Waters ACQUITY UPLC M-Class Columns User Manual

[ CARE AND USE MANUAL ]

ACQUITY UPLC M-Class Columns

TABLE OF CONTENTS

I. PREPARING ELUENTS

II. SAMPLE PREPARATION

III. PREPARING AND CONNECTING 75, 100, AND
150 µM I.D. ACQUITY UPLC M-CLASS COLUMNS

IV. PREPARING AND CONNECTING 300 µM I.D.
ACQUITY UPLC M-CLASS COLUMNS

V. CARE WHEN STOPPING FLOW

VI. HOW TO DIAGNOSE AND ADDRESS ABNORMALLY
HIGH BACKPRESSURE AND LEAKS

VII. CHECKING AND CLEANING A FOULED EMITTER

VIII. LONG TERM COLUMN STORAGE

IX. ORDERING INFORMATION

Note: Gloves should be worn for ALL operations
detailed in this document.

INTRODUCTION

Waters® ACQUITY UPLC® M-Class Columns are manufactured
to exacting specifications and are designed for use at pressures
up to 15000 psi. In order to achieve maximum performance, it is
important to ensure that proper connections are made to minimize
peak tailing and poor efficiency.

Each ACQUITY UPLC M-Class Column is individually tested to
ensure that it passes stringent quality control. Compared to
standard flow chromatography, successful day-to-day performance
of capillary-flow systems can require extra attention to detail.
This document provides several essential recommendations for
the successful use of ACQUITY UPLC M-Class Columns.

ACQUITY UPLC M-Class System.

ACQUITY UPLC M-Class Columns are available in 75, 100, 150, and 300 μm I.D.s.

[ CARE AND USE MANUAL ]

Nano-Tee with magnetic mount

Tubing to
HTM valve

outlet tubing

I. PREPARING ELUENTS

 Do not filter organic solvents. Use MS grade solvents directly

from the bottle.

 A solvent: 100% water with 0.1% formic acid.

 B solvent: 100% acetonitrile or methanol with 0.1% formic acid.

 Seal wash solvent: water, may contain small amount of

organic (no acid).

Inner compartment (swiveled open)

Analytical column

Tubing to MS sourceTrapping column

II. SAMPLE PREPARATION

 Samples for expression analysis must contain tryptic peptides

derived from proteins of interest at suitable concentrations.

 Samples may contain buffers and residual reagents from

approved digestion procedure.

 The sample must not contain other reagents, denaturants,

detergents, lipids, and must be free of particulates.

 Weak needle wash for peptides: 3% acetonitrile with 0.1%

formic acid.

Note:

 Good lab practices: no solvent bottles to go through the

dishwasher!

 Wear gloves when handling solvent lines and hardware.

 If system is contaminated with poor quality solvents,

flush with appropriate solvent.

Many report successful use of solution containing 25% water,

25% acetonitrile, 25% methanol, 25% IPA and 0.1% formic acid.

For PEG contamination, flush system with IPA.

 DO NOT USE strong bases, as they can strip fused silica.

 Where to source solvents:

 In North America: Fisher Optima or Burdick & Jackson.

 In Europe: Biosolve (Netherlands) provide very good solvents.

They offer smaller bottles (0.5 L) as well as “UPLC”
grade solvents.

III. PREPARING AND CONNECTING 75, 100 OR
150 µM I.D. ACQUIT Y UPLC M-CLASS COLUMNS

a) Preparation

Carefully remove your column from bag.

 The gold ferrule on the inlet of the column is placed snugly on the

tubing at the factory. The ferrule is lightly secured to reduce the
risk of it being lost as the column is removed from the packaging.
The ferrule is purposely set high on the tubing but will properly
seat itself in the correct position upon tightening.

 A small piece of Teflon® tubing is present on the outlet end of the

column for those who want to connect the device to a UV or PDA
detector rather than a mass spectrometer.

Note: The column is attached to a transfer tube with the use of a

zero dead volume union. This is hidden beneath the heat shrink

tubing. Care must be taken NOT to apply excessive force on the

ends of the nano column (e.g., when removing teflon tubing at

column outlet) that could cause this joint to separate.

ACQUITY UPLC M-Class Column Care & Use

2

[ CARE AND USE MANUAL ]

Wrong Correct

Carefully grab the teflon tubing with one hand and the BARE FUSED
SILICA capillary, not the PEEK tubing, with the other hand, to remove
the Teflon sleeve. Again, be careful NOT to apply unnecessary force
when removing the teflon tubing which could cause the fragile joint
between transfer tube and packed capillary to separate.

b) Connection

 Connecting and proper tightening of 75, 100, or 150 µm I.D.,

ACQUITY UPLC M-Class Columns to 15 K ACQUITY UPLC system.

 Flow mobile phase only in the direction indicated by the arrow

on the column label. Connect the column with the direction of
the flow arrow on the label pointing to the detector or Mass
Spectrometer.

1. For a column never installed on an ACQUITY UPLC
M-Class System.
 Turn nut until hand tight then an additional 1/2 turn

IV. PREPARING AND CONNECTING
300 µM I.D. ACQUITY UPLC M-CLASS COLUMNS

Carefully remove column from box.

 Connect the column inlet to the injector utilizing a Valco style

compression screw.

 Connect the column outlet to the detector or Mass Spectrometer

using a Valco style compression screw.

V. CARE WHEN STOPPING FLOW

 Do not stop the flow to your ACQUITY UPLC M-Class Column

suddenly. It is critical that the flow be slowly lowered via
the ACQUITY UPLC M-Class System controller to prevent
column damage.

2. For a previously installed and removed column from
ACQUITY UPLC M-Class System.
 Hand tight plus 1/8 turn

“Hand Tight” is when the ferrule bottoms in the M-detail fitting.
The ferrule is pre-staked higher than necessary on the tube, so
the ferrule must be seated by hand (hand tight) or lightly with
a wrench, then an additional turn. Note: In virtually every case,
gentle wrenching is required to get to the “hand tight” state
before the final half turn. Taking “hand tight” or “finger tight” too
literally can result in under tightening/leaks.

Note: DO NOT over tighten, leaking may occur and/or the ferrule

may become stuck in the port.

Ferrule Nut

VI. HOW TO DIAGNOSE AND ADDRESS
ABNORMALLY HIGH BACKPRESSURE AND LEAKS

 The inlet or the head of a chromatography column experiences

the largest amount of pressure in a LC system. The
ACQUITY UPLC M-Class Column inlet is designed to
operate at pressures up to 15 K psi.

 The fluidic pressure drops across the column at the outlet of the

column should only experience atmospheric pressure. The outlet
connection which connects the column to the transfer capillary is
designed to handle a maximum pressure of 800 psi.

 With a higher than normal system pressure, a small leak might be

detected at the outlet of an ACQUITY UPLC M-Class Column.

ACQUITY UPLC M-Class Column Care & Use

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