Desarrollo de Metodos en LC

Humax - 2015
 Esta presentacion….

http://exactas.udea.edu.co/~carlopez/desarrollo-metodos-humax-2015.pdf

Los Libros 2013-2015

http://dropcanvas.com/ar8qc
Evolución de la LC
 Contenido

Desarroolo de Metodos RP – Neutros y/o Ionizables

Seleccion de Columnas - Tecnologias

Seleccion del Proveedor de Columnas

Comparacion de Columnas

Troubleshooting on line

Software Comercial
Que hay detrás de cada pico?
 Términos Cromatográficos
Factor de Retención (k) = término usado para describir la migración de
los solutos (analitos) a través de la columna

Interpretando el factor de Retención

Si kA < 1;la elución es demasiado rápida para la determinación del tR.

Si kA > 20; la elución es demasiado lenta para ser practica

El rango preferido para kA está entre 1 y 10

 Factor de Retención (k) o Factor de Capacidad (k’)

k es una medida del número de volúmenes de columna requiridos para eluir un

Compuesto (proporcional al tiempo de residencia en la fase estacionaria).
Parámetro fundamental, sin dimensiones, que describe la retención
(independiente de la rata de flujo y la longitud de la columna

k = 1 to 20 - OK;
k = 3 to 10 - Mejor;
k = 5 to 7 - Ideal
 Factor de Retención (k)

Mide el número de volúmenes de columna que se requieren para eluir el

compuesto – (la fracción de tiempo gastado en la fase estacionaria (Cs) -

Un compuesto NO retenido – eluye sobre el frente del solvente, Ts= 0

Ts, Cs = 0 => k = 0
 Para un Componente retenido que eluya en 1 un volumen de columna

Para un componente retenido que eluya en 7 volumenes de columna

k = 1 to 20 - OK; k = 3 to 10 - Mejor; k = 5 to 7 - Ideal

Resolución vs k
Factor de Retención en Gradiente
 La ecuación General de Resolución

3. Eficiencia
2. Selectividad 1. Retención
Curva de van Deemter en HPLC
Efecto del tamaño de partícula

UHPLC
Ventajas de tamaño de partícula pequeño
Un nuevo invitado
Mas información en el mismo tiempo..!!!
Transferencia de Métodos: HPLC to UHPLC
UPLC in Life Science 2013
 Preservando la Resolución con la relación L / µm Constante

UHPLC
 Modos de Separación en LC

Fase Normal / Separaciones Quirales

HPLC- 2015

Fase Reversa / Fase Reversa Par-Iónico

Exclusión por Tamaño (GPC / GFC)

Intercambio Iónico – Exclusión

HILIC
Características generales de los modos de separación
Desarrollo y Optimizacion
de Metodos en HPLC
 Conocer muy bien la muestra
Cractersiticas de la muetsra que influencian la
seleccion del modo de sepracion y la columna

- La estructura de los componentes?

- El numero de componentes?
- La matrix de muestra?
- Los pKa de los componentes ionizables?
- El rango de Concentracion esperado?
- El peso (o rango) de peso molecular?
- La solubilidad?
- Otros datos pertinentes?
 Los Objetivos de la separación
Los fines de la sepracion tambien influencian la
seleccion
- Maxima resolucion para todos los componentes?

- Resolución parcial?

- Análisis rapidos?

- La economía de la separación(bajo consumo de solvente)?

- Estabilidad y vida media de la columna?

- Alta sensibilidad (analisis de trazas)?

- Otros objetivos?
 Desarrollo de Metodos
Metodos farmacopeicos Estudios de Estabilidad
Cuanto puedo Modificar? Que hay detrás de cada pico?
Condiciones partida para Moleculas Neutras?
- Modo de Separación
- Fase Estacionaria
- Fase Movil (ACN, MeOH, THF)
- Composición Inicial y Flujo
- Detector y Vol de Inyección
- Modo de Operación: Isocrático vs Gradiente
- Optimización – System Suitability
- Quality by Design

Condiciones partida para Moleculas Ionizables?

- Modo de Separación
- Fase Estacionaria
- Fase Movil (ACN, MeOH) y pH
- Composición Inicial y Flujo
- Detector y Vol de Inyección
- Modo de Operación: Isocrático vs Gradiente
- Optimizacion – System Suitability
- Quality by Design
https://www.aaps.org/uploadedFiles/Content/Sections_and_Groups/Regional_Discussion_Groups/Southern_California_Pharmaceutical/Resources/SCPDG%202014%20June%20Slides.p
Cambios Permitidos Usp (Agosto 22 de 2012)
Cambios permitidos Pharmacopea Europea
Desde Agosto 1 del 2014
Selecciones unas condiciones de Partida
para separar esta mezcla #1
Estructuras de los Compuestos

OH
N

(CH2)3CH3

Phenol 3-Butylpyridine

(CH2)5CH3

Anthracene 3-Hexylanthracene
 Selecciones unas condiciones de Partida
para separar esta mezcla #2
Estructures de los Compuestos

N N
N O N
O N O
N
O O O H H
S
H H H H H H NH
H2N S NH S S
NH S NH O N O O
N
O
N N N
H2N O O NH2
O HO O
O S N
HO O HO O HO O N
O S

N
O N O S O
NH H H H
H N N S O
O NH
O NH N S
O N N S N O
O HO O
HO O
HO
https://hplcfordummies.files.wordpress.com/2013/11/flowchart_2r2d5l5l.jpg
La variable mas importante?
Efecto del Solvente en RP
Impacto del %Organico sobre el Tr
 Fortaleza de la fase Móvil y la Retención

El aumento del porcentaje del modificador orgánico en al fase móvil tiene un profundo efecto sobre
la retención del analito debido al cambio en la polaridad de la fase móvil
Efecto del Solvente en RP
 Muestras Regulares e Irregulares

Regular Irregular
Nomograma de Fortaleza de Solvente
Phenyl Hexyl con Acetonitrile o Methanol
Selectvidad por Modificador
Orgánico
Planos Isoeluotrópicos
Cambio de selectividad: MeOH-ACN y THF
Selectividad por cambio de columna
Resumen
 Desarrollo de Métodos - Sustancias Ionizables

Alto Phenyl MeOH

Solventes
C18 C4

pH Columna

C8 CN
Bajo ACN
 Por qué el pH es tan crítico en el desarrollo de Métodos en LC?

- Cuales compuestos son sensibles al pH?

- Como afecta el pH la retención y la resolución?

- Qué tanto influencia el pH el desarrollo de mi método?

- Como influencia el pH la selección de la columna?

Factor de Retención vs pH
Sustancias Ionizables
Efecto del pH
Efecto del pH
Efecto del pH
 Marvin –Sketch On-line

http://www.chemaxon.com/marvin/sketch/index.php
Desktop - Descargar para Win32 , Win64 , Linux y Mac
http://www.chemicalize.org/structure/#!mol=OC%28%3DO%29C1%3DCC%3DCC%3DC1
Chemicalize

http://www.chemicalize.org/
 Ketoprofen
Lidocaine

Benzamide
La Química del analito y de la fase Estacionaria
 Isocratico vs Gradiente

Figure 1: Isocratic separation of a nitroaromatic

sample. Column: 100 mm× 4.6 mm, 5-µm particle
C18; flow rate: 2 mL/min; temperature: 25 �C; A-
solvent: water; B-solvent: methanol with %B Figure 2: Same sample as Figure 1. Simulated 0–100% B
shown on chromatograms. All chromatograms gradients with gradient times shown on chromatograms.
have the same x- and y-axis scaling. Simulated Same scaling as Figure 1.
chromatograms based on the data of reference 3.
Peaks: 1 = 2,6-dinitrotoluene, 2 = nitrobenzene, 3
= 2-nitrotoluene, 4 = 3-nitrotoluene, 5 = 2-nitro-
1,3-xylene, 6 = 4-nitro-1,3-xylene, 7 = benzene.
Fallos más frecuentes en LC
 El Culpable?

La columna?

El Metodo?

El Instrumento?

El Software?
 Recursos On-line

http://www.lcresources.com/tsbible/
 http://www.chromacademy.com/hplc_troubleshooting.html

Suscribase.!! Es Patrocinado por Waters..Free..!!!

 e-seminar Agilent

http://www.chem.agilent.com/en-US/Training-Events/eSeminars/Pages/default.aspx
http://www.sepscience.com/Techniques/LC
http://www.chromforum.org/
Forum USP – Registrese es Free..!!!!

http://www.usppf.com/pf/pub/index.html
http://www.chromedia.org/
Criterios Para seleccionar una Columna
Columnas en HPLC: Desde preparativas hasta Capilares
La tecnologia de la Fase Estacionaria
Grupos activos sobre la superficie de la silica
Resumen uso de Columnas RP
Efecto sobre la separación del tipo de columna
Ver Dos Archivos de las columnas de la USP

Columna L1 - C18
Columna L11 - Phenyl
Ejemplos de Selectividad
Cambios de Selectividad por Cambio de Columna
 Schematic of Hydrogen Bonding Interaction

Schematic of π-π interactions

Hydrogen bonding interaction is a type of dipole-dipole interaction that occurs when the interaction involves a
hydrogen atom bonded to 1 1a heteroatom, such as oxygen, nitrogen or sulfur. Hydrogen bonding interactions
occur between hydrogen atoms attached to heteroatoms and the electronegative fluorine atoms in
pentafluorophenyl (PFP) bonded phases, and they occur between heteroatom hydrogens and electronegative
groups, such as -CN groups in cyanopropyl bonded phases and amide (-NHC=O) groups in polar embedded
phases.
The point when selecting a phenyl column is that rather than using acetonitrile as the
mobile phase, methanol is used. This is because acetonitrile possesses a π electron (triple
bond), which would weaken the π electron possessed by the phenyl column.

This is illustrated by the type of separation that can be expected with methanol as the
mobile phase, shown in Fig. 2 (b), as compared with that using acetonitrile, shown in the
analysis example of Fig.

However, care is required due to the increased pressure during solvent delivery after
switching to methanol as the mobile phase
Columna Phenyl RP
Bonded Phase Structure Influences Method Selectivity
Different Surface Chemistry/Structure-Different Selectivity
Explore Selectivity with Bonded Phases
 Bonded phase

• Column Length

• Internal diameter

• Nature of the material used for column construction (typically stainless

steel)

• Metal passivation technique

• Interior tubing surface polishing

• Nature of the frit and spreaders used in the column end fittings

• Packing quality (including method of packing, packing pressure,

solvents used etc.)
El Formato
Metodos Ortogonales – Estudios de Estabilidad
Que hay detrás de un cromatograma?
https://www.youtube.com/watch?v=zDb_91QOKCA
https://www.youtube.com/watch?v=LyYAapT6BFo
 Common mistakes in analytical method development

• Inadequate formulation of method goals

• Insufficient knowledge of chemistry

• Use of the first reversed phase HPLC column available

• Use of wrong instrument set-up

• Trial and error with different columns, mobile phases

These mistakes often results in laborious, time consuming projects that lead to methods
which fail to meet the needs of the laboratory.

Getting started :
Listed below are some of the most common parameters.

• Nature of the sample

• Number of compounds/analytes present
• Chemical structure (functionality)
• Molecular weight of the compounds
• pKa values
• log P and/or log D values (hydrophilicity/hydrophobicity)
• Concentration
• Sample matrix
• Sample solubility
???????
Comparing the L/dp ratio as a function of separation index [easy-to-extremely difficult].
Columns with the same L/dp ratio will generate the same resolving power.
Silicas Totalmente porosas vs silicas superficialmente porosas
 Solid core
(also known as core-shell, fused core, solid core, or superficially porous)
El limite?
Core Shell – Difusion Eddy
Core Shell - Difusión
Core-shell: the best alternative to UHPLC
SEM Kinetex Core Shell
Columnas Core Shell
Carlos Lopez - udea
 Instrumentation for core-shell technology

Core-shell columns are hardly compatible with old generation HPLC system
Columnas / Compatibilidad con los Equipos
Transferencia de Metodos con Columnas 5µm a 2.7µm a 1.7µm

http://www.hplctransfer.com/
 Carlos
Lopez -
udea
 Calculate it for yourself –
the RRLC online method translator

http://www.chem.agilent.com/en-US/products-services/Instruments-Systems/Liquid-Chromatography/Pages/1200infinity_cost_calculator.aspx

http://www.chem.agilent.com/en-US/products-services/Instruments-Systems/Liquid-Chromatography/1290-Infinity-Binary-LC-
System/Pages/1290infinity_method_translator_cost_calculator.aspx

Descargar el software

http://www.chem.agilent.com/Library/software/Public/AgilentMethodTranslator1290.zip
http://www.phenomenex.com/tools/kinetexcalculator
Otro menos poderoso
http://www.dionex.com/static/documents/1111%20RSLC%20Method%20Transfer%20Tool%202.
3.xls
http://www.fortis-technologies.com/resources/Fortis+Method+Development+Calculator+V2.xls
http://www.acdlabs.com/resources/freeware/translator/
 Columnas Chromolith

Preparación

Si(OAlk)4
Polymer
H+

Starting Phase Separation Ageing Drying

Sol and Gelation

HPLC

Cladding
Caracteristicas de las columnas de silica monolitica Carlos
Lopez -
udea
 Columnas de silica Monolitica

25 mm
10 mm
4.6mm 3.0 mm
Carlos Lopez - udea
Columnas Monliticas vs Columnas Convencionales
 Velocidad y calidad en la Practica

1
4 Chromolith® Performance RP-18e
3 5 100 mm x 4.6 mm

5 ß-Blocker

1 mL/min - 17 bar
Mobile Acetonitrile/
0.1% TFA in
water
Phase (20(80; v/v)
5 mL/min - 85 bar Flow rate 1-9 ml/min
Detection 222 nm
Sample 1. Atenolol
2. Pindolol
3. Metoprolol
4. Celiprolol
9 mL/min - 153 bar 5. Bisoprolol
 Velocidad y calidad en la Practica

2
2
1
4 1
3 4
5 3 5

0.0 1.5
1 mL/min - 17 bar

9 mL/min - 153 bar

5 mL/min - 85 bar

12
9 mL/min - 153 bar
 Separación de 8 Esteroides – Dos Columnas en serie

6
Column 2 columns of
Chromolith Performance RP-18e
100-4.6mm
Mobile A: Acetonitrile
phase B: Water
Gradient Time/min %A %B
0,0 20 80
7,0 90 10
Flow rate 3 mL/min
3 Detection UV 220 nm
2
Temp. ambient
5 Inj.Volume 10 µL
8 Sample 1. Prednisolone
1
2. Cortisone
7 3. Nortestosterone
4 4. Estradiol
5. Testosterone
6. Corticosterone
7. Estrone
8. Progesterone

0,0 3,5 7,0 min

Columna de 1.4 Metros

6
7
5
108 000 N/column !!!
117 bar

4
2

0 30 60 min
1. Thiourea, 2. benzene, 3. toluene, 4. ethyl-,
5. propyl-, 6. butyl-, 7. pentylbenzene

ACN/ Water (80/ 20; v/v)

Limitaciones
 Carlos
Lopez -
udea
Como comparar columnas?
 USP Columns

http://www.usp.org/USPNF/columns.html
 Bonding Density

Chelating Tailing Factor

Capacity Factor Amitryptyline Tailing Factor Amitryptyline
Examples of separations of SRM 870 on commercial C18 columns - highly deactivated Type B silica with
polar embedded ligand (top), high silanol and metal ion activity phase (bottom).
Washing with phosphoric acid restores column performance
for chelating compounds
 Shape selectivity (bonding density)

The term ‘shape selectivity’ is used to denote a chromatographic quality exhibited by certain
stationary phases for which enhanced separations of geometric isomers may result based on
their molecular structure rather than other physical or chemical properties of the solute. SRM
870 does not characterize shape selectivity, however this property can be assessed by use of
other chromatographic test mixes, such as SRM 869a

Column Selectivity Test Mixture for Liquid Chromatography. This test uses benzo[a]pyrene
(BaP), phenanthro[3,4-c]phenanthrene (PhPh) and 1,2:3,4:5,6:7,8-tetrabenzonaphthalene (TBN) as
probes and the shape selectivity measurement is the selectivity between TBN and BaP. The
structures and space filling models of these compounds are shown in Figure

Testing columns for shape selectivity according to the USP classification model.
 Waters - Versión on-line

http://www.waters.com/waters/promotionDetail.htm?id=10048475&ev=10058108&locale=en_
US
http://www.acdlabs.com/resources/freeware/colsel/
 .

An early attempt at producing a generic set of probes for testing HPLC column characteristics
was made by Tanka and co-workers and since then work by the USP Working Group on HPLC
Columns, the Impurities Working Group of the PQRI Drug Substance Technical Committee in
collaboration with Dr Lloyd Snyder and work carried out by Euerby and Petersson to expand the
original probes designed by Tanaka have all undertaken to identify a definitive set of probes
which will allow the various important physico-chemical phase characteristics to be specified.
Most of these groups have also combined their data with various chemometric approaches to
produce quantitative databases based on principal component analysis (PCA) or tools to
visualize the relative groupings of commercially available columns according to their key
descriptors.

Spider diagram representing the various

characteristics of the stationary phase.

HR - hydrophobic retention
HS - hydrophobic selectivity
SS - steric selectivity
HBC - hydrogen bonding capacity
BA - base activity
C - chelation
IEX - ion exchange capacity at pH2.6 and 7.6
AI - acid integration
Test Hidrofobicos
Test de interacciones secundarias y de intercambio Iónico a bajo pH
Test de interacciones secundarias y de intercambio Iónico a alto pH
Carlos Lopez - udea
All HPLC C18s Are the Same Aren’t They?
There are several independent databases which assess the parameters deemed most likely to establish the
selectivity ‘character’ of a column, including factors such as hydrophobic retention, shape selectivity, acid and base
retention mechanisms. One example is the PQRI database being adopted by the United States Pharmacopoeia
(USP). For more explanation on the parameters assessed in this database :

H : Hydrophobicity. A measure of the ability of the stationary phase to retain hydrophobic compounds. Traditionally
‘carbon loading’ was used as a measure of this parameter, but this took no account of several key effects (e.g.
efficiency of ligand bonding process, ligand accessibility / performance and the degree / type of endcapping used.
The PQRI ‘H’ value provides a far more quantifiable practical value to this critical feature. ‘H’ gives a measure of
retention, but has only a minor effect on selectivity.

S: Steric or shape effect. The ability of the stationary phase to discriminate between molecules of similar size /
LogP, but with a different 3D shape. This can have a significant effect on selectivity. In some cases, this parameter
can be exploited to allow resolution of isomers.

A : Hydrogen Bond Acidity. Generally a desirable feature that gives a level of secondary interaction via low activity
silanol groups on the support surface. This influences selectivity, particularly with retention of weakly basic
molecules without excessive peak tailing.
Many phases with high A values provide the ability to use 100% aqueous mobile phases.

B: Hydrogen Bond Basicity. Desirable feature built into certain stationary phases (e.g. polar embedded phases) to
provide significantly modified selectivity. Larger ‘B’ values can have considerable effects in enhancing retention of
weak acids.

C (2.8) : Silanol Ionisation at pH 2.8. At this pH, all residual silanol groups should be in the less active (un-ionized ,
vicinal) form. Any acidic silanols that have been activated (e.g. by metal ions in stationary phase support) remain
charged and can have significant detrimental effect, particularly with excessive tailing of bases. More traditional
phases tend to have high C (2.8) values, with resulting poor peak shape.

C (7.0): Silanol Ionisation at pH 7.0. At this pH, all residual silanol groups should be fully ionized and the C (7.0)
value gives a measure of the total amount of ionized silanol groups available for secondary interaction with
analytes. For optimal alternative selectivity, ideally we should have a large C(7.0) – giving large amounts of silanols
available for secondary interactions, but a low C (2.8) – low amounts of activated silanols that cause peak tailing.
Desarrollo de métodos AutomatizadoAutomatizados

http://www.molnar-institute.com/
 Desarrollo de métodos Automatizados LC
y GC

http://www.acdlabs.com/products/com_iden/meth_dev/lc_sim/
Desarrollo de métodos
Automatizados

http://www.chromsword.de/

ChromSword Auto Method Development Software

Agilent
Automated HPLC Method Development: ChemStation with ChromSword
https://www.youtube.com/watch?v=zDb_91QOKCA
Desarrollo de métodos Automatizados

OSIRIS
HPLC dvelopment Method Software

http://www.kromatek.co.uk/pages/OSIRIS-software.html
http://www.smatrix.com/
http://www.perkinelmer.com/catalog/product/id/n2600403
Identification by retention time?
Calibracion por Estandar
externo
Como garantizar que hay pureza de pico?
UNO SOLO!!!!
Pureza - DAD
Pureza - MS
Lo ideal? Soñar no cuesta nada..!!
Estudios de degradacion forzada
 LC 2D

LC-LC LCxLC
LC 2D
http://www.chem.agilent.com/en-US/Promotions/pages/infinity-2dlc.aspx?CID=7024&ibt=2dlc
Selection of LCxLC conditions