Potentiometric method

A potentiometric flow-through electrode detector for use in high performance liquid chromatography and autoanalyzers. Said detector comprises a metal electrode which has the property that it provides a voltage response to the particular molecule to be detected thereby.

The present invention relates to detectors and more particularly to 
detectors for use in detecting the presence of chemicals. 
Present electrode detectors as are used in, say, High Performance Liquid 
Chromatography (HPLC) or Autoanalyzers are based upon voltammetric methods 
whereby a current resulting from an applied voltage is measured. Such 
electrode detectors are complex and subject to interference problems in 
use. Even more complex and expensive is the use of spectroscopic detectors 
which are most often supplied in association with present HPLC and 
autoanalyzers. 
The present invention proposes a potentiometric electrode detector for 
detecting amino acids and other organic copper complexing agents 
comprising a metallic tube copper electrode adapted to be flow-coupled to 
the effluent from a liquid chromatograph column or continuous flow 
analyzer, said electrode and a reference flow-through electrode being 
adapted for electrical connection to a voltage controller and recording 
means whereby changes in voltage at the metallic tube electrode due to 
copper-complexing are recorded. The essential requirement for the metallic 
tube electrode being such that it must provide a voltage response to the 
chemical substance to be detected. 
In a particularly preferred form of electrode detector the metallic tube 
electrode is formed from copper and the reference electrode is of platinum 
and with this form of electrode the following classes of chemicals have 
been found to be detectable. 
Biological Species: Amino acids, proteins, enzymes and enzyme substrates. 
Pharmaceuticals: Penicillins, sulphonamides, vitamins. 
Complexing Agents: EDTA, ethylenediamine, ammonia. 
Oxidising Agents: Hydrogen peroxide, nitric acid. 
Metals: All metals capable of reacting with EDTA can be indirectly 
detected. 
The embodiment of a copper tube electrode has advantages which include that 
it only provides a response to copper-binding molecules and strong redox 
reagents such that it suffers from fewer background interference problems 
than present spectroscopic or voltammetric detectors when used for the 
analysis of complex samples such as blood and urine, etc. Where the 
electrode detector is miniaturised say having a volume of 0.4 .mu.l, sharp 
HPLC peaks can be obtained.

Referring to FIGS. 1 and 2 there is shown an electrode detector 1 comprised 
of tubular flow-through components. The detector 1 has a Teflon inlet part 
comprising a Teflon tubing part 2 fitted into a Teflon mounting part 3. 
Fitted into part 3 is a platinum reference electrode tube 4 wrapped with a 
wire contact 5. The other end of platinum electrode 4 is mounted in a 
further Teflon mounting part 3' which is coupled via Teflon tube 2' to a 
flow-through copper electrode 6. The exit from copper electrode 6 is 
through a further Teflon tube 2". In use flow enters the detector from a 
flow absorbance detector via tube 2 and after passing through detector 1 
exits via tube 2". 
The following description concerns an example of the use of the detector of 
this embodiment from which its advantages will be readily appreciated. 
EXAMPLE 
In this example, we describe the use of a copper tubular electrode as a 
sensitive and selective detector in the analysis of amino acids by HPLC. A 
recent paper submitted by P. W. Alexander and C. Maitra to Anal. Chem. 
entitled "Continuous-flow potentiometric monitoring of .alpha.-amino acids 
with copper wire and tubular electrodes" (not yet published) reports the 
use of copper wire and copper tubular electrodes as sensitive universal 
detectors in continuous flow systems, where they were found to be superior 
to the copper selective membrane electrode for direct quantitation of 
amino acids. This system is shown to be useful as a detector for amino 
acids without post-column reaction of the eluted amino acids with 
Cu.sup.2+. 
Instrumentation 
A schematic diagram of the flow system used is shown in FIG. 3. In this 
system, a Waters model 6000 solvent pump, a Waters Model U6K injector, a 
Waters Model 450 variable wavelength detector and the electrode detector 
were linked serially in flow to each other. The electrode detector (FIGS. 
1 and 2) consisted of two tubular electrodes, one of copper and one of 
platinum, both of which were connected to an Activon voltage offset 
controller (.+-.1.5 V) and the platinum electrode was grounded. The 
outputs from the UV detector and the voltage controller were linked to a 
two pen Omniscribe Model B 5271 recorder, which had a 10 mV input. 
The components of the electrode detector system were as follows. The 
internal flow copper tubular electrode was constructed from 0.5 cm diam. 
copper rod, 2 cm in length. This rod was precisely drilled to give 
internal diameter of 0.75 mm for the flow path, the length of which was 
varied over the range 0.5-5.0, mm, however 1 mm was most frequently used. 
The platinum electrode was a 1 cm length of platinum tubing (0.75 mm ID) 
sandwiched between two pieces of Teflon tubing which served both to 
mechanically support the platinum electrode and to provide a means of 
attaching the flow tubing used to interconnect the various components of 
the system. 
Reagents and Stock Solutions 
Amino acids were obtained from various sources: glycine from BDH 
Biochemicals; valine from Koch Light and Co.; l-isoleucine from BDH Ltd; 
Methionine from NBC and phenylalanine from Merck. The amino acids were 
used without further purification and standard solutions were prepared 
immediately prior to use by dissolving weighed amounts in pH 6.7 buffer. 
This buffer solution was prepared from AR sodium hydroxide and potassium 
dihydrogen phosphate (BDH Chemicals). 
Volucon standard buffers were used to calibrate the pH meter. 
The mobile phase used for the HPLC separation of amino acids was prepared 
by mixing 50 ml of 1M NaH.sub.2 PO.sub.4, 19.2 ml of 1M NaOH and 10.0 ml 
of 40% w/v formaldehyde in a 1 l volumetric flask. The solution was then 
diluted to the mark to give a final pH of 6.7.+-.0.1. All water used for 
the chromatographic procedure was distilled and passed through a Millipore 
Q Water Purification System before use and methanol was triply distilled 
using all glass apparatus. 
The urine control sample was obtained from Travenol Laboratories (USA) and 
was freshly reconstituted prior to use. The pharmaceutical intravenous 
amino acid solution was also obtained from Travenol, under the trade name 
"Synthamin 17" and contained 9 essential and 6 non-essential amino acids 
in concentrations ranging from 400 mg-21 g per liter. This solution was 
diluted by a factor of 25 for chromatographic runs. Both urine and the 
intravenous solution were filtered through a 2.5 .mu.m Millipore filter 
before injection into the chromatograph. 
Chromatographic Procedure 
Separation by HPLC was accomplished using a .mu.-Bondapak C18 column (30 
cm.times.3.9 mm I.D., Waters Associates) maintained at 25.degree. C. The 
column was standardised using the manufacturer's recommended procedure and 
gave counts in the region of 4000 theoretical plates. The mobile phase 
flow rate was set at 1 ml/min (unless otherwise stated), producing a back 
pressure of 1000 psi. The absorbance detector was operated at 200 nm using 
a sensitivity setting of 0.1 AUFS. 
In this study, all eluted compounds were detected both by the UV detector 
and the copper electrode and the peaks displayed on the two pen recorder 
using a chart speed of 1 cm/min. Each data point shown in this paper 
represents the mean of triplicate injections. 
To prevent a decrease in sensitivity of the copper electrode due to 
poisoning, the electrode surface was periodically regenerated by rapid 
flushing with 5 ml of 8M HNO.sub.3 followed by 20 ml of distilled water 
and 20 ml of methanol. When the electrode was stored in the flow system, a 
solvent consisting of 50:50 v/v methanol/water adjusted to pH.about.5 was 
used to prevent deterioration of the electrode. 
Results and Discussion 
Mobile Phase 
The effect of pH on the response of the copper tubular electrode (CTE) was 
discussed in the previously referenced paper by Alexander and Maitra 
wherein it was shown that sensitivity for amino acid detection was 
greatest at high pH values. In this study the optimum pH for the mobile 
phase was determined to be 6.7 which represented a compromise between 
atttainment of maximum sensitivity and the prevention of electrode 
poisoning. 
FIG. 4 shows a typical chromatogram of a separation of five amino acids 
using the CTE as the detector system. 
A slightly acidic mobile phase was necessary to discourage the formation of 
insoluble cupric hydroxides, carbonates and phosphates on the inner 
surface of the CTE. The ready formation of such compounds in alkaline 
media would result in potential drift leading to error in the measured 
concentration. 
The purpose of formaldehyde in the mobile phase was to repress negative 
deviation of the baseline which occurred most prominently prior to the 
peaks due to sulphur containing amino acids such as methionine and 
cysteine. The exact mechanism whereby formaldehyde can eliminate such 
negative baseline deviations is not clear, but its reducing properties may 
assist in improving the rate of exchange of the amino acids on the 
electrode surface. 
Reference electrode 
The platinum tubular electrode shown in FIG. 1 was used for two purposes. 
Firstly, it acted as an auxiliary electrode to reduce electrical noise and 
secondly, it served as a reference electrode to the CTE. Since it was 
electrically grounded, the reference used was the earth potential. This 
configuration was adopted only after considerable experimentation with 
conventional reference electrodes, however, we found that the electrically 
grounded platinum electrode gave greatest sensitivity and least baseline 
noise. 
Electrode flow path 
Using glycine as a representative amino acid, the electrode response was 
studied as a function of the length of the electrode flow path for 
solutions of three different concentrations of glycine (5.8 .mu.g/ul, 11.5 
.mu.g/.mu.l and 17.4 .mu.g/.mu.l). In all cases, the electrode signal 
reached a maximum for a flow path of 1.0. mm. 
Poor response for very short flow paths is considered to be due to the 
relatively short residence times of the amino acid ligands in the 
electrode, whereas reduced response in the longer path length electrodes 
is due to sample dispersion effects. The optimum flow path length of 1 mm 
was adopted for all further work. 
Flow rate 
The effect of flow rate on response time and sensitivity was studied by 
removing the HPLC column, replacing it with a suitable connector and then 
injecting glycine (3.4 ug/ul) at various flow rates. Values of t.sub.max, 
the time required to reach the maximum of the chromatographic peak were 
measured for each flow rate and the results are shown in Table 1. 
TABLE I Response characteristics of the CTE and absorbance detectors at 
various flow rates. 
t.sub.max is the time taken to reach the peak maximum 
______________________________________ 
Flow rate 
Peak height (mm) 
t.sub.max (sec) 
(ml/min) UV detector CTE UV detector 
CTE 
______________________________________ 
0.5 122 87 24.2 27.0 
1.0 90 88 13.3 15.8 
2.0 57 74 6.9 8.5 
3.0 42 63 4.6 5.5 
4.0 37 58 3.5 4.0 
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In agreement with earlier findings, the CTE response improved with 
increasing flow rate, however this was accompanied by a decrease in 
sensitivity due to the decreased residence time of the amino acid in the 
electrode. The response time of the CTE compared favourably with that of 
the absorbance detector while the sensitivity of the CTE as measured by 
peak height, was less dependent on flow rate than was the sensitivity of 
the absorbance detector. It is clear that the optimum flow rate selected 
would represent a compromise between response and sensitivity; in this 
study we used a flow rate of 1.0 ml/min. 
The relationship between flow rate and resolution was also examined to 
provide additional information on the response of the CTE. Injections of 1 
ul of a solution containing 11.5 and 8.8 .mu.g/ul of glycine and 
isoleucine, respectively, were made at various values of solvent flow rate 
and the resultion (R.sub.s) of the two resolution peaks was calculated 
using the conventional formula. For values of R.sub.s &gt;0.8, peaks are 
considered to be only partially resolved, whereas R.sub.s &gt;2 indicates 
resolution with at least two peak basewidths between peak maxima. The 
results obtained are shown in FIG. 6 using the electrode detector 
(.DELTA.) and the UV absorbance detector (O). 
A decrease in resolution with increasing solvent flow rate was observed for 
both the absorbance detector and the CTE, however it was observed that 
resolution achieved with the absorbance detector declined much more 
rapidly than for the CTE. It is noteworthy however that at all flow rates 
tested, the UV detector gave slightly superior resolution. 
Calibration 
Calibration curves for glycine, valine and isoleucine were prepared using 
both the absorbance detector and the CTE. Linear plots were obtained with 
the UV detector for the three amino acids in the concentration range 0-25 
.mu.g/ul. The calibration plots obtained for the CTE were non-linear and 
are shown in FIG. 5; the electrode response follows the elution order 
glycine (A), valine (B), isoleucine (C). The shapes of the CTE calibration 
plots are similar to those obtained by Loscombe et al. J. Chromatogr. 166 
(1978) 403 in their work based on a copper selective membrane electrode. 
The precision of the electrode response was estimated by ten replicate 
injections of a solution containing glycine and isoleucine at 
concentrations of 1.74 .mu.g/ul and 1.30 .mu.g/ul respectively. 
Coefficients of variation of 1.4% for glycine and 2.5% for isoleucine were 
obtained and these results compared favourably with the absorbance 
detector which gave coefficients of variation of 4.0% and 3.6%, 
respectively, for the same 10 injections. 
Using a definition of detection limit as three times the baseline noise, 
the calculated detection limits for glycine, valine and isoleucine were 
75,200 and 300 ng, respectively, in a 1 .mu.l injection. 
We are currently developing a combined recorder offset and signal 
amplification system which will permit detection of smaller amounts of 
amino acids than stated above. Initial studies have shown that a ten fold 
increase in sensitivity over the above values can be easily attained. 
Applications 
The suitability of the CTE for analysis of urine and a pharmaceutical 
preparation was investigated. The freshly reconstituted urine sample was 
not pretreated in any way except for filtration through a Millipore filter 
prior to injection. The chromatograms obtained for the urine sample using 
both detectors are shown in FIGS. 7 and 8. These figures also show the 
chromatograms obtained from urine spiked with five amino acids. 
In FIGS. 7 and 8 the dark trace relates to an undiluted urine sample and a 
spiked urine sample (upper trace) containing the following amounts of 
amino acids added per ul of urine: (A) glycine (75..mu.g); (B) valine (5.3 
.mu.g); (C) methionine (7.6 .mu.g); (D) isoleucine (4.1 .mu.g); (E) 
phenylalamine (0.7 .mu.g). Conditions: 1 .mu.l injection with a flow rate 
of 1 ml/min. FIG. 7 concerns the use of the detector of this embodiment 
and FIG. 8 the use of a UV absorbance detector (at 200 nm). 
The chromatograms obtained using the UV detector are characterised by a 
profusion of unidentified peaks and this background renders recovery 
calculations of the added amino acids difficult. In contrast, the CTE 
chromatogram of the blank urine sample is very "clean" and the peaks 
produced by the added amino acids in the spiked sample are easily 
identified. From these chromatograms the recovery percentages for glycine, 
valine, isoleucine, methionine and phenylalamine were 96%, 106%, 107%, 
105% and 100% respectively. The amounts of amino acids added to the spiked 
sample are shown in the figure. FIG. 7 illustrated a marked advantage of 
the CTE, that is, its selectivity which eliminates the need for major 
pretreatment of the sample. 
An intravenous solution containing 15 amino acids and excipients such as 
sodium metabisulphite, sodium acetate and sodium chloride was analysed 
using the CTE detector. No pretreatment of the sample was involved except 
dilution and filtration. The chromatogram obtained is shown in FIG. 9. No 
attempt was made to identify or quantify each amino acid and the 
separation conditions were not optimised since the purpose was merely to 
demonstrate the utility of the CTE for pharmaceutical analysis. However, 
peaks due to 11 of the 15 components can be discerned and it is likely 
that the remaining amino acids are eluted together with the resolved 
components. 
This example was demonstrated that the CTE can be used as a selective 
detector in HPLC. In using such an electrode, problems associated with 
post column derivatisation procedures or absorbance detection of solutes 
having poor UV absorption are minimised. The performance of the CTE, as 
indicated by its response time and sensitivity, is comparable to that of 
the UV absorbance detector. Major advantages of the CTE of this embodiment 
are: firstly, the selectivity of the CTE is greatly superior to UV 
detection, and secondly the CTE can be manufactured at very low cost. 
In this example, amino acids have been used as solutes to demonstrate the 
application of the CTE to HPLC. Other copper binding compounds may also be 
detectable. Narang and Gupta Ind. J. Chem. 13 (1975) 705 have shown that 
sulpha drugs of sulphanilamide, sulphaguanidine, sulphathiazole, 
sulphamerazine, sulphadiazine and sulphapyridine readily formed complexes 
with copper and Evans et al "High Pressure Liquid Chromatography in 
Clinical Chemistry", Academic Press, London, 1967, P. 71-77, in their 
deiscussion of the analysis of porphyrins in biological materials by HPLC 
have reported that capro and meso-porphyrin esters readily complexed 
copper. We have found that a variety of solutes give response with the 
CTE. 
It will be appreciated that even though the embodiment described has been 
specific to a copper electrode detector, various metals or metal alloys 
are suitable for use in accordance with the present invention in relation 
to the detection of chemicals on the basis that the metal or metal alloy 
provides a potentiometric response to the molecule to be detected.