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during these critical days elicits a major improvement

in memory performance at all stages of training on a

12-arm radial maze. Choline deficiency during the

critical timeframe reduces memory performance. The

choline-induced spatial memory facilitation correl-

ates with changes in the birth, death, and migration

of cells in the hippocampus during fetal brain devel-

opment, with altered distribution and morphology of

neurons, biochemical changes, and electrophysio-

logical changes in the hippocampus. Human and rat

brains mature at different rates; rat brain is compara-

tively more mature at birth than is the human brain,

but in humans, hippocampal development may con-

tinue for months or years after birth.

Choline and Cancer

0009 Dietary deficiency of choline in rodents causes devel-

opment of hepatocarcinomas in the absence of any

known carcinogen. Choline is the only single nutrient

for which this is true. It is interesting that choline-

deficient rats not only have a higher incidence of

spontaneous hepatocarcinoma but also are markedly

sensitized to the effects of administered carcinogens.

Several mechanisms are suggested for the cancer-

promoting effect of a choline-devoid diet. A progres-

sive increase in cell proliferation that is related to

regeneration after parenchymal cell death occurs in

the choline-deficient liver. Cell proliferation and its

associated increased rate of DNA synthesis could be

the cause of the heightened sensitivity to chemical

carcinogens. Methylation of DNA is essential to

the regulation of expression of genetic information,

and the undermethylation of DNA observed during

choline deficiency (despite adequate dietary methio-

nine) may be responsible for carcinogenesis. Choline-

deficient rats experience increased lipid peroxidation

in liver. Lipid peroxides in the nucleus are a possible

source of free radicals that could modify DNA and

cause carcinogenesis. Choline deficiency activates

protein kinase C signaling, usually involved in growth

factor signaling in hepatocytes. Finally, a defect in

cell-suicide (apoptosis) mechanisms may contribute

to the carcinogenesis of choline deficiency.

Summary

0010 Choline in the diet is important for many reasons. As

our understanding of the importance of folate and

homocysteine nutrition increases, there should be

increased interest in how choline interacts with folate

and homocysteine metabolism. Recent findings about

choline in brain development should stimulate com-

parable studies in humans. During the next few years,

it is likely that food composition data will be available

for choline, and this will make it possible to examine

interactions between choline, folate, and methionine

when considering epidemiological data. In addition,

we should learn more about choline requirements in

women. For these reasons interest in choline as a

nutrient for humans should be sustained.

See also: Cancer: Diet in Cancer Prevention;

Cobalamins: Properties and Determination;

Phospholipids: Properties and Occurrence; Riboflavin:

Properties and Determination; Vitamin B

: Properties

and Determination

Further Reading

Albright CD, Friedrich CB, Brown EC, Mar MH and

Zeisel SH (1999a) Maternal dietary choline availability

alters mitosis, apoptosis and the localization of TOAD-

64 protein in the developing fetal rat septum. Brain

Research. Developmental Brain Research 115: 123–

129.

Albright CD, Lui R, Bethea TC et al. (1996) Choline

deficiency induces apoptosis in SV40-immortalized

CWSV-1 rat hepatocytes in culture. FASEB Journal 10:

510–516.

Albright CD, Tsai AY, Friedrich CB, Mar MH and Zeisel

SH (1999b) Choline availability alters embryonic devel-

opment of the hippocampus and septum in the rat. Brain

Research. Developmental Brain Research 113: 13–20.

Anonymous (1997) Betaine for homocystinuria. Medical

Letters on Drugs and Therapeutics 39: 12.

Banni S, Corongiu FP, Dessi MA et al. (1989) Free radicals

and lipid peroxidation in liver of rats kept on a diet

devoid of choline. Free Radical Research Communica-

tions 7: 233–240.

Bremer J and Greenberg D (1961) Methyl transfering

enzyme system of microsomes in the biosynthesis of

lecithin (phosphatidylcholine). Biochimica et Biophysica

Acta 46: 205–216.

Buchman AL, Dubin M, Jenden D et al. (1992) Lecithin

increases plasma free choline and decreases hepatic

steatosis in long-term total parenteral nutrition patients.

Gastroenterology 102: 1363–1370.

Buchman A, Dubin M, Moukarzel A et al. (1995) Choline

deficiency: a cause of hepatic steatosis during parenteral

nutrition that can be reversed with intravenous choline

supplementation. Hepatology 22: 1399–1403.

Buchman AL, Moukarzel A, Jenden DJ et al. (1993) Low

plasma free choline is prevalent in patients receiving long

term parenteral nutrition and is associated with hepatic

aminotransferase abnormalities. Clinical Nutrition 12:

33–37.

Cermak JM, Blusztajn JK, Meck WH et al. (1999) Prenatal

availability of choline alters the development of acetyl-

cholinesterase in the rat hippocampus. Developmental

Neuroscience 21: 94–104.

Chandar N, Amenta J, Kandala JC and Lombardi B (1987)

Liver cell turnover in rats fed a choline-devoid diet.

Carcinogenesis 8: 669–673.

1256 CHOLINE/Physiology

Chawla RK, Wolf DC, Kutner MH and Bonkovsky HL

(1989) Choline may be an essential nutrient in malnour-

ished patients with cirrhosis. Gastroenterology 97:

1514–1520.

Christman JK (1995) Dietary effects on DNA methylation:

do they account for the hepatocarcinogenic properties of

lipotrope deficient diets? Advances in Experimental

Medicine and Biology 369: 141–154.

da Costa K, Cochary EF, Blusztajn JK, Garner SC and Zeisel

SH (1993) Accumulation of 1,2-sn-diradylglycerol with

increased membrane-associated protein kinase C may be

the mechanism for spontaneous hepatocarcinogenesis in

choline deficient rats. Journal of Biological Chemistry

268: 2100–2105.

da Costa K-A, Garner SC, Chang J and Zeisel SH (1995)

Effects of prolonged (1 year) choline deficiency and

subsequent refeeding of choline on 1,2,-sn-diradyl-

glycerol, fatty acids and protein kinase C in rat liver.

Carcinogenesis 16: 327–334.

Drouva SV, LaPlante E, Leblanc P et al. (1986) Estradiol

activates methylating enzyme(s) involved in the conver-

sion of phosphatidylethanolamine to phosphatidylcho-

line in rat pituitary membranes. Endocrinology 119:

2611–2622.

Finkelstein JD, Martin JJ, Harris BJ and Kyle WE (1983)

Regulation of hepatic betaine-homocysteine methyl-

transferase by dietary betaine. Journal of Nutrition

113: 519–521.

Garcia-Perez A and Burg MB (1991) Role of organic osmo-

lytes in adaptation of renal cells to high osmolality.

Journal of Membrane Biology 119: 1–13.

Glueck CJ, Shaw P, Lang JE et al. (1995) Evidence that

homocysteine is an independent risk factor for athero-

sclerosis in hyperlipidemic patients. American Journal of

Cardiology 75: 132–136.

Goddijn-Wessel T, Wouters M, van de Molen E et al.

(1996) Hyperhomocysteinemia: a risk factor for

placental abruption or infarction. European Journal of

Obstetrics, Gynecology, and Reproductive Biology 66:

23–29.

Goshal AK, Ahluwalia M and Farber E (1983) The rapid

induction of liver cell death in rats by a choline-deficient

methionine-low diet. American Journal of Pathology

113: 309–314.

Gwee MC and Sim MK (1978) Free choline concentration

and cephalin-N-methyltransferase activity in the mater-

nal and foetal liver and placenta of pregnant rats. Clin-

ical and Experimental Pharmacology and Physiology 5:

649–653.

Handler P and Bernheim F (1949) Choline deficiency in the

hamster. Proceedings of the Society of Experimental

Medicine 72: 569.

Hershey JM and Soskin S (1931) Substitution of ‘lecithin’

for raw pancreas in a diet of depancreatized dog. Ameri-

can Journal of Physiology 93: 657–658.

Hoffbauer FW and Zaki FG (1965) Choline deficiency in

the baboon and rat compared. Archives of Pathology 79:

364–369.

Holmes-McNary M, Cheng WL, Mar MH, Fussell S and

Zeisel SH (1996) Choline and choline esters in human

and rat milk and infant formulas. American Journal of

Clinical Nutrition 64: 572–576.

Holmes-McNary MQ, Loy R, Mar M-H, Albright CD and

Zeisel SH (1997) Apoptosis is induced by choline defi-

ciency in fetal brain and in PC12 cells. Developmental

Brain Research 101: 9–16.

Horne DW, Cook RJ and Wagner C (1989) Effect of dietary

methyl group deficiency on folate metabolism in rats.

Journal of Nutrition 119: 618–621.

Institute of Medicine and National Academy of Sciences

USA (1998) Dietary Reference Intakes for Folate, Thia-

min, Riboflavin, Niacin, Vitamin B

, Panthothenic

Acid, Biotin, and Choline, vol. 1. Washington DC:

National Academy Press.

Jacob R, Jenden D, Okoji R, Allman M and Swendseid M

(1998) Choline status of men and women is decreased by

low dietary folate. FASEB Journal 12: A512.

Kang S (1996) Treatment of hyperhomocyst(e)inemia:

physiological basis. Journal of Nutrition 126: 1273S–

1275S.

Kim Y-I, Miller JW, da Costa K-A et al. (1995) Folate

deficiency causes secondary depletion of choline and

phosphocholine in liver. Journal of Nutrition 124:

2197–2203.

Kuksis A and Mookerjea S (1978) Choline. Nutrition

Reviews 36: 201–207.

Lieber CS, Robins SJ, Li J et al. (1994) Phosphatidylcholine

protects against fibrosis and cirrhosis in the baboon.

Gastroenterology 106: 152–159.

Lindblad L and Schersten T (1976) Incorporation rate

in vitro of choline and methyl-methionine into human

hepatic lecithins. Scandinavian Journal of Gastroenter-

ology 11: 587–591.

Locker J, Reddy TV and Lombardi B (1986) DNA methyla-

tion and hepatocarcinogenesis in rats fed a choline

devoid diet. Carcinogenesis 7: 1309–1312.

Lombardi B and Smith ML (1994) Tumorigenesis, proto-

oncogene activation, and other gene abnormalities in

methyl deficiency. Journal of Nutritional Biochemistry

5: 2–9.

Loy R, Heyer D, Williams CL and Meck WH (1991)

Choline-induced spatial memory facilitation correlates

with altered distribution and morphology of septal

neurons. Advances in Experimental Medicine and Biol-

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Lyman RL, Sheehan G and Tinoco J (1971) Diet and

-methionine incorporation into liver phosphati-

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McCully K (1996) Homocysteine and vascular disease.

Nature Medicine 2: 386–389.

Meck W and Williams C (1997) Simultaneous temporal

processing is sensitive to prenatal choline availability in

mature and aged rats. Neuroreport 8: 3045–3051.

Meck WH and Williams CL (1999) Choline supplementa-

tion during prenatal development reduces proactive

interference in spatial memory. Brain Research. Devel-

opmental Brain Research 118: 51–59.

Meck WH, Smith RA and Williams CL (1988) Pre- and

postnatal choline supplementation produces long-term

CHOLINE/Physiology 1257

facilitation of spatial memory. Developmental Psycho-

biology 21: 339–353.

Mudd SH, Ebert MH and Scriver CR (1980) Labile methyl

group balances in the human: the role of sarcosine.

Metabolism 29: 707–720.

Newberne PM and Rogers AE (1986) Labile methyl groups

and the promotion of cancer. Annual Review of Nutri-

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Pogribny IP, Basnakian AG, Miller BJ et al. (1995) Breaks in

genomic DNA and within the p53 gene are associated

with hypomethylation in livers of folate/methyl-deficient

rats. Cancer Research 55: 1894–1901.

Pomfret EA, da Costa K and Zeisel SH (1990) Effects

of choline deficiency and methotrexate treatment upon

rat liver. Journal of Nutritional Biochemistry 1: 533–

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Pyapali G, Turner D, Williams C, Meck W and Swartz-

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decreases the threshold for induction of long-term

potentiation in young adult rats. Journal of Neurophysi-

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Savendahl L, Mar M-H, Underwood L and Zeisel S (1997)

Prolonged fasting results in diminished plasma choline

concentration but does not cause liver dysfunction.

American Journal of Clinical Nutrition 66: 622–625.

Selhub J, Seyoum E, Pomfret EA and Zeisel SH (1991)

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1258 CHOLINE/Physiology

CHROMATOGRAPHY

Contents

Principles

Thin-layer Chromatography

High-performance Liquid Chromatography

Gas Chromatography

Supercritical Fluid Chromatography

Combined Chromatography and Mass Spectrometry

Principles

E Forga

cs and T Cserha

ti, Chemical Research

Center, Institute of Chemistry, Budapest, Hungary

Background

0001 Chromatography is a common name for techniques

based on the partition of the molecules to be analyzed

between a mobile and a stationary phase. Separation

is the result of different partitions of molecules be-

tween the two phases. Because the best separation of

any solutes can be obtained under equilibrium condi-

tions, analytical chemists prefer to use chromato-

graphic systems that are as near to the equilibium

state as possible. However, in the case of preparative

chromatography, where the main objective is not the

optimal separation of solutes but the maximum yield

of one or more solutes at a given purity, the situation

is entirely different. Preparation chromatographic

separations are generally not equilibrium processes.

The high sensitivity, selectivity, and reproducibility of

chromatographic methods have been extensively ex-

ploited in food and nutrition science and technology.

0002 This chapter overviews the theoretical back-

grounds, principles, and fundamental technical char-

acteristics of chromatographic separation processes

and instrumentations.

0003 Basic knowledge of the principles of chromatog-

raphy may help analytical chemists solve any separ-

ation problems, using chromatography as a tool.

Gas Chromatography

Principle

0004 Gas chromatography (GC) is the common name for

chromatographic methods in which the mobile phase

is gas, and the stationary phase is solid or liquid (gas–

solid chromatography (GSC) or gas–liquid chroma-

tography (GLC)). There are limitations to the type

of compound suitable for GC analysis. A compound

needs to have an appreciable vapor pressure at tem-

peratures below 350–400



C and has to be vaporized

rapidly without decomposing or reacting with the

components of the stationary and mobile phase

(instability at high temperatures usually renders a

compound unsuitable for GC analysis).

0005The common GC equipment consists of a carrier

gas system, injector, gas chromatographic column,

detector, and data-processing unit (Figure 1). The

carrier gas is generally a permanent gas with a low

or negligible adsorption capacity, i.e., hydrogen,

helium, argon, or nitrogen. The nature of the carrier

gas may influence the separation characteristics of

the GC system and can modify the sensitivity of the

detection.

0006As the stability and reproducibility of the carrier

gas flow rate are the prerequisites for a successful GC

analysis, they have a considerable influence on both

the efficacy of the separation and the quantification

of the results.

0007Injectors deliver the sample to the head of the GC

column and can be classified into two major groups:

vaporization and on-column injectors. Vaporization

injectors use high temperatures to rapidly vaporize a

liquid sample. Usually, a syringe is used to intro-

duce the sample into the thermostated injector. In

this case, the sample rapidly vaporizes, mixes with

the carrier gas, and is transported into the column.

On-column injectors deposit the sample directly into

the column without relying on vaporization of the

sample and its subsequent transport into the column.

Separation of volatile compounds of the injected

sample is performed in the GC column.

0008The columns used for gas chromatography can be

divided into two distinct groups: packed and capillary

columns. A packed column is a rigid metal or glass

column filled with small particles that are often

CHROMATOGRAPHY/Principles 1259

coated with a thin layer of high-molecular-mass poly-

mer. The most common solid supports are diatom-

aceous earth, fluorocarbons, graphitized carbon

black, and glass beads. The stationary liquid phase

has to comply with the following requirements: low

vapor pressure, high chemical stability, and relatively

low viscosity at the temperature of analysis; selectiv-

ity for the sample components under investigation;

and a good wetting capacity for the surface of the inert

support or for the possibly inert wall of the column. A

capillary (open tubular) column is a glass or fused-

silica tube of very small internal diameter (generally

between 0.20 and 0.53 mm). The inner surface of a

capillary column is coated only with a thin layer of

stationary phase, so it is still possible for the analytes

to come into contact with the inner wall of the tubing.

Stationary phases can be cross-linked and covalently

bonded to the inner wall of the capillary. The station-

ary phase in a capillary column is characterized by its

film thickness (0.1–5 mm). The advantage of capillary

columns is the considerably higher separation cap-

acity than that of packed columns. The decrease in

lateral diffusion, easier availability of the separation

liquid, and increase in column length account for the

enhanced separation efficacy of capillary columns.

Because of the better separation performance, capil-

lary columns have been used more frequently in gas

chromatography than packed columns.

0009 In order to achieve effective and reliable separ-

ation, a gas chromatographic column has to be kept

at a constant temperature (isothermal separation

mode), or the column temperature can be changed

according to a temperature program (temperature

gradient). As linear temperature programs consisting

of more than one step are usually sufficient for the

effective separation of analytes, nonlinear tempera-

ture programs are rarely used in the analysis of food

constituents. Detectors interact with the solute

molecules as they exit the column. This interaction

is converted into an electrical signal that is sent to a

recording or data-storage device. A chromatogram,

i.e., a plot of the intensity of the signal versus elapsed

time, is then created. The primary differences be-

tween the detectors are in the lowest amount of

a compound that is detectable (sensitivity) and the

compounds that produce the strongest detector re-

sponse (selectivity). Many different detectors (flame

ionization detectors (FID), nitrogen–phosphorus de-

tectors (NPD), flame photometric detectors (FPD),

electron capture detectors (ECD), thermal conduct-

ivity detectors (TCD), atomic emission detectors

(AED), electrolytic conductivity detectors (ELCD),

chemiluminescence detectors, mass spectrometry

(MS) detectors, etc.) and data-handling devices have

been developed for the sensitive and selective detec-

tion and quantification of sample components. The

FID uses a hydrogen flow mixed with the carrier gas.

The mixture is ignited, the analytes are burned, and

the ions formed during the burning process are

collected in a cylindrical electrode at a high voltage

applied between the jet of flame and the electrode.

The resulting current is amplified and detected. The

NPD is similar to the FID in its design. It contains

rubidium or cesium beads inside a heater coil near the

hydrogen jet. The partially combusted nitrogen and

phosphorus molecules absorb on to the surface of the

beads, thereby reducing the emission of electrons,

which increases the current. The FPD especially

detects sulfur and phosphorus compounds. Analytes

are burned in the flame, and because of the excitation

in the flame, light is emitted at 392 nm (sulfur) and

526 nm (phosphorus). A filter selects the wavelengths

reaching a photomultiplier tube. The ECD employs a

low-energy b-ray source for the production of elec-

trons and ions. Electron-capturing molecules (halo-

genated compounds) entering the detector decrease

the electron current, which can be amplified and

registered. The TCD responds to the changes in

thermal conductivity and specific heat, via a filament

under a current applied to the carrier gas flow.

Data storage

Detector

Oven

Column

Inlet

Flow regulators

Carrier gas

fig0001 Figure 1 Block diagram of a typical gas chromatogram.

1260 CHROMATOGRAPHY/Principles

Changes in the thermal conductivity and/or specific

heat of the current gas caused by the analytes change

the potential across the filament. The AED is suitable

for detecting selected atoms or groups of atoms, and

the ELCD can be used for detecting Cl-, N-, or S-

containing analytes. The chemiluminescence detector

is used mainly for detecting sulfur compounds. The

MS ionizes molecules at low pressures and then

separates them to create a spectrum of ion intensity

vs. mass-to-charge ratio. The good application par-

ameters of capillary GC/MS using the temperature

gradient is illustrated on the chromatograms of sterol

esters and free sterols in rapeseed and sunflower oils

(Figure 2). (See Chromatography: Gas Chromatog-

raphy.)

Fundamentals

0010 The distribution of solute molecules between the

stationary and mobile phases is defined by the

distribution constants (K

), i.e., the ratio of

the concentration of the solute molecules in the sta-

tionary phase to that in the mobile phase:

¼ compound concentration of stationary

phase=compound concentration of

mobile phase: ð1Þ

The general expression describing retention in terms

of column temperature and solute properties can be

expressed by:

ln K

¼G

=RT, ð2Þ

where, DG

is the change in Gibbs free energy for the

evaporation of the compound from the stationary

phase, T is the column temperature, and R is the

ideal gas constant. Equation (2) shows that the differ-

ences in the Gibbs free energy for the evaporation of

the solutes from the stationary phase result in differ-

ent degrees of solute retention.

0011The amount of time that the compound remains in

the stationary phase is called the retention time (t

15 28

Time (min)

(a)

15 28

(b)

Time (min)

15 28

Time (min)

(c)

15 28

(d)

Time (min)

fig0002 Figure 2 Gas chromatograms of sterol esters (a, c) and free sterols (b, d) in rapeseed (a, b) and sunflower oils (c, d). Peak

identification: 1, cholesterol (internal standard); 2, brassicasterol; 3, campesterol; 4, sitostesterol; 5, D

-stigmastenol; 6, betulin

(secondary internal standard). Reprinted with permission from Kalo P and Kuuranne T (2001) Analysis of free and esterified sterols

in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry. Journal of Chromatography

935: 237–248.

CHROMATOGRAPHY/Principles 1261

Dead time (t

) is the time required for a nonretained

solute to travel through a column.

0012 The partition ratio, also known as the capacity

factor (k

) is the amount of time a compound remains

in the stationary phase relative to the mobile phase.

¼ðt

 t

Þ=t

: ð3Þ

The resolution number (R) is used to describe the

efficacy of the separation of two peaks.

0013 Resolution numbers can be calculated using either

of two equations, as illustrated in Figure 3:

R ¼ 1:18ðt

 t

Þ=ðw

þ w

Þð4Þ

R ¼ 2ðt

 t

Þ=ðw

þ w

Þð5Þ

where t

and t

are the retention times of peaks

1 and 2, respectively, w

and w

are the peak widths

at half height of peaks 1 and 2, respectively, and w

and w

are the peak widths at the base of peaks 1 and

2, respectively.

0014 The theoretical plate number (N) characterizes the

separation capacity of a chromatographic column.

The number of theoretical plates is calculated by the

following equation:

N ¼ 5:545ðt

: ð6Þ

Liquid Chromatography

0015 The term liquid chromatography (LC) encompasses

chromatographic methods in which the mobile phase

is liquid and the stationary phase is organic or inor-

ganic solid. According to the shape of the solid-phase

support, liquid chromatographic methods can be

divided into planar and column chromatography.

Moreover, LC methods can be classified as normal

(adsorption or direct) and reversed-phase (RP) separ-

ation modes. When the chromatographic system

consists of a nonpolar mobile phase and a polar sta-

tionary phase, it is called normal chromatography. In

the reversed-phase separation mode, the mobile phase

is polar, and the stationary phase is nonpolar. (See

Chromatography: High-performance Liquid Chro-

matography.)

Thin-layer Chromatography

0016Thin-layer chromatography (TLC) is a planar chro-

matographic technique extensively used as a rapid

and straightforward analytical tool. The success of

TLC is due to its rapidity, cost-efficient optimization

of separation (mobile and stationary phases can be

changed easily), the high sample throughput in a

short time, and the suitability for screening tests. A

wide variety of inorganic and organic supports have

been used for separation in TLC; however, silicas

with different physicochemical characteristics (e.g.,

various surface pHs and pore diameters) and silicas

with hydrophobic ligands covalently bonded to the

surface (silanized silica, C

alkyl bonded

silica, amino, diol, cyano) are commonly used in

TLC. Besides silica and modified silica stationary

phases, other sorbents such as alumina (basic, neu-

tral, and acidic), diatomaceous earth (Kieselguhr),

celluloses and cellulose derivatives, polyamides have

also been used in TLC, because their separation char-

acteristics differ from those of silica-based layers.

0017Sample application Samples prepared for TLC are

dissolved in an appropriate solvent and applied in the

form of spots or narrow bands to the plates.

0018Development of chromatogram The most frequently

used developing technique is ascending TLC, which is

carried out in a chamber. Besides linear ascending

development, linear horizontal development and

anticircular development have sometimes been

employed. The use of multistep gradient elution and

two-dimensional development enhance the efficacy of

TLC separation.

0019Detection in TLC is generally based on the visible

or ultraviolet absorption of the solutes or on the use

of various detection reagents. One of the main

advantages of TLC is that a large number of detection

reagents can be used that are more or less selective. In

the last decade, many new instruments have been

developed for the quantitative evaluation of TLC

plates. Up-to-date chromatogram spectrophoto-

meters (densitometers) are suitable for the determin-

ation of the visible, ultraviolet, and fluorescence

intensity of solute spots. Characteristic densitograms

obtained after TLC separation of cholesterol in liquid

egg yolk are shown in Figure 4.(See Chromatog-

raphy: Thin-layer Chromatography.)

0020Fundamentals The retention factor, R

, charac-

terizes the position of spots in TLC and is determined

Detector

response

Direction of development

fig0003 Figure 3 Schematic illustration of the mode of calculation of

resolution numbers in gas and high performance liquid chroma-

tography.

1262 CHROMATOGRAPHY/Principles

by dividing the distance between the center of the

spots and the start line (z

) by the distance of the

eluent front from the start line (z

¼ z

ð7Þ

Other chromatographic parameters can be calcu-

lated, as described above.

High-performance Liquid Chromatography

0021 High-performance liquid chromatography (HPLC)

is the most frequently used liquid-chromatographic

technique. Separation is carried out using a liquid

mobile phase and a solid stationary phase filled

in columns. Similar to TLC, normal-phase and

reversed-phase separation modes can be employed.

Supports with different separation mechanisms, such

as ion exchange, size exclusion, gel-permeation

chromatography, etc. can be used in HPLC.

0022 Separation in ion-exchange HPLC is based on the

interaction of the charged solute with the oppositely

charged surface of the stationary phase. Ion-exchange

chromatography has been increasing in popularity

for the separation of inorganic species. Size-exclusion

chromatography (SEC) is a common name for liquid-

chromatographic methods that separate analytes

based on their effective shape and bulkiness at rela-

tively low pressures. SEC methods can be differenti-

ated according to the character of the mobile phase.

Gel-permeation chromatography (GPC) uses organic

mobile phases, and gel-filtration chromatography

(GFC) uses aqueous phases. The stationary phases

applied in this separation mode are porous particles

with closely controlled pore sizes. The molecules of

the solute may be able to enter the pores of the par-

ticles of the stationary, depending on their size and

shape. The choice of a mobile phase for this

separation mode is simpler than for other HPLC

techniques, because generally, only one solvent is

required. HPLC systems can be divided into two dif-

ferent parts: one part is for separation, and the other

part is for detection, including one or more detectors

and a signal output device (Figure 5). The part of the

HPLC responsible for separation consists of an injec-

tion device, a column, and the mobile-phase delivery

system.

0023Injectors There are several approaches to injection,

using a combination of a septum and syringe or valve

injection.

0024Columns The stationary phases for adsorption

HPLC are silica, alumina, porous graphitized carbon,

porous glass, etc., the mobile phase generally being a

mixture of relatively nonpolar organic solvents. The

RP mode requires the combination of a polar mobile

phase and a nonpolar stationary phase. Similar to

TLC, mainly silica with hydrophobic ligands co-

valently bonded to the surface have been used as the

stationary phase. The RP technique is popular

because of the reproducibility associated with the

use of the hydrocarbon-bonded phase. Octyl- and

octadecyl-bonded silica stationary phases are rela-

tively stable, with most aqueous eluents having a

pH value below 8. RP HPLC uses less toxic solvents

(water and an organic modifier miscible with water),

thus causing less environmental pollution. Typical

organic modifiers are methanol and acetonitrile.

0025The mobile-phase delivery system includes a pump

with a filter, a degasser and transfer tubing. The pump

delivers the mobile phase into the separation system

with a constant, precise, and reproducible flow rate.

Two different types of elution modes are used in

injection

device

pump

reservoir

SEPARATION

DETECTION

detector

output

waste

separation

column

fig0005Figure 5 Schematic diagram of a typical HPLC system.

(a)

(b)

10.1

22.2

5.7

10.0

14.7

22.7

fig0004 Figure 4 Typical densitogram obtained after TLC of choles-

terol: (a) standard solution (c ¼400 mgml

1

); (b) cholesterol

from egg yolk. Reprinted with permission from Pozar M, Wondra

AG and Prosek M (1999) TLC and HPLC determination of levels

of cholesterols before and after its reduction in liquid egg yolk.

Journal of Planar Chromatography ^ Modern Thin-layer Chromato-

graphy 12: 416–424.

CHROMATOGRAPHY/Principles 1263

HPLC: isocratic and gradient elution. The isocratic

elution mode uses a mobile phase of constant com-

position. The composition of the mobile phase is

changed according to a predetermined program in

gradient elution. The degasser removes the residue

of gases from the mobile phase. Gases can form

bubbles in the mobile phase, impairing separation

and sometimes precluding the reliable detection of

analytes.

0026 Detectors The ideal HPL detector has a high sensi-

tivity, a universal or specific response, a wide linear

dynamic range, and a stable response with tempera-

ture and flow rate. The most commonly used de-

tectors in HPLC are: ultraviolet-visible (UV-vis),

fluorescence, refractive index, and electrochemical

detectors.

0027 The characteristic chromatographic parameters

(separation factor, resolution, theoretical plate

number) in HPLC can be calculated in the same way

as in gas chromatography. (See Chromatography:

High-performance Liquid Chromatography.)

Supercritical Fluid Chromatography

Principle

0028 Supercritical fluid chromatography encompasses all

chromatographic methods in which the mobile phase

is supercritical under the conditions of analysis, with

the solvating properties of the fluid having a measur-

able effect on the separation. The physical state of

these conditions are illustrated in Figure 6 for CO

the most commonly used supercritical solvent. SFC

has several advantages over GC and HPLC: it extends

the molecular-weight range of GC, thermally labile

compounds can be separated at lower temperatures,

compounds without chromophores can be sensitively

detected, and the use of open-tubular and packed

columns is feasible. Figure 7 shows a schematic layout

of a SFC system.

Injectors

0029 The most common injectors in SFC are high-pressure

valve injectors similar to those used in HPLC. The

method used for injection for open tubular columns

is split injection. Several split-injection methods have

been employed, dynamic flow split being the simplest

and most popular.

Columns

0030 Open-tubular coated capillaries (50–100 mm internal

diameter (ID) ), packed capillaries (100–500 mmID),

and packed columns (1–4.6 mm ID) have been used

for SFC. Many types of stationary phases developed

for either GC or HPLC have been adopted for use

in SFC.

0031The separation process in SFC can be influenced by

changing the pressure or the density (pressure or

density programming). The programmed addition of

mobile phase modifiers can also be used to change the

solute retention. The mobile phase travels from the

pump to the heated zone, where it becomes super-

critical, and then to the injector and column.

Detectors

0032A major advantage of SFC over GC and HPLC is

that SFC is ‘detector-friendly.’ Both HPLC and GC

Column

Oven

Detector Recorder

High

pressure

pump

Injector

Microcomputer

fig0007Figure 7 Schematic diagram of a SFC system.

Sublimation line

−60−50 −40 −30 −20 −10 0 102030405060708090

= 31.06 ⬚C

Boiling line

Melting line

Supercritical fluid

= 73.8 bar

Liquid

Gas

350

300

250

200

100

150

Solid

Pressure (bar)

fig0006Figure 6 Phase diagram (pressure–temperature) for CO

1264 CHROMATOGRAPHY/Principles

detectors can be successfully interfaced to SFC. The

most frequently used SFC detectors are flame ioniza-

tion, electron capture, ultraviolet, and mass-spectro-

metric detectors. (See Chromatography: Supercritical

Fluid Chromatography.)

Fundamentals

0033 Carbon dioxide with or without a modifier is the

mobile phase used preferentially in SFC. CO

often used as the mobile phase in SFC, possibly be-

cause its physicochemical characteristics can be

changed easily under moderate temperature and

pressure conditions. The retention of solutes can be

changed by an order of magnitude by increasing the

fluid density during density programming.

0034 The chromatographic parameters (separation

factor, resolution, theoretical plate number, etc.) in

SFC can be calculated in a similar way to that in GC.

Capillary Electrophoresis

Principle

0035 Separation is based on the different electromigration

velocities of ionic solutes under the influence of a high

voltage in narrow-bore capillaries. The use of a capil-

lary has numerous advantages, particularly with

respect to the detrimental effects of Joule heating.

The high electrical resistance of the capillary enables

very high electrical fields (100–500 V cm

1

)tobe

used, with only minimal heat generation. Moreover,

the large surface area-to-volume ratio of the capillary

efficiently dissipates any heat that is generated. The

separation efficiency, which is often in excess of 10

theoretical plates number, is due in part to the plug

profile of the electroosmotic flow, an electrophoretic

phenomenon that generates the bulk flow of the solu-

tion within the capillary. This flow also enables the

simultaneous analysis of all solutes, regardless of

charge. The ideal properties of the capillary material

are its chemical and electrical inertness, UV-visible

transparency, flexibility, and low cost.

0036 CE is characterized by its simple experimental set-

up. A schematic diagram of a basic CE instrument is

shown in Figure 8. Both ends of a capillary filled with

buffer are dipped into two buffer reservoirs that are

maintained at the same level. A high voltage is then

applied to this capillary.

0037 Various modes of capillary electrophoresis can be

performed using the same intruments, including ca-

pillary zone electrophoresis (CZE), capillary gel elec-

trophoresis (CGE), micellar electrokinetic capillary

chromatography (MEKC), capillary isoelectring

focusing, and capillary isotachophoresis. Figure 9

shows the different modes of capillary electrophor-

esis. MEKC is the second most popular CE separation

technique (after CZE), because it can separate neutral

analytes too. A surfactant above its critical micellar

concentration is added to the background electrolyte.

Solutes with varying degrees of hydrophobicities par-

tition between the hydrophobic core of the micelles

and aqueous buffer. The interaction between the mi-

celle and the neutral solutes leads to separation. The

separation mechanism of neutral solutes in MEKC

can be described using modified chromatographic

relationships. The ratio of the total number of moles

of solute in the micelle (that is in the pseudostationary

phase) to those in the mobile phase, the capacity

factor k

, is given by:

¼ðt

 t

Þ=½t

ð1  t

Þ ¼ KðV

Þ, ð8Þ

where t

is the retention time of the solute, t

is the

retention time of unretained solute moving at the

EOF rate, t

is the micelle retention time, K is

the partition coefficient, V

is the volume of the micel-

lar phase, and V

is the volume of the mobile phase.

Injectors

0038Two different injection modes can be used: hydro-

dynamic and electrokinetic injections. With hydro-

dynamic injection, a pressure difference is applied

for a short period of time between the two ends of

the capillary. A pressure difference can be introduced,

depending on the design of the instrument, by pres-

sure, vacuum, or simply the height difference between

the levels of the buffer and sample reservoirs. Electro-

kinetic injection is performed by replacing the injec-

tion-end reservoir with the sample vial and applying

the voltage.

Buffer/Sample

−

Buffer

Capillary

High voltage

power supply

Detector

Data

acquisition

fig0008Figure 8 Schematic diagram of a CE system.

CHROMATOGRAPHY/Principles 1265