a spare
bottle or an Erlenmeyer flask filled with deionized water.

Proper Shutdown Procedures

Upon completion of the analyses, use one of the following two procedures to shut down the Pinnacle PCX
system properly. These procedures can prevent potential column damage, reaction coil blockage, high
background fluorescence, reagent precipitation, or other problems.

The Pinnacle PCX must be flushed out at the end of a series of injections, and the reactors must be cooled
down. If the instrument is simply stopped, with reagent inside the reactor in a hot state, with no movement, then
the heated reactor will become blocked. It is very important for a long useful life that the reactors be flushed
out until the temperature is cool.

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5.4

Section 5

Ma in te na nc e

MANUAL SHUTDOWN

You may shutdown the Pinnacle PCX manually by pressing/selecting the Disable function in the Control menu
of the PCX control software.
If you choose this function, allow the HPLC to pump for at least 30 minutes to allow for the reactor to cool and
to flush reagent from the reactor.

OR
AUTOMATIC SHUTDOWN

Create a Shutdown Method for Pinnacle PCX according to section 4 of this manual. Set it up as the last
method in the Pinnacle PCX sequence. Make sure pump flow rate is set to 0 mL/min and reactor temperature
is set close to room temperature. You can leave the column at the operating temperature or set it at room
temperature.

Create corresponding slowdown Method for HPLC. Set it up as the last method in the HPLC sequence. Make
sure HPLC flushes column and reactor with column storage eluant for at least 30 min.

RECOMMENDED ACCOMPANYING HPLC SLOWDOWN METHOD

Set the HPLC to 100% Storage Eluant (see application section for proper eluant for your column), and set the
HPLC pump at the normal analytical flow rate. Choose an eluant that elutes contaminants from the column; for
example, methanol for a reversed-phase column and regenerant for an ion-exchange column.

Time (min) % Storage Eluant Flow (mL/min)

0 100 Analytical flow rate*

30 100 Analytical flow rate*

30.1 100 0.0

*Follow instructions that come with the column.

LONG-TERM SHUTDOWN

The above methods are used if the Pinnacle PCX will be used again within 3 days of the last run. If you plan to
not use the Pinnacle PCX for more than 3 days, we recommend that you use the Long-term Shutdown.

In this procedure, the reactor is flushed free from reagent, thus ensuring that it will be free from blockage
upon your return.

There are two ways to perform this shutdown; automatic, or manual.

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MANUAL LONG-TERM SHUTDOWN

Make sure your HPLC pump is running storage eluant through the analytical column (see Application section
for proper eluant) and then remove the column for storage. Replace the column with a union and the eluants
with Water and Methanol. Set the HPLC pump to 80% Water and 20% Methanol at 1.0 ml/min. This will prevent
backflow into the column during Flush procedure.

Create a Flush method as described in section 4.9 under the Advanced Settings tab of this manual. Set up a
Sequence with one run of a Flush method and load it into Pinnacle PCX software.

Select Enable function in the Control menu.

Select Instrument – Maintenance- Flush Instrument. Follow directions on the screen to complete this
procedure.

5.5

After Flush Instrument is completed select Disable function in the Control menu of the Pinnacle PCX software to
turn OFF heaters and pumps.

Allow the HPLC to pump at least 30 minutes to allow for the reactor to cool.

AUTOMATIC LONG-TERM SHUTDOWN

Create a Flush method as described in section 4.9 under the Advanced Settings tab of this manual. Set it up as
the last method of your Pinnacle PCX sequence.

Create a corresponding method for your HPLC to flush analytical column with the storage eluant (see
Application section for proper eluant) and set it up as a last method of your HPLC sequence.

After sequence is completed select Disable function in the Control menu of the Pinnacle PCX software to turn
OFF heaters and pumps.

Allow the HPLC to pump at least 30 minutes to allow for the reactor to cool.

STORAGE

If the Instrument will not be used for a period longer than 2 weeks, it must be put into Storage state. This
becomes necessary because as reagents age, the chances of precipitation in the heated reactor increases.

Perform Manual or Automatic Long-Term Shutdown as described above.

Remove the reagents and replace them with a solution of 80/20 Water/Methanol.

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5.6

Section 5

Ma in te na nc e

Select Refill Pump(s) from Control menu of the Pinnacle PCX software. This will flush the reagent lines from
the bottles from the remaining reagent.

Select in the Pinnacle PCX software Instrument – Maintenance – Prepare for Storage/Shipping. Follow
directions on the screen to complete the procedure.

Pump the storage eluant through the analytical column (see Application section for proper eluant), and remove
the column and guard.
Cap both column and guard tightly.
Replace any buffers with water and flush the HPLC lines for 5 minutes.
Replace the water with 80/20 Water/Methanol and flush the lines.
Place a restrictor or union in the column oven in
place of the column.
Pump 80/20 Water/Methanol through the system.

3

Basic Maintenance Procedures

The following section will describe the procedure
for cleaning/replacing/rebuilding key elements of
the Pinnacle PCX instrument.
For the following procedures, refer to Figure 5-1
below, which calls out the various parts of the
Pinnacle PCX.

Starting at the valves, the parts of the Fluidics
Panel are numbered counter-clockwise.

1. Valve 2 (on dual systems only)

2. Pressure Transducer 2 (on dual systems only)

3. Gas is controlled by the toggle valve. Lever ON

pressurizes the manifold.

4. Pump 2 (on dual systems only)

5. Column Outlet

6. Mixing Manifold 2 (with integrated reagent

filter) (on dual systems only)

7. Heated Reactor

8. Mixing Manifold 1 (with integrated reagent

filter and over-pressure relief valve)

5

2

4

1

6

7

8

9

10

13

11

12

14

Figure 5-1

Pinnacle Operators Manual

Pickering Laboratories Inc.

9. Over pressure Relief valve

10. Pump 1

11. Pressure Transducer 1

12. Valve 1

13. Ambient reactor (on dual systems only)

14. Reactor outlet and union to connect to detector

Reagent Filter(s) Replacement

Refer to Figures 5-1 and Figure 5-2 for the location of the Reagent filter(s).

There are two filters in the Duplex system, and one filter
on the Simplex system. They are located in the reagent
manifolds; Items 6 and 8.

5.7

Section 5

Ma in te na nc e

Reagent Filter

1. Be sure that the Pinnacle PCX is shut down and
depressurized before changing a filter.

2. Remove the 1/16" fitting from the inlet of the reagent
filter.

3. Use a 7/16" wrench to remove the filter.

4. Replace the filter element. The correct filter element is
3102-9040, a 10 µm frit. Tighten with fingers first, then

Figure 5-2

give it a 1/4 turn with a 7/16" wrench
Reconnect the tubings to the filter. Use care not to strip the threads, and do not overtighten.
Start the LC pump and inspect for leaks.

Note: Always use a PEEK nut and ferrule to connect to the reagent filters. They are made of soft PEEK material,
and stainless steel nuts and ferrules will cut them, causing them to leak.

Pinnacle Operators Manual

Pickering Laboratories Inc.

5.8

Section 5

Ma in te na nc e

Ambient Reactor Replacement

The Ambient Reactor is Item 13 (on dual systems only).

1. Shutdown the post-column system and let the reactor cool for at
least 30 min.

2. Disconnect the two fingertight fittings at either end of the ambient reactor. These will be located on the
manifold (Item 6) and the inlet of the detector.

3. Loosen the screw that holds the black spindle to the fluidics panel with a 3/32" Alan wrench.

4. Remove the spindle and remove the used reactor.

5. Slide the new reactor onto the spindle and fasten to the fluidics panel.

6. Re-connect the fingertight fittings to the manifold and the union (Items 6 and 14). Do not overtighten the
fittings.

7. Start the LC pump and inspect for leaks.

Figure 5-3

Replacement of Heated Reactor Cartridge

The Heated Reactor is Item 7. Please refer to Figure 5-4 for removal of the Reactor Cartridge.

1. Shutdown the post-column system and let the reactor cool until it is a safe temperature for handling (below
30˚C).

2. Disconnect the fingertight fittings connected to the heated reactor. These will be located on the manifolds
(Items 6 and 8) for duplex systems or on the manifold and union in simplex systems (Items 8 and 14).

3. Loosen the retaining screw at the top of the reactor cartridge using a flathead screwdriver
(see Figure 5-4).

4. Gently slide the Reactor Cartridge out of the reactor
housing.

5. Replace the heated reactor cartridge with the proper
volume (see Appendix).

6. Reconnect the two fingertight fittings.
Do not overtighten. The inlet tubing of the
heated reactor is marked with a piece of
black shrink tube and connects to mixing
manifold I (Item 8).
Start the LC pump and inspect for leaks.

Retaining Screw

Figure 5-4

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 5

Ma in te na nc e

Valve Maintenance

Flushing the Valves is recommended before valves replacement or resealing to remove reagents from lines and
pumps. To flush the valve follow the steps below:

Remove the reagents and replace them with a solution of 80/20 Water/Methanol.

Select Refill Pump(s) from Control menu of the Pinnacle PCX software. This will flush the reagent lines from the
bottles from the remaining reagent. Select empty pump(s) to dispense liquid from pump.

Select Flush Pump(s) from Control menu of the Pinnacle PCX software.

5.9

Now Valves are ready for maintenance.

VALVE FACE REMOVAL PROCEDURE

Replace the post-column reagents with 80/20 water/methanol.
Flush the pump connected to the valve that requires maintenance (in the software, go to Control, then Flush
Pump).
When the flush is complete, pull the Flush tubing out of the liquid to prevent siphoning of the liquid back into
the flush line.

Power down the Pinnacle PCX and turn the power back on again. This will move the valve to the blocked
position. This is important for alignment of the new or repaired valve face.

Place some paper towels under the Pinnacle PCX to
absorb any excess liquid that is in the Flush line.
Disconnect the Waste line (position 6) from the
Valve face. Hold the end of the tubing upright for a
moment to allow any liquid to drain into the waste

1

2

6

container.
Disconnect the remaining fittings from the valve
face.

3

5

4

Flat-head screw

Flat-head screw

Using an Alan wrench and a flat-head screwdriver,
loosen the cap screw that holds the valve indicator
plate in the center of the valve, and the 2 flat-head
screws located between ports 1-2 and 4-5.

Cap screw

Figure 5-5

Pinnacle Operators Manual

Pickering Laboratories Inc.

5.10

Section 5

Ma in te na nc e

Pull the entire face horizontally forward. There will be some resistance.

If there has been any crystal build-up behind the valve, insert the jack screws
contained in the Valve Maintenance kit and tighten them until the face of the
valve comes forward and you are able to remove it (figure 5-7).

Body

Warning: When removing the valve face with the jack screws, it is important
to remember to remove the valve using equal turns on each side.
It is important that it come off of the instrument as straight as possible to
prevent deforming the soft PEEK material.

Remove the jackscrews after the valve is removed.

Inspect the face of the motor mount. Ensure that there is no excess crystal
build up. Carefully clean any crystals with a paper towel and some DI Water.

Using the Pinnacle Valve Seal tool, turn insert clockwise and push to remove
spool (figure 5-8).

O-RING REMOVAL

DO NOT use any sharp tools as this will scratch the soft plastic of the
insert.

For the 5 smaller O-rings (Size 4) – use a length of 1/16 OD Peek tubing to
remove.
For the 2 large O-rings (Size 18) – pinch o-rings with fingers and roll to remove.

Spool

Figure 5-6

jack
scews

Figure 5-7

Once O-rings are removed, sonicate PEEK ring and PTFE insert to remove any
crystals from valve. O-rings will be replaced.

O-RING REPLACEMENT

Clean the new O-rings first by sonicating them in a soapy water bath for 20
minutes and then rinse thoroughly with DI Water. Wear gloves and use care
not to get dust or dirt on them after cleaning.
Push the 5 new Size 4 O-rings into the ports of the PEEK ring. Port 6 (Waste
port) does not have an O-ring.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Figure 5-8

Figure 5-9

Section 5

Ma in te na nc e

Carefully slide the 2 new Size 18 O-rings onto the white center spool.

Note: Never reuse the O-rings. They take a shape after they are installed and will not conform to a new shape.

Note: Never use substitute O-rings.

Only use O-rings provided and approved by Pickering Laboratories.

INSERTING THE SPOOL INTO THE PEEK RING

Using the special Pinnacle Valve Seal tool (the same tool used to remove the
spool), carefully put the PTFE center back into place in the PEEK ring. Rotate
the insert clockwise to prevent slicing of the O-rings. Look into the valve, and
push insert toward you to ensure that no O-rings pop out.

1

2

6

5.11

Line up the holes in the spool so that when it is placed on the Pinnacle, the V is
between ports 2-3. This is the equivalent to the blocked position of the insert.

3

5

4

REPLACING THE NEW/REBUILT VALVE ON THE PINNACLE

Ensure that at the front face of the new valve, the body and spool are
completely flush. These two pieces must be level.

Slide the new valve onto the motor mount, so that the Number 1 is at the top.

Ensure that the dowels match the openings in the back of the valve.
Ensure that the V is between ports 2-3 (that is, ensure that the spool is in the home blocked position) refer to
figure 5-10.

Push the valve face firmly against the Pinnacle PCX. Ensure that the Valve Spool is firmly pushed against the
motor mount.

Replace the cap screws and tighten until you feel slight resistance. Then make 1/8 turn past snug.

Perform a pump flush to exercise the new valve and to properly position the spool within the body (remember
to put flush lines back into the solution).

Figure 5-10

Pinnacle Operators Manual

Pickering Laboratories Inc.

5.12

Section 5

Ma in te na nc e

Pump Seal Replacement

The Pinnacle PCX uses Pickering’s own pulse-free syringe pump with piston wash. The pump is specially
designed for extended seal life, however the piston seals will require periodic replacement.
The length of service to be expected from the seal depends on a wide variety of factors, including whether
proper shutdown procedures were followed, how often the system was turned on and off, and whether the
piston-wash system was wetted.

It is critically important that the seal be replaced immediately upon failure, or better yet, before failure, because
the reagent can leak into the mechanical housing of the pump and cause corrosion. When a leak occurs, you
may notice fluid on the side of the pump.

The pumps are Items 4 and 10.

SOFTWARE SET UP

To replace the seals, open the PCX control software and select Maintenance under the Instrument menu.
From the Maintenance sub-menu, select Change Seal, and follow the onscreen instructions.

Wait until the end of a run before performing this procedure to conserve reagent.
The Pinnacle will perform the Flush pump sequence, and will flush reagent out of the syringe.
Pull the Piston Wash and Flush lines out of the liquid, so they are just inside the caps. Leave enough space for
the pump to dispense any remaining liquid into the bottles.

When you are ready, click OK on the software.
The pump will retract the piston to dispense any piston wash back into the bottle.
Next, the pump will move the piston all the way forward to empty any remaining liquid into the Flush bottle.
The Valve will move to the blocked position to prevent any flow from the bottles.
Disconnect the Reagent lines from the pump head and plug the openings to prevent any spillage.
Open the panel on the right side of the Pinnacle PCX
by removing the captive screw located at the back of
the Pinnacle PCX, then push the cover back, and lift
it up.

TO REMOVE THE PUMP FROM PINNACLE PCX

Turn off Pinnacle PCX.
Disconnect the electronic pin connector from the
inside wall of the Pinnacle PCX.
Remove the two Alan bolts from the bottom of the
pump first. See Figure 5-11.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Figure 5-11

Section 5

Ma in te na nc e

Note: It is very important to remove the bottom bolts first. There are specially designed hooks in the chassis that
will support the pump.

Loosen and remove the third top screw. Use caution as this will cause the pump to lean toward you. Brace the
pump toward the wall of the Pinnacle PCX to prevent damage.
Carefully remove the pump from the chassis. If it is Pump 1, tilt the back end up and remove the pump in a
vertical position. If it is Pump 2, tilt the back end down, and remove the pump in a vertical position.
Place the pump on clean paper towels on the bench.

Wear gloves for this procedure.

REMOVE SYRINGE FROM BODY OF THE PUMP

Once the pump is on the bench, carefully remove the
syringe from the body of the pump:
Using a 3/32” Alan wrench, loosen the set screw (DO NOT
remove set screw) at the front of the syringe. It is important
to perform this step first, as it will remove the tension from
the bracket pins and cap screws (figure 5-12).

Set Screw

Once the set screw is loose, using the 3/16” Alan wrench, loosen the two cap screws that secure the bracket to
the pump base.

Figure 5-12

5.13

Cap Screw
Bracket

Cylinder Cap

Remove the bracket and cylinder cap by gently pulling the bracket ends out and away from the pins.

Carefully remove the ceramic syringe body by turning it in a
clockwise direction as you pull toward you (figure 5-13).
Warning: Never turn ceramic syringe body counter clockwise.
This will cause the Piston Head to unscrew and fall off inside of
the cylinder, making it very difficult to remove.

There will be some liquid inside the body, which will spill onto
the paper towels.
Set the syringe body aside on a clean dry surface.
The piston should be fully extended from the pump.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Figure 5-13

5.14

Section 5

Ma in te na nc e

PISTON HEAD UNIT DISSASEMBLY AND

Metal Piston Face

SEAL REPLACEMENT

Note: All items to be replaced in the Pump Seal
Replacement Procedure can be found in the the
Pinnacle Pump Seal Kit (PN 1452-0122).
In the following steps, you will replace 5 items:
Brass-ended set screw
Piston Head O-Ring
Secondary Cylinder O-Ring
Piston Rod O-Ring
Three, 4 - 40 x 1/4" capnut screws and washers

Using a 1/16” Alan wrench (do not use ball wrench
because it is too weak) remove the set screw from the
Piston Head (figure 5-15).

Note: Very Important! Never re-use the set screw. It has
a soft brass end that is designed to be used only once.
A new set screw is included with the seal kit. Re-use of
the set screw can cause the piston head to come loose.

Piston Head O-Ring

Set Screw

Secondary Cylinder O-Ring

PEEK Piston Head

Piston O-Ring
Guide

Piston Rod
O-Ring

Figure 5-14

O-Ring Guide

Pump Base

Lead Screw

Piston Head
Unit

Figure 5-15

Remove the piston head by turning it in a counter-clockwise direction. It is threaded onto the leadscrew. If
needed, use a cresent wrench to lossen the piston head.

Place the piston head on the bench with the PEEK side down. Using a Phillips Head screwdriver, remove the
metal plate by removing the 3, 4 - 40 x 1/4" capnuts and washers. These cannot be reused and should be
replaced. The piston head will disassemble into 3 parts:

1. Metal Piston Face

2. Piston Head O-ring

3. PEEK Piston Head

Blow out debris from brass-ended set screw that may be inside the piston head. Remove and discard the old
O-ring. Sonicate the metal plate, PEEK piston head, and new O-rings in a beaker of water.

Note: Do not over tighten cap screws. Over tightening will strip the threads on the PEEK piston head.

Rinse with DI water and reassemble the Piston Head using the new O-ring and cap screws. Set the reassembled
Piston Head Unit aside until Pump Reassembly.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 5

Ma in te na nc e

SECONDARY O-RING REPLACEMENT

To replace the secondary O-ring at the base of the pump,
first remove the piston wash connection and then “back
piston” by turning the lead screw 4 to 5 full turns manually.
Reverse direction of turn by 4 to 5 full turns by turning the
lead screw in the opposite direction. This will extend the
O-ring guide at the base of the pump (figure 5-16).
Note: O-ring guide will not come out with piston wash
connection still attached.

Slide the O-ring guide out and replce the small piston rod
O-ring that is located at the back of the guide (figure 5-17). Slide the guide back to the base of the pump and
replace the large secondary O-ring with the new one (figures 5-14
and 5-16).

Figure 5-16

5.15

Piston Wash
Tubing

O-ring Guide

Placement of
Secondary
O-ring

PUMP REASSEMBLY

Screw the newly assembled Piston Head Unit onto the head screw
(hand tight).

Using a new brass-ended setscrew, tighten the setscrew with the
1/16" Alan wrench. It is important to make this tight.

Figure 5-17

Lubricate the ceramic body with some methanol or isopropanol. Turning it in a clockwise direction, push it
back over the piston, using care to keep it horizontal. Ensure that the through holes are
orientated top and bottom (figure 5-18).

Place the cylinder cap onto the head of the cylinder and the
bracket on the pins, but do not tighten the cap screws. Reference
figure 5-12.

The set screw at front of cylinder has “adjusment” depression
to center bracket. Center the bracket by sliding it back and forth
a little bit until you feel it in the depression. Once it is centered,
tighten the set screw. Tighten the set screw so that the syringe is
tight against the base of the pump.

Figure 5-18

Finally, tighten the cap screws using the 3/16" alan wrench.

Pinnacle Operators Manual

Pickering Laboratories Inc.

5.16

Section 5

Ma in te na nc e

REPLACE THE PUMP IN THE PINNACLE PCX

Replace the pumps in the Pinnacle PCX by following the reverse of the directions in section titled “To Remove
the Pump From Pinnacle PCX” on page 5.12.

Be sure that the body of the pump rests on the support brackets, and that the front of the pump is protruding
through the front of the chassis.

Note: Be very careful not to pinch any of the other wires inside this area. Be especially careful of the reactor
wires and the valve wires when removing Pump 1.

Attach the reagent and piston wash lines.

FLUSH AND REFILL PUMPS

Turn ON Pinnacle PCX.
Select Control – Flush pump(s). Make sure all flush lines are immersed in solution.
After seals have been exercised for at least 60 minutes, perform Pump and Valve Seal Pressure Test followed by
a System Pressure Test. Follow the onscreen instructions.

Fuse Replacement

The fuse is on the back panel under the power switch.
Warning: The Pinnacle PCX must be shutdown to replace the fuse.
Remove the cord from the power inlet.
Use a small flat screwdriver to pry up the fuse holder then pull the fuse out.
Only use the correct type of fuse: 2 ea, 5mm x 20 mm, 5 amp, time lag
Reinstall the fuse holder and the power cord.

Pinnacle Operators Manual

Pickering Laboratories Inc.

1100-0500 Tubing Kit, Pinnacle, Dual Pump

TRANSDUCER 2

R23

COLUMN

PUMP 2

R25

5.17

Section 5

Ma in te na nc e

R2

R24

1

2

W2

6

FLUSH

R1

S2

FILTER

FILTER

HEATED

REACTOR

PUMP 1

R13

3

VALVE 2

R21

R22

AMBIENT
REACTOR

TRANSDUCER 1

R15

2

VALVE 1

3

R 11

R12

5

4

F2

R14

1

4

W1

6

5

F1

FROM

HPLC

WASTE

TO DETECTOR

Pinnacle Operators Manual

Pickering Laboratories Inc.

5.18

Section 5

Ma in te na nc e

1100-0501 Tubing Kit, Pinnacle, Single Pump

COLUMN

R1

S2

HEATED

REACTOR

TRANSDUCER

R13

FILTER

R15

2

FLUSH

R14

W1

1

6

FROM

HPLC

Pinnacle Operators Manual

Pickering Laboratories Inc.

PUMP

R 11

3

VALVE 1

4

5

F1R12

WASTE

TO DETECTOR

Section 6

appliCatiOns

6.1

Section 6

Ap pl ic At io ns

6.2

6.3

6.4

AMINO ACIDS
CARBAMATES
GLYPHOSATE

Pinnacle Operators Manual

Pickering Laboratories Inc.

6.2

Section 6

Ap pl ic At io ns

AMINO ACIDS

6.2 Introduction

6.2-1 Background

6.2-2 Basic Sample Preparation

6.2-3 Reagent Preparation

6.2-4 Analytical and Post-column Conditions

6.2-5 Procedure

6.2-6 Sample Chromatograms and Gradient Programs

6.2-18 Precautions

6.2-21 Notes

Introduction

High performance liquid chromatography (HPLC) with post-column derivatization is a technique for rendering
analytes more detectable than they would otherwise be in their native forms. Post-column derivatization gives
improved sensitivity or better selectivity (reduction of interference) leading to lower detection limits. The
Pickering Laboratories Pinnacle PCX was developed to facilitate the determination of amino acids in
using sodium ion-exchange or in native samples using lithium ion-exchange columns. There are two options

®

for post-column detection of amino acids. The first is the use of Pickering’s patented TRIONE
reagent, which will react with both primary and secondary amino acids. The second is the use of
o-phthalaldehyde (OPA), a fluorescent reagent that gives greater sensitivity but will detect only primary amino
acids.

A complete post-column analysis system for amino acids consists of the following components:

• HPLC ternary or greater gradient pump

®

• Manual injector or autosampler equipped with high pH compatible Tefzel

• Pickering Laboratories ion-exchange columns

• Pickering Pinnacle PCX post-column derivatization instrument

• Eluants, reagents, and standards

• Visible or fluorescence detector

• Chart recorder, integrator, or data system

Ion-exchange chromatography followed by post-column derivatization has been the method of choice for amino
acid analysis since S. Moore, D.H. Spackman and W.H. Stein published it in 1958—work which merited a
Nobel prize.

or PEEK™ seals

ninhydrin

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 6

Ap pl ic At io ns

Background

The separation is a multi-modal process wherein ion-exchange, ion-exclusion, and partition all take place.
The primary process is cation-exchange where a pH gradient mobilizes amino acids in order of their isoelectric
points; acidic amino acids such as glutamic acid elute early and basic amino acids such as lysine elute late.
Partitioning is affected by ionic strength and organic modifiers; for example threonine and serine are resolved
by partition effects. Ion-exclusion only occurs for highly acidic amino acids such as taurine.

Sodium ion-exchange is used for fast analysis of the 22 amino acids found in hydrolyzed protein or in simple
formulated products. Lithium ion-exchange is a slower technique with higher resolution to separate as many as
46 amino acids and compounds found in the complex mixtures of biological fluids or tissue extracts.

6.2-1

The most popular reagent for post-column detection is ninhydrin. Ninhydrin reacts with primary amines and
hydrindantin to form Ruhemann’s Purple (Figure 6.2-A) which is detectable at 570nm. Ninhydrin reacts with
secondary amines to form a yellow complex detectable at 440nm. The ninhydrin reaction is carried out at
130°C with a reactor volume of 500 µl. The elevated temperature is required because at room temperature,
the ninhydrin reaction is very slow and takes hours to go to completion.

Figure 6.2-A

Pinnacle Operators Manual

Pickering Laboratories Inc.

6.2-2

Section 6

Ap pl ic At io ns

An alternative reagent system based on o-phthalaldehyde (OPA) can be used for high-sensitivity detection of

™

primary amino acids. OPA reacts rapidly with primary amines and Thiofluor

mercaptoethylamine) under mild basic conditions to produce a strongly fluorescent isoindole derivative

(Figure 6.2-B). OPA does not react with secondary amines or aryl amines, so fails to detect Proline and other

secondary amino acids.

OPA

CHO

CHO

10 Amino Acid

OO

H

-

NH

R

+

3

pH>9, ambient T

(N,N-dimethyl-2-

CH

N

S

R

N

COOH

H

CH

3

3

HS

N

CH

Thiofluor

CH

3

3

Fluorescent Isoindole

Figure 6.2-B

However, it is possible to detect secondary amino acids by using a two-step reaction in which they are first

oxidized and then reacted with OPA. This technique has some disadvantages, and is not often used. Contact

Pickering for details.

The Pickering Pinnacle PCX derivatization instrument for fluorescent detection of amino acids is similarly

designed to the ninhydrin instrument, except that it contains a 150 µl reactor and the reacton is carried out

at 45°C.

Basic Sample Preparation

The following is a general sample preparation for Physiologic Fluid and Protein Hydrolysate samples. There are

many more types of samples that can be used for amino acid analysis. For details, consult the AOAC methods

and laboratory procedures.

Native amino acids are those found “free” in samples such as serum, urine and other physiological fluids, plant

extracts, foods and beverages. Although preparation of these samples for amino acid analysis is much simpler

and less time-consuming than protein hydrolysis, control of pH and normality, and removal of soluble protein

are critical factors which can affect the chromatography.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 6

Ap pl ic At io ns

The early-eluting amino acids — taurine, urea, aspartic acid, threonine, serine, etc. — are particularly
sensitive to pH and normality. Accordingly the samples must be held to a narrow pH range between 2.1 and

2.5, and the proper lithium ion concentration to ensure reproducibility in the early part of the chromatogram.
The later-eluting compounds are more tolerant of initial sample conditions, and their retention times are not

™

as likely to be affected. SERAPREP
as acetonitrile, perchloric acid and picric acid, and eliminate the need for dialysis, ultrafiltration, and repeated
centrifugation steps, followed by pH adjustment.

• Filter all samples through a 0.45µm membrane filter. Some samples may require even more stringent
filtration, especially if colloids are present.

• Samples must always be properly buffered. The ideal pH for sample injection is pH 2.3 ± 0.2.

• For native samples, be sure that all proteins have been removed before analysis.

and URIPREP™ replace commonly-used protein precipitation reagents such

6.2-3

PHYSIOLOGIC FLUID SAMPLES:

PREPARATION USING SERAPREP™ OR URIPREP

™

Use SERAPREP™ for preparing serum and other samples with a high buffering capacity, e.g. sardine oil. Use

™

URIPREP

for preparing urine and other samples with low buffering capacity, such as fruit juices, musts and
warts. The efficiency of protein precipitation and the need for post-centrifugation pH adjustment of the sample
determine which reagent is best for your particular sample.

™

1. In a microcentrifuge tube thoroughly mix equal portions of sample and SERAPREP

or URIPREP™.

2. Let stand for 5 minutes. Centrifuge the mixture at 13,000 rpm for 5 minutes. Check the supernate pH to
ensure that the range is pH 2.3±0.2. Adjust the initial mixing ratio as necessary.

3. Filter the supernate with a syringe filter (0.2 or 0.45 um).The filtrate is ready to be injected into an auto­sampler vial for amino acid analysis.

4. If further dilution is needed, use Li 220 to adjust the concentration of analyte.

Reagent Preparation

TRIONE PREPARATION

TRIONE® reagent requires little to no preparation, depending on what type you use.

T100: The one-part TRIONE

into the reagent reservoir and put the cap on the reservoir.

T200: To prepare two-part TRIONE

reservoir, and cap tightly under Nitrogen. Swirl until homogeneous.

®

(Cat. No. T100C) requires no preparation - simply pour the TRIONE® directly

®

(Cat. No. T200), pour Bottle 1 into the reservoir, add Bottle 2 to the

Pinnacle Operators Manual

Pickering Laboratories Inc.

6.2-4

Section 6

Ap pl ic At io ns

Note: TRIONE® is air sensitive, and must be kept under Nitrogen. The useful lifetime of T100 is three months*
unopened, and one month in the reservoir. The shelf-life of T200 is one year* unmixed, and one month in the
reservoir.
*From date of manufacture.

OPA PREPARATION

1. Pour 945ml of the OPA Diluent (Cat.No. OD104) into the reagent reservoir. Save approximately 5 ml for
Step 5.

2. Put the cap on the bottle, open the vent valve, and turn on the gas supply. Thoroughly deaerate the contents
by sparging with inert gas. Continue bubbling for at least 10 minutes.

3. Dissolve 300mg of OPA (Cat. No. O120) in 10mL of HPLC-grade methanol in a clean, dry container.

4. Turn off the gas supply and remove the cap from the bottle. Add the OPA solution to the deoxygenated diluent
in the reservoir. Wash any residual mixture into the reservoir with an additional 1–2 ml of methanol.

5. Dissolve 2g of Thiofluor™ (Cat. No. 3700-2000) in the reserved 5 ml of OPA Diluent and add to the
reservoir.

®

6. Add 3ml of 30% Brij-35

7. Replace the cap and close the vent valve. Gently swirl the reagent to complete the mixing. Turn on the inert gas.

(Sigma) solution.

Note: OPA reagent is sensitive to air oxidation and will degrade over time. The Pinnacle PCX system is designed
to minimize this oxidation. When the OPA reagent reservoir is maintained under inert gas pressure, the OPA
reagent can maintain its activity for up to one week without significant loss of activity.

Post Column Conditions

These are the recommended post-column conditions for the most common methods of amino acid analysis.
For the HPLC conditions, refer to the section titled Sample Chromatograms and Gradient Programs.

USING TRIONE NINHYDRIN REAGENT:

Reagent 1: TRIONE® (Cat. No. T100 or T200)
Pump 1 Flow Rate: 0.30 ml/min
Reactor 1 Volume: 500 µl
Reactor 1 Temp: 130°C

USING OPA REAGENT:

Reagent 1: o-Phthalaldehyde and Thiofluor™ in OD104 Diluent, plus 35% Brij-35
Pump 1 Flow Rate: 0.30 ml/min
Reactor 1 Volume: 150 µl
Reactor 1 Temp: 45°C

®

Pinnacle Operators Manual

Pickering Laboratories Inc.

USING SODIUM HYPOCHLORITE, FOLLOWED BY OPA REAGENT:

Reagent 1: 250 µl of 5% Sodium Hypochlorite in GA116 Diluent

™

Reagent 2: o-Phthalaldehyde and Thiofluor

in OD104 Diluent, plus 30% Brij-35
Pump 1 Flow Rate: 0.30 ml/min
Pump 2 Flow Rate: 0.30 ml/min
Reactor 1 Volume: 500 µl
Reactor 2 Volume: 100 µl
Reactor 1 Temp: 55°C
Reactor 2 Temp: Ambient

Procedure

6.2-5

Section 6

Ap pl ic At io ns

®

Pickering Laboratories recommends different gradient conditions depending on the column and type of sample.
Use the program recommended on the column data sheet for the initial testing. Do not change this
program until you are sure that the other aspects of the system are functioning properly.

The column oven temperature programming gives additional flexibility when optimizing methods. Using
temperature gradient allows to improve separation, shorten analysis time and fine-tune the method for detecting
compounds of interest. Please refer to page 4.8 for details on how to set up timetable for the column oven.

Set the maximum pressure limit on the HPLC to 220 bar to protect the column. Allow the column to equilibrate
for about 30 minutes under initial conditions. Inject 10µl of the Amino Acid Standard, and collect three
chromatograms. The first chromatogram will not be representative of the systems performance, so use the
second two to evaluate the performance.

Pinnacle Operators Manual

Pickering Laboratories Inc.

6.2-6

Section 6

Ap pl ic At io ns

Sample Chromatograms and Gradient Programs

METHOD 1: 0354675T 70-MIN PHYSIOLOGICAL FLUID LITHIUM (4.6 x 75mm)
GUARD COLUMN: GARD COLUMN TEMP: SEE GRADIENT HPLC FLOW RATE: 0.55ml/min
REAGENT FLOW RATE: 0.5ml/min INJECTION VOLUME: 10ul

HPLC PROGRAM

Time 1700-1125 % Li365 % Li375 % RG003 %

0 100 0 0 0
10 100 0 0 0
19 40 60 0 0
32 0 100 0 0
43 0 100 0 0

43.1 0 0 100 0
57 0 0 100 0

57.1 0 0 70 30
72 0 0 70 30

72.1 100 0 0 0

COLUMN OVEN PROGRAM

Time Temp ºC

0 34
6 34
17 65
25 70
70 70
71 34

1

20

23

15

17

16

18

21

24

2

3

456

0 10 20 30 40 50 70

8

7

11

44

10

9

12

19

25

22

26

27

13

:

14

30

29

28

31

45

36

37

39

35

34

32

33

38

40

42

41

60

PEAK IDENTIFICATION

1 Phosphoserine
2 Taurine
3 Phosphoethanolamine
4 Urea
5 Aspartic acid
6 Hydroxyproline
7 Threonine
8 Serine
9 Asparagine
10 Glutamic acid
11 Glutamine
12 Sarcosine

*Internal Standard

NOTE: This method utilizes column temperature gradient. Use Pinnacle PCX column oven or HPLC column oven with temperature
gradient capabilities.

13 -Aminoadipic acid
14 Proline
15 Glycine
16 Alanine
17 Citrulline
18 -Amino-n-butyric acid
19 Valine
20 Cystine
21 Methionine
22 Allo-Isoleucine
23 Cystathionine
24 Isoleucine

25 Leucine
26 Tyrosine
27 Phenylalanine
28 -Alanine
29 -Amino-i-butyric acid
30 Homocystine
31 -Aminobutyric acid
32 Tryptophan
33 Ethanolamine
34 Ammonia
35 Hydroxylysines
36 Ornitine

37 Lysine
38 1-Methylhistidine
39 Histidine
40 3-Methylhistidine
41 Anserine
42 Carnosine
43 Arginine
44 Glucosaminic Acid*
45 2-Aminoethyl-cysteine (AEC)*

Pinnacle Operators Manual

Pickering Laboratories Inc.

43

min

Section 6

Ap pl ic At io ns

METHOD 2: 0354100T ISOTHERMIC PHYSIOLOGICAL FLUID LITHIUM (4.0 x 100mm)
GUARD COLUMN: GARD COLUMN TEMP: 37°C HPLC FLOW RATE: 0.35ml/min
REAGENT FLOW RATE: 0.3ml/min INJECTION VOLUME: 10ul

CONDITIONS

Time (Min) Li275 % Li750 % RG003 % Comment

0 100 0 0 Inject
8 100 0 0 Isocratic
46 65 35 0 Linear Gradient
86 0 100 0 Linear Gradient
90 0 100 0 Isocratic
115 0 94 6 Linear Gradient
122 0 94 6 Isocratic

122.1 100 0 0 Step Change
140 100 0 0 Re-equilibration

6.2-7

2

1

3

20

8

5

7

4

6

17

18

16

19

10

15

9

12

13

11

14

PEAK IDENTIFICATION

1. Phosphoserine

2. Taurine

3. Phosphoethanolamine

4. Urea

5. Aspartic acid

6. Hydroxyproline

7. Threonine

8. Serine

9. Aspargine

10. Glutamic acid

11. Glutamine

12. Sarcosine

22

30

21

23

24

25

29

28

26

27

13. α-Aminoadipic acid

14. Proline

15. Glycine

16. Alanine

17. Citrulline

18. α-Amino-n-butyric acid

19. Valine

20. Cystine

21. Methionine

22. Cystathionine

23. Isoleucine

24. Leucine

44

40

39

41

31

42

37

38

35

34

33

32

36

43

45

12020 40 60 80 1000

25. Norleucine

26. Tyrosine

27. Phenylalanine

28. β-Alanine

29. β-Amino-i –butyric acid

30. Homocystine

31. γ-Aminobutyric acid

32. Tryptophan

33. Ethanolamine

34. Hydroxylysines

37. Ornithine

38. Lysine

39. Histidine

40. 3-Methylhistidine

41. 1-Methylhistidine

42. Carnosine

43. Anserine

44. α-Amino-β
guanidinopropionic acid

45. Arginie

35. Ammonia

36. Creatinine

Pinnacle Operators Manual

Pickering Laboratories Inc.

6.2-8

Section 6

Ap pl ic At io ns

METHOD 3: 0354100T TEMP GRADIENT PHYIOLOGICAL FLUID LITHIUM (4.0 x 100mm)
GUARD COLUMN: GARD COLUMN TEMP: SEE GRADIENT HPLC FLOW RATE: 0.4ml/min
REAGENT FLOW RATE: 0.3ml/min INJECTION VOLUME: 10ul

CONDITIONS

Time (Min) 1700-1125 % Li365 % Li375 % RG003 % Comment

0 100 0 0 0 Inject
15 100 0 0 0 Isocratic
27 40 60 0 0 Linear Gradient
45 0 100 0 0 Linear Gradient
60 0 100 0 0 Isocratic

60.1 0 0 100 0 Step Gradient
78 0 0 100 0 Isocratic

78.1 0 0 70 30 Step Gradient
95 0 0 70 30 Isocratic

95.1 100 0 0 0 Step Gradient
115 100 0 0 0 Equilibration

COLUMN OVEN PROGRAM

Time (Min) Temp. ºC

0 34
13 34
30 65
67 66
80 70
97 70
98 34

1

22

20

2

8

5

7

3

43

4

0 20 40 60

10

9

11

6

16

15

17

18

19

21

23

24

25

26

13

14

12

29

31

30

28

27

35

36

37

34

32

33

38

39

42

41

40

80

min

PEAK IDENTIFICATION

1 Phosphoserine
2 Taurine
3 Phosphoethanolamine
4 Urea
5 Aspartic acid
6 Hydroxyproline
7 Threonine
8 Serine
9 Asparagine
10 Glutamic acid
11 Glutamine

12 Sarcosine
13 a-Aminoadipic acid
14 Proline
15 Glycine
16 Alanine
17 Citrulline
18 α-Amino-n-butyric acid
19 Valine
20 Cystine
21 Methionine
22 Cystathionine

23 Isoleucine
24 Leucine
25 Tyrosine
26 Phenylalanine
27 β-Alanine
28 β-Amino-i-butyric acid
29 Homocystine
30 γ-Aminobutyric acid
31 Tryptophan
32 Ethanolamine
33 Ammonia

34 Hydroxylysines
35 Ornitine
36 Lysine
37 1-Methylhistidine
38 Histidine
39 3-Methylhistidine
40 Anserine
41 Carnosine
42 Arginine
43 Glucosaminic Acid*

*Internal Standard

NOTE: This method utilizes column temperature gradient. Use Pinnacle PCX column oven or HPLC column oven with
temperature gradient capabilities.

Pinnacle Operators Manual

Pickering Laboratories Inc.

Section 6

Ap pl ic At io ns

METHOD 4: 0393250 ISOTHERMIC PHYSIOLOGICAL FLUID LITHIUM (3.0 x 250mm)
GUARD COLUMN: GARD COLUMN TEMP: 40°C HPLC FLOW RATE: 0.30ml/min
REAGENT FLOW RATE: 0.3ml/min INJECTION VOLUME: 10ul

Time(min) %Li275 %Li750 %RG003 Comment

0 100 0 0 inject
17 100 0 0 isocratic
65 65 35 0 linear gradient
128 0 100 0 linear gradient
145 0 100 0 isocratic
185 0 94 6 linear gradient

185.1 100 0 0 step change
210 100 0 0 re-equilibration

6.2-9

1

2

3

22

20

5

8

7

10

9

4

6

020406080100 min120140 160180

18

17

16

19

23

15

13

12

11

14

24

21

30

25

26

27

31

29

28

35

33

34

32

36

40

39

41

42

37

38

44

43

PEAK IDENTIFICATION

1. Phosphoserine

2. Taurine

3. Phosphoethanolamine

4. Urea

5. Aspartic acid

6. Hydroxyproline

7. Threonine

8. Serine

9. Aspargine

10. Glutamic acid

11. Glutamine

12. Sarcosine

13. α-Aminoadipic acid

14. Proline

15. Glycine

16. Alanine

17. Citrulline

18. α-Amino-n-butyric acid

19. Valine

20. Cystine

21. Methionine

22. Cystathionine

23. Isoleucine

24. Leucine

25. Norleucine

26. Tyrosine

27. Phenylalanine

28. β-Alanine

29. β-Amino-i –butyric acid

30. Homocystine

31. γ-Aminobutyric acid

32. Tryptophan

33. Ethanolamine

34. Hydroxylysines

35. Ammonia

36. Creatinine

45

37. Ornithine

38. Lysine

39. Histidine

40. 3-Methylhistidine

41. 1-Methylhistidine

42. Carnosine

43. Anserine

44. α-Amino-β­ guanidinopropionic acid

45. Arginie

Pinnacle Operators Manual

Pickering Laboratories Inc.

6.2-10

Section 6

Ap pl ic At io ns

METHOD 5: 0354100T PKU AND MSUD SCREENING LITHIUM (4.0 x 100mm)
GUARD COLUMN: GARD COLUMN TEMP: 38°C HPLC FLOW RATE: 0.35ml/min
REAGENT FLOW RATE: 0.3ml/min INJECTION VOLUME: 10ul

Time (min) %Li275 %Li750 %RG003 Comment

0 86 14 0 Inject
25 73 27 0 Linear gradient

25.1 0 0 100 Step gradient
30 0 0 100 Isocratic

30.1 86 14 0 Step gradient
42 86 14 0 Re-equilibration

a-Amino-n-Butyric Acid

Cystathionine

Valine

Methionine

Isoleucine

Leucine

Norleucine

Tyrosine

Phenylalanine

5

10 2015

25 min

Pinnacle Operators Manual

Pickering Laboratories Inc.