hplc:&pastand&present presentation_jan2013.pdf · hplc:&pastand&present...
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HPLC: Past and Present
Dan Wang, Daniel Abate Harki Group Teach the Lab Presenta>on
01/10/2013
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Outline • Introduc>on and History • Components of HPLC – Sta>onary Phase – Detector – Examples
• Method Development – Parameters – Column Specifics – Method Valida>on
• New Advances in HPLC 2
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Introduc>on • HPLC: High-‐performance liquid
chromatography.
• A qualita>ve and quan>ta>ve technique, generally used for the es>ma>on of chemical, pharmaceu>cal and biological samples.
• Why high performance?
– Reduced par>cle size (<10 μm), densely packed. UPLC: par>cle size < 2 μm.
– Pressure stable column. – High and constant linear velocity of mobile phase.
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Brief History of LC
4 Angew. Chem. Int. Ed. 2010, 49, 2300 – 2312
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Brief History of LC • 19th century: adsorp>ve separa>on. • 1903: First “chromatography” by Tswed, for the isola>on of
chlorophyll cons>tuents. • 1910-‐1940: standardized and reproducible sta>onary phases
were developed (Al, Si…) • 1960s: HPLC was developed as an analy>cal tool. • 1974: LC-‐MS (fully developed in 1990s). • 2000s: development of new packing material and detector:
chiral stuff, micro-‐/nano LC.
Angew. Chem. Int. Ed. 2010, 49, 2300 – 2312 5
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Prepara>ve vs Analy>cal HPLC • HPLC is only method that offers equal performance in
analy>cal as well as in prepara>ve/process applica>ons.
Prepara>ve/Process: Food sciences and petrochemical area
Analy>cal: chemical, biological medical samples
6 Angew. Chem. Int. Ed. 2010, 49, 2300 – 2312
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Introduc>on
• Pump • Injector • Sta>onary Phase (column)
• Detector
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Types of Column
• Normal Phase • Reverse Phase • Size Exclusion • Ion Exchange • Bio-‐affinity • Chiral • Monolithic Silica • Chip-‐HPLC • 2-‐Dimensional-‐HPLC
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Types of Column
• Normal phase chromatography (NP-‐HPLC) : – separates analytes based on polarity. – a polar sta>onary phase, which retains polar analytes, and a non-‐polar mobile phase.
– Reten>on >me (adsorp>on strength) increases with increased analyte polarity.
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Types of Column
• Reversed phase chromatography (RP-‐HPLC): – a non-‐polar sta>onary phase and an aqueous, moderately polar mobile phase.
– Operates on the principle of hydrophobic interac>ons.
– Reten>on >me increases with increased non-‐polar surface area of analytes.
Journal of Global Pharma Technology 2010, 2, 22-‐26. 10
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Types of Column
• Size exclusion chromatography (SEC, gel filtra>on chromatography):
– Separates par>cles on the basis of size. – Useful for the separa>on of biopolymers such as proteins and polysaccharides.
11 Journal of Global Pharma Technology 2010, 2, 22-‐26.
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Types of Column
• Ion exchange chromatography (IEC): – Reten>on is based on the adrac>on between solute ions and charged sites bound to the sta>onary phase.
– Widely used in purifying water, ligand-‐exchange, purifica>on of proteins.
– Changing the pH of mobile phase enables the elu>on of proteins with different pI.
12 Journal of Global Pharma Technology 2010, 2, 22-‐26.
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Types of Column
• Bio-‐affinity chromatography: separa>on is based on specific reversible interac>on between proteins and ligands.
13 Journal of Global Pharma Technology 2010, 2, 22-‐26.
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Types of Column
• Chiral HPLC: – enan>oselec>ve packing adsorbents can be
prepared by adaching a suitable chiral compound to the surface of silica gel.
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Types of Column
• Monolithic silica columns: – An alterna>ve to par>cle packed columns. – High column performance with decreased column pressure.
RP-‐18: par>culate silica
Monolithic: rod structure with porous channels
J. Chromatogr. A 2007, 1168, 101 – 168 16
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Advantages of Monolithic Columns
– In silica based HPLC columns, the decrease of par>cle size results in decreases in run >mes, increase of selec>vity, but also drama>c increase of pressure.
– The porous structure provides monolithic with high permeability and high surface area.
– To achieve same separa>on, the pressure in monolithic column is much lower then silica column.
J. Chromatogr. A 2007, 1168, 101 – 168 17
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Types of Column
• Microchip-‐based HPLC (chip-‐HPLC): – Silicon-‐based LC microchips. – Has enhanced sensi>vity and reduced sample consump>on.
– The concept was first introduced in 1990. – The technology was largely developed amer 2000 due to the elevated demand on high throughput analysis and reducing the consump>on of organic solvent.
Sens. Actuators, B, 1990, 1, 249–255 Anal. Chem., 2009, 81, 2545–2554.
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Two-‐Dimensional HPLC
– The injected sample is separated by passing through two different sta>onary phases with two different separa>on mechanisms.
AnalyFcal Chemistry 2008, 80, 268–278
– The bands that are poorly resolved from the first column may be completely separated in the second column.
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Introduc>on
• Pump • Injector • Sta>onary Phase (column)
• Detector
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Types of Detector • Refrac>ve index (IR)
– Measures the change of refracFve index of the eluant from the column with respect to pure mobile phase.
– lack of high sensiFvity, non suitability for gradient eluFon, requires strict temperature control ±0.001oC to.
• Ultraviolet (UV) – depends on absorpFon of UV ray energy by the sample. – HPLC detectors used in general, are capable to detect very wide range
of compounds. – Moderate sensiFvity ranges Fll microgram quanFty of esFmaFon.
Prac>cal High-‐Performance Liquid Chromatography 21
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Types of Detector • Fluorescence
– Fluorescence rays emiUed by sample aVer absorbing incident light is measured.
– Xenon arc lamp is used to produce light for excitaFon. – Only suitable for compounds which can produce florescence. – High precision and sensiFvity. – Some compounds are not stable under fluorescent sFmulaFon.
• Photodiode Array (PDA) Detectors – the range of detecFon extends from UV, visible and to some extent to
IR region. – higher sensiFvity and measures the enFre absorpFon range . – it gives scan of enFre spectrum.
22 Prac>cal High-‐Performance Liquid Chromatography
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Types of Detector • Electrochemical detectors
– Specially suitable to esFmate oxidisable & reducible compounds. – When compound is either oxidized or reduced, the chemical reacFon
produces electron flow, which is measured as current. – Not universal, suitable for compounds which can't be assayed by UV
detector. Ex: primary amine. – super sensiFvity, which ranges Fll picograms measurement.
• Mass spectrometry – Universal. – High sensiFvity.
23 Prac>cal High-‐Performance Liquid Chromatography
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• HPLC in clinical chemical analysis: Case Study:
Transplant. Proc. 2006, 38, 1078 – 1082 24
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• HPLC in clinical chemical analysis: Case Study:
25 Angew. Chem. Int. Ed. 2010, 49, 2300 – 2312
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Case Study: • Two-‐dimensional chip-‐HPLC/MS/MS for the quan>ta>ve
analysis of 7-‐aminoflunitrazepam in urine samples.
Analyst, 2010, 135, 2737–2742 26
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Case Study: • Two-‐dimensional chip-‐HPLC/MS/MS for the quan>ta>ve
analysis of 7-‐aminoflunitrazepam in urine samples.
LC-‐ESI-‐MS
chip-‐HPLC-‐MS: Similar resolu>on 10 >mes faster Less solvent consump>on Chips are reusable
27 Analyst, 2010, 135, 2737–2742
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Case Study: • Human proteome analysis by using reversed phase monolithic
silica capillary columns – Tryp>c pep>des from 4 μg HeLa cell lysate proteins were directly injected onto a 4-‐m, 100 μm i.d. monolithic silica-‐C18 column
– 8-‐h gradient was applied at 500 nL/min, 41,319 non-‐redundant tryp>c pep>des from 5,970 proteins were successfully iden>fied.
– Best result yet reported without the use of exhaus>ve pre-‐frac>ona>on.
– The best result reported using conven>onal par>cle packed column iden>fied 10,183 pep>des.
J. Chromatogr. A 2012, 1228, 292– 297. 28
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Method Development & Valida>on
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Uses
• Check purity of new chemical en>>es • Monitor changes in synthe>c procedures/scale ups
• Evaluate new formula>ons • Carry out quality control
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Goals
• Op>mize detectability • Maximize efficiency, the chromatographic zone dispersion in column
• Efficiency dictated by mechanical and chemical separa>on parameters: – Mechanical: Column length, pore size, par>cle size – Chemical: Compe>>on for compounds between packing material and mobile phase
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Column Selec>on
• Challenging since more than 600 types/brands available
• Understanding of column features helps avoid problems of bad peak shape or poor resolu>on
• Column parameters provide informa>on regarding op>mal use
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Column Parameters
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Column Parameters
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Phase Selec>on
• C8 and C18 are ‘universal’ standard choices
• Carbamate bond offers alternate selec>vity
• Phenyl group extends column life >me
J. Sep. Sci., 2003, 26, 174-‐186 35
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Column Dimension Selec>on
• Internal diameter and column length • Diameter determines scale of separa>on (analy>cal vs. prepara>ve)
• Longer columns lead to more mechanical efficiency, but longer run >mes and more back pressure and solvent use
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Par>cle Size Selec>on
Sep. Sci. and Tech., 2007, 1, 145-‐187 37
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Pore Size Selec>on
• Choose pore size according to size of analyte to be tested
• Smaller pore sizes (10 to 100 Å) are used for small molecules and pep>des (3000 MW)
• Larger pore sizes (100 to 300 Å) provide larger MW analytes with beder efficiency and peak shape
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Column selec>vity
LC-‐GC, 1997, 2, 856-‐866 39
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Counteranion Effects
40 Sep. Sci. and Tech., 2007, 1, 145-‐187
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Orthogonal Screening
Sep. Sci. and Tech., 2007, 1, 353-‐371 41
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Op>mized Method
Sep. Sci. and Tech., 2007, 1, 373-‐405 42
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Method Valida>on
• “Valida>on is the process that helps establish, by laboratory studies, that the performance characteris>cs of a method meet the requirements for the intended applica>on.”
• Provides documented evidence that the method performs for the intended purpose
• FDA and other industry approved guidelines must be met
Sep. Sci. and Tech., 2007, 1, 441-‐458 43
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Method Valida>on Purposes
• Different valida>on procedures must be performed depending on method goal: – Iden>fica>on test: ensures iden>fy of an analyte in sample
– Quan>ta>ve impurity test: accurately reflects purity characteris>cs of a sample
– Quan>ta>ve analyte test: accurately quan>fies amount of analyte present in sample
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Valida>on tests • Specificity
– Unequivocally assesses an analyte response in the presence of components or mixtures
• Accuracy – Expresses closeness of agreement between the obtained value and its accepted or reference value
• Precision – Expresses closeness of agreement between a series of measurements of same sample under method condi>ons
• Linearity – Correlates ability to obtain results which are directly propor>onal to analyte concentra>on
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Valida>on tests, cont’d • Range
– Determines upper and lower concentra>on for which method has a suitable level of precision, accuracy and linearity
• Quan>ta>on Limit – Determines lowest amount of analyte which can be quan>ta>vely determined
• Detec>on Limit – Determines lowest amount of analyte which can be detected but not quan>tated
• Robustness – Assess whether typical parameter fluctua>ons are negligible on the outcome of the procedure
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Robustness
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Hyphenated techniques • HPLC coupled to different detec>on devices
• HPLC-‐ICPMS (induc>vely coupled plasma MS)
• ICPMS detects and measures concentra>on of certain elements (As, P, S, Br, etc)
Anal. Chem., 2011, 83, 3589-‐3595 49
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Hyphenated techniques
• HPLC-‐SPE-‐NMR • Combines post column trapping and enrichment
• Enriched sample elutes to NMR vial, system switches to deuterated solvent
J. Nat. Prod., 2012, 75, 876-‐882.
Sileshi G. Wubshet,
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