Waters nanoACQUITY UPLC Columns User Manual
[ Care and Use ManUal ]
WATERS nanoACQUITY COLUMNS
(FOR USE WITH 10,000 P.S.I SYSTEMS)
I. INTRODUCTION
Waters nanoACQUIT Y™ columns are manufactured to exacting
specifications, providing outstanding peak symmetry for maximum
sensitivity and accurate quantitation. As the columns are frit-
ted at both ends, they are able to withstand pressure changes
during injection and thus provide superior longevity. In order to
achieve maximum performance, it is important to ensure that proper
connections are made to minimize peak tailing and poor efficiency.
Each nanoACQUITY column is individually tested to ensure that
it passes stringent quality control. Compared to standard flow
chromatography, successful day-to-day performance of nanoflow
systems can require extra attention to detail. This document
provides several essential recommendations for the successful use of
nanoACQUITY columns.
CONTENTS
I. PREPARING ELUENTS
II. PREPARING SAMPLE
III. PREPARING AND CONNECTING 75, 100
AND 150 µM nanoACQUITY COLUMNS
IV. PREPARING AND CONNECTING 300 µM nanoACQUITY
CAPILLARY COLUMNS
V. CARE WHEN STOPPING FLOW TO COLUMN
VI. HOW TO DIAGNOSE AND ADDRESS ABNORMALLY
HIGH BACKPRESSURE AND LEAKS
VII. CHECKING AND CLEANING A FOULED EMITTER
VIII. LONG TERM, NANOACQUITY COLUMN STORAGE
IX. ORDERING INFORMATION
Note:GlovesshouldbewornforALLoperations
detailedinthisdocument
nanoACQUITYSystem ExampleofananoACQUITYcolumn.Availablein75,100,and150µm
nanoACQUITY UPLC Column Care and Use 1
[ Care and Use ManUal ]
Nano-Teewithmagneticmount
Tubingto
HTMvalve
Trappingcolumn
outlettubing
I. PREPARING ELUENTS
Do not filter 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)
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 contaminated, try 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.
Innercompartment(swiveledopen)
Analyticalcolumn
Tubingto
MSsource
II. PREPARING SAMPLE
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.
III. P REPAR ING A ND C ONN EC TING 75, 100 O R
150 µM nanoACQUITY 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. T he 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. T his is hidden beneath the heat shrink
tubing. Care must be taken NOT to excerpt force on the ends of the
nano column (e.g., when removing teflon tubing at column outlet)
that could cause this joint to separate.
nanoACQUITY UPLC Column Care and 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 put 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,
nanoACQUITY columns to 10 K 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 a nanoACQUIT Y system
Turn nut till snug then an additional 1/2 turn
2. For a previously installed and removed column from
a nanoACQUIT Y 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, the leak rate will increase and the ferrule
may get stuck in the port
IV. PREPARING AND CONNECTING
300 µM nanoACQUITY CAPILLARY COLUMNS
• Carefullyremovecolumnfrombox
• ConnectthecolumninlettotheinjectorutilizingaValcostyle
compression screw
• ConnectthecolumnoutlettothedetectororMassSpectrometer
using a Valco style compression screw.
V. CARE WHEN STOPPING FLOW TO COLUMN
• DonotstoptheflowtoyournanoACQUITYcolumnsuddenly.Itis
critical that the flow be slowly lowered via the nanoACQUITY system
controller to prevent column damage.
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 nanoACQUITY column
inlet is designed to operate at pressures up to 10 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 a nanoACQUITY column (see below).
Ferrule Nut
nanoACQUITY UPLC Column Care and Use 3
Leakatcolumnoutlet
Column Union Transfertubing
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