Running UPLC

1 

םירטליפה םג ןכו , HPLC 

Preparing to Run UPLC; Injection and Detection Paramters 

Running UPLC 

תונולוקב רשאמ הברהב םינטק םה UPLC תנולוק ךותב םיקיקלחה 

. ןהלש 

Typical HPLC conditions: 

• Flow rate: ~ 1 ml/mn 

• Column I.D.: ~ 4 mm 

• Inlet frit pore size: ~ 2 µ m 

Typical UPLC conditions: 

• Flow rate: ~ 0.6 ml/mn 

• Column I.D.: ~ 2 mm 

• Inlet frit pore size: ~ 0.2 µ m 

. UPLC ב תפטושה הדובעל עירפהל םילולע HPLC -ב 

הדובעל תידיימ םיעירפמ אלש םינטק םיקיקלח ןכל 

General Operating Practices 

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. 

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 

Dr. Shula Levin, Medtechnica 

Preparing to Run 

Preparing a Dry Instrument 

Strong and Weak Wash Solvents 

System and Loop Volume Determination 

Instrument Method Parameters 

General Operating Practices 

Make sure to cover bottles to protect it from dust! 

Do NOT use Parafilm ® to cover bottles – it dissolves. 

•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

2 


General Operating Recommendations 

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 µL mixer for TFA/ACN gradients at low wavelengths. 

Solvent Filters 

Critical Clean 

Stainless Steel (7 pack)700003616 

Stainless Steel (1/pk) 700003615 

Titanium (7 pack) 700003530 

Titanium (1 pack) 700003546 

Solvent filter assembly 289002172 

Solvent filter insert 5 µm 700002756 

PAT (PEEK Alloyed with Teflon) filter 

PSL901292 2 µm 

PSL901294 5 µm 


CORNING PYREX ® Type 1, Class A, Borosilicate Bottles 

SCHOTT DURAN ® Borosilicate Glass 3.3 Bottles 

Mobile Phase Preparation 

Filtration on membrane: 

Also: if Biolab solvents: Use ULC/MS grade only! 

Actions : Solvent degassing and filtration 

— Potential of impurities in filtration 

o GHP, PTFE, Nylon, or PVDF 

— Pores diameter : 0.2 µm. 

— Volatile additives may evaporate. 

NOTE: Refer to ‘Controlling Contamination in 

UltraPerformance LC/MS and HPLC Systems’ 

715001307D available from Waters Corp.

3 

Bottle Caps 


WAT062341 4 L bottle (large neck) 

WAT062479 1 L bottle (small neck) 

No Parafilm ® or other plastic films to cover solvent reservoirs 

Daily Startup 

Set up each module in turn: 

—Solvent Manager: 

o Prime the solvent lines (2 mins each). 

o Set analytical flow to equilibrate column. 

—Sample Manager: 

o Prime wash and sample syringes (6 cycles). 

o Characterize needle seal and system volume. 

—Detector: 

o Turn on lamp (needs >½ hr). 

o Check and record reference and sample energy. 

Can do these operations simultaneously 

Use the System Startup Button 


Startup the System According to Instructions 

http://www.forumsci.co.il/HPLC/UPLC_setup_21-9_09_Empower.pdf 

Recommended Solvents List 

ACQUITY UPLC System recommended Solvents 

Methanol Methanol/water mixtures 

Water Acetonitrile/water mixtures 

Acetonitrile (ACN) Isopropanol (IPA) 

Sample diluents (in addition to the solvents listed above) 

Dimethyl sulfoxide (DMSO) Dimethylformamide (DMF) 

Additives / Modifiers 

0.2% formic acid 0.1% trifluoroacetic acid (TFA) 

0.1% triethyl amine (TEA) 0.1% hexafluorobuteric acid 

10mM phosphate buffer 10mM ammonium bicarbonate 

50mM ammonium hydroxide 50mM ammonium acetate 

0.1% Ethylenediaminetetraacetic acid (EDTA) 

Cleaners 

Phosphoric acid (=30%) Sodium hydroxide (=1M) 

ACQUITY UPLC System non-recommended Solvents 

Methylene-Chloride Chloroform 

Strong acids >5% 

Ethyl Acetate Toluene 

Chlorinated solvents: (Trichlorobenzene)

4 

0.040 

0.030 

0.020 

AU 

0.010 

0.000 

-0.010 


Injection Parameters 

Mounting of the loop 

MUST BE PROPER!! 

5.605.705.805.906.006.106.206.306.40 

Minutes 

Typical problem with bad peek 

connections 

First blank 0.1 % 

Second blank 0.01 %. 

. 

With proper Peek 

connections 

First blank

5 


Recommendations for ACQUITY UPLC Sample 

Manager Injection Methods 

Wash Solvent description 

There are two wash solvents 

— Strong Needle Wash 

o Tubing flushing 

o Elimination of components injected 

o Never injected 

— Weak Needle Wash 

o Strong solvent elimination 

o Injected with the sample in partial loop pressure assist mode 


Wash cycle after injection 

needle 

VDD 

Sample Syringe 

BSM 

loop 

Wash block 

to column and 

detector 

Possible contaminated 

area 

VDD 

Sample Syringe 

Wash Solvent Considerations 

BSM 

loop 

to column and 

detector 

Strong needle wash 

followed by weak needle 

wash 

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

same organic species 

— This may not always be practicable, especially in the case of “sticky” 

samples. You may, however use a 100% organic strong wash solvent 

Do not use salt buffers in wash solvents 

Wash volume ratio (weak to strong) 

— Should be about 3:1, weak wash to strong 

— 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

6 

Strong Wash Solvent 


Flushes internal and external portion of the needle to prevent 

carryover 

Typically stronger than sample and mobile phase to dissolve 

sample residue 

Function performed in the wash station 

Strong solvent should be no stronger than the concentration 

needed to reduce carryover to an acceptable level 

Strong wash solvent does not contact the sample 

Weak Wash Solvent 

Purges needle and syringe fluid path 

Must be compatible with sample solvent 

For best results, weak wash solvent should be equivalent to 

the following (excluding buffers): 

— mobile phase composition (for isocratic separations) 

— 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 


Strong Wash Solvent 

Choose based on the chemistry application 

100% organic solvent is acceptable 

— Except THF 

— Do not add acid or base in 100% organic solvent 

Prime using the needle wash function 

Default value is 200 µL 

— 200 µL is a typical value for this function 

Weak Wash Solvent 

Compatible with initial gradient conditions and sample 

solubility 

Avoid buffers 

— increases the risks of precipitation at re-equilibration 

Five prime cycles fully replace wash solvents 

Default value is 200 µL

7 

THREE injection methods available: 


ACQUITY UPLC Sample Manager 

Injection Methods 

1. Full Loop Injection Method/Mode as the injection technique using 

“Pressure Assist” 

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 

Full Loop Injection 

Step 1 – Aspirate sample and air gap 



Example of a 20 µL injection with a 4X overfill 

20 µL Sample 

Weak Wash 

Solvent 

Air Gap 

Sample 

Sample 

Weak Wash 

Solvent 

Air Gap 

30 µL 

sample 

volume 

Sample Loop 

Volume Injected 

Overfill Factor 1 to 4 

30 µL 

sample 

volume 


Step 2 – Pressurize and position sample in valve

8 



Step 3 – Position sample and overfill the loop 

Sample Loop contains only sample 

No Air Gaps 

No weak wash 

Summary of Full Loop injection mode 

Best accuracy and precision performance 

Needs multiple loop volumes of additional sample to be used per 

injection 

Larger overfill factors are required for smaller loops 

Need to change injection loops to vary volume 



Step 4 – Sample Injection 

Summary of Full Loop 

Injection Mode 

BENEFITS 

— Best accuracy and precision performance 

TRADEOFF 

— Needs multiple loop volumes of additional sample to be used per 

injection 

o larger overfill factors are required for smaller loops (buffer zone 

improvement) 

— Need to change injection loops to vary volume

9 

Partial Loop Injection 

Weak Wash 

Solvent 


Sample 

Air Gap 

Sample Loop 

Volume Injected 

Pressure Assist Injection Sequence 

Step 1 – Aspirate sample and air gap 

Weak Wash 

Solvent 


Partial Loop (Pressure Assist) 

Loop Volume (µL) Needle Overfill 

1 Not Recommended 



10 1.0 – 5.0 

20 2.0 – 10.0 

50 5.0 – 25.0 


Step 2 – Pressurize and position sample in valve

10 



Step 3 – Position the sample 

Summary of Partial Loop 

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 



Step 4 – Injection valve injects sample 

Sample Loop contains 

Sample, air gaps and weak wash 

Partial Loop Using Needle Overfill

11 


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 

Step 1– Aspirate Sample and Air Gap 


Partial Loop Using Needle Overfill 


Step 2 – Injection Sequence

12 



Step 3 – Injection Sequence 







Step 1– Aspirate Sample and Air Gap

13 










Step 5 – Injection Sequence

14 



using Needle Overfill 

No weak wash injected onto column 

— mobile phase and sample injected 

Air gap not injected on column 

Sample does not come into contact with weak wash 

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 µL 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 

Automatic or manual 

Full loop or partial loop injection 

— Critical for partial loop injection 

Full loop 


— Air gap 

Manual settings 

(need to be set) 



Automatic settings 

(already set in the software 

See HELP) 

Good Repeatability: 

Setting Up the Sample Manager 

o 4 µL for 30 µL needle 

o 2 µL for 15 µL 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 µL 

o 100 µL per minute

15 

Strong Contaminations 


If the system is contaminated, some spare parts must be 

changed 

— Why: 

Air Gap 

o Due to previous bad or not appropriate washing, contamination 

has been accumulated and then released since the system is not 

able to compensate anymore 

o Manual washing is not strong enough to eliminate carry-over 

— How: Which spare parts must be changed 

o Seal wash of the wash station 

o Needle 

o VDD (bubble detector) 

o Loop 

Question: 

— 1) Should I use air gap? 

o Yes or No? 

o Which one? 

• Pre air, post air or both? 

— 2) What volume? 

Question: 

— Should I use? 

o Full loop 

o Partial loop injection 


Strong Washing 

Main Parameters Summary 

— Strong solvent 200 µL 

Weak Wash 

— Weak solvent 600 µL 

Full loop or partial loop injection 

— Use automatic settings 

Air gap 

— For partial injection 

— Automatic 

Draw speed 

— Automatic 

Pre and Post Air Gap Influence: Example 

4 4 1 140787 132577 96240 57406 136252 95577 45260 16142 

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% 

4 4 1 140787 132577 96240 57406 136252 95577 45260 16142 

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% 

4 4 1 140787 132577 96240 57406 136252 95577 45260 16142 

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%

16 

Air Gap Effects 


pre – air gap is more critical than post – air gap 

automatic 

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

Automatic (1) Automatic (1) 38889 27156 

0,0 

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 

Automatic settings 

— Pre and post air gap 

— 4 µL each 

— 1 µL for PLUNO 

Manual settings 

— Lower values: 

0,4 

o Sample diffusion providing peak area and peak height reduction 

— Higher values: 

o Can effect the chromatogram 

• Higher peak height RSD 

• Loss of resolution 

4,4 

4,0 

Air Gap 


Large air gap volume can effect the chromatogram. 

Detection Mechanisms 

System Configuration 

Detector Designs 

Importance of Sampling Rate 

Detector Requirements

17 


Designing for UPLC 

ACQUITY UPLC columns produce small volume peaks 

To avoid band spreading and maintain concentration the flow cell 

volume must be corresponding low 

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

which results in a loss of sensitivity 

UPLC detection requires cells that are designed for minimal 

dispersion, low cell volumes and optimum light throughput 

Importance of Sampling Rate 

Must ensure enough points are collected across a peak to 

adequately define the peak shape: 20-50 points across the 

peak. 

Peak detection algorithms require a minimum number of points 

across a peak to distinguish it from baseline noise and correctly 

determine peak lift off and touch down 

A peak which does not have enough data points will be difficult to 

integrate and therefore have irreproducible peak areas and 

heights. 


ACQUITY UPLC TUV and PDA 

– Features 

Light guided flow cell design 

Two flow cell options 

Low noise electronics 

— High brightness lamp 

Support for data rates up to 80 Hz 

— Independent data rate and filter constants 

Console Control 

Effect of Sampling Rate on Chromatography – 

TUV 

AU 

0.080 

0.070 

0.060 

0.050 

0.040 

0.030 

0.020 

0.010 

0.000 

1 pt/s 

5 pts/s 

40 pts/s 

0.50 0.52 0.54 0.56 0.58 0.60 0.62 

Minutes 

0.64 0.66 0.68 0.70 0.72 0.74

18 


Effect of Sampling Rate on Reproducibility 

Sampling 

Rate 

Points 

Across 

Peak 

Peak 

Area 

Peak Area 

%RSD 

Peak 

Height 

Peak 

Height 

%RSD 

1 pt/s 2 44125 2.436 27451 15.515 

2 pts/s 4 32822 1.790 41207 13.455 

5 pts/s 7 31554 0.971 67355 3.962 

10 pts/s 13 31321 1.129 73638 1.015 

20 pts/s 25 31284 0.603 76001 1.156 

40 pts/s 49 31534 0.284 77606 1.127 

Measured for Peak #5 - Phenacetin 

Filtering Constant At High Data Rates 

AU 

0.080 

0.070 

0.060 

0.050 

0.040 

0.030 

0.020 

0.010 

0.000 

No Filtering 

0.1s 

0.3s 

0.5s 

1.0s 

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 


Tunable Ultra Violet Specifications 

Wavelength Range 190 – 700 nm 

Increased data acquisition rates 

— Maximum 80 pts per second in single wavelength mode 

— Maximum 2 pts per second in dual wavelength mode 

Optimized time constant to filter smaller peak widths 

Two flow cells 

— Analytical 

— High Sensitivity 

ACQUITY PDA Detector 

Wavelength Range 190 – 500 nm 

MassLynx v4.0/4.1 

—80 pts/sec maximum data rate 

—0, 1, 2, or 3 second time constants 

—3D mode only 

Empower build 1154/2154 

—80 pts/sec maximum data rate 

—0.1 – 3.0 seconds- time constants 

—2D or 3D Mode 

Two flow cells 

—Analytical 

—High Sensitivity

19 


Conditions: 

Influence of Detector Settings: Sampling Rate 

Chromatographic Conditions : 

Column: ACQUITY BEH C 18 2.1 x 30 mm, 1.7 µm 

Mobile Phase A: 0.1% Formic Acid in H 2O 

Mobile Phase B: 0.1% Formic Acid in ACN 

Flow Rate: As indicated 

Isocratic: 97% A: 3% B 

Injection Volume: 2.0 µL 

Sample Diluent: 0.2% Formic Acid in water 

Strong Needle Wash: 50 µL 50 ACN: 50 H 2O 

Weak Needle Wash: 500 µL 0.1% Formic Acid in 

H 2O 

Temperature: 32°C 

Detection: UV @ 280 nm 

Time Constant: 0.1 

Sampling rate: as indicated 

Instrument: UPLC: Waters ACQUITY UPLC, with 

TUV detector 

Influence of Detector Settings: 

Sampling Rate 

Analytes (Caffeine and Metabolites): 

1. 1-methylxanthene 

2. 1,3-dimethyluric acid 

3. Theobromine 


5. 1,7-dimethylxanthene 

6. Caffeine 

Influence of Sampling Rate on Peak Height 

Sampling Rate 40 32 20 10 5 2 1 

1-methylxanthene 27145 27284 27525 26815 26694 21370 13530 

1,3-dimethyluric acid 41173 41191 41869 41173 41221 36798 26356 

theobromine 56726 56624 57589 56673 56578 51930 38274 


1,7-dimethylxanthene 18910 16717 18311 16739 17912 17829 15984 

caffeine 14405 14103 14024 14367 13901 14201 13529 

Influence of Sampling Rate on Resolution 

Sampling Rate 

1-methylxanthene 

40 32 20 10 5 2 1 

1,3-dimethyluric acid 6.9 6.98 6.91 6.96 6.7 5.84 4.79 

theobromine 1.9 1.86 1.93 1.87 1.87 1.78 1.44 

1,7-dimethyluric acid 7.81 7.75 7.71 7.84 7.67 7.23 6.73 

1,7-dimethylxanthene 2.36 2.29 2.36 2.42 2.37 2.43 2.09 

caffeine 16.29 16.39 16.27 16.14 16.24 15.98 15.5 



Sampling Rate 

AU 

AU 

AU 

0.05 

0.00 

0.05 

0.00 

0.05 

0.00 

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 

Minutes 

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 

Minutes 

Conditions: 

Influence of Detector Settings: Time Constant 

40 Hz 

5 Hz 

1 Hz 

Chromatographic Conditions : 

Analytes (Caffeine and Metabolites): 

Column: ACQUITY BEH C18 2.1 x 30 mm, 1.7 µm 1. 1-methylxanthene 

Mobile Phase A: 0.1% Formic Acid in H2O 2. 1,3-dimethyluric acid 

Mobile Phase B: 0.1% Formic Acid in ACN 

3. Theobromine 

Flow Rate: As indicated 


Isocratic: 97% A : 3% B 

Injection Volume: 2.0 µL 

Sample Diluent: 0.2% Formic Acid in water 

Strong Needle Wash: 50 µL 50 ACN : 50 H2O Weak Needle Wash: 500 µL 0.1% Formic Acid in 

H2O Temperature: 32°C 

Detection: UV @ 280 nm 

Time Constant: as indicated 

Sampling rate: 20 pts/sec 

Instrument: UPLC: Waters ACQUITY UPLC, with 

TUV detector 

5. 

6. 

1,7-dimethylxanthene 

Caffeine

20 

AU 

AU 

AU 

0.05 

0.00 

0.05 

0.00 

0.05 

0.00 



Time Constant 

0.00 1.00 2.00 3.00 4.00 

Minutes 

Resolution/Low Dispersion Flow Cell 

A light guiding flow cell is 

essentially an optical fiber 

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 

by the process of total internal 

reflection; 

— light rays that encounter the 

interface between the core and 

cladding are reflected back into 

the core with essentially 100% 

efficiency 

Light Path 

α Mobile Phase 

T c = 0 

T c = 1.5 

T c = 2.5 

TeflonAF 

TeflonAF 



Time Constant 

Influence of Time Constant on Peak Height 

Time Constant 0 0.1 0.5 1 1.5 2 2.5 

1-methylxanthene 27525 26592 19732 13094 9615 7475 5923 


Theobromine 57589 56123 47629 35066 27099 21813 18653 


1,7-dimethylxanthene 18311 18916 17631 14724 12382 10126 8674 

Caffeine 14024 14069 14253 13456 13057 12445 11338 

Influence of Time Constant on Resolution 

Time Constant 0 0.1 0.5 1 1.5 2 2.5 

1-methylxanthene 

1,3-dimethyluric acid 6.91 6.64 5.24 3.53 

Theobromine 1.93 1.87 1.53 1.08 

1,7-dimethyluric acid 7.71 7.58 6.79 5.19 3.68 

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 

Zooming In 

Mechanism of Total Internal Reflectance 

— The TIR condition requires that a component of the incident wave 

penetrates the AF, typically to a small fraction of the wavelength of 

incident light. 

90-α 

Teflon ® AF, n(w) 

Mobile Phase, n(f)

21 

Teflon ® AF light guiding 

flow cells may, with time, 

develop absorbance 

baselines that exhibit 

curvature. 

This is attributed to 

changes in optical 

characteristics of AF 

surface due to 

contaminants. 

These “changes” effect TIR 

efficiency. 


Why is Flow Cell 

Maintenance Important? 

Light Guided Flow Cell Maintenance 

Prevent build-up of contaminants: 

—Always maintain fluid flow through the cell 

o Avoid “Lamp ON - Flow OFF” condition 

0.2 

0.15 

0.1 

0.05 

o Make clean connections to cell 

—Flush new columns 

Dormant cells 

0 

06-Oct-2005 11:09:15 grd s wp1114.arw 

-0.05 

0 0.5 1 1.5 2 2.5 3 3.5 4 

Example of baseline curvature. The top plot is a chromatogram of a 

gradient separation. The bottom plot is zoomed vertically to show 

more clearly the magnitude of the baseline curvature. 

—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 


The Median Baseline Filter (MBF) 

— A processing tool for alleviating baseline curvature 

MBF Off 

MBF On 

— The MBF operates during acquisition in real time on the chromatogram 

whose baseline is to be corrected. 

— The MBF automatically extracts the baseline, smoothes it, and then 

subtracts the smoothed baseline from the original chromatogram. 

— Result is a chromatogram with greatly reduced baseline curvature. 

TUV Flow Cells 

High Sensitivity 

25 mm Pathlength 

2400 nL Volume 

Analytical 


500 nL Volume

22 

AU 

ACQUITY PDA 

Flow Cell Options 


High Sensitivity Analytical 


2400 nL Volume 

Flow Cell Signal Comparison 

0.0060 

0.0055 

0.0050 

0.0045 

0.0040 

0.0035 

0.0030 

0.0025 

0.0020 

0.0015 

0.0010 

0.0005 

0.0000 


500 nL Volume 

Analytical High Sensitivity Ratio 

Area 2995 6845 2.29 

Height 2368 5123 2.16 

Peak Width at 13.4% 2.02 sec 2.14 sec 1.059 

Noise: 

High Sensitivity Flow Cell = 0.000015 AU 

Analytical Flow Cell = 0.000012 AU 

0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 

Minutes 

1.10 1.20 1.30 1.40 1.50 1.60 1.70 


Flow Cell Comparison 

UPLC Conditions 

System: ACQUITY UPLC with TUV 

Column: 2.1 X 50 mm ACQUITY UPLC BEH C18, 1.7 µm 

Mobile Phase 7:3 Water/ACN + 0.1% Formic 

Flow Rate: 0.65 mL/min 

Injection Volume: 2 µL 

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 

2,4-D and Impurities 

AU 

0.20 

0.18 

0.16 

0.14 

0.12 

0.10 

0.08 

0.06 

0.04 

0.02 

0.00 

Analytical Flow Cell 

Flow Cell Comparison – Sensitivity 

Area 

Height 

Peak Width at 13.4% 

Analytical 

140955 

93942 

0.040497 

High Sensitivity 

326881 

211138 

0.042033 

Delta 

2.319 

2.248 

1.038 

High Sensitivity Flow Cell 

4.40 4.45 4.50 4.55 4.60 4.65 

Minutes 

4.70 4.75 4.80 4.85 4.90 

AU 

0.24 

0.22 

0.20 

0.18 

0.16 

0.14 

0.12 

0.10 

0.08 

0.06 

0.04 

0.02 

0.00 

-0.02 

-0.04 

-0.06 

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

Minutes

23 


Flow Cell Recommendations 

Analytical Flow Cell 

— Better chromatographic resolution 

High Sensitivity Flow Cell 

— For the highest sensitivity 

— Higher peak height (Beer’s law) 

— In general, better signal to noise 

Both Cells 

o Slightly higher noise because more flow perturbations to contribute to 

the noise, especially with absorbing mobile phases 

— Same linearity 

— Cover the full flow rate range 

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 


ACQUITY UPLC 

High Brightness Lamp 

High brightness deuterium lamp 

—Low noise characteristics 

—Nearly twice the brightness (energy) of ordinary deuterium 

lamps 

Benefits 

—Less Noise 

—Higher sensitivity 

Lamp Warranty 

— 2000 hours 

Lamp Firmware through Console 

—Hours of operation 

—Ignitions 

—Serial Number 

Recommendations

24 

Data acquisition 


Recommendations 

— Sampling rate 

— Filtering 

Tubing 

Vial Storage 

o Time constant 

— Use only the internal diameters for the 

ACQUITY UPLC system 

— Use connections with features designed for 

the ACQUITY UPLC system 

Bad storage could induce possible 

contamination 

—Ghost peaks 

—Unpredictable phenomena 

Storage condition 

—With cap 

—Away from dust 


Sources of Potential Contaminants 

Incompletely flushed samples, in stagnant condition 

—Formation of particulates 

—Formation of “thin film” layer 

—Photodegradation 

Column bleed, connection, tubing… 

Observations/Results 

—Progressive increase in gradient baseline curvature 

—Decrease in absolute energy (increased exposure time) 

Injections Recommendations 

Wash Process 

— Dedicated to sample to decrease carry over 

Full loop injection 

— Use overfill factor 

Partial loop injection 

— Use air gaps 

Air gap and Draw speed 

— Use automatic settings 

Sample loop volume determination 

— Best way to load the sample correctly in the loop

Running UPLC

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