Transcript Eksigent Tempo Demonstration Presentation

System Design and
Performance Overview
nano LC
nano MDLC
ht LC
nano LC
•Nanoscale binary gradient
chromatography
•No flow splitting and dynamic
flow control
•Additional loading pump for
high speed sample loading
and de-salting
Nano-Flow HPLC
• Active flow control of each mobile phase
– Measurement of flows with active feedback
– Accounts for changes in viscosity and back
pressure in the separation column
• No flow splitting
– Direct measurement of column flow and back pressure
– Reductions in mobile phase preparation and disposal
• Rapid system response
– Prompt step changes in flow or composition
– High flow loading/washing and “peak parking” integral to
system
Traditional Analytical vs. Capillary HPLC
Repeated Injections of BSA
digest: chromatograms and
column pressures
1300
10
1.2x10
Flowrate (nL/min) or Pressure (psi)
1200
9
TIC (cps)
9.0x10
9
6.0x10
9
3.0x10
0.0
1100
1000
900
Qa(nL/min)
Qa_profile(nl/min)
Qb(nL/min)
Qb_profile(nl/min)
Pc(psi)
Pc(psi)
Pc(psi)
800
700
600
500
400
300
200
100
5
10
15
20
25
Time (min)
• BSA tryptic digest
• 150 x 0.075 mm 5um ProteoPep2 C18
column
• water/acetonitrile gradient with 0.1%
formic acid
0
0
20
40
Time (min)
• 250 nL/min gradient
• column back pressure shows run to
run conditions of column
Dynamic Flow Control for Peak Parking
•Rapid flow rate reduction
(500-50 nL/min)
Flow rate and Pressure
1600
1400
•Gradient profile resumed
after parking
1200
1000
800
Qa(nL/min)
Qb(nL/min)
Pc(psi)
600
•Parking flow can be set at
any rate
•No additional hardware or
plumbing
400
200
0
0
10
20
Time (min)
30
40
•Triggered within software
or by external trigger
Peak parking example
214 nm Absorbance
Gradient separation of
Cytochrome c tryptic digest
Col: 150 x 0.100 mm
3 mm C18
Inj: 100 nL
Flow rate: 300 nL/min
Park rate: 50 nL/min
0
5
10
15
Time (min)
PARK
20
25
Phosphopeptide Identification with Peak Parking
Intensity, cps
XIC of +EPI: Exp 3, 784.0 to 784.9 amu
Max. 3.8e6 cps.
NO Peak
Parking
< 30 sec
0.0
8 9 10 11 12 13 14 15 16
Time, min
XIC of +EPI: Exp 3, 784.1 to 785.1 amu
Max. 9.5e6 cps.
Intensity, cps
1
2
3
4
5
6
7
Peak Parking
Activated
0.0
1
2
3
4
5
6
•Mixture of a and b casein
with BSA tryptic digests
•Q-Trap triple quadrupole
with linear trap
• Neutral loss (49 amu)
survey scan to identify
phosphopeptides
> 1.5 min
7
8 9 10 11 12 13 14 15 16
Time, min
• Peak parking from 500
nL/min to 50 nL/min
activated by MS software
when NL detected
Increased Phosphopeptide ID with Peak Parking
a-casein and its phosphopeptides identified with peak parking
VPQLEIVPNSAEER + Phos pho (ST)
YKVPQLEIVPNSAEER + Phos pho (ST)
FFVAPFPEVFGK
EPMIGVNQELAYFYPELFR
HQGLPQEVLNENLLR
m/z
830.7
976.3
692.8
772.6
880.8
alpha-Casein (overall score)
FQSEEQQQTEDELQDK + Phos pho (ST)
1030.3
beta-Casein (overall score)
KVPQVSTPTLVEVSR
DAFLGSFLYEYSR
TVMENFVAFVDK
VPQVSTPTLVEVSR
Bovine Serum Albumin (BSA)
820.3
784.5
700.3
756.2
Survey Scan
Peak Park
Mascot Search
MS
Neutral Loss
Neutral Loss
OFF
OFF
ON
Score
n/d
n/d
43 (17)
50
115
Score
n/d
n/d
n/d
n/d
n/d
Score
32
41
47
31
n/d
208
n/d
151
113
85
101
113
85
101
41
n/d
n/d
73
n/d
14
73
n/d
n/d
n/d
18
61
114
87
79
* Neutral Loss of 49 was used to target doubly charged phosphopeptides specifically
nano MDLC
•Dual binary gradient
chromatography (4 pumps)
• 20 – 1000 nL/min
• 1 – 20 microL/min
• No flow splitting and
dynamic flow control
Rapid Sample loading on
parallel traps to increase
throughput of dilute samples
Rapid Sample Loading Data
1st trap: rapid sample load
700
2nd trap: loaded while 1st
trap elutes
700
UV signal 214 nm
Flow A (nL/min)
Flow B (nL/min)
600
UV signal 214 nm
Flow A (nL/min)
Flow B (nL/min)
600
500
500
400
400
300
300
200
200
100
100
0
0
0
10
20
30
40
50
Time (min)
•5 mL of BSA tryptic digest loaded at 5
mL/min
•0.5mm x 2 mm CapTrap (Michrom)
•eluted at 500 nL/min onto 150mm x
0.1mm C18 analytical column
0
10
20
30
40
50
Time (min)
•5 mL of BSA tryptic digest loaded at
500 nL/min
•0.3mm x 5 mm PepMap C18 trap (LC
Packings)
•eluted at 500 nL/min onto 150mm x
0.1mm C18 analytical column
Microfluidic Flow Control Technology
•
Flow meters in each
mobile phase
measure the actual
flow rate
•
Active Flow Control
uses feedback to
control a rapid,
electronically adjusted
variable pressure
source
•
Flow rate is
maintained regardless
of system back
pressure or viscosity
•
Binary system
includes dual
controllers
Accurate Flow Control
402
Flow Rate (nL/min)
401
1 nL/min
step
400
399
2 nL/min
step
398
397
396
395
0
60
120
180
Time (sec)
240
300
ExpressLC
•Fast gradient chromatography
• 2-30 microL/min
• Optimized for 300 micoL
columns
•Direct real-time flow control
•Very fast, highly reproducible
gradients
ht LC System Layout
CTC autosampler- various
versions integrated with system
Temperature
controlled column
holder
Integrated injection
system- software
selectable, delivers
precise injection
volumes from
10 nl to 1 ul
Microfabricated
flow cell with
array-based UV
absorbance from
200 – 380 nm
ht LC Instrument Design
• Direct pumping system with precision flow control

• Reduce instrumental variance to achieve highest performance
allowed by column
– Instrumental variance includes
• Injection (dispersion of “plug”)
• Connection tubing
• Dispersion from fittings/connectors
• Detection cell
s2 = scol2 + sinj2 + stube2 + sfittings2 + sdet2
Loss in Efficiency Due to System Dispersion
for 150 x 0.3 mm Column
15,000
1
s2=150
2
Npl k  3700 nL
i
s2=1000
0.9
platescol 13,500
s =500
2 2
Npl k  2000 nL
i
platescol
0.8
12,000
s2=2000
2
Npl k  1000 nL
i
Plates
platescol
0.7
10,500
2
Npl k  500 nL
i
Assumptions
15,000 plates
s2col = 3700 nL2
platescol
s2=3700
2
Npl k  150 nL 9,000
0.6
i
platescol
0.5 0.5
7,500
0
0
0.5
1
1.5
k
i
K’
2
2.5
3
3
Time-Sliced Injections Provide Low Variance
60nl from variable injector
0.5
Intensity
0.4
0.3
60nl fixed internal loop
0.2
0.1
0.0
0
50
100
150
Volume (nL)
200
250
Variation of Injection Time / Volume
0.8
40 s
Relative Absorbance
12 s
8s
0.6
6s
4s
0.4
540 nL/min
2s
0.2
0.0
0
100
200
Volume / nL
300
400
Linear Variation of Injection Volumes
using Timed Injections
200000
Flow rate: 4.0 mL/min
Duplicate injections for each volume
Peak integral (mAU*nL)
180000
160000
140000
120000
100000
80000
60000
40000
20000
0
0
20
40
60
80
100
120
Injection volume (nL)
140
160
180
Conversion of Flow Rates and
Injection Volumes
Column ID
(Conversion)
Injection Size
Flow Rates
4.6 mm
(20X)
1.0 mm
(1X)
0.3 mm
(0.08X)
0.1 mm
(0.01X)
75 mm
(0.006X)
5 mL
250 nL
20 nL
2.5 nL
1.5 nL
10 mL
500 nL
40 nL
5.0 nL
3.0 nL
20 mL
1 mL
80 nL
10 nL
6.0 nL
0.5 mL/min
25 mL/min
2 mL/min
250 nL/min
150 nL/min
1 mL/min
50 mL/min
4 mL/min
500 nL/min
300 nL/min
5 mL/min
250 mL/min
20 mL/min
2.5 mL/min
1.5 mL/min
Low Dispersion / High Sensitivity
Detection Cell
• Chip-based flow cell
– 200-380 nm array detection
– Fiber optic coupling
– 4 mm pathlength, 45 nL detection cell
• Each separation column will have
independent detection cell and
detection array
Dual Beam Spectrometer Performance
Absorbance Baseline Drift and Noise Performance
• Temperaturecontrolled detector
arrays and light source
2.0
ch0
ch1
ch0/ch1
1.8
Absorbance / mAU
1.6
1.4
250-260 nm bandpass
1.2
• Methods developed
for stable fiber
illumination and
transmission
1.0
0.8
0.6
0.4
0.2
0.0
Drift  0.2 mAU/hr
-0.2
s = 0.030 mAU (0-1100s)
-0.4
0
2000
4000
Time / s
6000
8000
• Typical drift
 0.4 mAU/hr
• Typical short-term
noise  0.04 mAU rms
Detection Cell Variance
Time [s]
0
5
10
15
20
25
30
Eksigent : ~300 nL2
Absorbance
LC Packings : ~6500 nL2
Eksigent 45 nL Flow Cell
LC Packings 45 nL Flow Cell
0
500
1000
Volume [nL]
1500
Eksigent Microfabricated UV Flow
Cell Provides Excellent Linearity
2000
1800
Absorbance (mAU)
1600
Eksigent Absorbance [mAU]
Linear Fit
LC Packings Absorbance [mAU]
1400
1200
1000
800
600
400
200
0
0
100
200
300
400
500
600
700
Concentration (mM)
800
900
1000 1100
Full UV Data Displayed in
Eksigent Control Software
High Resolution: Isocratic Separation
Absorbance (mAU @ 255 nm)
500
400
300
200
Column:
Zorbax 300XDB-C18
3.5 mm
150 x 0.3 mm
Mobile Phase:
A: H2O
B: ACN
Gradient:
Isocratic 30% A : 70% B
Flow Rate:
4 mL/min
Injection:
40 nL
Sample:
1. Uracil
2. Acetophenone (19000)
3. Propiophenone (20300)
4. Butyrophenone (19700)
5. Valerophenone (19600)
6. Hexanophenone (19400)
7. Heptanophenone (19500)
100
0
0
2
4
6
Time (min)
8
10
Near Theoretical Efficiencies
8
Reduced Plate Height
7
Heptanophenone
fit to van Deemter
6
5
4
Small C-term
allows fast
analysis
3
2
0
2
4
6
8
10
12
Flowrate (mL/min)
14
16
18
20
High Resolution Gradient Separation
Absorbance (mAU @ 245 nm)
500
Column:
Zorbax 300XDB-C18
3.5 mm
150 x 0.3 mm
Mobile Phase:
A: H2O
B: ACN
Gradient:
Gradient 20  80% in 15 min
Flow Rate:
6 mL/min
Injection:
40 nL
Sample:
1. Uracil
2. Acetophenone
3. Propiophenone
4. Butyrophenone
5. Valerophenone
6. Hexanophenone
7. Heptanophenone
400
300
200
100
0
0
2
4
6
8
Time (min)
10
12
14
Rapid Gradient Separations
Method 2
Time (min)
2500
Absorbance (mAU)
2000
A (mL/min)
0
16
0.8
0.25
0.08
24
0.7
0.08
24
1500
1000
500
0
0
10
20
Time (s)
30
B (mL/min)
40
Chromatographic
Hydrophobicity Assay
600
Column:
Luna C18 5 mm
Acetophenoe
50 x 0.3 mm
Mobile Phase: A - 10 mM Ammonium Acetate
Absorbance (mAU @ 254 nm)
B - ACN
500
5-phenyl-1H-tetrazole
Flow Rate:
14 mL/min
Gradient:
2% -> 98% in 45 sec
Injection:
40 nL
Butyrophenone
400
Colchicine
300
Valerophenone
Propiohenone
e
Indole
Theophylline
200
100
0
0
10
20
30
Time (s)
40
50
60
Reduction of Analysis Time
Absorbance
2400
1900
1400
900
400
-100
0
1
2
3
4
5
6
7
Time (min)
Mixing
Conventional
ExpressLC
Gradient
Reset and Re-equilibration
500 ul
30 sec
3 min
2.5 min
1 min
28 sec
6 min
300 nl
2 sec
1.5 min
Ten Assays in 15 Minutes
Retention Time
6000
Absorbance (mAU @ 254 nm)
5500
Peak
% RSD
SD
(sec)
1
0.18
0.04
2
0.16
0.04
3
0.12
0.04
4
0.07
0.03
5
0.04
0.02
6
0.04
0.02
1000
7
0.00
-
500
8
0.06
0.03
5000
4500
4000
3500
3000
2500
2000
1500
0
0
10
20
30
Time (s)
40
50
60
2%  98% B in 45 sec. , hold 15 seconds, pre-run flush 30 sec.
A : 10 mM Ammonium Acetate pH 7.4
14 microL/min; 40 nL inject
B : 10 mM AA/ACN
Chromatographic Hydrophobicity Index
Simple Method Transfer with
Increased Gradient Speed
100
80
Gradient is both
reproducible and
linear
60
40
20
20
25
30
35
40
45
Retention Time (sec)
50
55
Anatomy of a Fast Gradient
A
stir
diffuse
B
tb = 2 td’
g(t)
t-dispersive
tb
tb = 0
td
td=td’+tb
stir & diffuse  mix mobile phase
td’
tb
• The delivered gradient is a function of delay volume and
system blur
• Re-equilibration also requires a similar transition
– Therefore the delay occurs 2 times per analysis
t
System Comparisons
• Vdelay divided by Vcolumn provides a relative comparison of delay
times between different systems- data is normalized for column
diameter
• Delay volumes based on manufacturer specifications
• Data below are for a 5 cm column
Eksigent
Agilent 1100
Aquity
Aquity
Column
diam.
(mm)
Delay
volume
(uL)
Vdelay/
Vcolumn
Vd/Vc,
normalized
to Agilent
2.1
0.3
2.1
2.1
1
0.35
560
150
150
0.099
3.234
0.866
3.821
3%
100%
27%
118%
Compared to Eksigent, next best system is 9X
worse in relative delay.
Analysis of Buspirone Metabolites LC/MS/MS
ExpressLC / API 2000
• Column: Zorbax SB C18, 3.5 mm, 0.30 x 50 mm
• Flow rate: 10 mL/min.
• Gradient: Multi-step
– A: Water / 0.1 % Formic Acid
– B: ACN / 0.06% Formic Acid
Time (min)
%B
0
5
0.1
5
0.6
25
1
60
1.1
98
1.4
98
MS Method
•
MS: AB/MDS Sciex API2000
–
–
Ionization mode: Turbo Spray with 150 mm
OD / 30 mm ID capillary inserted through
spray tip.
Polarity: Positive

Parameters

CUR: 10

GS1: 10

GS2: 20

IS: 5000

TEM: 150

ihe: ON

CAD: 8

DP: 80

EP: 10

CE: 50

CXP: 5
RLM Metabolites
4
6x10
418.1 / 137.9
402.1 / 121.9
Intensity (cps)
402.1 / 137.9
418.1 / 121.9
4
4x10
386.1 / 121.9
4
2x10
0
0.0
0.2
0.4
0.6
0.8
Time (min)
1.0
1.2
1.4
ExpressLC / API 2000 using Zorbax 1.8 micron C18
Time (min)
5
1.5x10
0
20
0.1
35
296/215
1.1
50
180/110
1.3
98
1.9
98
326/291
219/134
262/188
Intensity (cps)
278/186
5
1.0x10
Zorbax 1.8mm C18
4
5.0x10
50 x 0.3 mm
0.0
0.0
%B
0.5
1.0
Time (min.)
1.5
2.0
Long Term Repeatability
Gradient Separations
•
Column: Aquasil C18, 5 mm,
0.320 x 50 mm
– Column temperature: 30 C
Flow rate: 12 mL/min
•
Gradient: 30-95% in 1 minute
– A: Water (0.1% formic acid)
– B: Acetonitrile (0.1% formic acid)
– Injection: 80 nL
– Samples diluted in 70/30
water/acetonitrile
•
Detection: 300 nm @ ~ 5 Hz data
collection rate
•
Sample: compound in TPG5/Water
•
Each sample was injected 250 times
90
Mobile Phase Percent
•
100
80
70
60
50
40
30
20
10
0
0.0
0.2
0.4
0.6
0.8
1.0
Time (min)
1.2
1.4
System Repeatability: 250 Injections
Absorbance @ 300 nm (mAU)
.
400
200
200
100
10
0
0
20
40
60
Time (sec.)
80
100
Injection #
Retention Time: 250 Injections
Retention time RSD = 0.19 %
52.50
52.25
Retention Time (sec)
52.00
51.75
51.50
51.25
51.00
50.75
50.50
0
50
100
150
Injection Number
200
250
Peak Area: 250 Injections
Area RSD = 0.72 %
100
80
Area
60
40
20
0
0
50
100
150
Injection Number
200
250
Peak Width: 250 Injections
1.70
FWHM (sec)
1.65
1.60
1.55
1.50
0
50
100
150
Injection Number
200
250
Test System Linearity on Pfizer Test Mix
Reserpine
Absorbance @ 205 nm(mAU)
800
Dioctyl Phthalate
Column:
Zorbax C18 300SB
3.5 mm
50 x 0.3 mm
Mobile Phase:
A: H2O (0.05% TFA)
B: ACN (0.04% TFA)
Gradient:
Gradient 2%  98% B in 3 min
Flow Rate:
6 mL/min
Injection:
40 nL
600
Aspartame
400
Cortisone
200 µg/mL
200
0
0
1
2
3
Time (min)
4
5
Serial Dilutions
200 mg/mL
100 mg/mL
40 mg/mL
Absorbance @ 205 nm (mAU)
20 mg/mL
8 mg/mL
600
4 mg/mL
2 mg/mL
300
0
1.8
2.0
Time (min.)
Linearity Plots
2600
Aspartame
2400
Cortisone
2200
Reserpine
2000
Dioctyl Phthalate
Peak Area (a.u.)
1800
1600
1400
1200
1000
800
600
400
200
0
0
50
100
150
Concentration (mg/mL
200
250
Serial Dilutions
120
10 mg/mL
Absorbance @ 205 nm (mAU)
5 mg/mL
2 mg/mL
1 mg/mL
80
0.4 mg/mL
Blank
40
0
1.0
1.5
Time (min)
2.0
Linearity
100
Aspartame
40
Linear Fit
35
Cortisone
Linear Fit
30
80
Peak Area (a.u.)
25
Peak Area
60
40
20
20
15
10
5
0
0
-5
0
2
4
6
8
10
0
2
Concentration (mg/mL)
200
8
10
8
10
Dioctyl Phthalate
Linear Fit
Linear Fit
200
150
150
Peak Area (a.u.)
Peak Area (a.u.)
6
Concentration (mg/mL)
250
Reserpine
4
100
50
0
100
50
0
0
2
4
6
Concentration (mg/mL)
8
10
0
2
4
6
Concentration (mg/mL)
ExpressLC / API 4000 @ Foster City
• Applied Biosystems / Sciex Evaluation of ExpressLC-100
– Evaluate Performance for Rapid LC/MS
ExpressLC / API 4000
QC 1st Injection
Plasma (PP)
1st Injection
(Buspirone and Reserpine)
QC Last Injection
(after >900 injections)
Plasma (PP)
(after >900 injections)
ExpressLC / API 4000: Precipitated Plasma Injections
PP-Plasma Injection - Robustness (n=888)
0.600
Area Ratio (A/IS)
0.500
0.400
0.300
+/- (2xStd Dev)
0.200
Avg = 0.444+0.019
(Rel.Std.Dev.= 4.38%)
0.100
0.000
0
100
200
300
400
500
Injection #
600
700
800
900
1000
ExpressLC / API 4000: Precipitated Plasma Injections
PP-Plasma Injection - Robustness (n=888)
0.5
Retention Time (min)
0.49
0.48
IS R.T. = 0.484+0.001 min (+0.22%)
(or 29.03+0.06 sec)
0.47
0.46
Analyte R.T. = 0.430+0.001 min (+0.24%)
(or 25.79+0.06 sec)
0.45
0.44
0.43
0.42
0
100
200
300
400
500
Injection #
600
700
800
900
1000
100fg on Column(0.3x50mm) – Replicates Injections
Summary ht LC
• Microscale HPLC provide significant performance benefits over
conventional systems
– Gradient speed and minimal delay volumes
– Increased porosity (allowing higher flow rates / smaller particles)
– Exceptional resolution
• Multiplexed microscale HPCL can provide high throughput and high
performance
– No compromising data quality
• Amenable to direct method transfer, with adjustments to flow rate
and injection size
• Wide range of stationary phase commercially available