Transcript Welcome To SSI LabAlliance - Scientific Systems | HPLC Pumps

New Systems Based on the VersaGrad
Current Binary Configurations Available
Configuration
Flow and Pressure
Fluid Path
Series-III
5 ml/min @ 6,000 p.s.i.
10 ml/min @ 6,000 p.s.i.
40 ml/min @ 1,500 p.s.i.
Stainless Steel
PEEK
Model-1500 10 ml/min @ 6,000 p.s.i.
Stainless Steel
PEEK
Super
Series-III
Ultra High
Pressure
5 ml/min
(each channel)
12,000 p.s.i.
5 ml/min
(each channel)
18,000 p.s.i.
Stainless Steel Standard
Titanium Upon Request
Stainless Steel Standard
Titanium Upon Request
Detectors Available
 1200 UV/Vis 1 channel programmable
 1200 MWD 8 channel programmable
 1200 Diode Array Full wavelength spectral capability
 1200 Refractive Index
 1200 ELSD
 1200 Fluorescence
Automation and PC Reactors
 1200 Autosampler
 Alias Autosampler
 1200 instant pilot
 Post Column Reaction system
 Fract R1 and R2 Fraction collectors
 Column Oven
Versatility to Meet Any Application
 Custom build a system by line items:
1. Choose a pump
2. Add a detector (s)
3. Add an injector or Autosampler
4. Add the degasser if desired
5. Add a system accessory kit
6. Add the solvent tray
7. Add EZChrom with control options required
8. Add Instant pilot, PCR or Fraction collector if desired
9. Add a column oven
10. Quote as a package price not by line item
Pre Configured Systems
 Standard HPLC Systems



Series III Manual with EZChrom
Series 1500 with Autosampler and EZChrom
QGrad with Autosampler PDA and EZChrom
 Ultra High Pressure Systems


Series III UHP with Autosampler and EZChrom
Series UHP with Autosampler and EZChrom
 Application Based Systems



Advanced Protein Liquid Chromatography System APLC
Proteome UFFLC System
PCR system for Carbamates
Binary & Quaternary HPLC Systems
Features

Single and dual piston models available

Pressures up to 6,000psi SS 5,000psi PEEK

Manual or automated injection
Match the performance to the application flows
from 0.001ml/min to 100ml/min


Full range of detectors

EZChrom control and data Reduction

Elegance at an affordable price
VersaGrad Series III Manual
 Entry level system with
Shown with optional Instant pilot
EZChrom control & data
includes:
1. VersaGrad Series III
2. Degasser & solvent tray
3. EZChrom on preloaded
computer
4. Manual injector
5. Standard UV/Vis
detector
VersaGrad Series III System
with Automation Option
 Shown with optional
autosampler
 Add other options at any
time to upgrade a system
 Modularity allows
reconfiguration for
many applications
VersaGrad 1500 Binary System
 High end automated
Binary system
containing:
1. VersaGrad Model 1500
2. 1200 Autosampler
3. 1200 UV detector
4. 1200 Degasser
5. Solvent tray
6. PC with EZChrom pre
loaded
QGrad Quaternary Gradient System
 Quaternary high end
DAD based system with:
1. QGrad Pump
2. 1200 Diode Array
Detector
3. 1200 Autosampler
4. EZChrome Elite
Workstation with
DAD Pre installed
5. 1200 Degasser &
Solvent tray
Ultra High Pressure (UHP) Systems
Features

Single and dual piston models available
Pressures up to 12,000psi Single piston 18,000psi
dual piston


Automated injection

Full range of detectors

EZChrom control and data

Ultimate Performance at an affordable price
Series III HP Binary with EZChrom
 12,000psi entry level
UHP system with:
1. VersaGrad Series III
UHP, 12,000psi pump
2. Alias Autosampler
3. 1200 UV detector
4. 1200 Degasser
5. Solvent tray
6. PC with EZChrom pre
loaded
UHP Binary System
 18,000 psi ultimate UHP
system including:
1. VersaGrad UHP dual
piston pump
2. Alias Autosampler
3. 1200 UV detector
4. 1200 Degasser
5. Solvent tray
6. PC with EZChrom pre
loaded
Substantial Savings
Assay
Analysis
Time
Flow
ml/min
Test per
Year
Cost
per
test
Cost per
1000
Samples
Solvent Savings per
cost per
1000
1000
samples
Conventional
Isocratic
6
1
19200
$9.03
$9,025.63
$1,005
UHP Isocratic
1
1
115200
$1.50
$1,504.27
$167
Conventional
Gradient
30
1
3840
$45.13 $45,128.17
UHP Gradient
3.5
1
32914
$5.26
$5,264.95
$7,521.36
$5,024
$586
$39,863.21
The Ultra High Pressure Binary Systems
SAVINGS in METHOD DEVELOPMENT
 6-10 Times faster scouting runs
 System equilibration time is reduced
 Solvent changes are faster
 You can do in a morning what normally takes days
Retention Time
Detector 2-254nm
tmix dig 1ml 5 as
Detector 2-254nm
tmix dig 1ml 5 as
Retention Time
Retention Time
Detector 2-254nm
tmix dig 1ml 5 as
Detector 2-254nm
tmix dig 1ml 5 as
Retention Time
Detector 2-254nm
tmix dig 1ml 5 as
Detector 2-254nm
tmix dig 1ml 5 as
Detector 2-254nm
tmix dig 1ml 5 as
Detector 2-254nm
tmix dig 1ml 5 as
180
160
140
140
120
120
100
100
0
.2
50
0.
2508
0.
0
.2
2508
58
17
0. 252
160
80
0
.3
5
0.
0.
3508
3508
0
.3
50
18
7
0. 352
80
60
0
0.
0.
0.2
.21
2117
2117
11
27
5
0. 212
m AU
180 Retention Time
Detector 2-254nm
tmix dig 1ml 5 as
60
40
40
20
20
0
0
-20
0.000
-20
0.025
0.050
0.075
0.100
0.125
0.150
0.175
0.200
0.225
0.250
Minutes
0.275
0.300
0.325
0.350
0.375
0.400
0.425
0.450
0.475
0.500
m AU
Detector 2-254nm
tmix dig 1ml 5 as
0.
03108
01
.3
.3
71 1 7
0.1
3117
0. 312
UHP Reproducibility
Detector 2-254nm
tmix dig 1ml 5 as
Detector 2-254nm
tmix dig 1ml 5 as
4 Benzoate Esters
Column: 1.5micron
particle size 2.1mm X
10 cm C18 100
angstrom
Mobile phase: A Pump
Water B Bump
Acetonitrile 35%A
65%B
Flow: 1ml/min
Pressure: 12,500psi
Detector: UV at 254nm
UHP Reproducibility – Previous Data
4 component STD
RT Peak 1
RT Peak 2
RT Peak 3
RT Peak 4
Area Pk 1
Area Pk 2
Area Pk 3
Area Pk 4
0.212
0.252
0.312
0.352
29167
60869
159625
50108
0.213
0.253
0.313
0.352
28280
57873
155763
49578
0.212
0.252
0.312
0.352
27739
60399
158702
52029
0.212
0.251
0.312
0.351
27493
60128
158598
52144
0.212
0.251
0.312
0.351
28576
61495
160380
51906
0.212
0.251
0.311
0.351
27158
55929
156828
50080
0.212
0.252
0.312
0.352
27218
59682
156760
50817
0.212
0.252
0.312
0.352
26186
59227
157492
51729
RT Peak 1
RT Peak 2
RT Peak 3
RT Peak 4
Area Pk 1
Area Pk 2
Area Pk 3
Area Pk 4
Mean
0.212125
0.25175
0.312
0.351625
27727.125
59450.25
158018.5
51048.875
Standard Error
0.000125
0.00025
0.000188982
0.000182981
330.3848642
634.4822622
556.2327365
363.4748192
Median
0.212
0.252
0.312
0.352
Mode
0.212
0.252
0.312
0.352
0.000353553
0.000707107
0.000534522
0.000517549
1.25E-07
5E-07
2.85714E-07
8
-0.228571429
3.5
Standard Deviation
Sample Variance
Kurtosis
Skewness
27616
#N/A
59905
#N/A
158045
#N/A
51273
#N/A
934.4695115
1794.586841
1573.26376
1028.062038
2.67857E-07
873233.2679
3220541.929
2475158.857
1056911.554
-2.24
-0.076824027
1.132388831
-1.062808516
-1.995477475
2.828427125
0.404061018
0
-0.644061189
-0.052175524
-1.153921251
0.123682142
-0.304642966
Range
0.001
0.002
0.002
0.001
2981
5566
4617
2566
Minimum
0.212
0.251
0.311
0.351
26186
55929
155763
49578
Maximum
0.213
0.253
0.313
0.352
29167
61495
160380
52144
Sum
1.697
2.014
2.496
2.813
221817
475602
1264148
408391
8
8
8
8
8
8
8
8
0.000437435
0.00087487
0.00066134
0.000640339
1156.17554
2220.358592
1946.525867
1271.973207
0.17%
0.28%
0.17%
0.15%
3.37%
3.02%
1.00%
2.01%
Count
Confidence
Level(99.0%)
RSD %
Gradient Reproducibility UHP of Flavors –
Lemon Oils
Column: 1.5micron particle size
2.1mm X 10 cm C18 100
angstrom
Mobile phase: A Pump Water B
Bump Acetonitrile 30%B to
90%B in 3min
Flow: 1ml/min
Pressure: 9,000-12,500psi
Detector: UV at 214nm
Gradient Reproducibility UHP of Flavors –
Previous Data
2.948
2.956
2.959
2.945
2.959
2.942
2.947
3.25
3.26
3.262
3.248
3.262
3.243
3.252
RT Peak 1
Mean
Standard Error
Median
Mode
Standard Deviation
Sample Variance
Kurtosis
Skewness
Range
Minimum
Maximum
Sum
Count
Confidence Level(95.0%)
723758
789850
768130
757586
750768
751356
761623
2851392
3096385
3035568
3027459
2960960
2973226
3043120
RT Peak 2
Area Pk 1
Area Pk 2
2.9495
0.002665923
2.9475
2.959
0.007540368
5.68571E-05
-1.73539052
0.266571919
0.019
2.94
2.959
23.596
8
0.006303905
3.252625
0.0027577
3.251
3.262
0.007799954
6.08393E-05
-1.840542353
0.159965717
0.019
3.243
3.262
26.021
8
0.006520925
757223.375
6553.700609
756151
#N/A
18536.66457
343607933.4
2.083630001
-0.055388778
66092
723758
789850
6057787
8
15497.03939
2989253.875
27404.91773
3000342.5
#N/A
77512.81267
6008236127
0.085289083
-0.556248637
244993
2851392
3096385
23914031
8
64802.33306
0.26%
0.24%
2.45%
2.59%
UHP Binary Pump Retention Time Study at HPLC Pressure
Methyl
Benzoate
Ethyl
Benzoate
1.189
1.353
1.189
1.353
1.187
1.35
1.187
1.35
1.188
1.351
1.188
1.351
1.188
1.352
1.188
1.352
1.188
1.352
1.188
1.352
Zorbax
C18 5um
4.6 X 15
Solvent
H20 35%
CH3CN
65%
Flow
1.5ml/mi
Detection
UV
Pressure
1268psi
Column
254nm
New UHP Binary Pump Retention Time Study
Methyl Benzoate
Mean
Standard Error
Ethyl Benzoate
1.188 Mean
0.00021082 Standard Error
1.3516
0.00034
Median
1.188 Median
1.352
Mode
1.188 Mode
1.352
Standard Deviation
0.00066667 Standard Deviation
0.001075
Sample Variance
4.4444E-07 Sample Variance
1.16E-06
Kurtosis
0.08035714 Kurtosis
-0.88203
Skewness
-6.2444E-13 Skewness
-0.32201
Range
0.002 Range
Minimum
1.187 Minimum
1.35
Maximum
1.189 Maximum
1.353
Sum
11.88 Sum
Count
Relative Std Dev
10 Count
0.06%
0.003
13.516
10
0.08%
Application Specific Systems
Features

Tailored to a given application
18,000psi pumps advantages are exploited in the
UFFLC


EZChrom control and data
UFFLC of the Proteome
 Comes standard with
1. VersaGrad UHP
2.
3.
4.
5.
6.
7.
18,000psi pump
Alias Autosampler
1200 UV detector
1200 Degasser
Solvent tray
PC with EZChrom pre
loaded
Fract R1 Fraction
collector
UFFLC 2-Minute Hemoglobin Rapid Screen
SAMPLE: Hemoglobin FASC standard
COLUMN: PolyCAT A, 100x2.1-mm; 3-µm, 1500-Å FLOW: 1.2 ml/min A415 Pressure: 6938 psi
GRADIENT: 0-0.7’: 18-55% B; 0.7-1.1’: 55-85% B; 1.1-1.2’: 85-100% B; 1.2-1.3’: 100% B; 1.4’: Initial
A) 40 mM Bis-Tris + 2 mM KCN, pH 6.5 B) 40 mM Bis-Tris + 2 mM KCN + 200 mM NaCl, pH 6.8
A
80
S
mAU
60
C
40
F
A1c
A3
20
A2
0
0.0
0.2
0.4
0.6
0.8
1.0
Minutes
1.2
1.4
1.6
1.8
2.0
Mixed-bed ion-exchange HPLC of yeast lysate: Effect of flow rate
COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å
GRADIENT: 0 – 0.8 M NaCl in 20 mM HEPES, pH 7.0 A280
4
ml/min
60
mAU
1
ml/min
40
20
0
0
5
10
15
20
25
Minutes
30
35
40
45
Mixed-bed ion-exchange HPLC of yeast lysate: Effect of flow rate
A280
COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A280
GRADIENT: (1 ml/min) 0-30’: 0-50 %B; 30-40’: 50-100 %B; (4 ml/min) 0-2’: 0-8 %B; 2-7’: 8-100
%B
A) 20 mM HEPES, pH 7.0; B) 20 mM HEPES + 0.8 M NaCl, pH
7.0
2
4
Minutes
6
4 ml/min (4709
psi)
(X-axis
5x) 8 expanded
1 ml/min (1051 psi)
0
10
20
Minutes
30
40
Mixed-bed ion-exchange HPLC of E. coli lysate: Fast flow
COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A280 4 ml/min 4709 psi
GRADIENT: 0-2’: 0-8 %B; 2-5.5’: 8-95 %B; 5.5-6’: 95-100 %B
A) 20 mM HEPES, pH 7.0 B) 20 mM HEPES + 0.8 M NaCl, pH 7.0
25
20
mAU
15
10
5
0
0
1
2
3
4
Minutes
5
6
7
PROTEOMICS BY 2-DIMENSIONAL LC/ MS/MS
MudPIT: Multidimensional Protein Identification Technology
ADVANTAGES OVER 2-D
ELECTROPHOPHORESIS
1) Greater dynamic range:
Most abundant protein
detected
Least abundant protein
detected
2) Works better for
hydrophobic and very basic
proteins
3) Handling and automation
easier
- from Link et al., Nat.
Biotechnol.
17 (1999) 676-682 -
Acidic proteins;
Basic proteins;
Use anion-exchange Use cation-exchange
1200
# of proteins
1000
From: H. Wang et al.,
J. Proteome Res. 5
(2006) 361
800
600
400
200
0
4
5
6
7
8
9
10
11
12
13
pI value
pI Distribution of the Predicted Mouse Proteome
PROTEINS IDENTIFIED IN LOW-MOL.-WT. MOUSE SERUM
Method: SCX (96 fractions)-RPC
4000
Ref: B.L. Hood et al., J.
Am. Soc.
Mass Spectrom. 16 (2005)
1221
NOT ACCEPTABLE
3500
One Hit Wonders.
5000
Number of Proteins
4500
4356
3000
2500
ACCEPTABLE
Identifying proteins via
2 peptides or more
2000
1500
1115
1000
300
500
103
34
28
20
16
5
6
7
8
31
0
1
2
3
4
Number of Unique Peptides
>8
Fractionating Intact Proteins Increases
Detection
of Peptides from Proteins of Low Abundance
Low-abundance Protein
X is 0.1% of total protein
1
2
3
4
5
6
7
8
9
Now Protein X is 1.0 % of total protein in Fraction #6. After digestion, its
peptides will be 10x higher a percentage of the total in that fraction than would
have been true in a digest of the unfractionated mixture. That greatly increases the
chances of identifying Protein X through 2-3 of its fragments rather than just one.
10
Top-Down UHP Proteomics
Intact Proteins
Mixed bed Ion exchange
Fractions
MALDI TOF/TOF
Confirmation of Selected peaks
Tryptic Digestion
SCX
This step could be eliminated
due to pre-separations of Intact proteins
RPC
MS/MS
Informatics
Identify Proteins
Histone H4 Acetylation & Methylation Variants
COLUMN: PolyCAT A (104CT0315; HILIC mode)
VOLTAGE
0 Acetyl
The most minor
variants can be
the most
critical
METHYLATION
H2A
1 Acetyl
INTERPHASE
2 Acetyl
27.0
32.0
37.0
42.0
47.0
52.0
57.0
62.0
VOLTAGE
0-Acetyl
1-Acetyl
H2A
MITOSIS
METHYLATION
4-Acetyl
0 20
2-Acetyl
3-Acetyl
30
40
50
TIME (Min)
(courtesy James Pesavento - U. of Ill.)
60
70
UHP UFFLC
 Higher throughput than any other system
 Allows use of standard columns and UHP columns
 Allows normal chromatography and small scale prep
as well as UHP &UFFLC
 Highest pressure and flow limits on the market today.
Why limit yourself
 FPLC , is now obsolete with the speed and resolution
of UFFLC
 Top down proteomics is now much easier
PCR System
 Comes standard with
1. VersaGrad Series III
2. 1200 Fluorescence
3.
4.
5.
6.
7.
detector or UV
1200 Degasser
Solvent tray
PC with EZChrom pre
loaded
Sensivate post column
reaction system
Manual injector
Sensivate Post-Column Reactor
Applications
 Carbamates by OPA
 Glyphosates by OPA
 Aflatoxin by Iodine
 Amino Acids by OPA
 Amino Acids by
Ninhydrin
PCR System Applications
Application
Reactor 1
Temp.
Reactor 2
Carbamate
NaOH
100o C
OPA
Glyphosate
NaOCl
40o C
OPA
Aflatoxins
Iodine
40o C
(not used)
Carbohydrates
NaOH
40o C
(not used)
Lipids
Phenacyl
100o C
(not used)
Amino Acids Ninhydrin
Ninhydrin
140o C
(not used)
Amino Acids - OPA
NaOCl
40o C
OPA
Amino Acids - Primary
OPA
30o C
(not used)
State of the Art Separations
Aflatoxin
Glyphosate
Carbamate
Amino Acids
APLC System
 Going beyond FPLC
1. VersaGrad Series III
2. 1200 UV Detector
3. 1200 Degasser
4. Solvent tray
5. PC with EZChrom pre
loaded
6. Fract R1 Fraction
collector
Mixed-bed ion-exchange HPLC of yeast lysate:
A280
COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A280
GRADIENT: (1 ml/min) 0-30’: 0-50 %B; 30-40’: 50-100 %B; A) 20 mM HEPES, pH 7.0; B) 20 mM
HEPES + 0.8 M NaCl, pH 7.0
1 ml/min (1051 psi)
0
10
20
Minutes
30
40
Anion-Exchange of Ovalbumin Phosphorylation
Variants
COLUMN: PolyWAX LP (#104WX0510) SAMPLE: Sigma Grade VI (99%)
GRADIENT: 10 mM K-PO4, pH 7.0; 60-300 mM NaCl in 20’
160000
120000
A220
phosphorylation
variants
80000
40000
0
0.0
5.0
10.0
TIME (Min)
15.0
20.0
Cation-Exchange HPLC of BMP-14 (Bone Morphogenetic
Protein 14)
500
500
400
400
300
300
200
200
100
100
mVolts
mVolts
COLUMN: PolyCAT A, 100x4.6-mm; 3-µm, 1000-Å (item# 104CT0310; ser# A2562C)
MOBILE PHASE: A) 20 mM K-PO4, pH 6.0, with 20% ACN; B) Same + 0.6 M NaCl
GRADIENT: 20’ linear, 0-100% B, then 5’ at 100% B
1.0 ml/min
A220
SAMPLE: 25 µg BMP-14 in 20 µl of (Mobile Phase A:10 mM HCl = 2:1)
0
0
0
2
4
6
8
10
12
Minutes
14
16
18
20
22
24
BMP-14: Blowup to show deamidation variants
14
14
3
12
10
12
10
4
mVolts
8
mVolts
8
2
6
6
1
4
4
2
2
0
0
9.0
9.5
10.0
10.5
11.0
11.5
Minutes
12.0
12.5
13.0
13.5
14.0
BMP-14: More gradual gradient to improve separation of
variants; resembles preceding growth factor’s profile
QUESTION: Do all recombinant growth factors contain ~ 8%
Met[O] or mismatched -S-S- variants (@ cysteine knot motif)?
35
35
Another new
shoulder?
30
25
30
25
New shoulder;
deamidation variant?
20
mVolts
mVolts
20
15
15
10
10
5
5
0
0
0
2
4
6
8
10
12
Minutes
14
16
18
20
22
24
ERLIC of Phosphopeptides with Volatile Solvents
Column: PolyWAX LP (104WX0503)
Flow rate: 1 ml/min. Detection: A270 (blue) or A260 (red)
Gradient: 0-5’: 0%B; 5-45’: 0-100% B; 45-50’: 100%B
MP A: 20 mM NH4-Formate, pH 2.2, with
70% ACN
MP B: [TOP] 100 mM NH4-Formate, pH 2.2, w. 64% ACN; [BOTTOM] 20 mM NH4-Formate, pH 2.2, w.
10% ACN
________________KEY_________________
Which to choose:  [salt] or  [ACN]?
A: SLYSSSPGGAYVTR (Vimentin(51-64))
B: SVNFSLTPNEIK (MAP 1B(1271-1282))
C: WWGSGPSGSGGSGGGK (#1644)
A+P:
SLYSSS(p)PGGAYVTR
B+P:
SVNFSLT(p)PNEIK
C+P: WWGSGPSGSGGS(p)GGGK (#1645)
D+P:
GGAAGLGY(p)LGK (#1876)
200
A, B & C
150
D+P
A280
A+P
100
B+P
C+P
20  100 mM salt
260 nm
270 nm
50
70  10% ACN
0
0
5
10
15
20
25
Minutes
30
35
40
45
50
SPE OF PEPTIDES: Unexpectedly Selective
Extraction by Carbon and C18 NuTips
Urea
(1
M)
= Transferrin tryptic digest (30 µg)
75
= Peptides extracted/released by NT200C18 from 500 µg digest
= "
"
= "
(second set of releasing washes)
50
Millivolts
" NT200CAR "
"
25
0
-25
0.0
5.0
10.0
15.0
20.0
Minutes
25.0
30.0
35.0
Column: PolyHYDROXYETHYL A (item# 204HY0503), HILIC mode. Detection: A220
Gradient: 40’ linear, 0-70% B. A) 15 mM TEAP, pH 2.7, with 85% ACN
B) "
" 10
"
40.0
Comparison of ERLIC vs. HILIC of Amino Acids
MOBILE PHASE: 10 mM TEAP, pH 4.0, w. 65% ACN; 1 ml/min
L
M
I V
P
pGLU
+
Q N
A T
ABSORBANCE (215 nm)
G
A
T
E
E
D
H
D
PolyHYDROXYETHYL A
(neutral)R K
Cya
D
Q
+
S
H
T
N
I E
+V A
G
M
P
R
L
0
PolyWAX LP (basic)
H
K
S
N
V
pGlu
R
Cya
pGLU
P
Cya
10
20
TIME (Min)
TSKgel Amide-80
(acidic)
K
30
40
Systems For Every Application
 From HPLC to UHP in pressure
 From UFFLC to Prep in flow
 Versatile selection of detectors
 Full automation
 Data to meet your labs requirements
 Simplicity and functionality at an affordable price
LabAlliance has the system your customer needs