III / AMINO ACIDS / Liquid Chromatography 1999

will remain important for specialized applications Hus\ ek P and Macek K (1975) Gas chromatography of
requiring femtomole sensitivity. amino acids. Journal of Chromatography 113: 139.
KoK nig WA (1987) The Practice of Enantiomer Separation
See also: III/Amino Acids: Liquid Chromatography; Thin- by Capillary Gas Chromatography. Heidelberg: HuK thig.
Layer (Planar) Chromatography. Amino Acids and MacKenzie SL (1981) Recent developments in amino acid
Derivatives: Chiral Separations. Amino Acids and analysis by gas-liquid chromatography. In: Glick D (ed.)
Peptides: Capillary Electrophoresis. Methods of Biochemical Analysis, vol. 27, p. 1. New
York: Interscience.
Further Reading Weinstein B (1966) Separation and determination of amino
acids and peptides by gas liquid chromatography. In:
Gehrke CW, Roach D, Zumwalt RW et al. (eds) (1968) Glick D (ed.) Methods of Biochemical Analysis, vol. 14,
Quantitative Gas-liquid Chromatography of Amino p. 203. New York: Interscience.
Acids in Proteins and Biological Substances: Macro, Zumwalt RW, Kuo KCT and Gehrke CW (eds) (1987)
Semimicro and Micro Methods. Columbia, MO: Ana- Amino Acid Analysis by Gas Chromatography. Boca
lytical Biochemical Laboratories. Raton, FL: CRC Press.

Liquid Chromatography
I. MolnaH r-Perl, L. Eo( tvo( s University, LC of Underivatized AAs
Budapest, Hungary
Copyright ^ 2000 Academic Press To attain one of the main advantages of LC } separat-
ing the ‘classical 20’ as underivatized AAs } has ap-
The Rrst approach to the automatic liquid chromato- pealed to chromatographers. In spite of a number of
graphy (LC) of amino acids (AAs) } known today as efforts, the simultaneous LC of underivatized AAs
ion exchange chromatography (IEC) } was published has remained of secondary importance. Determina-
by Spackman et al. in 1958. In over 40 years later, it tion of a few selected AAs, such as tryptophan or
now takes less than 5 min (Figure 1) to separate and sulfur-containing AAs, has proved to be fruitful for
quantitate the essential protein AAs instead of 2 days. special tasks.
Early separations were carried out by post-column The aim of various investigations was to render
derivatization. unnecessary the time-consuming derivatization tech-
Over the last 20 years LC has offered unlimited niques. However, the characteristics of the free
possibilities in both the preparative and analytical AAs are considerably different from each other and
scale. The wide choice and sophisticated columns, their various structural properties did not permit their
detectors, derivatization procedures, development of easy resolution. Thus, in attempting to achieve better
modern instrumentation and data-handling systems separation of free AAs, further means of discrimina-
reduce time and costs, and give versatility and auto- tion were needed. For this purpose special techniques
mation in Good Laboratory Practice (GLP)- control- have been introduced, such as the use of various
led conditions for selectivity, sensitivity and repro- phase systems, ion pair and ligand exchange
ducibility. It is the responsibility of the researcher to chromatography, column-switching techniques or
choose the most appropriate method for the given anion exchange chromatography with electrochemi-
task. The most popular LC method for analysis of cal detection.
both free AAs (present in many natural matrices, The solvent-generated ion exchange phase system
biological Suids and tissues, feed and foodstuffs) and ensured the gradient elution of 19 AAs (Figure 2A):
of those constituents of protein hydrolysates is now some, but not all, are baseline-separated. A simple
reversed-phase (RP) chromatography after pre-col- isocratic method using aqueous, copper acetate/alkyl-
umn derivatization of the AAs. sulfonate additives containing acetate buffer (pH 5.6)
Numerous methods for derivatization are available as mobile phase, a conventional RP column and UV
in the literature. This article will discuss the advant- detection (230}240 nm) at different temperatures
ages and drawbacks of the commonly used deriva- and varying the concentrations of additives was un-
tives. able to separate the classical 20 protein amino acids.
Current trends in AA analysis identify the best SigniRcant improvement in the separation can be
conditions for enantiomer separation and the devel- obtained by column switching (Figure 2B), as well
opment of LC-mass spectrometry (LC-MS). as by using an anion exchange column, a quaternary
2000 III / AMINO ACIDS / Liquid Chromatography

Figure 1 Separation of the phenylthiocarbamyl AAs separated on TSKgel Super-ODS (for details see Table 3). Peaks: 1, ASP;
2, Glu; 3, Ser; 4, Gly; 5, His; 6, Arg; 7, Thr; 8, Ala; 9, Pro; 10, NH#
4 ; 11, Tyr; 12, Val; 13, Met; 14, Cys; 15, Ile, 16, Leu; 17, Phe; 18, Lys.
(Reproduced with permission from Tosohaas, The Bioseparation Specialist, (1995) Catalogue p. 157, Figure 9/2.)

gradient mobile phase and pulsed amperometric agent(s); special thermostable reactors (packed bed,
detection (Figure 2C). For tryptophan and the air-segmented and/or coil reactors) ensuring the ne-
sulfur-containing AAs (cysteine/cystine, methionine, cessary delay for quantitative reactions accompanied
glutathione, etc.), the fast isocratic elution of the with as small band broadening as possible. Last but
underivatized samples has gained wide acceptance not least, the mobile phase was probably incompat-
and is a powerful tool in their quantitation. ible with the derivatizing reagent. The preferred mo-
Tryptophan can be measured directly, within bile phase might be inappropriate for the optimum
8 min, in neutralized alkaline hydrolysates of feed conditions of derivatization reaction. The early and
and foodstuffs, using an RP column, 5% methanol current stages of post-column methods can be
containing acetate buffer (pH&4.0) and UV detec- illustrated by elution followed by post-column
tion at 280 nm. The pulsed amperometric detection reaction with ninhydrin (NHYD; Figure 3A), with
of sulfur-containing AAs, at the low pmol level, was o-phthaldialdehyde/
-mercaptoethanol (OPA/MCE;
carried out with an Au working- and an Ag/AgCl Figure 3B), or with 1,2-naphthoqunione-4-sulfonyl
reference electrode, subsequent to their separation chloride (NQS; Figure 3C). All three types of deriva-
on both cation exchange and on RP columns, ap- tives have been separated in most cases on ion ex-
plying as mobile phase 0.1 mol L\1 HClO4/0.15 mol change resin columns from the early 1970s. Recent
L\1 NaClO4/5% ACN. post-column methods are without exception, slow
separations (Table 1). However, the efRciency of the
LC of Derivatized AAs recently published methods of NHYD derivatives us-
ing short columns is superior, determining 59 com-
Derivatization studies have concerned the optimiza- pounds in 150 min and 40 compounds in 120 min
tion of parameters, such as the yield and stability of respectively.
derivatives, to separate and quantitate all AAs with
a simple and fast elution procedure. Pre-column Derivatization (Tables 2 and 3;
Figures 4^6)
Post-column Derivatization (Table 1, Figure 3)
Pre-column derivatization offers numerous advant-
Post-column derivatization was the Rrst develop- ages. It requires less equipment and allows the evalu-
ment of IEC in the area of RP/high perfor- ation of the derivatives in an easier way from the
mance liquid chromatography (HPLC), in its pioneer point of view of their selectivity, sensitivity, various
period. It took time to develop pre-column derivat- means of detection, derivatization yield, stability
ization concepts which resulted in considerable and storability. All of these phenomena can be con-
advantages. trolled and improved by use of modern instrumental
Drawbacks of the post-column techniques techniques and computerization, both individually
(Table 1) are long elution times and the need for and simultaneously. Potential disadvantages in pre-
costly devices, such as a delivery system for the de- column derivatization as procedures can be com-
rivatizing reagent (one or more extra pumps); (a) pletely avoided: contamination from the reagents
mixing chamber for the column efSuent and the re- (due to their insufRcient purity) and loss of analyte
 III / AMINO ACIDS / Liquid Chromatography 2001

Figure 2 LC of underivatized AAs. (A) Separation of a test mixture using n-propanol gradient. Column: 250;3 mm, RP-8;
temperature"253C. Peaks: 1, CySO3H; 2, Asp; 3, Ser; 4, Glu; 5, Thr; 6, Gly#Pro; 7, Ala; 8, Cys; 9, NH# 4 ; 10, Tyr; 11, Val; 12, Met; 13,
Ile; 14, Phe; 15, Leu; 16, His, 17, Lys; 18, Trp; 19, Arg. (Reproduced with permission from Kraak JC et al. (1977) Journal of
Chromatography 142: 671.) (B) Chromatogram of standard AAs using a column-switching technique. First column, Inertsil C3; second
column, Inertsil ODS-2. (Reproduced with permission from Hanai T and Hirukawa M (1988) Journal of Liquid Chromatography 11:
1741.) (C) Chromatogram of AAs obtained by pulsed amperometric detection. Peaks : 1, Arg; 2, Lys; 3, Gln; 4, Asn; 5, Thr; 6, Ala; 7,
Gly; 8, Ser; 9, Val; 10, Pro; 11, Ile; 12, Leu; 13, Met; 14, system peak; 15, His; 16, Phe; 17, Glu; 18, Asp; 19, Cys; 20, Tyr. (Reproduced
with permission from Frankenberger WT Jr and Martens DA (1992) Journal of Liquid Chromatography 15; 423.)
2002 III / AMINO ACIDS / Liquid Chromatography

Table 1 Advances in the LC of post-column derivatized AAs, obtained with o-phthaldialdehyde/
-mercaptoethanol (OPA/MCE),
with ninhydrin (NHYD) or with 1,2-naphthoquinone-4-sulfonate (NQS)

Author Column size Type Eluents (elution Detector Reagent Analyte RSD Matrix No. of
and temperature 3C) UV (nm) (3C) (nmol L\1) % AAs/
date cm ; mm, m FEx/Em elution
time (min)

Moore, 150 0.9 40 Amberlite Citrate buffers, UV NHYD 100I3000 * AAs in 20/
1958 IR-120, IE 0.2 mol L\1: pH 3.25 440, 570 (I) hydrolysates 24I48 h
for first day (303C),
and pH 4.25 for the
second day (553C)
Grunau, 15 3 5 Pickering Pickering Eluents UV NHYD 20 * Plasma 59/&150
1992 ‘fast run’ A (Li280), B (Li750), 570 (1303C) AAs
C (RG003) (423C)
Iwase, 6 4.6 3 2622, Five eluents: UV NHYD 50 (3 Plasma 40/120
1995 Hitachi, IE PF-1IPF-4, PF-RG, 440,570 (1303C) AAs
cont. Li salts, ethanol,
benzyl alcohol,
thiodiglycol, Brij-35
buffer with pH 2.8,
3.7, 3.6, 4.1, -;
(gradient programme:
28I403C)
Roth, 25 6 Aminex 6, Citrate buffers: F OPA/MC 10 * Model study 14/170
1973 IE pH 3.20, 4.25 and * E (553C)
6.40 for 40, 60 and
70 min (343C for
100 min, then raised
to 553C)
Elrifi, 60 9 * IE Pierce Pico-Buffer FH OPA/MCE 0.63I45.0 * AAs in 23/128
1986 system, Li citrate No data (403C) foods
buffers; pH of A,B,C,D
and E"2.9,
3.1, 3.5, 3.4 and 2.3;
temperature gradient:
0I44 min (343C),
44I128 min (633C)
Haginaka, 30 4.6 5 ODS-5 A: 15 mmol L\1 Na F OPA/MCE 0.25I2.5 (4.5 AAs in 18/&120
1988 #guard octane sulfonate/ 340/450 (603C) hydrolysates
3 4.6 5 21 mmol L\1
H3PO4/9 mmol L\1
NaH2PO4/CH3OH
(20/20/20/1, v/v),
pH 2.8;
B: as A, except
(1/1/1/6, v/v), pH 4.2
(603C)
MCller, 1993 15 3 5 Pickering, A: 0.24 mol L\1 F OPA/MCE 0.6 (11 Physio- 39/180
#guard IE Li citrate, pH 2.27, 340/448 (43C) logical AAs
2 3 5 B: 0.64 mol L\1
Li citrate pH"7.50
(503C)
Saurina, 15 4.6 5 Spherisorb A: 20 mmol L\1 UV NQS 32 (5 AAs in food 18/105
1994 ODS 2 H3PO4#20 mmol L\1 305 (653C) #feeda
NaH2PO4#
15 mmol L\1 SDS;
B: 25 mmol L\1
H3PO4#25 mmol L\1
NaH2PO4#18.5
mmol L\1 SDS/PrOH
(4:1, v/v); (503C)

No data available; IE, ion exchange resin; ahydrolysates.

 III / AMINO ACIDS / Liquid Chromatography 2003

from incomplete interaction, undesirable side reac- both UV and Suorescence, without the need to re-
tions and sample handling losses. move excess reagent, represented a great advance.
Although numerous pre-column derivatization Because of the different stability of the isoindoles
techniques have been introduced in the last 30 years, obtained from the reaction of AAs with OPA/MCE,
none complies with the criteria of an ideal procedure: pre-column derivatization with 3-mercaptopropionic
providing rapid and quantitative interaction in aque- acid (MPA) and several N-alkyl-L/D-cysteines was
ous media, permitting mild conditions, ensuring in- proposed. The OPA/MPA and OPA/N-acetyl-L-cys-
teraction with both primary and secondary AAs and teine (NAC) reagents provide more stable isoindoles
resulting in single and stable derivatives in the case of compared to those formed with OPA/MCE, and the
all AAs. optical resolution of enantiomeric amino acids with
OPA/NAC, as well as with other N-alkyl-L/D-cysteine
OPA Derivatives (Table 2 and Figure 4)
reagents, has opened a new area in enantiomer separ-
The pioneering work of Roth (1971) on the very fast ation of AAs. Due to robotic autosamplers which
reaction of AAs in aqueous solutions with ophthalal- provide excellent reproducibility for even moder-
dehyde mercaptoethanol (OPA/MCE), detectable by ately quantitative interactions, most AA analyses are

Figure 3 LC of post-column derivatized AAs (for details see Table 2). (A) Chromatographic profile of 59 AAs and related compounds.
Peaks: 3, o-phospho-DL-serine; 6, taurine; 9, o-phosphoethanolamine; 10, N -(1-D-mannityl)-L-glutamine (mannopine); 11, urea;
12,
-ciano-L-alanine; D, L-aspartic acid; 17, o-acetyl-L-serine; T, L-threonine; S, L-serine; N, L-asparagine; E, L-glutamic acid;
Q, L-glutamine; 27, L-homoserine; 29, sarcosine; 34, DL--aminoadipic acid; 36, S-methyl-L-cysteine; P, L-proline; G, glycine;
A, L-alanine; 42, L-citrulline; 45, L--aminobutyric acid; V, L-valine; 47, L-cystine; 50, -methyl-DL-methionine; M, L-methionine;
54, L-cystathionine; I, L-isoleucine; L, L-leucine; 62, L-norleucine; Y, L-tyrosine; F, L"phenylalanine; 67,
-alanine; 68; DL-
aminoisobutyric acid; 69, DL-homocystine; 70, -aminolevulinic acid; 71, 5-hydroxy-L-tryptophan; 72, -aminobutyric acid; 73, DL-
kynurenine; W, L-tryptophan; 76, ethanolamine; 77, -hydroxylysines (DL- and DL-allo); 78, ammonia; 79, -amino-n-caproic acid; 80,
creatinine; 81, L-ornithine; K, L-lysine; H, L-histidine; 85, 3-methyl-L-histidine; 87, 1-methyl-L-histidine; 89, L-carnosine; 90, L-anserine;
91, L-canavanine; 92, S-methyl-DL-methionine; 93, L--amino-
-guanidinopropionic acid; 94, L-leucinamide; 95, N G1-dimethyl-L-
arginine; R, L-arginine; 99, L-homoarginine. (Reproduced with permission from Grunau JA and Swiader JM (1992) Journal of
Chromatography 594: 165.) (B) Separation of OPA/MCE derivatives by gradient IEC chromatography. (Reproduced with permission
from MCller SE (1993) Journal of Chromatography 613: 223.) (C) Determination of AAs by ion pair liquid chromatography with
post-column derivatization using 1,2-naphtoquinone-4-sulfonate (NQS). Peaks: 1, Asp; 2, Ser; 3, Glu; 4, Gly; 5, Thr; 6, Ala; 7, Pro; 8,
Tyr; 9, Met; 10, Ile; 11, Phe; 12, Leu; 13, Nle; 14, Trp; 15, His; 16, Orn; 17, Lys; 18, Arg. Line"elution gradient profile. (Reproduced
with permission from Saurina J and HernaH ndez-Cassou (1994) Journal of Chromatography 676: 311.)
2004 III / AMINO ACIDS / Liquid Chromatography

Figure 3 Continued
 III / AMINO ACIDS / Liquid Chromatography 2005

Table 2 Advances in the LC of pre-column derivatized AAs, obtained with OPA/MCE, OPA/3-ethanethiol (OPA/ET), OPA/mercap-
topropionic acid (OPA/MPA), OPA/N-acetyl-L-cysteine (OPA/NAC), with OPA/isobutyryl-L /D-cysteine (OPA/NIBC) or with
OPA/MPA/fluorenylmethylchloroformate (OPA/MPA/FMOC)

Author Column size Type Eluents (elution Detector Reagent Analyte RSD Matrix No. of
and temperature 3C) UV (nm) (3C) (pmol L\1) % AAs/elution
date cm ; mm, m FEx/Em time (min)

Jones, 75 4.6 3 Ultrasphere A: THF/CH3 OH/NaAc F OPA/ 0.1I80 (1.5 AAs in 48/50
1983 #guard ODS (pH 7.2)"(5:95:900, v/v) 305I395 MCE hydrolysates
45 2.1 40 B: CH3 OH; (!) 420I650 (!)

Fekkes, 12.5 4.6 5 Spherisorb A: (pH 6.72I6.77 and F OPA/ 50 (2 Plasma 40/49
1995 ODS-2 B: (pH 5.95I6.00): 337/452 MCE AAs
250 mmol L\1 Na2HPO4/ (33C)
250 mmol L\1 propionic
acid/ACN/THF/H2O"
(20:20:7:2:51, v/v) C:
ACN/CH3OH/DMSO/
H2O"(28:24:5:43, v/v);
(25I353C)

Hill, 30 3.9 * -Bondapak A: 12.5 mmol L\L F OPA/ 5 * AAs in 20/40

1979 C-18 Na2HPO4 (pH 7.2) 229/470 ET (!) human
B: A eluent/ACN in serum
gradient; (!)

Eslami, 50 4.5 3 ODS IBM Buffer:&2 mol L\1 F OPA/ 40I100 * Model 22/14
1987 Na2 HPO4 (pH 7) 330/480 ET (!) study
A: ACN/H2O/buffer"
(50:425:25, v/v)
B: ACN/H2O/buffer"
(275:200:25, v/v);
(223C)

Godel, 25 4 4 Supersphere A: 12.5 mmol L\1 F OPA/ 1I10 (4.2 AAs in 28/40
1984 CH-8 Na2HPO4 (pH 7.2) 330/445 MPA (!) biological
B: 12.5 mmol L\1 fluids
Na2HPO4 (pH 7.2)/
ACN-(1:1, v/v); (!)

van Eijk, 15 4.6 2I3 Spherisorb A: 12.5 mmol L\1 F OPA/ 35 (3 Plasma 30/28
1993 #guard ODS-2 Na2HPO4 (pH 7.0)# 335/440 MPA (!) AAs
1 4 7 mL THF/1 l eluent
B: 12.5 mmol L\1
Na2HPO4 (pH 7.0)/
ACN/THF"(57:43:7,
v/v); (353C)

Teerlink, 10 4.6 3 Microsphere A: 4.5 mmol L\1 F OPA/ 100 (3.2 Plasma 25/17
1994 #guard ODS K2HPO4 (pH 6.9)# 230/389 MPA (!) AAs
1 2 2 mL THF/1L
B: 4.5 mmol L\1 K2HPO4
(pH 6.9)/CH3OH/ACN"
(50:35:15, v/v); (!)

Schuster, 20 2.1 5 Hypersil Protein hydrolysates, UV OPA/ UV: 2I5 (2.5 AAs in 19/20
1989 20 4.6 5 ODS A: 30 mmol L\1 NaAc 338/266 MPA/ F: protein 38/60
cont. 0.5% THF (pH 7.2); F FMOC 0.02}0.05 hydrolysates
ACN/0.1 mol L\1 NaAc" 230/455 (43C) Plasma AAs
(4:1, v/v); (423C) Plasma 266/310
AAs, A: 60 mmol L\1
NaAc cont. 0.6% THF
(pH 8.0);
B: ACN/0.1 mol L\1 NaAc/
CH3OH"(14:4:1, v/v);
(433C)
2006 III / AMINO ACIDS / Liquid Chromatography

Table 2 Continued

Author Column size Type Eluents (elution Detector Reagent Analyte RSD Matrix No. of
and temperature 3C) UV (nm) (3C) (pmol L\1) % AAs/elution
date cm ; mm, m FEx/Em time (min)

BartoH k, 10 4 3 Hypersil A: 18 mmol L\1 NaAc F OPA/ 50 (1.1 Plant AAs 21/8
1994 ODS (pH 7.2)# 0.02%(v/v) 340/450 MPA/
TEA#0.3% THF (v/v) 264/313 FMOC
B: ACN/CH3OH/NaAc (43C)
0.1 mol L\1 (pH 7.2)"
(2:2:1, v/v); (403C)

Indications as in Table 1.

performed with OPA derivatives. The essential short- tives has proved to be lasting, while the application of
age of an OPA/SH-group reagent (reactive toward the DANS and DABS derivatives is decreasing. However,
primary AAs only) was eliminated by Shuster’s prin- in the direct enantiomer separation of AAs, the use of
ciple } the automatic two-step pre-column derivatiz- DANS derivatives is preferred.
ation method applying the OPA/MPA/Suorenyl- The reaction of AAs with phenylisothiocyanate
methyl chloroformate (FMOC) reagent, which also (Table 3, Figures 1 and 5), in water-free media at
ensures derivatization of the secondary AAs. A high ambient temperature is quantitative and fast (10 min),
speed elution of OPA/MPA/FMOC derivatives was resulting in the highly stable single PTC derivatives
shown recently (Table 2, Figure 4: 19 compounds/ (except for cyst(e)ines in hydrolysates which elute in
8 min). Evaluating the improvements between the one to four peaks). The excess reagent is removed by
corresponding early and recent procedures, in the vacuum, and the PTC derivatives can be stored in the
newer methods shorter, thermostated columns of freezer for an unlimited time, and for a day after
smaller particle size with autosamplers are now used, dissolution in buffer at 43C. UV detection at 254 nm
giving greater sensitivity and reproducibility. allows their quantitation in the low pmol range. The
short PicoTag and the short TSK gel columns can
Phenylthiocarbamyl (PTC), FMOC, 1-N,N - separate 17 AAs within 12 min and 4.5 min, re-
Dimethylaminonapthalene-5-sulfonyl (DANS)
spectively.
and Dimethylaminoazobenzenesulfonyl (DABS)
The Rrst LC separation of the strongly Suor-
Derivatives (Table 3, Figures 1, 5 and 6)
escent DANS AAs has been used earlier in protein
Judging by the number of publications in the last chemistry and in thin-layer chromatography. The de-
decade, the interest in the PTC and FMOC deriva- creased popularity of this technique in LC can be

Figure 4 High speed RP-HPLC analysis of the OPA/MPA/9-fluorenylmethyl chloroformate derivatives. (Reproduced with permission
from BartoH k T et al. (1994) Journal of Liquid Chromatography 17: 4391.)
 III / AMINO ACIDS / Liquid Chromatography 2007

Table 3 Advances in the LC of pre-column derivatized AAs, obtained with phenylisothiocyanate (PITC), 5-dimethylaminonaphtalene-
1-sulfony1-CI (DANS), 4-dimethylaminoazobenzene-4-sulfonyl-CI (DABS) or with 9-fluorenylmethyl chloroformate (FMOC)

Author Column size Type Eluents (elution Detector Reagent Analyte RSD Matrix No. of
and temperature 3C) UV (nm), (3C) (pmol L\1) % AAs/elution
date cm;mm, m FEx/Em time (min)

Koop, 25 4.6 5 Ultrasphere A: 70 mmol L\1 NaH2 PO4 UV PITC 6000 * AAs in 18/130
1982 ODS (adjusted to pH 6.45 with 254 (!) protein
TEA) hydrolysates
B: ACN; (273C)
Tosohaas, 10 4.6 2 TSKgel A: 50 mmol L\1 NaAc UV PITC 250 * Model 17/4.5
1995 Super-ODs (pH 6.0)/ACN"(97:3, v/v) 254 (!) study
B: 50 mmol L\1 NaAc
(pH 6.0)/ACN"(40:60,
v/v); (403C)
Shang, 15 3.9 5 PicoTag A: NaAc (pH 6.4) UV PITC 5 (1.9 AAs in kelp 17/12
1996 ODS B: ACN; A and B 254 (!)
performed
in gradient (383C)
Bayer, 50 3 10 LiChrosorb, Eluent 10 mmol\1 F DANS 0.1 * Model 17/40
1976 RP 8 Na2HPO4/CH3OH" 340/510 (amb) study
(50:20, v/v) to which
1.5 mL CH3OH/min is
added (453C)
Martins, 15 3.9 4 Nova Pak A: 30 mmol L\1 phosphate F DANS 60 * AAs in 17/35
1996 C 18 buffer (pH 7.4)# 5 mL 338/445 (403C) polypetides
CH3OH#6.5 mL THF
adjusted to 100 mL with
distilled water
B: CH3OH/H2O"(70/30,
v/v); (253C)
Chang, * * 5 * A: 25 mmol L\1 NaAc UV DABS 5 * AAs in 17/40
1983 (pH 6.5) containing 4% 436 (703C) protein
dimethylformamide hydrolysates
B: ACN (403C)
Yang, 15 4.6 5 Hypersil A: 25 mmol L\1 NaAc UV DABS 50 * AAs in 17/40
1993 ODS (pH 6.35) containing 4% 436, (703C) polypetides
dimethylformamide 580
B: ACN (403C)
Einarsson, 50 4.6 3 Spherisorb Eluent: 20 mmol L\1 F FMOC (!) (6.6 AAs in 17/10 and
1983 500 2.26 5 ODS-2 NaAC buffer 265/315 (!) protein 33/100
(pH 4.08I4.31)/ACN hydrolysates,
gradient; (!) in urine

Qu, 15 4.6 5 Hypersil A: 30 mmol L\1 phosphate F FMOC 125 (1.0 AAs in 15/35
1996 ODS buffer (pH 6.5) in 15% 270/316 (!) protein
CH3OH (v/v) hydrolysates,
B: 15% CH3OH (v/v) biological
C: 90% ACN (v/v); samples
(383C)
Bank, 15 4.6 5 Micropak A: 20 mmol L\1 citric acid/ F FMOC 50 (3.6 AAs in 21/35
1996 ODS-80TM NaAc buffer (pH 2.85); 254/630 (!) protein
B: 20 mmol L\1 NaAc hydrolysates
(pH 4.5)/CH3OH"(80:
20, v/v); A, and B both,
cont. 0.01% (w/v) NaN3#
5 mmol L\1 (CH3)4 NCI;
C: ACN; (403C)

Indications as in Table 1.
2008 III / AMINO ACIDS / Liquid Chromatography

Figure 5 Separation of 27 phenylthiocarbamyl AAs. Column 150#(20 guard);4 mm, C18 Hypersil 5
m, temperature, 503C, eluent
A: 0.05 mol L\1. NaAc pH 7.2; B: A eluent/acetonitrile/methanol"46/44/10 (pH"7.2), flow rate: 2.1 mL min\1. Peaks:
1, aspartic, 2, glutamic acids; 3, hydroxyproline; 4, serine; 5, glycine; 6, asparagine; 7,
-alanine; 8, glutamine; 9, homoserine;
10, -aminobutyric acid (GABA); 11, histidine; 12, threonine; 13, alanine; 14, 1-amino-1-cyclopropane carboxylic acid (ACPCA);
15, arginine; 16, proline; 17, homoarginine; 18, tyrosine; 19, valine; 20, methionine; 21, cyst(e)ine; 22, isoleucine; 23, n-leucine;
24, phenylalanine; 25, tryptophan; 26, ornithine; 27, lysine. Hsystem peaks. (Reproduced with permission from Vasanits A and
MolnaH r-Perl (1998) Journal of Choromatography 832:109.)

explained by its two main disadvantages: long reac- stored for 4 weeks in solution at 253C, without any
tion times, or elevated temperatures for derivatiz- changes. In spite of the unique stability of DABS AAs
ation, and generation of Suorescent side products in aqueous media, and the improvement in their
(DANS hydroxide, DANS amide) and interference chromatographic conditions, the use of DABS AAs
from excess reagent. The disturbing effect of these is dwindling.
compounds cannot be completely eliminated and FMOC was introduced in 1983, as a Suorescent
they elute between the AA derivatives. No signiRcant labelling agent, reacting rapidly with both primary
improvement has been obtained and cannot be and secondary AAs, under mild conditions (borate
expected. buffer, pH 7.7}8.0) to give stable derivatives. The
DABS AAs were Rrst separated applying pre- excess reagent is extracted by pentane. Recent de-
column labelling. Derivatization was performed in rivatization studies have shown that, depending on
Na2CO3/NaHCO3 buffer, at pH&8.9 with DABS the time (2 and 40 min) and pH (8.0 and 11.4),
chloride dissolved in acetone under continuous considerable differences can be found. At pH &8,
stirring at 803C for 10 min. DABS AAs can be acidic AAs manifest low responses, and slow reaction

Figure 6 (for details see Table 3) HPLC of AAs derivatized with 9-fluorenylmethyl chloroformate (FMOC). Peaks labelled with
one-letter abbreviations for protein AAs, as well as: Hyp, hydroxyproline; R1, FMOC-hydroxylamine; R2, FMOC-hydroxyde; R3,
reagent peak present in blank derivatization. (Reproduced with permission from Qu K et al. (1996) Journal of Chromatography 723:
219.)
 III / AMINO ACIDS / Liquid Chromatography 2009

Table 4 Advances in the chiral separation of amino acids by LC: applying chiral mobile-phase additives (CMA), chiral stationary-
phase columns (CSP) and chiral derivatization reagents (CDR), such as OPA/ NACa and OPA/NIBCb

Author Column size Type Eluents (elution Detector Reagent Analyte RSD Matrix No. of
and temperature 3C): UV (nm) (3C) (pmol L\1) % AAs/elution
date cm;mm , m chiral recognition method FEx/Em time (min)

Takeuchi, 15 0.35 5 Develosil A: 40 mmol L\1 AmmAc F DANS (!) (!) Model 1 pair/30
1992 ODS-5 # 27 mmol L\1 -CD/ 315/539 (!) study
ACN" (3:1, v/v)
B. AmmAc/ACN"(72:28,
v/v); (253C) CMA

Marchelli, 10 8 5 Radialpak Isocratic: ACN/ F DANS (!) (!) Model 3 pairs/130

1996 15 4 5 C18 30 mmol L\1 NaAc 330/560 (!) study
(pH 7.0), containing
0.5 mmol L\1 N2-S-2-
hydroxypropyl-S-phenyl-
alaninamide#5 mmol L\1
Cu(II) Ac"(2:8, v/v);
(21.53C) CMA

Galli, 15 4 5 LiChrosorb Isocratic: ACN/ UV DANS (!) (!) Model 3 pair/30

1994 modified Si 100c 10 mmol L\1 NaAc 254 DABS study 4 pair/30
(pH 7.52), containing (!)
25 mmol L\1 Cu(III)
Ac"(7:3, v/v); (603C)
CSP

Iida 15 6 5 Home A: 100 mmol L\1 AmmAc UV PTC 1000 (!) Protein 18 pairs#
1997 maded (pH 6.5), 254 (!) sequencing 1 single/
B: 100 mmol L\1 AmmAc 150
(pH 65)/CH3OH"
50:50 (v/v); A and B
both contain 1 mmol L\1
butanesulfonate; (20I303C)
CSP

Nimura, 20 6 5 Develosil A: 50 mmol L\1 NaAc F OPA/ 5000 (2.3 D- and 14 pairs/70
1986 ODS-5 B: ACN (253C) CDRc 360/405 NAC L-AAs
(!) in protein
hydrolysates

BruK ckner, 25# 4 5 Hypersil A: 23 mmol L\1 Na F OPA/ 1I1000 (2 D- and 17 pairs#
1995 guard 2.1 ODS acetate (pH 5.95) 230/445 IBLC L-AAs in 5 single/70
2 B: ACN/CH3OH" (IBDC) food
(60:5, v/v); (253C) CDRd (!) hydrolysates

Indications as in Tables, as well as: CD cyclodextrin; aN-acetyryl-L-cysteine; bIBL (D) C, isobutyryl-L(D)-cysteine; c [(S )- and (R )-phenylalanine-
amide were covalently bonded to LiChrosorb Si100 silica gel; home maded silica support treated with PITC#
-CD; DANS, dansyl; DABS,
dabsyl; PTC, phenylthiocarbamyl.

is experienced; histidine and tyrosine give their mono- the presence of interfering substances are shown in
and disubstituted derivatives in varying ratios. With Figure 6.
longer reaction times, the amount of disubstituted
histidine decreases and that of tyrosine increases, Chiral Separations (Table 4 and
together with interfering hydrolysis products of the
reagent. At pH&11.4 faster reaction and less inter-
Figure 7)
fering hydrolysis products are found. After 40 min The knowledge of the distribution of AA enatiomers
reaction time, the monosubtituted histidine and the in different matrices, and/or the enantiomeric purity
disubstituted tyrosine are formed in quantitative of AAs, is of primary importance in the quality con-
yield. Also 30% less hydrolysis product is obtained, trol of peptide syntheses for pharmaceuticals/
favouring the resolution of the neighbouring alanine. medicines, as well as in various plant products and in
The separation of the FMOC derivatives and high AA-containing foods, including baby formulas.
2010 III / AMINO ACIDS / Liquid Chromatography

For separation and quantitation of enantiomers, phenylalanineamide as CMA is very time-consuming

HPLC is the method of choice. The application of the (Figure 7A): the separation of three AA pairs re-
three main approaches for enantiomer separation is quires more than 2 h. Thus, CMAs can be regarded
shown in Table 4 and Figure 7, including direct, as an inferior approach in the chiral recognition of
chiral mobile-phase additive (CMA) and chiral sta- AA enantiomers, due to the need for a continuous
tionary phase (CSP) and indirect methods (chiral de- supply of the often expensive CMA and to the dis-
rivatization reagent CDR). advantageous chromatographic conditions. Bonded
N 2-S-(N 2-R-)2-hydroxypropyl-S-phenylalanine-
Direct Methods
amide, CSP, allows the comparison of the CMA
Applying either -cyclodextrin (-CD) or Cu(II) and CSP protocols for the same enantiomer separ-
salts together with N2-S-(N2-R-)2-hydroxypropyl-S- ations. The CSP method resolved four pairs of

Figure 7 LC separation of AA enantiomers. (A) Enantiomeric separation of a mixture of three dansyl AAs. (Reproduced with
permission from Marcelli R et al. (1996) Chirality 8: 452.) (B) Separation of 37 phenylthiocarbamyl AAs. (Reproduced with permission
from Iida T et al. (1997) Analytical Chemistry 69: 4463.) (C) Aminogram of fir honey derivatized with (a) OPA/IBLC and (b) OPA/IBDC.
(Reproduced with permission from BruK ckner H et al. (1995) Journal of Chromatography 697: 229.)
 III / AMINO ACIDS / Liquid Chromatography 2011

Figure 7 Continued

dansylated AAs within 30 min, attesting to the Indirect Methods

superiority of CSP over CMA. Recently, the elut-
ion of the PTC AAs on a new CSP (Figure 7B) permit- Spectacular results have been achieved with the separ-
ted the partial separation of 18 AA pairs within ation of AAs derivatized by CDRs (Figure 7C).
150 min. Performing the separation with both OPA/N-L(D)-
2012 III / AMINO ACIDS / Thin-Layer (Planar) Chromatography

acetyl-cysteinyl and with OPA/N-L(D)-isobutyrylcys- See also: II/Chromatography: Liquid: Derivatization;

teinyl AA derivatives gave excellent resolution of en- Mechanisms: Reversed Phase.
antiomers. Consequently, the CDR technique is the
primary importance in a number of practical applica-
tions of the separation of enantiomeric AAs. The
Further Reading
interaction of AAs with the enantiomerically pure Blau K and Halket J (eds) (1993) Handboook of Deriva-
reagents takes place at ambient temperature, without tives for Chromatography. Chichester: John Wiley.
racemization, resulting in the formation of stable BruK ckner H, Langer M, LuK pke M, Westhauser T and Godel
diastereomer derivatives. H (1995) Liquid chromatographic determination of
amino acid enatiomers by derivatization with o-phthal-
Online LC-MS dialdehyde and chiral thiols. Journal of Chromatogra-
In the case of AAs, thermospray ionization has been phy 697: 229.
displaced by the milder techniques of electrospray Deyl Z, Hyanek J and Horakova M (1986) ProRling of
amino acids in body Suids and tissues by means of liquid
(ES) and atmospheric pressure chemical ionization
chromatography. Journal of Chromatography 379: 177.
(APCI), converting analyte molecules without frag- Grunau JA and Swiader JM (1992) Chromatography of 99
mentation into ions. The analyte should contain the amino acids and other ninhydrin reactive compounds in
AAs in a stable form: either in the free condition or in the Pickering lithium gradient system. Journal of
the form of stable derivatives, such as phenyl- Chromatography 594: 165.
thiohydantoins (PTH) or PTCs. SigniRcantly reduced McClung G and Frankenberger WT Jr (1988) Comparison
Sow rates are essential (100}300 nL min\1) for stable of reversed-phase high performance liquid chromato-
ES and APCI operation. In automated Edman micro- graphic methods for precolumn-derivatized amino
sequencing, the ES-MS of PTH derivatives. The pro- acids. Journal of Liquid Chromatography 11: 613.
tonated molecules were measured with a linear re- MolnaH r-Perl I (1998) Amino acids. In: Deyl Z, Tagliaro
sponse in the 50}1000 fmol level. F and Teserova E (eds) Advanced Chromatographic and
Electromigration Methods in BioSciences. Amsterdam:
Elsevier.
Future Trends Snyder LR, Kirkland JJ and Glajch JL (1997) Practical
Efforts are needed to extend the life time, plate HPLC Method Development. New York: Wiley Inter-
number and reproducibility of columns, and to science.
Spackman DH, Stein WH and Moore S (1958) Automatic
standardize testing methods. The extended use of
recording apparatus for use in the chromatography of
thermostated columns is desirable in order to obtain amino acids. Analytical Chemistry 30: 1190.
reproducibility in absolute and relative retention Zhou J, Hefta S and Lee TD (1997) High sensitivity analy-
times. LC-MS will be more widely used in laborator- sis of phenylthiohydantoin amino acid derivatives
ies as the cost of these instruments falls to the level of by electrospray mass spectrometry. Journal of the
GC-MS, and/or an all-purpose interface becomes American Chemical Society of Mass Spectrometry 8:
available. 1165.

Thin-Layer (Planar) Chromatography

R. Bhushan, University of Roorkee, suitable for use with strong corrosive reagents and
Roorkee, India one can perform many kinds of chemical reactions on
J. Martens, Universitat Oldenburg, Oldenburg, the plate, both from the points of view of detecting
Germany and locating the spot and of achieving improved
Copyright ^ 2000 Academic Press separation. Certain groups of interest can be chemic-
ally bonded to the reactive groups of support mater-
ial, e.g. silanization for reversed-phase studies. Im-
pregnation of the adsorbent with a variety of reagents
Introduction adds an additional feature for inSuencing the adsorp-
Thin-layer chromatography (TLC) is a simple and tion characteristics without covalently affecting the
inexpensive technique permitting a number of sam- inert character of the adsorbent. TLC is also success-
ples to be handled simultaneously, thus yielding ful in providing direct resolution of enantiomers of
a higher precision than sequential analysis. The inert a variety of compounds by the proper manipulat-
character of the thin-layer material makes it ideally ion of the support material. The analysis of amino