ASTM D5837 Standard Test Method for Furanic Compounds in Electrical Insulating Liquids by High-Performance Liquid Chromatography (HPLC)
14. Calibration
14.1 Calibration consists of development of a calibration curve for each furanic compound, development of extraction efficiencies for each extracted furanic compound, and daily single-point calibration of equipment. Determination of extraction efficiencies is not necessary for the direct injection method.

14.2 Calibration Curve:
14.2.1 As appropriate, for each furanic compound, inject an extraction standard in the solvent prepared in accordance with Section 10 to determine the retention time.

14.2.2 Develop a calibration curve for each compound at three separate points representing three orders of magnitude, such as 10 µg/L, 100 µg/L, and 1 mg/L (1000 µg/L) using calibration standards in oil prepared in accordance with Section 11.

14.2.3 Fig. 1 is an example of a calibration curve for each of the five furanic compounds. These curves were calibrated based on results for standards at 10 µg/L, 100 µg/L, and 1 mg/L (1000 µg/L).

14.2.4 Determine calibration curves periodically to check the linearity of the HPLC UV detector(s). Some laboratories have found intervals of 30 to 90 days between determinations to be acceptable.

14.3 Extraction Efficiencies:
14.3.1 To determine the extraction efficiency for each individual furanic compounds, run a 1-mg/L (1000 µg/L) extraction standard in solvent and a 1-mg/L calibration standard in oil, each three times. Prepare standards in accordance with Sections 10 and 11. The average integrated peak area for each compound is used to calculate the extraction efficiency from the appropriate equation as follows:
For liquid/liquid extraction:
EE, % = (Ro/Rs) x (VE/10) x Df x 100

For solid-phase extraction:
EE, % = (Ro/Rs) x (VE/10) x 100
where:
EE = extraction efficiency calculated and expressed as a percentage,
Ro = average integrated peak area of calibration standard in oil at 1 mg/L (1000 µg/L),
Rs = average integrated peak area of extraction standard in solvent at 1 mg/L (1000 µg/L),
VE = volume of solvent or solvent water mix used for extraction (generally 1 to 2.5 mL),
10 = constant (volume of oil standard used for analysis is 10 mL), and
Df = dilution factor for liquid/liquid extraction. This is the water to solvent ratio in the initial mobile phase of HPLC. If there is no dilution of the extract, Df = 1. If the extract is diluted with water before injection, Df = volume of dilution water/volume of extract.

14.3.2 Typical extraction efficiencies based on one laboratory's experience are in Table 1.

14.3.3 Determine extraction efficiencies periodically in accordance with 14.3.1, using the same time interval as that followed for determination of calibration curves as indicated in 14.2.4.

14.3.4 On a daily basis, run a single extraction standard in solvent to calculate extraction efficiency in conjunction with the daily calibration standard in oil run in accordance with 14.4 to ensure extraction efficiencies are remaining with the ranges established for the laboratory.

14.4 Single-Point Calibration - On a daily basis, run a calibration standard in oil. Use the integrated peak area for each compound of interest from this single-point, daily calibration to calculate results for unknown test specimens in accordance with 16.3.

15. Procedure
15.1 Inject a known volume of the diluted extract from Section 12 or 13 or the oil into the HPLC after stabilizing the apparatus at the initial conditions.

15.2 Analyze the test specimens under the same conditions as those used to analyze the standards in 14.3.4 and 14.4. Use the same aliquot volume (10 mL for extracted samples) for the unknown test specimen as that which is used for the calibration standard.

16. Calculation
16.1 Identify the furanic compounds represented by each individual peak by comparison of retention times with those obtained for the standards during the calibration process.

16.2 Determine the integrated peak area for each compound of interest.

16.3 Calculate the concentration of each furanic compound using the following equation:
CT = (RT/RS) x CS x (VT/VE)
where:
CT = concentration of furanic compound of interest in the test specimen,
RT = integrated peak area for the furanic compound of interest in the test specimen,
RS = integrated peak area for the furanic compound of interest in the daily calibration standard in oil,
CS = concentration of the furanic compound of interest in the daily calibration standard in oil for extracted samples,
VE = volume of extraction solvent used to extract the calibration standard in oil for extracted samples, and
VT = volume of extraction solvent used to extract the test specimen. For the direct injection technique, VENI is omitted as there are no extraction volumes to be considered.