Đao Tạo Hplc 20240517 - Giải Pháp Uplc Agilent-Dp - Version 2.Pdf

Highest retention time precision for reliable peak identificationThe 1290 Infinity II Flexible Pump is the only low- pressure mixing pump that combines the ultimate performance of a high

Agilent

Liquid Chromatography Technology

Phan Xuan Dai

Email: dai.phan@redstar-cms.vn

Tel: +84.333.936.562

Redstar-CMS Application team

Chromatographic resolution

Carryover

Retention time precision

• Overview pressure range

Agilent InfinityLab Series Pumps

1300

bar

1290 Infinity II High Speed Pump

1290 Infinity II Flexible Pump

800

600

bar

1260 Infinity II Binary Pump

1260 Infinity II Quaternary Pump

1260 Infinity II Isocratic Pump

400

High Speed Pump

Flexible Pump

• Microfluidics applied for the best mixing performance

Jet Weaver

The technology behind:

• Multi-layer technology

• Diffusion bonded stainless steel

• Etched structure (Microfluidics)

What it provides:

• Highest mixing efficiency

• Lowest UV detector noise

(35 µl & 100 µl in TFA applications)

Highest 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 high- pressure mixing UHPLC pump with the flexibility of a quaternary

pump This pump delivers superior gradient performance over the entire composition range from 1 to 99 %.

• Superior quaternary gradient delivery performance

Highest retention time precision

Specification (< 0.07 %)

mAU

0 5 10 15 20 25 30 35

3: Hydrocortisone acetate

1: Hydrocortisone

2: di-proprionate

MeCN + 1% TFA

H 2 O + 0.2% TFA

MeCN + 0.18% TFA

Run gradient!

Pure solvent + concentrated stock solution = any concentration you need

BlendAssist for automatic solvent blending

• Intelligent System Emulation Technology (ISET)

Intelligent System Emulation Technology

G7104A

G7104C

is available in

G7120A

Intelligent System Emulation Technology (ISET)

Programmed gradient

1290 gradient

1200 gradient

0 0.5 1 1.5 2 2.5 3 3.5 0

50 100 150 200 250 300 350 400

min

mAU

Injection

Same gradient conditions:

a) By moving the gradient b) By emulating the gradient mixing behavior

• Intelligent System Emulation Technology (ISET)

Intelligent System Emulation Technology

Agilent 1260/1290 Infinity II Flexible Pump Agilent 1260/1290 Infinity II Flexible Pump with ISET Agilent 1100 Series Binary LC

Chromatographic resolution

Carryover

Retention time precision

Agilent InfinityLab Series Autosamplers

Main Products & most important options

1260 Infinity II Hybrid Multisampler 1260 Infinity II Multisampler 1290 Infinity II Multisampler

• Max Operating pressure

• Injection Precision & Accuracy

http://t0.gstatic.com/images?q=tbn:WhqsIyusTkuS3M:http://z.about.com/d/graphicssoft/1/0/E/N/1/snow_flake_2.png

Wash inner needle

Flow-through principle washes inner needle thoroughly and constant with eluent

Wash outer needle Choice of 3 external

solvents to program

washing steps for the outer needle.

• Get you near-zero carryover

• Get you near-zero carryover

The 1290 Infinity II Multisampler with Multi-wash reduces carryover to less than 9 ppm!

Throughput: 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

Dual-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 & Regeneration

Injection

Injection

Run 1

Run 2

Analysis 1 Wash & Regeneration

Analysis 2 Wash & Regeneration

Injection

Injection

Alternating Column Regeneration runs with Dual-Needle

Alternating column regeneration: reducing idle-times

Optimizing throughput

Ideal conditions for a fast Dual-needle usage:

• Short runtimes

• Extensive sample load and prep procedures

• Extensive needle wash

• Alternating Column Regeneration

Overlay of 14 subsequent runs of the preservatives separation with dual-needle setup and automated column regeneration

Feed Injection feature: Overcome Solvent Effect

Metering device

Feed Injection

Pump

Sample Mobile phase

Feed solvent

Pain 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 exchange

Sample solvent Mobile phase

Big difference

in solvent strength

What do they look like?

• Breakthrough

• Poor peak shape

RPLC

LC/MS analysis

QuECheRS sample preparation

Challenge 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 µL Methanol / water [1:1 v/v]

UHPLC, vendor B UHPLC, vendor C

Feed 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

Feed Injection feature: Best solutions to mediate solvent effect

Classical Flow Through

Feed Injection

Feed Injection feature: Applied on Food safety

Mycotoxin

QuEChERS sample preparation

strong solvent matrix

Agilent InfinityLab LC Series Column Compartments

Increase daily throughput with focus on ease of use

“ 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

Loss of Peak Resolution

Thermal Mismatch Issues

• Centre of column below oven temperature

• Higher viscosity, lower linear velocity in centre

• Higher retention in centre

Concept available for

• 1290 Infinity II Multicolumn Thermostat MCT (G7116B)

• 1260 Infinity II Multicolumn 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 Valves InfinityLab Valve Solution Training Presentation

Ordering Information

Valve-Thermostat Clusters (VTC)

Position 1-8Position 1-8

Position 1-8Position 1-8

External valve drive (G1170A)

Up to four 1290 Infinity II MCT (G7116B) + mounted 8 column selectors (G4239C)

More automation capabilities

InfinityLab Quick Change valves for column regeneration, or

sample clean-up and enrichment And many more applications!

Agilent UV Detectors Portfolio

Sensitivity: Effects of Path Length Increase

Peak Dispersion

60 mm pathlength large geom cell volume (78 µL) lower light transmission

- Loss of resolution

- Loss of signal height

10 mm pathlength small geom cell volume (13 µL) high light transmission

Conventional flow cells:

60 mm pathlength

4 µL σV dispersion volume

Max-Light High Sensitivity cell:

Optofluidic waveguides (total internal reflection)

Data rate & Dispersion volume

• Ultra-low Dispersion flow cell and high data rate maintain the separation

min

0 0.1 0.2 0.3 0.4

mAU

0102030405060

The optimized ULD flow cell and data rates up to 240 Hz deliver

uncompromised chromatographic resolution – even for your fastest separations.

min

0 0.1 0.2 0.3 0.4

mAU

020406080100

Data rate = 240 Hz

Data rate = 20 Hz

• Increasing Linear UV Range by >30x

1290 Infinity II HDR Impurity Analyzer

60 mm

3.7 mm

Clustered DAD DAD 1

DAD 2

60 mm Cell – for low concentrations

3.7 mm Cell – for high concentrations

One, 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)

60 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 concentration

Detector Signal [AU/cm]

Conc [µg/mL]

30x Wider Linear Range with HDR Impurity Analyzer

• 3.7 mm and 60 mm and Max-Light flow cell

Sample:

Fixed 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

Chlorphenamine

Vitamine C

O H

NH CH3

N N

N

CH3

O

CH3O

O

O

OH O

H

O H O H

H

1µl injection to determine the first 3 peaks

min

mAU

0200400600800

3800

Detector saturation Outside the linear range

Conventional DAD vs HDR DAD, 5 µl injection

min

mAU

0500100015002000250030003500

HDR-DAD: All peaks within linear range (green)

Conventional DAD: 2 peaks out of linear range with 5µl injection (red)

THANK YOU