Page 1
Tips On Getting
More From Your
Liquid Chromatography
Carl Griffin
Application Engineer
Advanced Chromatography Media
Page 2
ZORBAX RRHT and
RRHD
POROSHELL 120
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
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
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
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
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
Page 8
USP Method-Naproxen
Page 10
NAPROXEN 50 mm Column
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
Page 12
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
Page 13
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
Page 14
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
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
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
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
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)
HPLC Instrument Components
Gradient Delay or Dwell Volume .
Extracolumn Volume
Data Sampling or Acquistion Rate
.
Number of Scans
or points
Effects of Delay Injection Program
Page 21
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
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
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
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
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
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
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
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.
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)