30x Wider Linear Range with HDR-DAD

Verbesserung der LC-Performance
durch Integration neuer
HDR-DA-Detektoren und
Hybrid LC-SFC-Systeme von
Agilent Technologies
APPLICA 2012
Dr. Holger Stalz
LC-MS Produktspezialist
Agilent Technologies Schweiz
Basel, 16. Oktober 2012
1
APPLICA 2012
16. Oktober 2012
30x Wider Linear UV range Quantification of widely different
concentration levels in one single run
Agilent 1200 Infinity High Dynamic Range
(HDR-DAD) Solution
Holger Stalz
LC/MS Produktspezialist
Agilent Technologies Schweiz
1260/1290 Infinity HDR-DAD
2
APPLICA 2012
16. Oktober 2012
History of DAD Linearity Gain
The last 30 years
45.0
> 30
40.0
1200 Infinity HDR-DAD
35.0
>30X Linearity
Linearity Gain
30.0
With HDR
25.0
20.0
15.0
10.0
5.0
1.4
1.7
2.0
1.4
1040 to 1090
1090 to 1050
1050 to 1100
1110 to 1200
(1995)
(2006)
2.9
0.0
(1986)
(1988)
1200 to 1290/60
(2010)
1200 to HDR
(2011)
Detector Model (Intro Year)
3
APPLICA 2012
16. Oktober 2012
Optofluidic Waveguides: Max-Light Flow Cells
Total-internal reflection in a non-coated fused silica fiber
1260 / 1290 Infinity DAD
Grating
Mirror
Programmable
or fixed slit
Deuterium
Lamp
Max-Light Cartridge Cell
10 mm or 60 mm path length
4
1024 element
diode-array
APPLICA 2012
16. Oktober 2012
Linearity, linear range and dynamic range
1200 Series Diode Array Detector, 10 mm flow cell
Detector Signal
[AU]
Linear _ range 
10 mm
Upper _ Limit
3  Detector _ Noise
 10
5
HDR- DAD
Upper limit
2.0
Slope = Response Factor
(Sensitivity)
HDR- DAD
Detector
noise
7*10-6
Linear range
Dynamic range
LOD
(3x S/N)
5
Conc (upper limit)
linear range
Conc (upper limit)
dynamic range
Conc [µg/mL]
APPLICA 2012
16. Oktober 2012
30x Wider Linear Range with HDR-DAD
3.7 mm and 60 mm and Max-Light flow cell
Detector Signal
[AU/cm]
30x lower Limit of Detection (LOD) for impurity analysis
- For samples with widely different concentration levels
- no time consuming
• reanalysis
• recalibration
• sample preparation
3.7 mm
60 mm
10 x higher sensitivity
~3x higher upper limit
Conc
[µg/mL]
30x lower Limit of Detection (LOD)
6
APPLICA 2012
16. Oktober 2012
1290/1260 High Dynamic Range – HDR
Increasing Linear UV Range by >30x
Technology
DAD 1:
60mm
Clustered DAD
 Cluster of two DADs

DAD 1: 60 mm Cell – for low concentrations

DAD 2: 3.7 mm Cell – for high concentrations

Output: One, combined signal, normalized to 10 mm
path length (HDR range: 0.6 x 10-6 to 6.7 AU/cm) vs
for 1200 Series: 7 x 10-6 to 2 AU/cm)
Control / Usability
 Like standard 1290/1260 DAD: Control,
data analysis, reporting
DAD 2:
Investment Protection
 Existing 1290/1260 DAD can be upgraded
(by 2nd DAD)
3.7mm
7
 Existing 1100/1200 Systems can be upgraded
(by two 1290 or 1260 DAD)
APPLICA 2012
16. Oktober 2012
1290/1260 High Dynamic Range – HDR
Increasing Linear UV Range by >30x
DAD 1:
60mm
For sample mixtures with widely
different concentration levels:
 All sample information in one run, e.g.
for fixed dose combinations
 shorter turnaround time
 higher instrument utilization
 reduced solvent and waste costs.
Clustered DAD
DAD 2:
 Up to 30x lower LOD, 15x lower LOQ
(compare to 1100/1200 Series)
 for more reliable automated integration
 higher area precision of low levels
impurities
 prepared for stronger regulations
3.7mm
8
 genotoxins
APPLICA 2012
16. Oktober 2012
Experimental
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
CH3
NH
CH3
Paracetamol
O
N
N
Caffeine
N
N
O
HO
CH3
H3C
O
Cl
CH3
Chlorphenamine
N
CH3
HO
Vitamine C
O
HO
N
H
HO
9
O
OH
APPLICA 2012
16. Oktober 2012
2 Injections needed if a conventional DAD is used
Paracetamol
mAU
Vitamin C
1000
800
1µl injection to determine the first 3 peaks
600
400
Chlorphenamine ?
Caffeine
200
0
0
1
2
3
4
5
min
mAU
3800
Vitamin C
3000
Paracetamol
Detector saturation
Outside the linear range
5µl injection to determine the last peak and further impurities
The first 2 peaks are out of linear range
2000
Chlorphenamine
1000
Caffeine
0
0
10
1
2
3
4
5
min
APPLICA 2012
16. Oktober 2012
Comparison of conventional DAD vs. HDR DAD,
5µl injection
mAU
Paracetamol
4000
3500
Vitamin C
Red conventional DAD: 2 peaks out
of linear range with 5µl injection
Detector saturation
Outside the linear range
3000
Green HDR-DAD:
All peaks within linear range
(up to 6AU)
2500
2000
1500
1000
Caffeine
500
Chlorphenamine
0
0
11
1
2
3
4
5
min
APPLICA 2012
16. Oktober 2012
Summary
With the Agilent HDR DAD the determination of low and high
doses drugs is possible in one run wheras the complete
determination of all drugs with a conventional DAD needs 2
injections with different injection volumes
Further improved precision and lower detection limits are
achieved compared to a conventional DAD
12
APPLICA 2012
16. Oktober 2012
13
APPLICA 2012
16. Oktober 2012
What is Supercritical Fluid
Chromatography?
Definitions:
1. Supercritical Fluid Chromatography is a chromatography
technique based on the use of supercritical carbon dioxide
as the mobile phase
2. Supercritical Fluid Chromatography (SFC) is a form of
normal phase chromatography that is used for the analysis
and purification of low to moderate molecular weight
molecules. Principles are similar to those in HPLC however
SFC typically utilizes CO2 as the mobile phase; therefore the
entire chromatographic flow path must be pressurized.
14
APPLICA 2012
16. Oktober 2012
Benefits of SFC vs. LC
• Unique selectivity (orthogonality to HPLC)
• Premier choice for chiral compounds
• Replacement for Normal-Phase-HPLC
• Complimentary to RP-LC or HILIC
• Speed of analysis (faster than LC)
• Cheaper than LC
• Green
• Broad detector compatibility
=>
15
APPLICA 2012
16. Oktober 2012
Unique Features of the
Agilent 1260 Infinity SFC
Performance
 HPLC-like UV-Sensitivity
 The only SFC with 600 bar power range
 Seamless MS and ELSD connection
Cost




10-15x lower operating costs with standard grade CO2
Lowest solvent consumption and waste generation
From entry LC upgrade to complete high-end MS/SFC
all in one hybrid SFC/UHPLC system
Time
 3 x faster than LC
 no significant equilibration to change from UHPLC to SFC with
the Hybrid SFC/UHPLC
Robustness
 Based on proven 1260 Infinity quality
19
APPLICA 2012
16. Oktober 2012
Product Overview: 3 Ways to get SFC from
Agilent ….
Hybrid SFC/UHPLC
System
Analytical SFC
LC Upgrade to SFC
• 1260 Infinity analytical SFC
• LC pump (Quat, Binary)
• 2pos/10port valve
• Hybrid SFC/UHPLC kit
• 1260 Infinity analytical SFC
• Fusion A5n (Aurora)
• 1260 SFC binary pump
• SFC accessory kit
Change from SFC to UHPLC
Orthogonal Separation
Power
17
Convert 1100/1200 to SFC
Best SFC performance
Most affordable
APPLICA 2012
16. Oktober 2012
The Agilent 1260 Infinity SFC/UHPLC
Hybrid System
Two orthogonal
techniques with a
single system
Simply switch the valve
18
APPLICA 2012
16. Oktober 2012
Hybrid System in SFC Mode
HPLC
Waste
Loop back
Restrictor
2
1
3
10
LC Pump out
9
4
8
5
6
BPR
7
LC Pump:
G1310B or
G1311B or
G1312B
SFC
Waste
SFC Pump outlet
Detector out
SFC Pump inlet
SFC-Binary Pump
19
APPLICA 2012
16. Oktober 2012
Hybrid System in UHPLC Mode
Waste
Restrictor
2
3
10
LC Pump out
9
4
8
5
BPR
LC Pump:
G1310B or
G1311B or
G1312B
1
6
7
Detector out
SFC Pump outlet
SFC Pump inlet
SFC-Binary Pump
20
APPLICA 2012
16. Oktober 2012
Why is orthogonal sample information important
and what are the advantages of the Agilent Hybrid
instrument?
• Orthogonal method screening in one single system
• Separate and detect impurities which co-elute when using a
single technique
• Resolve peaks of complex samples
• Switch from SFC to UHPLC forth and back in a single sequence
• No equilibration time between LC and SFC
• Significant cost saving, only one system has to be purchased
• Direct results comparison between SFC and LC
• No instrument to instrument variation
• Save lab space
• The only vendor who can offer both techniques in one system
21
APPLICA 2012
16. Oktober 2012
Performance: Orthogonal Separation: SFC Mode
1 - Caffeine
2 - Theophylline
3 - Cortisone
4 - Prednisone
5 - Hydrocortisone
6 - Prednisolone
7 - Sulfomerazine
8 - Sulfaquinoxaline
8 Components
SFC Conditions
Column: Zorbax Rx-Sil, 150 mm, 5 um, 4.6
Temp 27 C
Flow Rate 4ml/min
Gradient: CO2 v Meth
Time
%B
0
5
3.9
45
4
5
22
APPLICA 2012
16. Oktober 2012
Performance: Orthogonal Separation: UHPLC Mode
What to do if there is a resolution problem?
6 Components + 2 Co-Eluting Peak
LC Conditions
Column s–
ACE/Poroshell 3.0 x
150mm
Temp 45 C
Flow Rate 1ml/min
H20 v Meth
Time
%B
0
5
4.1
45
4.4
45
4.6
100
5.0
100
5.1
5
23
APPLICA 2012
16. Oktober 2012
Overlay SFC versus UHPLC mode
1 - Caffeine
2 - Theophylline
3 - Cortisone
4 - Prednisone
5 - Hydrocortisone
6 - Prednisolone
7 - Sulfomerazine
8 - Sulfaquinoxaline
LC
SFC
24
APPLICA 2012
16. Oktober 2012
Repeatability: SFC and LC in a single sequence
SFC-Mode
LC-Mode
Part of a sequence with alternating SFC and LC batches
25
APPLICA 2012
16. Oktober 2012
Ich danke Ihnen für Ihre
Aufmerksamkeit!
Haben Sie Fragen?
26
APPLICA 2012
16. Oktober 2012

Download Report