Designation: D 5837 – 99e1

Standard Test Method for

Furanic Compounds in Electrical Insulating Liquids by High-
Performance Liquid Chromatography (HPLC)1
This standard is issued under the fixed designation D 5837; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.

e1 NOTE—Equation 3 in 16.3 was corrected editorially in November 2003.

1. Scope D 923 Test Method for Sampling Electrical Insulating Liq-

1.1 This test method describes the determination in electri- uids2
cal insulating liquids of products of the degradation of cellu- D 3487 Specification for Mineral Insulating Oil Used in
losic materials such as paper, pressboard, and cotton materials Electrical Apparatus2
typically found as insulating materials in electrical equipment. D 3612 Test Method for Analysis of Gases Dissolved in
These degradation products are substituted furan derivatives, Electrical Insulating Oil by Gas Chromatography2
commonly referred to as furanic compounds or furans. This D 3613 Test Methods for Sampling Electrical Insulating
test method allows either liquid/liquid or solid phase extraction Oils for Gas Analysis and Determination of Water Con-
(SPE) of the furanic compounds from the sample matrix tent2
followed by analysis for specific furanic compounds by HPLC 2.2 International Electrotechnical Commission (IEC) Stan-
or direct injection for analysis of specific furanic compounds dard:
by HPLC. Method 1198 Furanic Compounds Analysis in Mineral Oil
1.2 The individual furanic compounds that may be identi- Insulating Oil3
fied and quantified include the following: 3. Terminology
5-hydroxymethyl-2-furaldehyde
furfuryl alcohol 3.1 Definitions of Terms Specific to This Standard:
2-furaldehyde 3.1.1 adsorbent, n—the stationary phase in solid-phase
2-acetylfuran
5-methyl-2-furaldehyde
extraction; silica is used as the adsorbent in this test method.
3.1.2 extract, n—the liquid phase of a liquid/liquid extrac-
1.3 The direct injection method generally has a higher limit tion containing the compound that has been extracted and that
of detection, especially for furfuryl alcohol. Greater interfer- will be analyzed.
ence for furfuryl alcohol may be expected when using the 3.1.3 liquid/liquid extraction, n—the preparative step of
direct injection method as opposed to extraction methods. extraction by mixing nonpolar test specimen with polar solvent
1.4 This test method has been used to successfully test for to preferentially partition and concentrate polar compounds of
furanic compounds in mineral insulating oil, silicone fluid, interest from an insulating liquid test specimen.
high fire point electrical insulating oils of mineral origin, 3.1.4 mobile phase, n—the carrier liquid phase in an HPLC
askarels, and perchloroethylene based dielectric fluids. analytical system used to transfer the prepared test specimen to
1.5 This standard does not purport to address all of the and through the analytical column and detector; the composi-
safety concerns, if any, associated with its use. It is the tion of the mobile phase affects elution time and separation of
responsibility of the user of this standard to establish appro- analytes.
priate safety and health practices and determine the applica- 3.1.5 solid phase extraction (SPE), n—a preparative step
bility of regulatory limitations prior to use. based on column chromatography, where intermolecular inter-
actions between adsorbent, solvent, and test specimen compo-
2. Referenced Documents
nents are optimized to effect retention of analytes on a
2.1 ASTM Standards: solid-phase extraction cartridge, followed by solvent elution
from the extraction cartridge.
1
This test method is under the jurisdiction of ASTM Committee D27 on
Electrical Insulating Liquids and Gases and is the direct responsibility of Subcom-
2
mittee D27.03 on Analytical Tests. Annual Book of ASTM Standards, Vol 10.03.
3
Current edition approved Oct. 10, 1999. Published December 1999. Originally Available from IEC, IEC Central Office, 3 rue de Varembe, P.O. Box 131,
published as D 5837 – 95. Last previous edition D 5837 – 95. CH-1211, Geneva 20, Switzerland.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.

1
 D 5837 – 99e1
3.1.6 ultraviolet (UV), adj—referring to that region of the 7.4 The analytical apparatus may be heated several degrees
electromagnetic spectrum including wavelengths from 10 to Celsius above ambient if necessary to reduce variance in
380 nm. The UV detectors of most HPLC systems operate in analytical results that may be caused by temperature fluctua-
the range of wavelengths from 190 to 380 nm. tions. Operation at ambient temperature or at a controlled
temperature of 30 to 40°C has been found satisfactory by some
4. Summary of Test Method laboratories.
4.1 Furanic compounds in electrical insulating liquids are 7.5 The following range of HPLC analytical conditions has
extracted from a known volume of test specimen by means of been found to be satisfactory for extracted test specimens
a liquid/liquid extraction or solid-phase extraction. A direct (specific examples are given in the appendix):
injection of the oil also may be used. Injection Volume 15 to 30 µL
Mobile Phase water/acetonitrile or water/methanol gradient
4.2 A portion of the extract or an aliquot of the oil is Flow Rate 0.5 mL/min to 1.5 mL/min
introduced into an HPLC system equipped with a suitable Column Temperature ambient to 40°C
analytical column and UV detector. Column 3.9 3 300 mm C18 60 to 125A, 4 to 10 µm or 4.1 3 150
mm PRP-1 100 A, 5 to 10 µm
4.3 Furanic compounds in the test specimen are identified Gradient see appendix
and quantified by comparison to standards of known concen-
tration. NOTE 1—Some laboratories have found it beneficial to filter all mobile
phase solvents with a 0.45-µm or smaller polytetrafluoroethylene or nylon
filter. Store water in containers shielded from light. Some laboratories use
5. Significance and Use 50 mL of methanol added to 4 L of water to inhibit biological growth.
5.1 Furanic compounds are generated by the degradation of
cellulosic materials used in the solid insulation systems of 7.6 The following HPLC analytical conditions have been
electrical equipment. found to be satisfactory for direct injection of the oil:
Injection volume 20 to 30 µL
5.2 Furanic compounds which are oil soluble to an appre- Mobile phase acetonitrile/water gradient
ciable degree will migrate into the insulating liquid. Flow rate, initial 0.5–1.0 mL/min
5.3 High concentrations or unusual increases in the concen- Column temperature ambient to 30°C
Column WatersT Nova-Pak C18 Reversed Phased 300 3 3.9
trations of furanic compounds in oil may indicate cellulose mm, 60A, 4 µm
degradation from aging or incipient fault conditions. Testing Gradient see Appendix
for furanic compounds may be used to complement dissolved
gas in oil analysis as performed in accordance with Test 7.7 For direct injection, a fixed wavelength between 274
Method D 3612. and 281 nm has been found to provide the best chromatography
for all compounds of interest, except furfuryl alcohol, which is
6. Interferences best measured with a separate test using a wavelength between
215 and 220 nm. Each furanic compound has a characteristic
6.1 Materials used in the manufacture of the polypropylene maximum light absorbance occurring within the indicated
tubes and polyethylene frits of some commercially prepared ranges of wavelengths. Use of variable wavelength or diode
solid-phase extraction columns may interfere with the deter- array detectors allows the selection of a specific wavelength for
mination of furanic compounds, such as furfuryl alcohol and each furanic compound. Each laboratory shall select the
5-hydroxymethyl-2-furaldehyde. specific wavelength to yield maximum absorbance for each
6.2 The use of acetone in any preparative or analytical step compound as follows:
will cause accelerated sample decay and may interfere with the
Furanic Compound nm
accurate determination of 5-hydroxymethyl-2- furaldehyde. 5-hydroxymethyl-2-furaldehyde 280 to 282
6.3 The use of cellulosic filtering media may serve to adsorb furfuryl alcohol 215 to 220
2-furaldehyde 272 to 280
furanic compounds yielding erroneous or unreproducible re- 2-acetyl furan 270 to 280
sults, or both. 5-methyl-2-furaldehyde 280 to 292

7. Apparatus 7.8 After the last compound of interest elutes through the
column, increase the acetonitrile or methanol to 100 % of the
7.1 High-Performance Liquid Chromatograph (HPLC)— mobile phase to remove all oil contamination remaining in the
The required analytical apparatus, an HPLC, consists of an analytical column.
injection device with sample loop, pumping system capable of 7.9 Readjust the solvent ratio of the mobile phase to the
mixing at least two solvents, reversed phase analytical column, initial conditions and allow 10 to 15 min for the column to
UV detector or detectors with the ability to operate at a come to equilibrium prior to the next injection.
minimum of two wavelengths, and a data recording device or
integrator.
8. Reagents and Materials
7.2 It is recommended that a precolumn packed with the
same material as the analytical column be used to increase 8.1 Acetonitrile, HPLC grade.
column life and remove interferences. 8.2 2-Acetylfuran, 99 % purity, CAS #1192-62-7.
7.3 Helium sparging of the mobile-phase solvents is recom- 8.3 Electrical Insulating Oil—Virgin oil of mineral origin.
mended in some cases and with some types of HPLC equip- 8.4 2-Furaldehyde, 99 % purity, CAS #98-01-1.
ment to displace atmospheric gases dissolved in the mobile- 8.5 Furfuryl Alcohol, 99 % purity, CAS #98-00-0.
phase solvents and to prevent the evolution of air bubbles. 8.6 Hexane, HPLC grade.

2
 D 5837 – 99e1
8.7 5-Hydroxymethyl-2-Furaldehyde, 99 % purity, CAS mobile phase. The resulting concentration is 1 mg/L (1000
#67-47-0. µg/L) for each of the five furanic compounds. Store as
8.8 Methanol, HPLC grade. indicated in 10.1.1.
8.9 5-Methyl-2-furaldehyde, 99 % purity, CAS #620-02-0.
8.10 Silica SPE Column—Solid-phase extraction column 11. Preparation of Calibration Standards in Oil
filled with 500 mg of silica. 11.1 Prepare standards of furanic compounds in new dielec-
8.11 Toluene—HPLC grade. tric liquid which has been tested and shown to have a flat
8.12 Vacuum Manifold—Device to pull vacuum on solid- baseline for the range of retention times for the compounds of
phase extraction column in order to pass sample and eluent interest. Mineral oil shall otherwise conform to Specification
through SPE column. D 3487. Other dielectric liquids should conform with appli-
cable ASTM specifications.
8.13 Volumetric Test Tube—Test tube designed to volu-
metrically measure in 0.10-mL graduations. NOTE 2—The same type of dielectric liquid should be used for standard
8.14 Vortex Mixer. preparation as the dielectric liquid found in the test specimen(s). This test
method has been developed for mineral oil, but has been found to be
8.15 Water—HPLC grade. applicable to other dielectric fluids.

9. Sampling 11.2 Volumetric Preparation:

11.2.1 Use a graduated 1-µL syringe to inject volumes of the
9.1 Obtain test specimens (insulating fluid samples) in five furanic compounds as listed in 10.1.2 into 8 mL of toluene.
accordance with the procedures for sampling in Test Method Dissolve the compounds and add quantitatively to a 1-L
D 923 or D 3613. volumetric flask. Make sure all compounds are thoroughly
mixed.
10. Preparation of Extraction Standards in Solvent 11.2.2 Dilute the 8 mL of toluene containing furanic com-
10.1 Prepare the extraction standards by dilution of a pounds to a total volume of 1 L with electrical insulating oil of
weighed standard compound to a standard volume or by mineral origin. The solution yields a concentration of 1 mg/L
volumetric addition of a standard compound to a standard (1000 µg/L) of each of the five furanic compounds. Store as
volume in accordance with either of the procedures described described in 10.1.1.
in 10.1.1 or 10.1.2. 11.3 Gravimetric Preparation:
10.1.1 Weight Procedure—Weigh out 0.100 g 6 5 % of 11.3.1 Weigh out 0.100 g 6 5 % of each of the five furanic
each of the five furanic compounds listed in this test method compounds and record the weight to the nearest 0.1 mg.
and record the weight to the nearest 0.1 mg. Dissolve weighed Dissolve the weighed portion in toluene and dilute volumetri-
portions into 100 mL of acetonitrile or methanol. Take 1 mL of cally to 100 mL in toluene. Mix thoroughly so that all five
this solution and add to a clean 1-L volumetric flask. Add 199 furanic compounds are dissolved completely.
mL of either acetonitrile or methanol, using the same solvent as 11.3.2 Volumetrically dilute 1 mL of the toluene solution
was used earlier to dissolve the weighed portions of the furanic from 11.3.1 to 1 L using electrical insulating oil of mineral
compounds. Bring the solution in the volumetric flask to 1 L origin. This solution of furanic compounds in oil yields a
with water. Other ratios of solvent to water may be used such concentration of about 1 mg/L (1000 µg/L) for each of the
as to match that of the initial mobile phase. This solution yields furanic compounds. Use the actual mass of each compound
a concentration of about 1 mg/L (1000 µg/L) of each of the recorded in 11.3.1 to calculate the exact concentration in the
furanic compounds. Use the actual mass of each compound to resulting solution. Store as described in 10.1.1.
calculate the concentration. Store in a clean, dark plastic
container. Do not store in glass. 12. Liquid/Liquid Extraction Procedure—Method A
10.1.2 Volumetric Addition—Furanic compounds that are 12.1 Measure 1 to 2 mL of the extraction solvent (methanol,
not liquid at ambient temperature should be heated to 35°C acetonitrile, or methanol/acetonitrile) into 10 mL of the test
where all of the compounds are in a liquid state. Use a 1-µL specimen in a test tube and cap securely. Mix using a vortex
syringe to add the indicated volumes of furanic compounds to mixer for 3 min for acetonitrile or acetonitrile/methanol
10 mL of acetonitrile or methanol. The volumes to be added are extractions or for 1 to 5 min for methanol extractions. Other
as follows: ratios of solvent to oil can be used as long as it is verified that
0.83 µL 6 1 % of 5-hydroxymethyl-2-furaldehyde the extraction efficiencies are unchanged.
0.88 µL 6 1 % of furfuryl alcohol
0.86 µL 6 1 % of 2-furaldehyde
12.2 Allow the two phases to separate. The top phase is the
0.91 µL 6 1 % of 2-acetylfuran extract, while the bottom phase consists of the nonpolar portion
0.90 µL 6 1 % of 5-methyl-2-furaldehyde of the test specimen. Separation may be enhanced by centrifu-
[Link] These volumes represent a mass of 1000 µg of each gation.
of the five furanic compounds. Add 10 mL of acetonitrile or 12.3 The extract may be run as is or may be diluted with
methanol containing the dissolved volumes of furanic com- water so that the resulting ratio of solvent to water is the same
pounds to 190 mL of the same solvent in a 1-L volumetric as that of the mobile phase used at the start of the HPLC run.
flask. Bring this solution to 1 L with water. Other ratios of NOTE 3—It has been found that filtering the extract prior to analysis by
solvent to water can be used such as to match that of the initial HPLC prolongs column life. Another effective method of cleanup is to

3
 D 5837 – 99e1
pass the extract through a precolumn.4 If a precolumn is used, the efficiencies for each extracted furanic compound, and daily
laboratory needs to verify by experimentation that there is no significant single-point calibration of equipment. Determination of extrac-
loss of furanic compounds. tion efficiencies is not necessary for the direct injection
13. Solid Phase Extraction (SPE)—Method B method.
14.2 Calibration Curve:
13.1 Insert SPE column(s) into the vacuum manifold and
14.2.1 As appropriate, for each furanic compound, inject an
pass 3 to 5 mL of hexane through each SPE column under
extraction standard in the solvent prepared in accordance with
vacuum. Do not dry the column.
Section 10 to determine the retention time.
13.2 Mix 10 mL of test specimen with 10 mL of hexane and
14.2.2 Develop a calibration curve for each compound at
pass through SPE column at a rate no faster than 3 mL/min.
three separate points representing three orders of magnitude,
Other quantities of oil can be used as long as it is verified that
such as 10 µg/L, 100 µg/L, and 1 mg/L (1000 µg/L) using
the extraction efficiencies are unchanged.
calibration standards in oil prepared in accordance with Section
13.3 Pass 10 to 20 mL hexane through the SPE column to
11.
rinse out residual oil and dry the column under vacuum for 5
14.2.3 Fig. 1 is an example of a calibration curve for each of
min. Discard all eluates.
the five furanic compounds. These curves were calibrated
13.4 Elute retained compounds from the SPE column using
based on results for standards at 10 µg/L, 100 µg/L, and 1 mg/L
an acetonitrile/water mixture composed of the same propor-
(1000 µg/L).
tions as in the HPLC system’s mobile phase. (20 % acetoni-
trile:80 % water has been found to be satisfactory.) Elute no 14.2.4 Determine calibration curves periodically to check
faster than 3 mL/min. the linearity of the HPLC UV detector(s). Some laboratories
13.5 Collect the first 2.0 to 2.5 mL of eluate from the SPE have found intervals of 30 to 90 days between determinations
column. Record the volume of eluate collected. to be acceptable.
13.6 Filter the eluate with a 0.5-µm or smaller polytetrafluo- 14.3 Extraction Effıciencies:
roethylene micro syringe filter prior to insertion for analysis in 14.3.1 To determine the extraction efficiency for each indi-
the HPLC system. Discard the spent SPE cartridge. vidual furanic compounds, run a 1-mg/L (1000 µg/L) extrac-
tion standard in solvent and a 1-mg/L calibration standard in
14. Calibration oil, each three times. Prepare standards in accordance with
14.1 Calibration consists of development of a calibration Sections 10 and 11. The average integrated peak area for each
curve for each furanic compound, development of extraction compound is used to calculate the extraction efficiency from
the appropriate equation as follows:
4
C18 Sep-Pakt (registered trademark of Waters Chromatography Division of For liquid/liquid extraction:
Millipore Corp.) has been found to be suitable. Available from Waters Chromatog- EE, % 5 ~Ro/Rs! 3 ~VE/10! 3 Df 3 100 (1)
raphy Division of Millipore Corp., 34 Maple Street, Milford, MA 01757.

Legend:
A. 5-hydroxymethyl-2-furaldehyde
B. 2-furfuraldehyde
C. 5-methyl-2-furaldehyde
D. 2-acetylfuran
E. furfuryl alcohol
FIG. 1 Example of a Calibration Curve

4
 D 5837 – 99e1
For solid-phase extraction: 16.2 Determine the integrated peak area for each compound
EE, % 5 ~Ro/Rs! 3 ~VE/10! 3 100 (2) of interest.
16.3 Calculate the concentration of each furanic compound
using the following equation:
where:
CT 5 ~RT/RS! 3 CS 3 ~VT/VE! (3)
EE = extraction efficiency calculated and expressed as a
percentage,
Ro = average integrated peak area of calibration standard where:
in oil at 1 mg/L (1000 µg/L), CT = concentration of furanic compound of interest in the
Rs = average integrated peak area of extraction standard in test specimen,
solvent at 1 mg/L (1000 µg/L), RT = integrated peak area for the furanic compound of
VE = volume of solvent or solvent water mix used for interest in the test specimen,
extraction (generally 1 to 2.5 mL), RS = integrated peak area for the furanic compound of
10 = constant (volume of oil standard used for analysis is interest in the daily calibration standard in oil,
10 mL), and CS = concentration of the furanic compound of interest in
Df = dilution factor for liquid/liquid extraction. This is the the daily calibration standard in oil for extracted
water to solvent ratio in the initial mobile phase of samples,
HPLC. If there is no dilution of the extract, Df = 1. If VE = volume of extraction solvent used to extract the
the extract is diluted with water before injection, calibration standard in oil for extracted samples, and
Df = volume of dilution water/volume of extract. VT = volume of extraction solvent used to extract the test
14.3.2 Typical extraction efficiencies based on one labora- specimen. For the direct injection technique, VENI is
tory’s experience are in Table 1. omitted as there are no extraction volumes to be
14.3.3 Determine extraction efficiencies periodically in ac- considered.
cordance with 14.3.1, using the same time interval as that
followed for determination of calibration curves as indicated in 17. Report
14.2.4. 17.1 Report the following information:
14.3.4 On a daily basis, run a single extraction standard in 17.1.1 A reference to this test method,
solvent to calculate extraction efficiency in conjunction with 17.1.2 Identification of the test specimen, and
the daily calibration standard in oil run in accordance with 14.4 17.1.3 Concentration in the test specimen of each furanic
to ensure extraction efficiencies are remaining with the ranges compound determined.
established for the laboratory.
14.4 Single-Point Calibration—On a daily basis, run a 18. Precision and Bias
calibration standard in oil. Use the integrated peak area for 18.1 Repeatability:
each compound of interest from this single-point, daily cali- 18.1.1 An estimate of repeatability by standard deviation for
bration to calculate results for unknown test specimens in liquid/liquid extraction of furanic compounds in mineral oil has
accordance with 16.3. been found by one laboratory to be as follows for ten
replicates:
15. Procedure 95 % repeatability interval as % of mean (nominal concentration of 100 µg/
15.1 Inject a known volume of the diluted extract from kg):
5-hydroxymethyl-2-furaldehyde: 11.7 %
Section 12 or 13 or the oil into the HPLC after stabilizing the furfuryl alcohol: 5.2 %
apparatus at the initial conditions. 2-furaldehyde: 8.0 %
15.2 Analyze the test specimens under the same conditions 2-acetylfuran: 5.5 %
5-methyl-2-furaldehyde: 3.3 %
as those used to analyze the standards in 14.3.4 and 14.4. Use 95 % repeatability interval as % of mean (nominal concentration of 1000
the same aliquot volume (10 mL for extracted samples) for the µg/kg):
unknown test specimen as that which is used for the calibration 5-hydroxymethyl-2-furaldehyde: 2.7 %
furfuryl alcohol: 5.3 %
standard. 2-furaldehyde: 2.4 %
2-acetylfuran: 0.6 %
16. Calculation 5-methyl-2-furaldehyde: 3.2 %
16.1 Identify the furanic compounds represented by each 18.1.2 An estimate of repeatability by standard deviation for
individual peak by comparison of retention times with those solid phase extraction of furanic compounds in mineral oil has
obtained for the standards during the calibration process. been found by one laboratory to be as follows for ten
replicates:
TABLE 1 Typical Extraction Efficiencies
95 % repeatability interval as % of mean (nominal concentration of 100 µg/
Liquid/Liquid Solid-Phase kg):
Extraction, % Extraction, % 5-hydroxymethyl-2-furaldehyde: 5.5 %
5-hydroxymethyl-2-furaldehyde 97–100 97–100 furfuryl alcohol: 16.7 %
furfuryl alcohol 76–83 95–100 2-furaldehyde: 6.0 %
2-furaldehyde 71–77 88–96 2-acetylfuran: 7.7 %
2-acetylfuran 66–72 89–97 5-methyl-2-furaldehyde: 6.0 %
5-methyl-2-furaldehyde 63–68 93–99 95 % repeatability interval as % of mean (nominal concentration of 1000
µg/kg):

5
 D 5837 – 99e1
5-hydroxymethyl-2-furaldehyde: 10.2 % 5-hydroxymethyl-2-furaldehyde: 3.9 %
furfuryl alcohol: 13.7 % 2-furaldehyde: 3.7 %
2-furaldehyde: 9.6 % 2-acetylfuran: 3.2 %
2-acetylfuran: 11.0 % 5-methyl-2-furaldehyde: 2.1 %
5-methyl-2-furaldehyde: 11.0 %
18.2 Reproducibility—A round-robin is planned to establish
18.1.3 An estimate for repeatability by standard deviation
reproducibility of this test method.
for the direct injection method of furanic compounds in
mineral oil has been found by one laboratory to be as follows 18.3 Bias—A round-robin is planned to establish bias of this
for ten replicates: test method.
95 % repeatability interval as % of mean (nominal concentration of 100 µg/
kg): 19. Keywords
5-hydroxymethyl-2-furaldehyde: 4.9 %
2-furaldehyde: 10.6 % 19.1 direct injection; furaldehyde; furanoid; furans; furanic;
2-acetylfuran: 12.6 %
5-methyl-2-furaldehyde: 6.0 %
furfuraldehyde; furfuryl alcohol; furfurylfurfurol; furyl; fur-
95 % repeatability interval as % of mean (nominal concentration of 1000 fural; HPLC; liquid/liquid extraction; solid-phase extraction;
µg/kg): SPE;5-hydroxymethyl-2-furaldehyde;2-furaldehyde;2-acetylfuran;5-methyl-2-f

APPENDIXES

(Nonmandatory Information)

X1. EXAMPLE CHROMATOGRAMS

X1.1 Fig. X1.1, Fig. X1.2 and Fig. X1.3 are sample
chromatograms of 100-µg/L calibration standards in oil differ-
ing in extraction procedures and operating parameters.

Retention Time, min Compound

2.22 5-hydroxymethyl-2-furaldehyde
4.11 furfuryl alcohol
Retention Time, min Compound 5.86 2-furaldehyde
2.91 5-hydroxymethyl-2-furaldehyde 8.71 2-acetylfuran
3.65 furfuryl alcohol 10.32 5-methyl-2-furaldehyde
4.32 2-furaldehyde
Extraction: Liquid/liquid extraction
5.27 2-acetylfuran
Column temperature: 40°C
6.42 5-methyl-2-furaldehyde
Injection volume: 20 µL
Extraction: Solid phase extraction (SPE) Column: 4.1 3 150 mm PRP-1, 75A, 5 µm
Column temperature: 30°C (Hamilton)
Injection volume: 15 µL Mobile-Phase Gradient:
Column: C18, 3.9 3 400 mm, 60A, 4 µm Mobile-Phase Composition Flow Rate, mL/min Time, min
(NovapakT—Waters) 60 % methanol, 40 % water to 70 % meth- 0.5 initial–14.00
Mobile-Phase Gradient: anol, 30 % water
Mobile-Phase Composition Flow Rate, mL/min Time, min 70 % methanol, 30 % water to 100 % 0.5 14.00–34.00
20 % acetonitrile, 80 % water 1.0 initial–7.30 methanol
100 % acetonitrile 1.0 7.30–7.50 100 % methanol to 60 % methanol, 40 % 1.5 34.00–45.00
100 % acetonitrile 1.25 7.50–8.00 water
100 % acetonitrile 1.5 8.00–14.00 60 % methanol, 40 % water 0.5 45.00—flat
20 % acetonitrile, 80 % water 1.5 14.00–23.00 baseline
20 % acetonitrile, 80 % water 1.0 23.00–28.00
FIG. X1.2 Chromatogram 2
FIG. X1.1 Chromatogram 1

6
 D 5837 – 99e1

FIG. X1.3 Chromatogram 3

X2. Mobile Phase Gradient

X2.1 The following gradient corresponds to the HPLC

conditions of 7.6 for direct injection:
Time (min) Flow Rate (mL/min) Acetonitrile, % Water, %

initial 1.0 20 80
3.0 1.0 50 50
5.3 1.5 50 50
7.0 2.0 100 0
9.0 3.0 100 0
10.0 3.5 100 0
18.8 2.0 100 0
20.0 2.0 50 50
22.0 2.0 20 80
25.0 1.0 20 80

7
 D 5837 – 99e1
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