3 - UPLC_Work_Practical_Considerations

Running U-HPLC: Practical Considerations 

Introduction to UPLC Work to 

HPLC/UPLC users: Practical 

Considerations 

Shula Levin, Waters (TC) Israel 

Shula_Levin@waters.com 

www.forumsci.co.il/hplc 

Migration From HPLC to UPLC: Scaling Down 

Shula Levin, Waters Israel 

1


The practice of UPLC operation is the same whether it is a Binary or a 

Quaternary System 

Solvent Management 

ACQUITY UPLC ACQUITY UPLC H-Class 

Solvent Manager Design Binary - 2 solvents Quaternary - 4 solvents 

Total Number of Solvents Solvents A1 or A2 and B1 or B2 

Solvents A, B, C, D 

(Solvents D 1-6 via SSV option) 

Type of Solvent Mixing High pressure mixing Low pressure mixing 

System Volume < 100uL standard configuration < 400uL standard configuration 

Column I.D. 1.0 mm to 4.6 mm 2.1 mm to 4.6 mm 

Pressure/Flow Performance 

15,000 psi up to 1mL/min 

9,000 psi up to 2mL/min 

Flow Rate Range 0.01 to 2mL/min 


2


Peaks are narrower in UPLC! 

Higher Sensitivity 

Separation between closely 

related compounds 

Higher peak capacity 

Shorter run times 

HPLC 

Typical Peak Width: 10-60 sec 

Solvents and cost saving 

UPLC 

Typical Peak Width: 2-10 sec 

Technology Changes from HPLC to UPLC 

Detector’s Optics 

& Fast Electronics 

Minimum Volume of Flow Cell 

a Mobile Phase 

TeflonAF 

TeflonAF 

Injector’s Low Volumes 

Power Switch 

LEDs 

Solvent Select Valves 

Seal Wash 

Independent drives for A1, A2, B1, B2 

Vent Filter/ Mixer/ 

Valve Tee Assembly 


Six 

cha 

nne 

l 

deg 

ass 

er 

Leak 

Detector 

High Pressure Pump 

With Low Delay volume 

High Speed Gradients 

Column-Over: Minimum Extra 

Column Band Broadening 

Strong & Smooth Tubings 

& connections 

OH OH OH OH OH H2 C CH 2 

OH OH 

Si Si Si Si Si Si Si Si Si 

O O O O O O O O O O 

O O O O O O O 

Si 

O 

O 

Si 

O 

H2 C 

CH2 

CH 

2 O 

H 

2 

C 

Si Si Si Si Si Si Si Si Si 

O O O O O O O O O 

H 

2 

C CH 

2 O O O O O O O O 

Column Packing Enduring high 

pressures & Temperatures 

OH 

3


םג 

ןכו 

, HPLC 

Running UPLC 

תונולוקב רשאמ הברהב םינטק םה UPLC תנולוק ךותב םיקיקלחה 

. ןהלש םירטליפה 

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 

Preparing to Run 

Preparing a Dry Instrument 

Strong and Weak Wash Solvents 

System and Loop Volume 

Determination 

Instrument Method Parameters 


4


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 

General Operating Practices 

• Make sure to cover bottles to protect it from dust! 

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

•Old 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 


5


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. 

CORNING PYREX ® Type 1, Class A, Borosilicate Bottles 

SCHOTT DURAN ® Borosilicate Glass 3.3 Bottles 

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


6


Solvent Filters 

Critical Clean 

Stainless Steel (7 pack)700003616 

Stainless Steel (1/pk) 700003615 

Titanium (7 pack) 700003530 

Titanium (1 pack) 700003546 

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 µm. 

— Volatile additives may evaporate. 

NOTE: Refer to ‘Controlling Contamination in 

UltraPerformance LC/MS and HPLC Systems’ 

715001307D available from Waters Corp. 

Solvent filter assembly 289002172 

Solvent filter insert 5 µm 700002756 

PAT (PEEK Alloyed with Teflon) filter 

PSL901292 2 µm 

PSL901294 5 µm 


7


Bottle Caps 

WAT062341 4 L bottle (large neck) 

WAT062479 1 L bottle (small neck) 

No Parafilm ® or other plastic films to cover solvent reservoirs 

Transferring Methods from HPLC to UPLC 

and from Binary to Quaternary UPLC 


8


Method Transfer Tools 

• New Columns Calculator 

– Now from HPLC to UPLC to HPLC 

– Localized 

– Ability to cut and paste conditions 

into Empower Methods Editor 

• Pre-injection volume and Gradient 

Smart Start 

– Useful for accommodating systems 

with different dwell volumes 

• New Method Transfer Chemistry Kits 

Transferring HPLC Methods between HPLC 

Systems of Multiple Vendors 

• Differences in system volume 

– keep the column/system volume ratio constant to ensure a similar ‘isocratic hold’ caused 

by the system at the beginning of the gradient. 

• An isocratic hold can be added to the gradient table if the system volume of the new 

HPLC is smaller 

• An injection hold may be added if the system volume of the new HPLC is larger 

(setting the pre-column volume to non-zero in the instrument method) 

• Differences in column heating 

– Column heaters have different heating efficiencies and the differences can impact 

absolute retention of the components and selectivity 


9


Transferring HPLC Methods between HPLC 

Systems of Multiple Vendors 

Transfer Methods With Ease 

Utilize existing Assets 

Transfer from 

UPLC-to-HPLC 

Original HPLC System 

Vendor A HPLC System 

Vendor B HPLC System 

ACQUITY UPLC System 

ACQUITY H Class System 

Future-proof your lab 

Run HPLC methods on 

ACQUITY UPLC H-Class 


10


AU 

AU 

Transfer between Binary to Quaternary UPLC 

2.00 

1.80 

1.60 

1.40 

1.20 

1.00 

0.80 

0.60 

0.40 

0.20 

0.00 

Peak1 - 1.937 

Peak2 - 2.301 

Auto-Scaled Chromatogram 

Peak3 - 5.074 

Peak4 - 5.308 

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 

Minutes 

System Name UPLC02; SampleName SST test 2; Acq Method Set DMAD12; Date 

Acquired 11/03/2010 09:57:18 IST; Channel Description ACQUITY TUV ChA 254nm 

2.20 

2.00 

1.80 

1.60 

1.40 

1.20 

1.00 

0.80 

0.60 

0.40 

0.20 

0.00 

Peak1 - 2.107 

Peak2 - 2.362 

Peak5 - 5.997 

Auto-Scaled Chromatogram 

3.865 

-0.20 

0.00 1.00 2.00 3.00 4.00 5.00 

Minutes 

6.00 7.00 8.00 9.00 10.00 

System Name H CLASS; SampleName SST PZP passive therm 400 ul-del; Acq 

Method Set Tevatest2_passive_Therm_del400; Date Acquired 13/06/2010 12:24:39 

IDT; Channel Description PDA Ch1 254nm@4.8nm 

Peak3 - 5.126 

Peak4 - 5.361 

5.668 

Peak5 - 5.970 

Peak6 - 6.132 

Differences in System Volume: 

Low vs. High Pressure Mixing 

Acquity UPLC (Binary-Mixing) 

Smaller System Volume = Smaller Dwell volume (~120 mL) 

A B C 

D 

Gradient 

Proportioning 

Valve 

Pump A 

Pump B 

Mixer 

Injector 


6.640 

Column 

Detector 

H-Class UPLC (Quaternary-Mixing) 

Larger System Volume = Larger Dwell volume (~400 mL) 

Pump 

Injector 

Column 

Detector 

11


Solvent Composition 

at Mixer 

Solvent Composition 

at Column Head 

System Delay (Dwell) Volume 

Timing Offset 

0 

Injection 

Actual mobile phase 

profile on original 

system measured at 

the column inlet 

System volume creates an offset before the solvent 

composition change reaches the inlet of column (i.e. an 

“isocratic hold” at the beginning of every gradient) 

Smaller Volume 

System Volume 0.35 mL 

{ 

} 

Target System with smaller volume 

(less isocratic hold time) 

t g 


x 

Time 

Different System Volumes 

Effect on Separation 

Original 

Instrument 

System Volume 0.0.7 mL 

Larger Volume 

System Volume 1.4 mL 

Target System with larger volume 

(longer isocratic hold time) 

12


Considerations in Method Development 

ACQUITY UPLC ® H-Class: 

Options for Method Development 

• Column Managers can be “banked” to 

support up to 6 columns 

• CM-A target is mid 2Q10 

• Can have CM-A + CH-A 

• Flow through needle injector 

• Quaternary multi solvent mixing 

• D line solvents 1-6 using internal solvent 

selection valve 

• SSV target is end 1Q10 


13


Automated Method Development 

LAN 

LAC/E Card 

Network 

4-Relay 

Panel 


Solvent Valve Assembly 


Target is end 1Q10 

Auto•Blend: Constitute the Mobile Phase on 

the Fly. 

Water Acetonitrile 

Alcohol Concentrated 

Modifier 

14


95% 

Water 

0% 

Isopropanol 

100% Water 

0% Acetonitrile 

0.05% TFA 

AU 

AU 

AU 

0.15 

0.10 

0.05 

0.00 

0.25 

0.20 

0.15 

0.10 

0.20 

0.15 

0.10 

0.05 

Auto•Blend Gradient 

0% 

Acetonitrile Water 

5% 

1%TFA 

45% 

0% 

Isopropanol 

50% 

Acetonitrile 

5% 

1%TFA 

Peptide Map Development 

Varying TFA Concentration - %D 


50% Water 

50% Acetonitrile 

0.05% TFA 

0.025% TFA – 2.5% D 

0.05% TFA – 5% D 

0.1% TFA – 10% D 

27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00 

Minutes 

15


AU 

AU 

AU 

AU 

AU 

AU 

AU 

0.10 

0.05 

0.10 

0.05 

0.10 

0.05 

0.10 

0.05 

0.10 

0.05 

0.10 

0.05 

0.10 

0.05 

Stability and Reproducibility 

Peptide Mapping 

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 

Minutes 


Sample Manager -FTN 

• Programmable Volume Injection 

– Up to 10uL injection standard 

– Up to 250uL with extensions 

• Needle in flow path design 

– Total volume sample injection 

– Low carryover performance 

• Full range of plates and vials 

• Supports load ahead and loop offline 


Friday Night 

Saturday Morning 

Saturday Afternoon 

Saturday Night 

Sunday Morning 

Sunday Afternoon 

Sunday Night 

16


SM-FTN Characteristics 

• Needle in flow path design 

– Injection volumes up to 250uL (with additional needles and/or extension loops) 

– Needle flushed with the entire separation gradient 

• Low carryover performance 

• Supports load ahead and loop offline 


Sample Manager -FTN 


17


Sample Manager –FTN 

Sample Loading 


Sample Injection 


18


Injection Performance 

2 Orders of Magnitude 

No re-configuration necessary across the entire UPLC injection volume range 

Area 

Area 

Area 

300000 

200000 

100000 

10.0µL @ 2µg/mL 

1.0µL @ 20µg/mL 

0.1µL @ 200µg/mL 

Same peak area 

Linearity Test: Injection Volumes 

0 

Calibration Plot 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Injection Volume uL 

Name: acetanilide; Fit Type: Linear (1st 

Order); Equation Y = 2.66e+004 X - 

8.70e+002; R^2 0.999997 

50000.0 

40000.0 

30000.0 

20000.0 

10000.0 

0.0 


0.00 2.00 4.00 6.00 8.00 10.00 12.00 


Name: propiophenone; Fit Type: Linear 

(1st Order); Equation Y = 3.47e+003 X - 

1.09e+002; R^2 0.999996 

250000 

200000 

150000 

100000 

50000 

0 


0.00 2.00 4.00 6.00 8.00 10.00 12.00 


Name: benzophenone; Fit Type: Linear 


5.74e+002; R^2 0.999993 

Area 

Area 

Area 

100000.0 

80000.0 

60000.0 

40000.0 

20000.0 

0.0 


0.00 5.00 10.00 


Name: acetophenone; Fit Type: Linear 


2.47e+002; R^2 0.999998 

250000 

200000 

150000 

100000 

50000 

0 


0.00 2.00 4.00 6.00 8.00 10.00 12.00 


Name: butylparaben; Fit Type: Linear (1st 

Order); Equation Y = 1.87e+004 X - 

6.19e+002; R^2 0.999991 

20000.0 

15000.0 

10000.0 

5000.0 

0.0 


0.00 2.00 4.00 6.00 8.00 10.00 12.00 


Name: valerophenone; Fit Type: Linear 


1.29e+000; R^2 0.999964 


19



Carryover Challenge 


3.5 AU Full Scale 

3.5 AU 

Full Scale 

Stress Injection @ 2mg/mL 

1st Blank Injection 

Carryover = 0.0002% 

Managing Column Temperature 

• Temperature affects separation selectivity, 

retention and resolution 

• Incoming solvent at room temperature 

• Passively heated in “Air Bath” 

• Transit time is 15-20 seconds 

• Temperature gradients from inlet to outlet and 

wall to center 

• Pre-heating solvent absolutely necessary for 

separation control, reproducibility, and transfer 

20



Active Pre-heating 

• Ensures consistent control of temperature 

• Eliminates environmental influences on 

temperature 

• Reduced volume to reduce extra column 

bandspreading 

• Passive pre-heaters supported for backwards 

compatibility 

Injection Parameters in Fixed Loop 

Autosamplers 


21


Injector design examples 

Flow-Through variable injector design 

0.040 

0.030 

0.020 

AU 

0.010 

0.000 

-0.010 

loop 

Alliance 2695 and H-Class UPLC 

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 %. 

. 

ACQUITY fixed loop injector design 

(Rheodyne) 


loop 

With proper Peek 

connections 

First blank


ACQUITY UPLC Sample Manager 

Injection Methods 

THREE injection methods available: 

1. Full Loop Injection Method/Mode as the injection technique using “Pressure Assist” 

• Injects 100% of actual loop volume 

2. Partial Loop Injection Method/Mode using “Needle Overfill” as the injection technique 

• Selectable: Recommended from 10% to 75% of total loop volume 

3. Partial Loop Injection Method/Mode using “Pressure Assist” as the injection technique 


Recommendations for ACQUITY UPLC 

Sample Manager Injection Methods 


23


needle 

VDD 

Sample Syringe 

Wash cycle after injection 

BSM 

to column and 

detector 

loop 

Possible contaminated area 

Wash block 

VDD 

Sample Syringe 

BSM 

loop 

Strong needle wash followed 

by weak needle wash 

Wash Solvent description 

• There are two wash solvents 

– Strong Needle Wash 

• Tubing flushing 

• Elimination of components injected 

• Never injected 

– Weak Needle Wash 

• Strong solvent elimination 

• Injected with the sample in partial loop pressure assist mode 


to column and 

detector 

24


Wash Solvent Considerations 

• 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 

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 


25


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 

• 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 


26


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 

ACQUITY UPLC Sample Manager 

Injection Methods 

THREE injection methods available: 

1. Full Loop Injection Method/Mode as the injection technique using “Pressure Assist” 

• Injects 100% of actual loop volume 

2. Partial Loop Injection Method/Mode using “Needle Overfill” as the injection technique 


3. Partial Loop Injection Method/Mode using “Pressure Assist” as the injection technique 



27


Full Loop Injection 

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 1 – Aspirate sample and air gap 


28



Step 2 – Pressurize and position sample in valve 


Step 3 – Position sample and overfill the loop 

Sample Loop contains only sample 

No Air Gaps 

No weak wash 


29



Step 4 – Sample Injection 

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 


30


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 

• larger overfill factors are required for smaller loops 

(buffer zone improvement) 

– Need to change injection loops to vary volume 

Partial Loop Injection 

Weak Wash 

Solvent 

Sample 

Air Gap 

Sample Loop 

Volume Injected 

Weak Wash 

Solvent 


31


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 

Pressure Assist Injection Sequence 

Step 1 – Aspirate sample and air gap 


32



Step 2 – Pressurize and position sample in valve 


Step 3 – Position the sample 


33



Step 4 – Injection valve injects sample 

Sample Loop contains 

Sample, air gaps and weak wash 

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 


34


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 


35


Partial Loop Using Needle Overfill 

Partial Loop using Needle Overfill 

Step 1– Aspirate Sample and Air Gap 


36



Step 2 – Injection Sequence 




37







38



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 

Manual settings 

(need to be set) 

Injection Parameters 

Injection Parameters 

Automatic settings 

(already set in the software 

See HELP) 


39


Injection Air Gap 

• Automatic or manual 

• Full loop or partial loop injection 

– Critical for partial loop injection 

Good Repeatability: 

Setting Up the Sample Manager 

• Full loop 

– Air gap 

• 4 µL for 30 µL needle 

• 2 µL for 15 µL needle 

– Overfill factor depends on loop size 

• 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 

• Pre and post air gap of 1 µL 

• 100 µL per minute 


40


Strong Contaminations 

• If the system is contaminated, some spare 

parts must be changed 

– Why: 

• Due to previous bad or not appropriate washing, contamination has been 

accumulated and then released since the system is not able to compensate 

anymore 

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

– How: Which spare parts must be changed 

• Seal wash of the wash station 

• Needle 

• VDD (bubble detector) 

• Loop 

Main Parameters Summary 

• Strong Washing 

– 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 


41


• Question: 

– 1) Should I use air gap? 

• Yes or No? 

• Which one? 

Air Gap 

– Pre air, post air or both? 

– 2) What volume? 

• Question: 

– Should I use? 

• Full loop 

• Partial loop injection 

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% 


42


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

difference 

Automatic 

(1) 

automatic 

Automatic 

(1) 

Air Gap Effects 

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

0,0 

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 

0,4 

Air Gap 

• Large air gap volume can effect the 

chromatogram. 


4,4 

4,0 

43


• Automatic settings 

– Pre and post air gap 

– 4 µL each 

– 1 µL for PLUNO 

• Manual settings 

– Lower values: 

Air Gap Settings 

• Sample diffusion providing peak area and peak height reduction 

– Higher values: 

• Can effect the chromatogram 

– Higher peak height RSD 

– Loss of resolution 

Chemistry of the Separations 


44


Second Generation Ethylene 

Bridged Hybrid Particles 

U.S. Patent No. 6,686,035 B2 Bridged Ethanes within a silica matrix 

Anal. Chem. 2003, 75, 6781-6788 


45

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