tips on getting more from your liquid chromatography · 2016-09-03 · 0 0.5 1 1.5 2 2.5 3 3.5 4...
TRANSCRIPT
![Page 1: Tips On Getting More From Your Liquid Chromatography · 2016-09-03 · 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min mAU 0 20 40 60 80 100 120 140 VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS](https://reader033.vdocument.in/reader033/viewer/2022060407/5f0f9c187e708231d44503b7/html5/thumbnails/1.jpg)
Page 1
Tips On Getting
More From Your
Liquid Chromatography
Carl Griffin
Application Engineer
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Advanced Chromatography Media
Page 2
ZORBAX RRHT and
RRHD
POROSHELL 120
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Poroshell 120 Columns for HPLC and UHPLC:
• 80-90% efficiency of sub 2um
• At ~40-50% lower pressure
• 2X efficiency of 3.5um (totally porous)
• A 2.7um particle size
• A 2um frit to reduce clogging
• A 600 bar pressure limit
• The particle has a solid core (1.7um) and
porous outer layer with a 0.5um diffusion
path
March 18, 2013
Confidentiality Label
3
Poroshell 120 columns have:
1.7um
0.5um
0.5um
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Page 4
Smaller Particles Improve Detection and
Integration
4.6 x 150, 1.8μm
490 bar
7 Impurities
All 7 Baseline
Separated!
4.6 x 150, 3.5μm
165 bar
7 Impurities
6 Not Baseline
Separated!
4.6 x 150, 5μm
93 bar
4 Impurities
2 Not Baseline
Separated!
Customer Sample, Impurity Method
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What is Sensitivity?
Limit of detection (LOD):
“The lowest concentration, or smallest mass flow, which can be distinguished
from the noise by a certain predefined probability (Signal/Noise).”
Noise
Signal
hSignal = 3(2) x hNoise
Limit of detection (LOD):
Page 5
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Analysis of 15 Analgesic Compounds Same Method for all Three Columns
ZORBAX Eclipse Plus C18, 3 x 100 mm, 3.5 µm
ZORBAX RRHD Eclipse Plus C18, 3 x 100 mm, 1.8 µm
Poroshell 120 EC-C18, 3 x 100 mm, 2.7 µm
Ibuprofen:
PW1/2=0.014
S/N=182
nc=43
Ibuprofen:
PW1/2=0.012
S/N=353
nc=54
Ibuprofen:
PW1/2=0.012
S/N=256
nc=56
2 min
June, 2011
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Van Deemter Comparision
7
van Deemter
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0 0.5 1 1.5 2 2.5 3 3.5
Flow rate (mL/min)
HE
TP
(cm
)
Eclipse Plus C18, 3.5 µm
Eclipse Plus C18, 1.8 µm
Poroshell 120 C18, 2.6um
Poroshell 120 C18, 2.6 µm
Poroshell 120 C18, 2.7 µm
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Page 8
USP Method-Naproxen
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Page 10
NAPROXEN 50 mm Column
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Group/Presentation Title
Agilent Restricted
Month ##, 200X
0 10 20 30 40
Changing Gradient Time to Affect Retention (k*) and Resolution
Time (min)
100% B
100% B
100% B
100% B
tg= 40
tg= 20
tg= 10
tg= 5
000995P1.PPT
1/k* = gradient steepness = b
tg F
S B Vm
k* =
= change in volume fraction of B
solvent
S = constant
F = flow rate (mL/min.)
tg = gradient time (min.)
Vm = column void volume (mL)
0% B
0% B
0% B
0% B
• S 4–5 for small molecules
• 10 < S < 1000 for peptides and proteins
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min0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
250
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\070809SBC180003.D)
1.4
74
3.3
23
5.0
64
5.6
51
6.1
14
6.9
64
8.3
37
9.6
90
10.9
82
Conventional Column - 4.6 x 150mm, 5µm, SB-C18
Flow Rate 1.0 ml/min
Injection Volume 15uL
Temperature 30°C
Wavelength 246nm
Sample rate 2.5 Hz
Time (min) % Acetonitrile
0 50
10 90
13.5 90
13.6 50
15 50
Initial Pressure: 69 bar
Final Pressure: 38 bar
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Shorten Column and Gradient Time by Same Factor 1/3 Column Length- 1/3 Gradient Time
RRHT Column – 4.6 x 50mm, 1.8µm, SB-C18
min0 1 2 3 4 5 6
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180009.D)
1.1
76
2.0
04
2.2
54
2.4
64
2.8
11
3.3
57
3.8
71
4.3
43
Flow Rate 1.0 ml/min
Injection Volume 5uL
Temperature 30°C
Wavelength 246nm
Sample rate 13.74 Hz
Time (min) % Acetonitrile
0 50
3.33 90
4.5 90
4.53 50
5 50
Initial Pressure: 132 bar
Final Pressure: 74 bar
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Increase Column Flow-Reduce Gradient Time Double Flow (2mL/min) – ½ Gradient Time
RRHT 4.6 x 50mm, 1.8µm, SB-C18
min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-10 08-22-16\070810SBC180004.D) 0
.587
1.0
20
1.1
46
1.2
60
1.4
37
1.7
15
1.9
70
2.2
01
Flow Rate 2.0 ml/min
Injection Volume 5uL
Temperature 30°C
Wavelength 246nm
Sample rate 13.74 Hz
Time (min) % Acetonitrile
0 50
1.67 90
2.25 90
2.27 50
3.34 50
Initial Pressure: 266 bar
Final Pressure: 146 bar
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UHPLC/TOF (1290/6230)Can Identify More
Compounds in Less Time
1 .5
min
Time Composition
0.0 10% ACN
1.5 100% ACN
224 pesticides at 50 pg each
217 ionized & detected in positive
mode
(97%, Find by Formula)
2.1 x 50 mm x 1.8 micron
Eclipse Plus C-18
900 bar
1.5 mL/min
1290 Infinity
TOF fast acquisition rates (20Hz) ensure maximum throughput
Page 15
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Ultrafast LC/MS Analysis for 15 Analyte Subset
1290 Infinity Applications
Peak Width 0.7 sec
RRHD Eclipse Plus C18
2.1x 50 mm, 1.8 um
750 bar
1 minute
Time Composition
0.0 10% ACN
1.5 100% ACN
Ultimate speed on a short column with ballistic gradient
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Data
Rate
Peak
Width
Resolution Peak
Capacity
80 Hz 0.300 2.25 60
40 Hz 0.329 2.05 55
20 Hz 0.416 1.71 45
10 Hz 0.666 1.17 29
5 Hz 1.236 0.67 16
min 0.1 0.2 0.3 0.4 0.5 0
80Hz
PW=0.30sec
40Hz
PW=0.33sec
20Hz
PW=0.42sec
10Hz PW=0.67sec
5Hz PW=1.24sec
Sample: Phenones Test Mix
Column: Zorbax SB-C18, 4.6x30, 1.8um
Gradient:: 50-100%ACN in 0.3min
Flow Rate: 5ml/min
80Hz versus 10Hz (20Hz) Data Rate
• Peak Width: – 55% (– 30%)
• Resolution: + 90% (+ 30%)
• Peak Capacity: + 120% (+ 40%)
• App. Column Eff.: + 260% (+ 70%)
Detectors For narrow peaks, high data rates!!! Maintaining Resolution at High Analysis Speed
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Decreased Column Volume May Require
Conversion for Injection Volume
2 col.
column1
column21 col. Inj.Vol.
Volume
VolumeInj.Vol.
2 col.column1
column21 col. 4
2.0
4.020 i.e. μl
ml
mlμl
Page 18
Zorbax column volume = 3.14 x r2 x L x 0.6 (r and L in cm)
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HPLC Instrument Components
Gradient Delay or Dwell Volume .
Extracolumn Volume
Data Sampling or Acquistion Rate
.
Number of Scans
or points
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Effects of Delay Injection Program
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0 10 20 30 40
Minor Dwell Volume Differences
Can Change Resolution
0 10 20 30 40
VD = 0.43
mL
Column: ZORBAX Rapid Resolution Eclipse
XDB-C8
4.6 x 75 mm, 3.5 µm
Mobile Phase: Gradient, 0 - 100 %B in 52.5 min.
A: 5/95 methanol/ 25 mM phosphate
pH 2.50
B: 80/20 methanol/25 mM phosphate
pH 2.50
Flow Rate: 0.5 mL/min
Temperature: 25°C
Injection: 5 L
Detection: 250 nm
Sample: Mixture of antibiotics and
antidepressants
Upper trace simulates actual run data
entered into DryLab® 3.0 software
Lower trace is simulated chromatogram
for larger VD
VD = 2.0
mL
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Effect of Extra-column Volume on a Gradient
Analysis of Alkylphenones
Default 1290, 8.6 µL Extra-column Volume
Optimized 1290, 3.0 µL Extra-column Volume
A: H2O; B: CH3CN
0.4 mL/min
t (min) 0 1.2
%B 25 95
1 µL injection of RRLC Checkout Sample (PN 5188-
6529) spiked w/ 50 µL 2 mg/mL Thiourea in
water/acetonitrile
TCC: ambient
DAD: Sig=254,4nm; Ref=Off
Sample:
1. Thiourea (v0 marker)
2. Acetanilide
3. Acetophenone
4. Propiophenone
5. Butyrophenone
6. Benzophenone
7. Valerophenone
8. Hexanophenone
9. Heptanophenone
10. Octanophenone
Agilent ZORBAX Eclipse Plus RRHD C18
2.1 mm x 50 mm, 1.8 µm, 959757-902
LC Rack System, 5001-3726
0.08 x 220 mm Capillary Tubing
V(σ)0.6 µL Flow Cell
Pmax=320 bar
Rs5,6=1.18
nC=35
Pmax=323 bar
Rs5,6=2.25 91% increase
nC=56 60% increase
Page 22
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System – Signal Height System volumes – System dispersion
min 0.5 1 1.5 2 2.5
mAU
0
100
200
350
400
550
600
System dispersion optimized
Peak width 0.018 min
Peak width 0.019 min
Resolution 1.902
min 0.5 1 1.5 2 2.5
mAU
0
100
200
300
400
System dispersion not optimized
Peak width 0.038 min
Peak width 0.037 min
Resolution 0.961
310
mAU
380
mAU
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System – Signal height System volumes – Delay volume
Delay volume ~ 700 μL
min 0.5 1 1.5 2 2.5 3 3.5 4 4.5
mAU
0
100
200
300
400
Delay volume ~ 120 μL
min 0.5 1 1.5 2 2.5 3 3.5 4 4.5
mAU
0
100
200
300
400
Column: ZORBAX SB-C18 2.1 x 50 mm, 1.8 μm
Flow: 0.42 mL/min
120 mAU
220 mAU
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Influence post-column capillary connections
min 0 0.1 0.2 0.3 0.4
mAU
0
20
40
60
80
100
120
140 One bad capillary connection!
min 0 0.1 0.2 0.3 0.4
mAU
0
30
60
90
120
150
180
210 Fixed!
130 mAU
160 mAU
Page 25
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What Happens If the Connections Poorly Made ?
Page 26
If Dimension X is too long, leaks will occur
Ferrule cannot seat properly
Mixing Chamber
If Dimension X is too short, a dead-volume,
or mixing chamber, will occur
Wrong … too long
Wrong … too short
X
X
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Page 27
Why Filter the Sample?
Extreme Performance Requires Better Sample “Hygiene”
• Prevents blocking of capillaries, frits, and the column inlet
• Results in less wear and tear on the critical moving parts of injection valves
• Results in less downtime of the instrument for repairs
• Produces improved analytical results by removing potentially interfering contamination
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A Dirty Sample: Precipitated Plasma Column
Life Test
Page
28
Column: 2.1x50mm, SB-C18
Sample: 1mg/ml plasma + 0.2mg/ml naphthalene in 75/25 ACN/H2O solution (filtered)
Method: flow = 0.8 ml/min; temp = ambient; detector = 210 nm; injection = 0.5 ul
Mobile phases: A – H2O + 0.1% TFA; B – ACN + 0.08% TFA
Gradient table: Time (min) 0 0.2 0.7 1.2 1.3 1.5
B% 20 20 90 90 20 Stop
400
500
600
700
800
900
1000
1100
1200
0 500 1000 1500 2000 2500
Pre
ssu
re (
bar)
Injections
RRHD Plasma Sample test
USCES00002
USCES01074
Sample was plasma, protein precipitated,
centrifuged, filtered through 0.2um filter.
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Page 29
In-Line Filters Provide Good Insurance
Against System OverPressure NEW RRLC In-line filter and fitting – max 600 bar
Protect RRHT columns with efficient in-line filter with 0.2 µm pore size frits
Description Part number Porosity
Frit
diameter
Flow
rate
Part number
Replacement
Frits
RRLC In-line
filter, 2.1 mm,
max 600 bar
5067-1551 0.2 µm 2.1 mm<1
mL/min
5067-1555
(10/pk)
RRLC In-line
filter, 3.0 & 4.6
mm, max 600
bar
5067-1553 0.2 µm 4.6 mm1 - 5
mL/min
5067-1562
(10/pk)