detector in hplc

8/10/2019 Detector in HPLC

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Detectors.The function of the detector in HPLC is to monitor the mobile phase as it emerges from the

column. The detection process in liquid chromatography has presented more problems than

in gas chromatography; there is, for example no equivalent to the universal flame ionisation

detector of gas chromatography for use in liquid chromatography. Suitable detectors can bebroadly divided into the following two classes:

(a) Bu lk property detectorswhich measure the difference in some physical property of the

solute in the mobile phase compared to the mobile phase alone, e.g. refractive index and

conductivity* detectors. They are generally universal in application but tend to have poor

sensitivity and limited range. Such detectors are usually affected by even small changes in

the mobile-phase composition which precludes the use of techniques such as gradient

elution.

(b) Solute property detectors, e.g. spectrophotometric, fluorescence and electrochemical

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, but the signal discrimination is usually sufficient to permitoperation with solvent changes, e.g. gradient elution. They generally provide high sensitivity

(about 1 in 109 being attainable with UV and fluorescence detectors) and 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 the important characteristics required of a detector are 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 the detector noise level. The lower the

value of Cn

for a particular solute, the more sensitive is the detector for that solute.b) A li near 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


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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 eluantfrom 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.

1. 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 areseparated by a diagonal glass divide. When both cells contain solvent of the same

composition,no deflection of the light beam occurs; if, 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 theconcentration 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 index ofthe 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 phasecontaining 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.

Ultraviolet 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

Fig. Refractive index detector.


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through a small flow cell held in the radiation beam. It is characterised by high sensitivity

(detection limit of about 1 x 10- 9 g mL -1 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 column effluent. The presence of air bubbles in the mobile phase cangreatly 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 detectorscovering the range 210~800

nm so that more selective

detection is possible. No

account of UV 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 narrowwavelength band. A microprocessor scans the array of diodes many times

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

stored in the instrument for subsequent print-out. An important feature of themultichannel detector is that it can be programmed to give changes indetection

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 environmentalcontaminants (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

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


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Electrochemical detectors. The term 'electrochemical detector' in HPLC normally

refers to amperometric or coulometric detectors, which measure the current associated

with the oxidation or reduction of solutes. In practice it is difficult to use electrochemical

reduction as a means of detection in HPLC 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 onoxidation of the solute.Examples of compounds 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. It may be noted here

that an anode becomes a stronger oxidising agent as its electrode potential becomes more

positive. Of course, electrochemical detection req uires the use of conducting mobile

phases, e.g. containing inorganic salts or mixtures of water with water-miscible organic

solvents, but such conditions are often difficult to apply to techniques other than reverse

phase and ion exchange chromatography.The amperometric detector is currently the most widely used electrochemical detector,

having the advantages of high sensitivity and 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; and

3. the auxiliary electrode is the current-carrying electrode and usually made of stainless

steel.

Despite their higher sensitivity and relative cheapness compared with ultravioletdetectors, amperometric detectors have a more limited range of applications, being often

used for trace analyses where the ultraviolet detector does not have sufficient sensitivity.

Effect of Temperature

EFFECT OF TEMPERATURE ON ANALYTE IONIZATION

The use of elevated temperatures for the reversed-phase HPLC separation of mixtures has

been used primarily for increasing column efficiency or shortening run time and

enhancing separation selectivity . Elevated temperatures also increase solute solubility

and diffusivity. Column efficiency is also expected to increase with temperature as

diffusion rate increases. However, temperature can also affect the dissociation constants

of the ionizable components, and this can lead to anomalous retention behavior of these

compounds as a function of temperature (i.e., increases in retention with increase in

temperature) . The pH of a phosphate buffer and acetate buffer are not significantly

affected by change in temperature . For acidic analytes, depending on the type of acid and

its intrinsic properties, the analyte pKa may not vary as a function of temperature.


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Phenolic and carboxylic acids pKas do not vary significantly with a change of

temperature. However, basic analytes may experience greater changes in their ionization

constants with increase of the temperature. The weaker an acid, the greater the change in

the analyte pKa (mainly seen for basic compounds) with a change in temperature.

Essentially for basic compounds the analyte is being analyzed in its more neutral form

with an increase in the temperature and may experience increases in retention at higheranalysis temperatures. Therefore the temperature of the separation should also be taken

into consideration when performing method development, especially for basic

compounds. Basic compounds that have pKa values >6 usually experience the greatest

changes in retention with increase in temperature. The pKa values of these basic

compounds decrease with an increase in temperature, thereby making them more neutral

when analyzed at higher temperatures.

SelectivityAlthough temperature has been proposed as a variable in altering selectivity, it has not

been widely used, because the majority of analytes show very similar changes onchanging temperature (especially over the limited conventional temperature range).

Significant differences may be observed if temperature can cause ionization changes or if

analytes with very different functional groups are present. However, care must be taken

in these situations because relative retention changes with column temperature could

result in a lack of method robustness, especially if caused by ionization changes.

Efficiency

One of the reported advantages of raising the temperature of a chromatographic

separation is an increase in peak efficiency. This is usually attributed to a reduction in

the viscosity of the eluent and an increase in the diffusion rate of the analyte as the

temperature is increased. A higher diffusion rate should reduces the mass transfer termeffect The improvement in efficiency is normally regarded as most significant for larger

analytes, such as biological and synthetic macromolecules, whose size reduces their

mobility. For smaller molecules the effects are relatively small, and often an increase in

efficiency can be attributed to a reduction in the retention factor on raising the

temperature

Antigens

The term antigen refers to any substance that is capable of eliciting the formation ofaspecific antibody directed against that substance. An antigen canbe a drug, peptide,

protein, carbohydrate, or lipid. Antigens can also be a combination ofthese substances,

such as a glycoprotein or lipoprotein.

The large number of substances that can act as antigens means that physicochemical

techniques like traditional chromatography or electrophoresis can be used to isolate

suchsubstances. However, the most efficient approach for this work is to use

immunoaffinity separations based on antibodies that are specific for these agents..


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Affinity Methods for Antigen IsolationAs described earlier, immunoaffinity methods are

probably the most practical approaches for isolating

specific antigens. This makes use of the immunological

activity of the antigens by binding them toimmunoaffinity columns containing immobilized

antibodies. The isolation of antigens by this approach

can employ polyclonal antibodies, affinity-purified

antibodies, or specific monoclonal antibodies to isolate

a sing|le compound from essentially any matrix. An

example of such a separation is given in Figure

Monoclonal antibodies have been used to isolate a large

number of antigens and are often the reagents of choice

when performing such isolations [13., 140].

Immunoaffinity separations have been successfullyapplied to many fields, including the isolation of

allergens, the purification of parasite antigens for

diagnostic purposes, and isolation of fungal or viral

components . Specific antigens associated with

receptors and other cellular components have been

successfully isolated using immobilized-antibody columns. In addition, antibody-based

systems have been used to purify recombinant proteins from expression systems such as

bacterial cultures . Along with whole antibodies, antibody fragments can be immobilized

and used as immunoadsorbents for antigen isolation. As early as 1981. Kennedy and

Barnes used immobilized F(ab)2 fragments to isolate class-specific IgG from humanserum. In the same manner, antibody Fab fragments and scFvs have been successfully

used to isolate whole bacteria and tagged recombinant proteins .

A technique that is becoming increasingly popular for immunoaffinity separations is a

method performed with antibody-coated paramagnetic panicles ( Figure). Although this

approach has been mainly used to isolate cells bearing specific antigens, there is great

potential for this to be used as a rapid technique for isolating immunologically specific

antigens . Immunomagnetic separations based on anti-immunoglobulin-coated particles

have been employed in the recovery of muscle tropomyosin, vimentin. and myosin heavy

chain . Similarly, Kausch et al. employed a procedure using streptavidin-coated particles

to isolate a number of cell organelles that had been previously labeled with biotinylated

antibodies. Angen et al. used antibody-coated Dynabeads to isolate pleuropneumoniae

serotype 2 bacteria from pure cultures and suspensions.

detector in hplc

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