vogel's textbook of quantita

8/10/2019 Vogel's Textbook of Quantita

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OLUMN ND

THlrH YER

LIQUID HROM TOGR PHY

packing techniques are used in which the particles

ar e

suspended in a suitable

solvent

an d

the suspension or slurry) driven into the

column under

pressure.

Th e essential features for successful slurry packing of columns have been

summarised.P Many analysts will, however, prefer to purchase the commercially

available

HPLC

columns, for which

the appropriate manufacturer s

catalogues

should be consulted.

Finally, the useful life of an analytical column is increased by introducing a

guard column. This is a

short column

which is placed between

th e

injector

an d

the HPLC column to

protect the

latter from

damage

or loss of efficiency caused

by particulate

matter

or strongly adsorbed substances in samples or solvents.

ma y also be used to saturate th e eluting solvent with soluble stationary phase

[see Section 8.2 2)J. Guard columns may be packed with microparticulate

stationary phases or with porous-layer beads; the latter are cheaper and easier

to pack than the microparticulates, bu t have lower capacities and therefore

require changing more frequently.

Detectors. Th e function

of

the

detector

in HPLC is to monitor the mobile phase

as it emerges from th e column. Th e detection process in liquid

chromatography

has presented more problems

than

in gas chromatography; there is, for example

no equivalent to th e universal flame ionisation detector of gas chromatography

for use in liquid

chromatography.

Suitable detectors ca n be broadly divided

into

th e

following two classes:

a

Bulk property

detectors

which measure the difference in some physical

property of the solute in the mobile phase compared to th e mobile phase

alone, e.g. refractive index and conductivity* detectors. T he y a re generally

universal in application bu t tend to have poor sensitivity an d limited range.

Such detectors ar e usually affected by even small changes in the mobile-phase

composition which precludes

th e use

of

techniques such as gradient elution.

b Solute

property detectors,

e.g. spectrophotometric, fluorescence an d electro

chemical detectors. These

respond

to a particular physical

or

chemical

property of

the solute, being ideally independent of the mobile phase. In

practice, however, complete independence of the mobile phase is rarely

achieved, bu t the signal discrimination is usually sufficient to permit

operation

with solvent changes, e.g.

gradient

elution.

They

generally provide

high sensitivity

about

1 in 10

9

being attainable with UV and fluorescence

detectors) an d a wide linear response range but, as a consequence of their

more selective natures, more than one detector may be required to meet

the demands of an analytical problem. Some commercially available

detectors have a number of different detection modes built into a single

unit, e.g. the Perkin-Elmer

3D

system which combines UV absorption,

fluorescence

and

conductimetric detection.

Some of th e important characteristics required of a detector

ar e

the following.

a Sensitivity,

which is often expressed as the noise equivalent concentration,

i.e. the solute concentration,

Cn,

which produces a signal equal to th e

detector noise level.

Th e

lower the value of C

for a particular solute, th e

more

sensitive is the detector for

that

solute.

The conductance detector is a universal detector for ionic species and is widely used in ion

chromatography see Section 7.4).

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EQUIPMENT FOR

HPLC

8

b A linear response The linear range of a detector is the concentration range

over which its response is directly proportional to the concentration of

solute. Quantitative analysis is more difficult outside the linear range of

concentration.

(c)

Type ofresponse

i.e.whether the detector is universal

or

selective. A universal

detector will sense all the constituents of the sample, whereas a selective

one will only respond to certain components. Although the response of the

detector will

not

be independent of the operating conditions, e.g. column

temperature

or

flow rate, it is advantageous if the response does

not

change

too much when there are small changes of these conditions.

A summary of these characteristics for different types of detectors is given

in Table 8.2.

Table 8.2 Typical detector characteristics in

HPLC

Type

Amperometric

Conductimetric

Fluorescence

UV

/visible absorption

Refractive index

Response

Selective

Selective

Selective

Selective

Universal

10

10

10

7

z

10

8

10

6

Linear range

10

4 - 1 0

5

10

3 -10 4

10

3 - 1 0

4

10

4 - 1 0

5

10

3 - 1 0

4

The range

over

which the response is essentially linear is expressed

as the factor by which the lowest concentration

en

must be

multiplied to obtain the highest

concentration.

A detailed description of the various detectors available for use in

HPLC

is

beyond the scope of the present text

and

the reader is recommended to consult

the monograph by Scott.55 A brief account of the principal types of detectors

is given below.

Refractive index detectors

These bulk property detectors are based on the

change of refractive index of the eluant from the column with respect to pure

mobile phase. Although they are widely used, the refractive index detectors

suffer from several disadvantages - lack

of

high sensitivity, lack of suitability

for gradient elution, and the need for strict temperature control 0.001 C

to operate at their highest sensitivity. A pulseless pump, or a reciprocating pump

equipped with a pulse dampener, must also be employed. The effect of these

limitations may to some extent be overcome by the use of differential systems

in which the column eluant is compared with a reference flow of pure mobile

phase. The two chief types of RI detector are as follows.

The deflection refractometer (Fig. 8.4), which measures the deflection of a

beam of monochromatic light by a double prism in which the reference

and

sample cells are separated by a diagonal glass divide. When both cells contain

solvent of the same composition, no deflection

of

the light beam occurs; i

however, the composition of the column mobile phase is changed because

of the presence of a solute, then the altered refractive index causes the beam

to be deflected. The magnitude of this deflection is dependent on the

concentration of the solute in the mobile phase.

2. The Fresnel refractometer which measures the change in the fractions of

reflected and transmitted light at a glass-liquid interface as the refractive

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COLUMN

ND

THIN t YER

LIQUID CHROM TOGR PHY

Mirror

i .

Light beam

~

Reflected beam

Reference

solvent

Fig. 8.4 Refractive index detector.

index

of

the liquid changes. In this detector both the column mobile phase

and a reference flow of solvent are passed through small cells on the back

surface of a prism.

When

the two liquids are identical there is no difference

between the two beams reaching the photocell,

but

when the mobile phase

containing solute passes through the cell there is a change in the

amount

of

light transmitted to the photocell,

and

a signal is produced. The smaller cell

volume about 3 ,uL in this detector makes it more suitable for high-efficiency

columns but, for sensitive operation, the cell windows must be kept

scrupulously clean.

ltraviolet detectors

The

UV absorption detector is the most widely used in

HPLC being based

on

the principle of absorption of UV visible light as the

effluent from the column is passed

through

a small flow cell held in the radiation

beam. is characterised by high sensitivity

detection

limit of

about

1 x

10-

9

g mL for highly absorbing compounds and, since it is a solute

property detector, it is relatively insensitive to changes of temperature and flow

rate. The detector is generally suitable for gradient elution work since many of

the solvents used in

HPLC

do not

absorb

to

any

significant extent at the

wavelengths used for monitoring the columneffluent. The presence of air bubbles

in the mobile phase can greatly impair the detector signal, causing spikes on

the chromatogram; this effect can be minimised by degassing the mobile phase

prior to use, e.g. by ultrasonic vibration. Both single

and

double beam

Fig. 8.5 instruments are commercially available. Although the original

detectors were single- or dual-wavelength instruments 254

and/or

280 nm ,

some manufacturers now supply variable-wavelength detectors covering the

range

2 ~ 8

nm so

that more

selective detection is possible.

No account

ofUV

detectors would be complete without mention of the diode

array multichannel detector, in which polychromatic light is passed

through

the flow cell. The emerging radiation is diffracted by a grating and then falls

on to an array of photodiodes, each photodiode receiving a different narrow

wavelength band. A microprocessor scans the

array

of diodes many times a

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\

Reference

photocell

Compound

UV filter

QUIPM NT FOR HPL

8 3

Hg lamp Quartz

Movable

source

lens \

calibrated

~

t

+

filter

Sample

~ h o t o e l l

+

Dual-

channel

cell

Fig. 8.5 Block diagram of a double-beam UV detector.

second

and

the spectrum so obtained may be displayed on the screen of a VDU

or s to red in the i ns tr um en t for subsequent print-out. An i mp or ta nt feature of

the multichannel detector is

that

it can be programmed to give changes in

detection wavelength

at

specified points in the chromatogram; this facility can

be used to clean

up

a chromatogram, e.g. by discriminating against interfering

peaks due to compounds in the sample which are not of interest to the analyst.

Fluorescence detectors These devices enable fluorescent compounds solutes)

present in the mobile phase to be detected by passing the column effluent through

a cell irradiated with ultraviolet light

and

measuring any resultant fluorescent

radiation. Although only a small proportion of inorganic and organic compounds

are naturally fluorescent, many biologically active compounds e.g. drugs)

and environmental contaminants e.g. polycyclic aromatic hydrocarbons) are

fluorescent and this, together with the high sensitivity of these detectors, explains

their widespread use. Because both the excitation wavelength

and

the detected

wavelength can be varied, the detector can be made selective. The application

of fluorescence detectors has been extended by means of pre- and post-column

derivatisation of non-fluorescent or weakly fluorescing

compounds

see

Section 8.4).

lectrochemical detectors The

term electrochemical detector in HPL

normally refers to amperometric or coulometric detectors, which measure the

current associated with the ox id ation or reduction of solutes. In practice it is

difficult to use electrochemical reduction as a means of detection in HPL

because of the serious interference large background current) caused by

reduction of oxygen in the mobile phase. Complete removal of oxygen is difficult

so

that

electrochemical detection is usually based on oxidation of the solute.

Examples of co mp ou nd s which can be conveniently detected in this way are

phenols, aromatic amines, heterocyclic nitrogen compounds, ketones, and

aldehydes. Since not all compounds undergo electrochemical oxidation, such

detectors are selective

and

selectivity may be further increased by adjusting the

potential applied to the detector to discriminate between different electroactive

species. may be noted here that an anode becomes a stronger oxidising agent

as its electrode potential becomes more positive. Of course, electrochemical

detection requires the use of conducting mo bile phases, e.g. containing inorganic

salts or mixtures of water with water-miscible organic solvents, but such

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COLUMN AND

THIN LAYER L nUID CHROMATOGRAPHY

conditions are often difficult to apply to techniques other than reverse phase

nd

ion exchange chromatography.

The amperometric detector is currently the most widely used electrochemical

detector, having the advantages of high sensitivity nd very small internal cell

volume. Three electrodes are used:

1. the working electrode, commonly made of glassy carbon, is the electrode at

which the electroactive solute species is monitored;

2. the reference electrode, usually a silver-silver chloride electrode, gives a stable,

reproducible voltage to which the potential of the working electrode is

referred;

nd

. the auxiliary electrode is the current-carrying electrode nd usually made of

stainless steel.

Despite their higher sensitivity nd relative cheapness compared with ultraviolet

detectors, amperometric detectors have a more limited range of applications,

being often used for trace analyses where the ultraviolet detector does not have

sufficient sensitivity.

8

ERIV TlS TlON

In liquid chromatography, in contrast to gas chromatography [see Section 9.2 2 J,

derivatives are almost invariably prepared to enhance the response ofa particular

detector to the substance of analytical interest. or example, with compounds

lacking an ultraviolet chromophore in the 254 nm region but having a reactive

functional group, derivatisation provides a means of introducing into the

molecule a chromophore suitable for its detection. Derivative preparation can

be carried

out

either prior to the separation pre-column derivatisation or

afterwards post-column derivatisation . The most commonly used techniques

are pre-column off-line

nd

post-column on-line derivatisation.

Pre-column off-line derivatisation requires no modification to the instrument

and, compared with the post-column techniques, imposes fewer limitations on

the reaction conditions. Disadvantages are that the presence of excess reagent

nd by-products may interfere with the separation, whilst the group introduced

into the molecules may change the chromatographic properties of the sample.

Post-column on-line derivatisation is carried

out

in a special reactor situated

between the column

nd

detector. A feature of this technique is that the

derivatisation reaction need not go to completion provided it can be made

reproducible. The reaction, however, needs to be fairly rapid at moderate

temperatures nd there should be no detector response to any excess reagent

present. Clearly n advantage of post-column derivatisation is that ideally the

separation

nd

detection processes can be optimised separately. A problem

which may arise, however, is that the most suitable eluant for the chromatographic

separation rarely provides an ideal reaction medium for derivatisation; this is

particularly true for electrochemical detectors which operate correctly only

within a limited range of pH, ionic strength

nd

aqueous solvent composition.

Reagents which form a derivative that strongly absorbs UV/visible radiation

are called chromatags;

n

example is the reagent ninhydrin, commonly used

to obtain derivatives of amino acids which show absorption at bout 570 nm.

Derivatisation for fluorescence detectors is based on the reaction of non

fluorescent reagent molecules ftuorotags with solutes to form fluorescent

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vogel's textbook of quantita

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