1966
Anal. Chem. 1981, 53, 1966-1967
Reversed-Phase Liquid Chromatographic Determination of Invert Sugar, Sucrose, and Raffinose c18
Gerard0 Palla Istifuto di Chimica Organica dell'Universit& 43 100 Parma, Ira&
Invert sugar, sucrose, and raffinose are the major components of juices and syrups, and their industrial analyses are carried out separately by polarimetry and Fehling reagents for reducing sugars ( I , 2). Several alternative chromatographic methods have recently been reported: silica (3),ion exchange resins (4-7), and other p packings (8)have been used and good results have sometimes been obtained. The packings which are most successful in separating mono-, di-, and trisaccharides are those with amine groups bound to inert silica or synthetic supports such as pBondapak carbohydrate (Waters Associates), Lichrosorb-NH2 (Merck), and Aminex resins (Bio-Rad Laboratories) (9). These methods involve elution with a water-acetonitrile system or water a t 80-85 "C. We have found that good results can be obtained by HPLC on C18 reversed-phase columns using only water as eluent for routine industrial analyses. Details of this method for the simultaneous determination of invert sugar, sucrose, and raffinose are reported. EXPERIMENTAL SECTION
We used a Waters Associates high-pressure liquid chromatograph with a M6000A pump, a U6K septumless injector, and a Series R 400 differential refractometer. The response of the refractive index detector was recorded on a strip chart recorder (Houston Instrument, Austin, TX) and on a Sigma 10 chromatography data station (Perkin-Elmer, Norwalk, CT). A microbonded silica packed column (Waters Associates, 10-ppore diameter, Bondapak Cle, 8 mm i.d. X 30 cm of length) was used, with a flow of 3 mL/min of distilled prefiltered water. Sugar standard solutions were prepared by dissolving pure compounds in distilled water; technical juices were prepared by mixing beet molasses and sucrose in different amounts. Impure solutions,before injecting, were passed below 10 "C through mixed ion exchange resin cartridges composed of analytical grade Amberlite IRA 93 and analytical grade Amberlite 200. One milliliter of the 1:l mixture of these regenerated resins could retain 100 mg of nonsugars. The samples were then filtered on Millipore membranes (0.5 pm) and analyzed. Crystal sugars and liquid sugars required filtration only. Twenty microliters of a 10% sugar solution was usually injected, but this volume could be increased to 200 p L when less than 0.1% of invert or other minor sugars had to be detected, without a decrease of resolution. The sugar concentrations were calculated by reference to peak areas of standard sugar solutions of similar and known content (external standards) or by adding to the samples known amounts of internal standards. RESULTS AND DISCUSSION
Chromatograms of several sugar samples are shown in Figure 1. The first profile (a) comes from a standard sugar solution of invert sugar (0.120%), sucrose (10.0%), and raffinose (0.210%). Relative standard deviations were 2.6 for invert, 0.8 for sucrose, and 3.2 for raffinose (10 experiments, 20 WLinjected). Resolution data (10) calculated on a solution containing 1% each of invert, sucrose, and raffinose were 2.54 between invert and sucrose and 2.46 between sucrose and raffinose. The detection limit tested for sucrose was 1pg and guarantees good performance also for trace analysis (entrainment in the evaporators). Other monosaccharides were tested (xylose, galactose, mannose) that had the same retention time of invert sugar; then peak area of invert can be considered
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Figure 1. Chromatograms of sugars on C,8 fiBondapak columns: standard solutlon (a); llquid sugar from sucrose (b); liquid sugar from beet molasses (c); liquid sugar from cane molasses (d). Numbered peaks are as follows: invert sugar (l), maltose (2), sucrose (3),trloses (4-6).
a measure of the monosaccharide content. Maltose eluted between invert and sucrose and could be easily detected; lactose had the same retention time of invert but is not present in vegetal juices. Profile b (Figure 1)refers to a liquid sugar from sucrose; profiles c and d show a detailed composition of liquid sugars obtained from beet and cane molasses. Sugar determination in molasses has always been a problem owing to the interference of trisaccharides (ketoses), that partially decompose during acid treatment required by the traditional analytical methods ( I ) . Instead sugars are well detected by HPLC: from profiles c and d we can easily calculate the exact percent of mono-, di-, and trisaccharides represented, respectively, by peak 1(invert and other monosaccharides), peak 2 (maltose), peak 3 (sucrose), and peaks 4, 5, and 6 (trisaccharides). Similar results can be obtained on amine packings that also permit the determination of the glucose/fructose ratio. We think, however, that the ClS packings show other advantageous characteristics: greater chemical inertness, longer life, and the possibility of using the most convenient and safe eluent, distilled water. Moreover, we found that the resolution can be easily corrected, when required, by increasing the length and the diameter of the column. ACKNOWLEDGMENT
The author thanks Giuseppe Casnati for helpful discussion during the course of this work.
0003-2700/81/0353-1966$0 1.2510 0 1981 American Chemical Society
Anal. Chem. 1981,53. 1967-1968
1987
(1) &ow. C. A,; Zerban.F. W. '"Ryricaland Charkal Memo60 k Sug a7 Analysis". 3rd 4.:Wlleq: New Y a k . 1941. (2) Meade. G. P. "Cane S w r Handbook". 5% mi.; m y : New Y a k .
(7) Lsnod. M. R.: Husbner.A. L.: Two. G.T. J. chomsfog. 1978. 147, 185-193. (8) Undan. J. L.: Lawhead. C. L. J. wmnultogr. 1975. 105, 125-133. (9) Canrad. E. C.: Palmer. J. K. Fmd Techrwl. (Chkam) 1978. 84-92. (10) Kirkland. J. J. "Modem Racl!-a in Liquid Chromatogaphy": W l l e y : New YOn. 1971;Chapta 1. pp 8-12.
(3) Roc&. J. L.; Rouchause. A. J. wmnulfog. 1978. 117, 216-221. (4) Jandera. P.: ChwaCek. J. J. ChomafqF. 1974. 98. 55-104. (5) Palmer. J. K.: Brandes. W. 8. J. Ag*. Fmd Chem. 1971. 22. 709-712. (8) Ladisd. M. R.: TMO. 0.T. J. chomafcq. 1970. 166. 85-100.
RECnvED for review Mareh 18,1981. Accepted June 15,1981. hi^ work was partially by the ~ ~ ~~~~~~h i ~ Council (C.N.R.).
LITERATURE CITED
lMR
Stainless Steel Cell for Infrared Spectrometrlc Analysis of Silicon and Germanium Halides D. L. Wood, J. P. Luongo: ell Lebwatwles.
m -n/n,~NSW
and S. S. DeBala ~enev07.974
The halides of silicon and germanium are important reagents for semiconductor and glass fiber manufacturing processes. Infrared spectrometry has been extremely useful for determining low-level impurities in these highly reactive reagents because group 4 halide molecules are intrinsically transparent especially in regions of the spectrum where hydrogen-containing impurities absorb (Z-3). Long pathlength cells which are insensitive to the corrosive properties of the reagents are required for these measurements. We describe here stainless steel liquid cells with AgCl windows which we have found to he useful for impurity determinations. Figure 1 shows some of the cells in assembled and unassembled conditions.
Suitable stainless steel tubing, such 89 25.4 mm o.d., 23 mm i.d. 304 alloy is cut to length with flat ends. Two no. 18 Luer-Lok syringe needles of 304 stainless steel with chromium-plated brass fittings are cut down and soft soldered with 60/40 Pb/Sn alloy into the cell body, as shown in Figure 1, for fill and vent ports. AgCl windows, 1 mm thick and 25.4 mm diameter are cemented with quick-setting (2 part, amine cure)epoxy adhesive over the ends of the cylindrical cell body. Teflon plugs are provided as stoppers for the Luer-Lok fittings. Smaller volume cells can he made as shown a t the left in Figure 1 by using 16 mm 0.d. stainless steel tubing and compressing the tube to an oval shape slightly larger than the crcea section of the sample beam when the cell is placed in the sample compartment. We find it useful, since the cells are easy to make, to have four to five cells of various path lengths from 1 to 100 mm to facilitate quantitative measurements of widely varying impurity levels. It is convenient to clean the cells by flushing with ACS reagent grade chloroform several times. Many solvents such as CC, contain enough moisture to cause objectionable precipitates with silicon tetrachloride, hut the chloroform seems to he free of this problem. If a film of silica with its strong infrared absorption a t 9.2 pm does form internally or externally on these cells, n quick rinse with 1 1 H F in water will restore the transmission without serious damage to the cell body or windows. The Luer-Lok fittings permit quick, airtight connections for transferring samples of the atmcepheric-sensitive materials from a sample container to the cell using a Luer-Lok tipped
Flgum 1. Assembled and unassembled cells.
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