UHP & UFFLC ChromatographyUHP & UFFLC Chromatography
High Performance Separations to High Performance Separations to 18,000 p.s.i. Saving You Time and 18,000 p.s.i. Saving You Time and
SolventSolvent
UHP Runs: 3-micron; 4.6 X150 vs. UHP Runs: 3-micron; 4.6 X150 vs. 1.8-micron; 2.1X 501.8-micron; 2.1X 50
Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
mA
U
0
20
40
60
80
100
120
140
160
mA
U
0
20
40
60
80
100
120
140
160Detector 1-254nmThermo150x4.6mm3um
Detector 1-254nmHypersil Gold 2_1 x 50
Ultra Fast Flow Liquid Ultra Fast Flow Liquid ChromatographyChromatography
UFFLCUFFLC
A New Era of UHP A New Era of UHP
UFFLC Scaling Factors for Column Diameters
Column Relative UFFLC
Diameter Normal Flow New Flow
mm Rate Rate
4.6 1.32 5.28
4 1 4
3.9 0.95 3.8
3 0.56 2.24
2.1 0.28 1.12
2 0.25 1
1 0.06 0.24
UHP, UFFLCUHP, UFFLC
18,000 p.s.i. Pump18,000 p.s.i. PumpAllows UHP UFFLC and HPLC in one Allows UHP UFFLC and HPLC in one
System System
15,000 p.s.i. Optimized 15,000 p.s.i. Optimized RecompressionRecompression
SpecificationsSpecifications
Flow Flow 0.001 – 5.000 ml/min0.001 – 5.000 ml/min
PressurePressure 18,000 p.s.i.18,000 p.s.i.
Pulsation >3000psiPulsation >3000psi 1.5%1.5%
Accuracy Accuracy < < – 1ml/min 10,000psi Methanol1ml/min 10,000psi Methanol
Precision Precision ++ 0.5% 0.5%– 30 sec average flow30 sec average flow
Pump Design InnovationsPump Design Innovations
True 18,000 p.s.i. pressure capabilityTrue 18,000 p.s.i. pressure capability
0.001 – 5.000 ml/min flow range0.001 – 5.000 ml/min flow range
EZChrom & Xcalibur driver availableEZChrom & Xcalibur driver available
Low volume UHP mixerLow volume UHP mixer
Maintaining HPLC capabilityMaintaining HPLC capability
Completing the systemCompleting the system
DetectorsDetectors
AutosamplerAutosampler
DataData
DegasserDegasser
DetectorsDetectors
UV, Diode Array, FluorescenceUV, Diode Array, Fluorescence
20-80 Hz data acquisition rate for fast 20-80 Hz data acquisition rate for fast chromatographychromatography
Standard and micro volume flow cellsStandard and micro volume flow cells
Full digital control and data acquisitionFull digital control and data acquisition
UHP AutosamplerUHP Autosampler
Allows injections to 18,000 p.s.i.Allows injections to 18,000 p.s.i.
Fully programmableFully programmable
Full or partial loop modeFull or partial loop mode
Available coolingAvailable cooling
Available sample prepAvailable sample prep
UHP AutosamplerUHP Autosampler
Sample capacity :2*96 well or 2*384 or 2*48 Sample capacity :2*96 well or 2*384 or 2*48 standard vials or 2* 12 10ml prep vialsstandard vials or 2* 12 10ml prep vials
Injection cycle time: <15 secInjection cycle time: <15 sec Injection: full loop, partial loop and µl pickupInjection: full loop, partial loop and µl pickup RSD<=0,3% at full loopRSD<=0,3% at full loop Carry-over Carry-over <0,05 %<0,05 %
Use Only What You Inject With Use Only What You Inject With µL Pick-up µL Pick-up
Zero sample lossFlush with transport solvent
inject
UHP: Transport liquid is in wash port
available
How µL Pick-up Works 1How µL Pick-up Works 1Pick-up sample
load
Inj.vol: (loop vol – 3x needle volume) / 2
How µL Pick-up Works 2How µL Pick-up Works 2Transport sample to the center of the loop
load
How µL Pick-up Works 3How µL Pick-up Works 3Switch valve to Inject
inject
Data and ControlData and Control
EZ Chrom & EZ Start DriversEZ Chrom & EZ Start Drivers
Xcalibur driversXcalibur drivers
Developing Analyst driverDeveloping Analyst driver
Data and ControlData and Control
EZ Start or EZ Chrom EliteEZ Start or EZ Chrom Elite
Full control and data analysisFull control and data analysis
From single instrument to enterprise based From single instrument to enterprise based systemsystem
CFR 21 11 is availableCFR 21 11 is available
Based on the Agilent/Scientific Software Based on the Agilent/Scientific Software industry standard platformindustry standard platform
TRUE Analysis TimeTRUE Analysis Time
UFFLC UHP ReproducibilityUFFLC UHP Reproducibility4 Benzoate Esters4 Benzoate EstersColumn: 1.5micron particle size Column: 1.5micron particle size 2.1mm X 2.1mm X
10 cm C18 100 angstrom10 cm C18 100 angstromMobile phase: A Pump Water B Bump Mobile phase: A Pump Water B Bump
Acetonitrile 35%A 65%BAcetonitrile 35%A 65%BFlow: Flow: 1ml/min1ml/minPressure: Pressure: 12,500psi12,500psiDetector: UV at 254nmDetector: UV at 254nm
Minutes0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.275 0.300 0.325 0.350 0.375 0.400 0.425 0.450 0.475 0.500
mAU
-20
0
20
40
60
80
100
120
140
160
180
mAU
-20
0
20
40
60
80
100
120
140
160
180
0.212
0.252
0.312
0.352
0.2117
0.2508
0.3108
0.3508
0.2125
0.2517
0.3117
0.3517
0.2117
0.2508
0.3117
0.3508
0.2117
0.2508
0.3117
0.3508
Detector 2-254nmtmix dig 1ml 5 as
Retention Time
Detector 2-254nmtmix dig 1ml 5 as
Retention Time
Detector 2-254nmtmix dig 1ml 5 as
Retention Time
Detector 2-254nmtmix dig 1ml 5 as
Retention Time
Detector 2-254nmtmix dig 1ml 5 as
Retention Time
Detector 2-254nmtmix dig 1ml 5 as
Detector 2-254nmtmix dig 1ml 5 as
Detector 2-254nmtmix dig 1ml 5 as
Detector 2-254nmtmix dig 1ml 5 as
Detector 2-254nmtmix dig 1ml 5 as
Detector 2-254nmtmix dig 1ml 5 as
Detector 2-254nmtmix dig 1ml 5 as
.3508min
Isocratic UFFLC UHP ReproducibilityIsocratic UFFLC UHP Reproducibility4 component STD
RT Peak 1 RT Peak 2 RT Peak 3 RT Peak 4 Area Pk 1
0.212 0.252 0.312 0.352 29167
0.213 0.253 0.313 0.352 28280
0.212 0.252 0.312 0.352 27739
0.212 0.251 0.312 0.351 27493
0.212 0.251 0.312 0.351 28576
0.212 0.251 0.311 0.351 27158
0.212 0.252 0.312 0.352 27218
0.212 0.252 0.312 0.352 26186
RT Peak 1 RT Peak 2 RT Peak 3 RT Peak 4
Mean 0.212125 0.25175 0.312 0.351625
Standard Error 0.000125 0.00025 0.000188982 0.000182981
Median 0.212 0.252 0.312 0.352
Mode 0.212 0.252 0.312 0.352
Standard Deviation 0.000353553 0.000707107 0.000534522 0.000517549
Sample Variance 1.25E-07 5E-07 2.85714E-07 2.67857E-07
K urtosis 8 -0.228571429 3.5 -2.24
Skewness 2.828427125 0.404061018 0 -0.644061189
Range 0.001 0.002 0.002 0.001
Minimum 0.212 0.251 0.311 0.351
Maximum 0.213 0.253 0.313 0.352
Sum 1.697 2.014 2.496 2.813
Count 8 8 8 8 Confidence Level(99.0%) 0.000437435 0.00087487 0.00066134 0.000640339
RSD % 0.17% 0.28% 0.17% 0.15%
6X Faster=Real Savings6X Faster=Real Savings
1 run/min vs 1 run every 6 min on normal 1 run/min vs 1 run every 6 min on normal column = 6X the capacity of conventional column = 6X the capacity of conventional HPLCHPLC
1ml/sample for UHP 6ml/sample for 1ml/sample for UHP 6ml/sample for conventional HPLC 1/6conventional HPLC 1/6thth the cost/analysis for the cost/analysis for solvents and solvent disposalsolvents and solvent disposal
Gradient Reproducibility UFFLC UHP Gradient Reproducibility UFFLC UHP of Flavorsof Flavors
Minutes
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6
mA
U
0
200
400
600
800
1000
1200
1400
mA
U
0
200
400
600
800
1000
1200
1400
0.24
1
0.38
8
0.44
80.
494
0.58
4
0.69
5
1.84
31.
886
2.05
2
2.15
5
2.27
9
2.34
7
2.51
2
2.88
2
2.95
0
3.04
2
3.13
03.
171
3.25
3
3.37
93.
424
Detector 2-214nmflavors
Retention Time
Detector 2-214nmflavors
Detector 2-214nmflavors
Detector 2-214nmflavors
Detector 2-214nmflavors
Detector 2-214nmflavors
Detector 2-214nmflavors
Detector 2-214nmflavorsLemon OilsLemon Oils
Column: 1.5micron particle size Column: 1.5micron particle size 2.1mm X 10 cm C18 100 2.1mm X 10 cm C18 100 angstromangstrom
Mobile phase: A Pump Water B Bump Acetonitrile 30%B Mobile phase: A Pump Water B Bump Acetonitrile 30%B to 90%B in 3min to 90%B in 3min
Flow: Flow: 1ml/min1ml/min
Pressure: Pressure: 9,000-12,500psi9,000-12,500psi
Detector: UV at 214nmDetector: UV at 214nm
3.424min
Gradient Reproducibility UFFLC UHP Gradient Reproducibility UFFLC UHP FlavorsFlavors
2.948 3.252.956 3.262.959 3.2622.945 3.2482.959 3.2622.942 3.2432.947 3.252
RT Peak 1 RT Peak 2
Mean 2.9495 3.252625Standard Error 0.002665923 0.0027577Median 2.9475 3.251Mode 2.959 3.262Standard Deviation 0.007540368 0.007799954Sample Variance 5.68571E-05 6.08393E-05Kurtosis -1.73539052 -1.840542353Skewness 0.266571919 0.159965717Range 0.019 0.019Minimum 2.94 3.243Maximum 2.959 3.262Sum 23.596 26.021Count 8 8Confidence Level(95.0%) 0.006303905 0.006520925
0.26% 0.24%
Savings in Gradient UFFLC UHPSavings in Gradient UFFLC UHP
3.5 min per run UHP vs. 30min per run 3.5 min per run UHP vs. 30min per run Conventional HPLC = 10 X the throughput of Conventional HPLC = 10 X the throughput of a conventional systema conventional system
3.5ml per assay vs. 30ml/assay in 3.5ml per assay vs. 30ml/assay in conventional HPLC reduces the cost per conventional HPLC reduces the cost per assay substantiallyassay substantially
Substantial SavingsSubstantial SavingsAssay Analysis
TimeFlow ml/min
Test per Year
Cost per test
Cost per 1000 Samples
Solvent cost per 1000
Savings per 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 $7,521.36
Conventional Gradient 30 1 3840 $45.13 $45,128.17 $5,024
UHP Gradient 3.5 1 32914.286 $5.26 $5,264.95 $586 $39,863.21
UFFLC Savings in Method UFFLC Savings in Method DevelopmentDevelopment
6-10 Times faster scouting runs6-10 Times faster scouting runs
System equilibration time is reducedSystem equilibration time is reduced
Solvent changes are fasterSolvent changes are faster
You can do in a morning what normally takes You can do in a morning what normally takes days days
Minutes0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
mA
U
0
20
40
60
80
UFFLC 2-Minute Clinical Hemoglobin Rapid Screen SAMPLE: Hemoglobin FASC standardCOLUMN: PolyCAT A, 100x2.1-mm; 3-µm, 1500-Å FLOW: 1.2 ml/min A415 Pressure: 6938 psiGRADIENT: 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’: InitialA) 40 mM Bis-Tris + 2 mM KCN, pH 6.5 B) 40 mM Bis-Tris + 2 mM KCN + 200 mM NaCl, pH 6.8
A1c
F
A
A2
S
C
A3
Minutes0 5 10 15 20 25 30 35 40 45
mA
U
0
20
40
60
Top Down Proteomics UFFLC of yeast lysate: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
1 ml/min
4 ml/min
Top Down Proteomics UFFLC of yeast lysate:
COLUMN: PolyCAT A + PolyWAX
Minutes2 4 6 8
Minutes0 10 20 30 40
A28
0
1 ml/min (1051 psi)
4 ml/min (4709 psi)(X-axis expanded 5x)
Minutes0 1 2 3 4 5 6 7
mA
U
0
5
10
15
20
25
Top Down Proteomics UFFLC of E. coli lysate:
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 %BA) 20 mM HEPES, pH 7.0 B) 20 mM HEPES + 0.8 M NaCl, pH 7.0
PROTEOMICS TODAY BY 2-DIMENSIONAL LC/ MS/MS
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 -
MudPIT: Multidimensional Protein Identification Technology
Typically identifies 2.5-3 peptides per protein; rugged method.
Good where sample size is limited
4 5 6 7 8 9 10 11 12 130
200
400
600
800
1000
1200
# o
f p
rote
ins
pI value
pI Distribution of the Predicted Mouse Proteome
From: H. Wang et al.,
J. Proteome Res. 5
(2006) 361
Acidic proteins; Basic proteins;Use anion-exchange Use cation-exchange
Complexity of the Proteome Complicate Bottom UP Analysis
4356
1115
300103 34 28 20 16 31
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
1 2 3 4 5 6 7 8 >8
Number of Unique Peptides
Num
ber
of P
rote
ins
PROTEINS IDENTIFIED IN LOW-MOL.-WT. MOUSE SERUM
Method: SCX (96 fractions)-RPC Shows Bottom Up LimitationRef: B.L. Hood et al., J. Am. Soc.Mass Spectrom. 16 (2005) 1221
NOT ACCEPTABLE One Hit Wonders.
ACCEPTABLEIdentifying proteins via2 peptides or more
UFFLC of Intact Proteins Solves the Problem & Increases Detection of Low Abundance Proteins
Low-abundance Protein X is 0.1% of total protein
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.
1 2 3 4 5 6 7 8 9 10
The Future Of Proteomics Top-Down The Future Of Proteomics Top-Down UFFLCUFFLC
M A L D I T O F /T O FC o n firm atio n o f S e le c te d pe a ks
In fo rm a ticsId e n tify P ro te ins
M S /M S
R P C
S C XT h is s te p cou ld be e lim ina ted
d u e to p re -se pa ra tio ns o f In ta c t p ro te ins
T ryp tic D ig e s tion
M ixe d be d Io n e xch an geF ra c tio ns
In ta ct P ro te ins
See The Whole Picture Histone H4 Acetylation & Methylation Variants
27.0 32.0 37.0 42.0 47.0 52.0 57.0 62.0
VO
LT
AG
E0 Acetyl
1 Acetyl
2 Acetyl
H2AMETHYLATION
TIME (Min)20 30 40 50 60 70
VO
LT
AG
E
0
4-Acetyl3-Acetyl
2-Acetyl
1-Acetyl
0-Acetyl
H2A
METHYLATION
(courtesy James Pesavento - U. of Ill.)
MITOSIS
INTERPHASE
The most minorvariants can bethe most critical
AdvantagesAdvantages
See lower abundance proteinsSee lower abundance proteins
Compare MS of intact proteins to proteins Compare MS of intact proteins to proteins found by bottom up methodfound by bottom up method
Allows further analysis of fractions of interest Allows further analysis of fractions of interest
High speed allows multiple runs to be set up High speed allows multiple runs to be set up giving higher utilization of the MSgiving higher utilization of the MS
Reduces number of One Hit WondersReduces number of One Hit Wonders
UHP UFFLC UHP UFFLC
Higher throughput than any other systemHigher throughput than any other system
Allows use of standard columns and UHP Allows use of standard columns and UHP columnscolumns
Allows normal chromatography and small Allows normal chromatography and small scale prep as well as UHP &UFFLCscale prep as well as UHP &UFFLC
Highest pressure and flow limits on the Highest pressure and flow limits on the market today. Why limit yourselfmarket today. Why limit yourself
HPLC, UHP, UFFLC Exclusively from HPLC, UHP, UFFLC Exclusively from
LabAllianceLabAlliance
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