changing the values of n and k in Equation 2. The procedure requires some time to complete. Hon-ever, the design and development of a new detector is a time-consuming project, and evaluation time would be rather negligible by comparison. The procedure could also be used to improve existing detectors by measuring the effect of design changes. NOMENCLATURE
-4i
= peak area, sq. em. = interval area of the interval i
c
=
.i
+
to i 1, m1.-mv. concentration of sample in mobile phase, mg./ml. = recorder sensitivity, mv. per cm. of chart (see definitions) = reciprocal chart spwd, mine/ em. = flow rate a t exit of column, ml./min. corrected to column temperature and atmospheric pressure
C1 Cr C3
maximum concentration in detector when a sample of weight Wa passes through a chromatographic column, assumed to be Wa divided by effective detector volume, mg./ml. i = integer such that 0 < 1 < n f l j ' , k , k' = integration limits defined by Equations 6 and 7 L = statistical confidence level = noise area, m1.-mv. = standard deviation of set (Nil,
CL
=
= mean of set ( N i ) ,i = 1 to = true standard deviation
n P
n of
the population (assumed normal) from which set { X i } was obtained = number of N i in set ( N i ) = probability = fiducial probability = detector sensitivity defined by Equation 1, mv./(mg./ ml.)
SL
= value of S a t concentration
1'
= volume of carrier gas passed
W
=
W,
= )\eight of sample coniponent
W6
=
CL
through detector, ml.
n eight of sample component, mg.
corresponding to N , , mg. limit of detection defined by Equation 2, mg. AP = distance between adjacent perpendiculars in ddinitions of interval area and noise area, ml. Ay = change in recorder output, millivolts (see definitions: recorder sensitivity) f ( ~ )= ~ a variate, x2, distributed by chi-square distribntion LITERATURE CITED
( 1 ) Dimbat, Martin, Porter, P. E., Stross, F. H.. H., ANAL.CHEM.28. 28, 290 11956). (1956). (2) Johnson, H. W.,Jr.; Jr., Strok, Strose, F . H.. Ibzd., 31, 357 (1959). (3) Porter, P. E., Deal, C. H., Stross, F. H., J . A m . Chem. SOC.78, 2909 (1956). (1956): RECEIVEDfor review August 26, 1058. Accepted I I a r c h 16, 1059.
Gas Chromatography Effect of Sample Size on Height of Equivalent Theoretical Plate and Retention Volume R. M. BETHEA and M O R T O N SMUTZ Chemical Engineering Department, Iowa State College, Ames, Iowa
b This research was conducted to determine the importance of sample size on the performance of gas chromatography columns a t low flow rates. The minimum value of HETP was found when a 4- to 7-pI. liquid sample was used for an alcohol test mixture in a dibutyl phthalate column. Minimum HETP values for the same mixture in a dibutyl sebacate column occurred a t a sample size of 10 to 12 pi. and also when an ester test mixture was used on the same two columns. Minimum values of retention time with sample size were obtained for all systems tested. Optimum values of sample size exist for some feed systems in certain substrates at low flow rates. This may aid other research workers in obtaining maximum resolution of mixtures.
T
best liquid sainple size for gasliquid partition chromatographic analysis in columns having an internal diameter of 2.5 to 7.6 mm. has been HE
determined by several investigators (10, 12) to be between 0.03 and 0.07 ml. and not over 0.10 ml. One of the most widely used methods of sample injection is with a microsyringe through B self-sealing rubber serum bottle cap. Lichtenfels et al. (9) have developed a micropipet for reproducibly injecting liquid samples in the range of 0.005 to 0.02 ml. Haskin et al. (?) have used the syringe method to reproduce peak heights within the rang" of zk0.5 to zk4.4% for known azeotropic. mixtures. The vaporized sample may reach the column in one of two ways, according to Keulemans (S),which represent two definable extremes: plug flow, defined as occurring when the sample reaches the column undiluted followed by tht, carrier gas at a sharp interface, and rxponential flow, defined as occurring when the sample and the carrier ga. are mixed before reaching the column. He stated two disadvantages to the syringe method. The first is that plug flow is not approached unless the syringe
tip is level 111th the top of the column packing, so that the sample may dissolve immediately in the partitioning agent. Many chromatographic systems use a sample bypass such as that reported by Davis and McCrea (1) for reproducibly injecting samples in approximately plug flow and thus avoiding this objection. The result of exponential flow is a superposition of a number of chromatograms whose time origins vary over the time required for the sample to be injected. Pollard and Hardy ( 3 ) have indicated that the column efficiency as measured by the number of theoretical plates is constant within = t l % for injection times up to 10 seconds. Keulemans' other objection to syringe injection is that the absolute size of the sample is subject to :I systematic error caused by expansion of the liquid in the needle or by creeping of the liquid. This error should be constant for a given sample injection system. Recently. Eggertsen and Groennings ($) have used the syringe injection VOL. 31, N O 7,JULY 1959
1211
4.0
-
3.5 -
-
0 7-
e
0
06-
3.0
METHbJdOL
2.5 -
i
0
2.0
-
1.5
-
0.5 I
I
am I-
W
I
5 0.4-
0
nI-
ETHANOL
1.0 -
-
W
'
L
A
A
r
/
-
0.3
A
x
a
01 0
I
I I 15 20 SAMPLE SIZE, MICROLITERS I
IO
I 25 I
0
Effect of sample size on HETP for primary
Figure 2. Series 3, dibutyl sebacate
technique for the resolution of the Cs to C, saturated hydrocarbons. They found that sample sizes of 10 to 15 mg. were small enough for good resolution and yet large enough to ensure reproducibility. Eggertsen, Knight, and Groennings (5) have indicated that in their particular system the sample size should not be larger than 3 mg. per component for closely boiling compounds. Larger samples caused broadening of the major peaks with a corresponding decrease in resolution of the neighboring peaks. This probably was due to partial occlusion of some peaks by the presence of a n excess of the more volatile component just preceding them. It has long been thought that the retention time for a given compound in a given chromatographic apparatus increases directly with increasing sample size because of the increase in time required for the zone maximum to appear in the effluent stream (11). The present authors have not found in the literature any indication of the presence of a minimum value of H E T P or retention time with sample size other than a t a sample size extrapolated to zero. This was demonstrated by van de Craats (8) and Pollard and Hardy ( 3 ) . The study reported here was designed to demonstrate the effect of sample size on the HETP (height equivalent to a theoretical plate) and on the retention time, TR,for the separation of various classes of organic compounds by several partitioning agents a t low flow rates. EXPERIMENTAL
Apparatus. The columns consisted of a packed length of borosilicate glass tubing 4.6 mm. in inside diameter, surrounded b y a vapor jacket com1212
b
ETHYL ACETATE
A VINYL ACETATE x i s o P m P y L ACETATE
5
Figure 1 . alcohols
0 ETHYL FORMATE
a
0.5 -
ANALYTICAL CHEMISTRY
I
I
5
I
I
IO 15 20 SAMPLE SIZE, MICROLITERS
I 25
Effect of sample size on HETP for esters Series 2, dibutyl phthalate
posed of a 135-cm. section of 5.1-em. glass tubing. T h e packed length was 128.2 cm. for Series 1, 130.0 cm. for Series 2 and 4, and 130.5 cm. for Series 3. T h e column assembly was made in t h e form of a condenser and was equipped with a stopcock at the bottom of the jacket to allow removal of condensate. The bottom of the column was connected to the detector through a ball joint with 1.5-mm. bore capillary glass tubing to reduce the volume available for component remixing. To ensure rapid and complete vaporization of the sample, the sample injection tee was kept at approximately 170' C. by the cone of a small radiant heater of the household variety. A Model TR-11-B Cow-Mac (CowMac Instrument Co., 100 Kinas Rd., Madison, N. J.) thermal condktivity cell, geometry 9193, with a Model 9293B Cow-Mac power supply control unit was used. The balancing bridge current was maintained at 175 ma. a t a sensitivity of 10.0, which corresponds to a pen deflection of 1inch per mv. potential difference between reference and measuring arms in the bridge circuit. The thermal conductivity cell was held a t 200' C. by means of an integral electric heater and thermoswitch. The recorder used was a Bristol ,Model IPH-570 Dynamaster with the range set a t 0 to 10 mv. Chart speed was 30 inches per hour. The helium flow rate was maintained constant by a Brooks Sho-Rate 150 rotameter with a n integral constant differential relay. The use of a twostage regulator on the gas cylinder with the constant pressure relay at the rotameter resulted in a constant carrier gas flow rate of 5.0 ml. per minute at column outlet conditions as the cylinder emptied. Test Mixtures. T h e test mixture for Series 1 and 3 was composed of methanol, ethanol, 2-propanol, 2methyl-2-propanol, 1-propanol, and 2butanol. T h e test mixture for Series
2 and 4 was composed of ethyl formate, vinyl acetate, ethyl acetate, and isopropyl acetate. T h e purity of each component was t h e highest normally available. This choice of systems was prompted b y t h e desire to have short total analysis times and t o obtain some indication of the generality of t h e results. Procedure. T h e column packing was 35 grams of substrate per 100 grams of T y p e C-22 Johns-Manville firebrick ground t o -48+65 Tyler mesh. T h e substrates used were dibutyl phthalate for Series 1 and 2 and dibutyl sebacate for Series 3 and 4. The packing was prepared by the method of Evans and Willard (6). Uniformity of packing was ensured by packing the column with the aid of an electric vibrator. A porous plug of glass wool was inserted at either end of the packed section to prevent carrying of solid material into the detector or loss of any packing if the sample injection assembly n-ere blown o f f by a sudden change in supply pressure. The instruments were allowed to warm up for 3 hours before the helium was turned on. At this time, the sample injection T heater and the column vapor jacket were started. The column was heated with methanol vapor a t 65.3" C. Column inlet pressure was adjusted to 1352 mm. of mercury, absolute, giving an outlet pressure of 755 mm. of mercury, absolute. The apparatus was then allowed to come to thermal equilibrium, as indicated by a straight horizontal line of 20-minute duration appearing on the recorder chart. Successively larger samples of the test mixture were then injected through a rubber serum cap, into the column with a microsyringe; about 4 minutes were allowed between the appearance of the last peak and the introduction of the next sample. Determination of Sample Size. T h e densities of the test mixtures u ere determined u-ith a pycnometer. Several
ISOPROPYL ACETATE
8
0-0 b 0-0-0-
'
8
0-
t
l4
-
I PRO PA MOL
-0
/
o
ETHYL ACETATE
9
0
0-
d--O,O-Q&Q
9
I-
4l-
ETHANOL
8-0-0-Q-0-0
0
5
4-
ME T n* NOL
~~o-o-Qyo-q
0
3
I
I 5
Figure 4. for esters
I
I
I 25
15 20 SAMPLE SIZE, MICROLITERS
0
IO
Effect of sample size on retention time Series 2, dibutyl phthalate 4.0
Series 1, dibutyl phthalate
sizes of hypodermic needles were calibrated by weighing 10 drops into a tared serum bottle through a selfsealing rubber stopper. This was repeated three times for each needle. T h e deviation in weight of a 10-drop sample was less than 0.3% for all needles. The volume of a drop from each needle was then calculated (Table I). Sample sizes of 20 and 30 pl, were obtained by using a tuberculin syringe with plunger spacers (Central Scientific Co., Chicago, Ill.). I n all cases, sample introduction was made in 1 second or less by counting drops from the appropriate needle.
Calculations. The retention times for t h e components were measured in minutes from the time of injection to t h e time of appearance of the component peak maximum. Values of H E T P were calculated from the equation adopted a t the Gas Chromatography Symposium in London ( 2 ) .
where T R = retention time of component, measured in inches, from injection t o appearance of the peak maximum for each component L = packed column length, em. x = distance between two inflection tangents measured at chart base line, inches RESULTS
Typical results of this investigation are presented graphically in Figures 1 to 4. The minimum H E T P for normal alcohols in the dibutyl sebacate (DBS)
3 .3
column was found a t sample sizes of 11 pl. for methanol and ethanol and 15 pl. for 1-propanol, as seen in Figure 1. Secondary alcohols exhibit similar minima a t 15 pl. in the same column. The minimum value of H E T P for normal alcohols in the dibutyl phthalate (DBP) column were obtained a t 4 to 7 pl. In the same column, secondary alcohols exhibit the minimum H E T P a t approximately 6 p1. As an average value for the six alcohols, a sample between 5 and 7 pl. for the dibutyl phthalate column and between 10 and 12 pl. for the dibutyl sebacate column will result in minimum values of HETP for each component. The difference in sample size producing these minima was due solely to differences in physical characteristics of the substrates and the solubilities of the sample components in them. To eliminate the possibility that the presence of these minima was specific for the alcohols and substrates used, identical tests were made using a fourcomponent ester test mixture in the two columns. I n the dibutyl phthalate column (Figure 2) the esters exhibited minima more pronounced than those of the alcohols. The minima for the alkane esters appeared at 5 to 6 pl. of sample in the dibutyl phthalate column and a t 8 to 10 pl. of sample in the dibutyl sebacate column. The minimum value of HETP for vinyl acetate was 5 pl. in the dibutyl phthalate column and 10 11. in the dibutyl sebacate column. The curves for the three normal alcohols in the dibutyl phthalate column (Figure 3) show that a marked de-
3 .O
u, I-
$ 2
2.5
-I -1
f
i a.
*'O
u)
W
a a
1.3
W
a a
00w a
1.0
0.5
0 0
I
2
3
RETENTION
4
5
TIME,
6
7
MINUTES
Figure 5. Effect of sample size on retention time and tailing for methanol in dibutyl phthalate Table
I.
Calibration of Hypodermic Needles
Seedle Size, BWG 27
26 25 24 2% 20
18
Drop Size, d. 3.23 3 78 3 97 4 08
5 05
5 67 7.17
VOL. 31, NO. 7, JULY 1959
1213
crease in sample size is required for minimum retention time as the molecular weight of the sample components increases. This is shown by the movement of the curve minima to the right as molecular weight decreases. This was also exhibited by the two secondary alcohols. The presence of similar minima vas observed with the dibutyl sebacate column. For this series of runs, the curve minima for the six alcohols occurred a t about the same sample size, 16 to 18 pl. The minimum values of retention times for the components of the ester test mixture occurred over a broader range. I n the dibutyl phthalate column (Figure 4), the optimum sample size was 10 to 15 pl. For the dibutJ.1 sebacate column, it was 6 to 7 , ~ l . KO overlapping or occlusion of peaks was observed \yith sample sizes under 20 111. Above that size, peak overlap became appreciable and increased with sample size. This had no effect on the esperimental results, as all minima n-ere observed a t sample sizes of 15 pl. or smaller. Some tailing was observed in all cases. This would indicate that the solutions formed betn-een substrate and sample constituent were nonideal or adsorption by the solid support for the substrate occurred. The change in
retention time with sample size for methanol in the dibutyl phthalate column is shown in Figure 5. The retention time-sample size curve for this series of runs has been shown in Figure 3. These chromatograms are typical of the ones obtained in this investigation. A curve connecting the points of maximum methanol concentration in Figure 5 shows a definite minimum a t approximately 13 to 15 pl. of sample. This curve has not been drawn in, to avoid crowding on the figure. As the theory cannot be expected to hold exactly for badly tailing peaks such as these (which are obviously nonideal), this could account lor the presence of the minima in the retention time-sample size curves. .Is is apparent from Figure 5 ) the principal effect of tailing is to broaden or enlarge the solute bgnd. As the sample size increased, the inflection points of the elution curves moved away from the base line, causing a change in the z intercept of Equation 1. The inflection tangents are intended to measure only the majority of the peak. I n the case of the lower sample sizes, the inflection tangents also measured part of the broadening of the peak caused by tailing. The percentage change in z with sample size was considerably greater than the variation
in retention time caused by the nonideality explained above. This may explain, in part, the presence of the minima in the HETP-sample size curves. LITERATURE CITED
(1) Davis, It. E., McCrea, J. hl., .4\.4~. CHEX 29, 1114 (1957).
(2) Desty, D. H., ed., ‘Yapour Phase Chromatography,” p. xiii, Butternorth, London, 1957. (3) Ibid., pp. 120-3. (4) Eggertsen, F. T., Groennings, S., - 4 ~ 1 . 4 ~CHEW . 30, 21-2 (1958). ( 5 ) Ewertsen. F. T.. Knight. H. 8..‘ . Grzhings,’S., Ibid.; 28, 385 (1956). (6) Evans, J. B., Willard, J. E., J. Am. Chem. SOC.78, 2809 (1956). (7) Haskin, J. F., Warren, G. W., Priestlev, L. J., Yarborouah, V. A4., ANAL.CHEX 30; 217-19 (1958). ( 8 ) Keulemans. A. I. M.. “Gas Chromatography,” pp. 61-7, ’Reinhold, Sew York. 1957. (9) Lichtenfels, D. H., Fleck, S. A, Burow, F. H., Coggeshall, N. D., AXAL. CHEJI.28, 1576-9 (1956). (10) Oil and Gas J . 54,126 (Dec. 17, 1956). (11) Porter. P. E.. Deal. C. H.. Stross. F. H., J . ’Am. Chem. Sic. 78, 2999-3000 (\ -19%). - - - I -
(12) Thomas, B. W., Znd. Eng. Chem. 47, 85-4 (June 1955). RECEIVED for review June 5, 1958. Accepted March 19, 1959. Investigation carried out as part of development work in gas-liquid partition chromatography sponsored by the Engineering Experiment Station, Iowa State College.
Qua nt ita tive Analysis of Commercial Bisphenol A by Paper Chromatography W. M. ANDERSON, G. 9. CARTER, and A. J. LANDUA Research laboratory, Shell Chemical Corp., Houston, rex.
b A method is presented which permits rapid determination of the impurities normally encountered in commercial grades of bisphenol A. The impurities are separated on two onedimensional paper chromatograms, with water and carbon tetrachloride OS solvents. Variations in the technique permit analyses over a fairly wide concentration range.
phenol], and codimer [4,4’ - hydroxyphenyl-2,2,4-trimethylchroman] are the principal impurities occurring in commercial bisphenol A. The pure compounds have been isolated from commercial bisphenol A by extractioii and fractional crystallization techniques and identified by means of their infrared spectra and physical properties. The structures of these materials are : Codimer
p,p’-BPA
CHI
B
A, 4,4-isopropylidenediphenol, [2,2-bis(4-hydroxyphenyl) propane, or p,p’-BPA], is used as a starting material in the manufacture of epoxide resins and other polymers. Work in this laboratory has shown that o,p ’-BPA [2-( 2-hydroxyphenyl) -2- (4hydroxyphenyl)propane], B P X [2,4bis ((Y,(Y- dimethyl - 4 - hydroxybenzyl) ISPHENOL
1 2 14
ANALYTICAL CHEMISTRY
I
CH, n , p ‘-BPA
-OH
OH
P
3
CHI
/
CHI
Because minor components may have some influence in syntheses invoh-ing
