1226
ANALYTICAL
CHEMISTRY, VOL. 50,
NO. 8, JULY 1978 DYNAPIC RANGE
point to restore the A spectra. This can also be done starting from the effective transmittance spectra. The appearance of the A, vs. A curve for r = 50 and 250 can be seen in Figure 2. For the regenerated A spectrum, we have now, by derivation
SNR,'
A h 10 (lowA+ r 2
=-
SNR,
10'
102
103
10'1
105
136,
lo\
(4)
and the limits of the A range are then the two or four roots of the transcendental equation:
where a' = (SNRA')MIN/SNRT. The limits of absorbance as a function of a'can be seen in Figure 3 for r = 50 and r = 250. Clearly, r should be kept small enough that the chosen a'can be exceeded over one continuous range of absorbance. Comparison with Figure 1 then shows that this range is far wider than a conventional cell's for any given a'. Higher improvements, probably not worth the effort, can be obtained from cells with > 2 simultaneous pathlengths. The computational capabilities of FT-IR instrumentation can thus be used to supplement their high SNR levels in order
Figure 3. Minimum and maximum absorbances and their ratio for a given minimum absorbance SNR relative to the 100 % transmission line SNR in a two-step cell for path ratios of 50 and 250
to greatly extend their dynamic range, by using specially designed sample cells. LITERATURE CITED (1) P. C. Hanst, A. Lefohn, and 6.Gary, Appl. Spectrosc., 27,188 (1973). (2) A. W. Mantz, Ind. Res., Feb. 1977. (3) A. W. Mantz, Appl. Spectrosc., 30, 459 (1976). (4) R. P. Baurnan, "Absorption Spectroscopy", Wiley, New York, N.Y., 1962, p 378.
RECEIVED for review October 25, 1977. Accepted April 13, 1978.
Two-Phase Sample Preparation and Concentration Technique for Sugar Derivatives Maria Martinez, David Nurok, and Albert Zlatkis" Chemistry Department, University of Houston, Houston, Texas 77004
There is a considerable advantage in preparing samples for gas chromatography in as concentrated a form as possible. This is of particular importance when determining trace components using an open tubular column where the sample is usually split before being introduced onto the column. In the analysis of nonvolatile compounds, such as sugars, it is necessary to derivatize before chromatography. This results in the compounds of interest being dissolved typically in 0.5 to 1 mL of reagent. Concentration can be effected by evaporation but this can lead to sample losses. These problems have been overcome by one of us ( 1 ) in the analysis of the kestoses, which are three isomeric trisaccharides that occur in low concentration in sugar cane molasses. Derivatives are prepared in a two-phase system such that essentially all of the silylated kestose is found in the top phase. The.& lylating reagent used is trimethylsilylimidazole which had previously been shown to be an excellent reagent for derivatizing sugars ( 2 ) . The top phase consists of hexamethyldisiloxane which is formed in situ or which may be added after silylation. The top phase occupies about 5 to 10% of the volume of the bottom phase and results in the sugar derivatives being concentrated by a factor of 10 to 20 times. For the kestoses, the error in using this technique as opposed to a single-phase technique is less than 3% when considering the same sample. This technique has proved useful for the analysis of trisaccharides in molasses (3) and also for the analysis of sugar impurities included in sucrose crystals ( 4 ) . T o illustrate the wider application of this technique, the analysis of sugars in beer, honey, sherry, and raisins is discussed in this note.
EXPERIMENTAL Reagents. The following were used Trimethylsylilimidazole 0003-2700/78/0350-1226$01 .OO/O
(Ohio Valley Speciality Chemicals, Inc., Marietta, Ohio). Hexamethyldisiloxane (Fluka AG, Tridom Chemical Inc., Hauppauge, N.Y.). Hexanes (Spectroanalyzed, Fisher Scientific Co., Fair Lawn, N.J.). Pyridine (Certified ACS, Fisher Scientific Co., Fair Lawn, N.J.), dried over Potassium Hydroxide (J. T. Baker Chemical Co., Phillipsburg, N.J.). Imidazole (99%, Aldrich Chemical Company, Milwaukee, Wis.) dried in a desiccator over silica gel. Samples. Samples were derivatized in the following forms: Honey in a 20% solution in distilled water, beer as a freeze dried solid, sherry and raisins without any prior treatment. Derivative Preparation. The silylating reagent consists of four volumes of trimethylsilylimidazole and one volume of dry pyridine. Reagent, 0.5 mL, is added to 8 mg of sample in a 1-mL mini-vial (Alltech Associates, Arlington Heights, Ill.) fitted with a Teflon-faced liner. The reaction is vigorous for samples containing appreciable quantities of water and care should be exercised. The mixture is shaken and allowed to stand for 10 min. The mixture is then saturated with imidazole and the double phase formed by adding 40 fiL to 100 fiL of either hexamethyldisiloxane or hexane. The vial is shaken and then centrifuged to separate the two phases. The top phase contains nearly all of the silyl derivative and is used for injection. The single-phase run refers to the derivitized sample before addition of imidazole. Chromatography. A Perkin-Elmer Model 900 gas chromatograph (FID) was used. Separation was on a 12 m X 0.25 mm glass open tubular column coated with SE 30. The carrier gas was nitrogen, introduced at a pressure of 5 psi. The inlet was set at 330 "C, the interface at 300 "C, and the oven was programmed at 4"/min from 150 t o 270 "C at which temperature it was maintained for 20 min.
RESULTS AND DISCUSSION The concentrating effect of the two-phase system is significant and is illustrated by a consideration of the chromatograms of a derivatized sample before and after formation 0 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
do
li0
IbO
TEMPERATURE,
Figure 1.
A0
2;o
is0
n;o
#C
-
Chromatogram of silylated honey sample prepared as a single
phase
J
I
I40
li0
1
I40
TEMPERATURE,
;IO
2iO
250
2;O
OC
Chromatogram of top phase of a silylated honey sample. Peak identification: (1) fructose, (2) glucose, (3) sucrose, (4) maltose, (5)trehalose, and (6) melezitose Figure 2.
of a two-phase system. Figure 1shows the chromatogram of a derivatized honey sample obtained by injecting 2 pL of the single-phase system. Large monosaccharide peaks are present together with trace disaccharide and trisaccharide peaks. Figure 2 shows a chromatogram of 2 pL of the same honey sample after conversion to a two-phase system. The monosaccharide peaks are off scale while the di- and trisaccharide peaks are about 10 times larger than in the single-phase system. Still larger peaks are obtained by arranging for a smaller top phase volume. There is no change in the shape of the chromatogram when the volume of the top phase is
do
li0
140
210
2iO
is0
1227
2;o
TEMPERATURE, -C
Figure 3.
Chromatogram of top phase of silylated beer sample
changed from 100 to 400 pL, apart from a uniform decrease in peak height due to sample dilution. The shape of the chromatogram is nearly identical when hexane is substituted for hexamethyldisiloxane in forming the two-phase system. Glucose, fructose, sucrose, trehalose, maltose, and melezitose were identified using standards. Some of the peaks may be due to compounds other than sugars, that are sufficiently apolar to be dissolved in the top phase. However, sugars are the most likely candidates for the unknown peaks both because of the nature of sample and reagent, and because of the clustering of peaks in regions corresponding to mono-, di-, and trisaccharides. The two-phase system gave significantly better chromatograms than the single-phase system for all samples analyzed. The two-phase chromatogram for beer is shown in Figure 3. There are several large peaks in the disaccharide region and small peaks in the mono- and trisaccharide regions. The two-phase chromatogram for sherry shows mono- and disaccharides, as well as traces of trisaccharides. The chromatogram for a sample of raisins shows very large monosaccharide peaks, about 12 disaccharide peaks, and no trisaccharides. The two-phase system forms spontaneously when samples containing sufficient water are derivatized with trimethylsilylimidazole. Such samples should be shaken for a few minutes to ensure rapid derivatization of the sugars.
LITERATURE CITED (1) D. Nurok, J . Chromatogr. Sci., 14, 305 (1976). (2) G. D. Brlttain, E. S. Sullivan, and L. R. Schewe, “Recent Advances In Gas Chromatography”, I. I. Domsky and J. A. Perry, Ed., Marcel Dekker, New York, N.Y., 223 (1971). (3) D. Nurok and T. J. Reardon, Proc. S . Afr. Sugar Tech. Assoc., 49, 94 (1975). (4) D. Nurok and T. J. Reardon, unpublished results.
RECEIVED for review March 13, 1978. Accepted March 31, 1978.
