Independent analysis of anions and cations using indirect photometric chromatography

An improved technique and apparatus for the independent measurement of anions and cations in solution, wherein the sample ions are chromatographically displaced in a plurality of ion exchange columns by an eluting ion which is or is made light-absorbing and wherein the sample ions are detectable from decrements in absorbance of the eluting ions as revealed by photometric monitoring; and the sample ions, cations and anions, are further detected independently by means for monitoring eluting anion and cation absorbance differences calibrated according to characteristic absorbance ratios of the selected eluting ion species at a plurality of wavelengths.

FIELD OF THE INVENTION 
This invention is in the field of ion exchange chromatography. It 
particularly concerns a method and apparatus for the independent analysis 
of coeluting sample anions and cations in a liquid effluent using Indirect 
Photometric Chromatographic (IPC) techniques. 
BACKGROUND OF THE INVENTION 
Liquid chromatography is used to separate the components of a sample 
substance by passing an eluent containing the sample through a column. The 
components of the sample in the eluent stream have different retention 
times within the column, and therefore exit the column in a particular 
sequence depending on the nature of the components of the sample and the 
nature of the column. The sequence of components is detected, for example, 
photometrically by measuring the intensity of light absorbed by the eluent 
stream. 
Developments in liquid chromatography have led beyond component separation 
and analysis to ion separation and analysis. Ion exchange liquid 
chromatography is often complicated by limitations in the capability to 
detect the eluted sample ions in the column effluent. For example, many 
inorganic and organic ions are non light-absorbing and difficult to detect 
using conventional photometric detectors. Even though the separation of 
such "transparent" ions may be conveniently effected using ion exchange 
resin columns, the detection and measurement of these transparent ions by 
conventional photometric means is ineffective since they are optically 
indistinguishable from the transparent eluents commonly prescribed by the 
art. Hence, practices using photometers to detect ions in effluent have 
been effective only when the ions to be analyzed either contain 
chromophores or can generate chromophores through post-column reactions 
with appropriate reagents. 
The problems of transparent ion detection are in part solved by the recent 
development of Indirect Photometric Chromatography (IPC). IPC has been 
described in detail in U.S. Pat. No. 4,414,842. IPC involves a method and 
apparatus for measuring ions in a sample undergoing chromatographic 
analysis wherein the ions of interest are transparent at the wavelengths 
monitored by the photometer. In the IPC method, these ions are first 
displaced from the ion exchange column in which they have been selectively 
adsorbed, by passing through the column, an eluent containing eluting ions 
which are or are made light absorbing. The displaced sample ions as they 
appear in the eluent are then detected in series and quantified by 
observing the decrements the ions cause in eluent absorbance, as revealed 
by photometric monitoring. For further details of IPC principles and 
techniques, see, "Indirect Photometric Chromatography", Anal. Chem., 1982, 
54, 462-469, written by Small and Miller. Both of the above references are 
incorporated herein. 
A problem with the known IPC method arises when an attempt is made to carry 
out independent analysis of both anions and cations, both of which are 
transparent. Since such ions in the mobile or eluent phase both contribute 
to the absorbance of UV wavelengths, it has not heretofore been found 
possible to measure independently, the eluent absorbance due to the 
individual anions and the individual cations. 
One attempt was made to detect and record anions and cations eluting 
simultaneously from a column; see Yamamoto, Yamamoto, Yamamoto, 
Matsushita, Baba and Ikushige, "Simultaneous Determination of Inorganic 
Anions and Cations by Ion Chromatography with Ethylenediaminetetraacetic 
Acid as Eluent" Anal. Chem, 56, 832-834, (1984). This attempt was not 
successful in independently determining the anion and cation 
concentrations as they eluted. The scientists noted that, "The retention 
times observed for Ca.sup.2+ and Mg.sup.2+, injected as metal cations, and 
those injected as EDTA chelate anions were not significantly different." 
To overcome this problem, the scientists converted the Mg.sup.2+ and 
Ca.sup.2+ cations to chelate anions using the EDTA eluent, separated the 
anion species for detection, and detected the anions using conventional 
chromatographic methods. The scientists specifically noted that Mg.sup.2+ 
and Ca.sup.2+ could not be found except as corresponding anion peaks in 
the chromatogram. 
Even though detection systems have been developed for differentiating among 
pure components of an eluent stream, e.g., see U.S. Pat. No. 4,367,041, no 
detection system presently exists which can distinguish between 
transparent anion and cation species having identical or nearly identical 
retention times. 
The present invention has as its object to provide a method and apparatus 
using indirect photometric chromatography techniques to detect 
independently anion and cation species of a sample within a single 
chromatograph even when the species have identical or nearly identical 
retention times. The technique of the present invention displaces sample 
anions and cations with photometrically monitorable eluent anion and 
cation species. The present invention teaches the use of an eluent 
containing a salt with chromophoric anion and cation species each having a 
known characteristic ratio of absorbance at predetermined wavelengths, and 
the use of certain mathematical equations to develop, independently two 
chromatograms, one for anions, and one for cations. 
Other objects, features and advantages of the present invention will become 
apparent upon reading the following detailed description and examples, 
when taken together with the drawing and appended claims. 
SUMMARY OF THE INVENTION 
The invention relates to a method and apparatus for detecting independently 
photometrically indetectable sample anions and cations using an anion 
exchange stationary phase and a cation exchange stationary phase, an 
Indirect Photometric Chromatography method of detection, a particular kind 
of anion and cation displacing eluent, a multiple analysis wavelength 
detector, circuitry for mathematically manipulating data, and a recording 
means. 
The method of the invention in a preferred form involves the steps of: 
(a) determining inherent relative absorbance ratios for the ions of the 
eluent, i.e., the ratio for the anions of a particular eluent at selected 
first and second wavelengths and the ratio for the cations of a particular 
eluent at the selected first and second wavelengths; 
(b) forming a stream of eluent using an eluent comprising a soluble salt 
solution having: (1) chromophoric monitor/displacing anions and cations 
capable of producing distinctive ratios of absorbance wavelengths and (2) 
a pH compatible with a first ion exchange stationary phase and a second 
ion exchange stationary phase and flowing the stream through the first ion 
exchange stationary phase and the second ion exchange stationary phase to 
a detector; 
(c) introducing a sample into the stream of eluent and flowing the sample 
with the eluent to the first ion exchange stationary phase; 
(d) displacing ions of the sample having a common charge with eluent ions 
having a similar change using said first ion exchange stationary phase and 
forming an effluent; 
(e) flowing the effluent from the first ion exchange stationary phase into 
the second ion exchange stationary phase and displacing the ions of the 
sample having a common charge opposite the charge of the sample ions of 
the preceding step (d) with eluent ions having a similar charge; 
(f) detecting the total absorbance values for the effluent at a first 
wavelength and at a second wavelength; 
(g) passing the detected total absorbance values from the detector to 
circuitry for determining the concentration of cations in coeluting 
anion-cation sample bands by multiplying the inherent relative absorbance 
ratio for the eluent anion with the total effluent anion and cation 
absorbance detected at the first wavelength and subtracting the total 
effluent anion and cation absorbance detected at the second wavelength to 
eliminate the effect of fluctuations in eluent anion concentrations and to 
reveal fluctuations in eluent cation concentrations and fluctuations in 
sample cation concentrations; and determining the concentration of anions 
in coeluting anion-cation sample bands by multiplying the inherent 
relative absorbance ratio for the eluent cation with the total effluent 
anion and cation absorbance value detected at the first wavelength and 
subtracting the total effluent anion and cation absorbance value detected 
at the second wavelength to eliminate the effect of fluctuations in eluent 
cation concentrations and to reveal fluctuations in eluent anion 
concentrations and fluctuations in sample anion concentrations; and 
(i) recording the individual responses corresponding to the anions and 
cations of the sample. 
Generally described, the present invention provides a method and apparatus 
for independently detecting photometrically indetectable anions and 
cations of a sample, regardless of the concentration of the sample, by 
chromatographically displacing the anions and cations of the sample using 
indirect photometric chromatography techniques. It should be noted that 
the phrase "regardless of the concentration of the sample" refers to 
concentrations of sample within normal chromatographic limitations. Eluent 
anions and cations, once photometrically detected, reveal the anion and 
cation of the sample by detection of the decrements caused in effluent 
absorbance of the eluent anions and cations when subjected to photometric 
monitoring at two preselected wavelengths, .lambda..sub.1 and 
.lambda..sub.2. 
This method and apparatus invention is predicated on the existence of 
characteristic absorption spectra for the components of a selected eluent. 
More specifically, this invention is based on the fact that for a set of 
wavelengths, an eluent containing a single salt will exhibit constant 
absorptivity ratios, each distinctive for the anion and cation of the 
eluent salt. Each constant ratio is referred to hereafter as the "inherent 
relative absorbance ratio." 
The general formula used in accordance with the principles and teachings of 
the invention is: 
EQU A.sub.d =RA.sub..lambda..sbsb.1 -A.sub..lambda..sbsb.2 
wherein A.sub.d, A.sub..lambda..sbsb.1, and A.sub..lambda..sbsb.2 and 
absorbance signals, and R is the inherent relative absorbance ratio for 
either an eluent anion or an eluent cation depending on the ion being 
measured. More specifically, if sample cations are to be measured then R 
is computed using the general formula: 
EQU R=(A.sub..lambda..sbsb.2.sup.- /A.sub..lambda..sbsb.1.sup.-) 
where A.sub..lambda..sbsb.1.sup.-, represents the absorbance value of the 
anion species of the eluent at a first wavelength and 
A.sub..lambda..sbsb.2.sup.- represents the absorbance value of the anion 
species at a second wavelength. If sample anions are to be measured, then 
R is computed using the general formula: 
EQU R=(A.sub..lambda..sbsb.2.sup.+ /A.sub..lambda..sbsb.1.sup.+) 
For example, if the salt copper ortho-sulfobenzoate (Cu o-SB) is used in 
the eluent, the general formula is written as follows: 
EQU A.sub.d =RA.sub.240 -A.sub.270 
wherein for sample anion detection 
EQU R=(A.sub.270.sup.+ /A.sub.240.sup.+); 
and for sample cation detection: 
EQU R=(A.sub.270.sup.- /A.sub.240.sup.-) 
with wavelength values given in nanometers. Solving for the factor R for 
sample anion detection using experimental data, R is about 0.45, i.e., 
EQU 0.45=(A.sub.270.sup.+ /A.sub.240.sup.+) 
and for sample cation detection R is about 0.74, i.e., 
EQU 0.74=(A.sub.270.sup.- /A.sub.240.sup.-) 
These R values are determined simply by ratioing cation peaks using 
detected absorbance at 240 and 270 nm and anion peaks using detected 
absorbance at these wavelengths. 
Further, when the absorbance difference signal (A.sub.d) for eluent anions 
is zero, then A.sub.d for sample cations is determined by the equation: 
EQU A.sub.d =(0.74)A.sub.240 -A.sub.270. 
Similarly, when the absorbance difference signal (A.sub.d) for eluent 
cations is zero, then A.sub.d for eluent sample anions is determined by 
the equation: 
EQU A.sub.d =(0.45)A.sub.240 -A.sub.270. 
A basic feature of the invention is in the use of eluent having light 
absorbing anion and cation species capable of chromatographically 
displacing anions and cations of the sample of interest. 
The eluent must contain light-absorbing anions and cations which: 
(1) selectively displace the sample anions and cations from the 
chromatographic columns or ion exchange media, and 
(2) reveal the sample anions and cations in the effluent. 
The eluent which performs these two functions contains anions and cations 
which displace the transparent sample anions and cations from the column 
and enable the anions and cations of the sample to be detected in the 
column effluent as dips or troughs in the baseline absorbance of the 
anions and cations of the eluent. 
The eluent must contain light-absorbing anions and cations which have UV or 
light absorption coefficients such that the anion will not completely mask 
the cation, or vice versa. Also, the eluent must contain anions and 
cations of appropriate displacing power to elute the sample ions from the 
column in a reasonable amount of time, i.e., allowing resolution of peaks 
without excessive peak broadening. An ideal eluent should contain anions 
and cations which exhibit absorbances of at least 0.1 AU at 10.sup.-3 
molar with a path length of 1 cm at 220 nm or above. 
The eluent should contain a "monitor/displacing" salt which is soluble in 
water and preferably either aromatic, organic, inorganic, polyvalent or 
monovalent, or a combination of these characteristics. Even more 
importantly, the eluent should not contain a second salt which may form 
spurious peaks upon photometric detection. 
The eluent should have a pH which does not affect chromatographic column 
packing not destroy the ionic nature of the chromatographic column. More 
specifically, the eluent must not have a low pH, such as pH 1, wherein 
eluent hydrogen ions would begin to function as the displacing ions in 
place of the intended eluent ions. Similarly, the eluent must not have a 
high pH, e.g., pH 12, such that the eluent precipitates metal in the 
eluent or alternatively causes the hydroxide to act as a displacing ion. A 
pH of less than 7 is preferred for most silica-based ion exchange columns. 
Eluents with a pH in the range of pH 4 to pH 5 are effective and useful 
within the teachings and principles of the invention. 
The apparatus of the invention involves a system for independently 
measuring anions and cations of a sample using indirect photometric 
chromatography, an eluent comprising a soluble salt solution having 
chromophoric anions and cations and having an inherent relative absorbance 
ratio for the anions of an eluent at selected first and second wavelengths 
and for the cations of the same eluent at the same first and second 
wavelengths. The invention further involves an anion exchange stationary 
phase, a cation exchange stationary phase and a detector connected in 
series. The invention includes means for forming a stream of eluent 
flowing through the anion exchange stationary phase and the cation 
exchange stationary phase to the detector. The invention involves means 
for introducing a sample of interest into the stream of eluent and flowing 
the sample and eluent through the anion exchange and cation exchange 
stationary phase means to produce an effluent. The detector is preferably 
a multiple wavelength detector with means for receiving the effluent from 
the ion exchange stationary phases and measuring the total absorbance of 
the anions and cations in the effluent at a first wavelength and a second 
wavelength. The apparatus invention also includes circuitry responsive to 
the detector with means for using the inherent relative absorbance ratios 
distinctive for the eluent anions and cations and the detected total 
absorbance values for the effluent at first and second wavelengths to 
determine separately sample anion and sample cation concentrations. The 
apparatus invention can further involve recording means for recording the 
individual responses corresponding to the anions and cations of the 
sample. 
The particular circuitry of the instant invention consists of means for 
using the inherent relative absorbance ratio for each eluent ion to scale 
the detected total absorbance value of the effluent ions at a first 
wavelength to produce a scaled value and subtracting the detected total 
absorbance value of the effluent at a second wavelength from the scaled 
detected total absorbance value at the first wavelength to produce 
individual absorbance values for the eluent anion and cation, eliminating 
the effect of fluctuations in eluent anion and cation concentrations and 
subsequently revealing fluctuations in sample anion and cation 
concentrations. 
Terms 
Throughout this specification terms particular to liquid chromatography and 
this invention will be used. The definitions of these terms follows: 
"Transparent"--This term refers to the inability or lesser ability of an 
anion or cation of a sample of interest to absorb light at the selected 
wavelengths or be photometrically detected relative to the light 
absorbance property of an eluent. 
"Eluent"--This term refers to the mixture of the specified salt in 
deionized LC quality water without sample, e.g., copper 
ortho-sulfobenzoate in LC quality water. 
"Effluent"--This term refers to the aqueous mixture which results from 
passing eluent through the medium capable of performing the ion exchange. 
"Wavelength"--This term refers to a wavelength or a wavelength band. 
"Inherent relative absorbance ratio"--This expression denotes the quotient 
of optical absorbances at two different specified wavelengths for a single 
ionic species employed in the eluent. Since spectral features are 
generally independent of species concentration, this ratio remains 
constant as the concentration of an absorbing species varies.

DESCRIPTION OF THE INVENTION 
Referring to FIG. 1, there is shown a typical diagram of an ion exchange 
chromatography system or apparatus which is desirably used in practicing 
the invention. The apparatus includes separating means such as two liquid 
chromatography columns 12 and 16 which are packed with media capable of 
performing ion exchange separations. Most chromatographic ion exchange 
media are in the pellicular or micro-particular form. Alternatively, the 
invention may employ a non-ion exchange chromatographic medium which is or 
can be rendered ion exchange-performing under the influence of the 
specified eluent. For example, a C.sub.18 reverse phase LC column could be 
used as one of the preferred separating means; such as the work reported 
by Skelly, N. E. Anal. Chem., 54, No. 4, 1982, pp. 712-715. 
Columns 12 and 16 are connected to a photometric detector 19. Detector 19 
is a flow-through UV photometer with micro-volume cell design for high 
performance liquid chromatography (HPLC) applications. Detector 19 is 
connected with waste vessel 38 for receiving the analyzed effluent. 
Sample may be placed into the eluent stream flowing into column 12 using 
any suitable device but preferably using a syringe 9 to load an injection 
valve 6 having a sample loop 35. The injected sample is swept through 
column 12 and then column 16 with eluent 3 containing monitor/displacing 
light-absorbing anions and cations. Eluent 3 is drawn through tubing 8 
from eluent reservoir 2 preferably using a chromatographic pump 4. The 
pressure of the eluent stream is preferably monitored by a pressure gauge 
5. A first ion exchange occurs in column 12. Effluent from column 12 is 
passed through tubing 14 to column 16 wherein a second ion exchange 
appears. Effluent from column 16, containing resolved anions and cations 
is flowed to the photometric detector 19 through tubing 8. Tubing 8 
preferably is conventional tubing with an 0.02 inch I.D. Tubing 14 is 
preferably a short length of 1/16 inch HPLC tubing. Photometric detector 
19 is in electronic communication with circuitry 34 and recording means 
36. 
Additionally, pump 4 of this embodiment is a standard liquid 
chromatographic pump capable of operation with non-pulsing flow rates in 
the appropriate range. As an example, pump 4 could utilize a flow rate in 
the range of greater than 0.5 cc/min and less than 5 cc/min. The pump 4 is 
capable of providing pressure on the system within the range of 100-6000 
psig. 
The columns of this embodiment are preferably anion exchange and cation 
exchange columns connected in series as columns 12 and 16. The particular 
columns useful in this method and apparatus are columns with a total 
capacity in milliequivalents (meq) preferably in the range of 10.sup.-3 to 
10 meq. 
Silica columns are particularly useful in this invention. Other columns 
which can be used within the principles and teachings of this invention 
include surface sulfonated cation exchange resins and surface agglomerated 
latex anion exchangers with resin substrates. 
These separating columns useful in this invention are preferably 
commercially available anion and cation exchange columns with low anion 
and cation exchange capacities. Preferably, the separating columns are 
strong anion and cation exchangers. As an example, a ZIPAX.RTM. SAX or SCX 
duPont column, with prepacked dimensions of 2.1 mm.times.500 mm is useful 
within the principles and teachings of this invention. 
Detector 19 is preferably an ultraviolet light detector; however, another 
detector can be used within the scope of the invention provided the 
detector offers a variable parameter to selectively enhance the 
detectability of eluent components. 
In the photometric detector 19, the absorbance of the light-absorbing 
eluent anions and cations is measured both at a first wavelength 
.lambda..sub.1 and at a second wavelength .lambda..sub.2. These measured 
absorbance values are then converted into signals which are passed by 
means 25 to circuitry 34 for mathematical manipulation. The circuitry 34 
determines the absorbance difference values for cations and anions 
individually using the general formula: 
EQU A=RA.sub..lambda..sbsb.1 -A.sub..lambda..sbsb.2 
for measuring ions in the sample, wherein 
A.sub..lambda..sbsb.1 is the total absorbance of the effluent: at a first 
wavelength; 
A.sub..lambda..sbsb.2 is the total absorbance of the effluent at a second 
wavelength; and 
R is the known inherent absorbance ratio for a given eluent ion. 
A computer may be used as the circuitry 34 to reveal the individual 
chromatograms or alternatively store and process the chromatographic 
patterns into sample ion data without recording the actual chromatograms. 
Similarly, the circuitry 34 can be used for sample cation chromatograms. 
The data resulting from these computations then can be transmitted over 
means 23 to recording means 36, such as a strip chart recorder with dual 
tracing capabilities, or alternatively to two recorders connected in 
parallel, each of which is capable of recording the respective absorbance 
information. Recording means 36 can consist of any form of storage device, 
such as a computer memory, chart paper or digital readouts. 
The concentrations of sample anion and cation can be determined 
independently and simultaneously using the above described apparatus. 
The method and apparatus of the present invention require a specified 
eluent to achieve the objects of the invention. The preferred 
characteristics of the eluent are described in the subsequent paragraphs. 
An ideal eluent is one which can exhibit UV absorbance spectra with a 
maximum absorbance wavelength for anion absorbance and a minimum 
absorbance wavelength for cation absorbance. Alternatively, an ideal 
eluent is one which can exhibit UV absorbance spectra with a minimum 
absorbance wavelength for anion absorbance and a maximum absorbance 
wavelength for cation absorbance. The eluent should have an absorbance 
within a range of 0.1 to 3.0 AU at the selected wavelengths. In the 
preferred embodiment, the eluent should exhibit an absorbance within the 
range of 0.5 to 1.5 AU at suitable wavelengths. 
The eluent should also have UV absorbing qualities capable of revealing low 
concentrations of transparent anions and cations during Indirect 
Photometric Chromatography (IPC). An ideal eluent should contain less than 
about 5.times.10.sup.-2 molar of displacing anions and cations for the 
purpose of sensitive detection using the IPC method of analysis. A 
preferred molarity for the eluent is within the range of b 
5.times.10.sup.-5 to 5.times.10.sup.-2 molar, ideally between 
5.times.10.sup.-5 to 5.times.10.sup.-3. 
An ideal eluent has an eluting power such that it produces a sample ion k' 
within the range of 0.5 to 50 at eluent concentrations ranging from 
10.sup.-5 to 10.sup.-1 molar. A preferred eluent develops k' within the 
range of 1 to 20 at concentrations ranging from 10.sup.-5 to 10.sup.-2 
molar. (k' refers to the expression: 
EQU (V.sub.E -V)/V, 
where V.sub.E equals sample ion elution volume and V is the void volume). 
In a preferred embodiment, the eluent of the instant invention should be 
capable of being easily prepared from analytical grade reagents and 
capable of being prepared so that no secondary salt is present. One such 
preferred eluent, which exhibits two different absorption spectra for its 
anion and cation, is easily prepared from analytical reagent grade 
ortho-sulfobenzoic acid cyclic anhydride (o-SBA) and copper (II) 
hydroxide. This preferred eluent comprises copper ortho-sulfobenzoate 
having the structural formula: 
##STR1## 
A particularly effective eluent salt is one which is multivalent, aromatic, 
and with a high molecular weight. Effective eluents are prepared by 
dissolving weighed amounts of the salts in deionized LC quality water. As 
an example, copper ortho-sulfobenzoate can be prepared by dissolving known 
weights of o-SBA cyclic anhydride and Cu(OH).sub.2 in deionized water to 
give the required equi-normal concentrations. 
Other eluents which meet these criteria include benzyl-trimethylammonium 
nitrate. Benzyl-trimethylammonium eluent cation, (BTA.sup.+), exhibits an 
inherent relative absorbance ratio of about 5.13 for sample anion 
detection, i.e., 
EQU R=(A.sub.260.sup.+ /A.sub.230.sup.+)=5.13 
For sample cation detection, nitrate eluent anion, (NO.sub.3.sup.-), 
exhibits an inherent relative absorbance ratio of about 0.025, i.e., 
EQU R=(A.sub.260.sup.- /A.sub.230.sup.-)=0.025 
It should be noted that the monovalency of both eluent species tends to 
reduce the sensitivity of detection compared to that of divalent Cu.sup.++ 
o-SB.sup.= which can be employed at lower eluent concentrations. Also 
BTA.sup.+ exhibits a disproportionately strong sample eluting power 
relative to nitrate. 
Another eluent is copper nitrate (Cu.sup.+2 (NO.sub.3.sup.-).sub.2). 
Cu.sup.+2 exhibits an inherent relative absorbance ratio of 0.248 for 
sample anion detection, i.e., 
EQU R=(A.sub.263.sup.+ /A.sub.233.sup.+)=0.248 
For sample cation detection, nitrate, the eluent anion, exhibits an 
inherent relative absorbance ratio of 0.031 at these same wavelengths, 
i.e., 
EQU R=(A.sub.263.sup.- /A.sub.233.sup.-)=0.031 
For this eluent, the equi-normal nitrate is very weak as a displacing ion 
relative to divalent copper and the sample anions are highly retained 
relative to sample cations. 
Additional eluents that have been considered include copper phthalate, 
copper trimesate, copper iodide, and pyridinium (aromatic cation) salts. 
In each of these cases, spectral data shows that one ion of the pair would 
exhibit an extreme degree of optical absorbance relative to the other, 
masking the counterion's absorbance at every wavelength. 
Benzyltrimethyl ammonium iodide (BTA.sup.+ I.sup.-) would be expected to be 
suitable using 250 and 270 nm detection wavelengths but monovalency would 
again be expected to limit sensitivity relative to divalent Cu o-SB. 
The following examples further detail the principles and teachings of the 
present invention. Additional objectives, aspects and advantages of the 
invention will be apparent from the following examples. 
EXAMPLE 1 
In this example, the apparatus includes an eluent reservoir, a Laboratory 
Data Control (LDC) Constametric I pump, a Rheodyne Model 7010 injection 
valve, a duPont ZIPAX.RTM. SAX column, and a duPont ZIPAX.RTM. SCX column 
both prepacked and 2.1 mm.times.500 mm, a Micromeritics Model 788 dual 
variable wavelength detector and a Linear Model 585 dual channel recorder. 
The preferred eluent is prepared from analytical reagent grade 
ortho-sulfobenzoic acid cyclic anhydride (o-SBA), and copper hydroxide to 
form a 5.times.10.sup.-4 molar mobile phase copper ortho-sulfobenzoate 
eluent. More particularly, the 5.times.10.sup.-4 molar copper 
ortho-sulfobenzoate eluent is prepared by dissolving 92.1 milligrams of 
ortho-sulfobenzoic acid cyclic anhydride (FW 184.17) and 48.8 milligrams 
of copper (II) hydroxide (FW 97.54) into each liter of water (LC quality 
water). The solution is stirred until all particles are dissolved and the 
pH of the eluent is measured to be in the range of pH 4 to pH 6. 
Eluent is placed in the reservoir and the LDC Constametric pump draws 
eluent from the reservoir and pumps it to a duPont ZIPAX.RTM. SAX column 
at a flow rate of about 1.0 milliliter per minute with an inlet pressure 
of 2200 psi for this first of the two columns in series. A 20 microliter 
sample aliquot containing 10.sup.-3 molar of NH.sub.4 Cl and 10.sup.-3 
molar K.sub.2 SO.sub.4 is injected into the stream of eluent between the 
pump and the first column using the Rheodyne Model 7010 sample injector 
valve. Anion exchange occurs in the first column and effluent from the 
first column then proceeds into a second ZIPAX.RTM. SCX column. The inlet 
pressure for the second column is approximately 1100 psi and approaches 0 
psig between the inlet and outlet of this ZIPAX.RTM. SCX column. Effluent 
from the SCX column is flowed into a Micromeritics Model 788 dual variable 
wavelength detector. In the detector, the effluent from the second column 
enters a cell having windows disposed at opposite ends. Light is passed 
from a light source through the first set of windows, the effluent, and a 
second set of windows to a variable angle concave grating which separates 
the light waves into at least two distinct wavelengths. The light beams at 
these wavelengths are then detected. In this example, UV detection is 
performed at two wavelengths, 270 and 240 nm. The instrument then passes 
the detected effluent absorbance values to circuitry or alternatively 
other means for mathematically manipulating the data relative to the 
general formula A.sub.d =RA.sub..lambda..sbsb.1 -A.sub..lambda..sbsb.2. 
The manipulated data is subsequently communicated to a Linear Model 585 
multichannel recorder. 
An interesting, useful feature of the invention is that sample anions and 
cations exhibit two different but constant ratios of peak heights or areas 
at 270 nm relative to 240 nm. These known inherent relative absorbance 
ratios are values equivalent to the ratios of the inherent molar 
absorptivities of Cu.sup.++ and o-SB.sup.= at the two wavelengths, 270 nm 
and 240 nm, in accordance with the principles of IPC. This feature allows 
immediate identification of peaks as anion or cation. 
At wavelengths 270 nm and 240 nm o-SB.sup.= exhibits a constant ratio of 
0.74 while Cu.sup.++ exhibits a constant ratio of 0.45. FIGS. 2-5 are the 
chromatograms resulting from using Cu o-SB in accordance with the 
parameters of Example 1. FIG. 2 is the chromatogram measured at 240 nm. 
FIG. 3 is the chromatogram measured at 270 nm. FIG. 4 is a chromatogram 
representing the detection of anions alone, i.e., A.sub.d =(0.45)A.sub.240 
-A.sub.270. FIG. 5 is a chromatogram representing the detection of cations 
alone, wherein A.sub.d =(0.74)A.sub.240 -A.sub.270. For purposes of 
illustration, the FIG. 4 chromatogram is inverted. A consequence of the 
method is to invert the anion chromatograms as seen in FIGS. 7 and 10. 
EXAMPLE 2 
This example illustrates the separation of anions and cations of a 
synthetic acid rain sample (Na.sup.+, NH.sub.4.sup.+, K.sup.+, Cl.sup.-, 
NO.sub.3.sup.-, SO.sub.4.sup.=) using an eluent of 2.5.times.10.sup.-4 M 
Cu o-SB. FIG. 6 is a chromatogram of two completely resolved peaks 
(NH.sub.4.sup.+, SO.sub.4.sup.=) and the coelution of the other compounds 
as only two peaks (Na.sup.+, Cl.sup.- and K.sup.+, NO.sub.3.sup.-) which 
are nonetheless also completely resolved by the method of the invention. 
Employing the method taught by this invention, the independent analysis of 
anions and cations results in the chromatograms of FIGS. 7 and 8, 
respectively. FIG. 8 shows the interference of a negative peak with the 
K.sup.+ peak. This interference is due to the absorbance of NO.sub.3.sup.- 
ion at 240 nm which results in an interference with the detection mode of 
IPC resulting in a peak height ratio different from 0.74. This kind of 
chromatographic result will occur for those few sample ions that absorb at 
either 270 nm or 240 nm wavelengths of detection. This peak interference 
difficulty is easily overcome if the sample ion is transparent at one of 
the detection wavelengths, as is the case with nitrite (NO.sub.2.sup.-) 
and nitrate (NO.sub.3.sup.-). Example 3, following, illustrates how this 
is accomplished. 
EXAMPLE 3 
This example shows the applicability of the present invention to the 
chromatographic analysis of anions and cations in complex mixtures 
including nitrite and nitrate. Using the apparatus of Example 1, a 20 
.mu.l 10.sup.-3 M sample containing Na.sup.+, NH.sub.4.sup.+, K.sup.+, 
Cl.sup.-, NO.sub.2.sup.-, Br.sup.-, and NO.sub.3.sup.- is injected into 
the system using a mobile phase of 2.5.times.10.sup.-4 M copper 
ortho-sulfobenzoate. FIG. 9 shows the combined chromatogram observed at 
270 nm, where both nitrite and nitrate are transparent. FIG. 10 
illustrates the anion chromatogram developed using the method of the 
invention. The potassium (K.sup.+) peak, coeluting with nitrate, is 
quantified from the difference in peak magnitudes between the combined 
peak (FIG. 9) and the nitrate peak alone (FIG. 10).