The acid value obtained using HC1methanol was 2.8, using diazomethane 1.4, and with BF3-methanol 1.1. Finally, the esters from each procedure were analyzed by GLC on an instrument designed in4this laboratory, using thermal conductivity detection and a 4-foot polyester column (Lac 1, R-296). The amount of each ester was calculated from the area under the curve using triangulation. Table I1 gives the average results obtained from four separate GLC runs on each sample. The results from the three methods agree fairly well. The esterification method described has been used for the routine preparation of methyl esters of fatty acids. Its main advantages are rapidity,
stability of reagent, and simplicity of apparatus. The procedure has been successfully scaled down to prepare esters on 1- to 10-mg. samples and has also been used on samples as large as 5 grams. Other acids that have been esterified successfully are 12-hydroxystearic acid and 12-ketostearic acid, Epoxy acids probably could not be esterified with this reagent without opening the oxirane ring. Fatty acids containing conjugated unsaturation have not been examined for changes after esterification with BF3-methanol. LITERATURE CITED
(1) Bottcher, C. J. F., Woodford, F. P.,
Boelsma-van Houte, E., van Gent, C. M., Rec. trav. chim. 78,794 (1959).
(2) Craig, B. M., Murty, N. L., J. Am. 021 Chemists' SOC. 36, 549 (1959). (3) Hornstein, Irwin,. Alford, John A., Ellion, L. E., Crowe, P. F., ANAL. CHEW32,540 (1960). (4) Hudy, J. A., Ibid., 31, 1754 (1959). (5) James, A. T., J . Chromatog. 2, 552 (1959). (6) Mitchell, J., Jr., Smith, D. M.,
"Aquametry, Chemical Analysis," Vol.
5, pp. 297-309, Interscience, New York, 1948. (7) Mitchell, J., Jr., Smith, D. M., Bryant, W. M. D., J. Am. Chem. Soc. 6 2 , 4 (1940).
(8) Schlenk, Herman, Gellerman, J. L., ANAL.CHEM.32, 1412 (1960). (9) Stoffel, Wilhelm, Chu, Florence, Ahrens, Edward H., Jr., Ibid., 31, 307 (1959).
RECEIVEDfor review July 25, 1960. Accepted Kovember 9, 1960.
Quantitative Determination of Sugars by Rapid Horizontal Paper Chromatography J. 6. HIMES, L. D. METCALFE, and HELMA RALSTON Armour Industrial Chemical Co., McCook, 111.
b Rapid, high-temperature paper chromatography was used to determine sugars in a mixture. By using methyl ethyl ketone, propionic acid, and water as a solvent, a number of sugars in a mixture were separated in 2 hours. For quantitative determination, densities of the colored spots were read on a densitometer.
T
utilization of several sugars by microorganisms in fermenting liquors was investigated in this laboratory. Three to seven sugars were used in these fermentation experiments. It was necessary to provide a rapid quantitative determination of each sugar in these broths. Paper chromatography appeared to be the solution t o this problem. A number of investigators have been separating and determining sugars by paper chromatography with good qualitative and quantitative results for a number of years, using various descending or ascending solvent techniques a t 20" to 25" C. However, the time required for the separations is 16 hours to 45 days. Some of these techniques have been investigated a t higher temperatures by Hough, Jones, and WadAlcock and Cannel1 ( I ) , and man (4, Counsell, Hough, and Wadman ( 2 ) . Elevated temperatures for paper chromatography require a development chamber of small volume to maintain equilibrium conditions, since the conHE
364
e
ANALYTICAL CHEMISTRY
stant relationship of liquid and vapor compositions cannot be maintained in a classical-type development chamber. Roberts and his coworkers (7) use a small horizontal rectangular chamber for paper chromatography of amino acids and sugars at 60" C. A modification of that apparatus (5)is used in our laboratory for amino acids, with methyl ethyl ketone, propionic acid, and water as the solvent system. Horizontal paper chromatography was successfully used with this solvent system to separate 7 sugars in 2 hours a t 60" C., the solvent front moving about 20 cm. The sugar spots were developed with a butanol solution of aniline phosphate. The colored spots of the chromatographed sugars were read with a densitometer for quantitative results. EXPERIMENTAL
Apparatus. Chromatographic Chamber. The chromatographic chamber has been described (3). A similar chamber is sold by Labline, Inc., Chicago, Ill., under the name Speedicell. Densitometer. The densitometer is a Photovolt densitometer, Model 525 (Photovolt Corp., Newark, N. J.), consisting of a light source unit and a meter unit with photocell scanning arm. A Varicord recorder and an automatic scanning motor, also supplied by Photovolt Corp., are connected to the basic two-unit densitometer to obtain an
automatic scanning and recording apparatus for paper strips. Filter Paper. Whatman No. 1 chromatographic filter paper sheets (18 X 22l/2 inches) were cut into 8 X l l l / r inch sheets for use. Reagents. Chromatographic Solvent. The solvent is prepared by first mixing 25 ml. of propionic acid and 75 ml. of methyl ethyl ketone, and then adding 30 ml. of distilled water to the mixture. High-grade solvents such as are available from Eastman will give good chromatographic separation without redistillation. Sugar Spot Indicator. One volume of 2N aniline in 1-butanol is mixed with 2 volumes of 2N HsP04 in 1-butanol, and the resulting precipitate removed by vacuum filtration. Sample Preparation. A solution of a sugar mixture or hydrolyzate is prepared in water so t h a t each sugar present will have a concentration of 10 to 20 mg. per ml. of solution. A solution containing a known mixture of sugars, resembling the unknown, is made in the same manner to be chromatographed on the same paper. The sugar solutions are kept frozen until ready for use. Procedure. Using a 1 4 . ultramicropipet, 1.0- and 2.0-~1. aliquots of both the known and unknown solutions were placed on the paper so that the spots were l/z inch apart, l 1 / 2 inches in from one of the &inch edges. As many as three known and three unknown solutions may be run per sheet. The first and last sample spots should
a70. 060
ARAB,hOSE
A
960'
RIBOSE
0.501 450LL
r 0 40-
w g0.40-
w
(L
c
B
0.3C
%0,0
-
~
0 201
0.20'
0.10.
I
8
9
IO
I1
olsrh2,cE , c M
15
I'
Figure 1. Absorbance of sugars vs. distances traveled on chromatogram
be 3/4 inch from the edges of the paper. After each I-pl. aliquot was applied t o the paper, a stream of warm air was used to dry it. Then another 1.0-p1. aliquot was overspotted for the 2-pl. sample. A l'/s-inch t a b was folded at a right angle to the paper, 3/8 inch below the line of sample spots. The opposite end of the paper was folded to form a l/p-inch tab. The paper was placed in the chromatographic chamber, which had been preheated for 1 hour at 60" C., and was positioned so t h a t i t lay over the glass rods, with the 11/8-inch tab extending down into the solvent trough at one end of the chamber. The plate glass was then clamped in place over the chamber. By means of a funnel, 25 ml. of solvent was delivered to the bottom of the chamber through one of the holes in the glass cover. Another 30 ml. of solvent was placed in the glass trough through the other hole in the glass cover. The holes were closed with cork stoppers. The chromatographic chamber was maintained a t 60" C. for 2 hours. The paper was removed from the chamber at the end of this time and hung in a n exhaust hood to airdry for 30 minutes. The dried chromatogram was dipped in the I-butanol solution of aniline phosphate and again hung in the exhaust hood for 30 minutes to dry. The paper was then placed in an oven a t 80' to 85" (3. for 30 minutes t o promote the color reaction of sugars and aniline phosphate.
Estimation of Sugars. The intensities of the colored sugar spots were read at 445 mp on the densitometer with a slit width of 1 mm. RIeasuring the maximum color density of the individual sugar spots ( 5 , 6 ) Kas found satisfactory. When this technique is used, the color density of the unknown must fall n-ithin the density color range of the known solution. Therefore, one of the two dilutions of the unknown was compared with the dilution of the known which it most closely resembled. The percentage of each sugar in each
Figure 2. Concentration curves of some sugars chromatographed on Whatman No. 1 filter paper and developed with aniline phosphate in 1 -butanol
A
Galactose Glucose Sucrose
unknown solution was calculated from the total weight of the sample in the original spot on the paper, from the weight of each individual sugar in the corresponding known mixture on the paper, and from the ratio of the color densities. Thus, %sugar
=
Ru 100 Ws X X Rs Wu
where
Ws
=
Ru
=
Rs
=
Wu =
weight of desired sugar in standard mixture on paper absorbance reading of sugar spot of unknown absorbance reading of sugar spot of standard mixture total weight of unknown sample on paper
If duplicate analyses were made, the samples were chromatographed on different papers. RESULTS AND DISCUSSION
The sugar solutions were ordinarily prepared in distilled water. However, it was found that HC1 up t o a concentration of 2.ON (as used for hydrolysis) did not interfere. If higher concentrations of HCl were present, the separation of the individual sugars was impaired. Variations in solvent ratio: temperature, or length of time for development did not improve Rf values. The RI values of chromatographed individual sugars (Table I) were consistent and reproducible when the same operating conditions were used. A synthetic mixture of sugars was chromatographed on six different papers along with a suitable known standard (Table 11). Estimation of the sugars
Ribose
I Fructose
V
lactose
in the mixture agrees well with the actual composition. To achieve this high accuracy it is necessary t o run a preliminary analysis on the unknown. From these data a standard is prepared t h a t closely resembles the composition of unknon-n. The analysis is again run, using the new standard. Figure 1 illustrates the type of graph obtained with the densitometer recorder. The distance each sugar
Table I. R, Values of Individual Sugars on Whatman No. 1 Filter Paper at 60" C.
(Solvent. Methyl ethyl ketone, propionic acid, and water. Color reagent. Aniline phosphate .) Sugar Rhamnose Ribose Arabinose Fructose Glucose Sucrose Lactose Average of Table II.
Rf
Valuea 0.68 0.60
Color of Spot Yellow-green Brown-red 0.53 Brown-red 0.50 Yellow 0.44 Gray-brown 0.38 Gray-brown 0.28 Brown four valuee.
Analysis of Known Mixture in Solution
Weight, Mg./Ml. Actual Foundc
Difference, Mg./Ml
Sugar Lactose 2 0 . 2 19.9 =k 0 . 3 Sucrose 2 0 . 3 20.6 d= 0 . 2 Glucose 10.1 1 0 . 4 f 0 . 5 Fructose 3 0 . 3 3 0 . 4 d= 0 . 4 Ribose 15.3 15.2 f 0 . 3 Arabinose 10.8 1 0 . 6 f 0 . 3 Rhamnose 10.0 10.0 =k 0 . 2 Average of six determinations.
VOL. 33, NO. 3, MARCH 1961
0.3 0.3 0.3 0.1 0.1 0.2 0.0
365
traveled on the chromatogram is shown on the abscissa; the density of each spot in arbitrary absorbance units is shown on the ordinate. The concentration of each sugar is calculated from its peak height. The arabinose and fructose peaks overlap in this graph. The change in slope at the lower shoulder of this peak was used to calculate fructose. Study had shown that this shoulder was actually fructose. However, a shoulder may be used as a peak height only when the known standard mixture is similar to the unknown. The color diffeiences of the various sugar spots (Table I) also facilitate the use of shoulders for density readings. Figure 2 shon-s the sugar concentrations plotted against the peak height
in arbitrary absorbance values. The curves were used to find the concentration range in which to prepare standards and samples. The most linear range is 10 to 20 pg. per ~ 1 .It is also evident that the color yields of the various sugars differ. During the preparation of these curves, other spots appeared on some chromatograms* Maltose and lactose contained small quantities of other identifiable sugars. However, the sucrose and fructose chromatograms had trace spots that were not attributable to sugars. I n general the procedure is a rapid, simple, and accurate method for quantitative analysis of sugar solutions. It should find many applications in biological studies and production control
requiring very rapid sugar determinations. LITERATURE CITED
(1) Alcock, Margaret, Cannell, J. S., Nature 177, 332 (1956). (2) Counsel1, J. p\T., Rough, L., Bradman, W. H., Research 4, 143 (1951). (3) Himes, J. B., Metcalfe, L. D., ANAL.
CHEM.31, 1192 (1959).
(4) Hough, L., Jones, J. K., Wadman,
H.p *’ 1950, (5)”. McFarren, E. F., Mills, J. A., ANAL. cHEY. 24, 650 (1952). (6) Roberts, H. R., Kolor, hI. G., Zbid., 29, 1800 (1957). (7) Roberts, H. R.,Kolor, M. G., N a t ~ 6 180, 384 (1957).
RECEIVEDfor review June 13, 1960. Accepted Xovember 2, 1960.
Automation of Ion Exchange Chromatographic Analysis of Condensed Phosphate Mixtures DANIEL P. LUNDGREN and NEIL P. LOEB Research and Development Division, Lever Brofhers Co., Edgewafer, N. J.
Rapid routine analysis of condensed phosphate mixtures in detergents i s possible when an ion exchange chromatographic column is coupled with a gradient elution system and connected to the Technicon AutoAnalyzer. This combination of techniques provides an automatic method which is relatively interference-free, and, consequently, no preliminary treatment of samples is required. The accuracy of the method is generally well within h 3 % of the amounts of the major components; minor components can b e detected down to levels of several hundredths of a per cent.
I
chromatographic analysis of condensed phosphate mixtures was simplified when Grande and Beukenkamp (1) published their gradient elution technique. Further improvements in speed, precision, and accuracy were developed by Kolloff (3). The procedures of these authors can be modified to yield still more advantages, especially in versatility and simplicity of operation, when flow rate and elution gradient are controlled by a n instrument. This instrument continuously analyzes the column effluent and graphically records its total phosphate concentration, thereby producing the elution curve. This arrangement permits automatic detection of the individual phosphate species which appear sequentially in the ef€luent. OX EXCHAXGE
366
ANALYTICAL CHEMISTRY
The sequence of operations required for the analysis is performed by the Technicon AutoAnalyzer (7). The instrument is programmed to conduct a quantitative acidic degradation of condensed phosphates; a portion of the resulting orthophosphate is isolated by dialysis and is then reacted with acidic ammonium molybdate to form phosphomolybdic acid. This compound is reduced with hydrazine sulfate and the concentration of the resulting heteropoly-blue complex is measured colorimetrically and recorded. The time required for an analysis depends upon the complexity of the phosphate mixture in the sample. -4 sample containing only ortho-, pyro-, and tripolyphosphate can be analyzed in a n hour. If the tetrameric phosphates and trimetaphosphate are also present, a complete analysis requires almost 2 hours. I n either case only 20 minutes of a n analyst’s time are needed. A calculation based on a weighted relative-area calculation of the recorded elution curre finishes the assay. ADAPTATION OF INSTRUMENT TO I O N EXCHANGE CHROMATOGRAPHY
The instrumentation and procedure described for total P205 determinations (4) are used essentially unchanged for monitoring an ion exchange column. The sample turntable is not used because the analysis is continuous. I n ion
exchange chromatography, the entire effluent from the column is pumped continuously into the AutoAnalyzer a t 2.0 ml. per minute to join with 6.66N sulfuric acid which is delivered continuously a t 0.7 ml. per minute. The steady states don-nstrearn from the proportioning pump are identical with those obtained in 20-per-hour sequential analyses (4) where sample solutions were pumped intermittently a t 0.7 ml. per minute to join with 3 . 0 5 sulfuric acid pumped steadily a t 2.0 ml. per minute. The switch from intermittent sampling to column monitoring can be made while the instrument is in operation, because the same set of tubes in the proportioning pump serves either application; only a momentnry perturbation in the base line is noted in making the change. The progress of a separation is controlled by the proportioning pump because the reservoir, constant-volume mixing chamber, and column are connected in series to the pump. The rapid response of the instrument permits the use of exponential elution gradients (6) which accomplish an analytical separation of condensed phosphate species in a minimum amount of time. A spare poly(viny1 chloride) tube is installed in the proportioning pump to facilitate regeneration and re-equilibration of an “off-stream” column. The AutoAnalyzer pump is powerful enough to handle with ease the additional load of the ion exchange accessories.
