My Biomedical Notebook: Chromatography

CHROMATOGRAPHY

I have thrown everything here and I will sort this note in a few days...

A little History

The word means “colour writing”, and the first

chromatographic separations were of coloured materials (dyes, inks)

Chromatography was
discovered by
M.S.Tswett in 1903

During chromatography three components interact

Sample (target analyte to be separated from matrix interferences)

- biological fluid such as urine or blood

- A mixture of proteins or enzymes

Stationary phase (immobilised)

- Paper (cellulose fabric)

- Thin plate covered in particles of different surface properties

- Particles packed into a column of glass or metal

Mobile phase (solvent)

- Typically a gas as in Gas chromatography (GC) or a liquid as in liquid chromatography (LC)

- Interactions between components vary according to the type of chromatography

Some main techniques

Paper:
Stationary phase is cellulose fibre.
Mobile phase passes by capillary action.

Thin layer:
Stationary phase is attached to matrix and coated on glass, plastic or metal plate.
Mobile phase passed over by capillary action (different samples can be run simultaneously)

Column:
Stationary phase attached to matrix packed into a glass or metal column.
Mobile passes through the column

Applications of chromatography in Biosciences

first, consider the importance of context

purify components (target analytes) from a complex mixture
urine, blood, plasma, tissue extracts, soil samples

identify components using qualitative analysis
proteins, peptides, endogenous molecules such as markers
of disease, xenobiotics, drugs of abuse, pesticides

size of molecules is all important. 100 or 10000 molecular weight?
how complex is the sample and what could interfere with measuring the target analyte?

measure components using quantitative analysis

A Target Analyte

The concept of a target analyte in an assay refers to a chemical constituent or constituents which need to be identified and quantified (characterised) in answer to a specific scientific hypothesis

Is a drink-driver suspect’s measured blood alcohol level >or < or = 80 mg ethanol/ml blood? (legal contexts)

Is an individual’s cholesterol level decreasing due to statin therapy, or does he need a higher dose? (clinical contexts)

Are levels of pesticides in London’s drinking water below the European guidelines on public health? (environmental contexts)

What are interfering constituents in the sample matrix?

Revise the constituents of blood ( and urine)

Main constituents of blood,

Plasma and urine are?

Hint: pH, density, cells, size, proteins, enzymes, buffering

What to do about the clotting process?

How is the sample prepared for chromatography?

The concept of partitioning

Separations are based on differential migration through a porous medium

Different chemical species prefer to occupy different physical environments (phases)
These chemical species will move to occupy their preferred environment if exposed to two different environments at the same time
This is known as partitioning and results in the separation of the two species

molecules separate (partition) on the basis of:
                Size, Shape
                Mass, Charge
                Solubility
                Adsorption properties

some basic techniques of separation

• filtration: select components by particle size

• floatation: select components by density

• crystallization: select components by solubility

• extraction: select components by solubility

• distillation: select components by boiling point

• chromatography : select components by affinity for a 'stationary phase'

Paper chromatography

Stationary phase is cellulose fibre.
Mobile phase passes by capillary action.

Thin Layer Chromatography
where stationary phase is polar (silica) and mobile phase is non-polar (hexane)
this is termed NORMAL PHASE CHROMATOGRAPHY

Yellow constituent has the highest affinity for the solvent

Green constituent has higher affinity for the stationary phase

Apply sample above

the mobile phase

Column Chromatography

Which mobile or stationary phase?

The choice of mobile or stationary phase depends on the characteristics of the compound (solute) being separated.

Methods are routinely available in labs, textbooks, references and scientific papers

Numerous combinations of Stationary and Mobile Phase are possible, separation depends on temperature, pH and other physical factors:

Adsorption equilibrium
Partition equilibrium
Ion-exchange equilibrium
Size equilibrium
Affinity equilibrium

Adsorption equilibrium:

between solid stationary and a liquid mobile phase (adsorption and hydrophobic interaction chromatography)

Partition equilibrium:

between stationary liquid phase and a mobile liquid or gas phase (Partition, reverse-phase, ion-pair, gas-liquid and countercurrent chromatography)

Ion-exchange equilibrium:

between stationary ion exchanger and mobile electrolyte phase (ion-exchange and chromatofocusing)

Next weeks practical uses ion-exchange TLC to separate ATP, ADP and AMP:

Adenosine = adenine + ribose, on the basis of different charges

Size equilibrium:

between liquid phase trapped in stationary porous structure and a mobile liquid phase (size exclusion chromatography)

Affinity equilibrium:

between stationary immobilised ligand and a liquid mobile phase (affinity, immunoaffinity, metal chelating covalent chromatography)

Solvent polarities (remember like dissolves like)

water                                    most polar
methanol
ethanol
propanone (acetone)

ethyl acetate
diethylether                       decreasingly polarity

dichloromethane

toluene
cyclohexane
petroleum ether
hexane                                 least polar

Liquid/liquid

The concept of partitioning

the toxic chemical constituents in a solution/mixture of mussels

If contaminated mussels

cause food poisoning

deaths, this experimental

strategy could be used

to determine the toxic

constituent

By testing solvents or

residues in mice, it is

possible to determine

which extract contains

the toxin

The two separation techniques chromatography (partition) and electrophoresis (size and charge) separate on different properties of the analytes. They are both used to locate the toxin, which is finally confirmed by characterising the chemical structure.

The chromatography process

load the analytes on top of the column or at the base of the thin layer.
Introduce the mobile phase
the components differentially eluted through the matrix material (development)
the separated analytes are separated into fractions or cuts

What determines how compounds separate?
Partition Coefficient a :
This is dependent on the relative affinity for the mobile and stationary phase
Partition Coefficient may be 0 and 1
The greater the value the more affinity for stationary phase

                molecules adsorb on the stationary phase
a =         ---------------------------------------------
                Molecules in mobile and stationary phase

Detectors (more in later lectures)

universal detector responds to all compounds eluting from a column
(John Wrights optical methods lectures)

selective/specific detector responds only to certain elements or functional groups

sensitivity: the ratio of detector signal to sample size (or detector response per amount of sample)

Minimal detectable level (MDL) the amount of sample in which the peak height is at least twice the noise height

Partition Chromatography

In Partition Chromatography there is a partition equilibrium between a stationary liquid phase and a mobile liquid or gas phase

Examples include:
Partition, reverse-phase, ion-pair and gas
liquid chromatography.

Partition can be predicted by the partition coefficient, a , of a substance
                                Concentration of compound in
                                solvent A at equilibrium
                a =         ---------------------------------
                                Concentration of compound in
                                solvent B at equilibrium

Two principle forms of partition chromatography:

Normal phase (as we saw in the earlier TLC example)

polar stationary phase such as silica
non-polar mobile phase such as organic solvent hexane, ethylacetate, dichloromethane

Separation results as the analyte displaces molecules of the mobile phase

The least polar molecules will elute first,

Polar elute molecules last

Highly polar molecules may require polar gradient elution (more on gradients later)

Reversed phase chromatography


Non-polar stationary phase

Only non-polar interactions with the stationary phase are possible

The bonded liquid stationary phase is usually alkylsilicane attached to silica

Common groups:
                                Butyl (C4)
                                Octyl (C8)
                                Octadecyl (C18)

Common mobile phases for reversed phase include:

                                                Water
                                                Aqueous buffers
                                                Acetonitrile
                                                Usually in a mixture

Separation is achieved by altering the mobile phase
Significant changes may be made by varying salt, pH or organic solvent

Polar molecules elute first

Non-polar molecules elute last

Non-polar molecules may also require a low polarity gradient in the mobile phase to remove them from the column

Ion exchange chromatography

definition of ion exchange

ion exchange chromatography is broadly defined as the separation of compounds based on the attraction of oppositely charged molecules

in other words, can be used to separate charged molecular species

This physical separation relies on differential partitioning of charged ions between 2 phases: stationary phase (solid support material) and mobile phase (solvent)

Ionization Equilibria for aqueous solutions

ion exchange in biology

many biological materials have ionisable groups: amino acids and proteins

the fact that they carry a net + or – charge can be exploited in separating them

the net charge depends on their pKa and the pH of the solution (Henderson-Hasslebach equation)

ionisable stationary phase

ion exchange materials are known as anionic or cationic according to their affinity for either negative or positive ions. (an) (Cap)

these materials can be grouped into 2 further groups

strongly ionised -SO₃H and –N⁺R₃

weakly ionised –COOH, -OH, -NH₂

Paper chromatography
Stationary phase is cellulose fibre.
Mobile phase passes by capillary action.

TLC

• Liquid chromatography system consists of a stationary phase, usually alumina, silica gel or cellulose and a mobile phase (solvent)

Resolution and spot size in TLC

Rs = X

0.5 (d1 + d2)

X is the distance between the centres of the 2 spots and d1 and d2 are the average diameters of the spots

Components are just separated when Rs = 1

So, it is possible to increase resolution either by increasing the separation of the spots or by decreasing the diameter of the spots (HOW?)

Stationary phases –
TLC plates

• Thin layer of adsorbent spread onto a glass, plastic or foil plate in a slurry form, then dried at high temperature

• Final surface of plate is almost chalk-like, so avoid touching or scraping the surface off

• Weak adsorbents: Sucrose, starch, talc

• Medium: calcium carbonate, magnesia, calcium hydroxide (surface area of 10-50 metres/gram)

• Strong: activated silica, silica gel and alumina (surface area of 100-500 metres/gram)

Strongly ionised groups

-SO₃H and –N⁺R₃

are completely ionised and are charged except at extreme pH values

acidic conditions -SO₃H

under basic conditions gives -SO₃^- + H⁺

basic conditions -NR₃OH

under acidic conditions gives –N⁺R₃ + OH^-

weakly ionised groups

–COOH, -OH, -NH₂

in ion exchange materials provide pH dependent groups whose maximum capacity is over a narrow pH range

-COOH reversibly gives – COO^- + H⁺

-NH3+ reversibly gives – NH₂ + H⁺

an ion exchange matrix (resin)

polystyrene cross-linked with divinyl benzene (insoluble resin)

selectivity can be modified by adding different

functional groups (give example in class)

properties of ion exchange resins

• PSDVB resin swells in contact with water (care in packing the column)

• extent of swelling depends on the degree of crosslinking

• the greater the DVB content the less water is retained

• cross-linking can be controlled to obtain a sieving effect as well as ion exchange

Cellulose

• Plant material, porous structure, smaller pores than paper

• Solvent flows evenly and spots are tight

• Used to separate hydrophilic compounds such as sugars, amino acids, soluble inorganic ions and nuclei acids (these would adhere too strongly to silica or alumina)

chemically-modified cellulose

• alternative to PSDVB

• available in gel and bead forms

• good flow properties

– carboxymethylcellulose = CM cellulose (weakly acidic)

– Polyethyleneimine = PEI cellulose (weakly acidic)

– diethylaminoethylcellulose = DEAE cellulose (strongly basic)

Chemistry of Adenosine

a simple
ion exchange column

• describe how to pack a column

• how to collect fractions

• commercial resins may contain iron or other heavy metals that must be removed by washing with appropriate solution

anion exchange mechanism

• positively charged groups

• attract negatively charged molecules

• use a cationic buffer: Tris, pyridine, alkylamines

5 steps

diffusion of ion to the exchanger surface
diffusion of ion through matrix to the exchange site
exchange of ions at exchange site
diffusion of the exchanged ion through the matrix to the surface
selective desorption of the target ion by the eluant

anion exchange TLC practical (SAX)

• separation of ATP, AMP and adenosine

• Polyethyleneimine (PEI)-cellulose is a weak anion exchange chromatographic packing material.

• At acid and neutral pHs the material is positive charged

• It will thus bind negative ions (ATP, ADP and AMP)

• Adenosine is neutral and does not bind to the plate

Beer lambert law

anion exchange HPLC (SAX)

Pepsins in Human Gastric Juice
The PL-SAX can be routinely used for the

identification of pepsins 1, 5, 3a, 3b and 3c

in peptic ulcer disease.

Excellent resolution facilitates quantitation, if required.

Sample: 250µl human gastric juice dialyzed against 0.05M sodium acetate, pH 4.1. Filtered 0.45µm.

Column: PL-SAX 1000Å 8µm, 50x4.6mm
Eluent A: 50mM Sodium acetate, pH 4.1
Eluent B: A + 1.0M NaCl
Gradient: 0-100% B in 20 mins
Flow Rate: 1.0ml/min
Detector: UV, 280nm

cation exchange mechanism

• negatively charged groups on exchanger

• attract positively charged molecules

• use an anionic buffer: acetate, barbiturate & phosphate

Sepharose type exchangers

• cross-linked agarose

• good for separating high molecular weight proteins and nucleic acids

• possibly some molecular sieving also occurs

cation exchange HPLC (SCX)

• Standard Protein Separation
Proteins which have a +ve charge at pH 6.0 will be retained by the cation exchanger. The more basic the protein, such as lysozyme (pl 11.8), the longer the elution time from the column under a typical NaCl gradient

Column: PL-SCX 1000Å 8µm, 50x4.6mm
Eluent A: 20mM KH2PO4, pH 6.0
Eluent B: A + 1.0M NaCl
Gradient: 0-100% B in 20 mins
Flow Rate: 1.0ml/min
Detector: UV, 280nm

KEY
1. Myoglobin
2. Chymotrypsinogen A
3. Cytochrome C

choice of ion exchanger 1

• stability of target analytes and sample matrix

• molecular weight

• biological components often need narrow pH range

• must select exchanger carefully on this basis

• if a sample is most stable below its isoionic point giving it a net + charge, use a cation exchanger

• conversely, if it is most stable above its isoionic point giving it a net – charge, use an anion exchanger

• samples stable over a wide pH range may be able to use either (see which works best)

weak electrolytes

• that need either a very low or high pH for ionisation can only be separated on strong exchangers

strong electrolytes

• use a weak exchanger

• less chance to denature the sample

• weak X does not bind weakly charger impurities

• enhanced elution characteristics

gradient elution

• continous or step-wise

• ionic strength gradients

• pH gradients

gradient elution for amino acids

• use a strong acid cation exchanger

• introduce sample at pH 1-2

• this ensures binding of all amino acids

• gradient elution using increasing pH and ionic concentration gives sequential elution of amino acids

• acidic AAs aspartic and glutamic elute first

• neutral AAs next: glycine and valine

• basic AAs lysine and arginine keep the net + charge up to pH value 9-11 and elute last

amino acid analyser

Proteins are first converted to their constituent amino acids by hydrolysis in constant boiling hydrochloric acid, at 110C, under vacuum for 24 hours.

After removal of the acid, the amino acids are separated by ion exchange chromatography, using a stepwise buffer gradient on a strong cation exchange column. The acidic amino acids elute first, followed by the neutrals, then the basic ones at the end.

After separation, amino acids are visualised by their reaction with ninhydrin.

4. They are quantified by comparison with a calibration mixture of amino acids.

TLC

• Liquid chromatography system consists of a stationary phase, usually alumina, silica gel or cellulose and a mobile phase (solvent)

Paper chromatography
Stationary phase is cellulose fibre.
Mobile phase passes by capillary action.

The concept of competitive equilibrium

• Partitioning of molecules of the sample between stationary and mobile phase

• 10 possible interactions:

• Solute-solute (association & dissociation)

• Solute-solvent

• Solvent-solvent

• Solute-adsorbent-solvent (sorption & desorption)

Adsorption is the basis of TLC

• Adsorption is the ability of a solid to attract other molecules to its surface and to hold them at the surface

• Adsorbents have porous surfaces, which increases surface area for adsorption

• No chemical reaction occurs between adsorbents and adsorbed materials

• Adsorbents can be chemically altered to give different selectivities

Stationary phases – TLC plates

• Thin layer of adsorbent spread onto a glass or foil plate

• Weak adsorbents: Sucrose, starch, talc

• Medium: calcium carbonate, magnesia, calcium hydroxide (surface area of 10-50 metres/gram)

• Strong: activated silica, silica gel and alumina (surface area of 100-500 metres/gram)

Properties of Stationary phases

• Inert material

• Reproducible (same chemical state each time?)

• Heat plates to deactivate, remove moisture

• Particle size is important – why?

• Layer thickness is important why?

– 0.25mm, 0.5 mm, 1.0 and 2.0 mm

– Up to 1 mg per plate is analytical scale

– >>> 1 mg overloads the plate and loses efficiency of separation

• Commercial plates – binder (G), no binder (H), fluorescent indicator (F254)

Silica gel

• Surface is covered in silanol groups (-Si-OH)

• Different gels have different numbers of –Si-OH groups, this gives different selectivity

• Slightly acidic in nature

• Used to separate steroids, amino acids, alcohols, hydrocarbons, lipids, bile acids, vitamins and alkaloids

Cellulose

• Plant material, porous structure, smaller pores than paper

• Solvent flows evenly and spots are tight

• Used to separate hydrophilic compounds such as sugars, amino acids, soluble inorganic ions and nuclei acids (these would adhere too strongly to silica or alumina)

Mobile phase (solvents)

• Cheap, analar grade

• Low boiling point (easy to remove solvent at low temperature)

• Unreactive with the adsorbent and the analytes of interest

• Must displace the solute from adsorbent into the mobile phase

• Must be able to separate a mixture of solutes so they can be identified - this is termed the solvent selectivity

• UV cutoff differs between solvents (dichloromethane 245 nm)

Simple starting point

• Start with a nonpolar solvent

• Add a more polar solvent in increasing steps

• 2, 4, 8, 16, 32% polar solvent

• This is equal to a increase in Eo of about 0.05 units

• Aim for the target Eo

Resolution and spot size in TLC

Rs = X

0.5 (d1 + d2)

X is the distance between the centres of the 2 spots and d1 and d2 are the average diameters of the spots

Components are just separated when Rs = 1

So, it is possible to increase resolution either by increasing the separation of the spots or by decreasing the diameter of the spots (HOW?)

TLC Detection/visualization

• Destructive versus non-destructive methods

• Visible radiation (coloured compounds, dyes, pigments in paints, food colourants)

• UV use lamp, (rarely some compounds may undergo chemical changes)

• Iodine vapour (iodine dissolves in the solutes and rapidly volatilises out of the spots leaving a blank plate within 30 mins

• Lipid compounds can be sprayed with water, leaving a colourless spot

Destructive TLC Stains

• Ninhydrin: 1^o or 2^o amines (sympathomimetics)

• Iodoplatinate: 3^o amines, alkaloids

• UV absorption at 254 nm: benzodiazepines, barbiturates, methaqualone

• Fluorescence at 366: Benzodiazepines, quinine, quinidine

TLC method development

• What is the sample matrix and how does it need to be prepared

• What are the likely analytes (individual or class of molecules)?

• Most appropriate visualization technique?

• What are the likely concentrations present in the sample? How much to load on plate?

• How to validate the method, using standards and widely accepted methods

Limitations

• Overloading of sample

• Must use reference standards

• Qualitative

• Semi-quantitative (10% error)

• Complex mixtures and interference

• Detection limits vary

• How to develop a method

Reproducibility

• Solvent chamber saturation time

• Quality and quantity of mobile phase

• Freshly prepared solution (volatiles)

• Temperature and pH

• Adsorbent activity

• Techniques and conditions

• Use of standards must always be employed

• STANDARDISE YOUR OWN PROCEDURE

Quantitation by TLC**

• For best results spots should have an Rf of between 0.3 and 0.7. Below 0.3 are too concentrated and above 0.7 are too difuse

• Visualise and scrape spot off plate

• Add solvent to centrifuge tube and mix

• Centrifuge and remove supernatant

• Analyse supernatant by UV or HPLC-UV

• Make up serial dilution series and always spot same volume of sample on plate

Level of sensitivity for quantitation

• Visible spectrometry 100 microgram

• UV spectrometry 50 microgram

• NMR spectrometry 10,000 microgram

• Gas-liquid chromatography

1 microgram

Chromatographic Column

HPLC Retention

Major parameters,

• V_R is retention volume, depends on the column type, size, and the instrument parameters

• V_o is dead volume, volume of the liquid phase inside the column

• k’ is retention factor (capacity factor),

independent of the column size and instrument setup

Retention Characteristics

Dead Volume

V_o, Dead volume is the volume of the liquid phase in the column

Simple rule:

Column dead volume = 65% of the volume of empty column

typical HPLC system

Retention Parameters**

HPLC Selectivity**

Resolution

Efficiency

Efficiency Parameters**

Column Efficiency

• Column length is a compromise between the efficiency and backpressure

• Column efficiency is proportional to the column length

• Specific efficiency (# of particles per one plate) decreases with an increase of column length

Silica

n rigid porous (or nonporous) particles

n wide variety of particle and pore sizes

n soluble in water at pH > 8

Column chemistry

silica particles (2-10 micron)

bonded chemistry: C18, C8, C6, C2, CN

tightly packed to minimise deadspace, requires high pressures to force solvent through (> 100 bar or >1 400 psi)

analytical scale- ng – mgs of material
preparative scale – mgs-grams of material

Factors Influencing HPLC Separation

Parameters affecting efficiency:

- Flow rate

- Column length

- Particle diameter

- Particle size distribution

Parameters affecting retention factor:

- Eluent type

- Eluent composition

- Stationary phase type

- Analyte nature

Parameters affecting selectivity

- Stationary phase type

- Analyte nature

- Eluent additives

- Temperature

- Eluent composition (ionisable analytes)

Reversed Phase
Separation Principle

• Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-C18, C8, Phenyl, C4)

• Difference in analyte sorption affinities results in their separation

• More polar analytes retained less

• Analytes with larger hydrophobic part are retained longer

• Almost no separation of structural isomers

Why use the term reversed-phase chromatography?

silica TLC plates were originally used by pharmaceutical chemists for separating compounds from organic solvents

As HPLC systems evolved in response to industry needs and advancing instrumentation, pharmacologists, toxicologists and biomedical scientists had an urgent need to identify analytes from biofluids. This required nonpolar stationary phases and polar mobile phases

The underlying principle was thus reversed, and the majority of analyses in bioscience today tend to be reversed-phase

Reversed-Phase HPLC Retention:

Neutral Analytes

Dependencies of retention of alkylbenzenes, alkylphenones, and alkylparabenes plotted against the number of carbon atoms in alkyl chain.

ln(k’)= m (#carbon atoms in alkyl chain) + b

High performance liquid chromatography

Choice of normal or reversed-phase system depends on the sample matrix and target analytes
Choice of mode depends on selectivity required and presence of interfering substances in the matrix
Main advantages:
                automation
                wide variety of parameters to achieve selectivity
                Wide variety of detectors: UV, MS, Fl, RI

Eluent Composition Effect on Selectivity

Gradient elution
alter a given chromatographic parameter over time
typically temperature, buffer strength, organic solvent composition
can retain sharp peaks throughout the separation, minimise band broadening

Opiate drugs by HPLC

Variety of column chemistries

Liquid-chromatography-mass spectromety results

Sample preparation

critical that solution contains no particulates that could block the column

centrifuge, filter

urine analysis – dilute and shoot

blood analysis – cannot direct inject, can inject diluted serum

References

Practical HPLC, Meyer, 1989

Practical skills in Forensic Science, Langford, A & colleagues (2005)

Any analytical biochemistry or chemistry book that covers the basics of chromatography

Chromatography Lecture 4

Learning Objectives:

to differentiate between the contexts of qualitative and quantitative analysis and validation terms accuracy and precision

to be able to compare the methods of detecting compounds: UV, colour reagents, sensitivity, selectivity

to be able to list methods for confirming identity;
use of standard compounds, reagents, mass spectrometry and nuclear magnetic resonance

performance of relevant calculations: of capacity factors and resolution

Qualitative analysis – chromatography can indicate the presence or absence of a compounds, elements, or ions in a sample. Eg: drugs in biofluids

Quantitative analysis – chromatography can also provide information on the chemical composition of a mixture, for example the proportion of an active ingredient in a tablet

Qualitative analysis

relies on the use of chemical standards for the target analytes, that is, available known compounds

there are many published methods for separation, in textbooks & catalogues, but mostly obtained from scientific journals

a major underlying assumption is that each spot (in TLC), or peak (in HPLC) has unique properties to other similar molecular structures

such as? Drug metabolites can be similar in structure to parent drug analytes

Quantitative analysis

depends on the context, what is the objective?

measure the amount in mg/mL or M of analyte

available standards?

available method

validate the method

the importance of precision and accuracy
example of the class analysing blood alcohol for Newham Police drink drive case. Half measure 79 mg/mL of ethanol in blood and the other half measure 82 mg/mL of ethanol in blood. Which is the right answer? What might be the source of errors in the analysis?

TLC – is it qualitative or quantitative?

Recall the PEI chromatography practical

what are the main limitations?
                Available detection methods
                sensitivity of detection limits
                overloading leads to band broadening

How do we know we have achieved a
successful separation?

The success of a separation can only be measured by using an appropriate detection method

Detection methods and their sensitivity ranges

UV/VIS variable wavelength down to 190 nm and 0.001 ABS        5 x 10^-10

UV/VIS scanning wavelength. A diode array detector measures all wavelengths together within 0.01 s, can then select the ones of interest
                                                                                                                                5 x 10 ^-10

Fluorimetry very sensitive, however limited to compounds that fluoresce
                                                                                                                                1 x 10 ^-11

Refractive index substances in solvent have different RI, not selective, but useful when there is no chromophore (ie sugars, steroids)                               5 x 10 ^-7

Conductive and amperometric an electical potential is applied to a cell this leads to molecules undergoing either an oxidation or reduction. This results in a current through the cell, which can be measured               10^-8-10-10

Mass spectrometry measures the masses of ions. Highly sensitive          > 1 10^-10

radioactivity very sensitive, not much used as need to incorporate isotopes
                                                                                                                                > 1 10^-10

How do we know we have achieved a
successful separation?

We need to mathematically state the result of a separation

The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time (t_R).

Each analyte in a sample will have a different retention time. The time taken for the mobile phase to pass through the column is called t_M.

A term called the retention factor (or capacity factor), k', is often used to describe the migration rate of an analyte on a column.

The retention factor for analyte A is defined as;

k'_A = t _R - t_M / t_M

t _R and t_M are easily obtained from a chromatogram.

When an analytes retention factor < 1, elution is so fast that accurate determination of the retention time is very difficult.

High retention factors (> 20) mean that elution takes a very long time.

Ideally, the retention factor for an analyte is between one and five.

We define a quantity called the selectivity factor, a , which describes the separation of two species (A and B) on the column;

a = k 'B / k 'A

When calculating the selectivity factor, species A elutes faster than species B. The selectivity factor is always greater than one.

Possible criteria for selection of a particular chromatography method

• The required level of accuracy and precision

• The number of samples to be analysed

• The amount of each sample available for analysis (eg: urine vs saliva)

• The physical form of the samples (solids, liquids, tissues)

• The expected concentration range of the analyte in the samples

– picograms, nanograms, micrograms, milligrams

• The sensitivity and detection limit of the technique

• The likelihood of interfering substances (preparation required?)

• The speed of the analysis

• The ease and convenience of the procedure

• The skill required by the operator

• Cost and availability of the equipment

Validation

• Is the process whereby accuracy and precision of a particular analytical method are checked in relation to specific standards, using an appropriate reference material containing a known amount of analyte

• is a method fit for purpose?

• under which conditions, limitations, margins of error

• instrumentation

• calibration

• validation

Terms of Validation

• Selectivity is the extent to which a method is free from interference due to other substances n the sample

• Sensitivity is the ability to discriminate between small differences in analyte concentration

• Detection limit is the minimum amount of concentration of an analyte that can be detected at a particular confidence level

• Sources of errors: method, instrument, materials, analyst

precision and accuracy

• Precision is the extent of mutual agreement between replicate data values for an individual sample

• (high accuracy but low precision)

• Standard deviation s and relative standard deviation % RSD see separate learning material

• Accuracy is the closeness of an individual measurement, or a mean value based on a number of measurements, to the true value

• (high precision but low accuracy)

precision and accuracy

Internal standard 1

where you add a known amount of a reference substance (not originally in the sample) to the sample to give an additional peak in the HPLC profile

then determine the response of the detector to both the test and reference substances, by analysing a standard containing known amounts of both substances to provide a response factor (r) where

r = peak area (or height) of test substance
      peak area (or height) of reference substance

use this response factor to quantify the amount of test substance (Qt) in a sample containing a known amount of the reference substance (Qr):

Qt = [peak area (or height) of test substance]          x    Qr
        [peak area (or height) of reference substance]        r

Internal standard 2

add to the sample at the first stage in the extraction so any loss or degradation of test substance during purification will have a similar effect on the internal standard

only holds true if the extraction characteristics are the same for the IS and the target analyte

IS should be chemically similar to the test substance

hopefully of similar polarity, but not identical WHY?

Typical HLPC chromatograms

LHS shows blank sample, note the Internal Standard (IS)

RHS shows 4 standard peaks added, labelled A-D

Example of a standard curve

Peak height ratio is used in this example

Use height or area under the peak, which is best and why?

Pharmacokinetic of Mebudipine (b) in Rabbits

Three adult male albino rabbit were each administrated single bolus intravenous doses of 0.50 mg/kg mebudipine dissolved in 60% PEG 400). Blood samples were collected from marginal ear vein at 5, 10, 20, 30,60, 120, 180,240 min after mebudipine administration

To one ml of plasma sample were added, 10 μl of internal standard (dibudipine, 20 mg/ml) solution and 0.5 ml of 1M NaOH.

The solution was mixed for a few seconds. 5 ml ethyl acetate was added to the solution which was subsequently shaken on horizontal shaker for 10 minutes. Evap under N2 and recon in 200 ul mobile phase

Intra and interday variation of mebudipine assay in rabbit plasma

mean (n=3) calibration curve for mebudipine was y=0.008x - 0.0022, r2 =0.9989 where, y and x are the peak height ratio and concentration (ng/ml), respectively. Mebudipine @ 10 ng/ml could be quantified.

Mean mebudipine plasma concentration-time profile in rabbits following IV administration of 500 mg/kg mebudipine. Each point represents the mean ± SE for three rabbits.

MHRA advisory April 2006

• As of 30 March 2006, seven reports of suspected adverse reactions associated with Polygonum multiflorum have been reported to the MHRA through the Yellow Card Scheme.

• All 7 reports are of liver reactions and comprise one report of abnormal liver function, 3 reports of jaundice, 2 reports hepatitis and one report of jaundice and hepatitis. The patients, 5 women and 2 men aged from 36 to 70 years old, were taking Polygonum multiflorum for hair loss (3 patients had taken the product Shen Min and 3 patients had taken the product Shou Wu Wan). All the patients had recovered or were recovering after stopping Polygonum multiflorum.

HPLC of methanol extracts from tablets

LC-ESI-Mass spectrometry

MHRA interest

Is it possible to discern which level of processed drug is present in a given OTC

Is the level of HSB interesting?

Is it possible to discern which level of processed drug is present in a given OTC

Is the level of HSB interesting?

J Gao, A Sanchez-Medina, BA Pendry, MJ Hughes, GP Webb and O Corcoran. Validation of a HPLC method for flavonoid biomarkers in skullcap (Scutellaria) and its use to illustrate wide variability in the quality of commercial tinctures. Journal of Pharmacy and Pharmaceutical Sciences, 2008, 11 (1): 77-87.

A Sanchez-Medina, CJ Etheridge, GE Hawkes, PJ Hylands, BA Pendry, MJ Hughes and O Corcoran. Comparison of rosmarinic acid content in commerical tinctures produced from fresh and dried lemonbalm (Melissa officinalis). Journal of Pharmacy and Pharmaceutical Sciences, 2007, vol 10, no 4, 455-463.

Find these scientific articles using Pubmed or Science Direct.

Test your understanding of the technical aspects of the paper, as related to the theory covered in these 6 lectures.

job descriptions requiring experience in chromatography

PDP, how do we get chromatography experience at UEL?

Hot job websites

www.newscientists.com
www.jobs.ac.uk

careful keyword search

London, UK, Europe, etc

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm

this is an appropriate academic website tutorial

Remember the story of the UEL graduate who won a job in LGC Ltd because the boss noticed her 3^rd year undergraduate project title?

It could be YOU!

Check out the Medicines Research Group at UEL and associated 3^rd year projects on HPLC and quality analysis.
http://www.uel.ac.uk/mrg

Section E: chromatography

1. which separation mode is typically used to separate drugs from proteins?

2. distinguish between the use of quantitative and qualitative chromatography

3. which equation defines the resolution of two HPLC peaks?

4. which detection methods are suitable for proteins, small drug molecules?

5. briefly describe gradient elution and how it can be used in chromatography

6. why is reversed-phase chromatography so named?
describe an application of reversed-phase chromatography in biofluid analysis

7. draw a flow chart for the main components of a HPLC system. Where in the system does the chemistry of separation takes place?