Agilent
Liquid Chromatography Technology
Phan Xuan Dai
Email: dai.phan@redstar-cms.vnTel: +84.333.936.562
Redstar-CMS Application team
Trang 2Retention time precision
Trang 3Pump● Binary pump● Quantary pump● Isocratic
Column
Auto sampler
● Fixed loop● Flow-throught
Overview UPLC System
Modular design!
Trang 4• Overview pressure range
Agilent InfinityLab Series Pumps
1290 Infinity II High Speed Pump1290 Infinity II Flexible Pump
1260 Infinity II Binary Pump
1260 Infinity II Quaternary Pump1260 Infinity II Isocratic Pump
400
Trang 5High Speed Pump
Trang 6Flexible Pump
Trang 7• Microfluidics applied for the best mixing performance
• Highest mixing efficiency
• Lowest UV detector noise
(35 µl & 100 µl in TFA applications)
With Jet Weaver35 µL
Other vendorw/ 50 µL mixer
TFA performance
Trang 8Highest retention time precision for reliable peak identification
The 1290 Infinity II Flexible Pump is the only low- pressure
mixing pump that combines the ultimate performance of a pressure mixing UHPLC pump with the flexibility of a quaternary
high-pump This pump delivers superior gradient performance over the entire composition range from 1 to 99 %.
Trang 9• Superior quaternary gradient delivery performance
Highest retention time precision
Specification (< 0.07 %)
3: Hydrocortisone acetate1: Hydrocortisone
2: di-proprionate
Trang 10MeCN+ 1%TFA
Pure solvent +concentrated stock solution = any concentration you need
BlendAssist for automatic solvent blending
Trang 11• Intelligent System Emulation Technology (ISET)
Intelligent System Emulation Technology
is available in
G7120A
Trang 12Intelligent System Emulation Technology (ISET)
Programmed gradient1290 gradient
1200 gradient
Same gradient conditions:
a) By moving the gradient
b) By emulating the gradient mixing behavior
Trang 13• Intelligent System Emulation Technology (ISET)
Intelligent System Emulation Technology
Agilent 1260/1290 Infinity II Flexible Pump
Agilent 1260/1290 Infinity II Flexible Pump with ISETAgilent 1100 Series Binary LC
Trang 14Retention time precision
Trang 15Agilent InfinityLab Series Autosamplers
Main Products & most important options
1260 Infinity II Hybrid Multisampler1260 Infinity II Multisampler1290 Infinity II Multisampler
• Max Operating pressure
• Injection Precision & Accuracy• Carryover
http://t0.gstatic.com/images?q=tbn:WhqsIyusTkuS3M:http://z.about.com/d/graphicssoft/1/0/E/N/1/snow_flake_2.png
Trang 16Wash inner needle
Flow-through principle washes inner needle
thoroughly and constant with eluent.
Wash outer needleChoice of 3 external
solvents to program
washing steps for the outer needle.
• Get you near-zero carryover
Trang 17• Get you near-zero carryover
The 1290 Infinity II Multisampler with Multi-wash reduces carryover to less than 9 ppm!
Trang 18Throughput: Same volumes left & right
Fast Injection for high throughput Use two identical loops for alternating injections in short sequence Lower overhead time for sample process steps (get sample, injector program steps, needle wash steps) Ideal for short runs and complex sample prep/loading steps
Additional time saving by alternating column regeneration
(up to 58% time saved from run to run vs sequentional runs)
Flexibility: Different loop volumes left & right
For injections e.g 20 µL at low dispersion and up to 500/900 µL loop for large volume Multi-load injections Maintain optimized dispersion without a need to change hardware from run to run
For safe reference or calibration use separate path for samples and for
contamination free checks or calibrations Also challenging applications or matrices can be safely separated using a defined path
Dual-needle: Usage
Trang 19Dual-needle: Throughput
Alternating column regeneration reduces cycle time for high-throughput applications by overlapping runs and wash/equilibartion steps Ideal for columns requiring long equilibration times (HILIC, NP, etc.)
Classic sequential runs
Analysis 1 Wash & Regeneration Analysis 2 Wash & Regeneration
Analysis 1 Wash & Regeneration
Analysis 2 Wash & RegenerationInjection
Run 1
Run 2
Analysis 1 Wash & Regeneration
Analysis 2 Wash & RegenerationInjection
Alternating Column Regeneration runs with Dual-Needle
Alternating column regeneration: reducing idle-times
Trang 20• Alternating Column Regeneration
Overlay of 14 subsequent runs of the preservatives separation with dual-needle setup and automated column regeneration
Trang 21Feed Injection feature: Overcome Solvent Effect
Metering device
Trang 22Pain point – Challenge of Solvent Effect
When do they happen?
What impact do they have?
• Not enough sensitivity for the analysis• Difficult manual peak integration
• Extra sample preparation: dilution, solvent exchangeSample
solvent Mobile phase
Big differencein solvent
What do they look like?
• Breakthrough• Poor peak shape
LC/MS analysis
QuECheRS sample preparation
Trang 23Challenge of Solvent Effect
• Early eluting compounds in Multi-Residue Methods
J Wong et al., J Agric Food Chem 2010, 58, 5897–5903
UHPLC, vendor A
Injection volume: 5 µLMethanol / water [1:1 v/v]
UHPLC, vendor BUHPLC, vendor C
Trang 24Feed Injection feature: Best solutions to mediate solvent effect
• Feed Injection
• Mediate strong solvent effect
• Agilent unique feature
• Strong sample (organic solvent)• Low component concentration
• Requirement of high Signal-to-Noise
• Flow Through Injection• Minimized dispersion• All other autosamplers
Solution for solvent effect
Run legacy methods
Trang 25Feed Injection feature: Best solutions to mediate solvent effect
Trang 26Classical Flow Through
Trang 27Feed Injection
Trang 28Feed Injection feature: Applied on Food safety
QuEChERS sample preparation
strong solvent matrix
Trang 29Agilent InfinityLab LC Series Column Compartments
Increase daily throughput with focus on ease of use
Trang 30“Forced air” versus “Still air” column compartment• A “Forced air” column compartment is not ideal for
UHPLC analysis, it enhances the effects of frictional heating Leading to broader peak, worse separation.
• A “Still air” column compartment, such as the
InfinityLab LC Multicolumn Thermostat module, is better suited.
Column Thermostat UHPLC Frictional Heating
Temperature Control and Related Effects
Trang 31Loss of Peak Resolution
Thermal Mismatch Issues
SAMPLE AT AMBIENT TEMPERATURE
SAMPLE ATAMBIENT TEMPERATURE
•Centre of column below oven temperature
•Higher viscosity, lower linear velocity in centre•Higher retention in centre
Trang 32Concept available for
•1290 Infinity IIMulticolumn Thermostat MCT (G7116B)•1260 Infinity IIMulticolumn Thermostat MCT (G7116A)•1290 Infinity Valve Drive (G1170A)
•1290 Infinity Flexible Cube (G4227A)
InfinityLab Quick Change Valves
More choices for automation
Valve drive and valve head as separate units
InfinityLab Quick Change ValvesInfinityLab Valve Solution Training Presentation
Trang 33Ordering Information
Valve-Thermostat Clusters (VTC)
Position 1-8Position 1-8
Position 1-8Position 1-8
External valve drive (G1170A)6 column selector (G4234C)
Clustering of up to 4 MCT gives you control over up to 32 columns
Up to four 1290 Infinity II MCT (G7116B) + mounted 8 column selectors (G4239C)
Trang 34More automation capabilities
InfinityLab Quick Change valves for column regeneration, or
sample clean-up and enrichment And many more applications!
Trang 35Agilent UV Detectors Portfolio
• Current Portfolio
1260 Infinity II DAD-HSG7117C1260 Infinity II MWDG7165A1260 Infinity II VWDG7114A
• Dispersion volume
Trang 36Sensitivity: Effects of Path Length Increase
Max-Light High Sensitivity cell:
Optofluidic waveguides (total internal reflection)
Trang 37Data rate & Dispersion volume
• Ultra-low Dispersion flow cell and high data rate maintain the separation
min0 0.1 0.2 0.3 0.4mAU
The optimized ULD flow cell and data rates up to 240 Hz deliver
uncompromised chromatographic resolution – even for your fastest separations.
min0 0.1 0.2 0.3 0.4mAU
Data rate = 240 Hz
Data rate = 20 Hz
Trang 38• Increasing Linear UV Range by >30x
1290 Infinity II HDR Impurity Analyzer
60 mm
3.7 mm
Clustered DADDAD 1
DAD 2
60 mm Cell – for low concentrations
3.7 mm Cell – for high concentrationsOne, combined signal, normalized to 10 mm
path length
(HDR range: 0.6 x 10-6to 6.7 AU/cm) vs for 1200 Series: 7 x 10-6to 2 AU/cm)
Trang 3960 mm path-length
3.7 mm path-length
Computing the signals from
60 mm path-length for the low concentration
3.7 mm path-length for the high concentrationDetector Signal
Conc [µg/mL]
30x Wider Linear Range with HDR Impurity Analyzer
• 3.7 mm and 60 mm and Max-Light flow cell
Trang 40Fixed Dose Combination drug was used:
Paracetamol (200mg) and Chlorphenamine (2.5mg ), (1:80),other compounds are Vitamin C (150mg)
and Caffeine (25mg) + further small impurities
Vitamine C
H
Trang 411µl injection to determine the first 3 peaks
Detector saturationOutside the linear range
Trang 42Conventional DAD vs HDR DAD, 5 µl injection
ChlorphenamineVitamin C
Detector saturationOutside the linear range
HDR-DAD:All peaks within linear range (green)
Conventional DAD: 2 peaks out of linear range with 5µl injection (red)
Trang 43THANKYOU