Table 11.
Interferences in Monitoring of UDMH
Response relative to UDMH AIIC __ 423
AIlC, Compound pa./p.p.m. X 100% Ethylene 0.57 +0.14 Isobutylene 3.19 +0.75 Nitrogen dioxide -0.33 -0.078 At equal concentrations, the response to nitrogen dioxide is over 1000 times less than that of UDhIH.
Stoichiometry in Batch Titrations.
T o confirm t h e apparent n % 8 observed in t h e continuous titrations, a series of variable current coulometric titrations of batch samples was performed. T h e circuit’, with generation current, integration, is shown in Figure 6. Titration curves at p H 7.0, 8.0, and 9.0 were first obtained and the apparent equivalence potentials found. Then control potentials corresponding to various bromine concentrations were chosen in the vicinity of the equivalence potential. These results are shown in Table I. Response Time, Noise Level, Drift,
and Amplifier Gain. T h e response time to incoming samples is constant throughout the pH range 7.0 t o 9.0 for t h e design center first’ order lag, approximately 1 minute to 90% of full scale. T h e response time is lengthened by using control potentials
greater t h a n about 100 mv. above the equivalence potential. At the equivalence potential, t h e rise time is less t h a n 1 minute, but there is a tendency to overshoot t h e steady state current. The noise level is judged to be k3 pa. which is quite satisfactory for typical base line currents of 50 pa. When the electrolytic cell is thermostated and the control circuit thermally stabilized, there is no base line drift. Care must be taken to avoid shorting together any electrodes when the instrument is in the “off” position. Resulting spontaneous electrolysis can occur leading to large start-up drift. The control amplifier gain is primarily affected by the se!ection of feedback resistor Ra in Figure 2. With R4omitted, the gain is about 30,000, while with R4at 100 meg. the gain is reduced to 1000. At all finite gains, there is a finite difference between the absolute values of control potential and the steady state solution potential (inner sensor value minus outer sensor reference value). Monitoring of the input shows this difference to be a few millivolts a t a gain of 1000. This imperfect potentiostating accounts for the drop-off in titration efficiency at high control potentials. Interferences. Predicted interferences are gaseous unsaturated hydrocarbons and nitrogen dioxide. The instrument responses to ethylene, isobutylene, and nitrogen dioxide have been investigated over a range of p H values and found to be negligible in t h e range pH 7 t o 8.5. T h e un-
saturated hydrocarbons consume bromine proportional to their concentrations while nitrogen dioxide oxidizes bromide t o bromine. The former produce positive errors while the latter leads to negative errors. The actual responses AZ/C determined from samples containing 13 to 51 p.p.m. by pressure are summarized in Table 11. LITERATURE CITED
(1) Bard, A. J., ANAL. CHEM.34, 57R (1962).
(2jIbid:, 36, OR (1964). (3) Barendrecht, E., Martens, W., Ibid., 34, 138 (1962). (4) Braman, R. S., DeFord, D. D., Johnston. T. N.. Kuhns. L. J.. Ibid.. 32. 1258 i1960).
‘
(5j-Biurneti, R. L., Klaver, R. F., Ibid., 35, 1709 (1963). (6) Eckfeldt, E. L., Eynon, J. U., I . S. A . Proc.. Part 3. Los Angeles, Calif.. Sept. 1955. (7) Kolthoff, I. M., Elving, P. J., “Treatise on Analytical Chemistry,” Part I, Vol. 4, Sec. D-2, Interscience, New York, 1963. (8) Landsberg, H., Escher, E. E., Ind. Eng. Chem. 46, 1422 (1954). (9) Lingane, J. J., “Electroanalytical Chemistry,” Second Edition, Interscience, New York, 1958. (10) AlcBride, W. R., Kruse, H. W., J . Am. Chem. Sac. 79, 572 (1957). (11) Olson, E. C., ANAL. CHEM.32, 1545 (1960). (12) Sakamaki, I., Yuke, S., Bunseki Kagaku 7,33-7 (1958). (13) Shaffer, P. A., Jr., Briglio, A., Jr., Brockman, J. A,, Jr., ASAL. CHEM.20, 1008 (1948). (14) Takahashi, T., Niki, E., Sakurai, H., J . Electroanal. Chern. 3, 373, 381 (1962). RECEIVEDfor review May 20, 1965. Accepted July 6, 1965. I
,
Analysis of Chromatographic Peak Displacement and Band Broadening by Impurities PETER D. KLEIN and BARBARA A. KUNZE-FALKNER’ Division of Biological and Medical Research, Argonne National laborafory, Argonne, 111. The relationship between the chromatographic purity of a compound, its characteristic retention value, and its dispersion b y the chromatographic column has been studied in synthetic, generated peaks. The computer effects of impurities have been evaluated for a variety of concentrations and mobilities on columns of widely differing efficiency. A general equation has been obtained which enables prediction of the displacement and band broadening of any chromatographic peak when contaminated a t any level with another component having a mobility different from the pure substance. A purity detection index for evaluating the performance of a column or thq purity of a compound isolated by chromatographic means is proposed on the basis of this equation.
-
I
dentification of a compound by its chromatographic mobility presupposes that its purity has been established. T o the degree that an uncertainty exists as to the precise purity of a compound, there will be a n associated uncertainty in the characteristic mobility of the compound. As yet no formulation of this relationship has been undertaken. Keulemans ( 1 ) has constructed a n analogue device that permits the contamination of one Gaussian peak with another of a n y size having any desired mobility. I t has been used to predict the separation a t which two maxima will be visible and some trial computations of the displacement of a given peak by a known contaminant have been carried out. As ingenious as this device is, i t does not permit a general assessment of the
effects of impurities on peak displacement when simultaneous variations of mobility, dispersion, and level of contamination take place. A recently developed computer program (@, used in predicting the behavior of isotope ratios in chromatograms of dual-labeled compounds (4, 6) appears to be a suitable tool for this investigation. This program permits the mathematical synthesis and combination of Gaussian peaks of any desired characteristics; a fraction-byfraction record enables detailed analysis of the resultant peak shape, displacement, and dispersion. Such an analysis, carried out on more than 60 chromato1 Present address, Department of Mathematics, University of Illinois, Urbana, Ill.
VOL. 37, NO. 10, SEPTEMBER 1965
1245
grams, has resulted in a general formulation of the peak displacement and bandbroadening produced by any level of contamination having a given mobility. This equation can be solved to obtain the minimum impurity which is present in a sample and the maximum difference in mobility or retention value from the pure compound that the impurity could possess. On the basis of these values, criteria of purity and identity from chromatographic measurements may be proposed.
IO
6
5
a 0
5
R
E! A
THEORETICAL
Descriptions of Real Chromatographic Peaks. The processes in a Chromatographic column operating under ideal conditions result in a peak which has the shape of a Gaussian distribution. Under such conditions, a peak may be described in terms of three dimensions: its displacement from the origin ( X ) , its dispersion ( u ) , and its area (n). These are obtained from the retention volume (= mobility), bandwidth, and sample size, respectively, and are sufficient to characterize the chromatographic behavior of any compound on a specific column. T o compare a given peak with another, however, i t is necessary to have the standard errors of these quantities as well so that tests for the significance of any difference can be made. The most appropriate way to obtain the information is by probit analysis (2). The algorithm for statistical estimation by this method is a lengthy procedure using a desk calculator (7). However, medium-speed computer programs have been developed that greatly facilitate the computation of the desired estimates and their standard errors. Therefore, the successive steps are illustrated schematically in Figure 1 and a detailed mathematical treatment will be omitted. The chromatographic peak (la) is converted into a plot of the cumulative per cent eluted ( l b ) which is then subjected to a probability transform. This transform converts the sigmoidal plot to a linear plot, enabling a regression analysis (IC) to be performed. The point a t which 507, of the peak has been eluted and the standard error of this value are obtained from the midpoint of the equation while the slope and its error yield the dispersion and its standard error. Construction of Synthetic Chromatograms. The equation for t h e shape of a n ideal chromatographic peak is given by
A x ; m, u )
=
1246
ANALYTICAL CHEMISTRY
Figure 1. Sequential transformation of experimental chromatographic peak (la) into cumulative per cent elution (Ib)followed by probability transform (IC). Line in
IC
has been computed by probit onalyris of points
The indefinite integral of this probability function cannot be expressed in closed form, nor is i t possible, in any simple fashion, to add two such functions with different parameters. By a variable transformation
x - ml Expression 1 is reduced to normalized form such that
lw
j ( z ; m, u)ciz =
where 9 ( a ) = 2/&J:
exp (-t*)dt,
O l a < m .
Hastings (3) gives the following approximation for +(cy) :
where = az = a3 = a4 = US
=
a6 =
0.070523078 0,042282012 0.0092705272 0.0001520143 0.0002765672 0.0000430638
The maximum error of this approximation is 0.0000003. The incremental area thus obtained may be multiplied by any desired sample size, and summations of values for a given increment can be carried out.
When increments of z (6x)of sufficiently small size are used, the discrete fractions approximate the continuous function closely, as shown in Figure 2u. Solution of the equation by this means also permits a direct visualization of the individual and composite curves in the compound chromatographic peak (Figure 2 b ) . EXPERIMENTAL
The basic Fortran program used in this study has been described by Kunze, Tyler, and IXpert ( 5 ) and is available upon request'. The input requires specification of the column load (nl, n3) the mohility of each component, (.Ifl, M 3 ) and their disperhions ( u ~ ,u s ) >the fraction size (Ax), and the limits of the chromatogram to be computed. The computer output obtained consists of the fraction number 5 , the increments of each component (Anl, Ana), and their sum (regarded here as An,). This output is in a format compatible with the requirements of the program that computes the required estimates by probit analysis from either experimental
or synthetic chromatograms. The data are then subjected to this a n a l y h . I n all chromatograms reported here, the following inputs were constant: total sample load, 100,000 units; reference mobility J i l , 100.00; fraction size, 1.00; limits of chromatogram computed, from Jil - 3u to *Us 3a where . I 1 3 > Ml-e.g., with Jf3 a t 108.00, u = 5.00, the chromatogram would be computed for each interval of 1.00 from 85.00 to 123.00. The reduced data for 20 chromatograms of this type are shown in Table I. I n the first series of chromatograms, j0,OOO units of component 1 were mixed
+
I2
a
5 5
a
c 2
0 V
LL
0 W
5 3
-J
0
>
W
s
c
a
-I W
P
0.01
0.10,
I
.o
IO .-
A M % OBSERVED Figure 3.
Relationship between fractional impurity (n3/. mobility of impurity, and resultant displacement of compound peak
F R A C T l ON
Figure 2.
nl
Chromatographic peak
+
n3),
A.
Exomple of synthetic chromatographic p e a k having a mean of 100.00, a dispersion of 5.00 and a n a r e a of 100,000 units B. Some p e a k together with secondary component of mean 1 1 0.00, dispersion 5.00, area 25,000 units. Shaded region, compound p e a k
with 50,000 units of a component that had a mobility or retention volume relative to component 1 of 1.01, 1.02, 1.04, 1.08, or 1.16. The column was considered to result in the same dispersion for each component-Le., u1 = u3. The emerging peak whose mobility and dispersion was determined b y probit analysis had the characteristics listed on the right side of the table. Inmeetion of the mobilities so obtained indicated that they were the weighted mean average of the two individual components multiplied by their respective mobilities,
.If* =
nlM1 n1
+ nJla + 723
(4)
This relationship is further illustrated by Figure 3, which compares the effect of various concentrations of contaminants a t various mobilities upon the displacement of the composite peak from the reference peak. Simply stat,ed, it demonstrates that for a given displacement from the reference peak, there is an inverse relationship between the possible concentrations and mobilities which can be assigned to the contaminant responsible for the displacement. There is no unique value for the contaminant producing a given displacement. On the other hand, Table I also illustrates that contaminants have a distinctive effect upon the dispersion of the composite peak, causing the peak to broaden. The displacement of the peak and its broadening are interrelated. When plotted in the form shown in Figure 4, each level of purityi.e., 50/50, 60/40, 70/30, 80/20--gave rise to a set of points which fell on the same line and which could be described by the equation
log A
+ B log [loO(u:l-
ul)]
(5)
T h e slope B was evaluated and found to be 0.545, and was identical for all four lines. The intercept log A proved to be deliendent both on the level of purity and on the dispersion of the component peaks ill-, and i l l a . Thus, to complete the evaluation of the intercept log A , the chromatograms in Table I1 were constructed according to the input parameters shown and the
Table I.
Synthetic Analysis
dispersions were obtained by probit analysis. These 47 values permit one to plot a linear equation for each of three levels of purity a t a given dispersion. When the purity was expressed as the area under the unit normal deviated curve (Figure 5 ) , log A was linearly related to the purity for a given dispersion and the intercept of this equation was inversely proportional t o the dispersion itself. When the constants for this equation were computed by linear regression, the resulting relationship obtained log
( ’) 41
= -0.877
- 1-e (iP(=>) (6)
These equations may be combined and rewritten in the form
of Two-Component Peaks by Probit Analysis
Input of component peaks
Output analysis of envelope peak Mz S.E. hfs Ul S.E. ~2 50/50 100.00 101.00 5.00 100.54 0.02 5.03 0.02 102.00 101.03 0.02 5.11 0.01 104,OO 102.03 0.02 5.39 0.01 108.00 104.02 0.04 6.40 0.04 116.00 108.05 0.25 9.87 0.29 60/40 100.00 101.00 5.00 100.44 0.02 5.03 0.02 102 00 100 84 0 02 5 11 0 02 n n2 101 64 n n2 .i 28 104 00 108 00 103 25 0 05 6 36 0 65 116 00 106 53 0 26 9 59 0 29 70/30 100 00 101 00 5 00 100 34 0 02 5 03 0 02 102.00 100.64 0.02 5.10 0.02 104.00 101.25 0.03 5.35 0.02 108,OO 102.47 0.07 0.06 6.24 116 00 105 14 0 27 9 15 0.29 ._ __ 80/20 100.00 101 00 5.00 100 24 0 02 5.. 02 n.. n2 102.00 100.45 0.62 5.08 0.02 104,OO 100.89 0.04 5.28 0.03 108.00 101.79 0.08 6.01 0.08 116.00 103.74 0.29 8.43 0.29 When dispersion of impurity peak was allowed t o increase in roportion to difference in mobility-e.g., 5.25 at 105, 5.50 at 110, or even 5.50 a t 105 anf6.00 at IlB-observed dispersion of mixed peak was not significantly altered. ndna
Mi
M3
a1.a’
VOL. 37, NO. 10, SEPTEMBER 1965
1247
5
IO
20 3040506070 80
IO
90 95
t
4.0
98
j
3.0 a W
> a
2.0
W
rn m 0
1.0
8
s
a
0.5 0.4
0.3 0.2 0.I
t
Figure 4. Relationship between peak displacement and band broadening for various levels of impurity
0.01'
'
-2.0 -1.5
'
'
-1.0 -0.5
'
0
'
0.5
'
1.0
I
1.5
1
2.0
U N I T NORMAL D E V I A T E
calculator.
] - 2.384
1.481 log
(7)
where q(z) is the purity function and is the area under the unit normal deviate curve that corresponds to the fractional purity X . The quantity X is given by
x=-
n1
n1
+ n3
and is deduced from the definition
X
=
Sm*'''=;i')
1/2, 1
exp(
dT (8)
DISCUSSION
The derivation of Equation 7 eliminates the necessity of repeated construction of trial chromatograms and permits direct computation of the effect of impurities on the displacement and band-broadening, using only conventionally available tables and a desk Table 11. MI = 100.00 nl+ n3 = ~OO,OOO
I t s use is threefold: first
it may be used to compute the purity of a sample whose mobility and bandwidth have been compared to a pure reference sample in the same system. Second, one may examine the equation for those parameters which most critically determine the instrument's ability to establish the purity of a chromatographic peak. Third, knowing the minimal displacement and minimal band broadening which can be detected, as well as the basic fractionation performance of a chromatographic system, one may compute the system's ability to establish purity, on the assumption that instrument or process errors are absent. The purity function Q(=) gives the maximum purity (or minimum impurity) of the sample in comparison with a known pure sample; this is, in fact, a pessimistic value, especially when the differences in means and dispersions are within the standard error of these measurements. However, from the displacement and purity, one can
Synthetic Chromatograms with Various Dispersions
u1,3
3.00 n
h
4.00
5.00
6.00
M*" 62 U2 U2 Q2 100.52 6.03 3.05 4.04 5.04 5.11 101.02 6.09 3.17 4.13 3.61 5.39 102.02 6.33 4.48 6.40 7.22 5.03 5.66 104.01 4.04 5.03 100.33 6.03 3.05 70/30 6.09 3.15 4.12 5.10 100,63 4.42 5.35 101.24 6.30 3.54 7.06 4.79 6.24 102.48 5.46 108,OO 90/10 101,oo 100.13 3.03 4.03 5.03 6.03 102.00 100.24 3.09 4.07 5.06 6.06 104.00 100.47 3.30 4.23 5.19 6.15 ... 6.55 108.00 100.88 4.05 4.84 Each envelope chromatogram synthesized by addition of two component chromatograms having proportions of nI and n2 shown, with mobility of n1 fixed at MI = 100.00. Withjn each set, mobil!ty of n3 varied a t mobilities M I shown. Resultant Mt values identical for all sets derived from same proportion. 50/50
1248
e
M 3
101.00 102.00 104.00 108.00 101.00 102.00 104.00
ANALYTICAL CHEMISTRY
Figure 5. Relationship of log A to dispersion and purity of compound peak
immediately assign a mobility to the contaminant and specify its minimum concentration using the relationship given in Equation 4. The characteristics of this contaminant include the possibility of a non-real (imaginary) component with either one of two properties: its mobility falls within the minimum 6M considered to be tantamount to identity for the comparison; or its concentration as obtained by the purity function is so large as to constitute a gross contamination which other criteria of homogeneity would have detected in prior analyses-i.e., i t appears to be as much as 50% of the test sample. Some trial computations and direct experiences with this equation show that i t contains some surprisingly stringent requirements in system performance. For example, resolution capacity ( u ~ ) in itself is not the sole determinant of the system's capability; in fact, if u decreases, there must be a corresponding improvement in the ability to determine differences between u2 and u1 if the performance is to remain unchanged. The equation shows that the quality of purity determinations is strongly dependent upon the basic ability to distinguish displacement in a peak and changes in its dispersion. Of the t n o , the ability to measure changes in peak dispersion appears to be the more difficult to extend, because it is limited by the ideality of the chromatographic peak itself. Any departure from ideality results in an increase in the error or uncertainty of the dispersion measurement and a concomitant decrease in the ability to measure a difference between two dispersions. This can be seen by reference to Figure IC. I n any comparison of systems, the performance characteristics will be
determined b y the three parameters ui, S.E.,M, S.E., and knowing these, a system-to-system comparison may be proposed. Replacing (M2-MI) b y S.E.,M and (Q-u,) by S.E.,, and expressing the error as a per cent of the mean or dispersion ( E X P , ESP) , Equation 7 becomes
1.481 log (ESP) - 2.384
(9)
This purity function may be used in the following fashion: Purity detection index
where is the mobility of the minimal impurity which can be detected by the system. This purity detection index would appear to be the logical basis on which to compare two systems for their ability to establish chromatographic purity. It also provides a single number for expressing the chromatographic purity of a compound in a given system and can therefore supply the basis for a rigorous purity criterion.
=
LITERATURE CITED
Ettre, L. S., ANAL.CHEM.36, 31A (1964). (2) Finney, D. J., “Probit Analysis,]’ Cambridge Univ. Press, London, 1952. (3) Hastings, C. B., Jr., “Approximations for Digital Computers,” Princeton (1)
Univ. Press, Princeton, N. J., 1955. (4) Klein, P. D., Simborg, D. W., Szczepanik, P. A., Pure Appl. Chem. 8 , 357 (1964). (5) Kunze, B., Tyler, S., Dipert, 31. H., “Semiannual Report of the Division of Biological and Medical Research,” p. 152, Arbonne Xational Laboratory, ANL-6823, January-July 1963. (6) ‘,‘%diochemistry Methods of Analysis, Proceedings of the IAEA Symposium in Radiochemical blethods of Analysis, Sulzburg, 1964, Vol. 2, p. 353, International Btomic Energy Agency, Vienna, 1965. (7) Tyler, S. A., Gurian, J., “Deterrnination of the LDSo, by Use of Probit, Angular and Logit Transformations,” Argonne National Laboratory Rept. ANL-4486, 1950. RECEIVEDfor review May 10, 1965. Accepted J ~ l 19, y 1965. Work supported by U. S. Atomic Energy Commission.
Structure and Behavior of Organic Analytical Reagents Some Aryl Azo 8-Quinolinols SUSUMU TAKAMOT0,l QUINTUS FERNANDO, and HENRY FREISER Department of Chemistry, University o f Arizona, Tucson, Ariz.
b The metal chelate formation constants of 5-benzeneazo-, 5-(o-hydroxyphenylazo)-, 5-(m-hydroxyphenylazo)-, and 5-(p-hydroxyphenylazo)-8-quinolinols have been determined potentiometrically in a 5070 v./v. dioxanewater medium a t 25” C. The acid dissociation constants of these ligands have been determined both spectrophotometrically and potentiometrically in a 5070 v./v. dioxane-water medium a t 2 5 ” C. The aryl azo substituents exert an acid strengthening effect on these reagents. The metal chelate formation constants are lower than the corresponding values for 8-quinolinol. As with other 8-quinolinols having electron-withdrawing substituents, the proton displacement constants are significantly higher. These reagents therefore have the added analytical advantage over the parent reagents, of forming metal chelates a t lower p H values.
of the chelates such as color and solubility. From the behavior of the alkylated and halogenated 8-quinolinols1 it would appear that electron-withdrawing substituents give rise to higher values of proton displacement constants. Since the chromophoric arylazo groups, wh n incorporated in a chelating agent such as 8-quinolinol, confer useful analytical properties to the reagent, i t is of interest to determine whether its electron-withdrawing behavior would have the predicted effect on the proton displacement constant of 8-quinolinol. An attempt has been made, by using the hydroxy substituted arylazo groups, to further modify the electron-withdrawing property of the arylazo group with a view to obtaining analytical reagents with large proton displacement constants.
0
Preparation of Azo Compounds. 5-Benzeneazo-, 5-( (o-hydroxyphenylazo)-, 5-(m-hydroxyphenylazo)-, and 5 - ( p - hydroxyphenylazo) - 8 - quinolinol were prepared b y a method similar to that used by Fox ( 2 ) for the preparation of 5-benzeneazo-8-quinolinol. 5Benzeneazo-8-quinolinol was purified by reprecipitation with acetic acid from a KOH solution and recrystallization from toluene, m.p. 189’ C. (dec.); lit. m.p., 170” C. ( 2 ) . 5-(o-Hydroxyuhenvlazo~-8-auinolinol was recrvstaliized’from’dioiane, m.p. 222’ C. (dec.); yo C, 67.82 found, 67.91 calcd. % H,
principal advantages of using organic reagents in analysis lies in the possibility of modifying the behavior of the reagent molecule by appropriate substitution. A wide range of electronic and steric effects of various substituents has been used t o bring about changes not only in those factors which affect the parameters that control the conditions of formation of metal chelates, such as the proton di placement constant ( 4 ) ,but also in those factors t h a t influence the properties NE OF THE
EXPERIMENTAL
4.37 found, 4.18 calcd. &(m-Hydroxyphenylazo)-8-quinolinol was recrystnllized from absolute ethanol, m.p. 210” C.; % C, 67.57 found, 67.91 calcd. % H 4.29 found, 4.18 calcd. 5-(pHydroxyphenylazo)-8-quinolinol was also recrystallized from absolute ethanol, m.p. 239’ C. (dec.); yo C, 67.72 found, 67.91 calcd. yo H, 4.27 found, 4.18 calcd. Reagents. 1,4-Dioxane was purified by refluxing over sodium metal for 48 hours and then fractionated through a 4-ft. column packed with glass beads. T h e distillate was collected between 99’ and 101” C., stored in t h e dark, and used within a week. All other compounds used were of reagent grade purity. Stock solutions of metal ions (0.01 to 0.002J1) were prepared from metal perchlorates and standardized gravimetrically. Apparatus. All potentiometric measurements were made with a glass and saturated calomel electrode pair and a Beckman Research p H Meter, standardized with a Beckman buffer solution a t p H 7.00 at 25” C. The titration vessel and the titration with carbonate free S a O H in a nitrogen atmosphere have been previously described in detail (3). Determination of pH M e t e r Correction and Ion Product of Water in 50% v./v. Aqueous Dioxane. A 0.01 J1 solution of HCIOl in 50% v./v. aqueous dioxane and at a constant ionic strength of 0.1, was titrated with ‘On leave from the Department of Physics and Chemistry, Gakushuin University, Tokyo, Japan. VOL 37, NO. 10, SEPTEMBER 1965
1249
