Waste water leaving the purification plant in Henriksdal near Stockholm was analyzed April 24, 1968. The sediment and the water were analyzed separately as described above. The results, given in Table 11, show that the water itself was almost free from residues in relation t o the amount found in the aluminium sulfate sediment, indicating the good effect if the plant used the aluminium sulfate precipitation in the cleaning procedure. The weight of sediment plus aluminium hydroxide was 265 mg per liter of water. A sample of 10 liters of waste water taken April 16 was impossible to analyze because serious artifacts were present. The amount of the artifacts was so large that the 10-ml extract from the water had t o be diluted 100 times to give peaks with the same height as 0.1 ng of aldrin. I n the same way the extract from one-fifth or 0.6 gram of the solid had to be diluted t o 1500 ml t o give the peak height corresponding to 0.1 ng of aldrin. The artifacts gave three peaks, (see Figure 2, peaks X,Y , a n d Z ) with the last peak having a retention time almost equal to aldrin on the S F 96 column and exactly equal to lindaneontheQF 1column. Anacetonitrilln-hexanepartition (7)
followed by elution with hexane from a silica gel column did not remove the artifacts from the chlorinated hydrocarbon fraction. When treating the extract with potassium hydroxide in methanol, the artifacts disappeared. Analysis of highly concentrated extract on a combined gas chromatograph-mass spectrometer (LKB 7 000) did not give any result. When this extract was cooled in carbon dioxide ice, crystals appeared which could be analyzed on the mass spectrometer (aia the direct inlet) and shown to be elemental sulfur as Ss molecules. Finally the three artifact peaks appeared on the gas chromatograph when a sulfur solution was injected giving proof that sulfur was responsible for these peaks.
(7) R. W. Storrherr and P. A. Mills, J. Ass. Ofice Agr. Chemists, 43, 81 (1966).
RECEIVED for review March 9, 1970. Accepted July 10, 1970.
ACKNOWLEDGMENT
The authors gratefully acknowledge the help of Professor G. Widmark, the head of the analytical institute at the University of Stockholm, and civil engineer B. Nucci.
Variable Selectivity Stationary Phases for Gas Chromatography Raymond Annino and P. F. McCrea Research Center, The Foxboro Company, Foxboro, Mass.
The performance of a new class of chromatographic stationary phases is described. Their selectivity can be manipulated over a wide range merely by changing the column temperature. By combining substances differing in melting point and solute selectivity, a stationary phase mixture is produced exhibiting an extended range of melting. Since both the effective volume and composition of the melt are strongly temperature dependent, absolute and relative retention volumes of the solutes are altered markedly by temperature over the transition region, resulting in varying degrees of specificity through choice of the appropriate column temperature. Positive relative retention shifts on the order of 100% have been observed with a 20 O C increase in temperature. It is shown that the ability of the novel stationary phase to identify various classes of solutes by a retention index method i s as good as the two-column method, and possesses the additional advantage that its specificity can be varied at will to obtain the best resolution of a complex sample.
A NUMBER OF gas chromatographic procedures have been devised which owe their success to the use of two different stationary phases, either singly or in combination. For example, a widely used technique for solute identification has been the determination of retention behavior of the unknown sample on two columns of different selectivity ( I , 2). ~~
(1) D. A. Leathard and B. C. Shurlock, in “Progress in Gas Chro-
matography,” J. H. Purnell, Ed., Interscience, New York, N. Y., 1968, pp 18,23. (2) G. Schomburg, in “Advances in Chromatography,” Vol. 6, J. C. Giddings and R. A. Keller, Ed., M. Dekker, New York, N. Y., 1968, p 211. 1486
F o r quantitative analysis, the separation of complex mixtures has been accomplished by combining different stationary phases either as series columns o r in a single column (3-5). All of these methods reflect a need for a stationary phase whose selectivity can be varied over a large range by manipulating some convenient parameter such as the column temperature. A recently reported method for peak identification (6) relies o n variations in the temperature dependences of the Kovats Indices (7) for various classes of solutes. These changes in selectivity with temperature, however, are relatively small for conventional stationary phases. This paper describes a class of stationary phases which undergo reversible temperature-dependent compositional changes to produce large variations in selectivity for various classes of solutes. These systems are not to be confused with the use of liquid crystals (8) o r conventional liquid sorbents operated near their freezing point (9), where alterations in selectivity for different solutes can be attributed primarily to a change in the physical state of the solvent. (3) R. A. Keller and G. H. Stewart, ANAL.CHEM., 36, 1186 (1964). (4) G. P. Hildebrand and C. N. Reilley, ibid., p 47. ( 5 ) A. B. Littlewood and F. W. Willmott, ibid., 38, 1031 (1966). (6) N. C. Saha and G. D. Mitra, J. Chromarogr. Sci., 8, 84 (1970). (7) E. Kovats, in “Advances in Chromatography,” Vol. 1, J. C. Giddings and R. A. Keller, Ed., M. Dekker, New York, N. Y., 1965. (8) H. Kelker and E. Von Schivizhoffen, in “Advances in Chromatography,” Vol. 6 , J. C. Giddings and R. A. Keller, Ed., M. Dekker, New York, N. Y., 1968, p 247. (9) R. Claeys and H. Freund, J. Gas Chromatogr., 6 , 421 (1968).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
THEORY
Stationary phase mixtures which possess a n extended range o f melting exhibit marked increases in solute retention as the temperature is raised through the transition region (IO). This behavior is a result of the transition from gas-solid adsorption to true partitioning and the subsequent increase in the volume of the partitioning medium with increasing temperature (11). One practical application of this general type of system, which is called a transition sorbent, has been recently described b y one of us (12) and utilizes the transitionregion thermal behavior of the multicomponent stationary phase t o achieve temperature-independent solute retention over spans of about 10 "C. The components of these stationary phases were chosen to possess similar solute selectivity and widely different thermal characteristics. F o r the present investigation, the stationary phase components were deliberately chosen to have not only a substantial thaw-melt region, but also a significant difference in solute selectivity. To illustrate the mechanism which results in variable solvent composition, Figure 1 depicts a hypothetical temperatureComposition phase diagram of substances A and B, in the form of a closed solid solution loop. F o r a mixture comprising X,mole % B in A , temperatures below TI will result in complete solidification of the melt. At TI the mixture begins to melt, yielding liquid with a composition corresponding to XI. Thus, at this temperature, the melt is very concentrated with respect to component B. At successively higher temperatures, more liquid is generated and its composition corresponds to the intersection of the Taxis with the liquidus curve. When temperature Tr is attained, all of the solid has melted to yield a liquid which has the original composition X2. Therefore, if the high-melting substance A possesses greater sorbing power for polar o r easily polarizable solutes than substance B, then a column which utilized a mixture of A and B as the stationary phase would, when the temperature was traversed between TI and T,, be expected t o exhibit solute selectivities commensurate with stationary phase mixtures ranging in composition from XIto X,. A consequence of the variation of liquid composition with temperature is the fact that the effective volume of liquid in the column is also a function of temperature over the transition range of the solvent mixture. At temperatures below TI, there is no liquid available for partitioning, so the primary retention mechanism is that of adsorption o n the solid organic surface. With increasing temperature, gas-liquid partitioning rapidly becomes dominant and a general increase in retention for all solutes is observed. AS the temperature approaches T2,positive retention shifts continue to occur because of both the increasing volume of the liquid phase and its changing composition. At temperatures above T2, the composition and volume of the stationary phase is constant, and solute retention assumes a normal GLC temperature dependence. The main effect of low liquid volume at temperatures near TI is the reduction of the effective range of stationary phase compositions which will yield satisfactory column performance. However, experience shows that this is not a serious limitation. EXPERIMENTAL
The thermal behavior of the pure and mixed stationary phases was determined with a Du Pont 900 Thermal Analyzer (10) J. H. Purnell, S. P. Wasik, and R. S. Juvet, Acta Chim. Acad. Sci. H m g . . 50, 201 (1966). (11) P. F. McCrea, Ph.D. Thesis, University of Wales, 1968, p 3. (12) P. F. McCrea and J. H. Purnell, ANAL. CHEM.,41, 1922 (1969).
t
I
0
I
I
/
XZ
I
I / I XI
100
MOLE % B IN A
Figure 1. Hypothetical solid solution phase diagram of substances A and B
and the Standard DTA cell, using dry helium as an inert atmosphere. The transition temperatures of all samples, including the organic standards used to verify the accuracy (+0.2 "C) of the temperature axis, were measured with the samples coated a t a 20 w/w loading o n the chromatographic support. After brief heating to a temperature above the sample melting range, all endothermal peak maxima were reproducible t o within 1 0 . 1 "C. Reduction of the rate of heating from the value of 10 "C/min normally used did not result in any shift in transition temperature. The chromatograph used was a n F & M Model 810R-12 with a thermal conductivity detector. The isothermal oven control circuitry was modified to allow setting the column oven temperature to within 1 0 . 1 "C. Subsequent measurements indicated stability to within ~ t 0 . 0 5"C. The injection port was maintained a t 200 "C and the detector oven temperature was 140 "C. It was experimentally verified that the temperature of the carrier gas was at the column temperature before entering the column, and that the maximum temperature gradient across the column was less than 0.1 "C. Column temperatures were potentiometrically measured by a calibrated copper-constantan thermocouple referenced to a distilled water ice bath. Carrier and reference helium flow rates were maintained by separate Brooks Model 8743 flow controllers. The stationary phases, stearic acid (octadecanoic acid) mp 69-70 "C, and 1,9-nonanedioic acid m p 105-107 "C, were obtained from Matheson Coleman & Bell and were used without further purification. Extrapolated literature vapor and pressure data (13) for the bulk liquids are 1.3 X 7 X IO-$ Torr at 100 "C for stearic acid and nonanedioic acid, respectively. The solid support, 80-100 mesh GC-22 (Coast Engineering Laboratory) was fluidized in dry air at 120 " C to remove most of the "fines" and moisture. After further drying a t 0.1 Torr, the support was coated with the stationary phase in a rotary evaporator using 90-120 "C ligroine as the slurry medium. During this procedure, the bath temperature was maintained above the stationary phase liquefaction temperature. No difference could be detected, either by thermal analysis o r by chromatographic measurements, between coating the solid support with transition sorbents which were prefused t o a homogeneous mixture, and coating with those which were not. The normal solvent weight loading was 20% by weight. Solvent-support ratios were determined as previously reported (12). All columns were 230 cm long except the stearic acid column which had a packed length of 1 meter. The 2-mm i.d. copper columns were packed straight with vertical tapping and then coiled to a 15-cm diameter. Sintered stainless steel plugs were used to retain the packing at the (13) T. Earl Jordan, "Vapor Pressure of Organic Compounds," Interscience, New York, N. Y.,1954, p 119.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
1487
z 0 I--
5 9 z-
a w
[LE
I-3
+
Ja
aa I W
aL W I I W
E+ z
\
W
I
20 MOLE % S IN N.
5 5 c
5 0
10
20
30 40 0 SO 60
70
80
90 uI
MOLE % STEARIC
ACID IN NONANEDIOIC ACID
Figure 2. Temperature-composition phase diagram for mixtures of stearic acid and nonanedioic acid (coated 20% w/won chromatographic support)
column outlet, and twisted glass fiber was utilized a t the inlet. All columns were conditioned a minimum of 12 hours a t a temperature a t least 15 "C above the melting range of the stationary phase. Sample introduction was made with a Hamilton 7001-N 1-p1 syringe and the maximum total sample size was 0.1 pl for a 5-component mixture. Air injected with the solutes served as a measure of the column dead volume. The usual corrections for temperature, soap bubble vapor pressure, and carrier gas compressibility were made to the measured flow rate. Solute retentions were measured from the air peak on the chart and converted t o time units with a knowledge of the true chart speed. F o r the determination of retention indices, injections of each class of solute-e.g., alcohols, aromatics, etc.-were alternated with injections of the n-alkane standards. The Kovats Indices of the solutes were obtained by first determining the best straight line fit, for multiple runs, of log retention volume for the n-alkanes as a function of 100'carbon number, using a linear regression computer program. Then, using the equation of this line, Kovats Indices for the solutes were computed from their retention volumes. Measurements were found to be reproducible t o within i l index unit from week to week. RESULTS AND DISCUSSION Thermal Analysis. Preliminary experiments involving the examination of a wide variety of potential stationary phases by D T A indicated that a mixture comprising mono- and dicarboxylic acids should have the required extended transition region as well as the necessary difference in selectivity. Stearic acid was selected as the slightly "polar" component of the mixture as its retention characteristics were well known (12). Nonanedioic acid was chosen as the relatively "high polarity" component on the basis of the favorable AI values shown later in Table I, as well as on the resulting temperaturecomposition phase diagram which is shown in Figure 2. Due to dilution effects and experimental limitations, low concentrations of stearic acid (less than 5 mole S in N ) ( S and N denote stearic acid and nonanedioic acid, respectively) and low concentrations of nonanedioic acid (greater than 80 mole % S in N ) result in poorly defined phase transitions a t the solidus and liquidus, respectively. This offers no practical difficulty, however, as the region of prime interest 1488
/ N. Y
IO MOLE % S IN
I \PURE io0
110
I
2.60
Ib
N
I
I
265
2.70
20 90
80
70
I
I
I
TEMP. ('C) I I 2.75
2 80
60 I
I I
I
1
2.85
2.90
2.95
3.00
IOYT (OK-') Figure 3. Log V,T (ml) as a function of T-l for elution of benzene on columns containing 0, 10, 20, 40, and 100 mole % stearic acid in nonanedioic acid
for this application lies in the range of 5 t o 80 mole % S in N. Examination of Figure 2 will show that a solid mixture of 80 mole S in Nmelts over a comparatively narrow temperature interval t o yield that same composition of liquid, whereas a 10 mole % solid mixture has a transition region from 62 to 102 "C,yielding liquid state compositions varying between 80 and 10 mole S in N . CHROMATOGRAPHIC RESULTS
The reproducibility of retention data obtained on the pure solvent columns was improved when the operating temperature was approached from below. This could be due to either a tendency of these stationary phases to supercool or to more rapid oven equilibration when operating in this manner. Therefore, this procedure was adopted for all measurements. Benzene retention behavior with increasing temperature on each of the pure solvent columns and on columns containing 10,20, and 40 mole S in N is given in Figure 3. The initial retention increase a t a temperature of 60 to 62 "C was predicted by the phase diagram and is a result of the onset of melting of the mixed stationary phase and the subsequent transition from gas-solid adsorption to gas-liquid partitioning. At elevated temperatures in the region of 85 to 93 "C, the rate of melting of the mixed stationary phases increases until, a t even higher temperatures, retention maxima occur a t the respective temperatures of complete liquefaction of each stationary phase. These latter transitions lie on the liquidus curve of Figure 2. Higher temperatures result in a normal temperature dependence of retention similar to that of the stearic acid column which is 100% liquid throughout the temperature range. It is of interest to note that the region from 90 t o 102 "C for the 10 mole %column has the greatest retention shift. Examination of the phase diagram indicates that it is this region
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
5.0 4.0
3.0 60 2.0
50
5
40
a 1.0 .9 -8
.7
1
2 IO
ill0
I
; 1
lO;,5
,
I00
9?,
I
90
,
87
I
I 8?
,
COLUMN TEMP. "C
.6 .5
.4
TEMP. ( O C ) 2.60
2.60
2.64
2.68
2.72
IOYT
2.76
2.80
2.64
2.68
2.84
(OK-')
Figure 4. Log VDT(ml) as a function of T-' for various solutes on a 10 mole % stearic acid in nonanedioic acid column Solute identification: (1) n-hexane; (2) methyl acetate; (3) n-heptane; (4) 2-propanol; (5) methyl propyl ketone; (6) n-octane where the greatest shift of composition, and hence volume of liquid, occurs. Since a large composition shift is required to achieve a meaningful alteration in column selectivity, the 10 mole S in N column was selected for further investigation, and Figure 4 illustrates the temperature dependence of retention for a variety of solutes on this column. Here one observes one type of retention response for the normal alkanes and a much more active behavior for the more polar acetate, alcohol, and ketone. Toluene, omitted on this plot for clarity, exhibits retention response intermediate to that of the alkanes and that of the polar solutes. Even at 80 "C,where the rate of change of stationary phase composition with temperature is relatively small, one can observe significant differences in the slopes of the curves for polar and nonpolar solutes. Specific retention volumes at temperatures above 80 OC were found to be constant with decreasing sample size and independent of the solvent weight loading in the range 9 to 20 wjw. Recalling the fact that observed retention volumes are a function of the volume of stationary phase as well as its composition, a much clearer illustration of solvent selectivity toward the various solutes can be obtained by normalizing all retention volumes to that of n-heptane. This is presented in Figure 5 as a plot of log a as a function of the reciprocal temperature where a is the relative retention with respect to n-C,. Now one observes the normal linear convergence of the alkane relative retentions with increasing temperature that occurs on conventional stationary phases. The magnitude of the decreasing shift in a is about 13% over the 80 to 102.7 "C temperature region. The more polar solutes, however, exhibit a very significant shift in a over the same temperature span, amounting to increases of 175 for methyl acetate and 2-propanol, 136z for methyl propyl ketone, and 5 1 z for toluene. Although the numbers quoted above have little value on their own-i.e., they will alter radically depending on the alkane used for normalization-they have meaningful
2.72
IOYT
2.76
2.80
2.84
(OK-')
Figure 5. Log relative retention as a function of T-l for various solutes on a 10 mole % stearic acid in nonanedioic acid column Solute identification: (1) n-hexane; (2) methyl acetate; (3) n-heptane; (4) 2-propanol; (5) methyl propyl ketone; (6) n-octane, (7) toluene
z
z
z
I
I
1
I
Figure 6 . Chromatograms illustrating variable selectivity stationary phase a t temperatures corresponding to the extremes of available polarity qualitative and quantitative significance in a relative sense as they attest to large positive shifts in selectivity for polar or easily polarizable solutes with increasing temperature, in contrast to the relatively small decreases in a with increasing temperature which is the case for most conventional sorbents (6). A graphic illustration of the wide variations in specificity exhibited by this column is given by the chromatograms shown in Figure 6 which were obtained at temperatures correspond-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
*
1489
~ _ _ _ _ _
~
~~
~ _ _ _ _ _ _
~
Table I. Retention Indices of Transition Sorbent and Pure Solvent Columns
Solute Ivia3 Islo$ 4 0 b I s '103 ''0 Me Acetate 797 557 737 Et Acetate 868 637 811 Pr Acetate 964 736 907 Acetone 831 542 761 MEK 911 638 845 MPK 996 730 931 1-Hexene 620 592 616 1-Heptene 723 694 716 1-Octene 826 793 815 2-Pr Ether 736 618 709 n-Pr Ether 826 704 798 n-Bu Ether 1021 900 990 Benzene 844 691 805 Toluene 955 801 914 Et Benzene 1041 889 998 rn-Xylene 1066 909 1022 o-Xylene 1101 93 1 1052 Pr Benzene 1124 975 1081 Methanol 882 509 788 Ethanol 935 585 851 2-Propanol 960 631 881 2-Butanol 1054 734 977 %-Butanol 1078 751 1000 n-Butanol 1143 797 1059 a Nonanedioic acid column; indices extrapolated from measurements at higher temperatures. * Stearic acid column. Transition sorbent column. 40a
Table 11. Index Difference Methods for Solute Identification
Solute
6 I.'% 3 0 a
61S2pb
Me Acetate Et Acetate Pr Acetate Acetone MEK MPK 1-Hexene 1-Heptene 1-Octene 2-Pr Ether n-Pr Ether n-Bu Ether
30 28 37 35 35
13 9 9 13 9 9
5
1
5 5 19 23 18
0
15
-1 1 2
70 75 72
Benzene Toluene Et Benzene m-Xylene o-Xylene Pr Benzene
24 26 26 25 27 25
8 8 8 7 7 7
91
Methanol Ethanol 2-Propanol 2-Butanol &Butanol n-Butanol
40 24 19 22 25 29
- 12
28
1
-8 -8 -6 -6 -5
6 A I s : . v ~ ~ Ao Ic. v - S 1 0 3 . 1 ~ d
132 127 126 149 147 147 13 15
240 23 1 228 289 27 3 266 28 29 33 118 122 121
91 88 90 97 86
153 154 152 157 170 149
137 156 155 155 155 162
373 350 329 320 327 346
a Nonanedioic acid column, AT = 23 "C (extrapolated from measurements over the range 107 to 124.5 "C). Stearic acid column, AT = 103.4-80.0 "C = 23 "C. Transition sorbent column, AT = 103.0-80.0 "C = 23 "C. Index difference between stearic acid column and nonanedioic acid column at 103.4 "C.
1490
ing to the two extremes of stationary phase polarity. Fully corrected flow rate was held constant to better than 2%. It is seen that the n-octane peak has a n identical 2-minute total retention time a t both temperatures, in marked contrast to the large retention shifts of the more polar solutes. The intermediate nature of the toluene retention shift as compared to that of n-octane and 2-butanol is also very evident. Column temperatures between the limits shown on the chromatograms yield intermediate values of selectivity, thus making it possible to "dial in" any desired amount of specificity for a given solute. Theoretical plate heights for n-octane varied between 0.86 mm a t 103 "C and 1.1 mm a t 80 "C, at an outlet carrier gas velocity of 23 cmjsec. These values could be improved considerably by operating closer to the minimum of the H us. uo curve, o r by reducing the solvent weight loading to about 10 to 15% w/w. Temperatures below 80 "C produced a small additional alteration in selectivity a t the expense of steadily worsening column efficiency. The choice of the lower operating temperature will be dependent on the volume of liquid present and on its rate of generation with increasing temperature. From the isothermal tie-line at 80 "C in Figure 2, and a knowledge of the solvent molecular weights, it was estimated that 18% of the total weight of solvent in the column was in the liquid state. F o r the transition sorbent columns studied, this value implies that an effective liquid loading of 3.6% is available for partitioning at 80 "C. This small amount of liquid probably exists in a matrix of solidified sorbent with a resulting high viscosity and, hence, large resistance to mass transfer contributing to the peak band-broadening. Retention indices at 103 "C for different classes of solutes in each of the pure solvent columns and in the transition sorbent column are shown in Table I. The difference in polarity between stearic acid and nonanedioic acid is evident from their respective indices and, as expected, the indices for the 10 mole % stearic acid in nonanedioic acid column lie numerically between those of the pure solvents and close to the nonanedioic acid column data. Table I1 gives a comparison of three methods of solute identification utilizing 6 1 and AI values for the different solutes, where 61 is defined as the difference in the retention index for a given solute/solvent pair over a defined temperature interval, and A I is the retention index difference for a solute o n two different columns a t a fixed temperature. The attractiveness of the latter method is vividly shown by the AZ data from the two pure solvent columns at 103.4 "C, defined as AFv-8 103 in Table 11. All classes of solutes are easily identified by the magnitude of the index difference. Such is not the case, however, with the 61 data on the pure solvent columns, namely 6 t v 2 3 0 and 6Zs23~,where it is impossible to distinguish between acetates, ketones, and aromatic hydrocarbons on these data. Of course, larger ranges of temperature than those employed for the 6 1 data of the present investigation might result in improved solute identification, but it must be remembered that retention times are very temperature dependent and a large (50 "C) increase in temperature could result in elution times so brief that their accurate measurement becomes difficult. The index difference for the solutes on the transition sorbent column at 80 and 103 "C, defined as 6AIs:.v23~, is also given in Table 11. It can be seen from these data that the variable selectivity stationary phase possesses the excellent specificity of the dual-solvent AZ method, in combination with the operational advantages of single-column analysis, as in the 61
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
method. The transition sorbent possesses the additional advantage that its specificity can be varied at will to obtain the best resolution of a complex sample. Thus, both qualitative sample identification and quantitative analysis with good peak resolution can, in most cases, be effected on a single column. There may be a number of other stationary phase mixtures which will give the same type of behavior reported here, such as : complexes of the solvent-solvent type which are thermally or pressure dependent, immiscible solvents, one of which undergoes a phase transition, etc. It appears that this novel technique would be a very profit-
able approach to tailoring a stationary phase to obtain the maximum separating power from the various solvent-solvent and solvent-solute interactions. The potential application of this technique to liquid chromatography has not been overlooked, and work is continuing in this area.
RECEIVED for review June 8,1970. Accepted August 10,1970. Presented, in part, at the 1970 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 3, 1970.
Estimation of the Active Site Normality of Papain Andrew Williams and E. C. Lucas ChemicaI Laboratories, University of Kent, Canterbury, U . K . A new active site titrator for papain has been synthesized via a cheap and efficient route. The substrate, p-nitrophenyl-a-N-CB,-D-norleucinate, is prepared by resolving N-CB,-D,L-nOt’leUCine using (-) ephedrine and coupling the D-acid with p-nitrophenol using dicyclohexylcarbodiimide. The titrant is shown to fulfill all the theoretical criteria necessary for a rigorous titration and estimate active site normalities accurately to less than 0.2 pM concentration. A low maximal deacylation rate constant allows titration of the enzyme over a pH range including the pH optimum for papain. THEACCURATE ESTIMATION of active site normality is essential to reliable studies of enzymes related to mechanism and involving kinetics. Enzyme molarity is usually determined by a rate assay using a suitable substrate but this technique suffers from the necessity of standardizing the rate assay by estimating the molarity of the protein. Such a procedure is suspect for obvious reasons ( I , 2 ) and a knowledge of the number of active sites per molecule is needed before normality can be calculated. A modern technique of protease assay utilizes the special mechanistic pathway of some of the enzymes where a n acylenzyme is formed which deacylates to regenerate active enzyme (Equation 1).
RCOX
+ EH RCOE
kl
E-S
-%
RCOE
k- 1
RCOzH
+ EH
+ HX (1)
Provided k a > ka, [Elo can be obtained from a plot of l / d ius. l/[Sl0. a is readily determined as it is the intercept (at zero time) of a plot of the concentration of leaving group expelled against time. This technique is called a “burst” or “titration” assay and has been applied by Bender (3) to a number of ester splitting enzymes. Although titrating agents exist for papain (3-9, this enzyme (and sulfhydryl proteases in general) suffers from a lack of convenient and sensitive assay procedures suitable for accurate work. We report in this communication a new substrate titrator for papain which is more versatile than existing agents. EXPERIMENTAL
Materials. Papain (EC 3.4.4.10) was prepared from the solidified papaya sap via the method of Kimmel and Smith (6) and was recrystallized twice. N-CB,-DL-norleucine(I). D,L-norleucine ( 1 3 grams) and sodium hydroxide (4 grams) were dissolved in 25 ml of water and the solution was cooled to 5 “C. A solution of sodium hydroxide (4.5 grams in 25 ml of water) and benzyl chloroformate (14.2 ml) were added simultaneously, dropwise, with stirring and cooling. After addition was complete, the solution was stirred at room temperature till there was no longer any smell of the chloride. The solution was cooled in ice, acidified with 10N HCl, and after it was allowed to stand for 15 min, the precipitate was filtered, washed with water, and dried in vacuo over KOH pellets. It was recrystallized from methanol/water. Yield, 80 of theoretical. N-CB,-D-norleucine. Ten grams of the D,L-acid and 3.3 grams (-)ephedrine were dissolved in 50 ml of hot ethyl(3) M. L. Bender, M. L. BeguC-Cantbn, R. L. Blakely, L. J. Brubacher, J. Feder, C . R. Gunter, F. J. Ktzdy, J. V. Killheffer, T. H. Marshall, C . G. Miller, R. W. Roeske, and J. K. Stoops, J. Amer. Chem. SOC.,88, 5890 (1966). (4) J. de Jersey, M. T. C . Runnegar, and B. Zerner, Biochem. Biophys. Res. Commun.,25, 383 (1966). ( 5 ) J. de Jersey and B. Zerner, Biochemistry, 8, 1967 (1969). (6) J. R. Kimmel and E. L. Smith, J . Biol. Chem., 207,515 (1954).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
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