Tert – Butyl Hydroquinone [TBHQ]

A. METHOD OF TEST FOR TERT BUTYL HYDROQUINONE (TBHQ)
A-1 PURITY
A-1.1 Reagents
A-1.1.1 Methanol
A-1.1.2 Sulphuric Acid - 1 N
A-1.1.3 Diphenylamine Indicator - 3 mg of p-diphenylaminesulphonic acid sodium salt per ml of 0.1 N sulphuric acid.
A-1.1.4 Ceric Sulphate - 0.1 N
A-1.2 Procedure - Transfer about 170 mg of the sample, ground to a fine powder and accurately weighed, into a 250-ml wide-mouthed Erlenmeyer flask, and dissolve in 10 ml of methanol. Add 150 ml of water, 1 ml of 1 N sulphuric acid, and 4 drops of diphenylamine indicator. Titrate with 0.1 N ceric sulphate to the just complete colour change from yellow to red-violet. Record the volume, in ml of 0.1 N ceric sulphate required. If HQ and DTBHQ are present in the sample they will be included in the titration and the correction as given in the formula shall be applied.

A-1.3 Calculation
Purity, as C₁₀H₁₄O₂, percent by mass (A) =

(8.311 x N (V - 0.1)) / M, where
V = volume, in ml, of 0.1 N ceric sulphate required;
N= normality of the ceric sulphate solution; and
M = mass, in grams, of the sample taken

NOTE - 0.1 ml represents the volume of ceric sulphate solution consumed by the primary oxidation products of tert-butyl hydroquinone ordinarily present in the sample.
Purity, as C₁₀H₁₄O₂, corrected percent by mass =
A - (percent HQ x 1.51) - (percent DTBHQ x 0.75)

A-2 t-BUTYL-p-BENZOQUINONE
A-2.1 Apparatus
A-2.1.1 Spectrophotometer - A double-beam infrared spectrophotometer with matched 0.4 mm liquid sample cells with calcium flouride windows.
A-2.2 Reagents
A-2.2.1 Mono tertiary-butyl-p-benzoquinone Reference Standard
A-2.2.2 Carbon Tetrachloride
A-2.3 Procedure
A-2.3.1 Preparation of Standard - Transfer about 10 mg of accurately weighed mono tertiary-butyl-p-benzoquinone Reference Standard, into a 10 ml volumetric flask. Dissolve in carbon tetrachloride, dilute to volume with the same solvent and mix.

A-2.3.2 Preparation of Sample - Transfer about 1 g of the sample, accurately weighed and previously reduced to a fine powder in a high speed blender, into a 10 ml volumetric flask. Dilute to volume with carbon tetrachloride, and shake for 5 minutes to extract the t-butyl- benzoquinone. Filter through a millipore filter or equivalent before use.

A-2.3.3 Fill the reference cell with carbon tetrachloride and the sample cell with the standard preparation [A-2.3.1]. Place the cells in the respective reference and sample beams of the spectrophotometer and record the infra spectrum from 1600 to 1775 cm-1. On the spectrum draw a background line from 1612 to 1750 cm-1, and determine the net absorbance (As) of the standard preparation at 1659 cm-1. Similarly, obtain the spectrum of the sample preparation [A-2.3.2], and determine its net absorbance (A_u) at 1659 cm ^-1.

A-2.4 Calculation
t-butyl-p-benzoquinone, percent by mass =

(A_u / A_s) x (W_s / W_u) x 100, where
A_u = net absorbance of the sample preparation;
A_s = net absorbance of the standard preparation;
W_s = exact mass, in mg, of the Reference Standard taken; and
W_u = exact mass, in mg, of the sample taken

A-3 2,5 Di-t-BUTYL HYDROQUINONE AND HYDROQUINONE
A-3.1 Apparatus
A-3.1.1 Gas Chromatograph - of a suitable type equipped with thermal conductivity detector, containing a 0.61 m x 6.35 mm (O.D.) stainless steel column packed with 20 percent Silicone SE-30, by mass and 80 percent Diatoport S (60/80 - mesh), or equivalent materials.

A-3.1.1.1 Operating conditions - The operating parameters may vary depending upon the particular instrument used, but a suitable chromatogram may be obtained using the following conditions:

a) Column temperature - programmed from 100 to 270°C at 15°C per minute;
b) Injection port temperature - 300°C;
c) Carrier gas - helium or nitrogen flowing at a rate of 100 ml per minute;
d) Bridge current - 140 ma; and
e) Sensitivity - 1 x for integrator, 2 x for recorder

A-3.2 Reagents
A-3.2.1 Hydroquinone, 2, 5 di-t-butyl hydroquinone and Methyl Benzoate stock solution - Weigh accurately about 50 mg each of hydroquinone (HQ) 2,5-di-t-butylhydroquinone (DTBHQ), and methyl benzoate (internal standard). Transfer into separate 50-ml volumetric flasks, dilute to volume with pyridine and mix.

A-3.3 Procedure
A-3.3.1 Calibration Standard - Into separate 10 ml volumetric flasks, add 0.50, 1.00, 2.00 and 3.00 ml of the HQ stock solution. Then to each flask add 2.00 ml of methyl benzoate (internal standard) stock solution; dilute each to volume with pyridine and mix, In the same manner prepare four DTBHQ calibrating solutions. Prepare the trimethylsilyl derivative of each solution as follows - add 9 drops of calibration solution to a 2 ml serum vial, cap the vial, evacuate with a 50 ml gas syringe, add 250 ml of N,O-bis-trimethylsilyl-acetamide, and heat at about 80°C for 10 minutes. Chromatograph 10 ml portions of each standard in duplicate, and plot the concentration ratio of HQ to internal standard (X-axis) against response ratio of HQ to internal standard (Y-axis). Plot the same relationship between DTBHQ and the internal standard.

A-3.3.2 Sample Preparation and Procedure - Transfer about 1 g of the sample, accurately weighed, into a 10 ml volumetric flask. Add 2.00 ml of the methyl benzoate internal standard stock solution, dilute to volume with pyridine and mix, Prepare the trimethylsilyl derivative as described in [A-3.3.1] and then chromatograph duplicate 10 ml portions to obtain the chromatogram. The approximate peak times in minutes are: Methyl benzoate-2.5; trimethylsilyl derivative of HQ-5.5; trimethylsilyl derivative of tert-butylhydroquinone-7.3; trimethylsilyl derivative of DTBHQ-8.4.

A-3.4 Calculation
A-3.4.1 Determine the peak area (response) of interest by automatic integration or manual triangulation. Calculate the response ratio of HQ and DTBHQ to internal standard, From the calibration curves determine the concentration ratio of HQ and DTBHQ to internal standard, and calculate percent HQ and percent DTBHQ as follows:

HQ or DTBHQ, percent by mass =

(Y x I x (10/S)), where
Y = concentration ratio (X-axis on calibration curve);
I = percent (w/u) of internal standard in the sample preparation; and
S = mass, in grams, of the sample taken

A-4 HEAVY METALS
A-4.1 Reagents
A-4.1.1 Ammonia Solution - Dilute 400 ml of ammonium hydroxide (28 percent) to 1000 ml with water.

A-4.1.2 Hydrochloric Acid - 10 percent.
A-4.1.3 Lead Nitrate, stock solution - Dissolve 159.8 mg of lead nitrate in 100 ml of water containing 1 ml of nitric acid. Dilute with water to 1000 ml and mix. Prepare and store the solution in lead-free glass containers.

A-4.1.4 Standard Lead Solution - Dilute 10 ml of lead nitrate stock solution, accurately measured, with water to loo mI. Each mI of the solution so prepared contains the equivalent of 10 mg of lead ion (Pb). Prepare the solution on the day of use.

A-4.1.5 Nitric Acid - 10 percent (u/u)
A-4.1.6 Sulphuric Acid - 94.5 to 95.5 percent (u/u)
A-4.1.7 Acetic Acid -6 percent (m/u)
A-4.1.8 Hydrogen Sulphide - A saturated solution of hydrogen sulphide made by passing H₂S in cold water.

A-4.2 Procedure
A-4.2.1 Solution A – Take 2 ml of the standard lead solution in a 50 m Nessler tube and add 23 ml of water. Adjust the pH to between 3.0 and 4.0 by addition of acetic acid or ammonia solution. Dilute with water to 40 ml and mix.

A-4.2.2 Solution B – Place 500 mg of the sample, accurately weighed in a suitable crucible, add sufficient nitric acid to wet the sample, and carefully ignite at a low temperature until thoroughly charred, covering the crucible loosely with a suitable lid during the ignition. After the substance is thoroughly carbonised, add 2 ml of nitric acid and 5 drops of sulphuric acid and cautiously heat until white fumes are evolved. Then ignite, preferably in a muffle furnace at 500 to 600°C until the carbon is all burnt off. Cool, add 4 ml of dilute hydrochloric acid; cover and digest on a steam bath for 10 to 15 minutes. Uncover and slowly evaporate on a steam-bath to dryness. Moisten the residue with one drop of hydrochloric acid, add 10 ml of hot water and digest for 2 minutes. Add drop wise ammonia solution until the solution is just alkaline to litmus paper, dilute with water to 25 ml and adjust the pH to between 3.0 and 4.0 (pH indicator paper) by the addition of diluted acetic acid. Filter, if necessary. Wash the crucible and the filter with 10ml of water. Transfer to a 50 ml Nessler tube. Dilute the combined filtrate and washing with water to 40 ml and mix.

A-4.2.3 To each tube add 10 ml of freshly prepared hydrogen sulphine, mix and allow to stand for 45 minutes and view down over a white surface. The colour of [Solution B] shall not be darker than that of [Solution A].

A-5. TOLUENE
A-5.1 Apparatus
A-5.1.1 Gas Chromatograph - of a suitable type equipped with a flame ionization detector containing a 3.66 m X 3.18 mm (O.D) stainless steel column packed with 10 percent Silicone SE-30, by mass, and 90 percent Diatoport S (60/80-mesh), or equivalent material.

A-5.1.1.1 Operating conditions - The operating parameter may vary depending upon the particular instrument used, but a suitable chromatogram may be obtained using the following conditions:

a) Column temperature - programmed from 70 to 80°C at 15°C per minute and held.
b) Injection port temperature - 275°C
c) Carrier gas - helium or nitrogen flowing at a rate of 50 ml per minute
d) Cell temperature - 300°C
e) Hydrogen and air settings - 138 kPa each
f) Sensitivity - 1 x 10²

A-5.2 Reagents
A-5.2.1 Toluene
A-5.2.2 Octyl Alcohol
A-5.3 Procedure
A-5.3.1 Preparation of Standard Toluene Solution - Prepare a solution of toluene in octyl alcohol containing approximately 50 mg per ml and , calculate the exact concentration (Cr) in percent (m/u).

A-5.3.2 Preparation of Sample Solution - Transfer about 2 g of the sample accurately weighed, into a 10 ml volumetric flask. Dissolve in octyl alcohol. Dilute to volume with the same solvent and mix. Calculate the exact concentration of the solution (Cs) in percent (m/u).

A-5.3.3 Inject a 5 ml portion of the standard solution into the chromatograph, and measure the height of the toluene peak on the chromatogram. The toluene retention time is 3.3 minutes. Similarly obtain the chromatogram on a 5 ml portion of the sample solution, and measure the height of the toluene peak. Calculate the percentage of toluene in the sample.

A-5.4 Calculation
Toluene, mg/kg =

(H_s/ H_r) x (C_r/ C_s) x 106, where
H_s = height of the toluene peak of the sample solution;
H_r = height of the toluene peak of the standard solution;
C_s = concentration in percent ( m/u ) of the sample solution; and
C_r = concentration in percent ( m/u ) of the standard solution

A-6 ULTRA VIOLET ABSORBANCE
A-6.1 Reagents
A-6.1.1 L-Ascorbic Acid
A-6.1.2 Ethanol
A-6.1.3 Isooctane
A-6.1.4 Anhydrous Sodium Sulphate
A-6.1.5 Hexadecane
A-6.2 Procedure
A-6.2.1 Dissolve 1 g of L-ascorbic acid in 100 ml of ethanol and 100 ml of water contained in a 500 ml separator (S-1). Transfer about 50 g of the sample, accurately weighed into the separator. Shake to dissolve, then add 50 ml of isooctane and extract for 3 After the phases have separated, drain the lower aqueous phase into a second 500 ml separator ( S-2 ), then after 1 minute of separating, drain the lower layer into the separator (S-2). Add a second 50 ml portion of isooctane to the aqueous solution in repeat the extraction procedure as previously described, drawing lower, aqueous layer into a third 500 ml separator (S-3). Add third 50 ml portion of isooctane to the aqueous solution and repeat the extraction procedure as previously described, drawing off and discarding the lower aqueous layer.

A-6.2.2 Extract each isooctane solution (that is, the solution in S-1, S-2, S-3), with two 100 ml portions of a 0.5 percent solution of ascorbic acid in ethanol-water (25:75). Shake each mixture for 1 minute, phases to separate, and discard the lower, aqueous layer: extract each isooctane solution with two 100 ml portions of a 5 percent solution of ethanol in water, and discard the lower, aqueous layers. Finally, wash each solution twice with 100 ml of water, and discard the washes.

A-6.2.3 Lightly pack a standard size chromatographic tube with 100 g of anhydrous sodium sulphate, and wash the packed with 75 ml of isooctane, discarding the wash. Filter the solution from S-1 through the column, and collect filtrate in distillation flask. Wash S-1 with the isooctane solution contained in S-2, and then pour the solution onto the column, collecting the filtrate in the flask. Wash S-2 and S-I, successively, with the isooctane in S-3, and filter the solution through the column as before. Wash S-3, S-2 and S-1 in that order and in tandem with two successive portions of isooctane, and pass the washings individually through the column and into the flask. Let the column drain completely.

A-6.2.4 Add 2 ml of hexadecane and 2 boiling stones to the 500 ml distillation flask containing the combined isooctane extracts, and attach the flask to a suitable vacuum distillation assembly. Evacuate the assembly to about one-third atmosphere, then immerse the flask in a steam bath, and distill the solvent. When isooctane stops dripping the receiver, turn off the vacuum, wash down the walls of with 5 ml of isooctane added through the top of the distillation, then replace the thermometer and again evacuate. The isooctane should distill over in about 1 minute. At the end of this distillation add another 5-ml portion of isooctane and repeat the stripping procedure.

A-6.2.5 Quantitatively wash the residue from the distillation flask into a 50 ml volumetric flask with isooctane, dilute to volume with isooctane, and mix. Determine the ultraviolet absorption spectrum of the solution in a 5 cm silica cell from 400 mm to 250 mm, with a suitable spectrophotometer, using isooctane as the blank. Determine the absorbance of a solvent control by following the above procedure in a every detail, but with the sample omitted. From the sample spectrum determine the maximum absorbance per cm path length in each of the following wavelength intervals: (a) 280-289 mm; (b) 290-299 mm; (c) 300-359 mm; and (d) 360-400 mm. Calculate the maximum net absorbance per cm in each interval by subtracting from the sample absorbance the corresponding absorbance per cm of the solvent control. The following net absorbance values are not exceeded at the indicated intervals: (a) 0.15; (b) 0.12; (c) 0.08; and (d) 0.02.