Journal of Chromatographic Science, Vol.
18, October 1980
Practical Aspects of
Normal-Phase Chromatography
Seth R. Abbott
Varian Instrument Group, 2700 Mitchell Drive, Walnut Creek, California 94598
Abstract
Normal-phaaa chromatography in HPLC typically
sncompasssa adsorption ohromatography on silica
and partition chromatography on cyano and amino
bondad phasas. Uniqua aaparationa ars provldad for
saccharidas, iaomara, ataroids, and clasa aaparatlont
of lipida and polynuclaar aromatic hydrocarbons.
Introduction
The purpose of this review is to organize the applications of
normal-phase chromatography into a coherent structure so as
to allow the chromatographer to make a rational decision as to
when normal-phase chromatography should be applied and to
review the advantages and disadvantages of normal-phase vis
a vis reversed-phase chromatography. The latter task seeks to
aid the chromatographer in making his own judgement as to
whether the recent trend (stampede) to apply reversed-phase to
90% of HPLC applications represents a true picture of reality
or an error in intellectual perspective. The author's opinion on
this matter will be withheld.
A normal-phase separation is characterized by:
(A) Increased retention with increased solute polarity
(B) Decreased retention with increased mobile phase polar-
ity
(C) Solute retention mechanism dominated by interaction
of polar stationary phase sites with solute polar (hydro-
philic) groups
(D) Mobile phase usually less polar than the stationary
phase
Normal-phase separations encompass adsorption chroma-
tography on silica and alumina and partition chromatography
on polar chemically bonded phases. The use of alumina is too
rare to warrant inclusion in this report. Commercially avail-
able polar bonded phases offer a wide range of selectivity
(Table I). Listed in order of increasing polarity are diol<cyano
<dimethylamino< amino <diamino. Most polar bonded
540 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
phase separations are performed on amino and cyano phases.
Diol phases have been used predominantly in steric exclusion
chromatography; however, their use could increase with
future development of multistage methods (1) in normal-
phase chromatography.
Table 1. Polar Bonded Phase Structures
OHOH
I 1
Diol -(CH2)3OCH2CHCH2
Cyano -(CH2)3C=N
Amino -(CH2) n NH2 n = 3 or 4
Dimethylamino -(CH 2 )3N(CH 3 ) 2
Diamino -(CH 2 )3NH(CH 2 ) 2 NH 2
Bonded phase synthesis has been reviewed by Majors and
Hopper (2) and by Cox (3). Preparation of a polar, chiral
bonded phase has been described by Giibitz, et al. (4). A thor-
ough study of amino phase synthesis has been described by
Jones, et al. (5).
The major advantages of reversed-phase chromatography
have been reviewed by Karger and Giese (6). These advantages
are oriented toward biomolecule separations and are based
predominantly on the use of an aqueous mobile phase and on
the weak surface energies of interaction between the solute
and the nonpolar stationary phase.
The advantages of normal-phase chromatography are:
(A) Ability to obtain class separations which cannot be ob-
tained in reversed-phase (see the section on Class Se-
parations)
(B) Unique ability to separate isomers (see the section on
Isomer Separations), which is of great significance in
natural product and pharmaceutical chemistry
(C) Ability to separate highly hydrophilic species which
cannot be retained on reversed-phase (e.g., saccha-
Journal of Chromatographic Science, Vol. 18, October 1980

rides, see the section on Separation of Saccharides) Geometrical Isomers

(D) Use of predominantly organic solvent-based mobile
Normal-phase chromatography on silica is used for the
phases, avoiding silica dissolution problems experienced
separation of complex mixtures of porphyrin and bile pigment
in reversed phase
isomers. Rasmussen and coworkers (9) resolved a series of
(E) Mobile phase volatility, allowing simpler, more effi-
biliverdin-dimethyl ester isomers (bile pigments) on 5 ^ silica.
cient concentration and transfer steps in off-line frac-
Hajibrahim and coworkers (10) demonstrated that porphyrin
tion collection and structural characterization of solute
resolution is greater on silica than on a C18 reversed phase.
peaks
The volatility of the mobile phase (organic) is advantageous
(F) Ability to differentiate solutes based on differences in
in porphyrin research since fraction collection and concentra-
hydrophilic structure rather than hydrophobic structure
tion prior to off-line structural analysis is often required (11).
(see the section on Separation of Hydrophobically
However, one should note that for analyses in which relatively
Labelled Compounds)
simple separations are required and direct aqueous injection is
(G) Compatibility of organic phase with molecules having
desirable, such as clinical analysis for erythrocyte porphyrins
either low stability (see the section on Separation of
(12), the reversed-phase technique is preferable.
Compounds which are Unstable in Aqueous Solution)
Normal-phase chromatography on silica has been combined
or aggregation problems (e.g., phospholipids) in
off-line with reversed-phase to allow separations of a wider
aqueous phases
range of prostaglandins than would be possible with either
(H) Lower viscosity (~ 2.5-fold) of organic mobile phases,
technique alone. Whorton and coworkers (13) found that
resulting in lower operating pressures (see the section on
whereas reversed-phase chromatography readily separated
Instrumental Considerations)
PGEa and PGE2 (which differ by the presence of an additional
(I) Greater compatibility of organic mobile phase with on-
double bond in the alkyl chain of E2), it did not separate the
line coupling to electron-impact mass spectrometry (see
geometrical isomers PGE2 and PGD2 (Figure 1). Silica separa-
Instrumental Considerations)
ted the PGE2-PGD2 geometrical isomers but failed to separate
(J) Availability of wide range of organic solvents to capital-
PGEi-PGE2. Thus, silica fractions were collected manually
ize on special (2° or 3°) solvent effects
(K) Availability of a wide range of stationary phase selecti- and then analyzed by reversed-phase chromatography (Figure
vities, which can be utilized to obtain high separation 2).
performance in multistage chromatography
It should be noted that with the recent commercial develop-
ment of 5 \i normal bonded phases to complement the already
available 5 \i silica, there should be no significant differences
in efficiency between normal phase and reversed-phase
columns. The aforementioned normal-phase advantages have
reinforced its widespread use in class separations, isomer sepa-
rations, saccharide separations, and separations of complex
mixtures of steroids and steroidal type vitamins.
Factors that could increase the use of normal-phase chro-
matography in the future are: use of 2-3 \i particles; increased Figure 1. Prottaglandln structures. E2 and D2 arc geometrical
convenience of multisolvent mobile phase development with Isomers.
recently developed ternary high performance liquid chromato-
graphic (HPLC) systems; on-line LC/mass spectrometric (MS)
development; development of a viable lipid detector to inter-
face to successful silica separations; recent development of
automated multistage or multidimensional chromatography in
which different selectivities of polar (normal) phases are
coupled together or in which normal phase is coupled to
reversed phase (subject to mobile phase compatibility require-
ments); development of micro (capillary) LC, which reduces
cost and the toxicity/waste disposal problem of organic
solvent mobile phases.

Isomer Separations
Normal-phase chromatography of silica is unique in provid-
ing excellent separation of geometrical isomers. This has been
attributed to lock-key type steric fitting of solute molecules
with the discrete adsorption sites of the silica surface (7).
Recent work has demonstrated excellent selectivity to dia-
stereomeric derivatives of optical isomers for the polar hydro-
gen-bonding aminopropyl bonded phase and for silica (8).
I i
! A
Figure 2. A. Normal phase separation of prostaglandins on ^Porasll.
Linear gradient from chloroform to 6% methanol, 0.6% acetic acid In
chloroform, 1 ml/mln. B. Reversed-phase separation of prostaglan-
dins on ^Bondapak fatty acid (phenethyl phase). Isocratlc mobile
phase: 76.7:23.0:0.2:1 water-acetonltrlle-benzene-acetlc acid, 2
ml/mln. (Reprinted from reference 13 by permission of Elsevler
Scientific Publishing Company.)

541
 Journal of Chromatographic Science, Vol. 18, October 1980

Szepesi and coworkers (14) recently demonstrated the substitution on solute retention in reversed-phase chromatog-
separation of the stereoisomers of ergot alkaloids in plant raphy precludes its use for most class separations.
extracts and fermentation products on 5 \i silica. Slais and The development of multistage chromatography (1,20,21),
Subert (15) separated the cis- and /rcws-isomers of the tricyclic in which a fraction from one column is automatically switched
benzothiepine drugs on an aminopropyl bonded phase (Figure onto a second column for further separation, could capitalize
3). The separation was significant in that the cis- and trans- on the superior class separations of normal-phase
configurations differ in biological activity. chromatography and the superior "within-class" separations
of reversed-phase chromatography. For example, one could
separate lipids into classes such as sterols, triglycerides, fatty
acids, and phospholipids by adsorption chromatography (22)
on 5 \x silica and through the use of a switching valve direct
B these class fractions onto a reversed-phase column, which
would then separate the components of each class which differ
from each other by alkyl group substitution. The feasibility of
© such a system will be contingent on compatibility of the
Ji-trans •
normal-phase eluent with that of the reversed-phase column.

PFCCADER
100 mV

(wr)
0 4 8 12 16

Figure 3. A. Structure of benzothiepene isomers. B. Separation of

isomers of A, on aminopropyl phase, 10 n Silasorb column, 4 mm x
20 cm. 56:30:13:1.0:0.25 n-hexane-methanol-n-butanol-water-70%
HCIO4. (Reprinted from reference 15 by permission of Elsevler Scien-
tific Publishing Company.)

Tamegai and coworkers (8) extensively reviewed the HPLC

separation of optical isomers as their diastereomeric deriva-
tives, citing many applications of normal-phase chromatog-
raphy on silica and on the aminopropyl bonded phase. Souter
(16,17) demonstrated separation of the camphorsulfonamide
diastereomers of a series of racemic amphetamines and of the
analgesic drug propoxyphene. Furakawa, et al. (18) demon-
strated separation of the camphorsulfonamide diastereomers A
of D,L amino acids on 5 \i silica (Figure 4). Wickly and co- U)
workers (19) studied the normal-phase separation of cortico-
steroid acetal epimers and concluded that selectivity increased 40
with increased polarity of the stationary phase (NH2> silica >
CN). Thus, amino phases appear to have great potential for
separation of diastereomers. 20

20 60 nin

Class Separations
Normal-phase chromatography on silica or polar bonded
phases is commonly used for class separations such as lipid
separations on silica and the separation of polynuclear
aromatic hydrocarbons based on the number of condensed
rings on amino bonded phases. The marked effect of alkyl
Figure 4. Gradient elution separation of N,d-10-camphorsulfonyl p-
nitrobenzyl D and L-amino acids on amino phase. Column: MicroPak
N H 2 , 1 0 ^ . 2 m m x 2 S c m ; solvent: A * 70:15:15 Isooctane-dlchloro-
methane-isopropanol, B = 90:10 Isooctane-dlchloromethane; flow
rate: 0.5 ml/min; gradient as shown. (Reprinted from reference 18 by
permission of Elsevier Scientific Publishing Company.)

542
Journal of ChromatographicScience, Vol. 18, October 1980
Polynuclear Aromatic Hydrocarbons
Separation of polynuclear aromatic hydrocarbons (PAH) is
necessary for characterization of fuel sources and trace analy-
sis of these potential carcinogenic and mutagenic species in
air, wastewater, and industrial process streams. Sixteen PAHs
containing from 2-6 condensed rings were recently identified
by the EPA as priority pollutants.
Reversed-phase chromatography offers considerable selecti-
vity (23) for PAHs. However, alkyl substitution significantly
increases PAH retention so that alkyl substituted PAHs with
low condensed ring content elute in the same region as un-
substituted high ring content species. Thus, in reversed-phase
chromatography a separation sequence according to the
number of condensed rings is not possible for complex
samples containing alkylated PAHs. This interferes signifi-
cantly with qualitative and quantitative characterization of the
PAH mixture.
Wise, et al. (24) showed that aminopropylsilica bonded
phases yield a separation sequence based on condensed ring
number, regardless of alkyl substitution. A subsequent study
by Chmielowiec and George (25) on several polar bonded
phases showed superior PAH selectivity in terms of ring
number mediated separation sequence for a diamino (-(CH^j-
NH(CH2)2NH2) phase. Alkyl substitution less than decyl did
not significantly affect PAH retention. Phenyl substitution
increased PAH retention, but to a considerably lesser extent
than in reversed-phase chromatography. Chmielowiec and
George (25) found the diamino phase separation sequence to
be determined by condensed ring number for up to 4-ring aro-
matics with partial breakdown of the ring number-elution
sequence for higher condensed ring structures. The diamino
phase appears promising for characterization of PAH mix-
tures. A PAH separation on a 10 ^ diamino column is shown
in Figure 5. Development of a 5 \i analog and use of gradient
elution should extend the utility of this technique.
S I L I C A - ( OIAMINE)
n-H«pton« 6%CK | CL ( in-H«pton«
20 30 10 20 30
RETENTION VOLUME (ml)
Figure 5. Separation of PAH on 10 p, 4.6 mm x 25 cm dlamino-slllca
column. Flow rate: 2 ml/mln; ambient temperature. Single ring: Peak
1: benzene. Two rings: Peak 2: Indene; 3: napthalene; 4: 2,3,5-trl-
methylnaphthalene; 5: blphenyl; 6: azulene. Three rings: Peak 7:
fluorene; 8: o-terphenyl; 9: anthracene; 10: phenanthrene; 12:
m-terphenyl. Four rings: Peak 14: fluoranthene; 15: pyrene; 16:
benzo(b)fluorene and benzo(c)fluorene; 17: benzo(a)anthracene.
Five rings: Peak 19: benzo(a)pyrene; 20: perylene; 2 1 : anthanthrene;
23: dlbenz(a,h)anthracene; 24: dibenz(a,c)anthracene. Six rings:
Peak 22: benzo(ghi)perylene; 25: dlbenzo(e,h)pyrene. (Reprinted
from reference 25 with permission of the American Chemical Society.)
Mourey, et al. (26) showed that a pyrrolidone bonded phase
on 5 \i silica (-(CH2)3- ^\) also provides a normal phase PAH
ii
O
separation according to ring number; for PAHs of a given ring
number, separation occurs according to type of condensation.
Thus, cata-condensed ring systems (e.g., chrysene, 4 rings) are
more retained than peri-condensed ring systems (e.g., pyrene,
4 rings). The PAH separation mechanism on amino, diamino,
and amide (pyrrolidone) type phases is believed to involve a
charge transfer type interaction of the lone pair electrons of
the stationary phase nitrogen atoms with the condensed aro-
matic nuclei (24,26).
SARA
Separation of liquid fuels into saturates, aromatics, resins,
and asphaltenes (SARA) is used by the petroleum industry in
monitoring refinery processes and characterizing crude oils. In
the method of Galya and Suatoni (27) the hexane soluble
portion of the sample containing saturates, aromatics, and
resins (phenols, aromatic aldehydes, N-heterocycles) is sepa-
rated on an aminopropyl phase in the normal-phase mode
with a hexane mobile phase (Figure 6). Saturates elute as a
single low k' peak, followed by aromatics eluting in sequence
as to condensed ring number. The resins are then backflushed
in ~ 7 min. Refractive index (RI) detection is used. The
asphaltene content is determined nonchromatographically by
the difference between the weight of hexane insolubles and
toluene insolubles (the hexane-insoluble asphaltenes will dis-
solve in toluene). A separation of asphaltenes along with aro-
matics and resins in bitumen and heavy petroleum crudes has
been achieved by Reichert and Johnson (28) using a cyano-
propyl phase and step gradient elution.
Aromatics
PEAKS
Saturates: 1.08 min.
Aromatics: 1.30-5.24 min.
Backflush: 6.67 min.
Figure 6. Separation of saturates, aromatics, and resins on 4 mm x
30 cm, 10 micron ^Bondapak NH2 column. Flow rate: 3 ml/mln
hexane; RI detection. (Reprinted from reference 27 with permission
of Marcel Dekker Publishing Company.)
543
 Journal of Chromatographic Science, Vol. 18, October 1980

Alfredson and Apffel (29) pointed out that the reactivity of precolumn derivatization step prior to separation on silica
stationary phase primary amino groups with aldehydes and (32,33). Detection of the benzoyl label is optimal at 230 nm. A
ketones could limit the utility of the aminopropyl phase for typical separation is shown in Figure 9.
carbonyl-containing fuel samples. In addition, the amino
column did not separate saturates and olefins. Alfredson and
Apffel (29) used a cyanopropyl phase column to obtain SARA
separations similar to those obtained on an amino phase. In
addition, series coupling of the cyano column to a 5 \x silica
column allowed separation of saturates and olefins while
preserving the separation of aromatics and polar compounds
(resins) (Figure 7). The cyano column was located before the
silica column since polars are more easily backflushed from
the less retentive cyano phase.

a UV(254nm) 1.0AUFS

r ±

\\

AROMATIC*
Figure 8. Separation of soybean lipids on silica. Mobile phase as in-

S dicated; peaks 1-3: hydrocarbons, waxes, pigments, and unknowns;

Ilk •
34 32 30 28 26 24 22 20 It 1« 14 12 10|
Figure 7. Separation of saturates, olefins, aromatics, and resins
(polars) on MicroPak CN-10 + MicroPak Si-S columns (4 mm x 30
cm) in series. (Reprinted from reference 29 by permission of Varian
Instrument Group.)
4-5: sterol esters; 6: triglycerides; 7-11,14,18, 20, 25, 28: unknowns;
12: sterols; 13: free fatty acids; 15: esterified sterol glucosldes; 16:
monogalactosyl diglycerides; 17: sterol glucosldes; 19: cerebro-
sides; 21: phosphatidyglycerols; 22: digalatocsyl diglycerides; 23:
phosphatidylethanolamines; 24: phosphatidylinositols; 26: phos-
phatidylcholines; 27: phosphatidic acid; 29: lysophosphatidyl-
choline and other very polar lipids. (Reprinted from reference 22 by
permission of Elsevier Scientific Publishing Company.)
1
1 ' 1 ' I ' i
A

Lipids 2

Lipid mixtures are complex, cover a wide polarity range,

\A A A .

and require class separation (22). Within each lipid class, the -A. A_- CONTROL
molecules differ as to alkyl chain character. Thus, lipid class
separation requires separation based on polarity, favoring I 2 3 4
adsorption chromatography and ruling out reversed-phase - KRABBE'S
chromatography. 1
A silica separation of soybean lipids into 18 classes is shown

VJ
O
in Figure 8. Note the use of 5 solvents in the mobile phase Q.
tn 2
gradient. Mobile phases based on more than 2 solvents are UJ 1 3 4
used in lipid separations in order to exploit special solvent •N - GAUCHER'S
effects. One should also note that phospholipids tend to form 3

1
A .J\A -A-
micelles in nonpolar solvents. Phospholipids elute in a polar
organic mobile phase on silica, avoiding micelle formation.
However, reversed-phase chromatography would require a
nonpolar mobile phase which could introduce micelle
u\ 4
- FABRY'S

uI
3 4
formation problems.
Nielson (30) showed that variability in the retention of
acidic phospholipids can occur due to trace divalent cations k- — STANDARD

(Ca*2, Mg*2) on the silica surface. A study of contamination of . 1 1 . 1

. 1 . I . I .
silica with trace metal ions and a means (acid washing) to 8 10 12
remove such contamination was recently reported by Verzele, TIME (min)
etal. (31). Figure 9. Separation of plasma glycollplds of different disease states
Analysis of glycosphingolipids in plasma is of importance in on silica. Glycollpld fractions from 1 ml of plasma were Isolated,
benzoylated, and dissolved In 100 H' of CCI4 and 40 pi analyzed by
hereditary disease detection, plasma membrane research, and
HPLC. Peaks are Identified as the derivatives of: 1: GLC-Cer; 2:
antigen and tumor research (32). These species absorb too Lac-Cer; 3: Gal-Lac-Cer; 4: globoside. (Reprinted from reference 32
weakly in the UV to allow detection at plasma levels. Thus, the by permission of the Journal of Lipid Research.)
carbohydrate (glyco) portion of the lipid is perbenzoylated in a

544
Journal of Chromatographic Science, Vol. 18, October 1980

Research in HPLC analysis of lipids is currently limited by silica gel column using an aqueous acetonitrile mobile phase to
detection problems. Lipids have only weak UV absorption, which an amine modifier was added at 0.1% level in a pre-
and in addition, UV discriminates components too severely liminary in situ coating step, followed by a reduction of the
based on double bond content in the alkyl chains of the lipid. amine level to 0.01% in the mobile phase used for analysis.
Certain lipid classes can be detected adequately after derivati- Saccharide retention was somewhat lower on the in situ amine
zation; for example, the aforementioned detection of glyco- modified silica than on a chemically bonded aminopropyl
lipids at 230 nm absorbance as their perbenzoyl derivatives phase. Retention decreased in the sequence: amino chemically
(14), bile and fatty acids detected as their fluorescent BMC
derivatives (34,35), and phosphatidyl ethanolamines and
phosphatidyl serines, which should be detectable by fluor-
escence after post-column derivatization with o-phthalalde-
hyde. However, many other lipid classes of significance are
not amenable to currently available pre- and post-column
labelling reagents.

Separation off Saccharides

Alkylamine bonded phases are used for the separation of

saccharides and oligosaccharides in foods (36,37) and in bio-
logical mixtures (38,39). The stationary phase alkyl group is
typically w-propyl or w-butyl, and the amino group is primary
(an exception is the Partisil-10-PAC column which contains
cyano and secondary amine groups in a 2:1 ratio in the
stationary phase). The mobile phase consists of acetonitrile-
water or acetonitrile-dilute phosphate buffer.
Although the separation mechanism has occasionally been
misidentified as reversed-phase (38), it is a normal-phase
mechanism by virtue of the fact that increased aqueous levels
of the mobile phase reduce carbohydrate retention and elution
order follows carbohydrate polarity. A typical saccharide
separation is shown in Figure 10.
Reducing saccharides exist in aqueous solution as equilib-
5 10 15 20
rium mixtures of cyclic hemiacetal and acyclic aldehydes
(aldoses) or cyclic hemiketal and acyclic ketones (ketoses). It is TIME lmln.J
well known that reducing saccharides react with primary and
secondary amino groups by carbonyl-amine condensation to Figure 10. Separation of caramel mono- and dlsaccharldes on amino-
yield a Schiff base (40). Reactivity order (41) tracks the equilib- propyl phase. Column: fiBondapak carbohydrate, 3.9 mm x 30 cm;
rium concentration of the acyclic form (42) and is ribose> flow rate: 2 ml/mln; mobile phase: 80/20 acetonitrlle/water; Rl
detection. Peak 1: fructose; 2: glucose; 3: sucrose; 4: maltose; 5:
arabinose > xylose > mannose ~ galactose > glucose » fructose lactose. (Reprinted from reference 36 by permission of the Journal of
>sorbose. Thus, pentoses are more reactive than hexoses, and Food Science.)
aldoses are far more reactive than ketoses. Nonreducing sac-
charides such as sucrose, raffinose, and stachyose are non-
reactive. The condensation is faster in alcohols than in less
polar organic solvents (e.g., acetonitrile) and is known to be
catalyzed by high levels of acetic acid (> 5%). Table II. Saccharide Reactivity with Amino Phase
An unpublished study by this author of reaction of saccha-
rides with a primary amino phase during chromatography in On-Column Sample Loss
acetonitrile-water demonstrated significant on-column sample Ribose Glucose Maltose
reaction (> 10%) for reducing saccharides higher than glucose
in the aforementioned reactivity scale (Table II). Thus, galac- Amino Phase 1 ml/min 60% 12% 0%
tose and the aldopentoses (e.g., ribose) should not be analyzed 2ml/min 43% 10% —
on primary amino phases. Protonated
Amino Phase 1 ml/min 48% 8% —
Secondary amines are significantly less reactive towards car-
bonyl groups (43) than are primary amines, and thus the
(Phosphate form) 2ml/min 27% 4% —
secondary amino phase of the Partisil-10-PAC column could 4 mm x 30 cm MicroPak NH2-10.80% acetonitrile-20% water for ribose,
be more amenable to aldopentose separation. glucose studies. 70% acetonitrile-30% water for maltose. Peak area for
saccharide eluting from primary amino column referenced to that from a
An interesting variant of the amino phase separation of sac- quaternary amine column (MicroPak SAX-10). Amino phase converted to
charides was demonstrated by Aitzmuller (44) and by Wheals phosphate form by 0.1 M H3PO4 wash. Same mobile phase as for unprotonated
phase was used for analysis.
and White (45). These authors separated saccharides on a

545
 Journal of Chromatographic Science, Vol. 18, October 1980
bonded phase >in situ polyamine>m situ diamine>m situ
amine>silica. A viable separation was obtained with in
situ-coated tetraethylenepentamine (Figure 11).
JLJ
i—i—i—r~~i—r
Figure 11. The separation of sugars on 1: 3-amlnopropyl bonded
phase; 2: silica using an acetonitrile-water (75:25) eluent. Separation
3 was obtained on silica with 0.01% TEPA added to the eluent. Rl
detection; 4.9 mm x 12.5 cm columns; 2 ml/min flow rate. (Reprinted
from reference 45 by permission of Elsevier Scientific Publishing
Company.)
Wheals and White (45) observed that the in situ coating
method yielded columns more tolerant of nonsugar co-
extractives than the chemically bonded amino phases. If the
lifetime limiting process for the primary amino bonded phase
were gradual reaction of the amine sites with sample car-
bonyls, one would expect the in situ columns to last longer. In
the in situ method, the amine groups (adsorbed on weakly
acidic surface silanols) could be desorbed upon reaction with
carbonyls and then continuously replaced (regenerated) by the
mobile phase amine modifier.
Separation of
Hydrophobically Labelled Compounds
Functional group specific precolumn fluorescent labelling
reagents have been developed for primary and secondary
amines, sulfhydryls, phenols, aldehydes, ketones, and carbox-
ylic acids (46,47). These labels, in order to confer high fluor-
escence sensitivity (thus, high absorption and high fluores-
cence quantum yield) onto the aforementioned compounds,
contain condensed aromatic ring systems (e.g., dansyl). The
relatively hydrophobic ring system of the label tends to
dominate reversed-phase chromatography of mixtures of der-
ivatives of hydrophilic substrates, resulting in reduced selecti-
vity. Complex mixtures of such derivatives are often more
readily resolved on silica or polar bonded phases, on which the
solute structure should determine retention to a greater degree
than the attached fluorescent label (48).
For example, Avigad (49) demonstrated fluorescent label-
ling of reducing carbohydrates with dansyl hydrazine prior to
thin layer chromatographic (TLC) analysis. Reversed-phase
chromatography would be expected to be dominated by
interaction of the relatively hydrophobic dansyl group with
the stationary phase. The hydrophilic carbohydrate nucleus
should have little interaction with a nonpolar reversed phase.
546
Dansylated carbohydrates should thus be separated on a polar
stationary phase such as silica [used in TLC by Avigad (49)].
Naider and coworkers (50) separated protected hydrophobic
oligopeptides on silica. The ter/-butoxy carbonyl (BOC)
group used to block oligopeptides was relatively hydrophobic
and thus straight-phase was preferable to reversed-phase
chromatography for this application. In addition, BOC-
protected isomeric peptides were readily separated on the silica
stationary phase (Figure 12).
0.08 J B
20.04
o
0.0
4 8 4
TIME WIN) TIME(MIN)
Figure 12. Separation of isomeric dlpeptides Boc-Met-X-OCH3 and
Boc-X-Met-OCH3. A. 1 = Boc-Met-Gly-OCH3; 2 * Boc-Gly-Mtt-
OCH3. B. 1 = Boc-Met-Ala-OCH3; 2 = Boc-Ala-Met-OCH3. (Reprin-
ted from reference 50 by permission of Elsevier Scientific Publishing
Company.)
Steroids and Vitamins
Steroids comprise a wide variety of active compounds which
have a fused, 4 member reduced ring system in common:
The plethora of natural and synthetic steroids differ as to
isomeric conformation, ring substitution (e.g., hydroxyl, car-
bonyl), and ring double bond content. Steroids are thus
frequently separated by normal-phase chromatography since
the separation mechanism is determined by the difference in
ring substituents or isomer conformation rather than by the
hydrophobic 4-ring nucleus.
25-Hydroxyvitamin D3 (25-OH-D3) is the first major
metabolite of vitamin D in the biogenesis of the calcium and
phosphorus-mobilizing hormone 1,25-dihydroxyvitamin D3.
Serum levels of 25-OH-D3 reflect vitamin D status or defective
Journal of Chromatographic Science, Vol. 18, October 1980

25-hydroxylation. Because 25-OH-D can be derived fron
either vitamin D2 or D3, separation of serum 25-OH-D2 from
25-OH-D3 (Figure 13) is necessary in research on vitamin D
metabolism.
Eisman, et al. (51) demonstrated that such a separation is
readily achieved on silica (shown in Figure 14). Reversed-
HO
Vitamin D, Vitamin D,
Figure 13. Vitamin D2 and D3 structures. Metabolism consists of
hydroxylatlon at C-25 (25-OH-D) and at both C-| andC 2 (1,25-dl0H-D).
phase chromatography has had difficulty in separating vita-
min D2 and D3 analogs (52). However, DeVries, et al. (53)
was recently able to achieve D2-D3 separations on reversed-
phase with a 3° mobile phase (acetonitrile-propionitrile-water).
VanHaelen-Fastre and VanHaelen (54) recently demonstrated
silica separation of hydroxyvitamins D3 and their corres-
ponding prehydroxyvitamin D3 isomers. These species could
not be resolved by reversed-phase chromatography in the same
study.
Normal-phase chromatography has also been used to pro-
vide excellent separations of complex mixtures of androgens
(55) and testosterone and its metabolites (56). A precaution to
be considered in the normal-phase separation of steroids in-
volves avoidance of ketosteroid analysis on amino phases.
Ketosteroids react with amino stationary phases (57). Thus,
for ketosteroids one should use either silica or cyano phase
columns.
Tocopherols (vitamin Es) represent another class of struc-
turally similar compounds which are separated by normal-
phase chromatography on silica. Jansson and coworkers (58)
demonstrated resolution of a, p, y, and d tocopherols on silica
(Figures 15 and 16).

0.004 Solvent
ront (a)
0.003 25-0 H03

0.002

0.001

£ 0.000
3 10 15 20
« 0.004 m 1 n 16
O Solvent
(b)
b Front Figure 15. Tocopherol structures. Note that p and y forms are
Isomers.
0.003

0.002

25-OHOo CH» 3 CH 3
0.001 25-OHO2

a-tocopherol (Vitamin E) Rj , R2 =
0.000 3
5 10 15
6-tocopherol R
l = CH, \ ? = H
y-tocopherol = H / R2 = CH,
Time (min) 6-tocopherol
Flgurt 14. Typical HPLC profiles obtained with human blood plasma R »H
samples with (a) high and (b) low 25-0H-D2 and 25-OH-D3 elutlon Figure 16. Separations of tocopherols In serum on 10 n silica, 4 mm x
positions are Indicated. Column: 4 mm x 3 cm, 10 micron ^Porasll; 30 cm ^Porasll column. Flow rate: 2.5 ml/mln; mobile phase: 92:8
flow rate: 2 ml/min; mobile phase: 2.5% isopropanol In hexane. n-hexane-diisopropylether; fluorescence detection. (Reprinted from
(Reprinted from reference 51 by permission of Analytical Bio- reference 58 by permission of Elsevler Scientific Publishing
chemistry.) Company.)

547
 Journal of Chromatographic Science, Vol. 18, October 1980
Separation of Compounds which are
Unstable in Aqueous Solution
Compounds which are unstable in water are not compatible
with the aqueous mobile phases typically used in reversed-
phase chromatography and thus must be separated by normal-
phase chromatography. An interesting example of such a
compound is benfurodil hemisuccinate, a vasodilator which is
stable in plasma due to fixation to plasma proteins but is un-
stable in aqueous solutions. Turcant, et al. (59) thus analyzed
this drug in plasma by chromatography on silica with a non-
aqueous mobile phase.
Instrumental Considerations
Pressure Considerations
Typical normal-phase mobile phases have viscosities (rj) of
~ 0.5 cp (25°C). Reversed-phase mobile phases such as
water-acetonitrile and water-methanol have rj ~ 1.3 cp (60).
Thus, normal-phase chromatography is run at ~ 2.5-fold less
pressure than reversed-phase.
Recent developments in column technology have led to the
introduction of highly efficient (~ 8000-10,000 plates) 15 cm,
5 \i normal and reversed-phase columns. At a typical flow
velocity of ~ 0.3 cm/sec (1 ml/min on 4 mm i.d. column), 5 p
column operating pressures are ~ 50 and 125 atm for normal-
and reversed-phase chromatography. These pressures are well
within the normal operating range of most commercial pumps.
However, if one is to use coupled columns to attain high effi-
ciencies for complex separations, such as coupling four 15 cm
columns in series to achieve 40,000 plates, the respective
normal- and reversed-phase pressures increase to 200 and 500
atm. At 500 atm, the lifetime of pump components (e.g., seals)
are shortened and the bed compression lifetime of columns
typically packed by chromatographers at ~ 400-600 atm is
also shortened. It should also be noted that at 500 atm one is
approaching the safety rating of many column end fittings.
The 2- to 5-fold lower viscosity of normal mobile phases
could become a factor in spurring normal-phase development
for the use of 3 \i columns. Studies by Halasz, et al. (61) have
suggested that 1-3 \i is the optimum particle size for HPLC. A
single 3 \x, 4 mm i.d. x 15 cm column will generate operating
pressures of ~ 140 and 350 atm in normal- and reversed-phase
chromatography.
Halasz, et al. (61) hypothesized that axial thermal gradients
in columns due to frictional heating of the mobile phase could
conceivably act to limit the efficiency inherent in sub-5 \A
HPLC supports. Halasz, et al. (61) derived an approximate
expression for the frictional heating of the mobile phase:
AP
AT =
41.3pC
where AP is the pressure drop across column, p is the mobile
phase density, and C is the mobile phase heat capacity. Al-
though AP will be ~ 2.5-fold lower in normal- vs. reversed-
phase chromatography, the lower density and heat capacity of
organic solvents relative to water tend to balance this effect so
that these AT effects will be similar in the normal- and
reversed-phase modes.
548
Extra-Column Band Broadening Considerations
Extra-column variance due to injector valve volumes and
coupling line volumes (injector-* column, column-•detector)
are due to laminar flow band broadening (62). The Golay
equation for the variance due to laminar flow band broaden-
ing through these components at typical LC flow rates is
written below:
nr4Q
2 _
LF ~ 24D,
m
where r is the inner radius of component and Q is the flow rate
of mobile phase. The solute diffusion coefficient, D m , is
related to the mobile phase viscosity, rj, by the empirical
Wilke-Chang expression:
where M2 is the mobile phase molecular weight, v 2 is the
mobile phase association factor, Vx is the solute molar
volume, and r\ is the mobile phase viscosity. The lower
viscosity and higher molecular weight of organic solvents used
in the normal-phase mode yield an ~ 3-fold higher diffusion
coefficient, thus reducing laminar flow variance terms relative
to those of the reversed-phase mode. This should become
important in coupling instrumentation to 3 \A and 5 \JL short
columns (< 15 cm lengths) so as to preserve the inherent effi-
ciency of these columns.
Detector Considerations
Flow sensitivity—absorbance and refractive index detectors.
Most commercial HPLC pumps are of the reciprocating type
and commonly use hydraulic capacitor (RC) type dampers
whose damping efficiency increases with downstream
pressure. Thus, flow pulsations are greater at the lower
operating pressures of normal-phase mobile phases. The
extent to which a periodic flow pulsation is translated to a
periodic detector baseline fluctuation for optical absorbance
detection is a function of the flow sensitivity of the detector.
For any given absorbance detector design, flow sensitivity is
increased for the case of solvents of high thermal diffusivity.
Organic solvent thermal diffusivity is greater than that of
water and thus detector flow sensitivity with organic solvents
is ~ 3 times higher than that for water.
This potential problem in normal-phase chromatography
has been reduced by several trends:
(A) Use of 5 \i particles, increasing typical pressures 4-fold
over previous 10 \i columns
(B) Advances in detector design to reduce flow sensitivity
(tapered flow cells, thermostatting of detector inlet line
and flow cell, optical imaging to ensure that incident
light beam passes through the flow cell without striking
cell walls)*
(C) Use of restrictor precolumns inserted before the injector
to ensure operating pressure > 50 atm at which hydrau-
lic dampers act with good efficiency
'Although use of capillary heat exchangers between column and flow cell will
significantly reduce detector flow sensitivity, this design option Is less
attractive than a-c since it can introduce extra-column band broadening.
Journal of Chromatographic Science, Vol. 18, October 1980
The flow sensitivity of RI detectors is a function of the
pressure across the detector flow cell. Although the lower
operating pressures of normal-phase chromatography result in
a less efficient damping system, a given flow pulsation will
translate into a lower pressure change across the detector flow
cell (lower solvent viscosity). These effects tend to balance so
that normal-phase chromatography should not have a dis-
advantage in RI flow sensitivity relative to reversed-phase
chromatography.
Detector compatibility. The lower polarity, higher
volatility, lower gas expansion volumes, and wetting
characteristics of organic mobile phases confer an advantage
in coupling normal-phase HPLC to moving belt-type LC/MS
interfaces (63). These problems led Karger, et al. (64,65) to
couple a modified Technicon (Tarrytown, New York) auto-
extractor into such an LC/MS system. The reversed-phase
mobile phase was continuously extracted with dichlorometh-
ane, which was then transported to the belt interface.
Normal-phase LC/MS with a moving belt interface does not
require the auto-extractor (66,67), which is a source of extra-
column band broadening and is characterized by -solute
extraction solvent-dependent yields of 10-50% and additional
mechanical complexity.
Electrochemical detection. Electrochemical detection re-
quires a conductive solvent. Typical normal-phase mobile
phases are nonconductive. Thus, a supporting electrolyte such
as 0.01 M lithium perchlorate must be added to the mobile
phase, either before or after its passage through the column
(68). In addition, the distance between the working electrode
and counter electrode of the detector must be kept small to
minimize solution resistance. Thin-film electrode cells satisfy
this requirement.
Addition of a supporting electrolyte after the column avoids
problems due to (A) adsorption of the ionic electrolyte species
onto the normal phase with a resultant change in chromato-
graphic properties and (B) insufficient solubility of the sup-
porting electrolyte in a mobile phase such as hexane. One can
resolve this problem by the post-column addition of the sup-
porting electrolyte as a dilute solution in a polar hexane-
miscible organic solvent such as isopropanol.
Fluorescence. Thefluorescenceof certain classes of fluoro-
phores is quenched by polar solvents. For example, dansyl
derivatives of phenols and amines fluoresce strongly in
solvents of low dielectric constant such as those used in
normal-phase chromatography and fluoresce weakly in high
dielectric solvents (69,70) such as those used in reversed-phase
chromatography. Thefluorescence-of other classes of com-
pounds is quenched by nonpolar solvents and hence are more
compatible with reversed-phase chromatography. Examples
are chlorophylls, quinine, and aflatoxins Bx and B2. Solvent
effects on fluorescent solutes should be investigated before
deciding on a separation mode for these solutes.
A solution to mobile phase/fluorescence yield incompati-
bility is the use of packed fluorescence flow cells. Thus, to
detect aflatoxins Bx and B2 in normal-phase chromatography
using a relatively nonpolar mobile phase, Johnson and
Abu-Shumays (71) packed thefluorometerflowcell with silica
gel. The polar environment of the silica enhanced the B, and
B2 fluorescence.
Temperature Effects
Temperature effects in adsorption chromatography have
been studied by Maggs (72) who showed these effects to be
modifier-dependent. Scott and Reese (73) showed that as a
general rule in adsorption chromatography, retention time
precision of 1 % and 0.1 °7o requires column thermal control to
within ±O.35°C and ±0.04°C, respectively. Recently, Sisco
and Gilpin (74) showed that for the case of a partially soluble
modifier such ~ 0.1 % water in a hexane mobile phase, drama-
tic temperature effects on the distribution of modifier between
mobile phase and stationary phase can occur. The effect is
most pronounced for highly polar surfaces such as silica and
for the typical column temperature range of 25-35°C. The
data suggest (74) that retention time precision of 1 °Io and 0.1 °7o
require thermal control to within ± 0.1 °C and ± 0.01 °C,
respectively.
Thermal control of column temperature to ± 0.05°C is with-
in the state-of-the-art of commercial HPLC technology (air
ovens or aluminum thermal blocks). Control to ± 0.01 °C or
better requires column thermostatting in a liquid bath. Solvent
thermal control is also required for this extreme case.
A practical implication of Sisco and Gilpin's (74) article
would seem to be that use of alcohol modifiers such as iso-
propanol would be preferable to water for high retention
reproducibility.
Ternary HPLC Systems
Normal-phase chromatography on silica has historically
utilized mobile phases based on more than two solvents,
capitalizing on special solvent effects available from a wide
variety of miscible organic solvents. A firm basis for pre-
dicting these effects exists in the Hildebrand solubility
parameter theory (75). The recent development of micro-
processor-controlled ternary HPLC systems (76) should
expand the capability of HPLC to utilize these multisolvent
effects. Mobile phase optimization in methods development
should become faster and more convenient with these systems.
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