Buffered Superheated Water as an Eluent for Reversed-Phase
High Performance Liquid Chromatography
O. Chienthavorn / R. M. Smith*
Department of Chemistry, Loughborough University, Loughborough, Leicestershire, LE11 3TU, U K
Key Words
Column liquid chromatography
Superheated water eluents
Sulfonamides
Buffers
pKa determination
Summary
Inorganic buffers have been used to control the pH o f a
superheated water eluent for reversed-phase liquid
chromatography. Using a temperature gradient from
70-190 ~ the relative order of elution of a series of
sulfonamides on a polystyrene-divinyl benzene column
was determined at pH 3, 7 and 11. The separations were
compared with conventional reversed-phase separa-
tions on polymer and ODS-bonded silica columns. The
apparent pKa values of selected sulfonamides at ele-
vated temperatures were determined from their reten-
tion factors across in a range of superheated buffers and
the values were compared to those reported at room
temperature.
Introduction
Previous studies have demonstrated that because the
polarity of water decreases markedly as its temperature
is raised, superheated water under moderate pressure
can be used as the sole eluent component in reversed-
phase liquid chromatography [1-4]. It can possess a
polarity similar to methanol-water mixtures and has
been used to separate a wide range of compounds, in-
cluding phenols, barbiturates and analgesic drugs.
However, many analytes can ionize and frequently in
conventional reversed-phase chromatography the mo-
bile phase is buffered to suppress or control ionization in
order to improve peak shapes and reproducibility. It was
therefore important to determine if buffers could also be
Original Chromatographia Vol. 50, No. 7/8, October 1999
0009-5893/99/10 485-05 $ 03.00/0 9 1999 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
used at elevated temperatures and if they would have an
equivalent effect as in ambient separations.
A number of studies [5] have examined the dissociation
of water at elevated temperatures and pressures, al-
though the latter has only a small effect. The dissocia-
tion of water increases steadily as the temperature is
raised at the saturated vapour pressure from pKw = 13.3
at 50 ~ to pKw = 11.6 at 150 ~ The pressure would
have to be increased to 10,000 bars to achieve the same
change suggesting that the pressures of less than 50 bar
employed for superheated water separations would have
little effect.
This study investigates the potential of buffered super-
heated water as the mobile phase for reversed-phase
chromatography. Changes in the retention of a series of
sulfonamides on a polystyrene-divinylbenzene (PS-
DVB) column with the pH of the eluent are compared
with separations in conventional eluents. These drugs
have been of considerable analytical interest over many
years and a large number of methods have been reported
for their determination, including reversed-phase liquid
chromatography with buffered eluents on both ODS
silica [6-10] and PS-DVB columns [11-13] and more
recently capillary electrophoresis [14-16]. Changes in
retention over a limited pH range are used to determine
if the apparent pKa of the analytes is changed sig-
nificantly by the elevated temperature.
Experimental
Materials and Reagents
The sulfonamides (Table I) were obtained from various
sources: sulfanilic acid, sulfacetamide and sulfisomi-
dine (Sigma, Poole, UK); sulfanilamide (Hopkins and
Williams, Essex, UK); sulfaguanidine and sulfathiazole
(BDH, Poole, England); sulfapyridine (May and Baker,
Dagenham, UK); and sulfamethazine (K and K La-
boratories, USA). N4-acetylsulfanilamide and N 1, N 4-
diacetylsulfanilamide were synthesized in the labora-
tory. THF was of HPLC grade from Fisons Scientific
Apparatus (Loughborough, UK) and buffer salts were of
485
TableI. Structure and pKa of test sulfonamides 0.01-inch I.D. stainless steel tubing. The column and
coil were placed in a GC oven (Series 104, Pye Unicam,
UK) and the temperature was controlled by a pro-
grammer controller (Series 104, Pye Unicam, UK).
Samples (10 #L) were injected into an injection valve
Sulfonamide RI R2 pK,a pK,,z
(Model 7125, Rheodyne, Cotati, USA), fitted with a
3.2116]
Sulfanilic acid (NHxC~IaSO3H) 20 #L sample loop, mounted outside the oven. A set of
copper fins (3 cm • 12 cm • 0.05 mm) was attached to
Sulfa~anidine H NM
I 11.3116] the outlet tubing between the column and detector to
~--NH2 cool the mobile phase. The peaks were detected using a
Sulfanilamide H H 2.4116] 10.4116] Jasco UV/Visible detector (Model 870, Jasco, Japan) at
254 nm and the results were recorded on a HP-3395
N*-Acetylsulfanilamide integrator. The pressure in the detector was maintained
HBC--C-- H
with a Jasco 880/81 back pressure regulator set at
Sulfacetamide o 35 kg cm -2. The mobile phase was purged with nitrogen
H --C--CH3 1.8116] 5.4116] gas during the experiment. The temperature of the oven
during a separation was maintained at 70 ~ for 30 rain
N],N%Diaeetylsulfanilamide O o
HsC--C-- --C--C~B and was then increased at 2 ~ min 1 to 190 ~

Sulfathiazole _ ~
H 2.1114] 7.2116]

Sulfisomidine / N_~\ CH~

H --~.~N
x polystyrene-divinyl benzene column gave good peak
Sulfapyfidine
H 2.6116]
/ cHz
Sulfametbazhle fi~---(
H
analytical grade from a range of suppliers. Deionised
water was prepared in an Elga Purification unit (Wy-
combe, Bucks, UK). Approximately 1 mg mL -1 sample
solutions of each compound were prepared in 20 % tet-
rahydrofuran (THF)-water.
For the measurement of pKa values, buffer solutions of
phosphate, citrate, carbonate, acetate or borate, were
prepared at concentrations between 1-3 mM in deio-
nized water. The pHs of the solutions were measured
with a digital pH-meter (Model 520A, Orion Research,
Boston, USA).
Apparatus
Superheated WaterChromatograph.
The superheated water chromatographic system com-
prised a LC- 10AD Shimadzu pump (Shimadzu, Ja]gan),
which delivered the mobile phase at 1.0 mL rain- to a
PRLP-S (polystyrene-divinylbenzene, PS-DVB) (5 #m,
15 cm x 4.6 mm I.D.) column (Polymer Laboratories,
Shropshire, UK) through a preheating coil of 100 cm x
Results and Discussion
2.7114] 7.3114] Although the separation of selected sulfonamides on a
CHz
shapes with unbuffered superheated water, there were
N~
run to run variations in retention times and elution order,
8,4[16] suggesting that a buffered system was necessary. Be-
cause of the decreasing polarity of water with increasing
temperature, there was concern that inorganic buffer
2,4116] 7.4[16] salts might be insoluble at elevated temperatures. How-
ever, Marshall and coworkers [17] have reported that
CH3 dipotassium hydrogen phosphate will readily dissolve in
water at 100-400 ~ A set of phosphate buffers was
therefore prepared as eluents and their pH were mea-
sured at ambient temperature. Low concentrations (1-3
mM) were used to minimize the possibility of corrosion
of the HPLC system.
Separation of Sulfonamides at pH 3.0.
In trial isothermal separations using superheated pH 3.0
phosphate buffer the retention times of the sulfonamides
covered a wide range. The temperature of the column
oven was therefore held at 70 ~ for 30 min, then pro-
grammed at 2 ~ m i n I to 190 ~ The increasing tem-
perature during the separation reduced the polarity of
the eluent and so speeded up the elution of the later
analytes [2, 3]. Using these conditions the ten test sul-
fonamides (Table I) were eluted in a single run with
good separation and symmetrical peaks, except for sul-
fisomidine and sulfapyridine, which slightly overlapped
(Figure 1A and Table II). The retention times of all the
sulfonamides were now consistent in successive injec-
tions, unlike the variations found with unbuffered water.
A faster rate of temperature programming would short-
en the separation time further but the separation showed
an undesirable baseline drift and some compounds were
not resolved.

486 Chromatographia Vol. 50, No. 7/8, October 1999 Original

0009-5893/99/10 485-05 $ 03.00/0 9 1999 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
The selectivity of the separation was similar to those Table II. Retention factors (k) of sulfonamides on elution with
reported for conventional buffered organic modifiers at different pH buffers. Conditions as Figure 1
room temperature on a similar PS-DVB column. For
Compound pH
example, using an acetonitrile-buffer pH 3.1 eluent on a 3.0 7.0 11.0
PRP-1 column, Lee [12] found the elution order: sulfa-
nilic acid, sulfaguanidine, sulfanilamide, sulfathiazole, Sulfanilic acid 1.86 0.97 1.02
sulfamerazine, and sulfamethazine. An earlier study on Sulfaguanidine 15.62 15.92 15.28
a PS-DVB (XAD-2) column with an acetonitrile-pH 2.8 Sulfanilamide 23.48 23.69 11.01
buffer eluent reported the same order although the effi- N4-Acetylsulfanilamide 51.27 50.88 11.01
Sulfacetamide 85.19 2.26 1.02
ciency was much poorer [ 11 ]. NI,N4-Diacetylsulfanilamide 103.99 2.26 2.63
On an ODS-bonded silica column with acetonitrile-pH Sulfathiazole 108.90 87.83 2.23
2.8 buffer (10:90), Ricci and Cross [9] reported the Sulfisomidine 122.50 102.91 2.63
Sulfapyridine 126.77 123.35 11.01
order: sulfanilic acid sulfaguanidine, sulfanilamide, Sulfamethazine 147.93 130.91 11.01
sulfisomidine, sulfacetamide, sulfathiazole/sulfapyr-
idine (unresolved), and sulfamethazine. The order was
the same with methanol-buffer pH 2.8 (85:16) except
that sulfaguanidine and sulfanilamide were unresolved idine and sulfamethazine. In this case the main change
and sulfisomidine was now retained more than sulface- compared to the lower pH eluent was again the ioniza-
tamide This is possibly because sulfisomidine has a tion of sulfacetamide. When Ricci and Cross [8] ex-
pKa,~ of 2.7 and at pH 2.8 its ionization and hence amined the elution of the sulfonamides on an ODS col-
retention will be sensitive to the conditions. They also umn over the range pH 2.75 to pH 6.0 with methanol-
examined the effect of changing the proportion of me- buffer, they also found that the retention of sulfaceta-
thanol but there was little effect from 10 to 16% for mide decreased but except for small changes at pH 6.5,
these analytes. Wieling and coworkers [7], also found a the other analytes in the present study showed no
similar order of retention at pH 3.0, except that in both change, although they did not examine N 1, N4-diace -
methanol and acetonitrile as the modifier, sulfanilamide tylsulfanilamide.
was the most highly retained of this group of com-
With a further increase in the pH of the superheated
pounds.
eluent to 11, the retentions of almost all of sulfonamides
were greatly reduced (Figure 1C and Table II) as all are
Effect of Higher pH. largely ionized. Only sulfaguanidine, which has a high
pKa,2 = 11.3, remained unchanged. Similar effects were
On increasing the pH of the superheated buffer solution found for the XAD-2 column at pH 11.7, when all the
to pH 7.0, with the same temperature program, the re- sulfonamides had retention factors of less than one [11].
tentions and elution order of the sulfonamides altered Because of the instability of ODS silica stationary pha-
(Figure 1B and Table II). Some compounds changed ses at high pH no comparable results can be obtained for
markedly, in particular the Nl-acetyl derivatives, sulfa- these columns.
cetamide and N 1, N4-diacetylsulfanilamide, which were
eluted after 51.7 and 63.0 min with the pH 3 eluent but
were virtually unretained at pH 7. Sulfacetamide has a Determinations of pKa Values of Sulfonamides in
reported pKa 2 of 5.4 [ 16] and although a value could not Superheated Water Conditions
be located for N ,1 N 4 -diacetylsulfanilamide, it would be
expected to undergo a similar ionization of the SO2-NH- It was of interest to determine if the temperatures used in
COCH3 group. Thus both compounds should be largely superheated water chromatography had a significant
ionized at pH 7.0, markedly increasing their polarity. effect on the effective pKa of the analytes or altered their
Small decreases in retention, compared to the separation degree of ionization by changing the pH of the eluent
at pH 3.0, were found for sulfathiazole, pKa, 2 = 7.2, solution. Four selected sulfonamides, representing a
sulfisomidine, pKa, 2 = 7.3, and sulfamethazine, pKa,2 = range ofpKa,2 values were therefore examined in detail.
7.4. Although these compounds are less acidic than In each case the eluent pH was determined at room
sulfacetamide, all would be partially ionized under these temperature before the solution was heated. Acetate,
conditions. A smaller change was found for sulfapyr- carbonate, borate and citrate buffers were used to cover a
idine, pKa,2 = 8.4, which would be only slightly ionized. wide pH range and no noticeable inconsistencies were
The remaining compounds; sulfanilic acid, sulfaguani- noticed between the separations in the different buffer
dine, pK~,2= 11.3, sulfanilamide, pKa,2 = 10.4, and N 4- salts. In some cases the different buffer ranges over-
acetylsulfanilamide (assumed to have a similar pKa,2) lapped and at the same pH the retention factors were the
maintained approximately the same retentions as at pH same irrespective of the buffer salt.
3.0. The separation order again reflects that found pre- To determine the apparent pKa,2 values, the retention
viously with acetonitrile-buffered eluents. On a XAD-2 factors were correlated with the pH (Figure 2) eluent
column at pH 6.73 [1 1], the order was sulfacetamide, according to equation 1 [18] using a non-linear regres-
sulfaguanidine, sulfanilamide, sulfathiazole, sulfapyr- sion.

Original Chromatographia Vol. 50, No. 7/8, October 1999 487

 40.00

A 30.00 9
pH 3.0
89 25.00
* sulfacetamide
o~ 20.00 9 9 sulfathiazole
67 ~ 15.00 ~ 9 sulfanilamide
2 4 ~,\ / Oo~',~ = sulfamethazine
3 10.00

5.00

0.00
lb 20 3b 4'0 5'0 6b 7'0 8'0 9'0 160 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min) pH

5,6 Figure 2
Changes in retention factors (k) (points) of sulfonamides with pH of
the aqueous eluent and fitted regression curves (lines) from equa-
B tion (1) used to calculate the pKa,2. Compounds: @-sulfacetamide;
A-sulfathiazole; O-sulfanilamide; II-sulfamethazine.

9
pH 7.0

2 8 It 10
Table Ill. Comparison of experimental pKa values for selected
sulfonamides measured in hot and superheated water and literature
~Kavalues

Compound Temperature(~ pKa,2(exp.) pKa,2 (lit.[16])

Sulfanilamide 60 10.71 10.4

Sulfacetamide 90 5.20 5.4
Sulfathiazole 120 6.06 7.2
1'0 2'0 30 40 50 60 7'0 80 9b Sulfamethazine 150 6.28 7.4
Time (min)

6,7,8
1,5 /
3,4,9,10
ko L1
k= + (1)
K~ [H+]
1+ - - 1+ - -
[H + ] Ka

Where, k = retention factor at a given hydrogen ion

concentration, k0 = retention factor o f the neutral spe-
cies, k_1 -- retention factor o f the dissociated species, Ka
= ionization constant, and [H +] = hydrogen ion con-
centration. In each case there was a good fit between the
experimental and theoretical relationships (Figure 2)

0
l 10 20 30 4'0 5'0 6'0
" 7'0
Time (min)
Figure 1
The separation of 10 sulfonamides using with different superheated
aqueous buffers as the eluent. Conditions: column, PRLP-S (4.6 x
150mm); mobile phase, ~hosphate buffer; flow rate, 1 mL min-1;
back pressure, 30kg cm- ; detection, UV absorption, 254 urn; oven
temperature, 70 ~ for 30 min, then increased at 2 ~ mini to 190 ~
Eluent: A, pH 3.0 buffer; B, pH 7.0 buffer; C, pH 11.0buffer.Peaks: 1-
sulfanilic acid; 2-sulfaguanidine; 3-sulfanilamide; 4-N4-acet-
ylsulthnilamide, 5-sulfacetamide; 6-N1,N4_diacetylsulfanilamide; 7-
sulfathiazole; 8-sulfisomidine; 9-sulfapyridine; 10-sulfamethazine
S0 90
=
and the regressions yielded an apparent pKa value which
could be compared with the value at room temperature
(Table III).
As the p H o f the eluent was successively reduced, the
retention factor o f sulfacetamide rose to a m a x i m u m at
pH 4.0 and then decreased from pH 3.5 to p H 3.0. This
effect was assumed to represent the protonation o f the
amino-group (pKa,~ = 1.78) and reflects similar results
on the XAD-2 column [11]. As this ionization is not
reflected in the correlation equation (1) the retention at
pH = 3.0 was omitted from the analysis. From the re-
gression equation the calculated pKa, 2 for sulfacetamide
at 90 ~ was 5.2, which is very similar to that reported
pKa,2 = 5.4 at r o o m temperature [ 16], which also agreed

488 Chromatographia Vol. 50, No. 7/8, October 1999 Original

closely with the equivalent experiment on the XAD-2
column [ 11 ].
A similar close relationship was found between the ex-
perimental value for the pKa,2 = 10.71 for sulfanilamide
at 60 ~ and the literature value of 10.4 [16]. For these
two compounds the apparent values at elevated tem-
peratures were within experimental error of the litera-
ture values.
However, larger differences were found between the
literature values and the experimental values measured
at higher temperatures for sulfathiazole (at 120 ~ ex-
perimental pKa,2 = 6.06, at room temperature literature
pKa,2 = 7.2 [16]) and for sulfamethazine (at 150~
experimental pKa,2 = 6.28 and at room temperature lit-
erature pKa,2 = 7.4 [ 16]). In both cases the measured pKa
was lower than expected by about 1 pH unit. Thus under
these conditions it appeared that there was either a
change in the effective pH of the mobile phase or in the
degree of ionization of the analytes. However, although
the dissociation of water changes from a pKw of 13.3 at
50 ~ to 11.6 at 150 ~ Kryukov and coworkers [19]
found that from 25 ~ to 150 ~ there was only a slight
increase in the pH of phosphate, phthalate, tartrate and
tetra-oxalate buffers, although the pH of a borate buffer
declined slightly. This suggests that we can assume that
the hydrogen ion concentration remains approximately
constant over this temperature range. Thus the experi-
mental results suggest a change in the effective pKa,2 of
these sulfonamides at elevated temperature.
Conclusions
This study has demonstrated that it is possible to employ
inorganic salts as buffers in superheated eluents for re-
versed-phase liquid chromatography. Some change in
the degree of ionization of the analytes was observed
above 100 ~ and the dissociation constants in buffered
superheated water were apparently lower than at room
temperature.
Original ChromatographiaVol. 50, No. 7/8, October 1999
Acknowledgements
The authors wish to thank the Government of Thailand
for a scholarship to O. Chienthavorn.
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Received: May 14, 1999
Revisedmanuscript
received: Jun 22, 1999
Accepted: Jun 30, 1999
489