Adlard E.R. (ed.) Chromatography in the Petroleum Industry

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412

Chapter

are expensive and fragile, they are easily poisoned by the non-selective adsorp-

tion of analyte molecules to the gel material. Sieving buffers are less fiagile and

less expensive but have not been sufficiently investigated to determine their po-

tential for the analysis of industrial polymeric materials.

14.2.4

Capillary isoelectric focusing (CIEF)

This technique

also derived from classical electrophoresis in which sub-

stances are separated on the basis of their isoelectric point or PI values. The

capillary

filled with an ampholyte solution which forms a pH gradient in the

capillary when the voltage

applied. The analyte molecules (almost exclusively

proteins) migrate to the point where they have a net neutral charge, at which

point they stop migrating. The focused zones of the sample can be eluted

from

the capillary using pressure or the electro-osmotic flow of a different, non-

ampholytic buffer. This method provides very accurate determination of PI val-

ues and has a large sample-loading capacity; up to

pg of material can be loaded

onto the capillary, separated, and the purified fractions collected.

14.2.5

Instrumentation

As described briefly in the introduction to this chapter, CE systems have a

common architecture whatever their application. The different parts of the sys-

tem must be optimized to achieve reproducible analyses, ease of use, sensitive

detection and automatic operation. The main components of a

system are

discussed

detail and the important parameters highlighted.

14.2.6

Capillary

Although

possible using glass or polymer capillaries, silica capillaries

are the most widely used

they give the best combination of properties for

CE.

Alternatives to silica are usually explored in order to overcome difficulties with

the separation; in these circumstances it

usually more productive to investigate

an alternative technique. Alternatively, the chemistry of the electrolyte can be

adapted to dynamically coat the capillary surface.

A considerable amount of ef-

fort

being put into improving the stability and nature

coating materials; a

number of significant developments are expected in the near future.

The dimensions of the capillary are important parameters. The internal diame-

ter is usually between

and 150pm. Smaller diameters can improve separa-

Capillary electrophoresis

the petroleum industry

413

tions due to an increase in efficiency, but at the expense of detection limits

(injected volumes are smaller and the detector pathlength is shorter). The use of

larger capillary diameters can improve detection limits, but at the expense of the

separation. The recent development of Z-Flowcells for most commercial instru-

ments has improved detection limits

without the need to use large-bore capillar-

ies. The square of the diameter of the capillary is directly proportional to the cur-

rent generated in the capillary at a given potential. This limits the diameter of the

capillary since applying sufficient field strength to separate the analytes may

generate an excessive current if the capillary has

large internal diameter. This

can be manifest in

two

ways, the current generated exceeds the safety limits of

the system and it shuts down, or the heat generated by the current causes boiling

of the electrolyte and a break in the electrical continuity of the capillary. Before

either of these

two

points are reached, detector baseline instabilities will be ob-

served because of bubble formation.

The separation capacity of the capillary is proportional to its length. Keeping

other analytical parameters constant, increasing the length of the capillary de-

creases the electric field strength leading to longer run times.

14.2.7

High

voltage power supply

The most important feature of the power supply is stability; as the “pump”

the CE system, it is important to have a good-quality power supply. Most devices

will operate at up to

+30

with a high degree of accuracy and precision. They

can run at constant voltage, constant current, or a mixture of both. Safety pre-

cautions are built into the CE system to prohibit inadvertent hazards. CE systems

cannot be considered spark-proof; this may limit the extension of their use in

hazardous areas such as oil platforms or refineries, unless they are operated in a

purged environment.

14.2.8

Temperature control

Applying a potential difference across the capillary and the resulting current

generates heat, this has to be dissipated to avoid boiling within the capillary. Dif-

ferent approaches are feasible, using either air flow across the capillary, or ex-

ternally cooled fluid circulation. With the latter approach, the capillary has to be

inserted into a cassette-type device to facilitate the coolant. Good temperature

control is necessary for reproducible migration times.

change of 1°C causes a

change in ionic mobility (Fig. 14.9). Temperature control of the electrolyte

and sample compartments is also important. The selectivity of MEKC separa-

References pp.

425-426

414

3.0

2.9

Chapter

carbonate

phosphote

fluoride

nitrate

nihite

sulfate

chloride

bromide

Fig.

14.9.

Influence

temperature on the migration times

anions. Analytical conditions: electro-

lyte,

mM chromate,

0.5

CIA-Pakm

OFM

anion-BT. Courtesy

Millipore-Waters.

tions can be significantly altered by changing the temperature, while the stability

of temperature labile samples will be enhanced by low

(4°C)

temperatures.

14.2.9

Injection

There are

two

modes of injection found on commercial instruments, hydrody-

namic injection and electrokinetic (or electromigrative) injection. A pressure

differential is used to load the sample into the capillary in the hydrodynamic

mode. This can be obtained by different means; gravity or air pressure

the in-

jection side,

vacuum at the detection side.

the capillary is held in

cassette,

pressure or vacuum has to be used for hydrodynamic injection. Air-cooled capil-

laries can be raised and lowered to facilitate gravimetric injection. Where direct

pressure or vacuum is used, it is important that there

accurate control of the

Capillary electrophoresis in the petroleum industry

415

pressure and the sample vials are well sealed, so that reproducible injections can

be made. When using gravimetric techniques for injection, the capillary is placed

in the sample vial and elevated to a given height above the detection end of the

capillary. An aliquot of the sample will syphon into the capillary. The injected

volume is proportional to the pressure differential, sample viscosity, capillary

diameter and time. If any of these parameters cannot be controlled, the injections

will be irreproducible. Normally, the capillary diameter will stay the same, as

will the pressure differential if all seals are in good condition. One can manipu-

late the time of injection to facilitate loading; viscosity is more difficult to con-

trol, particularly if the sample matrix is subject to change. Temperature control

is important here because of its effect on the viscosity of solutions.

Injection volumes are very small, less than

common, and should be

considered if sample homogeneity is not reliable. The resolution of the separa-

tion can be compromised if the injection volume is too large. It is difficult to de-

fine exactly the volume of sample that will disrupt a CE separation.

Electromigrative injection is used to selectively introduce sample components

into the capillary by generating a potential difference between the capillary and

the ions in the sample. The injection of negatively charged species is obtained by

making the end of the capillary positive with respect to the sample. The negative

ions migrate to the capillary where they can accumulate. Positively charged spe-

cies are injected by making the end of the capillary negative with respect to the

sample. Differing amounts of sample are injected by varying either the injection

voltage or the length of time the voltage is applied. Unlike hydrodynamic injec-

tion, electromigration is a selective injection technique, only the positive or

negative ions in the sample will be injected, neutral species will not. This can be

significant benefit if the sample matrix is complex and the operator knows

which ionic species are of interest.

disadvantage of this technique is the non-

linear relationship between the field-strength used and the mobility of the ions in

the sample. The most mobile ions will migrate through the sample and into the

capillary faster than slower ions. There will therefore be an enhanced accumula-

tion of the most mobile ions that can lead to inaccuracies in quantitation unless

the sample matrix is well defined and stable. A good use of electromigration in-

jection is in the analysis of very low levels of anions and cations in ultra-pure

water (Fig.

14.10).

14.2.10

Detection

There are a number of detection techniques available for

systems; all are

taken from HPLC, although a number of modifications are necessary for opti-

mum

performance. The detector types and their uses are presented here.

References

pp.

425-426

416

Chapter

,,,~IIl~I~l~~~~~~~~~"'

3.60 3.80

4.00

4.20

4.40

4.60

4.8

minutes

Fig.

14.10.

Analysis

anions at trace level in deionized water containing

2000

ppm boron (as

boric acid). Electrolyte

mh4

sodium chromate, pH

7.8,

0.5

mh4

CIA-PakTM anion-BT. Detection

indirect

254

nm. Injection by electromigration

(45

kV).

Courtesy

Millipore-Waters.

Peak

chloride

ppb);

sulfate

ppb);

nitrate (n.q.);

chlorate

(30

ppb);

fluoride

PPb).

I4.2.10.1

detection

is the most widely used detection technique in CE. The detector flow-cell

normally obtained by removing the polyimide sheath from the capillary at a

predetermined distance from the sample end. The flow cell thus created

mounted into an appropriate optical assembly. The very short optical pathlength,

corresponding to the internal diameter

the capillary, limits the sensitivity

the technique, although it does lead to sharp analyte peaks. Recently

two

variants

flow-cell design have been introduced, both of which aim to increase the opti-

cal

pathlength and increase sensitivity. The Z-Flowcell increases the pathlength

mm, while the Bubble cell increases the pathlength to 300pm. Both result

increased sensitivity but with a decrease in resolution.

Detection wavelengths are selected either by lamp and filters (discretely vari-

able) or by

diffraction grating and polychromator lamp. Changes

absorbance

due to the analyte are detected by photodiode, or photomultiplier,

photodiode

Capillary electrophoresis

the petroleum industry

417

array (Fig. 14.1 1). Each of these approaches has advantages. Discretely variable

detectors have the advantage of simplicity and unmatched sensitivity at specific

wavelengths. The tunable detectors offer more flexibility as they can be tuned to

the appropriate wavelength for the analyte, but they often lack sensitivity at ul-

tra-low wavelengths (lower than 190 nm). Photodiode array detectors for CE

have only recently been introduced and offer increased information about the

sample. The main benefit of

CE,

however, is the ability to resolve closely related

species. The benefits of photodiode array detection in

are not as obvious as

they are for the lower resolution techniques such as liquid chromatography.

alternative use of

detection is termed indirect

UV.

The technique re-

lies on the use of an electrolyte having a strong chromophore at a specific

wavelength such that non-UV absorbing analytes can be detected. (Fig. 14.12).

.........

...

..................

....

,..'

*.."

.....

.:-.

-9"

.......................................................

o'..

........................

.....

Fig.

14.11.

Four most common

visible detectors in capillary electrophoresis:

(A)

fixed-

wavelength detection;

(B)

variable-wavelength detectors;

(C)

scanning monochromator detector;

(D)

photodiode array detector.

light source; 2a and 2b, deuterium

tungsten switchable lamp

sources;

mirror;

capillary;

filter;

grating;

photodiode

photomultiplier;

diode array.

Reprinted with permission

[4].

References pp.

425-426

Chapter

418

-c

CIP-Sullonate

ppnl

CtO-Sullonnte 25

ppiii

C9-Sulloriale 25

ppiii

CB-Stillonale 25

ppi~

C7-Sullonate 25 ppit~

C6-Sulloiiale 25

ppiil

7 CS-Sulfonate 25

ppiit

C4-Sullonale

ppri~

6.0

8.0

10.0

12.0

Migralion

lime

(Mnules)

Fig.

14.12.

Indirect UV detection of sulfonic acids. Electrolyte: naphthalene sulfonic acid and ace-

tonitrile. Capillary

em. Injection

hydrostatic. Voltage

kV. The naphthalene

sulfonic acid provides

the

background absorbance necessary for the detection of the non-

absorbing analmytes. This electrolyte

was

also

chosen

its

mobility

close to the mobility of the

analytes.

This

necessary

for

symmetrical peaks

[4].

Courtesy of Millipore.

The use of indirect

detection for ion analysis by CE is described extensively

by Jandik and Bonn

[

121. This detection mode

universal and very sensitive

with electrolytes such as chromate

parahydroxybenzoate. Due to differences

in the separation mechanism, there are no “system peaks” in CE, unlike the use

indirect

in HPLC. The

flow cell or bubble cell do not offer significant

advantages for indirect

as they do not improve the signal to noise ratio.

14.2.10.2

Fluorescence, indirect fluorescence,

laser-inducedfluorescence

Direct fluorescence detection allows very low detection limits for compounds

containing fluorophores,

those which can be derivatized to form fluorescent

compounds. Most applications concern peptides, amino acids and proteins. The

most

sensitive detectors are those using lasers as the excitation source. The exci-

tation wavelengths are 442 and

326

nm with a helium-cadmium laser, 488 nm

with a cadmium laser

argon ion laser. It is usually necessary to derivatize the

sample to be able to use these wavelengths. Classical derivatives such as ortho-

phthaldehyde or dansyi chloride are used with normal fluorescent detection for

amino compounds, but special reagents have ben developed for use with laser

induced fluorescence detection. Using these compounds, very good detection

limits can be obtained. Ten attomoles

amino acids can be detected [25]. Deri-

Capillary electrophoresis

the petroleum industry

419

vatization at such low levels is very difficult and these results were obtained by

dilution of the sample from higher (nanomole) concentrations.

Indirect laser induced fluorescence detection permits the detection

non-

fluorescent analytes by using an electrolyte containing a fluorescent compound.

Results have been published for the determination

both cations and anions

using indirect laser fluorescence. The detection limits, however, are in the same

order of magnitude as the detection limits by indirect

with a less expensive

instrument.

14.2.10.3

Amperometric detection, conductometric detection,

detection

All the detection modes used in WLC will be available for CE in the near

future. The following three modes

detection are still in the development stage

but will certainly soon be in routine use.

Amperometric detection.

This technique is often termed electrochemical detec-

tion, it requires that the analyte molecules have a redox potential, i.e. that they

can be either oxidized or reduced by applying a low voltage. Classical examples

are catecholamines, carbohydrates and some inorganic compounds such as sul-

phite, sulphide, cyanide and iodide. Detection of metal ions has also been de-

scribed. The design of a cell is shown Fig.

14.13.

Working electrodes can be

made

carbon fibre (catecholamine analysis), copper (carbohydrates), silver

(sulphur species), mercury, gold or platinum. Interest in this technique lies in the

selectivity and sensitivity it can offer, for example attomoles

catecholamines

can be detected

[26].

Compared to HPLC, amperometric detection in

gives

better coulombic efficiency (percentage

the electroactive analyte that is oxi-

dized or reduced in the cell).The main difficulty to overcome in CE is that rou-

tinely the detector is set to approximately

1.0

(in order to oxidizeheduce the

analyte) which generates a current

nanoamps, in the presence

the high volt-

age and microamp current generated by

CE.

It is therefore necessary to ground

Fig.

14.13.

Amperometric detection cell design. Reprinted with the permission of ref.

[25].

fused silica capillary;

drop of carrier electrolyte;

stainless steel plate;

RE,

reference electrode;

WE,

working electrode;

AE,

auxiliary electrode.

References pp.

425-426

420

Chapter

the high voltage at the detection end of the capillary before the amperometric

detector flow cell

[27].

A similar approach

required for conductometric detec-

tion.

Conductometric detection.

This mode of detection works in exactly the same

way as the HPLC detector and it

attractive detector to use for measuring

ions. The detection principle is to measure the change in conductance of a solu-

tion due to the presence of an ion. The conductance can either be enhanced or

reduced by the presence of the ion. It is difficult to measure small changes in

conductance

the presence of the highly conducting CE electrolyte and high

voltage. An interesting experimental approach

the use

chemical suppression

1281.

Using this method, good detection limits were presented for anion analysis,

but a number of problems still need to be resolved. It is difficult to assemble the

suppressor and conductivity cell on the capillary and the lifetime of the suppres-

sor is compromised by the cationic surfactants used to control the EOF. If the

EOF

not controlled, the separation becomes very unstable. There are difficul-

ties therefore in the selection of electrolytes which are compatible with both

separation and suppressioddetection.

Once these problems have been overcome, conductometric detection of ions

separated by CE should offer advantages against present methods

of detection.

Mass

spectroscopy detection.

The mass spectrometer is an attractive detection

technique as it can give absolute identification of the separated analytes based on

their molecular mass and, if the appropriate technique is used, mass spectrum. It

for this reason that

LC-MS

a fast growing technique. One of the difficulties

with LC-MS is the incompatibility between the optimum flow rates for LC

analysis versus the optimum flow rates for most MS interfaces. One possible

solution attempted by MS manufacturers is to modify the interface to accommo-

date high flow rates. This can, however, compromise the performance of the MS.

alternative solution is to use capillary LC with microbore columns and flow

rates in the microliter per minute range.

CE, the EOF usually ranges from

0.1

,uI/min, which makes it more suitable for

interfaces than HPLC.

A review of CE-MS published recently

[28]

comments that the electrospray

ionization interface (ESI) is more frequently used than fast atom bombardment

(FAB). This is probably due to the extensive use of ESI for protein and peptide

analysis, for which it

well suited, as is CE.

There are compatibility problems with CE electrolytes and

MS,

this can be

reduced by the use of a sheathing liquid which effectively dilutes the CE electro-

lyte to ionic levels that the

can tolerate (Fig.

14.14).

The combination of the

resolving capacities of

and CE are synergistic. The only issue

sensitivity

since the injected amounts

CE are so small, and the need to dilute the CE

Capillary electrophoresis in the petroleum industry

lo,

Coowr

E5~

rotcogr,

elate

Hoc

Fund

sllka

ESI

buff-

flow

t+lOpl/mln)

-r

Inol

Purp

Fig.

14.14.

CEMS

interfacing. Reprinted with the permission

ref.

[26].

electrolyte. Reducing the elution speed (by controlling the CE voltage) and by

using optimized injection techniques (sample stacking, isotachorphoresis pre-

concentration), it is possible to improve detection limits. Selected ion monitoring

(SIM) also brings an additional gain in sensitivity.

14.3

APPLICATIONS

CAPILLARY ELECTROPHORESIS

Capillary electrophoresis can be applied to most of the areas previously asso-

ciated with liquid chromatography. The following review is not intended to be

exhaustive and new applications are being published almost daily.

The majority of applications published to date relate to the pharmaceutical in-

dustry. Most new pharmaceutical compounds are now subjected to various

modes of CE analysis during their development stages and as these become for-

mulated into new drug products, it is apparent that CE will play an increasing

role in this field.

area of particular importance is the use of CE to provide

elegant separation of optical isomers in the presence of electrolyte additives such

as cyclodextrin (Fig.

14.15).

Most pharmaceutical drugs are cationic and are as-

sociated with a counter-ion such as sulphate or chloride. The quantitation of the

counter-ion and of possible anionic impurities is readily achieved by capillary

ion analysis

(CIA)

which allows rapid and accurate mass balance problems to be

resolved (Fig.

14.16).

Within the chemical industry in general, the most common application of CE

is the analysis of mixtures of small ionic species where it is used for process

monitoring, quality control product formulation and environmental monitoring

References pp.

425426