ACQUITY UPLC SAMPLE MANAGER
INJECTION MODE Quick Reference Card
goals of analysis
Goal
Mode
Comments
n
Flexibility for
a wide range
of samples
Partial-loop with
needle overfill
(PLNO)
Fast overall
speed of analysis;
lowest cycle time;
lowest sample
consumption
Partial-loop,
pressure
assisted
(PLPA)
Best precision
and accuracy;
lowest
dispersion;
best resolution
Full-loop
(FL)
n
n
n
n
Provides superior partial-loop accuracy (including between instruments), precision, and linearity for
samples including strong and weak acids and bases, hydrophilic, and hydrophobic compounds.
Best general-purpose mode for partial-loop injection; default mode for ACQUITY UPLC® Systems.
Examples: Performing typical analyses with a single standard concentration;
developing a calibration curve.
Use when throughput is your primary consideration, or if the sample is precious.
Examples: Testing bioanalytical assays; running three or more 384-well plates;
using a runtime less than 1.0 minute.
n
Use for method development projects or QC assays.
n
Can use small loops – 1.0 or 2.0 µL – with the 15-µL needle.
n
Examples: Performing a plate count study; using 1.0-mm I.D. columns.
See also: “Selecting an ACQUITY UPLC injection technique” in ACQUITY UPLC Console online Help.
Recommendat ions
Characterizing the system volume is critical to good sample manager performance. If the sample needle, loop, or syringe changes, configure the
volumes and characterize the seal, needle, and loop volumes. When changing weak-wash solvents, characterize the needle and loop volumes.
Observe the maximum-recommended sample volumes for typical sample vials and microtiter plates. For example, with a nominal 50-µL loop,
six injections in full-loop mode with default settings use 900 µL of sample, which is close to the recommended maximum volume of a standard
2-mL vial (approximately 1200 µL).
To prevent sample carryover, ensure the injection valve fittings are installed correctly. For gradients, ensure the final gradient hold is long enough.
For viscous samples (PLNO and partial-loop modes):
n If sample % relative standard deviation (RSD) is in the range of 2% to 10%, reducing the syringe draw rate
usually reduces %RSD to 0.5% or less.
n If the area %RSD varies significantly, ensure sample temperatures are fully equilibrated.
ACQUITY UPLC SAMPLE MANAGER
INJECTION MODE Quick Reference Card
Summa ry of Modes
Partial-loop using needle overfill mode
Partial-loop mode (with pressure assist)
Sample volumes
n The sample manager injects up to 75% of sample loop volume with
good linearity. The minimum for 1-µL, 100-µL, and 250-µL loops
is 20%, and the minimum for 2-µL through 50-µL loops is 10%.
n Air gaps bracket the “pre-sample + sample + post-sample” volume.
n The sample needle aspirates the specified injection volume plus
15.0 μL (14.0 μL pre-sample volume and 1.0 μL post-sample volume).
n Sample passes through needle, loop, and volume detection
device (VDD).
n Only the initial mobile phase (gradients) and the sample
(in its diluent) are injected.
Sample volumes
n The sample manager injects up to 50% of sample loop volume
with good linearity. The minimum for 1-µL, 100-µL, and
250-µL loops is 20%, and the minimum for 2-µL through
50-µL loops is 10%.
n Air gaps bracket the sample volume, reducing dispersion.
The sample’s leading edge is positioned at the end of the loop
closest to the column.
n Because the sample passes through the needle and loop but
not the VDD, it can show different carryover characteristics
than PLNO or FL.
n Select a small needle (15 µL) when using partial-loop
(pressure assist) mode, or when using the smallest loops.
Characteristics
n Good overall accuracy, precision, linearity, recovery, and low dispersion.
n Strong-wash solvent can be very strong.
n If needed, you can customize the needle overfill flush volume in
the sample manager instrument method > Advanced button.
Use at least 15 μL for best performance.
Limitations
n Carefully select strong-wash and weak-wash solvents to ensure minimum carryover. Because strong-wash and weak-wash solvents are
not co-injected, ensure both are strong enough to prevent carryover.
Also, select solvents with different selectivities, such as high organic
in the strong-wash solvent, with acid or base in the weak-wash solvent.
n Cycle time is slightly slower than in partial-loop mode.
n PLNO is not available for nanoACQUITY UPLC® Systems.
Characteristics
n Good linearity.
n Accuracy requires optimizing sample viscosity versus draw rate
and sample volume versus loop volume, where larger volume
is better.
Limitations
n The initial mobile phase (gradient), sample (in its diluent),
and weak-wash solvent are co-injected. To ensure that the
weak-wash solvent does not interfere in the chromatography,
observe the early eluters carefully.
n Using a smaller needle increases cycle time.
n The “map” of volumes of sample needle, sample loop,
and connecting tubing must be accurate.
n Poor or infrequent sample manager characterization
affects results.
Full-loop mode
Sample volumes
n The sample manager injects 100% of sample loop volume. It injects the center of the larger sample volume drawn from the sample vial.
n Use an appropriate overfill factor to ensure that the sample fills the entire sample loop. The default number of overfills is increasingly
larger for smaller loop sizes.
Characteristics
n Full-loop injections yield better recoveries (lower area-count losses) than partial-loop injections.
n Accuracy is determined by the square of the tubing I.D. tolerance of the loop used.
Example: For a 5-µL loop, typical tubing I.D. is 0.009 inch, typical tolerance in 316 SS tubing is ± 0.001-inch, and volumetric tolerance is ± 22%.
n The volumetric tolerance of a 20-µL loop made with 0.015-inch I.D. tubing can vary ± 13%.
Limitations
n Higher sample consumption.
n Higher loop-to-loop variability.
ACQUITY UPLC SAMPLE MANAGER
INJECTION MODE Quick Reference Card
Samp l e injec t ion and usage volumes
PLNO mode
Partial-loop mode
Full-loop mode
Loop
volume
(µL)
Injection
volume (µL)
Sample used
(µL)
Injection
volume (µL)
Sample used
(µL)
Injection
volume (µL)
Default overfill
factor
Sample used
(µL)
1.0
0.1 to 0.8
15.1 to 15.9
NR²
NR²
~1.0
5.8
6
2.0
0.2 to 1.5
15.2 to 16.5
NR²
NR²
~2.0
5.6
12
5.0
0.5 to 3.8
15.5 to 18.8
NR²
NR²
~5.0
5.0
25
10.0
1.0 to 7.5
16.0 to 22.5
1.0 to 5.0
1.0 to 5.0
~10.0
4.0
40
20.0
2.0 to 15.0
17.0 to 30.0
2.0 to 10
2.0 to 10.0
~20.0
3.0
60
50.0 ³
5.0 to 37.0
20.0 to 45.0
5.0 to 25.0
5.0 to 25.0
~50.0
2.0
100
100.0
10.0 to 75.0
25.0 to 90.0
10.0 to 50.0
10.0 to 50.0
~100.0
2.0
200
250.0
25.0 to 188.0
39.0 to 203.0
25.0 to 125.0
25.0 to 125.0
~250.0
1.5
375
1
NR = Not recommended
1 The 50-μL and 100-µL loops require a 250-μL sample syringe. The 250-μL loop requires a 500-μL sample syringe.
Loo p offline o pt ion
Use the Loop Offline option to:
(1) Reduce dispersion from the injector.
(2) Reduce the contribution of the sample loop to the dwell volume.
(3) Save cycle time; with full-loop and PLPA, Loop Offline is required when Load Ahead is used. (PLNO can load the Loop Ahead without Loop Offline.)
Reducing dispersion is better accomplished by using full-loop injections with a small loop.
Decreasing dwell volume is only significant if you have a large sample loop installed.
Loop Offline can cause missing and wandering peaks when the contents of the sample loop are not consistently flushed onto and out of the column.
Solutes deposited in the sample loop because of poor solubility are not transferred to the column until the gradient composition dissolves the
sample and flushes it onto the column. At that point, the solute has a very low retention factor (k’) and elutes from the column in one column
volume. To ensure that all peaks are flushed out of the sample loop, pump the gradient volume + one column volume before taking the loop offline.
Loop Offline also changes the injection sequence for PLNO (although the needle washes are the same). Because PLNO with Loop Offline enabled
requires two additional injection valve moves, there is no prewash before aspirating the sample. Samples that would normally be washed from
the loop by the prewash will elute when the loop comes back on-line.
ACQUITY UPLC SAMPLE MANAGER
INJECTION MODE Quick Reference Card
Load ahead o pt ion
This option increases sample throughput and minimizes the injection cycle time of samples through the ACQUITY UPLC System. While injecting the
current sample, the sample manager begins the sample preparation portion of the injection cycle for the next injection.
Recommendations
n
n
If you select Load Ahead in PLNO mode, do not set Loop Offline.
If you select Load Ahead in partial-loop or full-loop mode, you must set a Loop Offline time that is less than the runtime.
Spa re pa rt s
Sample needles
Type
Characteristics
Part number
(15-µL)
Part number
(30-µL)
PEEK
Caution: Do not use with hexane or tetrahydrofuran
700002708
700002644
205000370
205000369
205000363
205000362
FEP tubing with
stainless steel tip
Stainless steel
tubing with
stainless steel tip
n
n
Greater strength
More robust operation
Greater strength
More robust operation
n Preferred for samples that are known to be attracted to hydrophobic polymers
n Can use with hexane and tetrahydrofuran
n
n
Sample loopsSyringesAdditional information
Sample loop (µL)
Part number
Type
Size (µL)
Part number
1
430001516
Sample
50
410001348
2
430001264
Sample
100
700002570
5
430001311
Sample
250
410001347
10
430001326
Wash
2500
700002569
20
430001320
50
430001325
100
430002209
250
430002211
n
n
n
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is a trademark of Waters Corporation. All other trademarks are the
property of their respective owners.
©2009 Waters Corporation. Produced in the U.S.A.
May 2009 715002108EN LB-PDF
ACQUITY UPLC System Quick Start Card
(part number 720002688EN)
ACQUITY UPLC Console online Help
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