Lecture 7: 1.Advanced Separations Methods: HPLC vs. UPLC vs ...
Transcript of Lecture 7: 1.Advanced Separations Methods: HPLC vs. UPLC vs ...
Lecture 7:
1.Advanced Separations Methods: HPLC vs. UPLC
vs. HILIC
2. Nanoflow vs. ESI
3. Applications;
4.Laser capture miscrodissection (LCM)
1 ME 330.80: Role of Chromatography & Mass
Spectrometry in Biological Research
http://www.hopkinsmedicine.org/mams/
Adriana Bora, PhD
Nov 19th 2012
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Technologies for Proteomics
Nature biotechnology 28, 695-709, (2010)
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“omics” Workflow Tissue Blood CSF Plasma Urine Serum Sample
Sample preparation
Peptide/Protein Extraction, Desalting, Abundant Protein Depletion, Detergent Removal, etc.
Separation HPLC/UPLC, HILIC, SEC, IEC, etc.
Mass Spectrometry
LC-MALDI, MALDI-TOF, QTOF, IT, Orbitrap, etc.
MASCOT, SEQUEST, PROTEIN DISCOVERER, etc. Data Analysis
Liquid Chromatography
• Defined as separation of components of a mixture based upon the rates at which they elute from a stationary phase typically over a mobile phase gradient.
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• Ion exchange chromatography • Size exclusion chromatography • Adsorption chromatography
Different Phases
• Normal Phase – This is where the stationary bed is strongly polar (silica gel) and the mobile phase is largely non-polar such as hexane.
• Reverse Phase – The stationary phase is non-polar and the mobile phase are polar liquids such as methanol, acetonitrile, or water. The more non-polar substances have longer retention.
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Elution Types
• Isocratic – where the eluent is at a fixed concentration.
• Gradient – where the eluent concentration and strength are changing.
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Types of Liquid Chromatography
Gravity Chrom. Tsvett, 1903
Flash Chrom. 1978
HPLC 1952 UPLC 2004 (TLC) Paper Chrom.
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HPLC Characteristics • Columns have small internal diameters (2-10 mm)
usually made with a reusable material like stainless steel
• High inlet pressures of several thousand psi’s and controlled flow of mobile phase
• Precise sample introduction and small sample requirements
• Special continual flow detectors that use small flow rates and low detection limits
• Some are equipped with automated sampling devices
• Rapid analysis with high resolution
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Stationary Phase in HPLC
• Particle size 3 to 10 µm packed tightly with a pore size of 70 to 300 Å
• Surface area of 50 to 250 m2/g
• Bond phase density – number of adsorption sites per surface unit (1 to 5 per 1 nm).
• Typical surface coatings:
Normal phase (-Si-OH, -NH2)
Reverse phase (C8, C18, Phenyl)
Anion exchange (-NH4+)
Cation exchange (-COO-)
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Mobile Phase in HPLC
• Purity of the solvents
• Detector compatibility
• Solubility of the sample
• Low viscosity
• Chemical inertness
• Reasonable price
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Path of Mobile Phase
Mobile Phase degassing
Mobile Phase reservoir
Mobile Phase mixing
HPLC Pump Rotary Sample Loop injector
HPLC Column
HPLC Detector
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How does it work HPLC/UPLC Technology?
Phytochem. Analysis, 2010, 21, 33-47, Allwood &Goodacre
Pumps
A B Line wash
Syringe wash
Solvent proportioning valve
Flow valve and monitor
Autosampler injector
HPLC/UPLC column
Waste
Detector
Secondary Detector: UV/Vis MS NMR
Chromatogram
Solvent A 5% Aq, ACN +0.1% FA Solvent B 95% Aq, ACN +0.1% FA
HPLC Columns
• HPLC Columns come in various sizes and many factors involving your analyte or the function of the column should be considered when selecting the appropriate one.
• Some common dimensions: 10, 15, and 25 cm in length;
• 3, 5, or 10 mm diameters;
• 4 to 4.6um internal diameters
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HPLC Detectors
• Most HPLC instruments are equipped with optical detectors.
• Light passes through a transparent low volume “flow cell” where the variation in light by UV Absorption, fluorescent emission, or change in refractive index are monitored and integrated to display Retention Time and Peak Area.
• Typical flow rates are 1 mL/min. and a flow cell volume of 5-50 µL.
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Common HPLC Detectors
• Refractive Index (RI) - universal
• Evaporative Light Scattering Detector (ELSD) – universal
• UV/VIS light – selective
• Fluorescence – selective
• Electrochemical (ECD) selective
• Mass Spec (MS) - universal
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Mass Spectrometer
• Thermospray – mobile phase is directed to a capillary column that is heated and points at a skimmer cone. (Too much build up on orifice)
• Electrospray (ESI) – analytes are charged upon exiting the capillary tube and cross sprayed with nitrogen. The charge particles cause a “Coulomb explosion” making smaller droplets of analyte to enter the skimmer cone.
• Atmospheric Pressure Chemical Ionization (APCI) – Analyte is heated by a ceramic tip on the column, cross flow of nitrogen decreases the droplet size, and a “corona discharge” charges the particles to enter the detector.
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Why HPLC?
• HPLC works with compounds of higher molecular weights and polarity.
• Many biological samples are charged such as DNA and proteins.
• HPLC can be used with larger sample sizes and sample recovery to continue synthesis
• Good at separating stereoisomers; techniques that employ heat (GC) can cause racemization during analysis.
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UltraPerformance Liquid Chromatography
(UPLC ) Technology • In 2004, further advances in instrumentation and
column technology were made to achieve very significant increase in:
RESOLUTION
SPEED
SENSITIVITY
Increase separation EFFICIENCY
• Columns with smaller particles [<1.7um]
• Mobile phase delivery is done at >15,000psi
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Contrasting HPLC and UPLC
• UPLC gives faster results with better resolution
• UPLC uses less of valuable solvents like acetonitrile which lowers cost
• The reduction of solvent use is more environmentally friendly
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UPLC columns
an ethylene bridged hybrid (BEH) structure • Superior mechanical strength • Efficiency • High pH stability and peak shape for
bases • C8; C18;Phenyl; HILIC • pH range 1-12 • Max pressure 15,000psi • Particle size 1.7um • Pore diameter/volume 130A 0.7 mL/g • Surface Area 185 m^2/g
• Peptides • Proteins • Oligonucleotides DNA/RNA • Amino acids
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UPLC principle
The evolution of particle sizes over the last three decades.
Pla
te h
eig
ht
Why is UPLC more efficient
• Peak capacity (P) is the number of peaks that can be resolved in a specific amount of time.
• P is proportional to the inverse of the square root of the Number of theoretical plates (N): N = L/H
• Lower plate heights generate a smaller number of plates
• Plate heights are correlated through the Van Deemter equation
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Why we need UPLC Technology?
• Metabolomics is the comprehensive assessment of endogenous metabolites of low-molecular weight (<1,000 Da)of a biological system.
• These small molecules, including peptides , amino acids , nucleic acids , carbohydrates , organic acids, vitamins , polyphenols , alkaloids and inorganic species act as small-molecule biomarkers that represent the functional phenotype in a cell , tissue or organism.
• Applications: drug discovery, toxicology, nutrition, cancer, natural product discovery, etc.
• These large-scale analyses of metabolites are intimately bound to advancements in ultra-performance liquid chromatography–electrospray (UPLC) technologies and have emerged in parallel with the development of novel mass analyzers and hyphenated techniques.
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Chromatograms of simvastatin
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• hypolipidemic drug • control elevated cholesterol
Chromatogram showing separation of Telmisartan and its degradation products in a mixture of stressed samples a) UPLC
b) HPLC
Angiotensin II receptor antagonist used to control hypertension
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Hydrophilic Interaction Liquid Chromatography (HILIC)
• The principle of HILIC was described by Samuelson and Sjostrom (1952) for the separation of monosaccharides using anion exchange rasin as stationary phase; Samuelson O, Sjöström E. 1952. Utilization of Ion Exchangers in Analytical Chemistry. XXIV. Isolation of Monosaccharides. Sven Kem Tidskr64: 305–314.
Cubbon et al, Jun 2009, Mass Spec. Reviews; Metabolomic applications of HILIC–LC–MS
• The mechanism of retention is based on the hydrophilic partitioning of the analytes into the water-enriched stationary phase, and weak electrostatic interactions with either the positive or negative charge of the functional group. • HILIC separates polar molecules.
• A sulfoalkylbetaine zwitterionic stationary phase.
• Like the SCX method mentioned above, HILIC chromatography can be used to enrich
PTM-modified sub-populations from complex biological samples.
• Specifically, HILIC has been shown to enrich for glycosylation, N-acetylation, and phosphorylation. Peptides with N-acetyl modifications will elute as an early sub-fraction during a HILIC separation at relatively low water/organic mobile phase conditions. Phosphopeptides will elute within the middle of a HILIC separation, but with little contamination from non-phosphorylated species. Glycosylated peptides will be retained on the HILIC column while most sample components elute off; once high water/organic mobile phase ratios are reached, the remaining glycosylated peptides will elute as a purified sub-proteome.
• HILIC offer a good alternative to RP chromatography for the analysis of highly polar metabolites such as carbohydrates, their phosphorylated derivatives, and glycolytic intermediates, which are poorly retained on RPs.
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HILIC application
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HILIC applications
In real life we mostly use orthogonal separations
Gauci et al, J. Proteome Res. , 2009, 8(7), pp 3451-3463
2000
Nucleic proteins
• NanoLC (nLC) is named after the low flow rate (200-300 nL/min).
• This uses very low sample volumes and (1µL) very high selectivity and sensitivity are possible.
• nLC-ESI-IT-MS/MS is mostly used for the identification of proteins from very complex mixtures.
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NanoLC- ESI –MS
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Membrane Trap
SDS PAGE
Column
Gel Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE)
J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573
+ -
Anode
Chamber
Cathode
Chamber
Collection
Chamber
Courtesy of John Tran
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+ Anode – Cathode
Membrane Trap
SDS-PAGE Column
Collection Chamber
Top Down Proteomics
Front-end separation
Mass spectral data acquisition
Protein identification by database search
Intact protein separation based on molecular weight:
Gel Eluted Liquid Fraction Entrapment Electrophoresis
(GELFrEE)
Tran, J. C.; Doucette, A. A., Anal. Chem. 2008, 1568-1573
Time (60-90 min)
MW
Courtesy of Neil Kelleher
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Tran, J.C.; Doucette, A.A., Anal. Chem. 2008, 80, 1568-1573
Gel Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE)
Courtesy of John Tran
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15 15.5 16 17 18 20 22 24 26 28 30 35 40 45 60 75 90 std
collection time (min)
25 27 30 33 36 42 48 54 60 66 72 87 102 112 157 202 std
212 100
54
39
30
20
7.3
212
100
54
39
30
20
7.3
J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573
Mol W
t (k
Da)
Mol W
t (k
Da)
collection time (min)
1 cm Column
3 cm Column
GELFrEE Separation of Bacterial Proteome
•1cm column gives faster and better separation over a broad mass range
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J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573
GELFrEE Recoveries
BSA
Cytochrome C
Ubiquitin
40 ng Loading
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gel
columns
collection
chamber
gasket gasket
cathode
chamber
anode
chamber
plate
bolts
dialysis
membrane
plate
plate
plate - +
power terminals
Multiplex GELFrEE
J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209
Load Collect
Courtesy of John Tran
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Load Collect
Multiplex GELFrEE
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Increased Loading Capacity
250 150 100
75 50 37
25
15
10
kDa
250 150 100
75 50 37
25
15
10
kDa
20
collection time (min)
16 17 18 19 20 21 22 24 26 28 30 35 40 45 60 75 90 std
16 17 18 19 20 22 24 26 28 30 35 40 45 60 75 90 std
Overloaded Column
Pooled Fractions from Equivalent Overloaded Amount
800 µg /column
800µg loaded in 8 different channels :100 mg / column
J.C
. T
ran
; A
.A. D
ou
ce
tte
, A
na
l. C
he
m. 2
00
9, 8
1, 6
20
1-6
209
0.4mm id
0.4mm id
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Increased Throughput Fractions 1-16
collection time (min)
16 17 18 19 20 22 24 26 28 30 35 40 45 60 75 90 std
250 150 100
75 50 37
25 20
15
10
kDa
250 150 100
75 50 37 25
15
10
250 150 100 75 50 37 25 20
15
10
kDa
kDa
Yeast
Bacteria
Urine
J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209
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High reproducibility of different in channels
J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209
800ug per column
100ug per column
S.cerevisiae Proteome
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•1120 proteins •428 unique proteins •1% false positive rate
S.cerevisiae Proteome
Identified proteins with LC MSMS
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Adriana Bora; Carol Anderson; Muznabanu Bachani; Avindra Nath; Robert J. Cotter; J. Proteome Res. 2012, 11, 3143-3149.
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• Analyzing CSF using GelFrEE –LC-MS/MS
Bottom Up Proteomics
400
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Unseparated human CSF proteome
Adriana Bora; Carol Anderson; Muznabanu Bachani; Avindra Nath; Robert J. Cotter; J. Proteome Res. 2012, 11, 3143-3149.
A) Silver stained images showing GelfrEE fractions for visualization of the protein
separation found in each fraction.
B) and C) show the reproducibility of the separation technique.
Separated human CSF proteome with GelFrEE
Sample Preparation
Affinity Column
4 min Tryptic
Digestion
On-Column
Desalting
C18 Reverse Phase HPLC
SRM-MS Detection
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• Reduces variability to under 10% • Cutting sample prep time down to 10min • Reducing operating cost by 50%
Perfinity – Shimadzu 8030 triple quadrupole MS
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14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 min
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uV
1min
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4min
6min
8min
Insulin protein digested on trypsin column using
Perfinity System
Undigested protein
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CSF 30min gradient- 6min digestion-60 %B -40C
Separation of CSF proteins using the Perfinity
System
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Laser capture miscrodissection (LCM)
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Applications of LCM
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Cont…
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Cont….