Large-scale Enrichment of Sulfur-containing Acid in Acetonitrile Using Triazine Analog as the Derivatizing Reagent for Desorption from Humic-fraction-modified Silica Gel

Article abstract of DOI:10.1002/jccs.201500281

The analog of triazine is used as the derivatizing reagent for enriching large-scale acid (e.g. amino acid) containing a sulfur atom on the humic-fraction-modified silica gel in acetonitrile and desorbing from the adsorbent in hexane, respectively. The percent yield of the chemical derivatization under alkaline conditions, ranging from about 8 to almost 100 %, is pH dependent, and varies significantly among these examined analytes, believed to be due to the structure of the analyte, not the derivatizing reagent. The percentage of enrichment, not reproducible under an aqueous environment and independent of the type of triazine analogs, reaches almost 100 % in all cases. The force leading to the adsorption is the complexation between carboxyl groups on analyte and a humic-fraction-modified adsorbent based on the adsorption equilibrium results. Consequently, these results are not reproducible under ethyl ether or methanol environments due to the competition for binding sites from solvent molecules.

Full text of DOI:10.1002/jccs.201500281

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Large-scale Enrichment of Sulfur-containing Acid in Acetonitrile Using Triazine
Analog as the Derivatizing Reagent for Desorption from Humic-fraction-modified
Silica Gel
Shun-Wei Hu and Shushi Chen*
Department of Applied Chemistry, National Chiayi University, Chiayi 600, R.O.C.
(Received: Jul. 20, 2015; Accepted: Nov. 30, 2015; Published Online: Jun. 30, 2016; DOI: 10.1002/jccs.201500281)
The analog of triazine is used as the derivatizing reagent for enriching large-scale acid (e.g. amino acid)
containing a sulfur atom on the humic-fraction-modified silica gel in acetonitrile and desorbing from the
adsorbent in hexane, respectively. The percent yield of the chemical derivatization under alkaline condi-
tions, ranging from about 8 to almost 100%, is pH dependent, and varies significantly among these exam-
ined analytes, believed to be due to the structure of the analyte, not the derivatizing reagent. The percent-
age of enrichment, not reproducible under an aqueous environment and independent of the type of triazine
analogs, reaches almost 100% in all cases. The force leading to the adsorption is the complexation be-
tween carboxyl groups on analyte and a humic-fraction-modified adsorbent based on the adsorption equi-
librium results. Consequently, these results are not reproducible under ethyl ether or methanol environ-
ments due to the competition for binding sites from solvent molecules.
Keywords: Humic fraction; Enrichment; Desorption; Carboxyl group; Triazine analog;
Sulfur-containing acids.
INTRODUCTION
Humic acid (HA) is a product of the microbial degra-
bearing no chlorine atom, are worldwide used pre-emer-
gent weed herbicides for animal feed crops in the United
1
6-18
dation of dead plant matter and can be found in soil nearly
States.
This is the first time they are used as the de-
1
everywhere at variable content. Its structural complexity,
rivatizing reagents in an adsorption-oriented enrichment
process. How the structure of the analyte and triazine ana-
log affect both the chemical derivatization yield and the
percentage of enrichment was explored, and discussed as
well. Finally, the chemical derivatization reaction and ad-
sorption mechanisms were carefully studied by examining
the FTIR results, and by comparing the adsorption equilib-
rium data and the adsorption behavior in different liquid
phases with those obtained in previous work.
being a mixture of numerous acids with various compo-
nents, such as quinone, phenol, catechol and sugar moi-
eties, leads to many agricultural benefits and applica-
2
-6
tions. Other than that, the sorption-desorption of quater-
nary herbicides in vineyard-devoted soils, for example, has
7
-11
been described.
Applications that are adsorption-ori-
ented, using humic-fraction-modified silica gel as the ad-
sorbent, have been implemented recently in carboxyl-con-
taining pesticides and biogenic amines in acetonitrile,
organophosphorus pesticides, phthalate-based plasticizers,
triazine analogs and pesticidal carboxylate esters in hex-
EXPERIMENTAL PROCEDURES
Apparatus: The HPLC system used in this study was a
Hitachi Model L-7100 coupled to a D-2500 Chromatopac data
station and a UV detector. The detection wavelength for the ad-
sorption evaluation, enrichment and derivatization yield mea-
surements was set at 275 nm in all cases. A Hitachi spectrometer
Model U-3900 was used to acquire the UVspectra. FTIR spectra
1
2-15
ane.
In these studies, analytes are dissolved and ad-
sorbed under suitable conditions. Additives that present in
the matrix negatively affect the adsorption. No additive
was involved in the process. Note, adsorption for analytes
not soluble in acetonitrile or hexane will not occur.
-
1
In this work, a humic-fraction-modified silica gel was
used as the adsorbent for enriching several sulfur-contain-
ing acids (e.g. cysteine) in large scale in acetonitrile after
chemical derivatization with a triazine analog containing
chlorine atom. These triazine analogs, along with those
with a resolution of 4 cm were obtained by scanning samples 10
times on a Shimadzu Model FTIR-8400 system.
Chemicals: All chemicals employed in this study, includ-
ing the organosilane reagent used as a linker in chemical immobi-
lization reactions, the sulfur-containing acids, triazine analogs
*
Corresponding author. Tel: +886-5-271-7997; Fax: +886-5-271-7901; Email: schenphd@mail.ncyu.edu.tw
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Hu and Chen
and the reagents used in enrichment evaluation and chemical
derivatization reactions were purchased from Sigma (St. Louis,
MO, USA) and the Aldrich Chemical Co. (Milwaukee, WI,
USA), respectively. The silica gel (5 mm particle diameter, 100 Å
diameter) under the elution of an acetonitrile solvent at a flow
rate of 1.0 ml/min. The percentage of enrichment was calculated
in triplicate to obtain an average in all cases, based on the differ-
ence in peak areas obtained before and after the enrichment evalu-
ation. The enrichment was also evaluated in the liquid phase of
acetonitrile and ethyl ether, respectively. These data were then
used in the discussion of a mechanism involved in the adsorption
process.
2
porosity with a specific surface area of 400 m /g), used as the sup-
porting matrix of the solid phase in the enrichment evaluation at
ambient temperatures, was a product of Silicycle (Quebec City,
QC, Canada) and was chemically modified with a humic fraction
collected under acidic conditions before being used as the adsor-
bent according to the derivatization procedures reported previ-
The chemical derivatization reaction was carried out ac-
2
2
cording to the procedure in previous work. The amount of
1
9,20
ously.
The solvents, such as toluene, acetonitrile, methanol,
derivatizing reagent used in the reaction was deliberately in ex-
-
3
triethylamine, methylene chloride, hexane and ethyl ether, were
of an HPLC grade and purchased from Fisher Scientific (Pitts-
burgh, PA, USA) and Merck Taiwan Ltd. (Taiwan, ROC). They
were used to wash the humic-fraction-modified silica gel before
the adsorption evaluation, and the adsorption followed by the en-
richment evaluation and also used as the mobile phase in HPLC
analysis. In all cases, filtered (0.2 mm) and distilled water were
used.
cess of 2.00 × 10 M, the concentration of analyte, in order to
drive the reaction in the right direction for completeness.
RESULTS AND DISCUSSION
The derivatizing reagents dominating the solid-phase
peptide synthesis strategies for many years for amino pro-
tection are acid chloride typed. The most widely used of
them include N-tert-butyloxycarbonyl (t-BOC) and flu-
2
3-26
Conditions for Percent Yield Measurement and Enrich-
orenylmethyloxycarbonyl (FMOC) chlorides.
As de-
ment Evaluation: A standard solution of a triazine analog was
scribed previously, the generated reagent cations, due to
the leaving of highly electronegative chlorine anions, will
react with the nucleophilic side chains of amino acids to
-
3
prepared at a concentration of 2.57 ´ 10 M, and used as the refer-
ence for estimating the percent yield of derivative in the 3-h
chemical derivatization reaction by comparison based on the
chromatographic peak area. The UV spectra for atrazine and its
cysteine derivative, purified through an adsorption procedure de-
scribed in this work, are shown in Figure 1. As can be seen, a simi-
larity between the two spectra was observed. The molar absorp-
2
7-31
form a peptide bond under alkaline conditions.
This
mechanism for chemical reaction is also applicable to the
other class of derivatization reagents, such as isocyanate
3
2-37
and isothiocyanate.
However, these compounds were
mainly used to create additional attractive (e.g. cyanate
group) or repulsive forces (e.g. sulfur atom) for enanti-
oresolving analyte containing amino groups through the
tivity of the atrazine derivative of cysteine was calculated to be
5
2
.13 ´ 10 L/mol cm and found to be reasonably close to that for
2
1
atrazine. As a result, the estimation by comparison based on the
chromatographic peak area was acceptable.
3
An HNO concentrate solution was diluted to obtain the de-
sired pH, which in turn was used in the 3-h derivatization reaction
to evaluate the pH effect on the percent yield of a derivatization
reaction.
In the enrichment evaluation, a matrix solution was sam-
pled for HPLC analysis first before a weighed amount of humic-
fraction-modified adsorbent (10 mg) was added to it for a 3-h ad-
sorption after the derivatization reaction under the alkaline condi-
tions was completed. The adsorbent was collected by decanting
the liquid phase, and it was washed several times with acetonitrile
before being left to dry under a reduced pressure and lowered
temperature. 100 mL of fresh hexane was then added to the dried
adsorbent to release the derivative. The resulting solution was
sampled immediately without further treatment for the HPLC
analysis with a C18 column (150 ´ 4.6 mm i.d.; 5-mm particle
Fig. 1. The UV spectra of derivatizing reagent atrazine
and cysteine derivative of atrazine obtained by
purification method described in this study.
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Enrichment of Triazine Tagged Sulfur-containing Acid
HPLC approach under polar-organic elution instead of be-
ing the group protector for solid-phase peptide synthesis.
In this study, the reaction mechanism for the derivative
product of sulfur-containing acid by reacting with atrazine
analogs remains the same, as shown in Figure 2. It was be-
lieved that a positively charged carbon atom at Position 1
was induced as a result of strong electronegativity of the
chlorine atom attached to it under alkaline conditions and
was subject to the attack of a nucleophile-containing elec-
tron-rich functional group with the elimination of one HCl
molecule. This proposed mechanism is also supported by
the FTIR spectrum in Figure 3. The characteristic stretch-
-
1
ing frequency centered at 2547.39 cm for S-H of cysteine
-
1
(
top) and for C-Cl of atrazine at 990.09 cm (middle) was
disappeared. However, the vibrational frequency centered
-
1
at 805.89 cm for C-S-C of atrazine derivative of cysteine
bottom) was observed. These FTIR results, along with the
(
percent yield results summarized in Table 1, clearly indi-
cated that it was the sulfur, not the nitrogen or oxygen atom
on the analyte, that conducted the nucleophilic attack. This
happened because the nitrogen or oxygen atom in the
amino or hydroxyl group attracts electrons more tightly
making these groups weaker nucleophiles than the thio
2
8-30
group in an aqueous phase.
Furthermore, the nitrogen
in an a-amino acid was not ready to donate its lone pair of
electrons due to the nearby electron withdrawn carboxylic
group. As a result, the percentage yield of derivatization for
those analytes bearing no amino groups, such as 3-mer-
captopropionic acid in Table 1, was affected negatively. In
addition, the percentage yield of derivatization for an
analyte with a methylated thio group, such as methionine,
was also expected to be low because the nearby hydrogen
atom for producing HCl molecules in the derivatization re-
action was absent. The aforementioned description clearly
accounts for the significantly varied percent yield of de-
rivatization under alkaline conditions ranging from about 8
to almost 100%, as shown in Table 1.
Fig. 3. FTIR spectra for cysteine (top), atrazine (mid-
dle) and atrazine derivative of cysteine (bot-
tom).
This mechanism of derivatization involving a triazine
analog was further investigated by changing the pH value
Fig. 2. The proposed mechanism for the derivatization reaction involving in atrazine and cysteine under the alkaline condi-
tions.
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Table 1. The percent yield of derivative resulted from the reaction of sulfur-containing acids with three triazine-type
a
derivatizing reagents under alkaline conditions
Atrazine
Propazine
Cl
Trietazine
Cl
Analyte, reagent
Cl
N
b
Structures vs. percent yield ,
N
N
CH3
N
N
CH3
N
N
CH3
CH3
C2H5
Enrichment (%)
N
N
N
CH3
H3C
N
N
CH3
H3C
N
H
N
N
H
H
H
H
C2H5
Cysteine
NH2
8
8.18% (~100)
~100% (~100)
~100% (~100)
6.05%
90.01% (~100)
~100% (~100)
7.89%
HS H2C C COOH
H
Homocysteine
NH2
~
100% (~100)
HS H C H C C COOH
2
2
H
Methionine
NH2
7
.11%
H C S H C H C
C
H
COOH
3
2
2
Penicillamine
SH NH2
1
5.29%
1.22%
13.31%
H C C C COOH
3
CH H
3
Thiolactic acid
CH3
4
8.01% (~100)
51.00% (~100)
8.80%
47.31% (~100)
7.66%
HS C COOH
H
3
-Mercaptopropionic acid
8
.50%
HS CH2 CH2 COOH
a
The saturated Borax solution was used as the liquid phase for dissolving analyte in all cases. The data were an average of
three measurements. The chemical derivatization reaction was carried out for a 3-h time period.
b
The data in parentheses represent the percentage of enrichment. Note that only the derivatives with sizable percent yields
were measured. All the data were an average of three measurements.
under a chemical reaction. Results summarized in Figure 4
show the effect of pH change on the percent yield for the
derivatization of cysteine using atrazine as the derivatizing
reagent. As can be seen, lower percent yields were ob-
served as the pH values were lowered. This is because the
concentration of hydrogen ions at a lower pH is expected to
be high, and thus, hinder the production of HCl in the
derivatization process, which in turn would lower the per-
cent yield of derivatization reaction.
for easy detection in HPLC analysis. The other purpose for
using triazine analogs as the derivatizing reagent was their
specific desorptive characteristic in acetonitrile on humic-
1
2,15
fraction-modified silica gel.
This controllable adsorp-
tion/desorption process by simply switching liquid phases
on the same adsorbent is the key for this method to be appli-
cable to enrich sulfur-containing acids in a large scale. In
other words, selective adsorption/desorption for a particu-
lar component (e.g. derivatizing reagent or derivative) and
the removal of residual reagents (e.g. salt from the alkaline
Borax solution, and the remaining, unreacted analyte) in
the derivatization reaction can be easily achieved just by
changing the liquid phase. As can be seen, the percentage
of enrichment in Table 1 for these selected derivatives
Table 1 also lists the percentages of enrichment for
several selected derivatives through a 3-h adsorption on
humic-fraction-modified silica gel in acetonitrile. In the
enrichment evaluation, sulfur-containing acids were first
derivatized with triazine analogs to obtain chromophore
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Enrichment of Triazine Tagged Sulfur-containing Acid
fully remove the residual atrazine before dissolving the ad-
sorbent in hexane to release the adsorbed derivative for se-
quential HPLC analysis. The enrichment was also exam-
ined in other liquid phases, such as ethyl ether and metha-
nol, under the same conditions. Unfortunately, repro-
ducibility of the aforementioned results was not observed
owing to the competition for the limited number of binding
1
2-15
sites on the adsorbent from solvent molecules.
The mechanism for adsorbing pesticidal compounds
bearing the single carboxyl functional group by humic-
fraction-immobilized silica gel in acetonitrile has been re-
1
2
ported. Complexation interaction between carboxylic
groups of the analyte and the adsorbent was the force re-
sulting in the adsorption. However, the triazine analogs,
used as the derivatizing reagents in this work, were ad-
sorbed in hexane through a dipole-dipole oriented mecha-
Fig. 4. The dependence of the percent yield of deriva-
tive on the pH for a 3-h derivatization reaction.
1
5
reached almost 100% in all cases. A typical chromatogram
showing the enrichment results for an atrazine-cysteine de-
rivative is shown in Figure 5. Since the supernate was only
decanted after a 3-h adsorption in acetonitrile, the chro-
matographic peak for atrazine still existed. However, the
peak intensity was reduced dramatically revealing the im-
portance of washing the adsorbent with fresh acetonitrile to
nism characterized by a faster adsorption equilibrium. In
the present work, an atrazine-cysteine derivative was first
enriched through the proposed method, and then, used in
the adsorption equilibrium study in acetonitrile. Upon
comparison, a high similarity in the profile was observed in
the 3-h adsorption evaluation as shown in Figure 6. This
clearly indicated that the resulting adsorption was due to
the interaction between the carboxylic groups of analyte,
not the triazine moiety, and the adsorbent molecule.
CONCLUSIONS
Enriching derivative of the sulfur-containing acid in
Fig. 5. The HPLC chromatograms with UV detection
showing the enrichment of atrazine-cysteine
derivative before and after the process.
Fig. 6. The 3-h adsorption equilibrium of cysteine de-
rivative of atrazine in acetonitrile on the hu-
mic-fraction-modified adsorbent.
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1
1
1
1
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1
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companied with the switching of liquid phases on the
humic-fraction-modified silica gel is demonstrated to be
plausible. The chemical derivatization is pH dependent and
the corresponding percent yield varies significantly among
these examined analytes under the alkaline conditions by
ranging from about 8 to almost 100%, and is believed to be
due to the structure of the analyte, not the derivatizing re-
agent. The percentage of enrichment as a result of the
complexation between carboxyl groups on analyte and an
adsorbent reaches almost 100% in all selected cases, and is
not reproducible under aqueous, ethyl ether and methanol
environments.
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This research was conducted with the financial sup-
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