Coulometric flow analyzer for use with immobilized enzyme reactors

Mar 29, 1978 - Present address, American Enka Co., Enka, N.C. 28728. 2 Present .... samples were obtained from Saint Mary's Hospital. (Athens .... 1.3...
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944

ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978

1978. Work supported in part by NIH Grant AM-18352; the Inc., New City; the Diabetes Foundation, Inc., New York City; and the Joslin Diabetes Foundation, Inc., Boston, Mass. D. A. Gough was supported by NIH Postdoctoral Fellowship 21 F326M05335-01.

(10) J. R. Guyton, K. W. Chang. S.Aisenberg, and J. S.SoeMner, Med. Instrum., 9, 227 (1975). (11) p, L, Altman and D, S,Dittmer, Ed,, "Biology Data book", Voi, 111, Fed, Am. SOC.Exp. Biology, Bethesda, Md., 1974.

RECEIVEDfor review September 6, 1977. Accepted March 29,

Coulometric Flow Analyzer for Use with Immobilized Enzyme Reactors Robert E. Adams' and Peter W. Car?" Department of Chemistry, University of Georgia, Athens, Georgia 30602

A flow coulometric immobilized enzyme analyzer based on the use of a fast totally electrochemical pH-stat has been developed. The insolubilized urease had a K,,, of 43 mM and an activity of 1000-1200 units per gram of glass at its pH optimum of 6.8 in 0.2 M sodium perchlorate. I n the absence of phosphate buffer, urea adsorbed on the immobilized enzyme, causing broad peaks. Peak width was decreased by adding 1,3-diaminopropane to the electrolyte. It serves as a competitive inhibitor ( K , = 79 pM) and blocks adsorption of urea. Urea could be analyzed with good precision (3 % ) in simulated sera and in quality control reference sera. Recoveries of urea added to quality control sera were 100 % The major problem involved in determining urea in human serum was a high and irreproducible blank which limited the precision to about 5 YO.

.

A variety of devices have been used in conjunction with immobilized enzyme reactors as the basis for specific analyzers (1-3). Photometric flow systems using immobilized enzyme reactors have been developed for the determination of glucose (4-6), pyruvic acid ( 7 ) ,L-aspartic acid (a), penicillin G (9), urea (10-12), nitrate (13),phosphate and sulfate ( 2 4 ) ,and uric acid ( 1 2 ) . Horvath and co-workers have recently described continuous flow analyzers in which glucose, glycerol, and ATP were measured by the use of multiple enzyme tubular reactors (15). Crouch and co-workers have described a stopped-flow colorimetric analyzer for glucose (26). Potentiometric ( 1 7 ) and amperometric (18, 19) electrodes have many virtues as detectors for immobilized enzyme reactors. These devices generally do not require t h e use of auxiliary color development reactions, thereby permitting simpler flow systems and improved throughput due to decreased sample dispersion. Several groups, including Mosbach (20) and ourselves (22), have developed calorimetric analyzers which detect the heat generated in adiabatic columns packed with immobilized enzyme. An extremely sensitive chemiluminescent detection system has been developed (22). Other types of detectors, including refractometers (23), conductometers (24),and pH-stats (25),have been used to study the kinetics of immobilized enzyme reactors but as yet have not been used as t h e basis for flow analyzers. Potentiometric enzyme electrodes have been developed, based on several types of ion selective electrodes including hydrogen ion sensitive glasses ( I , 26-28). Papariello and

co-workers have had some success in designing a penicillin selective sensor based on the detection of local p H changes generated by an immobilized enzyme. Mosbach has also used hydrogen ion sensitive glass electrodes in conjunction with immobilized enzymes. The most attractive feature of using a p H sensor with immobilized enzymes is t h a t they are, in principle, nearly universal detectors since so many enzyme catalyzed processes will generate an acid or a base. In practice this approach is limited because the electrode will be sensitive to the sample p H per se. This could be overcome by using a differential pair of electrodes (29, 30),which has not as yet been done, or by adjusting the sample p H before the measurement is made. A very insidious source of error is that the measured p H change will depend upon the buffer capacity of the material which surrounds the sensor (27) which must depend at least in part on the sample. A pH-stat, Le., a device which measures the total quantity of acid or base produced by a chemical reaction, while holding p H constant, is applicable to the measurement of the extent of many reactions. These devices have the advantage over simple p H measurements since the amount of reagent added will be independent of buffer capacity. Such instruments have been quite valuable in the measurement of enzymatic reactions (32) and in kinetic analysis (32, 33). Recently we developed a totally electrochemical pH-stat in which p H changes were measured with a glass electrode and coulometrically restored to a pre-set value (34). We felt that this device could be used with immobilized enzyme analyzers. In addition, it serves as a model system to test the behavior of a mass flow sensitive immobilized enzyme analyzer. T h e test enzyme and substrate chosen for this study was t h e urease catalyzed conversion of urea to ammonia and carbon dioxide, which is shown below: NH,-C-NH, II

+

H,O-

urease

+

CO,

(1)

It should be recognized that at p H 6 to 8 the actual products are predominantly, but not exclusively, ammonium and bicarbonate ions; therefore the actual amount of acid or base generated will depend upon pH. A detailed analysis indicates t h a t upon complete conversion to the above products (vide infra) the amount of acid or base produced would be given by Equation 2:

A m o l e acidlbase [H'] m o l e s urea added

'Present address, American Enka Co., Enka, N.C. 28328. Present address, Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, Minn. 5545j. 0003-2700/78/0350-0944$01 0010

2NH,

0

6 1978 American

Chemical Society

[H'] + 2KIK2 * + K 1[H'] + K , &

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978

945

Figure 2. Block diagram of the flow coulometric immobilized enzyme anatyzer. (A) Supporting electrotytebuffer reservoir, (B)peristattic pump, (C) sampling valve, (D) reactor column cortaining active enzyme, (E) reactor column containing inactive enzyme, (F) pH-stat cell assembly, (G) pH-stat electronic assembly and readout, (H) waste outlet-aspirator, (I) nitrogen inlets

I

P

a

5

6

I

7

I

pH

I

9

10

11

,

I 12

Plot of moles of acid or base produced per mole of urea hydrolyzed vs. pH. The data shown are calculated from Equation 2 with K , = 4.5 X lo-’, K , = 4 . 7 X IO-”. and K , = 5.8 X IO-’’ Figure 1.

-

.

.

Flow Out

~..

.

: .,

.

.-

NH, -C-NH,

+

2 0 H - -.ZNH, + CO, * -

A

2 H , 0 + 2H’

0

NHI-C-NHZ I1

- 2”:-

(3)

H,CO,

(4)

0

In the p H range 6-9, where the enzyme is active, the amount of product detected by the pH-stat is a strong function of pH. Equation 2 is based on the assumption that reaction products are in fact ammonia and carbon dioxide. Jespersen (35)has shown that in certain buffers the actual product is primarily ammonium carbamate. In phosphate buffer the products are as given in reaction 1. Addition of phosphate buffer to the flow stream should cause reaction 1 to occur, but as shown previously ( 3 4 ) ,the electronic control time constant of any pH-stat is directly proportional to the system buffer capacity; thus for fast response the total buffer capacity must be kept reasonably low. The urea-urease system was chosen for this study because urea is present in relatively high concentrations in serum (3-9 m M (36))and is frequently measured in the clinical laboratory (37). Urease has been immobilized on controlled pore glass and other materials (38). Such preparations are stable for more than one month and have been well characterized.

EXPERIMENTAL Instrumentation. The electrochemical pH-stat employed in this work has been described previously ( 3 4 ) . Electronic noise

was reduced by replacing the original input electrometers with AD 515 operational amplifiers (Analog Devices. Norwood, Mass.). This resulted in a fourfold decrease in noise (referred to the system input) from 20 to 5 pL‘ peak to peak. Analysis of the signal-time curves as described previously (34) upon injection of dilute sodium hydroxide directly into the pH-stat vessel indicates a response time of 7 s under the actual analytical conditions. Baseline signal widths were less than 35 s when tested by injection into the cell. Although this is fast, relative to a conventional pH-stat, it is a substantial contributor to peak width (vide infra). A block diagram of the flow system is given in Figure 2. A peristaltic pump (B, Masterflex Model 7545.10 with a Model 17013 head, Cole-Parmer, Chicago, Ill.) delivers the supporting electrolyte-buffer mixture from reservoir (A) to either a column containing active enzyme (D) or an inactive enzyme (E). These

G

-~

I

r--D ~

-

~

where K , and K , are the first and second acid ionization constants of H,C03, and K 3 is the acid ionization constant of ammonium ion. This function is plotted in Figure 1, which indicates that a t high pH. two moles of base (OH-) are consumed per mole of urea, but at low p H , two moles of acid (H+) are removed per mole of urea as shown by reactions 3 and 4 respectively.

.

fA

._ Flow In

13

Figure 3. Schematic diagram of pH-stat cell assembly for flow analysis. (A) Water jacketed electrolysis cell (20 mL), (B) stirring bar, (C) combination pH-reference electrode,(D) auxiliary electrode compartment, (E) Ag/AgCI auxiliary electrode, (F) Pt generaling electrode, (G) Pt solution ground electrode, (H) nitrogen inlet. (I) solution inlet, (J) aspirator solution

outlet columns have a volume of 0.5 mL and are fabricated to accept Chromatronix plastic connectors. Samples are injected (C) into the flow stream via a Chromatronix sampling valve (Chromatronix Model SV8031, Berkeley, Calif.). Fluid flow flushes the reaction products from the columns into a glass electrochemical cell (F) which serves as the pH-stat vessel. The cell is blanketed with nitrogen (I) and excess solution is continuously aspirated out of the cell (H). Figure 3 is a diagram of the electrochemical cell used in the flow analyzer. It consists of a 20-mL container with a water jacket for temperature control. The response time of the pH control system is proportional to the cell volume. Due to mechanical restraints. such as the size and location of the coulometric electrodes, we adopted a 20-mL volume, which is much less than the volume of our previous design 150 mL), for the sake of operational convenience. It should be possible to reduce this to less than 10 mL, thereby improving the sensitivity and the response time. A Beckman 39505 (Irvine, Calif.) combination glass electrode was used as the pH sensing element. A platinum electrode was used as the solution ground. The platinum generating electrode had an area of about 0.15 cm2, The auxiliary generating compartment consists of a silver/silver chloride wire in saturated sodium chloride. It was separated from the sample solution by a “thirsty glass” thimble (Corning tbpe 7930 porous glass. Corning, N.Y.). This thimble provided a low impedence path (60 Q ) between the two generating electrode:;. Current and voltage measurements were made with a 4l/, digit Newport Model 2000 BS (Santa Ana, Calif.). digital voltmeter. An analog trace of the signal was provided by a Iinear Instruments 255 (Pasadena, Calif.) strip chart reccrder. Reagents. All solutions were prepared from freshly boiled deionized water. Aqueous urea stanmdards were prepared by dilution of a 0.1 M stock solution which contained 0.6 g of urea (Mallinckrodt. Analytical Reagent, St. Louis, Mo.1 in 100 mL of an appropriate electrolyte (vide infra). Seven percent by weight bovine serum albumin (BSA)4.970sodium chloride was prepared by diluting 32 mL of 22% BSA (Miles Laboratory, Elkhart, Ind., Lot No. 1 2 ) to 100 mL with deionized water and adding 0.9 g of sodium chloride (Baker. Analyzed. Phillipsburg, N.J.). A stock solution of urea in 7 7~BSA-0.970 NaCl was prepared by adding 0.06 g of urea to 50 mL of 7 % BS4 -0.9% NaCl solution (0.02 >I

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978

urea). Ammonium carbonate (Baker, Analyzed) was prepared as a 0.02 M solution. Versatol, Versatol-A, Versatol-A-Alternate (General Diagnostics, Morris Plains, N.J.) and Calibrate 1,2,3 (General Diagnostic) were prepared as per the label instructions. Human serum samples were obtained from Saint Mary’s Hospital (Athens, Ga.) and refrigerated until use. In all but some preliminary experiments the flow electrolyte was composed of 0.2 M sodium perchlorate (Fisher Purified, Pittsburgh, Pa.), 0.1 mM disodium ethylenediamine tetraacetate (Baker, Analyzed), 0.1 mM sodium dihydrogen phosphate (MC/B Manufacturing Chemists, Norwood, Ohio), and 5 mM 1,3-diaminopropane (Fluka, Hauppauge, N.Y.). Sodium perchlorate was purified by making a concentrated solution (about 4 M) adding an excess of concentrated sodium hydroxide, to remove heavy metals, and finally filtering. EDTA, phosphate, and diaminopropane were added and the pH was adjusted to the final value after dilution to the desired concentration. A series of preliminary experiments were conducted with a variety of substitutes for 1,3-diaminopropane in the flow electrolyte (vide infra). The following substitutes were tested at a concentration of 5 mM unless otherwise indicated: arginine, lysine, guanidine hydrochloride, and glycinamide from Sigma Chemical Co. (St. Louis, Mo.); butylamine, sodium acetate, sodium propionate, hexamethylene tetraamine, and acetone from Baker; 1,6-diaminohexane from Eastman, and polyethylene imine from Dow (PEI-12, a t 0.1% by weight.) Column Packing Materials and Enzyme Immobilization. Various solid carriers were tested including: controlled pore glass (CPG-10, 550-A pore size, 200-400 mesh, Electronucleonics, Inc., Fairfield, N.J.), Glycophase-G (250-A pore size, 100-200 mesh, Corning Biological Division, Medfield, Mass.), and nonionic cellulose (Bio-Rad Laboratories, Richmond, Calif.). In addition, a Nylon tubular reactor containing immobilized urease, which was graciously supplied by Miles Laboratories, Slough, U.K., was also tested. A y-aminopropyltriethoxy silane derivative of CPG-10 was prepared by the toluene-reflux method (38) and by the acetone solution method of Robinson (39). The method of Robinson et al. yields a product glass containing 80-90 pmol of amino groups per gram of glass, as opposed to the 30-35 pmol per gram obtained by the toluene-reflux method. The acetone silanization method which was used in this work ultimately leads to an increased yield of enzyme activity and greater specific activity. Urease was immobilized on the y-aminopropyltriethoxy silane derivatized CPG-10 using a glutaraldehyde linkage. The immobilization proceeds as follows: 5% by volume glutaraldehyde is prepared by diluting a 25% solution of glutaraldehyde (Sigma) with sodium dihydrogen phosphate (Baker, Analyzed) buffer (0.1 M, pH 7.0) to provide 100 mL of solution, 1.0 g of silanized CPG-10 is covered with the glutaraldehyde solution and reacted under vacuum (aspirator) for 1.0 h at room temperature; the mixture is allowed to stand at atmospheric pressure for 2 more hours; the glass is washed five times with the phosphate buffer, five times with 0.5 M NaCl (Baker, Analyzed) and five times with phosphate buffer; 1 mL of urease (Sigma Type IV, 10.7 IU/mg) in the above buffer at a concentration of 100 mg/mL is added to 1 g of the derivatized glass, a vacuum applied for 5 min with swirling, and then refrigerated ( 5 “C) for 18 h. The glass is then washed as indicated above to remove unbound enzyme. A hydrophobic glass was obtained by treating the glass with SC-87 (Pierce Chemical Co., Rockford, Ill.) according to the manufacturers’ directions. Procedures, Aqueous urea samples were injected via a 120-pL sample loop through the immobilized urease column with no dilution of the sample. The aqueous solutions required no blank correction. BSA-urea, reference serum, and human serum samples were injected with a 260-pL sample loop after diluting the samples by a factor of four with the flow electrolyte. Blanks were obtained by directly injecting 260 pL of sample into the electrochemical cell with either a 500-pL syringe or through an inactive urease column (E, in Figure 2) via a sampling valve. The characterization of the immobilized enzyme was accomplished with an immobilized enzyme stirrer (40) which consisted of a “star-shaped” magnetic stirrer which contained 5 mg of the glass immobilized enzyme.

RESULTS Initial experiments with a flow electrolyte (containing only 0.2 M sodium perchlorate, 0.1 m M phosphate, and 0.1 mM

0.8

1

T

4.0

0.5

I

1.0

1.5

2.0

1

T

(mini

0.5

1.0 [mini

1.5

4.0

T

0.5

1.0 (mini

1.5 (rnin,

Current-time curves from the coulometric analyzer in preliminary experiments. (A) Urea injected through column: 120 pL of 10 mM urea; flow electrolyte, 0.2 M sodium perchlorate; 0.1 mM EDTA, 0.1 mM sodium dihydrogen phosphate; flow rate 3 mL/min; peak width at half height 7 mL. (B) Ammonium carbonate injected through column: 120 pL of 10 mM ammonium carbonate; same flow rate and electrolyte as (A); peak width at half height 0.7 mL. (C) Peak shape obtained with some of the additives used in the adsorption studies (see text). (D) Ammonium carbonate injected directly into the pH-stat cell: 120 pL of 10 mM ammonium carbonate; electrolyte 0.2 M sodium perchlorate, 5 mM 1,3diaminopropane,0.1 mM EDTA; 0.1 mM sodium dihydrogen phosphate. (E) Urea injected through column: 120 pL of 10 mM urea; same electrolyte as (D); flow rate 1.6 mL/min; peak width at half height 0.5 mL. (F) Ammonium carbonate injected through column: 120 pL of 10 mM ammonium carbonate: same electrolyte and flow rate as (E); peak width at half height 1.0 mL Figure 4.

EDTA) were quite surprising. The current-time curves generated by the pH-stat upon injection of 120 pL of urea were very irreproducible, quite broad (7 m L a t half height), and so highly skewed that baseline recovery was not obtained for as long as 5 min. This behavior should be contrasted with t h e narrower peaks of t h e thermometric and refractometric detectors (23). Although these flow systems were geometrically very similar to those employed in the present work, peak half-widths were typically only 0.3 mL and not badly tailed. We attribute these phenomena to the differences in solution conditions between the present system and the other systems which generally contained phosphate a t high concentration (0.5 M). An increase in the sodium perchlorate concentration from 0.2 to 0.5 M had no appreciable effect on the results. Figure 4 gives a qualitative indication of the results obtained under a variety of conditions. Injection of a n equivalent amount of ammonium carbonate gave a more symmetric and narrower peak which was still relatively broad compared to t h e thermometric analyzer. Papariello and co-workers observed a similar phenomenon in their analyzer which employed immobilized penicillinase (27). We felt t h a t urea and its reaction products were adsorbing to the immobilized enzyme and/or glass support. A series of experiments were conducted to assess the effect of the nature of the support on peak shape. T h e results of these studies are detailed in Table I. In all cases ammonium carbonate was the test species and, with the exception of the glass derivative coated with a hydrophobic

ANALYTICAL CHEMISTRY, VOL. 50, NO. 7 , JUNE 1978

e

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Table I. Investigation of Column Packing Materials and Their Effect on Peak Shape and Half Width Type of sample Peak width at Relation to Figure 4 Column packing injected" half height, mL A or B (",),CO, (",),CO, SC-87 derivatized glass (",),CO, DEAE cellulose (NH,),CO, (",),CO, Nylon-tube immobilized urease Nylon-tube immobilized urease Urea " The indicated sample was present at a concentration of 1 0 mM. y-Aminopropyltriethoxysilane glass Glycophase G glass

Table 11. Lineweaver-Burke Plots" and pH Profile of Immobilized Ureaseb Steady state current, PHC Enzyme units mAd 6.2 0.40 2.0 6.4 0.50 2.5 6.6 0.56 2.8 3.0 6.7 0.60 6.8 0.62 3.1 6.9 0.54 2.7 7.0 0.48 2.4 7.2 0.28 1.4 7.4 0.18 0.9 " Lineweaver-Burke Analysis: Without diaminopropane: yintercept = 1 . 7 1 (min/mM) t 0.093, slope = 95.9 (min) t 0.6, correlation coefficient = 0.999 with Student's t = 99.7. With 5 mM diaminopropane: yintercept= 1.40 (minimM) t 0.066, slope = 6 4 . 1 (min) t 0.6, correlation coefficient = 0,999 with Student's t = 99.7. Performed at 25 'C in 0.2 M sodium perchlorate, 5 mM 1,3-diaminopropane, 0.1 mM EDTA, 0.1 mM sodium dihydrogen phosphate, and 0.05 M urea with an immobilized enzyme stirrer ( 4 0 ) containing 5 mg of glass. The pH was varied by manually adjusting the pH-set point dial of the pH-stat. Measured after letting the cell current decay to its steady state value. material (SC-87), the peak half-widths were considerably greater than those obtained with the thermometric detector. Since the glass could be chemically modified to prevent t h e problem, we postulated that it should be possible to add some material to t h e electrolyte which would block adsorption of urea and its reaction products. Ideally, such a material must have no buffering action, not be hydrolyzed by urease, and not overly inhibit the catalytic action of the enzyme. Of t h e materials listed above only 1,3-diaminopropane fulfilled all of these criteria. Guanidine hydrochloride, sodium propionate, sodium acetate, and acetone gave the double peaks shown in Figure 4, curve C; hexamethylene tetramine and polyethylene imine had very high buffer capacities and may have inhibited the enzyme; glycinamide spontaneously hydrolyzed to glycine and ammonia; t h e remaining materials had very little effect. Only 1,3-diaminopropane improved the system behavior to t h a t shown in Figure 4, curve E (half-width 0.5 mL). T h e concentration of this diamine is very important: 1 mM had almost no effect and 10 mM showed smaller peaks (thereby indicating some inhibition of the enzyme) which were not appreciably narrower than with 5 m M solutions. The immobilized enzyme preparation was characterized by use of the immobilized enzyme stirrer configuration of Guilbault and Stokbro (40). These results are summarized in Table I1 which indicates a p H optimum a t 6.8 in the presence of diaminopropane, and the linear Lineweaver-Burke plots (not shown) indicate t h a t diaminopropane is a competitive inhibitor with a K , of 79 p M . These same plots also show t h a t the Michaelis constant of immobilized urease, in t h e absence of diaminopropane, is 43 m M and t h e specific

0.8 0.6 0.3 0.6 0.6 5.0

N o improvement

Some improvement Very much improved Some improvement Some improvement Some improvement

25

5c

5

URtA C O N C ~ N l R I 1 l l O I [PI ~

Figure 5. Calibration curve with aqueous urea standards. All results were obtained using 120 pL of urea at the indicated concentration. The flow electrolyte was 0.2 M sodium perchlorate-0.1 mM EDTA-0.1 mM phosphate-5 mM 1,3-diaminopropaneat pH 6.8. Flow rate was 1.6

mL/min. The intercept of the data up to 15 mM is negligible. The least squares slope and standard deviation were 1.41 f 0.02 C/mM

activity obtained from measurement of V,, is 100+1200 units per gram of wet glass. The above K , is in good agreement with other measurements for this enzyme in these laboratories (23). Although 5 mM diaminopropane does in fact inhibit the enzyme, it is not so effective as to prevent complete hydrolysis of reasonable amounts of urea. A calibration curve, Le., plot of coulombs vs. concentration of aqueous urea standards, is shown in Figure 5. These results were obtained with a column packed with 0.2 mL of urease immobilized on CPG-10 glass derivatized by y-aminosilanization and glutaraldehyde cross-linking. The front and back of the 0.5-mL column was packed with SC-87 derivatized nonporous glass (80-120 mesh). This figure conservatively indicates a lower limit of detection of 0.05 m M (41). Above about 15 m M the calibration curve becomes distinctly nonlinear. The nonlinearity is due to incomplete conversion. This was shown by injecting 50 m M urea, collecting t h e column effluent, diluting it to a final volume of 5 mL, and reinjecting. Previous work (23) with 0.50-mL columns of urease and the thermometric detector indicated complete conversion u p to 200 m M urea. We attribute the early onset of nonlinearity to three differences: First, a smaller amount of enzyme (0.2 mL vs. 0.5 mL) was used, but this can account for at most a factor of 2.5; second, there is an inhibitor present; finally, the enzymatic reaction takes place under conditions which are more basic that the p H optimum of the enzyme due to t h e low buffer capacity of t h e flow electrolyte. This represents a distinct disaduantage to this approach and t o any approach wherein p H changes are measured in enzymatic reactions. A similar effect related to low buffer capacity was observed with a flow analyzer for penicillin based on direct observation of the p H change (27). The data given in Table

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978

Table 111. Precision and Fractional Conversion of Aqueous Urea Standards Coefficient of Percent Coulombs variation converConcn,mMa x lo’ (%)b sionC 85d 0.104 1.25 4.0 0.522 7.4 3.6 100 1.04 15.2 103 2.7 5.22 96.2 70.8 1.8 99.9 146.9 10.43 1.0 12.51 0.78 100 176.4 103 15.07 0.80 219.5 98.6 20.86 0.83 290.0 46.9 52.22 1.2 345.1 104.3 26.6 391.7 1.3 Data were obtained under the following conditions: flow rate 1.6 mL/min; electrolyte 0.2 M sodium perchlorate, 5 mM 1,3-diaminopropane, 0.1 mM EDTA, 0.1 mM phosphate; temperature 25 “C; pH 6.8; 120-pL samples were injected into the column. Relative standard deviation of triplicate runs. % conversion E 100 (coulombs found)/(coulombs expected). This value is probably due to a poor current efficiency at low concentration and the difficulty in blank correction.

TIME 1111ni

Figure 7. Peak shape as a function of sample volume, plot of current vs. time upon injection of indicated volume of urea into the reaction column. The time of injection is shown by the arrow. A 6 mM sample of urea was injected into the electrolyte whose flow rate was 3.0 mL/min. All other conditions are the same as Figure 6. (A) 260-pL sample, (6) 2.5-mL sample, (C) 3.5-mL sample

I

5.0

I

IO

2 0

30

40

SAMPLt VOLJMl lmli

Figure 8. Peak half width as a function of sample volume. All conditions are identical to those of Figure 7 I

IO

20

30

40

53

60

7 6

I

FLOK. RAT[ In1 m i l l

Figure 6. Dependence of peak current and area on flow rate. A 120-pL sample of 6 mM urea was injected at the indicated flow rate. All other conditions are as given in Figure 6. (W) Peak height (rnv), ( 0 )peak area

111show that quite precise results can be obtained even when conversion is not complete, and that in the range of normal serum urea concentration we can obtain a precision of 1-2% for a 1 2 0 - ~ Lsample. T h e precision of the results a t low concentration (0.1 mM) indicates that the flow system does not have much more noise than the nonflow system (34). T h e slope of the calibration curve corresponds to 1.22 f 0.02 mol of base generated per mole of urea injected, in good agreement with the data of Figure 1 which were calculated based on literature values of the ionization constants. This also indicates that conversion of low concentrations of urea is complete and very little ammonium carbamate (35) is formed under our experimental conditions. The major difference between a concentration sensitive and a mass flow detector is related to the variation in peak height and peak area with flow rate (42). As shown in Figure 6, the peak area, i.e., coulombs needed to neutralize the column effluent, is independent of flow rate up to about 4 mL/min while the peak height is a linear function of flow rate. This is in agreement with the expected behavior of a mass flow detector and contrasts with previous results with concentration sensitive detectors. At a flow rate of 4 mL/min, the area under the peak decreases and the peak height is no longer a linear function of flow rate. This is attributed to both incomplete conversion of urea to products in the enzyme reactor and to removal of some of the products from the electrolysis cell before they are completely neutralized by the pH-stat. The

sweep-out effect would not be improved by a decrease in cell volume since both the electrolysis time constant and the “flush-out” time constant are proportional to cell volume. We believe that the principal effect is incomplete reaction of the 6 mM sample in the enzyme column since the calibration curve and other experiments indicate t h a t incomplete conversion (vide supra) occurs a t concentrations in excess of 15 m M a t a flow rate of only 1.6 mL/min. The effect of sample volume on the flow reactor is shown in Figures 7 and 8. Up to a limit of about 260 FL the peak half-width is essentially independent of the injected volume and ultimately becomes a linear function of the volume injected. This experiment showed that considerable urea was present in the column effluent when concentrations greater than 15 m M were injected. As given in Figure 7, the peak height will increase continuously with sample volume until a flat-topped peak is obtained, indicating that that sample completely fills the column. This occurs at a sample volume of about 1.0 mL with the present system. This is in qualitative agreement both with our previous observations (23) and with a peak width model based on Sternberg’s calculations on the effect of extra column factors on peak broadening (43). This study also indicates t h a t the sample response time (through-put) would not be improved by use of sample volumes of less than 260 pL. The analytical sensitivity (slope of the calibration curve) would be decreased. For this reason we inject samples of 100-250 FL. As shown in Figure 8, the limiting peak half-width a t low sample volume (