UHP & UFFLC Chromatography

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


Retention Time


Retention Time


Retention Time


Retention Time








.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 2-214nmflavorsLemon 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

UHP & UFFLC Chromatography

Documents

Transcript of UHP & UFFLC Chromatography