(29) Swift, E. H., Anson, F. C., “Advances in Analytical Chemistry and Instrumentation,” pp. 293-345, C. N. Reilley, ed., Interscience, Kew York, 1960. (30) Swift, E. H., Anson, F. C., l‘alantu 3, 296 (1960). (31) Swift, E. H., Butler, E. A., ANAL. CHEM.28, 146 (19.56). (32) Tanaka, N., Kyuno, E., Sato, G.,
Tanamushi, R., J . Phys. Chem. 66, 2706 11962’1. (33) Waggoner, W. H., J . Chem. Educ. 35. 339 (1958). ~. ~~. (3:) ’Vozza, J. F., Zbid., p. 145. ( 3 0 ) Walton, A. G., ANAL. CHEM.35, 917 (1963). \ - - - - ,
RECEIVEDfor review April 1, 1964. Accepted June 24, 1964. A joint con-
tribution from Hall Laboratory of Chemistry, Wesleyan University, Middletown, Conn., and Gates and Crellin Laboratories (Contribution number 3119), California Institute of Technology, Pasadena, Calif. Work done at the California Institute of Technology was supported by the National Science Foundation. One of the authors (D. C. T.) held a Connecticut Water Resources Commission Fellowship during the summer of 1962.
Rapid, Accurate Microanalysis of the Lower Fatty Acids with Particular Reference to Seria Determination F. A. VANDENHEUVEL Animal Research Institute, Canada Department o f Agriculture, Ottawa, Ontario
b Isolation of the lower fatty acids by steam distillation is efficiently carried out with the described apparatus which permits the simultaneous processing of ten 0.5-ml. samples under preset, controlled conditions. About 20 minutes are required to recover over 99% of all acids except formic acid of which a precisely known percentage is obtained. Desiccated aliquots of the neutralized distillates are mixed with measured volumes of a standard solution of formic acid in carbon disulfide which quantitatively releases the acids from their salts. The resulting solutions are injected into a chromatograph equipped with a flame ionization detector. Statistical analysis of results obtained with standard solutions of mixed acids indicates a relative for injected solutions error of f containing 2 pmoles of acid per ml. when xylene is used as an internal standard. Adjustment of concentrations often reduces the number of calibration values required for computation in serial analyses to only two. Formic acid, when present, is determined on 0 . 5 ml. of distillate by the precise micromethod of Grant.
170
A
of a recent monograph on the biochemical application of gas liquid chromatography (GLC) is devoted to the description of methods for the analysis of the lower fatty acids ( 4 ) . Alost of the progress achieved in this field since the first remarkable results obtained by James and Martin (21) followed from the availability of imliroved GLC equipment including more sensitive detertors. An appreciable, though lesser, contribution was made by the discovery of column materials better suited to this type of analJ-sis ( 2 , 3, 10, 1 1 , 18, 25, 26, 29). Efforts made to improve the iircparative stqis leading from the N IMPORTANT sEcrIoN
1930
ANALYTICAL CHEMISTRY
test material to injected solutions were not nearly as successful. Thus the method as a whole still lacks the speed, efficiency, and simplicity so desirable in many applications, particularly those requiring the serial determination of many samples. I n this respect, the classical procedure consisting of steam distillation and subsequent transfer of the acids from aqueous distillate to some organic solvent (12, 1 4 , 16, 18, 19, 21, 23, 28) is very cuinbersome and several attempts have been made to avoid these steps altogether. First Emery and Koerner (10) and then Erwin, hlarco, and Emery ( 1 1 ) proposed the direct injection of aqueous sample solutions taking advantage of the flame ionization detector’s insensitiveness to nater (24). However, Ackman and Burgher ( I ) showed that aqueous solution of the lower acids could not be analyzed reliably in this way unless formic acid was added to the carrier gas. Application of their procedure to aqueous biological samples will not, however, prevent substances which the samples contain from interfering with the GLC analysis of the lower acids. Hence a quantitative method for the preliminary isolation of the acids is still required. Steam distillation offers a basically simple, potentially effective means for this piirpose. However, the necessity for a strict control of conditions ( 6 , 7 , 16) has imposed serious difficulties on its application in serial determinations. Our efforts in improving this method in speed, efficiency, and its adaptability to the simultaneous processing of many samples under the same conditions have led to the apparatus and the simple routine procedure described below. On the other hand, the transfer of the distilled acids from water to organic solvents has often been carried out in many complicated, time-consuming steps (12, 14, 16, 21, 25). The use of dichloroacetic arid for this purpose
(20) is not free of complications (20, 30). Several authors have forsaken the direct analysis of acids, preferring a conversion to esters (8, 17, 28, 32). A method is described below in which the neutralized steam distillate is evaporated to dryness and a measured volume of carbon disulfide containing formic acid is added to the residue. The resulting solution is then injected edirectly into the chromatograph equipped with a flame ionization detector. I n this extremely simple and rapid procedure, advantage is taken of the insensitivity of the detector to both carbon disulfide (9, 31) and formic acid (24). The latter liberates all other acids from their sodium salts quantitatively. I n addition, carbon disulfide offers many advantages in this analysis, permitting a speed and reproducibility unattainable with other solvents (10, S I ) . Corrosion by formic acid of metal parts of GLC equipment is entirely avoided in the absence of water and “ghosting” (1) does not occur. Formic acid, if present in the original material, is determined independently on 0.5 ml. of the distillate. The first section of the following account describes a multiunit microsteam distillation apparatus, the related operating procedure, and performance study. I n the second is given a GLC method starting from steam distillate which could be obtained through the use of any distilling unit. While the two procedures can be applied independently, many advantages arise from their combination. EXPERIMENTAL
Multiunit Micro-Steam Distillation Apparatus. DESCRIPTIOK. Fig-
ure 1, I, shows a n end view of this apparatus. Boiler A carries two rows of -$- 24/40 necks B and C. During operation, each neck holds a microstill while nitrogen is admitted through inlet tube D . The boiler is
during operation in ice-cold water filling Plexiglass container Ji7 to level X. Figwe I,111, shows the flat. sides of Teflon st,opper 0, which has been designed to improve finger hold. The dimensions of microstill K , which are shown exactly to scale in Figure 1, I, are designed to ensure complete tightness and to prevent entrainment of sample during operation. The still body and side-arm are made of sections of borosilicate glass tubing. Inlet tube L is made of 0.5-mm. bore, 5-mm. 0.d. capillary tubing. This has a 45" bevel at one end, and a IO-mm. length of standard tubing at the other. Sidearm N is made of thick-walled tubing selecaed for outside diameter uniformity. The latter is chosen about five- to eightthousandths of an inch larger than the drill used to bore the corresponding hole in Teflon stopper 0. The endsection of the sidearm is first ground flat, and only the ground part is then carefully fire-polished (the outside diameter of the end should not be modified), Sidearm N at first fits very tightly in stopper 0. Heating and subsequent cooling during the first operation will reduce the tightness to that of a snug sliding fit. Figure 2 shows a side view of the borosilicate glass manifold. In Figure 3, a photogmph of the 10unit apparatus in operating condition shows box A containing the manifold which carries two rows of microstills B on each side. On the right, the microstills are seen connected to their respective condensers immersed in bath C. The Plexiglass rack holding the
7 , : : : ; : , : , - Jj* l [ u
0 1 2 , . , b I B O l O C D I
Figure 1. apparatus I. Still.
11.
End
view
Receiver.
111.
of
multiunit
Side view of Tef-
lon stopper
heated by coil E , connected t o a powerstat not shown. The coil is embedded in porcelain cement filling carriage F which can slide within metal box G . Metal box G and the boiler are held in metal box H which is filled to level J , with shock- and heat-insulating silicone foam (Dow Corning R-7002 resin) generated in situ. Nitrogen saturated with steam enters microstill K through inlet tube L , bubbles through sample 111, and entrains distillate through side-arm N . The latter is held in T 24/40 Teflon stopper 0. The distillate leaves the microstill through side tube P (l&gaugehypodermic tubing) connected by a length Q of Teflon tubing (16gauge-Teflon Spaghetti, Fluorocarbon Products h e . , Camden 1, N. J.) to condenser tube R (Figure 1, 11). The condenser tube (16gaugehypodermic tubing) is held in the head of receiver S by silicone rubber cement (General Electric RTV-20). The outside of tube R is scored at the level of narrow tube U and the extremity of the latter is flared to strengthen this joint. Nitrogen escapes throuxh side-arm V . The whole receiver system is immersed
Figure 2.
Side view of IO-unit borosilicate glass monifold
Figure
condensers is visible. Manometer D used to adjust the nitrogen inlet prcssure is seen at the front. Adapter E holding a thermometer is fitted with side-arm F , which is closed with a pinchclamp during distillation.. AnJusTMENTs. Several operational parameters of the above apparatus are adjusted only once as follows. AMicrostills. (Figure 4, I). All but one (A) of the standard necks of the manifold B are tightly closed with rubber stoppers C. The manifold is connected on the one hand to a simple U-tube manometer D filled with water, and on the .other. hand to nitrogen cylinder E equipped with a regulating valve system. An empty microstill is then tightened in the open neck A of the manifold, and connected to condenser F which is not cooled. Outlet tube G of the condenser is connected as shown to glass flowmeter H (Fischer and Porter Tri-Flat No. 9142 equipped with steel ball). Nitrogen is slowly admitted to the manifold and the position of stainless steel screw J is adjusted to obtain a 50 ml. per minute flow (reading 65 on flowmeter H ) under 40mm. water pressure read on manometer D. This is repeated for all microstills. Fore Pressure. (Figure 4, 11). All microstills are filled with 0.5 ml. of water, fitted to the manifold, and connect,ed to condensers. The nitrogen is then connected as shown in Figure 4, 11, which describes the arrsngement used in normal operat,ion. This arrangement compriscs collar Af whish tightens the rubher pressure tube on the inch tuhe of female Luer-lock adapter
3. Ten-unit micro-steam distillation apparatus VOL. 36, NO. 10, SEPTEMBER 1964
1931
K
D
I Figure 4. I. Connections for microstill flow rate adjustment. for fore pressure adjustment
N , and 2-inch 22-gauge hypodermic needle 0 squeezed in the connecting pressure tubing by screwclamp P . I t serves the purpose of raising the fore pressure to a level which corresponds to the required low flow rate, yet is high enough to be reproducibly adjusted with the help of simple U-tube mercury manometer K . A 7-inch lengt,h (not shown) of No. 22 Teflon Spaghetti (Fluorocarbon Products Inc.) is connected to the inlet needle and immersed in the boiler water. The free extremity is located at' about the middle of the boiler. Thus preheated nitrogen bubbles through the water and is evenly mixed with steam. The gas is admitted slowly until manometer D indicates 40-mm. water pressure. Each condenser outlet is then connected in turn to flowmeter H . which should in-
(I. Connections
dicate somewhat less than 50 mm. per minute gas flow. The fore pressure read on manometer K (around 100-mm. Hg) should be reproduced in all subsequent operations. Manometer K is permanently attached to the apparatus (see Figure 3, D ) . Heating Rate. Manometer D (Figure 4, I) is removed from outlet L and the cooling tanks are filled with ice water. The heater is then turned on. With a 55-ohm heater in the 10unit apparatus, water is brought to boil in the manifold in 20 to 22 minutes under 115 volts (250 watts). Outlet L is then closed with a pinchclamp and the powerstat connected to the heater is set a t 55 volts (130 watts) which should ensure a distilling rate of 15 ml. per hour per microstill. Under these conditions the steam temperature will be 97" C. and a flow of 70 nil. per minute will be indicated on flowmeter H connected to each condenser in turn. Final adjustment of stainless steel screws J are made a t this time to ensure a uniform 70 ml. per minute nitrogen flow through all stills. Steam temperatures in excess of 100" C. correspond to unnecessarily high pressures.
latter is clamped shut and the powerstat set for operational heating rate. About 5 minutes later, a check of the flow rates is made by connecting each condenser in turn to flowmeter H (Figure 4, I). Condensers are removed in succession when filled and optimal distillate volumes are determined as follows. Optimal Distillate Volume. A curve showing amounts of steam-distillable solute entrained for given volumes of total distillate can be established for any type of material. The use of such curves will considerably simplify the routine distillation of many samples of the same type provided they 'are obtained under .operating conditions reproducible in all subsequent runs. Curves answering this criterion are easily established with the present apparatus by loading all microstills with 500 pl. of test solution and collecting conveniently scaled-up volumes of distillates by removing the receivers in succession. The weighed contents of individual receivers are then analyzed and per cent recoveries plotted against distillate volumes. Performance Study. This point is demonstrated by the data given in Figures 5, 6, and 7 . In these examples, titrations were carried out with standard solutions of sodium methoxide matching the concentrations of initial test solutions and delivered under nitrogen from microburets to the magnetically stirred distillates. The minimum fresh 0.01% alcoholic phenolphthalein solution that gave a visible end point, was employed. Carbon dioxide-free distilled water was used and blank corrections were applied. Total acid values were determined directly on the test solutions
60
ROUTINE OPERATIXG PROCEDURE.
@PROPIONIC
-
0 5%
~ P R O P l O N l C-... 0 05% BUTYRIC 0 5%
'OI
@BUTYRIC
0.05%
1 I
Figure 5. Elimination curves for the lower fatty acids
1932
ANALYTICAL CHEMISTRY
The required volumes of test solutions are introduced in microstills with calibrated syringes. If necessary, the num.her of stills needed to occupy all positions in the manifold is completed by filling stills with 0 . 5 ml. of water. Each microstill is then fitted with the corresponding Teflon stopper and inserted in the manifold. Moderate pressure is exerted in tightening the stoppers which expand upon heating. Each microstill is then connected to a condenser and the tanks are filled with ice water and some chipped ice uii t o the level X indicated in Figure 1. The nitrogen fore pressure is set a t the ol~erational level and the heater turned on full. ;is a steady flow of steam forms a t the steam outlet, the
''\0~7,-PRO PIONIC
1
1
'
1
'
1
5
VOLUME
RATIO IO
Figure 6. Semilogarithmic plot steam distillation data
1 of
Insert. Variation of formic acid yield in IO volumes of distillate with initial concentration
MATERIALS.With the exception of formic acid (Baker Analyzed Reagent) pure fatty acids (hlatheson Coleman & Bell, East Rutherford, X. J.) were used. -411 were analysed individually by GLC. By far the largest impurity content was found in valeric acid (valeric, 90.707,; isovaleric, 6.107,; butyric, 2.047,; propionic, 0.5%; acetic, 0.58%; formic, traces). These analytical data were taken into account in expressing the composition of standard mixtures. Fisher C-184 reagent grade carbon disulfide was employed in preference to P I FROM 0 0 5 % the spectroscopically pure grade. The 840TO I O % 5 3 20% 20% detector flame was not snuffed out I ACETIC I ACID when 4 bl. of CS2 were injected under 30- , i E ....20% the proposed conditions, and the recorder pen returned almost to base I line less than 1 minute after injection. Xylene (Fisher X-5) was used as an internal standard in preference to other compounds tried for this purpose. The behavior of compounds emerging ahead of xylene-i.e., on the tail end Figure 7. Influence of salinity, curves of the sol\-ent band-was understandI, 11, and 111; elimination of mixed acids, ably erratic. For those emerging after xylene and before acetic acid-it., in curve IV the strong, undetected formic acid band--the response was inconsistent and per cent recoveries calculated from also. them. ,Inhydrous formic acid was prepared Analysis of Distillates. APPARATUS by adding to 100 grams of ice-cold 90% A N D TEcHxIQuEs. The Perkin-Elmer formic acid (cf. above), 30 grams of 154 vapor fractometer, the related P205 by small portions while stirring ionization detector and electrometer under a blanket of dry nitrogen. unit, and the 1-mv. P h i l i p P R 2216 Fractional distillation (Vigreux) of the A/21 recorder, were installed accordresulting clear mixture under 100-mm. ing to manufacturers' instructions pressure (b.1). 43" to 44" C,)! yielded a except t h a t t h e chroniatograph (oven, 50% middle fraction containing 0.37, fan, etc.) was electrically dissociat'ed water. Both the 10% forerun and the from the rest of the apparatus undistilled remainder were discarded. (elertrometer, recorder, etc.) by conFORRIIC ?ICID-XYLEKEIK CS2 SOLUnecting the latter to a separate line TIOK (FXC). To 400 ml. of CS2 kept free from disturbances from other placed in a dry separatory funnel were equipment. h t 123" C. and attenuaadded 5 ml. of anhydrous formic acid. tion x4, less than l(% drift per hour This \vas shaken for 1 minute and left was consistently ob standing for an hour. The bottom tained operation. layer was then collected in a 500-ml. rubber septa (WilE;en!i Instrument and dry glass-stoppered bottle. If necesResearch, lnc.) were dsed ( S I ) , and in sary, a small volume of CS2 was added all cases 4-pl. injections of the carbon to remove a slight haze. To the clear disulfide solutions were made using a solution, volumes of a 1:20 dilution of 10-pl. Hamilton syringe. The syringe xylene in CS2 were added with a microwas fitted with the Chaney adapter and syrinqe. The mixture was tested by also with the needle guard described injecting 4 pl. into the chromatograph in a previous article 1:31), No stream olierated under the conditions previsplittrr was used ; the ,samples were sent ously described. Enough of the xylene in toto to the detector. The 42-inch solution was added to produce a xylene long, 1, 4-inch o.d. stainless steel column peak height (Figure 8) of about 80 was filled with 207, diethylene glycol chart divisions. Because of the variaadipate ~,olyesterl)hos].ihoricacid (Dega, tions in isomer composition of comZ 101 from Lachat Chemical Inc.) mercial xylene, the volume required in on 60- to 60-mesh acid-washed Chroour experiments, 160 p l . , should be mosorb IT (Johns-3.Ianville Corp.). considered as a guide only. Before use, the column had bee; The formic acid titer of F X C solugradually heated under helium from 100 tions was determined by repeatedly to 150" C. over a period of 6 days. extracting 25 ml. with water and titratThis column was used to perform over ing the extract volumetrically. The 1800 analyses.. Over a period of 2 FXC solution prepared as described >-ears the most notable change was abore was at least 0.15&Y. I t could a slow increase in porosity. Peak be kept unchanged for many months elution times were adjusted to their when stored a t 5" C. in 60- to 100-ml. original values simply hy adjusting the glass-stoppered (silicone grease) bottles. carrier gas flow rate. using the peak GEKERAL PROCEDURE. The steam elution time of valeric acid (10.76 distillates are neutralized to a faint minutes) a:: reference. Consequently, pink coloration to phenolphthalein with the required flow rate changed from standard S a O H solution. - i n aliquot 80 to 60 nil. per minute over the 2-year is then evaporated to dryness in an oil period. .I slight increase in resolution bath (110' C.) under a jet of nitrogen. power was also observed. I
DISTILLATE/SAMPLE 2 3 4 5 6 7
VOLUME RATIO 8 9 IO I I I2
~
~
1
Figure 8. Chromatogram obtained by injecting 4 PI. of solution M (Table
11). Carrier gas-helium, 60 ml. p e r minute. Temperature-column a n d detector, 1 2 3 " C.; flash heater, 200°C. Settings-electrometer, XI, X 1 6 ; recorder, 0 to 1 mv. Insert. Sample flask. Evaporation of neutrali z e d steam distillate a n d transfer of acids to FXC solvent w e r e both carried out in the same flask
The residue is heated in an oven at 150" C. for 30 minutes and cooled in a vacuum desiccator. The final test solution is obtained by adding a measured volume of F X C solution and shaking. From three to four 3-mm. diameter glass beads added before evaporation help in the mixing. Four microliter injections of these solutions are made into the chromatograph operated under described conditions (Figure 8). The concentrations are computed from calibration curves established by treating in exactly the same manner measured volumes of standard aqueous solutions of mixed sodium salts prepared as described below. K i t h the simple flasks exemplified in Figure 8, final volumes from 0.1 to 1 ml. can be used in accurate estimations (Tables 111, IF'). Eva1)oration and acid release are carried out without loss in the same flask. SERIALDETERMINATIONS. With samples which do not differ too much in relative composition, appreciable time and accuracy are gained by observing t'he following simple protocol. Initial neutralization of steam distillates is effected by volumetric titration using the method described in the first section. By weighing, or by transferring the neutralized distillates to volumetric flasks and bringing up to volume, it is then possible to take aliquots that Kill contain a given uniform total of micromoles of acids. Evaporating to dryness and adding the same volume of F X C solution to the desiccated residues will VOL. 3 6 ,
NO. 10,
SEPTEMBER 1964
1933
Table I.
Relative Composition of Standard, MC, and Test Mixtures, MQ, MS, MT Acid content of 25-ml. stock solutions
Acid mixtures
Acetic,
Propionic,
Butyric,
%
%
9%
MC
28 06
$8 MT 4
20 14 46 36
6 02 5 22 39 49
52 61 40 50
18 29 4 15
Weighta in 25-ml. stock solution,
Valeric,
9% 33 50 43 8
10 35 53 38
mg. 279 1 255 9 204 2 153 4
32 02 85 63
Neutralized and brought to 25-m1. volume.
distillate (Figure 6, insert). The reproducibility was +l%--i.e., within the precision limits of the titration method. Obviously then, it is possible to arrest the distillation a t this point, thus completely recovering all acids except formic acid for which a correction can be applied. Figure 6 shows the data fitting a straight line when plotted semilogarithmically, a property which indicates the simple relation
c Table
II.
Dilution of a stock solution
Acid Content of Standard Solutions M, N, P, Q, and Test Solutions
M
N
MC/25
MC/50
R, P
S, T
MC/100
Q
R
MC/200
hIQ/25
S
T
hIS/25
hIT/25
42 70 379.0 37.03 358.0
24 20 223.9 94.3 53.1
Acid content, Wg./ml. _ _ 5. ~ 229.0 201.8 372.0
234 6
A r e_ t i c_ _ _ .312 _ _
_.
Propionic Butyric Valeric
171.9 151.4 279.0
~
158 2 114.5 100.9 186.0
then produce concentrations of acids falling within a restricted range. When t,he concentration of distillates is very low, the securing of sufficient material for GLC analysis may require evaporation of the whole titrated distillate. A uniform concentration in total acid is then obtained by using calculated TTolumes of F X C solution. STANDARDSOLUTIONS.A standard mixture of pure acids is obtained by weighing. A standard stock solution (Table I) is prepared by weighing aliquots in 25-ml. volumetric flasks. The contents are then neutralized (faint pink to phenolphthalein) with aqueous NaOH solution, and brought to the mark with distilled water. Standard solutions of various strengths are prepared (Table 11) by placing aliquots of the standard stock solution in 25-ml. volumetric flasks and bringing to the mark. These solutions will keep for many months when stored at 5 " C. with the stoppers silicone-greased. DETERMINATION OF FORMIC ACID. This analysis is effected on 0.5 ml. of steam distillate by the colorimetric method of Grant ( I S ) which can be carried out in the presence of other acids. I n this method, formic acid is reduced to formaldehyde by Mg ribbonHCl, and the intensity of the color developed after addition of chromotropic acid reagent, is compared to that obtained with a series of similarly treated standard solutions. The sensitivity and accuracy of this simple method are comparable to that of the present GLC method for the other acids. With the proposed multiunit apparatus, it is convenient to arrest the distillation when 10 volumes have been collected. This corresponds to the essentially complete recovery of all acids except that of formic acid, for which a t least 20 volumes would be 1934
ANALYTICAL CHEMISTRY
78 10 57.25 50.45 93.00
61 85 149.60 300.40 512.00
required. The percentage fd of formic acid recovered in 10 volumes varies linearly with the concentration of initial solutions from 0.05 to 1% (Figure 6). It is given by the empirical expression fd = 89 0.14d, where d is the concentration of the distillate in micromoles of formic acid per milliliter as determined by the colorimetric method. The value f d , which is reproducible within +l%, is then used to calculate the formic acid content of the starting material.
+
DISCUSSION
Steam Distillation. As expected, elimination proceeded considerably more slowly for formic and acetic acid (Figures 5 and 6) than for higher acids. Ten volumes of distillate per volume of test solution were needed for >99% recovery of acetic acid as against only four volumes for propionic acid, for example. Elimination of butyric acid was even faster, the rate increasing in the series up to a maximum for nonanoic acid (33). A practically complete recovery of formic acid required about 20 volumes of distillate. Interestingly, recoveries of acetic acid were equal for equal distillate volumes over a fairly wide range of test solution concentrations. Small differences observed in this regard for other acids were confirmed in triplicate experiments. The recovery of formic acid from solutions of different concentrations was studied with particular care (20 determinations for each concentration). A linear relationship between per cent recovery and initial concentration was found for 10 volumes of
=
Ky
(1)
where c is the concentration of acid in the condensate running to the receiver, and y the concentration of acid in the sample flask. The value of K , a constant characteristic of each acid, is given by the slope, - K , of the straight line. Verification of the above thermodynamic property by the data demonstrates a theoretical efficiency for all microstills and the efficiency of the system as a u hole. The thermodynamic property was not altered, and the complete elimination of acids required the same volume of distillate when changes up to lOOyo were made in the rate of steam production. Attempts to improve the efficiency of acid removal by adding large amounts of sodium chloride to test solutions failed with all acids except acetic for which a distinct salting-out effect was observed. I n this case, competition for water did result in lessening the extent of association of the acid with the solvent. Curve 111, Figure 7 , shows a somewhat faster rate of elimination than curve I obtained with a salt-free solution. Addition of salt to the manifold water-Le., increasing the temperature-resulted in a lessening of this effect (curve 11). The modifications described did not bring considerable improvement in acid distillation efficiency and can therefore be omitted. The use of more effective dehydrating agents is limited in many cases by their effects on other constituents of samples, particularly those of biological origin, in generating undesirable steam-distillable decomposition products. KO difference in the rate of elimination mas observed for any acid when 1% octyl alcohol !$as added to the sample. The antifoaming properties of this compound can thus be used safely when required. Subsequent evaporation of neutralized distillate (see Experimental, Analysis of Distillates) completely eliminates the alcohol entrained. I t can be concluded therefore that the >99% recovery of mixed acids excluding formic acid will require 10 volumes of distillate. This is confirmed by experiments with mixed acid solutions (Figure 7 , curve IV) and various
Table 111.
Statistical Calibration Data for Standard Solutions M, N, PI Q
Soh.
No. of trials
Compound
Concn., dml.
M
10
XY .4 c Pr Bu Va Total
...
P
10
Va Total XY
0
R
10
=
...
AC
2.60 1.54 1.15 1.82 7.11
2 Pr
...
Pr Bu T-a Total
Q
5.20 3.09 2.29 3.64 14.22 ... 3.90 2.32 1.72 2.73 10.67
1.30 0.77 Bu 0.57 0.91 Va Total 3.55 arithmetic mean of peak heights H .
Quant. injected, lrg.
fl,.
chart div. 88 3 86 1' 63 2 67 7 67 6
2cBRjc =!= 7 c
3.6 4.1 3.5 2.9 3.1
97.5 71.5 76.7 76.6
87 7 66 8e 46 0 49 6 49 6
1.53 1.21
3.5 3.6 4.8 4.8 4.2
76.1 52.4 56.5 56.5
8
1.81
l e
0.458 0.404 0.744
87 43 30 32 32
5 1 6
1.0;
0.71 0.62 0.73
4.1 4.1 4.5 3.9 4.5
49.0 34.7 36.5 36.0
0:3i3 0.229 0.202 0.372
88 21 15 16 16
0
1.73 0.80 0.59 0.45 0.55
3.9 7.3 7.6 5.5 6.7
23.8 17.7 18.5 18.5
:iio 0.916 1
0,807 1.487
:
0 937 0,688 0.604 1,115 0: 625
06
6 3 3
1.11 1.11
1.03
...
...
...
...
0.31 0.26 0.42
0.34
...
0.63 0.73 1.10
0.83
...
...
0.30 0.32 0.41 0.37 I
.
0.40
1.20 1.45 1.31
...
.
0.26 0.20 0.21 0.23
1.06 1.18 1.16 1.28
...
...
0.20 0.17 0.12 0.16
1.70 1.92 1.30 1.73
SH/SR
...
4.1 3.5 3.7 3.1 ... 4.0
3.7 2.7 2.2
... 4.1 3.5 3.0 3.2
...
4.0 3.5 3.8 3.4
d%HaA)
s = with 'I'= 10; s = estimated standard deviation. .1' - 1 2 C V X = 2 x coefficient of variability = 95% confidence limits approximately. d M R H = mean ratio of H to xylene peak height. e Corrected for acetic acid content of 4 pl. FXC solvent (0 7 chart divisions). b
...
1.61 1.27 0.91 1.24 1.06
c
fluids of biological origin with or without the addition of known amounts of standard mixed solutions. This rule applies to any sample which does not contain relatively large amounts of the higher (>C12) acids (33). The examples above show the type of information which can be swiftly and reliably obtained through the use of the present apparatus. Following the determination of optimal conditions, the routine processing of many samples requires less time and attention t'han are involved in a single operation with a single-unit apparatus of former design. I n the present design the sample flask (microstill) is located in the boiler. This compact arrangement permits a multiplicity of stills .to be connected readily to the boiler. As many as 20 microst'ills can be conveniently grouped on the same manifold. The microstills are robust units which are easily disconnect'ed for loading or cleaning. The scope of applicat'ion of this type of apparatus obviously extends to any type of material which can be successfully isolated by steam distillation. The use of microstills further extends the range of application to cases where only very small sampies are available or where a reduction in preparative scale results in appreciable economy of time and effort. The described microstills will handle volumes of test solution from 250 to 750 p l . In most cases this range of volumes corresponds to
2 C V H = 200 SH/R; 2 CVB
amounts of distilled solutes which can be conveniently and accurately estimat,ed by flame ionization GLC, spectrophotometric, or other sensitive analytical method. Simple modifications of the present design have led us to stills of 50 pl. capacity. The low steam output per still has permitt'ed the grouping of condenser and receiver in a single unit of simple design. Tube R , Figure 1, sufices to condense most of the distillate. I n t.he case of t'he lower fat'ty acids, a very small amount of condensation occurs a t the base of side-arm 1'. This is washed down to the receiver by placing a few drops of water in tube V and blowing through this tube while the joint is pulled slightly apart. High nitrogen flow rates tend to increase condensation in the side-arm. The recommended rate is sufficient to permit the smooth operation of all stills and to prevent backing up of sample fluid into the boiler should a decrease in heat input occur accidentally. GLC Analysis of Distillates. T h e concentrations of volatile fatty acids in steam distillates obtained from biological materials often fall below the range for accurate determination by direct injection with t'he flame ionization detector. The procedure, consisting in evaporating neutralized steam distillates, is rapid when carried out under the conditions already described and provides a
=
200 S R / M R H .
mean3 of concentrating and storing, aithout loss, many samples prior to their serial determination by GLC. I n contrast with previous methods, the present one proposes a simple quantitative procedure combining the release of acids from their salts and their transfer to organic solvents. While water or acetone containing an excess of formic acid can also be employed for this purpose, the use of carbon disulfide is more advantageous in the subsequent GLC analysis. K i t h this solvent, a greatly reduced initial disturbance (10, S I ) permits the use of shorter elution times. Furthermore, detector signal stability is appreciably greater throughout. Consequently, more accurate analyses can be carried out a t lower signal attenuation. The data in Tables 111 and IV show the degree of reproducibility and accuracy attainable a t the submicrogram level. -1short column and a relatively ,-mall number of normal acids were used to simplify the task of aecuring enough data for a statistical anal? sis The results (Table 111) indicate ( s I I / s R ) that the precision of direct calibration ( S H ) was about one-quarter that with an internal standard ( s R ) . I t further establishes that duplicate analyseq by the results procedure will yield estimated values (2CT.'R/ d2) comprised within +lyGof the calculated one. 95% of the time, for acid concvmtrations exceeding L'OL.36, NO. 10, SEPTEMBER 1964
1935
(3) Averill, W J Gas Chromatog. 1, 22 (196%). ( 4 ) Burchfield, H. P., Storrs, E. E., "Biochemical Alauhcations of Gas Chroma 1 oerauhv." Academic Press. X P ~ ~
Table iV.
Results Obtained with Test Solutionsa
Acid, pmoles/ml. _ ~ _.___ Propionic__ _ Butyric-AceticCalcd. Found Calcd. Found Calcd. Found 1 03 1.02 2 02 2 03 3 41 3 43 1 02 2 02 :i15 (1 05) (2 00) (3 30) s 0 71 0 70 5 12 5 10 0 42 0 43 0 70 5.13 0.43 (0.67) (5.10) (0.45) T 4.03 3.99 3.02 3.00 1.07 1.06 4.04 3.03 1.08 (4.05) (3.04) (1.08) a Average of duplicate determinations. Quantities in parentheses direct calibration. Test soh. R
2 pnoles per ml. Means of adjusting the final concentrations have been described. With an int,ernal standard, duplicate injections are sufficient in determining ca1il)ration values for most ~)url)oses. When adjustment of the final concrntrations to a restricted range is feasible, only two calibration values arc necrssary. Confirmation of the st'atistical data was obtained by analysis of test solu( T a b l ~IT). l'hrsc rrsults were ned by using peak heights. -\s previously demonstrated ( S I ) , peak heights constitutc rtdiable and Iiracltical means of nicasuring This is not suriirisin of pr:& height by adjusted ]leak elution time is an accurate substitute for the use of Ileal; arcas ( 5 . 2 2 . 2 7 ) , and since the rrproducibility of peak elution times is exccllcnt ( 1 0 . 2 7 , in the present' work). These conclusions are undoubtrdly applicabl(~ to columns permitting a complete separation of both normal and isoacids. A 6-foot column filled with the .Ilctcalfe-type packing employed ( 2 ~ 526) ~ will completely sel~arateall but isobutyric from iiropionic acid. Our expcricncc, n-it11 more selective packing mat,crial; ( 2 , S, 1 2 ) i. not estcnsive enough t o afford a com1)arison on the basis of 5taI)ility. The 1)resrnt coluinn s h o w iininipaircd properties after countlws hour.* of operation. The forniic acid of FXC solvent dots not modify thc dctcctor rosl)onse to t'he lower fatty acids. l'he rel(,ase of acids from theii, salts results, not only from
1936
ANALYTICAL CHEMISTRY
I-aleric Found 5 02 .i-22 ( 5 00)
Calcd. 5 01
I
3 02
3 02 3.02 (3.10) 0.52 0.51 0 53 (0.54) obtained through
the higher acid strength, but also from the large excws of formic acid used. In the Iiresent experiments, the FXC solvent rontaintd 164 pniolcs formic acid 1)er nil. and the conccntration of other acids nri-cr exceeded 5.2 pnioles per inl. When this 1)roportion was decreased to 20: l , the releasc of acetic acid was only 95YG. I n the i~rescnt case ('Table H I ) , a slight but definite nonlinearity of the dettctor response was observed below the microgram range ( S I ) . These results con>titute a further demonstration of thc necessity for calibration of the flame ionization detector in quantitativc \vork. and of the usefulnw of a statisticd anal! GLC data. Previously reached by the present author ( S I ) , these conclusions can tie extended to all methods of detection following the recent publication of a report on annl~-ticalGLC accuracy in q~ccializcdlaboratories ( 1 5 ) . ACKNOWLEDGMENT
The technical assi*tance of IT. Grant Layng in distillation exlieriments is gratefully acknowledged. LITERATURE CITED
( I ) .Ickman, It. G . , Burgher, R. I)., X N A I . CHEM. . 35, 647 (1!)63). ( 2 ) Averill, \I.., "3rd International Gas
Chromatography Synip(isiuni,'' S . Brennei, J. 1:. Callen, 11, 1). Weiss, eds., p. 1, hcadeinic Press, S e w I-urk, 106'2.
Yorkr 1665. ( 5 ) Caroll, K. K., Satitre 191, 377 (1961). (6) Clague, J. A . , Food Res. 7, 56 (1942). ( 7 ) Clark, E. P., Hillig, F., J . .-lssoc. O f i c . .tgr. C'hemists 21, 685 (1938). (8) Craig, E. M., Nurty, S . L., J . A m . Oil C'hmist:3 ' SOC.36, 549 (1959): ( 9 ) Ikwvnr, R. .I.,J . C'hroniatog. 6, 318 (1961 ~ . i, . j~
(10) Emery, E. XI,, Koerner, IT. E., ANAL.CHEM.33, 146 (1961). (11) Erwin, E. S., hlarco, G. L., Emery, E. AI., J . Dairy Sci. 44, 1768 (1961). (12) Gehrke, C. E., Lamkin, IT, M., J . =Igr. Food Chem. 9, 85 (1961). (133 Grant, IV. M.,,Isar,. CHEM. 20, 267 ( 1048). (14) Hankinson, C. L., Harper, IT. J., Nikalajcik, E., J . Dairy Sci. 41, 1502 (19%).
(15) Homing, E. C., J . L i p i d Res. 5 , 20 11964). (16) Hnghes, R . B., J . Sci. Food A q r . 11, 47 (1960). (17) Hunter, I. R., J . C'hronmtog. 7, 288 11962)). (18) Hunter, I. R., Xg, H., Pence, J. IT., ASAL. CHEM.20, 1757 (1960). (19) Hunter, I. R., Ng, H., Pence, J. \T., J . Food Sci. 26, 578 (1961). (20) Hunter, I. R., Ortegren, \-, H., Pence, J. IT., ASAI.. CHESI.32, 682 (1960). (21) James, A. T., Martin, A. J. P., Biocheni. J . 50. 682 11960). (22) Kateman, G., J . Chromafog. 8, 280 i1061
( 2 3 ) Iiunieno, F., J . .tgr. ~ ' h e n i . SOC. J a p a n 36, 181 (1962). (24) Lovelock, J. E., Asar.. CHEY. 33,
16.'
[ 1961).
123) .\letcalfe. L. 11.. J . Gas Chronzatoo. 1, 7 (1962).' (26) lletcalfe, L. U., Suture 188, 112
(1960). (2TJ Pecsok, 11. L., "Principle and Practice of Gas Chromatography," p. 145, ITiley, S e w York, 1959. (28) Kalls, J. It-., AYAI..CHEJI. 32, 332 il960). (2G) Smith, E.I>., Gosnell, A. B., Ibid., 34, 438 (1962). (30) I?auppj G., ilngew.. ('hem. 71, 284 ( 1!l50 ) . (31) Vandenheuvel, F. A , , .4SrzI.. CHEM. 35, 1186 (1'363). (32) \-orbeck, 11, L., llattick, 12. R., Lee. F. A , . Pederson. C. S.. Sature 1871 689 ( I i 6 0 ) . (33) \I'eeninc,k, R . O.>.\-ew Zealand J . Sci. 1, 18 (1958).
RECEIT h D for revlev January 13, 1964 Resubmitted April 3, 1061 Accepted June 12, 1961 Cont~ibut~on So 166, Anirnal Research Institute
