Preparing to Run UPLC; Injection and Detection Paramters

Running UPLC
,HPLC UPLC Preparing to Run
.

Typical HPLC conditions: Typical UPLC conditions:
Preparing a Dry Instrument

Flow rate: ~ 1 ml/mn Flow rate: ~ 0.6 ml/mn
Strong and Weak Wash Solvents
Column I.D.: ~ 4 mm Column I.D.: ~ 2 mm
System and Loop Volume Determination

Instrument Method Parameters
Inlet frit pore size: ~ 2 m Inlet frit pore size: ~ 0.2 m

.UPLC HPLC -

General Operating Practices

General Operating Practices
Make sure to cover bottles to protect it from dust!

Prepare fresh solvents daily. Do NOT top-up aqueous solvents

use a fresh reservoir bottle. Bacterial contamination is a real
problem.

Make up only 500 mL at a time.

Water obtained from a properly-maintained Milli-Q system does
not need to be re-filtered unless buffer salts are added.
Preferably: use a recognized UPLC or LC-MS grade solvent.
Do NOT use Parafilm to cover bottles it dissolves.

HPLC-Grade ACN or MeOH that has already been filtered
(certified on the bottle) does not need to be re-filtered.
Preferably: Use a recognized UPLC or LC-MS grade solvent Solvents and buffers are filtered
Reservoirs should be fully covered with Teflon stoppers and NOT Parafilm
Use filters to block dust from the air (Photo)
Bottles should be made of Type 1 glass

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Dr. Shula Levin, Medtechnica
 Preparing to Run UPLC; Injection and Detection Paramters
General Operating Recommendations CORNING PYREX Type 1, Class A, Borosilicate Bottles
SCHOTT DURAN Borosilicate Glass 3.3 Bottles

Use only Pyrex (Borosilicate 3.3) bottles. DURAN bottles

dissolve at high pH (>9).

Use highest quality solvents, water, buffers and additives.

Flush buffers out of system with water after use (use 10-20%
organic in water for storage).

Keep all 4 solvent lines primed (use 10-20% organic in water for
unused lines).

Keep seal wash primed.

Re-prime solvent lines before starting.

Use 100 L mixer for TFA/ACN gradients at low wavelengths. Also: if Biolab solvents: Use ULC/MS grade only!

Solvent Filters
Mobile Phase Preparation

Filtration on membrane:
Actions : Solvent degassing and filtration

Potential of impurities in filtration

o GHP, PTFE, Nylon, or PVDF

Pores diameter : 0.2 m.

Critical Clean Volatile additives may evaporate.

Stainless Steel (7 pack)700003616 Solvent filter assembly 289002172
NOTE: Refer to Controlling Contamination in
Stainless Steel (1/pk) 700003615 Solvent filter insert 5 m 700002756 UltraPerformance LC/MS and HPLC Systems
715001307D available from Waters Corp.
Titanium (7 pack) 700003530
Titanium (1 pack) 700003546

PAT (PEEK Alloyed with Teflon) filter

PSL901292 2 m
PSL901294 5 m

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Dr. Shula Levin, Medtechnica
 Preparing to Run UPLC; Injection and Detection Paramters
Bottle Caps Startup the System According to Instructions
http://www.forumsci.co.il/HPLC/UPLC_setup_21-9_09_Empower.pdf

WAT062341 4 L bottle (large neck)

WAT062479 1 L bottle (small neck)

No Parafilm or other plastic films to cover solvent reservoirs

Daily Startup Recommended Solvents List

ACQUITY UPLC System recommended Solvents

Set up each module in turn: Methanol Methanol/water mixtures
Water Acetonitrile/water mixtures
Solvent Manager: Acetonitrile (ACN) Isopropanol (IPA)
o Prime the solvent lines (2 mins each). Sample diluents (in addition to the solvents listed above)
o Set analytical flow to equilibrate column. Dimethyl sulfoxide (DMSO) Dimethylformamide (DMF)
Additives / Modifiers
Sample Manager:
0.2% formic acid 0.1% trifluoroacetic acid (TFA)
o Prime wash and sample syringes (6 cycles). 0.1% triethyl amine (TEA) 0.1% hexafluorobuteric acid
o Characterize needle seal and system volume. 10mM phosphate buffer 10mM ammonium bicarbonate
50mM ammonium hydroxide 50mM ammonium acetate
Detector: 0.1% Ethylenediaminetetraacetic acid (EDTA)
o Turn on lamp (needs > hr). Cleaners
o Check and record reference and sample energy. Phosphoric acid (=30%) Sodium hydroxide (=1M)
ACQUITY UPLC System non-recommended Solvents

Can do these operations simultaneously Methylene-Chloride Chloroform

Use the System Startup Button Strong acids >5%
Ethyl Acetate Toluene
Chlorinated solvents: (Trichlorobenzene)

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Dr. Shula Levin, Medtechnica
 Preparing to Run UPLC; Injection and Detection Paramters
Injector design examples
ACQUITY fixed loop injector design
Flow-Through variable injector design
(Rheodyne)

loop

Injection Parameters loop

Alliance 2695 and H-Class UPLC

Mounting of the loop ACQUITY UPLC Sample Manager

MUST BE PROPER!! Injection Methods
0.040
0.030
THREE injection methods available:
0.020
AU

0.010
0.000 1. Full Loop Injection Method/Mode as the injection technique using
-0.010 Pressure Assist
5.605.705.805.906.006.106.206.306.40
Minutes o Injects 100% of actual loop volume

2. Partial Loop Injection Method/Mode using Needle Overfill as the

injection technique
o Selectable: Recommended from 10% to 75% of total loop volume

3. Partial Loop Injection Method/Mode using Pressure Assist as the

injection technique
o Selectable: Recommended from 10% to 50% of total loop volume

Typical problem with bad peek With proper Peek

connections connections
First blank 0.1 % First blank <0.005 %
Second blank 0.01 %. .
4 . .

Dr. Shula Levin, Medtechnica

 Preparing to Run UPLC; Injection and Detection Paramters
Recommendations for ACQUITY UPLC Sample Wash cycle after injection
Manager Injection Methods

to column and
detector
needle loop
Possible contaminated
area
VDD

BSM

to column and
Sample Syringe detector
Wash block loop

VDD

BSM
Strong needle wash
Sample Syringe followed by weak needle
wash

Wash Solvent description Wash Solvent Considerations

As a general principle, strong and weak solvents should include the

There are two wash solvents same organic species
Strong Needle Wash
This may not always be practicable, especially in the case of sticky
o Tubing flushing
o Elimination of components injected samples. You may, however use a 100% organic strong wash solvent
o Never injected
Do not use salt buffers in wash solvents

Weak Needle Wash

Wash volume ratio (weak to strong)
o Strong solvent elimination Should be about 3:1, weak wash to strong
o Injected with the sample in partial loop pressure assist mode Sufficient to ensure the weak wash flushes the strong from the needle
and sample loop

For more details on solvents, see the section titled Selecting weak
wash and strong wash solvents in the ACQUITY Operators Guide

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Dr. Shula Levin, Medtechnica
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Strong Wash Solvent Strong Wash Solvent

Choose based on the chemistry application

Flushes internal and external portion of the needle to prevent

100% organic solvent is acceptable
carryover
Except THF

Typically stronger than sample and mobile phase to dissolve
Do not add acid or base in 100% organic solvent
sample residue

Prime using the needle wash function

Function performed in the wash station

Default value is 200 L

Strong solvent should be no stronger than the concentration
200 L is a typical value for this function
needed to reduce carryover to an acceptable level

Strong wash solvent does not contact the sample

Weak Wash Solvent Weak Wash Solvent

Compatible with initial gradient conditions and sample

Purges needle and syringe fluid path
solubility

Must be compatible with sample solvent

Avoid buffers

For best results, weak wash solvent should be equivalent to
increases the risks of precipitation at re-equilibration
the following (excluding buffers):

Five prime cycles fully replace wash solvents
mobile phase composition (for isocratic separations)

Default value is 200 L
initial gradient condition (for gradient separations)

If you dilute the samples, match the weak wash solvent to the
sample diluent

Degassed for good hydraulic properties

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Dr. Shula Levin, Medtechnica
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ACQUITY UPLC Sample Manager Full Loop Injection
Injection Methods Example of a 20 L injection with a 4X overfill

THREE injection methods available:

20 L Sample
Weak Wash Weak Wash
1. Full Loop Injection Method/Mode as the injection technique using Solvent
Pressure Assist Solvent
Sample Sample
o Injects 100% of actual loop volume Air Gap Air Gap

2. Partial Loop Injection Method/Mode using Needle Overfill as the

injection technique 30 L 30 L
o Selectable: Recommended from 10% to 75% of total loop volume sample sample
volume Sample Loop volume
3. Partial Loop Injection Method/Mode using Pressure Assist as the
injection technique
o Selectable: Recommended from 10% to 50% of total loop volume Volume Injected

Overfill Factor 1 to 4

Full Loop Injection Full Loop Injection

Step 1 Aspirate sample and air gap Step 2 Pressurize and position sample in valve

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Dr. Shula Levin, Medtechnica
 Preparing to Run UPLC; Injection and Detection Paramters
Full Loop Injection Full Loop Injection
Step 3 Position sample and overfill the loop Step 4 Sample Injection

Sample Loop contains only sample

No Air Gaps
No weak wash

Summary of Full Loop

Summary of Full Loop injection mode
Injection Mode

Best accuracy and precision performance

BENEFITS

Needs multiple loop volumes of additional sample to be used per Best accuracy and precision performance
injection

Larger overfill factors are required for smaller loops
TRADEOFF

Needs multiple loop volumes of additional sample to be used per

Need to change injection loops to vary volume injection
o larger overfill factors are required for smaller loops (buffer zone
improvement)
Need to change injection loops to vary volume

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Partial Loop Injection Partial Loop (Pressure Assist)

Loop Volume (L) Needle Overfill

1 Not Recommended
Sample
2 Not Recommended
Weak Wash Weak Wash
Solvent Solvent 5 Not Recommended

10 1.0 5.0

20 2.0 10.0
Air Gap
50 5.0 25.0
Sample Loop

Volume Injected

Pressure Assist Injection Sequence Pressure Assist Injection Sequence

Step 1 Aspirate sample and air gap Step 2 Pressurize and position sample in valve

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Dr. Shula Levin, Medtechnica
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Pressure Assist Injection Sequence Pressure Assist Injection Sequence
Step 3 Position the sample Step 4 Injection valve injects sample

Sample Loop contains

Sample, air gaps and weak wash

Summary of Partial Loop

Partial Loop Using Needle Overfill
using Pressure Assist

Shorter Cycle Time than PLUNO

No sample cushion

Sample volume is conserved

Recommended for large sample loop injections

Performance dependency on weak wash solvent matching to mobile
phase

Air Gaps injected onto Column

Accuracy is generally lower compared to Needle Overfill

Has lower injection range, within a given loop for partial loop
injections. Injection range is from 10 50% of the loop volume

Accuracy and Precision are lower than Full Loop Mode

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Dr. Shula Levin, Medtechnica
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Summary of Partial Loop
Partial Loop Using Needle Overfill
using Pressure Assist

BENEFITS:
Short Cycle Time
Sample volume is conserved
Recommended for large sample loop injections

TRADEOFF:
Accuracy and Precision are lower than Full Loop Mode
Performance dependency on weak wash solvent matching to mobile
phase
Accuracy is generally lower compared to Needle Overfill

Partial Loop using Needle Overfill Partial Loop using Needle Overfill
Step 1 Aspirate Sample and Air Gap Step 2 Injection Sequence

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Dr. Shula Levin, Medtechnica
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Partial Loop using Needle Overfill Partial Loop using Needle Overfill
Step 3 Injection Sequence Step 4 Injection Sequence

Partial Loop using Needle Overfill

Partial Loop using Needle Overfill
Step 1 Aspirate Sample and Air Gap
Step 5 Injection Sequence

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Dr. Shula Levin, Medtechnica
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Partial Loop using Needle Overfill
Step 2 Injection Sequence Partial Loop using Needle Overfill
Step 3 Injection Sequence

Partial Loop using Needle Overfill Partial Loop using Needle Overfill
Step 4 Injection Sequence Step 5 Injection Sequence

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Dr. Shula Levin, Medtechnica
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Summary of Partial Loop Injection Parameters
using Needle Overfill

No weak wash injected onto column
Injection Parameters
mobile phase and sample injected

Air gap not injected on column Automatic settings

Manual settings
(already set in the software

Sample does not come into contact with weak wash (need to be set)
See HELP)

Recommended for partial loop injections, especially from small
loops because accuracy is improved compared to Pressure Assist
Partial loop injections

Has wider linear range, can inject from 10 75% of the loop
volume

Cycle time depending on aspiration rate (with smaller loops the
time increases)

Needs 15 L additional sample to be used for cushion volume per
injection irregardless of injection size

Accuracy and precision lower than full loop mode

Injection Air Gap Good Repeatability:

Setting Up the Sample Manager

Automatic or manual

Full loop or partial loop injection

Full loop
Critical for partial loop injection
Air gap
o 4 L for 30 L needle
o 2 L for 15 L needle
Overfill factor depends on loop size
o see Help for Automatic parameters
Metering device compatible with sample volume

Partial loop
Low draw speed for low volume
Pre and post air gap are essential
Automatic parameters
o Pre and post air gap of 1 L
o 100 L per minute

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Strong Contaminations Main Parameters Summary

If the system is contaminated, some spare parts must be
Strong Washing

changed
Strong solvent 200 L
Why:
o Due to previous bad or not appropriate washing, contamination
Weak Wash
has been accumulated and then released since the system is not Weak solvent 600 L
able to compensate anymore
o Manual washing is not strong enough to eliminate carry-over
Full loop or partial loop injection
Use automatic settings
How: Which spare parts must be changed

Air gap
o Seal wash of the wash station
o Needle For partial injection

o VDD (bubble detector) Automatic

o Loop

Draw speed
Automatic

Air Gap
Pre and Post Air Gap Influence: Example

Question:
4 4 1 140787 132577 96240 57406 136252 95577 45260 16142
1) Should I use air gap? 0 0 1 90362 85040 62005 42553 92886 64077 30046 12199
Diffusion effect 55.80% 55.90% 55.21% 34.90% 46.69% 49.16% 50.64% 32.32%
o Yes or No?
4 4 1 140787 132577 96240 57406 136252 95577 45260 16142
o Which one? 0 4 1 89291 84372 63720 40764 85074 61209 29754 11578
Diffusion effect 57.67% 57.13% 51.04% 40.83% 60.16% 56.15% 52.11% 39.42%
Pre air, post air or both?
4 4 1 140787 132577 96240 57406 136252 95577 45260 16142
2) What volume? 4 0 1 120864 113953 81763 52170 122771 85128 39046 14816
Diffusion effect 16.48% 16.34% 17.71% 10.04% 10.98% 12.27% 15.91% 8.95%

Question:
Should I use?
o Full loop

o Partial loop injection

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Dr. Shula Levin, Medtechnica
 Preparing to Run UPLC; Injection and Detection Paramters
Air Gap Effects
pre air gap is more critical than post air gap Air Gap

automatic

Large air gap volume can effect the chromatogram.
4,4
4,0

0,0
0,4

Pre air gap Post air gap Peak area Peak height % area difference

Automatic (1) Automatic (1) 38889 27156

4 0 38488 26796 98.97

4 4 38179 26592 98.17
0 4 30866 21404 79.37
0 0 30748 21413 79.07

Air Gap Settings

Detection Mechanisms

Automatic settings
Pre and post air gap
System Configuration
4 L each
Detector Designs
1 L for PLUNO

Importance of Sampling Rate

Manual settings

Detector Requirements
Lower values:
o Sample diffusion providing peak area and peak height reduction

Higher values:
o Can effect the chromatogram
Higher peak height RSD

Loss of resolution

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Designing for UPLC ACQUITY UPLC TUV and PDA
Features

ACQUITY UPLC columns produce small volume peaks

Light guided flow cell design

To avoid band spreading and maintain concentration the flow cell
Two flow cell options
volume must be corresponding low
Low noise electronics
High brightness lamp

If you use conventional flow cells the path length must be reduced

Support for data rates up to 80 Hz
which results in a loss of sensitivity
Independent data rate and filter constants

UPLC detection requires cells that are designed for minimal
Console Control
dispersion, low cell volumes and optimum light throughput

Importance of Sampling Rate Effect of Sampling Rate on Chromatography

TUV

Must ensure enough points are collected across a peak to 0.080

1 pt/s
adequately define the peak shape: 20-50 points across the 5 pts/s
0.070
peak. 40 pts/s
0.060

Peak detection algorithms require a minimum number of points

0.050
across a peak to distinguish it from baseline noise and correctly
determine peak lift off and touch down 0.040

AU
0.030

A peak which does not have enough data points will be difficult to
integrate and therefore have irreproducible peak areas and 0.020

heights. 0.010

0.000

0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74
Minutes

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Effect of Sampling Rate on Reproducibility Tunable Ultra Violet Specifications

Sampling Points Peak Peak Area Peak Peak
Wavelength Range 190 700 nm
Rate Across Area %RSD Height Height

Increased data acquisition rates
Peak %RSD
Maximum 80 pts per second in single wavelength mode
1 pt/s 2 44125 2.436 27451 15.515
Maximum 2 pts per second in dual wavelength mode
2 pts/s 4 32822 1.790 41207 13.455

Optimized time constant to filter smaller peak widths
5 pts/s 7 31554 0.971 67355 3.962

Two flow cells
10 pts/s 13 31321 1.129 73638 1.015 Analytical

20 pts/s 25 31284 0.603 76001 1.156 High Sensitivity

40 pts/s 49 31534 0.284 77606 1.127

Measured for Peak #5 - Phenacetin

Filtering Constant At High Data Rates ACQUITY PDA Detector

Wavelength Range 190 500 nm

0.080
No Filtering

MassLynx v4.0/4.1
80 pts/sec maximum data rate
0.070 0.1s
0, 1, 2, or 3 second time constants
0.3s
0.060 3D mode only
0.5s
0.050 1.0s

Empower build 1154/2154
0.040 80 pts/sec maximum data rate
AU

0.1 3.0 seconds- time constants

0.030
2D or 3D Mode
0.020

0.010

Two flow cells
Analytical
0.000 High Sensitivity
0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80
Minutes

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Dr. Shula Levin, Medtechnica
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Conditions: Influence of Detector Settings:
Influence of Detector Settings: Sampling Rate Sampling Rate
0.05
Chromatographic Conditions : Analytes (Caffeine and Metabolites):
Column: ACQUITY BEH C18 2.1 x 30 mm, 1.7 m 1. 1-methylxanthene

AU
Mobile Phase A: 0.1% Formic Acid in H2O 2. 1,3-dimethyluric acid 40 Hz
3. Theobromine 0.00
Mobile Phase B: 0.1% Formic Acid in ACN
4. 1,7-dimethyluric acid 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Flow Rate: As indicated
5. 1,7-dimethylxanthene Minutes
Isocratic: 97% A: 3% B 6. Caffeine 0.05
Injection Volume: 2.0 L
Sample Diluent: 0.2% Formic Acid in water

AU
Strong Needle Wash: 50 L 50 ACN: 50 H2O
0.00 5 Hz
Weak Needle Wash: 500 L 0.1% Formic Acid in
H 2O
0.05
Temperature: 32C

AU
Detection: UV @ 280 nm
Time Constant: 0.1
0.00 1 Hz
Sampling rate: as indicated 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Instrument: UPLC: Waters ACQUITY UPLC, with Minutes
TUV detector

Influence of Detector Settings: Conditions:

Sampling Rate Influence of Detector Settings: Time Constant

Influence of Sampling Rate on Peak Height Chromatographic Conditions : Analytes (Caffeine and Metabolites):
Column: ACQUITY BEH C18 2.1 x 30 mm, 1.7 m 1. 1-methylxanthene
Sampling Rate 40 32 20 10 5 2 1
Mobile Phase A: 0.1% Formic Acid in H2O 2. 1,3-dimethyluric acid
1-methylxanthene 27145 27284 27525 26815 26694 21370 13530 3. Theobromine
Mobile Phase B: 0.1% Formic Acid in ACN
1,3-dimethyluric acid 41173 41191 41869 41173 41221 36798 26356 4. 1,7-dimethyluric acid
Flow Rate: As indicated
theobromine 56726 56624 57589 56673 56578 51930 38274 5. 1,7-dimethylxanthene
Isocratic: 97% A : 3% B
1,7-dimethyluric acid 25182 25015 25501 24917 25009 23586 20093 6. Caffeine
Injection Volume: 2.0 L
1,7-dimethylxanthene 18910 16717 18311 16739 17912 17829 15984
caffeine 14405 14103 14024 14367 13901 14201 13529 Sample Diluent: 0.2% Formic Acid in water
Strong Needle Wash: 50 L 50 ACN : 50 H2O
Weak Needle Wash: 500 L 0.1% Formic Acid in
Influence of Sampling Rate on Resolution H 2O
Sampling Rate 40 32 20 10 5 2 1 Temperature: 32C
1-methylxanthene
1,3-dimethyluric acid 6.9 6.98 6.91 6.96 6.7 5.84 4.79
Detection: UV @ 280 nm
theobromine 1.9 1.86 1.93 1.87 1.87 1.78 1.44 Time Constant: as indicated
1,7-dimethyluric acid 7.81 7.75 7.71 7.84 7.67 7.23 6.73 Sampling rate: 20 pts/sec
1,7-dimethylxanthene 2.36 2.29 2.36 2.42 2.37 2.43 2.09 Instrument: UPLC: Waters ACQUITY UPLC, with
caffeine 16.29 16.39 16.27 16.14 16.24 15.98 15.5
TUV detector

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Dr. Shula Levin, Medtechnica
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Influence of Detector Settings:
Time Constant Influence of Detector Settings:
Time Constant

0.05 Influence of Time Constant on Peak Height

Time Constant 0 0.1 0.5 1 1.5 2 2.5
AU

Tc = 0 1-methylxanthene 27525 26592 19732 13094 9615 7475 5923

0.00 1,3-dimethyluric acid 41869 40811 33662 24129 18370 14791 15241
Theobromine 57589 56123 47629 35066 27099 21813 18653
0.05 1,7-dimethyluric acid 25501 25121 23014 18704 15325 12503 10522
1,7-dimethylxanthene 18311 18916 17631 14724 12382 10126 8674
AU

Caffeine 14024 14069 14253 13456 13057 12445 11338

0.00
Tc = 1.5
Influence of Time Constant on Resolution
0.05
Time Constant 0 0.1 0.5 1 1.5 2 2.5
AU

1-methylxanthene
1,3-dimethyluric acid 6.91 6.64 5.24 3.53
0.00 Tc = 2.5 Theobromine 1.93 1.87 1.53 1.08
0.00 1.00 2.00 3.00 4.00
1,7-dimethyluric acid 7.71 7.58 6.79 5.19 3.68
Minutes
1,7-dimethylxanthene 2.36 2.37 2.18 1.8 1.45 1.22 1.02
Caffeine 16.27 16.5 16.19 15.09 13.47 12.18 10.87

Mechanism of Total Internal Reflectance

Resolution/Low Dispersion Flow Cell

Zooming In
The TIR condition requires that a component of the incident wave

A light guiding flow cell is
Light Path penetrates the AF, typically to a small fraction of the wavelength of
essentially an optical fiber
incident light.
whose core material is a fluid

The optical fiber consists of a
core material surrounded by a
cladding layer (Teflon AF).

Light is guided through the core TeflonAF Teflon AF, n(w)
by the process of total internal Mobile Phase
reflection;
light rays that encounter the TeflonAF
interface between the core and
cladding are reflected back into 90- Mobile Phase, n(f)
the core with essentially 100%
efficiency

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Dr. Shula Levin, Medtechnica
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Why is Flow Cell The Median Baseline Filter (MBF)
Maintenance Important?
06-Oct-2005 11:09:15 grds wp1114.arw
0.2 MBF Off

Teflon AF light guiding
0.15 MBF On
flow cells may, with time,
0.1
develop absorbance
0.05
baselines that exhibit
0
curvature.
-0.05
0 0.5 1 1.5 2 2.5 3 3.5 4

This is attributed to
changes in optical
characteristics of AF
surface due to
contaminants.
A processing tool for alleviating baseline curvature

These changes effect TIR
efficiency. The MBF operates during acquisition in real time on the chromatogram
whose baseline is to be corrected.
Example of baseline curvature. The top plot is a chromatogram of a The MBF automatically extracts the baseline, smoothes it, and then
gradient separation. The bottom plot is zoomed vertically to show
more clearly the magnitude of the baseline curvature. subtracts the smoothed baseline from the original chromatogram.
Result is a chromatogram with greatly reduced baseline curvature.

Light Guided Flow Cell Maintenance TUV Flow Cells

Prevent build-up of contaminants: Analytical

High Sensitivity
Always maintain fluid flow through the cell
o Avoid Lamp ON - Flow OFF condition
o Make clean connections to cell
Flush new columns

Dormant cells
Pre-flush with clean mobile phase, dry with clean air purge, then
plug ports

Certain low strength, acid-based, wash solutions can help
reduce baseline curvature if contamination occurs
Example 1% weak concentration formic acid solution

25 mm Pathlength 10 mm Pathlength
2400 nL Volume 500 nL Volume
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Dr. Shula Levin, Medtechnica
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ACQUITY PDA
Flow Cell Options Flow Cell Comparison
UPLC Conditions
System: ACQUITY UPLC with TUV High Sensitivity Flow Cell
Column: 2.1 X 50 mm ACQUITY UPLC BEH C18, 1.7 m
Mobile Phase 7:3 Water/ACN + 0.1% Formic
Flow Rate: 0.65 mL/min
High Sensitivity Analytical Injection Volume: 2 L
Strong Wash = 75% ACN/25% Water (250 uL)
Weak Wash = 5% ACN/95%Water (1000 uL)
Sample Temperature: 10 C
Column Temperature: 35 C
Detection: ACQUITY TUV @ 231nm (0-1.7 min)
280 nm (1.7 3.0 min) 20 pps, FTC=0.30
Sample = 080 mg/mL2,4-D in 50% ACN/50% Water
Data: Empower 2

Analytical Flow Cell

25 mm Pathlength 10 mm Pathlength
2400 nL Volume 500 nL Volume

2,4-D and Impurities

Flow Cell Signal Comparison Flow Cell Comparison Sensitivity

0.24

0.22

0.0060 Analytical High Sensitivity Delta

Analytical High Sensitivity Ratio 0.20
0.20

0.18

0.0055
Area 2995 6845 2.29 Area 140955 326881 2.319
0.16

0.14

Height 2368 5123 2.16 0.12

0.18 Height 93942 211138 2.248 0.10

AU
0.0050 Peak Width at 13.4% 2.02 sec 2.14 sec 1.059 0.08

0.06

0.04

0.16 Peak Width at 13.4% 0.040497 0.042033 1.038 0.02

0.0045 0.00

-0.02

-0.04

0.0040 0.14 -0.06

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9 .00
Minutes

0.0035
0.12
AU

0.0030

AU
0.10
0.0025

0.08
0.0020
Noise:
0.0015 High Sensitivity Flow Cell = 0.000015 AU 0.06
Analytical Flow Cell = 0.000012 AU
0.0010
0.04

0.0005
0.02
0.0000

0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 0.00
Minutes

4.40 4.45 4.50 4.55 4.60 4.65 4.70 4.75 4.80 4.85 4.90
Minutes

22
Dr. Shula Levin, Medtechnica
 Preparing to Run UPLC; Injection and Detection Paramters
Flow Cell Recommendations ACQUITY UPLC
High Brightness Lamp

High brightness deuterium lamp

Analytical Flow Cell Low noise characteristics
Better chromatographic resolution Nearly twice the brightness (energy) of ordinary deuterium
lamps

High Sensitivity Flow Cell

Benefits
For the highest sensitivity
Less Noise
Higher peak height (Beers law) Higher sensitivity
In general, better signal to noise
Lamp Warranty
o Slightly higher noise because more flow perturbations to contribute to 2000 hours
the noise, especially with absorbing mobile phases
Lamp Firmware through Console

Both Cells Hours of operation
Same linearity Ignitions
Cover the full flow rate range
Serial Number

Back Pressure Regulator

Maintains a constant 250 psi

back pressure on the flow cell

Not used with a mass
spectrometer

Used with both TUV and PDA
Recommendations

23
Dr. Shula Levin, Medtechnica
 Preparing to Run UPLC; Injection and Detection Paramters
Recommendations Sources of Potential Contaminants

Incompletely flushed samples, in stagnant condition

Data acquisition Formation of particulates
Sampling rate Formation of thin film layer
Filtering Photodegradation
o Time constant
Column bleed, connection, tubing

Tubing
Use only the internal diameters for the
Observations/Results
ACQUITY UPLC system Progressive increase in gradient baseline curvature
Use connections with features designed for Decrease in absolute energy (increased exposure time)
the ACQUITY UPLC system

Vial Storage Injections Recommendations

Wash Process

Bad storage could induce possible Dedicated to sample to decrease carry over

contamination
Full loop injection

Use overfill factor
Ghost peaks

Partial loop injection
Unpredictable phenomena
Use air gaps

Air gap and Draw speed

Storage condition Use automatic settings
With cap
Sample loop volume determination
Away from dust Best way to load the sample correctly in the loop

24
Dr. Shula Levin, Medtechnica