Application of flowcell HPLC-FTIR technique to the analysis

of benzoylated saccharides and compounds with similar polarity

Rudolf Galensa, Klaus Landt, and Werner Herres
Institut ffir Lebensmittelchemie der Universit/it Hannover, Wunstorfer Strasse 14, D-3000 Hannover, Federal Republic of Germany
Bruker Analytische Messtechnik, Wikingcrstrasse 11, D-7500 Karlsruhe, Federal Republic of Germany

Anwendung der HPLC-FTIR-Kopplung used, those applying molecular spectroscopy (MS, IR)
zur Analyse benzoylierter Saccharide are unique because they not only detect a compound
und Verbindungen mit fihnlicher Polaritfit eluting at a retention time t - but they are also capable
to give a positive identification of this compound. But
Zusammenfassung. Der Einsatz eines Fourier-Trans- in contrast to the MS and FTIR couplings to gas chro-
form-Infrarotspektrometers (FTIR) in der Verbin- matography - which have achieved a high degree of
dung mit der HPLC errn6glicht die simultane Detekti- optimization and operational ease - there are many
on bei mehreren Wellenzahlen. Wegen der grogen Ei- difficulties to overcome in the case of liquid chroma-
genabsorption der bei RP-Chromatographie meistens tography. Most of the problems encountered with IR
ben6tigten FlieBmittel ist eine IR-Detektion dort flowcell detection are related to the nature of the sol-
nicht sinnvoll. Das ternfire, in einem weiten Bereich vents employed. Solvent self-absorptions can obscure
variable Elutionsmittelgemisch Isooctan/Diethyl- considerable parts of the useful spectral region. The
ether/Acetonitril bietet gute UV- und IR-Detektions- on-line coupling of reversed-phase HPLC (MeOH,
m6glichkeiten bei der Verwendung von Kieselgelsfiu- water etc.) with IR is still unrealized. The situation is
len. Es werden HPLC-FTIR-Analysen von Reinsub- much better in case of adsorption and size exclusion
stanzen (PHB-Ester) und ffir benzoylierte Extrakte (GPC) chromatography. Here HPLC-FTIR has al-
(Sojamehl, Hautbrfiunungsmittel) gezeigt. ready established itself as a very valuable tool (cf. [1-
7]).
Summary. Fourier transform infrared (FTIR) spec- The present communication deals with adsorption
trometry in combination with liquid chromatography HPLC-FTIR investigations on derivatized (mainly
provides nonspecific detection and highly specific benzoylated) natural extracts. The preparation of
multichannel analysis simultaneously. Standard re- such derivatives is very simple and rapid, as has been
versed-phase eluents are incompatible with IR-flow- demonstrated in many applications (cf. [8-11]). In this
cell detection because of their strong self-absorptions. way not only standards but various samples and dif-
The ternary solvent system isooctane/diethyl ether/ ferent classes of compounds may be analyzed with
acetonitrile, which exhibits adequately adjustable po- FTIR detection.
larity, has already revealed its potential in the separa- Good chromatographic conditions of this type of
tion of derivatized food extracts on silica columns us- extracts have been reported for a ternary solvent sys-
ing UV detection. In the HPLC-FTIR analyses re- tem isooctane/diethyl ether/acetonitrile [8, 9]. This sol-
ported in this investigation different compositions of vent system is well-suited for this purpose because of
this ternary solvent system were applied to the analysis its transparency to infrared radiation in the spectral
of an artificial mixture, of benzoylated soy flour and regions 4000-3300 cm- 1, 2600-I 550 cm- 1, and
benzoylated tanning product. 1000-500 cm-1 (1 mm NaCl-window). Concentra-
tions of ca. 0.5 mg/ml are required (20 gl injection vol-
ume ca. 10 gg substance). LC-FTIR is capable of de-
tecting sub-microgram quantities with microbore
Introduction equipment [6].
"Detection" is still a field of ongoing research and de- HPLC-FTIR analyses are reported for benzoyl-
velopment in HPLC. Among the various methods ated alcohols, esters of p-hydroxybenzoic-acid and
saccharides with varying compositions of the solvent
Offprint requests to: R. Galensa system.

z Lebensm Unters Forsch (1987) 185:36-38

© Springer-Verlag1987
05 055 05

0 2 5 - ~ 0275j ~

a
0
¢000
'&,
3000 2000
WaveFIUFflbefs [m-I
1000
b
0/+000 , A3000 2000
Wavenumbers cm-I
L
1000
C
0
~,000
/k,
3000
~ ,
2000
Wavenumbers cm"I
,
1000

Fig. 1 a-e. Single beam background of solvent compositions, see Experimental

methanol (0.5 ml) is added and the solution is allowed to stand for
5 rain or longer, the benzoates are then precipitated by the addition
of water (50 ml). Allow to stand for at least 20 min then pass the so-
lution through a 20 ml syringe connected to a reversed phase car-
tridge (SEP-PAK RP-18; Millipore, Eschborn, FRG). Pyridine and
polar other substances are removed by washing with water
(4 x 5 ml). The benzoates are eluted with isooctane/diethyl ether/ace-
tonitrile (150 + 80 + 20) (50 ml). For the benzoylation procedure see
also [8, 9].

' £
i

8
i

12
L

16
i

20 mln
i , i i ,
Results and discussion
Fig.2. HPLC-FTIR chromatogram; eluent B see Experimental; Derivatization introduces new groups with new IR
a PHB ethyl ester, b benzoylated PHB ethyl ester, c naphthoylated
bands into the molecules and alters the spectra of the
PHB ethyl ester
original molecules. This can be illustrated with the
ethyl ester of p-hydroxybenzoid acid. Figure 2 repro-
Experimental duces the HPLC-FTIR chromatogram of an artificial
HPLC mixture of the original ester and its benzoylated and
Bruker LC 21; FTIR-Spectrometer IFS 85 (Bruker, Karlsruhe, naphthoylated derivatives (1.5 mg/ml). Chromatogra-
FRG) combined with a broadband MCT detector; spectral resolu- phy with nonderivated molecules is possible but takes
tion was 4 cm- 1 and zero filling was applied to generate spectra with longer.
one data point every wavenumber; 1 mm NaC1 flow cell (20 Ixl vol- Figure 3 illustrates the changes to the spectrum of
ume); silica column: 125 x 4.6 mm I.D. of SC-Hypersil 3 gin; injec-
tion valve: Rheodyne 7•25 with a 20 pl sample loop; Eluents: Isooc- PHB ethyl ester (a), upon benzoylation (b) and naph-
tane, max. 0.005% H20 (Uvasol ®, Merck, Darmstadt, FRG); di- thoylation (c) (chromatogram see Fig. 2).
ethyl ether, max. 0.1% HzO (p.A. Merck); Acetonitrile, max. 0.01% Figure 4 reproduces a HPLC-FTIR chromato-
H20 (Baker analyzed HPLC ®, Gross Gerau, FRG); solvent compo- gram of soy flour (40 mg benzoylated and concen-
sitions: Isooctane/diethyl ether/acetonitrile; A: 150 + 80 + 20 (0.8 ml/
min); B: 1 5 0 + 1 0 + 2 (0.7 ml/min); C: 1 5 0 + 2 0 + 4 (0.7 ml/min). The
trated to 1 ml). Esterification with benzoyl chloride
single beam background of A, B, and C are reproduced in Fig. 1. also makes detectable sugars and other compounds
containing hydroxyl groups. Of course only scanty
Benzoylation spectral data are produced but after benzoylation all
Benzoates were produced by the action of benzoyl chloride (0.5 ml) saccharides exhibit constant ratios between the carbo-
on the homogenized sample (30-50 mg), in 4 ml pyridine solvent. nyl and aromatic bands and have identical spectra. So
The mixture is heated in an ultrasonic bath (60 °C) for 1 h, then saccharides can be distinguished by other substances

% f ......

, , , , , , , , , , , J R , i R , i , ,

3900 3000 2100 1200 3900 3000 2100 1200 3900 3000 2100 1200
a Wavenumbers cm-I b Wavenumbers cm-I c Wavenumbers cm4

Fig.3a-e. HPLC-FTIR spectra of PHB ethyl ester (a), benzoylated PHB ethyl ester (b), and naphthoylated PHB ethyl ester (e) (HPLC chro-
matogram see Fig. 2)

37
 . . . . . . y----~- . ~ e-( d . . . . . . .

/
1

178o 1~o 1~o

35bo soo 15 oo
rain Wavenumbers cm-I
Fig.4. HPLC-FTIR chromatogram of a benzoylated soy flour; Fig.7. HPLC-FTIRspectrumofdihydroxyacetone (HPLCchroma-
eluent A, see Experimental; 1 saccharose, 2 raffinose, 3 stachyose togramseeFig.6)

diode array detector [10]. These results will be de-

. . . . . . . . . . . . scribed in a subsequent publication.
Figure 6 illustrates a HPLC-FTIR chromatogram
ofa benzoylated cosmetic tanning product, containing
the self tanning agent dihydroxyacetone (DHA). Be-
cause of the high reactivity of DHA the stability of
such a cosmetic is limited and a quantitative method
is required [11]. The amount (Fig. 6) of DHA injected
was 8 gg. The carbonyl group of the original molecule
3900 ' 30'00 ' 21bo ' 12;o is recognizable (Fig. 7).
Wavenumbers cm-~
Fig.5. HPLC-FTIR spectrum of stachyose (HPLC chromatogram Conclusion
see Fig.4)
The use of mixture of isooctane/diethyl ether/acetoni-
trile allows the separation and detection of many dif-
ferent substances even in biological or pharmaceutical
extracts by means of a HPLC-FTIR system. Limiting
factors include the considerable concentrations of
substances necessary. Improvements are possible by
use of the microbore technique.

Acknowledgements. This work was supported by Prof. Herrmann,

Institut ffir Lebensmittelchemie, Universit~it Hannover, and by
grants from Arbeitsgemeinschaft industrieller Forschungsvereini-
gungen (AIF), Cologne, and from the Forschungskreis der Ern/ih-
rungsindustrie, Hannover, which are gratefully acknowledged.
' ½ ' /~ ' 6' 8' 10 12
rain
Fig. 6. HPLC-FTIR chromatogram of a benzoylated cosmetic tan- References
ning product; eluent C, see Experimental; 4 methanol, 5 glycerine,
6 dihydroxyacetone 1. Kuehl DT, Griffiths PR (1980) Anal Chem 52:1394-1399
2. Jinno K, Fujimoto C (1983) Chromatographia 17:259-261
3. Combellas C, Bayart H (1983) J Chromatogr 259:211-225
in natural extracts. Figure 5 shows the FTIR-spec- 4. F61ster U, Herres W (1983) Farbe + Lack 89:417-420
trum of stachyose (chromatogram see Fig. 4). The 5. Johnson CC, Taylor LT (1983) Anal Chem 55:436-438
benzoylated soy flour contained 1.5 percent stachyose, 6. Brown RS, Amateis PC, Taylor LT (1984) Chromatographia
18:396-400
corresponding to 12 gg injected. 7. Jinno K, Fujimoto C, Nakaniski S (1985) Chromatographia
Aromatic structures present before benzoylation 20:279-282
e.g. flavonoids present different adsorption ratios 8. Galensa R (1983) Z Lebensm Unters Forsch 176:417-420
than do saccharides differing carbonyl groups will be 9. Galensa R (1984) Z Lebensm Unters Forsch 178:199-202
10. Siewek F, Galensa R (1984) J Chromatogr 294:385-389
observable in the spectrum. Therefore the reproduc- 11. Galensa R, Schuster B (1985) Dtsch Lebensm Rundsch 81:273-
ibility and high resolution of the FTIR-spectrometer 275
allow the determination of the degree of glycosidation
of flavonoids such as has been reported using a UV Received November 5, 1986

38