hplc:&pastand&present - medicinal chemistry presentation_jan2013.pdf · –...

HPLC: Past and Present

Dan Wang, Daniel Abate Harki Group Teach the Lab Presenta>on

01/10/2013

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

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.

3

Brief History of LC

4 Angew. Chem. Int. Ed. 2010, 49, 2300 – 2312

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

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

Introduc>on

•  Pump •  Injector •  Sta>onary Phase (column)

•  Detector

7

Types of Column

•  Normal Phase •  Reverse Phase •  Size Exclusion •  Ion Exchange •  Bio-‐affinity •  Chiral •  Monolithic Silica •  Chip-‐HPLC •  2-‐Dimensional-‐HPLC

8

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.

9

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

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.

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.


Types of Column

•  Bio-‐affinity chromatography: separa>on is based on specific reversible interac>on between proteins and ligands.


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.

14

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

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

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.

18

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.

19

Introduc>on

•  Pump •  Injector •  Sta>onary Phase (column)

•  Detector

20

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

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

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

•  HPLC in clinical chemical analysis: Case Study:

Transplant. Proc. 2006, 38, 1078 – 1082 24

•  HPLC in clinical chemical analysis: Case Study:

25 Angew. Chem. Int. Ed. 2010, 49, 2300 – 2312

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

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

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

Method Development & Valida>on

Uses

•  Check purity of new chemical en>>es •  Monitor changes in synthe>c procedures/scale ups

•  Evaluate new formula>ons •  Carry out quality control

30

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

31

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

32

Column Parameters

33

Column Parameters

34

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

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

36

Par>cle Size Selec>on

Sep. Sci. and Tech., 2007, 1, 145-‐187 37

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

38

Column selec>vity

LC-‐GC, 1997, 2, 856-‐866 39

Counteranion Effects

40 Sep. Sci. and Tech., 2007, 1, 145-‐187

Orthogonal Screening

Sep. Sci. and Tech., 2007, 1, 353-‐371 41

Op>mized Method

Sep. Sci. and Tech., 2007, 1, 373-‐405 42

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

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

44

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

46

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

47

Robustness

48

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

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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