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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to a method of rapid and precise analysis, both
quantitatively and qualitatively, for carbohydrates, and in particular,
this invention relates to a method for regular syrups, fructose syrups,
and blends of syrups or carbohydrates with sucrose to determine
constitutional makeup.
There have been four main procedures in the past for the quantitative
analysis of fructose and dextrose in syrups or for the analysis of other
syrup and starch compositions.
(1) The method of polarimetry is described in U.S. Pat. No. 3,694,158 and
is believed to be the first feasible system for the continuous analysis of
a process stream to detect the amounts of dextrose and fructose in
mixtures simultaneously. This procedure has the disadvantage, however, of
producing imprecise results if the composition contains appreciable
amounts (over two percent) of higher homologs of dextrose, i.e. degree of
polymerization of 2-10 or higher, e.g. maltooligosaccharides. Furthermore,
the method becomes inaccurate if the tested composition contains mixtures
of three or more types of sugars. The method is also sensitive to
variations in temperature, thus requiring a controlled atmosphere.
(2) High pressure liquid chromatography has been developed to where it is
capable of analyzing carbohydrate solutions containing fructose, dextrose,
higher homologs of these sugars, and still other sugars. The method is
capable of providing an automatic analysis in a period of time of 10-30
minutes. A discussion of this procedure may be found in AACC Paper No. 48,
Oct. 5-8, 1976, Annual Meeting In New Orleans, La. by H. D. Scobell. Other
than the inordinate length of time to obtain the analysis results, this
process appears to be a suitable technique except where there are blends
containing sucrose or maltose, or where there are appreciable quantities
of maltulose, etc. These components elute simultaneously with other
constituents of the analyzed sample and yield imprecise results.
(3) Gas/liquid chromatography is an earlier procedure than high pressure
liquid chromatography and it is capable of quantitative analysis of a
carbohydrate composition to determine the various sugars present in one
analytical scan. The difficulty with this procedure is that it requires a
precise technique in the preparation of samples and in the derivatization
of samples. Furthermore, the analytical time, not including the sample
preparation time, is inordinantly long, i.e. about 20 minutes. See K. M.
Brobst and C. E. Lott, Am. Soc. Brew. Chem. Proc. 71-75 (1966).
(4) There are many chemical procedures for the quantitative analysis of
dextrose, fructose, higher sugars, and mixtures of sugars. Illustrative of
such analytical techniques are:
(a) Cystein carbazole method for the determination of ketose sugars, Corn
Refiners Tentative Method E-1, Jan. 6, 1976;
(b) "The Quantitative Determination of Glucose, Fructose, and Sucrose in
Fruits and Potatoes", E. S. Della Monica et al, J. Food Sci. 39, pp.
1062-3 (1974);
(c) "Spectrophotometric Analyses Of Glucose And Mixture Of Glucose,
Fructose, And Sucrose", E. Garrett and J. Young, J. Pharm, Sci. 58, pp.
1224-7 (1969); and
(d) "Dextrose Equivalent",
Corn Refiners Method E-26. These are all wet chemical analyses which have
substantially the same disadvantages of the possibility of technician
error, the requirement of large amounts of bench space and analytical
equipment, and the large amount of time involved in the analysis.
Furthermore, these techniques are either so specific that they are capable
of analyzing only a single type of sugar or they are so non-specific that
they are incapable of distinguishing one type of sugar from another.
The present invention provides an analytical process which overcomes these
disadvantages. It is extremely fast in that it can provide a complete
analysis in 1-3 minutes, frequently less than 1 minute. There is no
necessity for derivatization nor is there a need to carefully control the
amount of solids in the sample. It is preferable to have anomeric
equilibrium in the mixtures analyzed. This technique is capable of
analyzing samples containing from less than 1 percent up to 100 percent
solids and it is capable of installation on a production line for
continuous or semi-continuous analysis of process streams. The technique
is capable of handling higher sugars as well as mixtures of sugars. It is
capable of providing information as to carbohydrate complex formations,
such as fructose-H.sub.3 BO.sub.3. It is also capable of providing an
analysis of the degree of anomerization and the rate of anomerization. The
process is also able to provide information as to the extent of
derivatization and other behavorial questions such as carbohydrate
anomerization in acids, bases, and organic solvents.
The present invention is based on infrared (IR) analysis of carbohydrates.
While IR spectra of carbohydrates have been studied and characterized for
many years [two reviews have been published: "Infrared Spectra of
Carbohydrates" by W. Brock Neely, in ADVANCES IN CARBOHYDRATE CHEMISTRY,
12 13-33 (1957) and "Infrared Spectroscopy and Carbohydrate Chemistry" by
H. Spedding in ADVANCES IN CARBOHYDRATE CHEMISTRY, 19 23-49 (1964)] it was
not immediately apparent that IR could be used as a
qualitative/quantitative analytical tool. This position was taken in a
paper by W. J. Hoover, et al. in J. FOOD SCIENCE, 30 253-261 (1965)
"Isolation and Evaluation of the Saccharide Components to Starch
Hydrolysates II. Evaluation" where it was stated with reference to
hydrolyzed starch, "No trends were noted in shifts of peaks or development
of peaks with increase in molecular weight (from dextrose) . . . The
spectra of all of the saccharides were so similar that they could not be
used in identifying or distinguishing between the various
malto-oligosaccharides." Furthermore, a paper "Infrared Spectra of
Carbohydrates in Water and a New Measure of Mutarotation" by Frank S.
Parker in BIOCHIM. BIOPHYS. Acta. 42 513-519 (1960) indicates that the
absorbance at 1143 cm.sup.-1 is the most indicative band for observing
mutarotation of .alpha.- and .beta.-glucose. In accordance with the
present invention absorbances at 1038 cm.sup.-1 and 1080 cm.sup.-1 provide
much more useful information.
Very little infrared work has been done specifically for fructose. Typical
of that work is that which is described in "Identification of the
Anhydrides of D-Fructose from the `Fingerprint` Region of their Infrared
Spectra" by W. W. Binkley, et. al., INTERNATIONAL SUGAR JOURNAL 73 259-261
(1971), which clearly is not directed at the analytical purposes of the
present invention.
SUMMARY OF THE INVENTION
The present invention provides a process for qualitatively and
quantitatively analyzing a chemical composition for its content of starch
or sugars comprising:
(1) Subjecting a sample of a chemical composition to infrared spectroscopy
to produce a spectrum;
(2) Noting the amplitude of the absorbances at the known frequencies for
each species or family of species (wherein "family of species" means, for
example, dextrose, maltose and higher homologs, e.g.
maltooligosaccharides) of carbohydrate; and
(3) comparing each noted amplitude with known amplitudes for standard
samples of that same specie or family of species to determine the amount
of that specie or family of species in the composition.
Two basic types of infrared instruments and associated techniques are
utilized for this work. One infrared analytical technique employed in this
process is infrared Fourier transform spectroscopy which has been known as
an instrumental technique for the past few years after the first
commercial instruments were developed in the late 1960's. The preferred
type of instrument utilizes a Michelson Interferometer which produces an
interferogram of the sample being analyzed. The interferogram is then
subjected to a calculation by a digital computer in accordance with a
mathematical procedure known as "Fourier transform" to yield the frequency
spectrum. It is an alternative procedure to employ the Hadamard transform
to produce the frequency spectrum. This type of spectroscopy has already
found wide and diversified applications in areas such as a quality control
technique for determining impurities in semi-conductors, and it is also an
excellent detector for use in connection with the various chromatography
analyses. Other applications for this type of spectroscopy are the remote
monitoring of hot gas emission from smoke stacks wherein the technique
involves a telescope to observe the emissions from a remote location, the
identification of microscopic particles, the detection of minor additives
in a composition, and routine spectroscopy performed by conventional
grating spectrophotometers.
This type of spectroscopy is applicable over a very wide spectral range
from about 10,000 cm.sup.-1 in the near infrared frequencies to 10
cm.sup.-1 in the far infrared frequencies. This method is capable of
providing precise finger printing of organic molecules exhibiting fine
absorbances in the mid range of 4,000 cm.sup.-1 to 400 cm.sup.-1.
Resolution may be obtained over the entire spectral range to a value
better than 0.1 cm.sup.-1.
Conventional infrared dispersive spectrophotometers using prisms or
gratings and filters to scan the various wavelengths of interest may also
be used as the second basic type of spectrophotometer. Modern
microprocessor and computer controlled infrared spectrophotometers
utilizing scale expansion can scan from 4000 cm.sup.-1 to 32 cm.sup.-1
with better than 0.2 cm.sup.-1 resolutions. These instruments are capable
of scanning spectra to produce the resolution required for this analysis
in 8 minutes. Furthermore since the analysis for the present invention
requires scanning from about 1800 cm.sup.-1 to 700 cm.sup.-1, preferably
1200 cm.sup.-1 to 800 cm.sup.-1, or the region of 700 cm.sup.-1 to 50
cm.sup.-1, the analysis may be performed in less than 2 minutes. With the
computer and microprocessor controlled instruments the spectrum need not
be scanned, since the instrument can report the amplitudes of absorbances
at discrete wavelengths. The time for such an analysis is much less than 1
minute, and a computer makes the necessary computations needed to
complete the analysis.
The present invention provides a method of rapid quantitative and
qualitative analysis of starch, starch derivatives, and all of the
specific sugars, examples of which are dextrose, fructose, lactose,
galactose, and sucrose whether separate or mixed with each other. This
method yields information as to the composition of regular corn syrup, the
Dextrose Equivalent (DE), amount of dextrose, etc. The analysis may be
performed on water solutions of the compounds, glassy amorphous solids or
pelletized solids. Further types of information which can be obtained
include the solids content, anomeric conformation, complex formation with
other compounds, reaction and anomerization rate studies, degree of
derivatization, behavior of carbohydrates in solutions of acids, bases,
salts, or organic solvents, and othe/similar information.
The process involves the analysis of standard samples containing known
amounts of known compounds to determine the absorbances and frequencies of
such samples to serve as standard samples against future analyses of
unknown compositions. The standard samples are normally correlated into a
graph for purposes of interpolation. The unknown sample is analyzed by
infrared spectroscopy and the amplitude of the absorbance and the
frequency are compared with measurements on standard samples to determine
the amount of a particular carbohydrate which is present in the
composition.
The preferred apparatus and procedures used for analyses in accordance with
this invention are as follows:
(1) Cell Windows -- Irtran 2 windows of ZnS and BaF.sub.2 windows for the
Fourier transform spectroscopy and conventional infrared spectroscopy.
Cell windows may be of other materials, such as ZnSe.
(2) Cell Thickness -- 12.5, 15, or 25 microns
(3) Number Of Scans -- 250, 125, 30, and 1
(4) Resolution -- 4 wave members
(5) Word No. -- 32 bit words
(6) Instrument -- Digilab IRFTS 15 equipped with a double beam head; Perkin
Elmer 283 with 580 infrared Spectrophotometer; or Nicolet 7199 FT-FR
System
(7) Light -- monochromatic, e.g. by employing filters, lasers, etc.
(8) Concentration -- 10-30% solids preferred, wider ranges if desired
(9) Sample Preparation -- Anomeric equilibrium attained prior to analysis
of sample. Equilibrium obtained either by allowing solution to reach
equilibrium by natural action or by the addition of several drops of
ammonium hydroxide when speed is necessary.
(10) Analysis Of Unknowns -- Samples of fructose corn syrup and regular
corn syrup analyzed by high pressure liquid chromatography for
carbohydrate composition and by CRA Method E-26 for DE in regular corn
syrups.
In order to more fully understand the present invention the following
examples of the operation of this invention serve to illustrate the scope
and nature of the invention. Parts and percentages are by anhydrous weight
unless otherwise noted.
EXAMPLE 1
Mixtures of fructose and dextrose ranging from 2% fructose/98% dextrose to
98% fructose/2% dextrose prepared such that when distilled water was added
a 30% solids solution was obtained. Several drops of ammonia solution were
added to each sample to ensure anomerization to equilibrium. After a
complete solution was obtained, each sample was placed in a cell made from
Irtran II windows with a cell thickness of approximately 12.5 microns. An
infra-red Fourier transform spectroscopic analysis was run on each sample
using as the primary parameters 250 scans, 4 wave number resolutions and
32 bit words. The instrument employed was Digilab IRFTS 15 with a double
beam head. The ratios of the absorbances of fructose at 1068 cm.sup.-1 and
of dextrose at 1037 cm.sup.-1 were computed and plotted against the known
values for the proportion of fructose to dextrose in each sample. The
result was a nearly linear plot as shown in FIG. 1 in the attached
drawing.
Unknown samples were then analyzed by the same process, the ratios of
absorbances were computed and the proportion of fructose to dextrose
determined from this plot. When this proportion was checked by other known
procedures, such as chromatography the results were found to be accurate.
EXAMPLE 2
In the same manner described in Example 1 tests were performed on other
sugars, such as lactose, galactose, maltose, maltotriose, and sucrose, and
even including starch. In each instance, the analytical technique was
found to be accurate and the analytical time varied from less than one
minute to not more than 3 minutes.
EXAMPLE 3
In the same manner described in Example 1 analyses were made on known
samples and mixtures of samples of alpha-D-glucose and beta-D-glucose to
determine the applicability of this technique for the study of
anomerization rates. Thirty scans were run by IRFTS, thus allowing an
analysis time of 27 seconds. Analyses were made at time intervals so as to
obtain a value for the reaction rate.
EXAMPLE 4
Employing the same procedures as in Example 1 an experiment was run to
determine the applicability of the technique for establishing the Dextrose
Equivalent (DE). Pure compounds of dextrose, maltose, maltotriose, and
starch were tested by this spectroscopic technique to establish their
frequencies and absorbances. Syrups having a known DE were also tested in
the same fashion. By computing ratios of the amplitude of absorbances
against values of DE a linear plot was obtained. Unknown syrups were then
subjected to the same analytical technique and the ratios of the amplitude
of absorbances were computed and applied to the plot. The technique is
applicable not only to mixtures of two sugars but also to mixtures
containing at least five sugars. Data from the plot yielded a good
correlation of IR-computed DE with actual DE of the unknown syrups
determined by chemical analyses.
EXAMPLE 5
The analyses described in Examples 1-4 inclusive were performed on a Perkin
Elmer 283 and 580 Infrared Spectrophotometer. The resolution was 3.7 cm.
The scan time from 1500 cm.sup.-1 to 900 cm.sup.-1 was about 2 minutes.
Solutions of 30% solids were employed and 2 drops of ammonium hydroxide
were added to produce complete anomerization. The analytical results were
substantially identical to those produced in Examples 1-4.
Although the invention has been described in considerable detail with
reference to certain preferred embodiments thereof, it will be understood
that variations and modifications can be effected within the spirit and
scope of the invention as described hereinabove and as defined in the
appended claims.
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