ANALYTICAL LETTERS

Vol. 37, No. 9, pp. 1981–1989, 2004

ENVIRONMENTAL ANALYSIS

Direct Chromatographic Method for

Determination of Hydrogen Cyanamide and
Dicyandiamide in Aqueous Solutions

Maciej Turowski* and Balasaheb Deshmukh

The Dow Chemical Company, Analytical Sciences, Midland,

Michigan, USA

ABSTRACT

A simple high-performance liquid chromatographic method to separate

and determine hydrogen cyanamide and dicyandiamide was developed.
A reversed-phase chromatographic system consisted of 100% purified
water as a mobile phase and highly hydrophobic triacontyl (C30) station-
ary phase used at ambient temperature with UV detection at 200 nm.
The linearity was excellent (R . 0.999) for both analytes, in the range
of 500 ppb to 5000 ppm for cyanamide and 100 ppb to 100 ppm for
dicyandiamide. Immediate formation of dicyandiamide was observed
when dissolving pure cyanamide in water. Sample solvent appeared
very important for successful component separation, as its variation may
cause peak deterioration and loss of the stationary phase selectivity.

*Correspondence: Maciej Turowski, The Dow Chemical Company, Analytical

Sciences, 1897 Building, Midland, Michigan 48667, USA; Fax: þ1-989-636-6432;
E-mail: mturowski@dow.com.

1981

DOI: 10.1081/AL-120039440 0003-2719 (Print); 1532-236X (Online)

Copyright # 2004 by Marcel Dekker, Inc. www.dekker.com
1982 Turowski and Deshmukh

The chromatographic procedure can also be employed to detect urea in

the presence of cyanamide and dicyandiamide.

Key Words: HPLC; Hydrogen cyanamide; Dicyandiamide; Reversed

phase chromatography; pH; Urea.

INTRODUCTION

Hydrogen cyanamide (H2N–C;;N; CAS # 420-04-2) is a vital com-

ponent of many formulations in agricultural industry. Its 50% aqueous
solution is used as a plant growth regulator concentrate (e.g. Dormexw
from BASF or Cyanamid L500w from Degussa), effective in breaking
dormancy and resulting in an increase of the flowers and fruits produced.[1,2]
Hydrogen cyanamide also serves as a substrate in synthesis of other phar-
maceutical and agrochemical active compounds, biocides, dyes, and fine
chemicals. It is the starting point in the so-called “cyanamide route,” industrial
method of synthesis of creatine monohydrate[3]—an amino acid that is
believed to improve sport performance. Although that belief may seem
scientifically controversial and is widely discussed,[4 – 6] the fact remains
that creatine production and sales have been established worldwide in a
very large industrial-scale. Cyanamide salt—calcium cyanamide is an inhi-
bitor of aldehyde dehydrogenase, hence it has been widely used in aversion
therapy for alcoholism.[7,8] An interesting work of Wollin and Ryan describes
the so-called primordial organic synthesis of protein, nucleosides, and other
organic compounds solely from cyanamide and potassium nitrite in the
presence of small amounts of oxygen, imitating possible primitive earth
conditions.[9]
Joji et al. reported that cyanamide is stable for more than 7 hr in aqueous
solutions in the pH range of 2.5–8.5. The report also states that at the
pH
2.5 cyanamide is converted into urea, while in the pH range of 8–12 it
begins to form dicyandiamide.[10] The dimer formation was also observed at
prolonged storage in the aqueous solutions. Dicyandiamide (CAS # 461-58-5)
is a synonymous name for the dimer of cyanamide or cyanoguanidine, which
itself is used broadly in the production of melamine[11] and as a curing agent
for epoxy resins.[12,13] In the agricultural industry, dicyandiamide is used as a
nitrification inhibitor in mixtures with urease inhibitors and urea to prevent
nitrogen loss in the soil and keep nitrogen in the stable ammoniacal form for
a longer period of time.[14]
Interestingly, despite its wide spectrum of applications and adverse
effects, the methods for monitoring or determination of hydrogen cyanamide
and its derivatives are far from simplicity. A paper chromatography method
Determination of Hydrogen Cyanamide and Dicyandiamide 1983

was reported in 1956 for cyanamide, its derivatives, and urea.[15] A few high-
performance liquid chromatography (HPLC) methods have been described,
but most of them employ either a pre-column derivatization (usually with
dansyl chloride)[16 – 20] or a sophisticated detection, such as pulsed electro-
chemical detector[21] and may be considered either tedious or expensive.
Therefore, it seemed necessary to develop a simple and inexpensive
method for analysis of cyanamide and dicyandiamide, separately or simul-
taneously in a mixture. Many formulations contain hydrogen cyanamide
solution in water. Such solutions are often stored for the long periods of
time (such as weeks or months) before entering a synthetic process or before
being applied as an agricultural product. Herein, we describe a rapid HPLC
method for direct UV detection and determination of hydrogen cyanamide
and dicyandiamide. The method does not require any derivatization and is
also capable of detecting urea, if present in a sample.

EXPERIMENTAL

Solid hydrogen cyanamide (99%, ACS grade, catalogue # 18,736-4) and

its 50% (wt/wt) solution in water (catalogue # C87908) were supplied from
Aldrich. Dicyandiamide (sold under commercial name Amicure CG-1200)
was from Air Products and Chemicals, Inc., Performance Chemicals Division.
Urea (grade Ultra, catalogue # U0631) was from Sigma. Ammonium
hydroxide (30%, ACS Plus grade, catalogue # A669) and hydrochloric acid
(6 N solution/certified, catalogue # SA56) were from Fisher Scientific.
Nanopure water (18.2 MV) for mobile phase and sample preparations was
produced in-house using MilliQ system from Millipore. The cyanamide stan-
dard solutions used for linearity study were prepared by serial dilutions in
water, immediately before injections. Three separate solutions (2 mg/mL)
of cyanamide—in water, in 1% ammonium hydroxide, and in 1% H3PO4
were prepared and stored for 24 hr before the injections. The 50% solution
of hydrogen cyanamide in water was diluted 1 : 100 with water directly
before the injection. Urea standard solution (2 mg/mL) was prepared in water.
For method development, a Hitachi D-7000 HPLC system was used
equipped with quaternary gradient pump, autosampler, column thermostat,
UV-VIS detector, and data collection system.
Analytical HPLC conditions were as follows: (a) HPLC column: Prontosil
200-5-C30 (250  4.6 mm, 5 mm, 120 Å) from Mac-Mod Analytical/Bischoff
Chromatography; (b) mobile phase: 100% freshly produced nanopure water;
(c) flow rate: 0.8 mL/min; (d) column temperature: 258C; (e) UV detection
wavelength: 200 nm (1.0 AUFS); (f) injection volume: 20 mL, samples injected
at least in triplicate.
1984 Turowski and Deshmukh

Linearity of the system was investigated for cyanamide and dicyandia-

mide. Six standard solutions were prepared for cyanamide and four for dicyan-
diamide, by serial dilutions (1 : 10) and each was injected in triplicate. The
concentration range for cyanamide was from 500 ng/mL (500 ppb) to
5000 mg/mL (5000 ppm) and for dicyandiamide from 100 ng/mL (100 ppb)
to 100 mg/mL (100 ppm). Peak area of standards was used to derive the
correlation plot and linear regression equations. Instrument precision was
calculated from triplicate injection of a selected cyanamide standard solution.

RESULTS AND DISCUSSION

Cyanamide as well as dicyandiamide and urea are significantly hydrophilic

rather than hydrophobic in nature.[22] The logarithms of n-octanol/water
partition coefficient (log P) for these three compounds are 20.82, 21.15, and
22.11, respectively. Since an approach was taken to develop a convenient
reversed-phase chromatographic method, it appeared quite difficult to find a
proper stationary/mobile phase combination that would provide reasonable
retention factors. After some preliminary attempts, a highly hydrophobic
triacontyl (C30) stationary phase appeared most successful. It is based upon
high-purity silica with covalently bonded C30 alkyl chains on the surface and
is offered by various commercial sources. In addition, C30 phase is claimed
to work well and provide good reproducibility in highly (up to 100%)
aqueous mobile phases.[23] Indeed, in case of this work the 100% purified
water appeared to be the most appropriate mobile phase. Acidified mobile
phases (e.g. 0.01% phosphoric or acetic acid) did not appear any better than
just purified water. Ultrapure water has the additional advantages because it
is the simplest and cheapest mobile phase to prepare and is perfectly compatible
with LC-MS. The C30 stationary phase used in this study demonstrated
excellent reproducibility over several hours of continuous analysis in pure
water. As the analysed compounds are basic, one would expect a significant
tailing in pure water, however, during this study no noticeable peak deterio-
ration was observed.
Using a chromatographic system with 100% water as mobile phase, a
special attention should be given to solvents in which the analytes are injected
onto the column. During this work, it was observed that mixtures of cyanamide
and dicyandiamide dissolved in methanol and injected onto the column, eluted
together with the solvent front. Evaporation of methanol solution and recon-
stitution of residuals in water generated the chromatograms where these two
compounds were very well resolved. Since pure methanol is significantly
stronger solvent than pure water for the employed reversed-phase system,
this observation is in agreement with the previous reports that addressed
Determination of Hydrogen Cyanamide and Dicyandiamide 1985

this problem.[24,25] In this case, retention loss rather than peak shape deterio-
ration was observed, which could be attributed to relatively weak interaction
of the analytes with C30 stationary phase as well as extreme difference in
solvent strength used for injection (100% methanol) and elution (100%
water). A proper sample preparation, therefore (e.g. evaporation and reconsti-
tution), resulting in injection of 100% aqueous solutions of cyanamide and/or
dicyandiamide should be performed in case the analytical matrices contain
solvents different than water.
Figure 1(a) represents a sample chromatogram of 50% cyanamide solu-
tion in water, diluted 100 times with water (direct injection of 50% solution
saturated UV detector). As supplied commercially, the 50% solution con-
tained dicyandiamide. Also, there is a small peak appearing upon the right

Figure 1. Representative chromatograms of hydrogen cyanamide: (a) commercial

50% solution in water, diluted 100 times with water; (b) freshly prepared solution in
water; (c) solution in water stored for 24 hr at ambient temperature; (d) solution in
1% ammonium hydroxide stored for 24 hr at ambient temperature; and (e) solution
in 1% hydrochloric acid stored for 24 hr at ambient temperature. (View this art in
color at www.dekker.com.)
1986 Turowski and Deshmukh

side of cyanamide peak, which indicates that this particular commercially

available solution may contain impurities. Dicyandiamide was also formed
immediately upon contact with water, which is demonstrated in Fig. 1(b),
representing a chromatogram of freshly prepared solution of pure cyanamide
(one of the calibration standards), and Fig. 1(c) representing pure cyanamide
solution in water (2.5 mg/mL) stored for 24 hr at ambient temperature.
Fig. 1(d) and 1(e) are the chromatograms of the 24-hr stored hydrogen
cyanamide solutions in 1% ammonium hydroxide and 1% hydrochloric acid,
respectively. It must be noted here that previously reported more-than-7-hr
stability of cyanamide in water solutions, cited above[10] cannot be supported,
because the dicyandiamide peak was observed in the standard solutions that
were prepared directly before the injections. Storage in basic conditions for
24 hr caused a significant formation of dicyandiamide, however, no other
decomposition was observed. Another possibility is a formation of dicyan-
diamide in solid state, due to the contact of stored solid hydrogen cyanamide
with moisture. Further study to investigate this phenomenon is recommended.
Separately prepared solutions of pure urea and dicyandiamide in water did not
demonstrate any extra peaks.
One-day storage in acidic conditions resulted in a significant decom-
position of hydrogen cyanamide to more degradation products, including
urea. Urea generated a significant response; however, another (unidentified)
degradation product generated the largest peak at 3.25 min.
Linearity of the system was studied for cyanamide and dicyandiamide in the
range of 500 ppb to 5000 ppm and 100 ppb to 100 ppm, respectively. Observed
correlation coefficient R was equal or greater than 0.999 for both compounds.
An interesting observation was that dicyandiamide generated much
stronger UV response than cyanamide at 200 nm. For example, a relative
area percentage of the two peaks in Fig. 1(c) is 74% cyanamide to 26% dicyan-
diamide; however, the actual concentration proportion is roughly around
173 : 1. Concentrations calculated according to the respective calibration
curves are 2.779 and 0.016 mg/mL, respectively. Apparently, having one
additional double bond dicyandiamide is a significantly stronger chromophore
at 200 nm despite the similarity of structures.
The observation of significantly different responses should be kept
in mind when analyzing commercial formulations containing aqueous
solutions of hydrogen cyanamide. For example, a commercial 50% cyan-
amide solution obtained from Aldrich [Fig. 1(a)] demonstrates the response
generated by cyanamide being actually smaller than that of dicyandiamide
(peak area proportion of ca. 44 : 56). However, the actual concentration
of two components as determined from the respective calibration curves is
significantly different: 5.931 mg/mL for cyanamide and 0.132 mg/mL for
dicyandiamide.
Determination of Hydrogen Cyanamide and Dicyandiamide 1987

CONCLUSIONS

The HPLC method described in this report demonstrates feasibility of

analysis and determination of hydrogen cyanamide and dicyandiamide in
the aqueous solutions. The method could possibly be used for analysis of
other formulations containing the analytes, after the certain sample prepara-
tions that lead to 100% aqueous sample solution injected onto the C30
column. Also, the method seems to be an interesting starting point to
develop a tool to study the acidic and basic degradation process of hydrogen
cyanamide. The presence of urea can be noted, however, given conditions
do not allow its determination. The advantage of the method is absolute
compatibility with LC-MS techniques and a very low cost of analysis, due
to use of pure water as the mobile phase.

ACKNOWLEDGMENTS

The authors wish to thank Joe Becker of The Dow Chemical Company for
the ideas and a preliminary HPLC work on the hydrogen cyanamide.

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Determination of Hydrogen Cyanamide and Dicyandiamide 1989

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Received December 30, 2003

Accepted March 20, 2004