Spectrophotometric measurement of pH gradients in continuous-flow systems

Analytaca Chumca Acta, 268 (1992) 29-38 Elsevler Science Publishers B V , Amsterdam

Spectrophotometric measurement of pH gradients in continuous-flow systems S Sagrado Vlves, M J Medma Hemlndez,

J L Martin Herrera and G Rarms Ramos

Lkpartament de Quhuca Analituza, Facultat de Quimrca, Unrversrtatde Valha,

46100 Bugassot, VaGncta (Spam)

(Recerved 30th August 1991, revlscd manuscript received 24th Apnl 1992)

Abstract In the presence of an acid-base mdlcator and usmg fast diode-array spectrophotometrlc scans, pH gradients m the spectrophotometnc cell can be measured at the same time as any other colonmetnc flow-mjectlon (FI) determmatlon 1s performed The effects of refractwe index changes, adsorptlon-desorptlon processes at the tube walls and assoclatlon of the mdlcator species with the system to be Investigated are considered Effectlve correction for the first two effects 1s demonstrated Indicators with different molecular structures and polantles gave slrmlar pH gradients, suggestmg adequate accuracy for most potential apphcatlons Apphcatlon to the detection and correction of systematic and random errors m pH-sensitive FI procedures ISdemonstrated using the o-phthalaldehyde-iv-acetylL-cysteme method for bovme serum albumin Keywords Flow mjectlon, UV-Visible

spectrophotometry, Gradlent scanning, pH, Trlstlmulus colorlmetry

Routme pH measurements are usually performed potentlometrlcally vvlth a glass electrode Also, new types of pH sensors based on modified optical flbres (optrodes) and semiconductor deaces have been developed [ 1,2] Spectrophotometry m the presence of acid-base mdlcators 1s not a usual means of measurmg pH values, however, under optnnum conditions, it can be as precise as potentlometry [31 Moreover, it can be very useful when other devices cannot be used, such as m maccesslble sampling sites or m corrosive envlronments In addition, electrodes and other devices that rely on the establishment of an equlhbrmm at a surface or through mterfaces are also hnuted m speed, thus, for instance, they cannot accurately measure the rapld pH changes that are found m many flow-mJectlon (FI) sltuatlons Instead, mdlcator acid-base reactions m solution are fast Correspondence to G Ramls Ramos, Departament de Quinuca Analitsa, Facultat de Quinuca, Umversltat de Valbncla, 46100 Bmyassot, Valbncla (Spam)

enough to follow closely any FI pH gradient, and the pH m the cell can be measured at the same time as any other spectrophotometrlc assay 1s performed The use of the dlsperslon coefficrent of a dye m the presence of an acid-base buffer has been proposed as an indirect means of measuring pH gradients m FI systems [4] This approach assumed the identity of the dlsperslon coefficients of the species mvolved, measurements being hmlted to the front and back parts of the bolus where the dye and the buffer systems undergo dlsperslon Apphcatlon to the determmatlon of the pK’ values of coloured substances was demonstrated In this work, pH gradients m FI experunents were directly and contmuously measured usmg an acid-base Indicator added to both sample and carrier solutions The pH values are determmed on the basis of the spectral changes of the mdlcator system The effects of refractive index changes, adsorptlon-desorptlon processes at the tube walls and association of the indicator species with those

0003-2670/92/$05 00 Q 1992 - Elsemer Science Pubhshers B V All rights reserved

30

S Sagrado fives et al /AnaL Chm Acta 268 (1992) 29-38

of the system to be mvestlgated were considered, and effectwe correction of the two former effects 1s demonstrated In addrtlon, spectrophotometrlc momtormg of the pH throughout an FI expenment 1s proposed as a means of revealmg systematic or accidental errors m determmatlons and of correcting the value of the analyte signals, thus lmprovmg the rehablhty and accuracy of pH-sensltlve determmatlons Many pH-sensltlve procedures have been reported for FI systems Thus, for instance, the fluornnetrrc and spectrophotometrlc o-phthalaldehyde-2-mercaptoethanol (OPA-ME) procedure to determine ammomum ion has been adapted to the FI methodology [S], however, the recommended carrier pH of 6 8 1s crltlcal as a signal vs pH plateau does not extst Consequently, to analyse acid samples routinely, small sample volumes or large dilution factors should be used The use of large buffer concentrations 1s not advisable owmg to the systematic errors mtroduced by changes m the refractive index at the sample/ carrier interfaces Many other analytical procedures exhibit a short optimum pH range and a large dependence of the sensltlvlty on the pH outside that range In this work, the pH gradients and the FI peaks for bovme serum albumm (BSA) after derlvatlzatlon with OPA-N-acetyl+cysteme (NAC) reagent [6] were sunultaneously measured Throughout an FI peak, many spectra of the indicator and the derlvatlzed BSA were acquired using a diode-array spectrophotometer, the pH gradient being calculated from the Indicator spectra Finally, to increase the precision of the pH measurements, and to facilitate unmedlate apphcation to all substances that exhibit colour changes wlthm the vlslble region, complementary tnstnnulus colorlmetry [7] was applied THEORY

In the presence of an acid-base mdlcator, the pH of the medium can be related to the absorbance of the solution by means of the equation A, -A

pH = pK’ + log-

A -A,

(1)

where A IS the absorbance of the mdlcator at a given pH and concentration, A, and A, are the absorbances given by solutions of the acid and base conjugated forms of the mdlcator at the same concentration, respectively, and pK’ 1s the protonatlon constant of the mdlcator As absorbances are related to concentrations rather than to actlvltles, a condltlonal constant should be used Instead of usmg single absorbances, A, A, and A, can be replaced with a lmear combmatlon of absorbances, taken at two or more than two wavelengths, A(h), A,(h) and A,(h) This permits a substantial improvement m the precision of a determmatlon Precision 1s optlmlzed by increasing the difference A, -A,, which can be necessary m some instances, e g , when the sensmg system would exhibit a small spectral change However, m most practical situations the Judicious selection of a pair of smgle wavelengths, or a pair of different weighted sums of wavelengths, will give satisfactory precision Complementary trlstunulus [7] provides a sultable general-purpose combination of absorbances which should give good preclslon when a chemlcal indicator that shows a colour change wlthm the visible region 1s used as the pH-sensmg system In complementary tnstrmulus colorunetry, weights given to the absorbance values account for the different sensltlvltles of the human eye m relation to the three prnnary colours The resultmg three functions, X,, Y, and Z,, are genentally represented by R, Equation 1 can be written using an R, function pH=pK’+logR

Rcl -Rc _R

c

CO

It should be emphasized that the use of the trlstunulus functions 1s not necessary, however, using these functions the same software package can be unmedlately applied to a large number of sensing substances tradrtlonally used as visual acid-base mdlcators without selecting wavelengths and without changing the weights assigned to the wavelengths In most instances, the spectrophotometrlc determmatlon of organic substances, including many drugs, 1s performed m

31

S Sagradoviueset a.?/Anal Chm Acta 268 (1992)29-38 the UV region and the use of a pH mdlcator gnnng colour changes m the vlslble remon is desirable In these mstances, trlstmmlus functions have the advantage of bemg universally apphcable Obviously, other functions should be nnplemented when sensmg substances exhlbltmg spectral changes 111the UV region are used As stated above with Eqns 1 and 2, the theory which follows 1s developed comparatively, usmg both genenc and tnstnnulus functions A drawback of Eqns 1 and 2 is that A or R, must be measured at the same concentration at which the spectra of the acid and basic conjugated forms of the indicator were measured This 1s a problem m FI determmatlons where the indicator concentration can be changed unpredictably by adsorption-desorptlon processes at the tube walls To overcome thrs dlfflculty, the two followmg approaches can be used First, If cahbratlon and measurements are also done at a second wavelength, or using a different linear combination of absorbances, B, the following expression can be deduced

Al - (A/w4

pH=pK’+log(A,~)&-A, In Eqn 3, the spectra of the sample can be taken using any concentration, independent of the concentration at which the values of A,, A,, B, and B, were obtained Equation 3 can also be expressed using a par of tnstunuh, or even a linear combination of trlstlmuh Thus, for mstance, one can write xc1

-xKY,,

+Zd/(Y,

+a1

Alternatively, the value of A at the calibration concentration can be wrltten as a function of two absorbances taken at different wavelengths, or as a function of two dtierent linear combmatlons of absorbances, A* and B *, which can be measured at any other concentration, the same or different of that used to obtain A (44l

whtch can be also expressed m relative terms, using the complementary chromatic coordinates, Q,, Q,, and Q, (generically, Q,> and the optical concentration parameter, J The expresslon proposed by Flaschka [81 results pH = pK’ + log

J,(Qr, - Qr) J,( Qr - Q,o>

(5)

where Q, = R,/(X, + Y, + Z,) and J = k(X, + Y, + ZJ, k being a constant

(6) 1

Analogously, using a pair of tnstlmuh, X2 and Yc*,

xc=x,* I

(Xc&

X,*(y,,--Xc,)

such as

-&Y,1)

-y,*(x,,-x,,)

1

(7)

Equation 6 or 7 can be used m combmatlon with Eqn 1 or 2, respectively, to establish the pH of a solution using measurements taken at any mdlcator concentration Moreover, Eqn 6 or 7 can be used to find the relative variation of the indicator concentration, f = A*/A = X,*/X,, where the cahbratlon concentration at which A or X, were measured 1s taken as the reference value

EXPERIMENTAL

Apparatus and reagents A pH meter (McropH 2000, Cmon, Barcelona) and a diode-array spectrophotometer (Model 8452A, Hewlett-Packard, Palo Alto, CA) connected to a Vectra computer via an HPIB protocol (Hewlett-Packard) were used The FI assembly was built using a peristaltic pump (Model Mmlpuls 3, Gdson, Mlddleton, WI), an mjectlon valve (Model 5020, Rheodyne, Berkeley, CA), a 1%~1 flow-cell (Model 178012-QS, Hellma, Mulheun/ Baden) and 0 5 mm 1 d PTFE tubes The cod reactor used to evaluate pH gradients was constructed using a 50-cm PTFE tube, and a cod reactor built up with a 150~cm tube, followed by a lOO-cm smgle-bead string reactor (SBSR), was used m the experiments done m the presence of BSA

pH=pK’+logxc[(y,o+Z,,)/(Y,+zc)] -xc, (4)

-Alw

A=A* A*(Bo-Al)-B*(Al-B(J [

32 The followmg stock solutions were prepared 0 04% bromothymol blue (BTB) (Rledel, Hannover), thymol blue (TB) (Probus, Barcelona), phenol red (PR) (Probus) and neutral red (NR) (Adlershof, Berhn) m water, 5 X lo-* M OPA (analytlcal reagent grade, Serva, Heldelberg) prepared weekly m ethanol and stored m the dark, 5 x lo-’ M aqueous NAC ( > 99%, Fluka, Buchs) prepared weekly and stored m a refrrgerator, 125 x 10v4 M BSA (for mlcroblology, Fluka) m water, 0 1 M phosphate buffer solution, and boric acid-borate buffer solution, prepared from 6 2 g of orthoborlc acid and 15 g of NaOH m 1 1 of water The OPA-NAC-buffer reagent conslsted of 30 ml of the OPA and NAC solutions and 5 ml of the borrc acid-borate buffer diluted to 500 ml Distilled, delomzed water (Barnstead delomzer, Sybron, Boston, MA) was used throughout

Software Data acqulsltlon and control of the spectrophotometer were performed by the HewlettPackard MS-DOS UV-VIS package The program PHSENSOR, written m QmckBASIC, was designed to make direct use of the * WAV and * TIM files generated by the MS-DOS UV-VIS package The program should be provided with the pK’ value of the mdlcator, its nominal concentration (optional), the * WAV spectra of the aad and base conjugated forms of the indicator taken at the same arbitrary cahbratlon concentration from which the Rcl, R,, Q,, and Q,, parameters of the mdlcator are to be calculated and a * TIM file contammg a series of spectra measured at different time values and at any mdlcator concentration durmg a smgle FI mjectlon If an FI acid-base pseudo-tltratlon 1s performed, the entlre colour transltlon of the mdlcator ~111 be obtained from the mformatlon provided by the * TIM file The program uses absorbance values within the 380-770 nm range to calculate tnstrmulus, chromatic coordmates and the J parameter, in addition to pH values on the basis of Eqns 2 and 7 or, optionally, of Eqn 5 When the program default optlon 1s used, the Q, chromatic coordlnate showing the largest range of values 1s selected to apply Eqn 5 Slmllarly, to apply Eqn 7,

S Sagraab vivesetal /Anal Chm Acta268(1592) 29-38

the program selects the combmatlon of the two trrstlmuh showing the largest reversed correlation (z e , the closest to a correlation coefficient r = - 1) In both mstances, the proposed choice should lead to the most precise pH values The ASCII output matrix can be formatted to contam times, pH values, X,, Y, and 2, tristnnuh, the relative varlatlons of the mdlcator concentration, f, and absorbances at any given wavelength The appropriate columns of this matrix file can be plotted using general-purpose plottmg software Evalzuztwn of pK’ values The pK’ values of the mdlcators used were evaluated by continuously pumping 1 X 10e5 M BTB, PR, NR or TB solutions through the flow cell The spectra of the acid and base forms of the mdlcators were measured by pumping solutions of the mdlcators containing 1 X low3 M HCl and NaOH, respectively The spectra of series of Indicator solutions contammg 1 X 10m3 M phosphate buffer of pH 7 30 were also measured From Eqns 2 and 7, and Eqn 5, pK’(BTB) = 732fO01, pK’(PR)=802+004 and pK’(NR) = 6 78 f 0 04 (SIXmeasurements) For TB m the OPA-NAC-boric acid-borate buffer medium, pK’ = 9 15, but when the determmatlon was made m the presence of 2 5 X 10m5 M BSA, pK’ = 7 7 was obtained The spectra of the TB acid and base conjugated forms also showed changes m the presence of BSA These changes were observed to be pH dependent, 1 e , bathochromlc shifts produced by the presence of BSA were larger m weakly acldlc or weak basic solutions than m more basic and more acidic media Spectral changes together with the apparent higher acid@ of TB suggested that, wlthm some pH range, BSA 1s bound to the mdlcator, the mteractlon being stronger wth the amomc basic TB species than wrth the non-lomc actdrc species Evaluatzon of pH gradzents The pH gradlent measurmg procedure was studled by mJectmg 1 X 10m3 M HCl or NaOH solutions containing 1 x 10e5 M of each mdlcator on a carrier contammg the same indicator con-

S Sagmdo viues et aL /And

centratlon and 1 X 10m3 M phosphate buffer of pH 7 30 Other FI condltlons were flow-rate 1 ml mm-‘, sample volume 150 ~1 and number of spectra taken per mjectlon 80 (one spectrum per second) BSA detennmnutwns A carrier contammg the OPA-NAC-bone acid-borate buffer of pH 9 5 was used InJected samples contamed 2 5 X lo-’ M BSA at several pH values All these solutions were made 1 X 10m5 M m TB Other FI condltlons were flow-rate 19 ml mm-‘, sample volume 180 ~1 and number of spectra taken by mjectlon 100 (one spectrum per second)

RESULTS AND DISCUSSION

Study of the pH gradaent measunng procedure The colour transltlons produced when acldlc and basic BTB solutions were mjected on a buffered carrier solution of pH 7 25 are shown m Fig 1 usmg a 2, vs Y, plot The corresponding [ Y,(pH), Z,(pH)] straight hne theoretical tranatlon, between the
20000 zc

1 @of*

33

Chm. Acta 268 (1992) 29-38

Ycd

15000 -

10000 -

5000 -

Fig 1 Z, vs Y, plot for BTB 0 = Theoretical &our transition using pomts spaced every 0 25 pH units, o = ~qlect~on of an acidic solution mto the buffered tamer of pH = pK’, q = lnlectlon of a basic solution mto the same earner

007,

!

“,,,0 03

b

A=498

nm

h=770

nm

i 0 01

0

10

20

30

40

50

60

70

80

t(s) Fig 2 Absorbance vs tune durmg the myzctlon of (a) an acidic and (b) a basic sample Absorbance was measured at 770 nm (basehe) and 498 nm (BTB lsosbestlc porn0

presence of background dnft due to refractive mdex changes and, probably, changes m the mdlcator concentration These undesirable effects have been described elsewhere [9,10], and were also observed wth the other mdlcators used Refractive mdex and concentration effects for BTB can be dlstmgulshed m Fig 2, where the correspondmg absorbances at two wavelengths, 770 and 498 nm, are plotted against tnne The mdlcator did not absorb at 770 nm and, therefore, absorbance variations were attnbuted only to changes m refractive index In addition, 498 nm 1s the lsosbestlc pomt of BTB and, consequently, absorbance changes at this wavelength were attnbuted to the sum of both refractlve index and total mdtcator concentration changes It has been shown that the effects produced by refractive mdex changes are mdependent of wavelength, which makes the correction of refractive index changes possible by subtractlon [ll] The corrected points, [A(h) -A(770), t], were used to calculate the (R:, t) or (Q,, t) data which are needed to establish the pH gradient usmg Eqns 2 and 7, or Eqn 5 The pH gradients for the mJectlons of acidic and basic solutions of BTB, calculated by means of Eqns 2 and 7, and also Eqn 5, usmg uncorrected [A(h), t] pomts, are given m Fig 3 In the

S Sagrado viws et al /AnaL

34

absence of systematic error, the same series of (pH, t) pomts should be obtained However, dlfferences between the pomts obtained by the two methods can be observed m Fig 3 These dlfferences were higher when the mJectlons were performed using more concentrated solutions However, when the correction for refractive index changes was applied, Eqns 2 and 7 and Eqn 5 lead to exactly the same (pH, t) values Figure 4 shows the vanatlon of the concentrations of several mdlcators m FI experunents, m which HCl or NaOH solutions containing an mdlcator at a given concentration were mjected into a buffered carrier containing the same indicator concentration These experunents were duphcated usmg mdependent solutions, the results being the same After correctlon of the refractive index changes by subtractlon, the relative vanatlons of indicator concentration were calculated as f= (R,*/R,) The mdlcators used here belong to different chermcal farmhes, and show large differences m hydrophoblclty Thus, BTB and PR are tnphenylmethane denvatlves, with two phenohc groups and a slmrlar structure, however, the hydrophoblclty 1s much larger for the non-lomc acid form of BTB than for the correspondmg form of PR, owing to the additional bromme, methyl and SOpropyl groups In addltron, BTB and PR have anionic base forms, whereas NR 1s a dlphenyldr-

8:

PH

g.

a

80

75

l

. 70

t

j,

* *

>-

,~t[,

0

, , , , , , , , , , , ,

20

40

8ot(s)

8o

Fig 3 Calculated pH gradients durmg the mecfion of acldx and basic samples 0, 0 = Eqn 5, *, l = Eqns 2 and 7

180

1 f(w

160

140

9’

1 1

0

6

d

f

I

801’,“,,,,,,,~,,,,,,,, 0

Chtm Acta 268 (1992) 29-38

20

**

60

40

80

t(s) Fig 4 Relatwe vanatlon of the BTB ( -1, PR (---_) and NR (- - - - - -1 concentration, (R:/R,)x 100 = fC%), durmg the mJectlon of (01 an acidic and ( * 1 a basic sample mto a buffered earner All solutions contamed the same indicator concentration

azme derivative wth a protonated ammo group as the catlomc acid form When the basic BTB and PR solutions were inJeCted, the mdlcator concentration mcreased at the bolus front and decreased at the back The opposite behavlour was observed when the acldlc BTB and PR solutions were mJected This can be ratlonahzed m terms of the adsorptlon-desorptlon processes at the tube walls It seems reasonable to assume that the non-lomc acidic forms of the mdlcators are more strongly adsorbed on the non-polar PTFE tube walls than the amonrc basic forms [6] Both conlugated forms of the indicators are present m slgmficant concentrations at the pH of the carrier, which 1s close to the pK’ values, therefore, any change m pH lead to large variations m the concentrations of the two species When an acldlc solution is mjected, an mcrease m the concentration of the acid form leads to an increase of the amount of adsorbed mdlcator, and the mdlcator concentration m solution decreases When the pH returns to its former carrier value, the indicator excess 1s desorbed and Its

S Sag&o

Eves et aL /Anal

35

Chm Acta 268 (1992) 29-38

concentration m solution Increases The opposite can be argued for an rnlectlon of basic solution Rapid and small pH changes (less than 1 pH unit) should be enough to produce these reversible processes In comparison with the PR, the larger adsorption-desorptlon effects observed m Fig 4 for BTB can be attnbuted to the higher hydrophobrclty of its acid form Other differences between the BTB and PR curves can be attnbuted to the higher pK’ value of PR Differential migration effects through the hquld-liquid bolus/carner interfaces could also be produced, probably bemg much smaller than the adsorption-desorptlon effects Concerning NR, the large concentration mcrease mltlally produced by the mjectlon of acidic solution can be attributed to protonatlon of the ammo group producmg desorptlon Adsorption of the indicator to restore equrhbnum can explain the lower concentration observed after the bolus No explanation was found for the concentration increase produced by the basic mn]ectlon, which suggests that desorptlon 1s also produced However, the use of Eqns 2 and 7 or Eqn 5 should filter out all concentration varlattons m the calculation of pH gradients Accuracy of the pH gradlent measunng procedure Finally, to establish an adequate procedure to evaluate the accuracy of the pH gradients obtamed 1s not a sunple matter However, a comparison of the results obtamed wth different mdlcators can be an indirect means of evaluating the accuracy of the pH measurements Such a comparison relies on the dtierent physical and chemical behavlours of the indicators and, therefore, indicators of different chemical farmlies, or wth large differences m hydrophoblclty, as occurs wrth BTB, PR and NR, should be used A comparison of results obtamed Hrlth the three mdlcators 1s shown m Fig 5 The pH gradlents were obtained by mjectmg HCl and NaOH solutions mto a buffered carrier, all solutions containing the same indicator system Dtierences wlthm the optunum sensmg regon of pH = pK’ f 1 were below 0 2 pH umts, which mdlcates the absence of large systematic errors The accuracy

90

1

80

Fig 5 pH gradients obtamed wth BTB (1, PR (- - -_) and NR (- - - - - -1 by 1nJectmg HCl (pH decreases) and NaOH (pH Increases) solutlons mto a buffered tamer

of the pH gradients obtamed should rely on both accurate cahbratlon of the pK ’ and absorbance functions of the conjugated forms of the mdlcator and effective correction of refractive mdex changes and adsorption-desorptlon effects

Appltcatlon to the unprovement of the reluabhty and accuracy of BSA determmatwn As shown above, BSA produces changes m the TB spectral and acid-base charactenstlcs and, therefore, cahbratlon of the TB parameters under the same expernnental conditions at which the BSA determmatlon 1s to be performed 1s not possible An appropriate BSA cahbratlon concentration does not exist for the followmg two reasons First, the BSA concentration decreases as longtudmal murmg and dlffuslon progress along the FI expenment and, consequently, contmuous-flow or batch expenments cannot adequately reproduce the FI mlectlon conditions Second, the BSA concentration ill obviously change when dtierent BSA standards and samples are used

36

Calibration of TB parameters at two extreme sltuatlons was tned, 1 e , absence of BSA and maxunum BSA concentration to be used for the cahbratlon graph The TB cahbratlon spectra and pK’ values obtained m an OPA-NAC-boric acid-borate buffer medium m the absence of BSA and wth 2 5 X 10m5 M BSA were used to calculate the pH gradients during a series of mjectlons of 2 5 x low5 M BSA samples contammg dtierent amounts of HCl and NaOH InJectlons were made mto the OPA-NAC-boric acid-borate carrier of pH 9 5 The resulting derlvatlzed BSA peaks, measured at the wavelength of maxunum absorbance (336 nm), together with the corresponding pH gradients, are shown m Fig 6 As can be observed m Fig 6 (upper part), small changes m the pH gradient produced large changes m the height and shape of the derlvatlzed BSA peaks The differences between the pH gradients calculated using both cahbratlon conditions (middle and lower parts of Fig 6) obey two independent factors first, the different cahbratlon pK’ values which cause a shift of the ordinate scale, and second, the differences m the absorbance cahbratlon functions used, which are due to the changes m the spectra of the indicator acid and base forms, and which determine the different shapes of the pH gradient curves Owing to the presence of different BSA concentrations, the pH gradients calculated usmg any set of cahbratlon parameters are not accurate, however, both the middle and lower parts of Fig 6 show sets of well separated (pH, t) curves, which suggests that both can be reliably used for quahtatwe purposes, to reveal an accidental change m the pH of a BSA sample In addition, quantitative correction of systematic or,accldental errors of the BSA concentration due to pH

Fig 6 Absorbance and pH gradients for mJectlons of BSA solutions havmg various pH values mto an OPA-NAC-bone acid-borate buffer carrier of pH 9 5 Samples were prepared m (1) 1 X 10d3 M NaOH, (2) 6x 10m4 M NaOH, (3) water, (4) 2~ lo-’ M HCl, (5) 4~ low5 M HCI and (6) 6 x 10e5 M HCl The pH gradients were calculated using the TB cahbratlon parameters obtamed m the absence of BSA (nuddle part) and m a 2 5 x 10e5 M BSA solution (lower part)

S Sagrado f&es et al /Anal Chm Acta 268 (1992) 29-38

001, 0

20

40

60

100 ““t(s)

11 0

PH 10 0

08

1

90

80 0

20

40

60

““tcS

joo

95

PH 85

75

651,,,,,“,,,,,,.‘,“,1 20 0

40

60 ““t( Sfoo

S Sag&o

vives et aL /Anal

37

Chm. Acta 268 (1992) 29-38

changes can be performed by applymg secondorder modellmg of the BSA sensltwlty-pH dependence The proposed model IS A,=

4 1 - k,S,

- k&

where A, and A, are corrected and experunental (uncorrected) functions of the absorbances at the BSA peak, respectrvely, S, 1s a function of the pH and k, and k, are constants To estabhsh k, and k, by hnear least-squares, Eqn 8 1s rearranged as follows

A, -4.z A*S,H,

where an “optunum” or reference value of the slgnal, A,, has been wrltten mstead of the corrected slgnal, A,, and where (A,,, SpH,l) represents the signal and the respective value of the pH function of the lth cahbratlon standard Modelhng usmg Eqns 8 and 9 can be applied usmg absorbances and pH values measured at a given time (e g , the tune at which maxunum absorbance of the reference solution IS obtamed) for the A and SpH functions, respectively The problem of choosmg an adequate trme value IS avoided by usmg A x t and pH X t areas The latter approach was used to model the series of experiments m Fig 6 The area between the A(t) curve for mJectlon 1 and the basehne was used as the reference value, A, Analogously, the A x t areas of the other curves were measured to provide the A,, cahbratlon values The correspondmg S,,$ values were obtained as the areas measured between the pH gradient curve of mJectlon 1 (reference) and the other pH gradtent curves Using the TB cahbratlon parameters obtamed m the absence of BSA, the values k, = 3 49 X 10m2 and k, = -6 5 X 10m4 were calculated In add&ion, usmg the TB parameters obtamed m the presence of BSA, k, = 177 X lo-* and k, = -3 9 X 10e5 were obtained The errors obtained by using these constants and Eqn 8 to correct the BSA concentrations for the same series of mJectlons, together with the errors obtamed by using the correspondmg “uncorrected” values of the signal, A,,, are plotted

a

0

i 0

-50,

= k, + k2SpH,r

8‘

0

-30

0

s

v

a

0 s 10

r

r

I I 20

0

I

1

8 30

S PH Fig 7 Relative errors obtained for the senes of Injections in Fig 6 Errors were calculated as (0, o) lOO(A, - A,)/A, for the uncorrected and COTand (0, A) lOWA,- A,)/A, rected values, respectively CorrectIons were performed usmg TB cahbratlon parameters obtamed in an OPA-NAC-buffer solution m (0, 0) the absence and (0, A) the presence of 25~10-~ MBSA.

agamst S,, m Fig 7 It can be deduced that, mdependently of the expernnental conditions used to obtam the TB parameters, apphcatlon of the model pernuts satisfactory correction of the BSA concentrations, gwmg flse to average errors of about 15% Concluswns The FI gradient scanmng technique

described here permits effective momtormg of the pH mside the spectrophotometrlc cell at the same tune as an FI determmatlon 1s performed The procedure 1s rapld and snnple and gives reproducible results Apphcatrons m detectmg unexpected pH changes and m correctmg the analyte slgnal, lmprovmg the rehablllty and accuracy of pH-senatlve determmatlons, have been demonstrated Accurate pH values can be obtamed only wlthm the range of 2-3 pH units where the indicator exhibits its spectral change, however, samples glvmg pH values outside the optimum sensmg range can be rehably detected as outhers A hrmtatlon IS posed by the need to avoid chemlcal reaction between the species of the selected mdlcator and those of the sensed system, how-

S Sagrado F&es et al /Anal

38

ever, as it has been shown, this can be tolerated m some extent and, even m these instances, the analyte signal correctlon procedure proposed here can be apphed successfully This work was partially supported CYT, Project DEP89/429/C2

by the CI-

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